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Leaf-Tissue Freeze-Tolerance Mechanisms in Bahiagrass (Paspalum notatum Fluegge)


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L EA F -T I SSU E F REE Z ETO L ERA NC E M ECH AN I SMS I N B AH I AG RA SS ( Paspalum notatum Flegge) By J ACQUE WI L L I AM BREMAN A DI SSER TATI ON PRESENTED TO THE G RADUATE SCHOOL OF T HE UNI VERSI TY OF FL ORI DA I N PARTI AL FUL FI L L MENT OF T HE REQUI REMENTS FOR THE DE GREE OF DOCTOR OF PHI L OSOPHY UNI VERSI TY OF FL ORI DA 2006

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Copy rig ht 2006 by J acque Wil liam Bre man

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To my pare nts, J anet a nd J ohn Br eman

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iv ACKNOWL EDGMENTS We all stand on the shoulders of those w ho prec ede us. We tra vel trails blazed by others. I hope that this five-y ear eff ort provides support a nd information for othe rs who trave l this t rail of investig ation. Ac kn ow le dg me nts a re imp or ta nt i n r e c og nizi ng tho se ke y pe op le wh o ma de thi s e f f o r t p o s s i b l e : T h e s u p e r v i s o r y c o m m i t t e e m e m b e r s t h e g r a d u a t e c o o r d i n a t o r f a m i l y, friends a nd staff. The super visory committee g uides the students lea rning and re sear ch proje ct. Eac h committee member leave s a leg acy in the way the student is molded. A students standing in the aca demic wor ld refle cts the quality of the c ommitt ee. I d like to thank Dr. Que senber ry who taug ht me skills i n cy tology and plant bre eding and shar ed his love of tea ching Dr. B lount renew ed my enthusiasm for f ield work a nd plant exploration. Dr. Sinclair c halleng ed me to think critica lly about re sults, as well as the nee d to quantify and re cord a ll data for r efle ctive thinking. D r. B arne tt taught me pla nt bree ding strateg ies to shorten the time nee ded to provide improve d cultivars. Dr Coleman taug ht me the joy of re sear ch, the e x citement of que sting a fter ne w knowledg e, and the deep sa tis fa c tio n o f o bta ini ng re su lts D r. Col e ma n a nd I we re tr uly a te a m on ou r p ro je c ts with animalscienc e applica tions. A g ood g radua te coor dinator also provide s g uidance and mentorship. I was fortunate to have a n excellent g radua te coor dinator. Dr. Woffor d taug ht me the importance of under standing and quantify ing the mode of tra it inheritance to develop

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v eff icient bre eding prog rams. Additionally Dr. Wofford ope ned my vision to l arg er horizons during our visits. I hope we can c ontinue those cha ts in the future. Fa mily is crucia l for emotional support. My study was pe rhaps more taxi ng since it was underta ken while wor king f ull tim e for the Flor ida Coopera tive Ex tension Service. Gra duate sc hool and independe nt resea rch ta x es the e nduranc e of the soul when experiments go a wry equipment fa ils, and examination and other dea dlines loom omi no us ly Co nti nu ing the e ff or t un a ide d a t ni g ht a nd on we e ke nd s w a s ma de po ssi ble by family support. My brothers Jim and J eff Br eman ha ve bee n there for me. I want to espe cially thank and r ecog nize my pare nts, J ohn and Janet Br eman. Despite their a g e, they at times worked phy sically beside me w hen experiments had to be prepa red or completed. My pare nts were alway s available by phone whe n I neede d an emotional lifeline. Their emotional support and pra y ers we re unw aver ing in spite of the experience d darkne ss of the times. My pare nts deserve the hig hest respe ct and honor for their dedic ation. I tha nk my da ug hte r, L a ur e l Sc ha a fs ma a nd my so n, Jona tha n B re ma n, fo r t he ir respe ct, love, and e motional support. Their spouses, Ke ith Schaafsma a nd J ennife r Br eman, unde rstood my need to f ind respite by spending time with my g randc hildren (Na than B rema n, Gavin B rema n, and Alec Schaaf sma). Som e on e wh o h a s w a lke d w ith me thr ou g ho ut t he la st p or tio n o f t his pr oje c t is my fianc e, A licia. My best frie nd and my life compa nion-to-be ha s cele brate d the projec ts succe sses, g rieve d the fa ilures, and stea dfastly been the re e ach ste p of the long way reg ardle ss of the cha lleng es. Without Alicia, it would have be en diffic ult to gathe r my self up fr om setbacks to continue towa rd completion. We both look forwa rd to the

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vi day when we can f inally be tog ether as husband a nd wife instea d of 1,500 miles apar t. Al ic ia is m y joy a nd ha pp ine ss. I have to r ecog nize a friend w ho was a lso a staff me mber in the Animal Scienc e depar tment. J ohn Funk, biolog ical scie ntist, i s one of those spe cial people who went above a nd bey ond his job to m ake sur e the students r esea rch w as succ essful. John m ade sure that e quipment was working materia ls were available technique w as prope r, and results wer e satisfa ctory befor e letting me work on my own. During the long night a nd we e ke nd ho ur s, I kn e w I c ou ld c ou nt o n Joh n to c he c k o n me so me tim e s to ma ke su re my rese arc h was g oing w ell. J ohn taug ht me the prope r microtome tec hnique which made the ana tomical study succe ssful. I n the proc ess of the r esea rch w ork, I g ained a friend f or whom I have so much respect. Ge tti ng e qu ipm e nt t o o pe ra te a nd de ve lop ing a c on sis te nt t e c hn iqu e a re c ru c ia l to ob ta ini ng c on sis te nt r e su lts T wo sp e c ia l pe op le wh o h e lpe d me in t he fa tty a c id a na ly sis work we re Rober t B ob Que rns, B iologica l Scientist, and Georg e Person, labor atory technicia n. Bob w as helpful with sug g esting various proc edure s. Georg e wa s a true partne r in revie wing curr ent literature with me and compa ring differ ent proc edure s. He made sure that the Hew lett-Packa rd g as chr omatog raph w as working proper ly which produce d data that ha d the lowest possible coe fficie nt of varia bility The thre e of us cele brate d the completion of the f atty acid e x periments as ha ppily as if we had bee n a team wor king f or y ear s. R i ch ar d Fe t h i er e, co o rd i n at o r o f R es ea rc h P ro gra m s i n t h e A gro n o m y Fo ra ge E v a lu a ti o n L a b o r a to r y w a s in s tr u me n ta l i n le tt in g me ta k e p r io r it y s o I c o u ld a n a ly ze two y ear s worth of nitrog en and in v itr o or g a nic ma tte r d ig e sti bil ity re su lts T his

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vii expedited the animal-scie nce r esea rch pr ojects with Dr. Coleman in time to prese nt an abstrac t and poster f or the I nterna tional Grasslands Cong ress in I rela nd. Edwin Ed B ow e rs b iol og ist a t B ro ok sv ill e Sub tr op ic a l A g ri c ult ur a l Re se a rc h St a tio n, he lpe d me g et throug h some of the ne ar infr are d scanning quirks of the softwa re a nd equipment in a most patient and supportive manne r. This helped us a chieve results in time for the imp os e d d e a dli ne s. Ac kn ow le dg me nts of g ra du a te c omm itt e e f a mil y f ri e nd s a nd sta ff ha ve on ly cover ed the hig h points of this project. This is in no way meant to minimiz e or e x clude fa c ult y f a mil y me mbe rs f ri e nd s, a nd sta ff wh o h a ve be e n s up po rt ive a nd he lpf ul i n s o many way s. We are not islands. We do not stand alone. A s we tra vel the cour se of our day s, we tra vel trails forme d by others and hope fully leave signs a long the way to make t h e j o u r n e y e a s i e r f o r o t h e r s I h o p e t h a t t h i s p r o j e c t h e l p s o t h e r s a l o n g t h e w a y. M a y I help others a s kindly as those who ha ve g rac iously offe red the ir assistance .

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vii i TAB L E OF CONTENTS p age A C K N O W L E D G M E N T S ................................................ iv L I S T O F T A B L E S ...................................................... xi L I S T O F F I G U R E S .................................................... xiv A B S T R A C T .......................................................... xv CHAPTER 1 I N T R O D U C T I O N .................................................... 1 P l a n t C o l d i n j u r y a n d C o l d t o l e r a n c e ..................................... 1 C h i l l i n j u r y ....................................................... 2 F r e e z e S t r e s s ..................................................... 5 F r e e z e P r o c e s s .................................................... 5 S u g g e s t e d S c h e m e ................................................. 7 P l a n t F r e e z e t o l e r a n c e S u m m a r y ..................................... 15 B a h i a g r a s s i n t h e S o u t h e a s t U S ......................................... 16 I m p o r t a n c e a n d U s e ............................................... 16 Adaptation and G eog raphic Distribution . . . . . . . . . . . . . . . 16 Calendar For ag e Dr y Matter Produc tion . . . . . . . . . . . . . . . 17 Com me rc ia l Cu lti va r F re e zetol e ra nc e Va ri a bil ity . . . . . . . . . . . 17 Pot e nti a l B a hia g ra ss L e a f F re e zetol e ra nc e Tr a it M e c ha nis ms . . . . . . . . 18 A n a t o m i c a l ...................................................... 18 P h y s i o l o g i c a l .................................................... 21 G e n e t i c ......................................................... 21 O u t l i n e o f t h e R e s e a r c h P l a n ........................................... 23 2 LE A F T IS S U E F R E E Z E T O LE R A N C E T R A IT D IV E R S IT Y IN B A H I A G R A S S ..................................................... 27 I n t r o d u c t i o n ........................................................ 27 L e a f t i s s u e F r e e z e t o l e r a n c e ........................................... 28 M a t e r i a l s a n d M e t h o d s ................................................ 30 L e a ftis su e F re e zetol e ra nc e Tr a it Sc re e nin g Expe ri me nt 1 . . . . . . . . 30 L e a ftis su e F re e zetol e ra nc e Tr a it Sc re e nin g Expe ri me nt 2 . . . . . . . . 33

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ix L e a f v s R o o t E f f e c t s E x p e r i m e n t 3 ................................... 34 R e s u l t s a n d D i s c u s s i o n ............................................... 35 L e a ftis su e F re e zetr e a tme nt S c re e nin g Expe ri me nt 1 . . . . . . . . . . 35 L e a ftis su e F re e zetol e ra nc e Sc re e nin g Expe ri me nt 2 . . . . . . . . . . 41 L e a f v s R o o t E f f e c t s E x p e r i m e n t 3 ................................... 43 S u m m a r y .......................................................... 45 3 AN AT OM Y R EL AT ED TO L EA F -T I SSU E F REE Z ETO L ERA NC E . . . . . 47 I n t r o d u c t i o n ........................................................ 47 M a t e r i a l s a n d M e t h o d s ................................................ 50 I n i t i a l T w o l i n e E x p e r i m e n t ......................................... 50 E i g h t l i n e E x p e r i m e n t ............................................. 53 R e s u l t s a n d D i s c u s s i o n ............................................... 54 I n i t i a l T w o l i n e E x p e r i m e n t ......................................... 54 E i g h t l i n e E x p e r i m e n t ............................................. 57 S u m m a r y .......................................................... 61 4 PHYSI OL OGI CAL MECHANI SMS ASSOCI ATED WI TH L EAF -TI SSUE F R E E Z E T O L E R A N C E .............................................. 63 I n t r o d u c t i o n ........................................................ 63 F a t t y A c i d C o m p o s i t i o n ............................................ 66 M a t e r i a l a n d M e t h o d s ................................................ 71 O s m o l a l i t y ...................................................... 71 F a t t y A c i d C o m p o s i t i o n ............................................ 73 R e s u l t s a n d D i s c u s s i o n ............................................... 76 O s m o l a l i t y ...................................................... 76 F a t t y A c i d C o m p o s i t i o n ............................................ 77 S u m m a r y .......................................................... 82 5 GE NE TI C B EH AV I OR OF TH E L EA F -T I SSU E F REE Z ETO L ERA NC E T R A I T ............................................................ 83 I n t r o d u c t i o n ........................................................ 83 T e t r a p l o i d R e p r o d u c t i o n .............................................. 83 D i p l o i d R e p r o d u c t i o n................................................. 84 M a t e r i a l a n d M e t h o d s ................................................ 89 S e e d P r o d u c t i o n F r o m C l o n e s ...................................... 89 E m e r g e n c e f r o m S e e d ............................................. 92 D i a l l e l ......................................................... 94 R e s u l t s a n d D i s c u s s i o n ............................................... 98 S e e d P r o d u c t i o n f r o m C l o n e s ....................................... 98 E m e r g e n c e f r o m S e e d ............................................. 99 D i a l l e l 2 0 0 4 ................................................... 101 D i a l l e l 2 0 0 5 ................................................... 103 S u m m a r y ........................................................ 109

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x 6 C O N C L U S I O N S ................................................... 112 I n t r o d u c t i o n ....................................................... 112 S u m m a r y o f O b j e c t i v e s .............................................. 113 Rang e of L eaf -tissue Fr eezetoleranc e Tra it Ex pression . . . . . . . . . 113 Confirmation of L eaf or Root-tissue Fr eezeTolera nce . . . . . . . . . 115 An a tom ic a l D if fe re nc e s B e tw e e n F re e zeSe ns iti ve a nd F re e zeTo le ra nt L i n e s ....................................................... 116 Phy siologica l Differ ence s Between Freeze-sensitive and Freeze-tolerant L i n e s ....................................................... 117 Her edity and Mode of Gene Action of L eaf -tissue Fr eezetoleranc e . . . . 120 S u g g e s t e d F u r t h e r R e s e a r c h .......................................... 121 R E F E R E N C E S ....................................................... 123 B I O G R A P H I C A L S K E T C H ............................................. 131

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xi L I ST OF TAB L ES T ab l e p age 2 -1 C an o p y l ea fd am age ra t i n g s y s t em s h o ws p er ce n t age o f c an o p y l ea fd am age a n d c a n o p y g r e e n l e a f ............................................... 32 2-2 Mean simple ef fec ts across 26 ba hiag rass lines subjec ted to targ et temper ature trea tments of prog ressively colder fre ezing e vents, L TFT sc ree ning e x p e r i m e n t 1 ...................................................... 36 2-3 Mean simple ef fec ts across a ll bahiag rass lines with ac tual tempera ture trea tments of prog ressively colder fre ezing e vents, ca nopy leaf -dama g e, L T F T s c r e e n i n g e x p e r i m e n t 1 ......................................... 37 2-4 Prog ressively lower f ree zing temper ature events e ffe cts on ca nopy fre ezeda ma g e of se le c te d s e xua l di plo id a nd a po mic tic te tr a plo id li nes L TFT s c r e e n i n g e x p e r i m e n t 1 .............................................. 38 2-5 Whole plant recove ry rating s as a pe rce nt of control plants, 18 d af ter trea tment of selec ted sexual diploi d and apomictic te traploid lines by pr og re ssi ve ly low e r f re e zing te mpe ra tur e e ve nts . . . . . . . . . . . . . 40 26 Ca no py fr e e zeda ma g e of se le c te d s e xua l di plo id a nd a po mic tic te tr a plo id lin e s, a s a ff e c te d b y a sin g le fr e e ze t re a tme nt ( -6 C) in a n e nv ir on me nta lly c o n t r o l l e d c h a m b e r ................................................. 42 27 Ca no py fr e e zeda ma g e of se le c te d s e xua l di plo id a nd a po mic tic te tr a plo id lines while root sy stem was kept a bove fr eezing (5C) F ebrua ry 2002 . . . . 44 2-8 Whole-plant rec overy rating s as a pe rce nt of control plants af ter 18 da y rec overy in gr eenhouse afte r fr eeze tre atment (-3.2C) w hile root sy stem was kept above fre ezing ( 5C) Fe bruar y 2002 . . . . . . . . . . . . . . . . 45 3-1 Comparison of vessel diame ters of tropic al climbing pla nts compare d to trees . 49 3-2 Mean midrib xy lem diameter acr oss three le af positions of two lines sampled 1 J a n u a r y 2 0 0 3 .................................................... 55 3-3 Mean midrib xy lem diameter of three leaf positions across two lines (F L 9, FL 67) var y ing in L TFT, sa mpled 1 J anuar y 2003 . . . . . . . . . . . . 55

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xii 3-4 Mean simple ef fec ts of vessel and va scular bundle diameter s of four f ree zetolerant vs. four fre eze-sensitive ba hiag rass c lones sampled 1 F ebrua ry 2003 . . 57 35 An a ly sis of va ri a nc e c omp a ri ng me a n v e sse l a nd va sc ula r b un dle a re a sim ple eff ects of f our hig hvs. four lowleaf -tissue fre eze-tolera nt clones sampled 1 F e b r u a r y 2 0 0 3 ..................................................... 58 3-6 Mean midrib aba x ial (fa cing the bottom side of the leaf ) xy lem and vasc ular bu nd le pa ra me te rs fr om a F e br ua ry 20 03 sa mpl ing da te . . . . . . . . . . 59 4-1 Fa tty acid c ompositi on of g razed orc hard g rass (T able f rom L oor et a l., 2002) . 69 4-2 Osmolality of bahia g rass sexual diploid li nes re prese ntative of fr eezet o l e r a n c e a n d f r e e z e s e n s i t i v i t y ........................................ 77 4-3 Ba hiag rass lea f blade fatty acids, a s a fr action of total e x trac ted fa tty acids . . 78 44 L e a f b la de do ub le bo nd ind e x (D B I ) a nd un sa tur a te d f a tty a c id: sa tur a te d f a tty a c i d r a t i o ......................................................... 81 5-1 Ger mination test via emerg ence of 100 see d scar ified with conc entra ted sulphuric ac id for 15 minutes, 23 J une 2004 . . . . . . . . . . . . . . 93 5-2 Crosses and self -pollinations made Aug ust 2003 through D ece mber 2003 of fre eze-tolera nt, intermediate, a nd fre eze-sensitive ba hiag rass c lones . . . . . 99 5 -3 P er ce n t em er gen ce o f c ro s s es an d s el fp o l l i n at i o n s m ad e A u gu s t 2 0 0 3 t h ro u gh Dec ember 2003 of fr eezetolerant, interme diate, and f ree ze-sensitive b a h i a g r a s s c l o n e s.................................................. 100 1 5-4 Mean e merg ence of cr oss F s from nine c lones vary ing in the le af f ree zetoleranc e tra it and self-pollination prog eny of nine c lones compar ed to an o p e n p o l l i n a t e d s t a n d a r d ............................................ 101 55 Pa re nt c lon e c a no py fr e e zeda ma g e ra tin g s u se d to de ve lop a dia lle l c ro ss rate d afte r a f ree ze event 17 De cembe r 2004 . . . . . . . . . . . . . . 102 5-6 Mean c anopy leaf fre eze-da mag e ra tings (1 to 9) of prog eny rate d afte r a fre eze eve nt (17 Dec ember 2004) as a result of fe male par ent and pollen s o u r c e .......................................................... 102 5-7 Analy sis of varianc e of c anopy damag e ra tings made afte r 17 De cembe r 2004 fre eze using the fixed model Griffing s Method 3, and 6 ba hiag rass c lones vary ing in L TFT tr ait, and their pr og eny . . . . . . . . . . . . . . . 104

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xiii 58 Pa re nt c lon e c a no py fr e e zeda ma g e ra tin g s u se d to de ve lop a dia lle l c ro ss r a t e d a f t e r a 2 2 D e c e m b e r 2 0 0 5 f r e e z e ................................. 105 5-9 Prog eny canopy fre eze-da mag e ra tings (0 to 100% ) ra ted af ter 22 De cembe r 2005 as a r esult of fe male par ent and pollen sourc e . . . . . . . . . . . 106 5-10 L TFT phe noty pe ef fec ts partitioned into their varianc e sourc es fr om a fixed six pare nt diallel mating de sign a naly zed with Griffing s Method Three . . . 107

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xiv L I ST OF F I GURES Fi gu re p age 11 Chi llinj ur y sy mpt om r e sp on se of c hil lse ns iti ve pla nts le a din g to r e ve rs ibl e o r i r r e v e r s i b l e c e l l a n d p l a n t i n j u r y ...................................... 3 12 F re e zeinj ur y sy mpt om r e sp on se of pla nts le a din g to r e ve rs ibl e or ir re ve rs ibl e c e l l a n d p l a n t i n j u r y .................................................. 8 21 Wat e rba th e xpe ri me nt s ho wi ng tub s, he a te rs a nd we ig hte d p ots . . . . . . 35 3-1 FL 67 bahiag rass (f ree ze-tolerant) section showing bundle shea th, gir der sy ste m of sc le re nc hy mou s ti ssu e su pp or tin g the va sc ula r b un dle . . . . . . 56 3-2 FL 9 (fr eezesensitive) sec tion showing lar g er va scular bundle are a than F L 67 . 56 3-3 Fr eezesensitive line within hours of being place d in full sunlight afte r a c on tr oll e d f re e ze e ve nt, 10 h a t 6 C, s ho ws da ma g e d mi dr ib r e g ion s in iti a lly . 60 4-1 Relationship of osmolality fre ezing tempe rature osmotic pressure and re la tiv e hu mid ity of a n a qu e ou s so lut ion wi th a pu re so lut e . . . . . . . . . 65

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xv AB STR AC T Abstrac t of Dissertation Prese nted to the Gra duate School of the Unive rsity of F lorida in Partial Fulf illment of the Requirements for the Deg ree of Doc tor of Philosophy L EA F -T I SSU E F REE Z ETO L ERA NC E M ECH AN I SM I N B AH I AG RA SS ( Paspalum notatum Flegge) By J ACQUE WI L L I AM BREMAN May 2006 Chair: Ann R. Blount Cochair: Ke nneth H. Que senber ry Ma jor De pa rt me nt: Ag ro no my B a hia g ra ss ( Paspalum notatum Flegge) is the forage base that Florida livestock pr od uc e rs de pe nd on L e a ftis su e fr e e zetol e ra nc e (L TF T) is a tr a it f ou nd in s ome ba hia g ra ss p la nts a ft e r a fr e e ze e ve nt. Un de rs ta nd ing the L TF T m e c ha nis m c ou ld h e lp expand Florida s fora g e base into the winter months to financially benef it a $1 billionplu s in du str y tha t su sta ins a n e sti ma te d 1 8, 07 6 jo bs A ne e d a ro se to f ind the me c ha nis m that allowed plants with L TFT to survive fre eze eve nts. The ra ng e of the L TFT tr ait expression was quantified using controlled fr eezing trials. Eig ht clones we re se lected f or ana tomical study Four clones that we re f ree zetolerant a nd four c lones that wer e fr eezesensitive wer e compa red a nd contra sted for anatomica l and phy siologica l differ ence s. L eaf x y lem diameter was a n indicator of L TFT. T he lines with fre eze-tolera nce had xy lem diameter s rang ing f rom 157 m to 187 m. The lines that wer e fr eezesensitive had xy lem diameter s rang ing f rom 209 m to 242 m.

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x vi L eaf osmolality was not a me chanism involved in hig h-L TFT c lones. Clones with free ze-toleranc e ra ng ed fr om 545 to 817 mmol kg Clones that were fre eze-1 sensitive rang ed fr om 489 to 859 mmol kg -1 L eaf fatty acid ( FA) composition was not a mecha nism invol ved in L TFT. Seve n FAs ( C14:0 my ristic, C16:0 palmitic, C16:1 palmitoleic, C18:0 stearic, C18:1 oleic, C18 :2 l ino le ic C1 8:3 lin ole nic ) a c c ou nte d f or the ma jor c omp os iti on in t he ba hia g ra ss li nes tes ted L eaf fat ty aci d co mp os it io n wa s n ot a me chan is m i nv ol ved in hi gh-L TFT clones. Unsa turated F A profile, unsa turated F A:saturate d FA, a nd double bond index we re c on tr a ste d f or nin e c lon e s th a t va ri e d in L TF T. No ne of the F A p a ra me te rs c ou ld predic t bahiag rass L TFT. The L TFT he ritability was c alculate d from a dia llel mating de sign using Griff ing s fix ed model for Method 3. The LTFT heritability was low (H = 25%, h = 22 8%). Dominant g ene a ction acc ounted for most L TFT tr ait-expression heritability

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1 CH APT ER 1 I NTRODUCTI ON P lant Cold-in jury and Cold -toler ance Florida ag riculture a nd natura l-resour ce industries in 1997 a ccounte d for $31.4 billion i n sales, $18.2 billion in ex ports, $12.3 billion i n valueadded r evenue and 314,000 jobs (Hodg es et a l., 2000). Erra tic and unpre dictable f ree ze and above -fr eezing events ha ve historically damag ed cr ops as fa r south on peninsular F lorida as Miami-Da de County Milli ons of dollars of c rop losses have resulted f rom these f ree ze events. Entire ag ricultural industries ha ve bee n chang ed or move d as a r esult of seve re f ree ze events. T h e F lo r id a c it r u s in d u s tr y p e r ma n e n tl y mo v e d to th e s o u th e r n p e n in s u la to e s c a p e f r e e ze events that c ould not be manag ed using traditional wind machine s, g rove he ater s or sp ri nk le r i rr ig a tio n ( Cr oc ke r, pe rs on a l c omm un ic a tio n, 20 00 ). Th e or na me nta l, transplant, and hy droponics industry in Florida ha s used over head spr inkler irrig ation, as well as c overe d or hea ted g ree nhouses, to protec t their hig h-value crops ( Hochmuth, persona l communication, 2005). Any thing that puts ag riculture of this economic sca le at ri sk is s ome thi ng tha t ne e ds to b e stu die d a nd ma na g e d. F re e zete mpe ra tur e tol e ra nc e in crop pla nts needs to be studied so cr op damag e and loss risk ca n be re duced. The F lorida for ag e-ba sed livestock industry (dairy horses, be ef c attle, shee p and g oats) ac counted f or $1.009 billion in sales and 18,076 jobs in 1997 (Hodg es et a l., 2000). F or the F lorida livestock industry to remain e conomically viable, it must grow the bu lk o f t he re qu ir e d f or a g e s w ith in t he sta te F or a g e s in F lor ida ha ve e c on omi c va lue in

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2 addition to forming the fee d foundation for the fora g e-f ed livestock industry Florida hay and pasture outputs were e stimated at an a dditional $55.9 mil lion, crea ting a n additional 2,910 jobs in 1997. B a hia g ra ss ( Paspalum notatum Flueg g e) is the pa sture base livestock produce rs depend on dur ing the war mer months, on an estimated 2.5 million acre s in Florida (Chambliss, 2002). L ivestock produc ers suppleme nt, during the winter months, with hay fr om n or thc e ntr a l F lor ida no rt hw a rd s a s a dir e c t r e su lt o f f ro st i nju ry to b a hia g ra ss pastures. F rost injury has occ urre d on bahiag rass a s far south as L ake O keec hobee (Mislevy 2005, persona l communication). Alterna tive small-g rain winter fora g es pla nte d f ro m no rt hc e ntr a l F lor ida thr ou g ho ut t he pa nh a nd le ha ve be e n h ist or ic a lly damag ed fr om severe fre eze eve nts. Sm all g rain winter fora g es ar e costly to sow. They ar e a l s o at ri s k fr o m fr ee z e a n d d ro u gh t (W ri gh t et al 2 0 0 5 ) b ec au s e t h ey p ro d u ce fo ra ge Dec ember throug h April, while bahia g rass is not available for g razing Conserved fora g es ar e an importa nt and costly require ment for F lorida livestock produc ers f rom L ake O keec hobee thr oug h peninsular F lorida and e ncompassing the panha ndle re g ion. Fr eezeinjury during cool winter months puts bahiag rass a t risk in Florida. B eca use of thi s r isk fa c tor it i s im po rt a nt t o u nd e rs ta nd pla ntle a f f re e zeinj ur y a s it pe rt a ins to fora g e produc tion. Chill-in jury Be cause some plant leaf chill-injury sy mptoms phy sically rese mble leaf fre ezeinjury sy mptoms, understanding the main mecha nisms invol ved in plant-lea f chill-injury may provide insig ht into plant freezeinjury and its avoidanc e or toler ance Chill -injury a nd pla nt t ole ra nc e of tha t in jur y c a n b e a su bje c t of c on fu sio n in the lit e ra tur e T he re is

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3 Not reversible Reversible Chill stress 0 0 C Time 20 0 C Reversible Chill stress 0 0 C Time 20 Critical temperature a need to define some terms used in the literature and review basic mechanisms that apply to cold injury and plant cold-tolerance. Chill-injury is foliar, and whole-plant damage, as a result of temperatures above 0C and below some threshold temperature unique for that species and even for a specific genotype. Freeze-injury is plant damage at temperatures below 0C or when radiative frosts occur with ice formation (defined later). Chill-injury can be conceptualized as the plant responding with increasing damage to cold stress as ambient temperature approaches 0C, and depending on the duration of that stress (Figure 1-1). Damage to bahiagrass foliage from chill stress has not been documented. However, there may be mechanism continuums that may apply to further our understanding of freeze-injury in bahiagrass. Figure 1-1.Chill-injury symptom response of chill-sensitive plants leading to reversible or irreversible cell and plant injury (Adapted from Nilsen and Orcutt, 1996) Temperature ranges where damage to chill-sensitive plants occurs vary. Values such as from 12 to 0C (Buchanan et al., 2000.) and from to 20 to 0C (Lyons et al.,

PAGE 20

4 1979; Hudak and Sala j, 1999) have been r eporte d. I n contra st, chill-resistant plants can tolerate chilling tempe rature s without irrever sible injury Chill -sensitive plants tend to be of tr op ic a l or su btr op ic a l or ig in o r h a ve the ve g e ta tiv e po rt ion of the ir lif e c y c le on ly du ri ng the wa rm e st p or tio n o f t he y e a r ( Gu y 2 00 3) Ch ill -s e ns iti ve pla nts c omp ri se ma ny ma jor fi e ld c ro ps s uc h a s c ott on s oy be a n, ma ize a nd ri c e Ch ill -s e ns iti ve pla nts rar ely survive a f ree ze event in which ice forms in the tissue. Sy mptoms of chilling injury de pe nd on c hil lstr e ss s e ve ri ty (p ro ximity of te mpe ra tur e to 0 C) a nd du ra tio n o f t he c hil l stress temper ature (Nilsen and O rcutt, 1996). Chill stress sever ity and dura tion (Fig ure 11) ma y de te rm ine pla nt d a ma g e Ch ill str e ss m a y de te rm ine wh e the r p la nt d a ma g e is reve rsible. Chill st ress sy mptoms m ay include Chang es in membrane structure and composition Dec rea sed protoplasmic stre aming E l ec t ro l y t i c l ea k age Pla smo ly sis I nc re a se d o r r e du c e d r e sp ir a tio n ( de pe nd ing on se ve ri ty of c hil l st re ss) Production of abnor mal metabolites Reduce d plant g rowth Sur fa c e le sio ns on le a ve s a nd fr uit Abnormal c urling lobbing, a nd crinkling of lea ves Water soaking of tissues Cr a c kin g s pli tti ng a nd die ba c k o f t e rm ina l g ro wt h Ra pid le a f w ilt ing fo llo we d b y wa te rso a ke d p a tc he s th a t de ve lop int o s un ke n p its of collapse d tissue, that usually dry up on war ming, lea ving ne crotic a rea s of leaf tissue Pla nt d e a th Perturbation of the membrane may be the f irst sign of c hill-injury (L y ons et al., 1979). Whether or not the plant has suffic ient time to become a cclimated to c hilling tempera ture a ffe cts the seve rity of the da mag e (B uchana n et al., 2000). A cclimation rese mbles lowering the cr itical tempera ture for a plant to experienc e injury sy mptoms.

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5 Acc limation si g nals ca n include shortening day -leng th, cooler te mpera tures (Toma show, 1999), cha ng es in nutrition, water r elation, and stag e of g rowth (B uchana n et al., 2000). F r e e z e St r e ss Fr eeze stre ss is sim ilar to chill stress, but the tempera tures ar e below 0C a nd may i n c l u d e t h e p r e s e n c e o f i c e ( N i l s e n a n d O r c u t t 1 9 9 6 ) Ic e f o r m a t i o n m a y n o t a l w a ys occur at temper ature s below 0C, but if formed, ic e is forme d outside the ce ll membrane, in the apoplast. F r e e z e P r oc e ss The fr eeze pr ocess include s dehy dration stress to the living cells in the sy mplast. B e c a us e the os mot ic po te nti a l of ic e is l ow e r t ha n th a t of wa te r, c e ll w a te r e xits th e c e ll tow a rd the g ro wi ng ic e c ry sta l. A s th e fr e e ze p ro c e ss c on tin ue s, the c e ll d e hy dr a te s, c e ll volume is reduce d, and the osmotic conc entra tion in the cell incre ases ( Rajasheka r, 2000). The e x tra c ellular a nd intrace llular fre ezing pr ocess a nd injury to plant cells have been la rg ely descr ibed and visualized by microscopy (Asac hina, 1978). I nf ra re d th e rm og ra ph y ha s b e e n u se d to vis ua lize a nd re c or d th e fr e e zing pr oc e ss. When water fre ezes, hea t is given of f fr om the phase c hang e fr om water to ice. I nfra red ph oto g ra ph y (t he rm og ra ph y ) h a ve be e n u se d to ob ta in r e a ltim e da ta F ro m da ta g ather ed fr om thermog raphy superc ooling a s well as the f ree zing temper ature of the plant tissue can be identified (Ta iz and Geig er, 2002) The a ctual proc ess of ice nuclea tion on leaf blade s of var ious species ha s been visualiz ed and r ecor ded with infra red the rmog raphy (Wisniewski et al., 1997). I nfra red thermog raphy rec orded f ree zing temper ature s of -1.5 to -2.1C for three specie s of b a r l e y ( Hordeum sp .) (Pea rce and F uller, 2001) unde r natura l free ze conditions. The order of fr eezing for uprooted plants plac ed in a c ontrolled environment c hamber ,

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6 occur red f irst for a dr oplet of wa ter plac ed on the ba rley leaf blade, whic h froze and a cted as the ice nuclea tion sit e. Sequential a nd succe ssive infrar ed ther mogr aphy of the nuclea ted leaf blade showe d the midrib x y lem vessel re g ion froze from the site of e xter na l ic e nu c le a tio n. F re e zing of the ba rl e y le a f p ro g re sse d d ow n th e e nti re mid ri b x y lem vessel re g ion. Once the entire midrib vesse l reg ion was fr ozen the rema inder of the leaf blade f roze. The ne x t org an that fr oze was the e x posed roots, followed by older leave s, then y oung er le aves, a nd finally the sec ondary tillers. The fr eezing order thus fo llo we d ic e nu c le a tio n o uts ide of liv ing pla nt, fo llo we d b y le a f i nv a sio n o f t he a po pla st throug h the xy lem, then the mesophy ll of the lamina. I n potted Lolium perenne L and Poa supine Schrad. in controlled environme ntal chambe r, the orde r of f ree zing re corde d by infrar ed ther mogr aphy was fr om the roots to crown throug h connec tive tissue, followed by fre ezing of the shoots and leave s (Stier et al., 2003). Nu c le a tio n is on e of the va ri a ble s in de te rm ini ng a t w ha t te mpe ra tur e pla nts fr e e ze. Pla nt i c e nu c le a tor s c a n b e div ide d in to t wo c la sse s ( Pe a rc e 2 00 1a ): e xtri ns ic and intrinsic. Ext rinsic nuclea tors are outside of the plant which a id ice for mation and are also ref err ed to as he terog eneous nuc leators. He terog eneous nuc leators a re substances tha t cataly ze the formation of a stable ice nucleus. When wa ter molec ules come tog ether spontaneously to form a stable ice nuc leus the term homog eneous nuclea tion is used. Ex trinsic heter og eneous nuc leators ( L al and L al, 1990) c an be w ater droplets, dust particles, ic e nucle ation-ac tive (I NA ) b a c te ri a o r w ind a g ita tio n. I ntr ins ic nuclea tors are substances w ithin the cell which ca taly ze ice for mation. An example of intrinsic nucleator s might be those f ound in ry e ( Se c ale c e re ale L .) ce lls (Brush e t al., 19 94 ). I n r y e in tr ins ic nu c le a tor s w e re fo un d to be c omp le xes o f p ro te ins ,

PAGE 23

7 car bohy drate s, and phospholipids in which both disul fide bonds and f ree sulfhy drol g roups wer e important for nuclea ting a ctivity S u g g es te d S ch eme Fr eezeinjury can oc cur to fr eezesensitive plants at or just below 0C when ice nuclea tion occurs. F ree ze-tolerant plants c an withstand fr eezing at temper ature s -3C lower tha n fre eze-sensitive plants. F ree ze-tolerant plants will thaw, re hy drate and fu nc tio n n or ma lly by va ri ou s me c ha nis ms. Th e re is a ne e d to or g a nize pla nt r e sp on se and published mec hanisms of fre eze-tolera nce. Ma ny sc he me s h a ve be e n u se d in a tte mpt s, to i nte g ra te the va ri ou s me c ha nis ms of fr eezeavoidanc e and f ree ze-toleranc e with observe d plant response s and bioche mical adjustments. Earlier attempts such as those r efe renc ed (L evitt, 1978) did not have the c u r r e n t b o d y o f k n o w l e d g e a v a i l a b l e f r o m p l a n t m o l e c u l a r a n d b i o c h e m i c a l s c i e n c e ( G u y, 2003). A sche me for c lassify ing w hether plants are fre eze-sensitive or f ree ze-tolerant was propose d by Sakai and L arc her ( 1987). The c oncept of a continuum in plant injury in response to stre ss duration and intensity was propose d by Nilsen and Or cutt (1996) a nd illust rate d previously (F igur e 1-1) These c oncepts c an be integ rate d with established fre eze-stre ss mechanisms as shown in F igur e 1-2. The proposed sc heme of plant toleranc e to fre eze-stre ss can be useful in org anizing the litera ture. F igur e 1-2 c an helpful in classify ing pla nts based on fr eeze intensity and dura tion. helpful in classify ing plants based on f ree ze intensity and dura tion. This conce ptual scheme would classify plants as fre eze-sensitive if they wer e damag ed by shallow fre ezes down to -3C. Fr eezetolerant plants would be those which could withstand fre ezing tempe rature s below -3C. B oth shallow and dee p fre ezes occ ur

PAGE 24

8 0 C -10 C -3 C -50 C Freezesensitive Freezetolerant Heterogeneous nucleation Homogenous nucleation Dehydration tolerance + Membrane fatty acids + Cryoprotectants Heterogeneous nucleation after supercooling due to solute accumulation Time in peninsular Florida. Bahiagrass genotypes with freeze-tolerance may have more than one protective mechanism. Figure 1-2.Freeze-injury symptom response of plants leading to reversible or irreversible cell and plant injury (Conceptualized by the author based on data from Sakai and Larcher, 1987; Nilsen and Orcutt, 1996; Guy, 2003) One protective mechanism postulated (Levitt, 1978; Pearce, 2001) for shallow freezes (-1 to -3C) has been freezing-point depression of cell sap by heterogeneous ice nucleation after transient supercooling. Therefore, freeze-tolerant bahiagrass genotypes may be transiently protected by supercooling during short freeze events. Supercooling is simply defined as water or a solution below the equilibrium freezing point of water (0 to -1C) and above the homogeneous nucleation temperature of water (-40 to -41C) (Chen et al., 1995). Supercooling is a mechanism that allows plants to avoid what could be lethal intracellular freezing by reducing the freezing point of the cell solution (Hudak, J. and J. Salaj, 1999). Supercooling is an unstable thermodynamic situation when a liquid solution is not in phase equilibrium with the solid ice phase. The process of

PAGE 25

9 c on ve rs ion fr om a n u ns ta ble sta te s uc h a s su pe rc oo le d li qu id, to a sta ble sta te su c h a s a fr oze n p ha se is ini tia te d b y nu c le a tio n ( Va li, 19 95 ). Nu c le a tio n o c c ur s w he n a sma ll volume of the ne w ice pha se occ urs. Nucle ation is followed by g rowth of the ne w thermody namically stable ice phase. G rowth of the sta ble ice pha se is controlled by the latent hea t of wate r. The sy stem experience s equilibrium when the ra te of ice formation equals the r ate of ice melting and the r ate of water vaporization equals the r ate of water vapor c ondensation. Palta and Weiss (1993) sug g est that herba ceous pla nts nucleate as a re sult of bacter ial flora on the surfa ce of their lea ves at tempe rature s betwee n -5 a nd 0 C. B a c te ri a l f lor a c ou ld c on fo un d s up e rc oo lin g e ff e c ts. Fa ctors af fec ting ice formation include the prese nce of intrinsic ice nucle ators and extrinsi c ice nuclea tors (dust, bacte ria, fung i, wind ag itation, dew or ice cry stals) (L al and L al, 1990; Palta and Weiss, 1993; Vali, 1995). Fa ctors af fec ting ice g rowth ra te include cooling rate cell wa ll porosity and membra ne porosity Fr eezetolerant plants ca n preve nt protoplast injury by using e x trinsice nucle ators to form e x tra-c ellular ice in the apoplast (c ell wall spac e, interc ellular spac e, and xy lem). Protoplast injury at te mpe ra tur e s b e tw e e n 5 a nd 0 C, c a n b e pr e ve nte d b y fo rm ing ic e c ry sta ls w ith intrinsic ice nucle ators. F ree ze-tolerant plants lowe r their f ree zing temper ature by acc umulating solutes during deepe r fr eezes (3 to -10C). Fr eezetolerant plants may e xpe ri e nc e tr a ns ie nt s up e rc oo lin g a ft e r h e te ro g e ne ou s ic e nu c le a tio n e ve n th ou g h th e ir fre ezing point has be en depr essed by acc umulating solutes. Under shallow fre ezing tempe rature conditions (to -3C), superc ooling may have an ada ptive advanta g e. Eve n though e ventual ice nuclea tion may occur with shallow fr e e zing th e sp e e d o f i c e c ry sta l g ro wt h ma y no t be ra pid e no ug h to c a us e pr oto pla st damag e. Unde r colde r temper ature reg imes, superc ooling c an ca use more pla nt tissue

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10 damag e once heter og eneous ic e nucle ation occur s due to the hig h rate of fr eezing (Wolfe a nd B ry a nt, 20 01 ). Th is m a y be the c a se fo r h e rb a c e ou s ti ssu e fr e e zeinj ur y fr om r a pid tissue free zing, but the opposite ca se may be made for woody tissue where deep superc ooling is a me chanism of de ep fr eezetoleranc e (Wisniewski, 1995; Quamme, 1995). Plant osmoreg ulation can be done by two methods. Metabolic sy nthesis of so lut e s su c h a s su g a rs a nd oth e r o smo tic a c a n o c c ur du ri ng the a c c lim a tio n p ro c e ss. Water ca n be moved f rom one tissue to another in a proc ess that dehy drate s the tissue, incre ases the osmotic c oncentr ation and depr esses the f ree zing point. The mec hanism of osmotically reduc ing the fre ezing point of c ell sap by incre asing solute conce ntration has been summar ized by Sutcliffe (1977) The tempe rature at which a solution of cell sap f fr e e zes (T ), ha s b e e n r e la te d to os mot ic po te nti a l ( in kPa) (Sutcliffe, 1977) f T= /12.2 I f plant sap osmotic potential ra ng es betwe en -3000 to -4000 kPa the n fre ezing de pr e ssi on a s a str ic t r e su lt o f o smo tic po te nti a l w ou ld r a ng e fr om 2. 5 to 3. 3 C. T his is not much protection. The a ctual osmotic potential of the tissue ca n be dete rmined experimentally by the fre ezing point-de pression method using thermocouple s and extracted plant sap using the Van t Hoff f ormula, then c orre cting for supe rcooling and room tempera ture (Saupe 2004). An e ven more acc urate calc ulation can be deter mined if the solute composition can be deter mined and tabular solute values could be used. Thermody namic and me chanic al ef fec ts have be en integ rate d into a scale which re lates the hy draulic pr essure of wa ter in Pasca ls, the equilibrium free zing temper ature os mol a lit y a nd the re la tiv e hu mid ity of wa te r a t th a t pr e ssu re te mpe ra tur e a nd os mol a lit y (Wo lf e a nd B ry a nt, 20 01 ). Ho we ve r, fr e e zing po int -d e pr e ssi on fr om i nc re a se d o smo tic

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11 conce ntration and subseque ntly more ne g ative osmotic potential is only for a few deg ree s, as ca lculated a bove. Fr eeze stre ss-induced c ell dehy dration happe ns once ic e nucle ation occur s at colder tempera tures than c an be pr otected by osmotic conce ntration and tempora ry su pe rc oo lin g O the r p la nt m e c ha nis ms m us t pr ote c t th e pla nt f ro m de sic c a tio n. Ce ll dehy dration prog resse s when wa ter lea ves the c ell throug h the ce ll membrane to the apoplast whe re the ice c ry stal continues to g row. The ice c ry stal will continue to gr ow until the ice phase comes to thermody namic equilibrium with the liquid and g as wa ter phases within the sy mplast + apoplast sy stem. At prog ressively colder below-f ree zing tempera tures, mec hanisms that preve nt cell dehy dration bec ome incre asing ly important. Membrane integ rity is important in preventing cell dehy dration. Membra ne integ rity is damag ed whe n fre ezinginduced de hy dration occ urs (Pea rce 2001). Membrane flexibi lity become s an important fa ctor in dete rmining w hether or not membrane integ rity will occur a s the ce ll volume contrac ts through loss of w ater and the n r e -h y dr a te s o n th a wi ng to f ull tur g or pr e ssu re (B uc ha na n e t a l., 20 00 ). Re se a rc h in ry e protoplast showe d fre ezing to below 10C without acclimation cause d membrane failure and ice nuclea tion that invaded the protoplast (Steponkus et a l., 1981). I t was membrane failure that prec eded inva sion of the protoplast with ice. I n non-ac climated pr oto pla sts in tr a c e llu la r i c e fo rm a tio n w a s st ro ng ly de pe nd e nt o n th e c oo lin g ra te wi thi n the ra ng e of 5 to -3C. The g rea ter the c ooling r ate the mor e intrac ellular ice was formed. U nder slowcooling rate experiments, ry e protoplast injury resulted f rom c on tr a c tio n d ur ing c old -s tr e ss t re a tme nt a nd ly se s o n w a rm ing M ic ro sc op ic inv e sti g a tio ns of the pla sma le mma ha ve c or re la te d d e le tio n o f t he me mbr a ne int o

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12 vesicles a s the ce ll volume is reduced thr oug h dehy dration during slow fre ezing ( Hudak and Salaj, 1999). Plasmalemma inflexibi lity and dele tion may be re lated to lower flexibi lity of membrane s with highe r conc entra tions of saturated f atty acids. See dling whe at cultivars acc limated at low, above -fr eezing tempera tures, showe d an incr ease in and -l ino le nic fatty acid a s a per cent of the membra ne composition. These c ultivars had lower lethal fre eze temper ature s (as much a s 10C lower for some cultivars) tha n unacc limated seedling s (St. J ohn, 1979). Cold acc limation duration increa sed the membra ne ra tio of poly unsaturate d fatty acids (C18:3/C18:2) from 0.37 to 0.86. Species in which c oldacc limated plasma membra nes have the hig hest ratio of di-unsa turated phospholipids and lowest proportion of g lucoce rebr osides tend to be the most cold-ha rdy (B uchana n et al., 2000). Membrane fluidity may be the r esult of the membra ne fa tty acid ( FA) c omp os iti on F a tty a c id c omp os iti on c a n c ha ng e the te mpe ra tur e tha t me mbr a ne ph a se chang es occ ur (B uchana n et al., 2000). The more unsatur ated the F A content the lowe r the temper ature the membra ne re mains in the liquid-cry stal phase. Shifting to the g el stag e has be en assoc iated with more sa turated F A content a nd membrane damag e at war mer tempe rature s. Phase transition tempera tures of g el to liquid of phosphatidy lcholine (PC) specie s which diffe red by the FA g roup ra ng ed fr om 55C for disteroy l-PC (16:0-PC ) to -19C for dioleoy l-PC (18:1-PC ) (Palta a nd Weiss, 1993). Cool-tempera ture a cclimated pota to ( So lan um c om me rso nii Dun.) plasma me mbrane extraction showed a 28.5% incre ase in 18:2 F A and a 10.1% dec rea se in 16:0 FA. A n incre ase in plasmale mma ATPase a ctivity and an a dditional 5C freeze tolera nce (fr om to -9C) was the result of c ool-tempera ture a cclimation.

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13 As long as the membra ne re mains in the liquid-cry stal stag e, the c ell can f unction no rm a lly a lbe it a t a slo we r r a te a s te mpe ra tur e s d e c re a se O nc e the me mbr a ne g e l ph a se occur s, membrane functions, such a s osmoreg ulation and transloca tion, become impaire d when c ry stal patche s of membra ne for m, and membra ne per meability incre ases (B uchana n et al., 2000). Plants have desatura se enzy mes, which c an cha ng e the position of the double bonds on a fatty acid c hain. Desa turases a re a ctivated f or normal membrane FA tur nover. A fter a cclimation sig nals (cool tempe rature s, short day s, etc.) a re e xpe ri e nc e d, de sa tur a se s a re up -r e g ula te d w ith the ne t e ff e c t of a n in c re a se in unsaturate d fatty acid c ompositi on of the membra ne PC. The ac tual mecha nism can simply be moving the location of the double bond on the F A ca rbon cha in. For e x ample, moving the double bond from the se cond and third c arbon position (melting 18 point = 40C) on an 18-c arbon c hain FA (C ) to betwe en the ninth and the te nth car bon position (melting point = -20C) c an cha ng e the melting tempera ture or pha se shift 60C. Th is m e c ha nis m w ou ld i mpa c t f re e zetol e ra nc e T he lon g e r t he me mbr a ne wo uld re ma in functional a nd in the liquid-cry stalline phase, the long er it could re main fre eze-tolera nt. Once the phase shift occur red f rom liquid-cry stalline to a g el at a te mpera ture be low -0 C, if a po pla sti c ic e e xiste d, me mbr a ne s w ou ld a llo w r a pid ic e c ry sta l g ro wt h leading to increa sed dehy dration stress a nd eventua l cell damag e. Ge l phase membra nes ma y a lso be mor e pr on e to s he a ri ng a nd fo rm ing e nd oc y tic ve sic le s, wh ic h d e c re a se membrane materia l during cell volume re duction during fre eze eve nts, as mentioned previously Upon thawing and attempting to rehy drate the integ rity of the c ell, the membrane is compromised and c ell death oc curs. The role of ac climation in prepar ing the plasmalemma for fre eze-stre ss has bee n me nti on e d. Che mic a l a nd str uc tur a l c ha ng e s in the pla sma le mma wh ic h h e lp r e sis t

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14 fre eze dehy dration, mec hanica l stress, molecular packing and other fre eze eve nts, can be induced by tempera tures as low a s -3C in Robinia pseudacacia L Dura tion of acc limation temperature tends to increa se fr eezetoleranc e (H udak and Sala j, 1999). Ha rd e ne d ( c he mic a l a nd str uc tur a l c ha ng e s th a t oc c ur thr ou g h a c c lim a tio n) pla sma membrane become s highly folded in some spec ies, invag inated in others, a nd associate d with poly phenolic anti-f ree ze compounds in stil l other spec ies. Chang es in ac climated plasma membra ne ar e assumed to a id in plasma membrane integ rity under se vere fre ezestress. Protein metabolism is i mportant in cold ac climation and fre eze-tolera nce, but perha ps not for the historica l rea son L evitt had postulated (Guy and Carte r, 1982). G lu ta th io n e w a s th o u g h t t o p r o te c t m e mb r a n e p r o te in s u lf h y d r o l g r o u p s d u r in g f r e e ze stress there by preve nting pr otein denatur ing, w hich was be lieved to lead to c ell fre ezedamag e. Hig h levels of g lutathione had bee n found to be c orre lated with ac climated fr e e ze h a rd ine ss i n s e ve ra l pl a nt s pe c ie s. Th e pr ote in m e ta bo lis m th a t ma y fa c ili ta te understanding plant cold ac climation and ac climated plant ability to withstand more se ve re fr e e ze s tr e ss i s th e g e ne tic up -r e g ula tio n a nd do wn -r e g ula tio n o f e nzy ma tic proteins controlling cell proc esses (G uy 1990). A pa rtial listing of low tempe rature re sp on siv e g e ne s a ff e c te d p ro c e sse s w hic h in c lud e d r e sp ir a tio n, c a rb oh y dr a te me ta bo lis m, l ipi d me ta bo lis m, p he ny lpr op a no id m e ta bo lis m, a nti oxida nt m e ta bo lis m, reg ulatory enzy mes in ten diffe rent plants, and te n functionally unknown proteins (G uy et al., 1994). Spinach pla nts acc limated to low tempera ture c ompare d to control and droug ht-stressed pla nts at low tempera ture showe d g ene pr oducts that wer e expressed either to low tempe rature acc limation or to droughtstress. Fr eezing toleranc e wa s e nh a nc e d w he n th e se pr ote ins a c c umu la te d in the le a f. Sin c e pa rt of fr e e zestr e ss

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15 toleranc e is toleranc e to desicc ation these re sults support previous literature r eviewe d. The ove rlapping and quantitative na ture of g enes involved in fr eezestress tolera nce ha s b egu n t o b e s u b s t an t i at ed wi t h m i cr o ar ra y re s u l t s u s i n g Ar ab ido ps is thaliana (L .) He y nh (Thomashow, 1999; Wanner and Junt tila, 1999). Perhaps a s many as 1,000 to 7,000 g e ne s in the e nti re Ar a bid op sis g e no me of 28 ,0 00 to 3 0, 00 0 g e ne s ma y be inv olv e d in fre ezing stre ss toleranc e (G uy 2003). F re e zetol e ra nc e a c c lim a tio n ma y be ind uc e d b y sh or t da y s, c oo l te mpe ra tur e s, application of a bscisic ac id (AB A) droug ht-stress, potassium fer tiliz ation and occa sionally phosphorous fer tiliz ation (McKe rsie and L eshem, 1994). A cclimated pla nt g e ne ra liza tio ns inc lud e inc re a se d o smo tic c on c e ntr a tio n, de c re a se d ti ssu e wa te r c on te nt, incre ased star ch and pr otein conce ntration, lipid accumulation, fa tty acid unsa turation, incre ased soluble pr otein content, incr ease d mRNA, increa sed poly somes, incre ased tRNA and incre ased A BA decr ease d respira tion. Som e fr e e zetol e ra nt p la nts c a n a lso lim it t he g ro wt h o f a po pla sti c ic e c ry sta ls with proteins and poly sacc haride s, limi ting the e x tent of protoplast dehy dration fr om ice cry stal g rowth. I n ry e the de velopment of fr eezetoleranc e to -30C by acc limation was acc ompanied by a tenf old increa se in protein extracte d from the e x trac y toplasmic reg ions of the le a ve s ( Gr if fi th e t a l., 19 93 ). Pho tom ic ro g ra ph y of pr ote in e xtra c ts s ub je c te d to low tempera tures showe d ice c ry stals were nuclea ted at temper ature s approa ching 0C and then the ic e cr y stal g rowth wa s restricte d. P lant Fr eez e-t olerance Summ ary Un de rs ta nd ing c old te mpe ra tur e tol e ra nc e me c ha nis ms c a n b e he lpf ul i n developing fre eze re sistant crop plants. Genoty pe diffe renc es in plant chill or fre ezetol e ra nc e ha ve be e n s ho wn to b e re la te d to ma ny fa c tor s. Cha ng e s in wa te r r e la tio ns ,

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16 so lut e a c c umu la tio ns me mbr a ne fa tty a c id c omp os iti on a nd nu me ro us me ta bo lic e ve nts a n d p a th w a y s a r e u n d e r g e n e ti c c o n tr o l. T h u s b r e e d in g a n d s e le c ti o n f o r c h il l o r f r e e ze re sis ta nc e a pp e a rs to b e a re a lis tic ob je c tiv e Pl a nt b re e de rs a nd mol e c ula r g e ne tic ist s could utiliz e this increa sing body of informa tion to develop chill and fre eze-tolera nt crop plants. Bahi agrass in the Sou theast US Im por ta nc e and U se Ba hiag rass is the major pa sture g rass used by the livestock industry in Florida a s w e l l a s t h e S o u t h e r n C o a s t a l P l a i n s o i l s o f G e o r g i a a n d A l a b a m a ( B l o u n t e t a l 2 0 0 1 ) In Florida alone, ba hiag rass is estimated to cove r over 1 milli on hecta res ( Chambliss, 2000). Ba hiag rass uses inc lude pasture hay sod and see d. Adaptation an d Geogr aphi c Distr ibu tion B a hia g ra ss i s a da pte d to sa nd y so ils in w a rm h umi d tr op ic a l a nd wa rm te mpe ra te reg ions. This gra ss tolerates soils with low fertility low pH and pe rsists under continuous stocking ( Gates e t al., 2004). B ahiag rass c an survive on dr oug hty soils and soils with i ntermittent flooding. I t was introduce d in Florida a s an improved f orag e and has bec ome natura liz ed (e stablished as par t of the loca l flora) throug hout the Southern Coastal Plain and the Gulf Coast of the Souther n USA. Pensac ola, a more c old-hardy dip loi d c ult iva r h a s b e e n f ou nd g ro wi ng a s f a r n or th a s so uth e rn Ok la ho ma a nd in Virg inia and g rows fr om Texas to North Carolina, extending into Arka nsas and Tenne ssee. F ree ze-toleranc e appa rently limit s the rang e of ba hiag rass to those re g ions wher e brie f and sha llow below-fr eezing tempera tures ar e experienc ed.

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17 Calendar F orage D ry Mat ter P roduction Th e ba hia g ra ss g ro wi ng se a so n in the so uth e a ste rn Un ite d St a te s is fr om A pr il t o October The g rowing season be comes shorte r moving from the Coastal Plain to the Pie dmo nt ( Ga te s e t a l., 20 04 ). F or a g e g ro wt h d ist ri bu tio n th ro ug ho ut t he y e a r a pp e a rs to be influenc ed by tempera ture a nd photoperiod with more dr y matter (D M) produc tion during the war mer sea son, longe r day leng ths and at lower latitudes. Twice a s much fora g e wa s clipped fr om Pensacola ba hiag rass plots in J uly compar ed to Octobe r at 32 N in the southeaster n United States. I n southeast B razil (22 45S) the g ener al trend w as for 90% of the total annua l DM acc umulation to occur during the war m summer ha lf of the y e a r a nd 10 % d ur ing the wi nte r ha lf Sh or te nin g da y le ng ths ha ve be e n s ho wn to re du c e ba hia g ra ss g ro wt h e ve n w he n te mpe ra tur e s w e re wa rm E xten din g da y le ng th with artificial lig hting r esulted in incre ased ba hiag rass g rowth (Sinclair e t al., 2001; Sinclair et al., 2003). Com m erc ial Cultivar Fr eez e-t olerance Variability As ea rly as 1942 lea f tissue toleranc e to frost a nd fre ezing wa s rec og nized as an imp or ta nt t ra it o f b a hia g ra ss c ult iva rs (B ur ton 1 94 6) A sc a le of 1 to 5 w ith 1 = mos t frost tolera nt and 5 = most frost sensitive wa s used to rate four ba hiag rass c ultivars afte r a fr eeze e vent of -5C. Common tetra ploid (4 x ) wa s rated 5, Wallace (4 x ) wa s rated 3, Para g uay (4 x ) wa s rated 2, Wil mington ( 4 x ) w a s r a te d 2 a nd P e ns a c ola d ipl oid (2 x ) wa s rated 1. Tifton 9 is a diploid cultivar, that is considere d to be cold-toler ant. Tifton 9 is a Geor g ia re lease develope d from a c ollection of Pensac ola see d collecte d from 16 fa rms and subjec ted to nine y ear s of re stricted re curr ent phenoty pic selec tion (RRPS ) (Ga tes et al., 2004).

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18 Po te n ti a l B a h i a g ra s s L ea f Fre ez eto l er a n ce T ra i t M ec h a n i s ms Anatom ical F re e zestr e ss p ro du c e s o smo tic str e ss. Se ve re os mot ic str e ss d ur ing fr e e ze e ve nts followed by immediately hightranspira tion demands in Florida f ield conditions might be minimi zed by leaf desicc ation protec tion. L eaf desicc ation protec tion could be enha nced by a thicker wax lay er. I f lea f wa x would preve nt leaf de sicca tion under seve re tempera ture stre ss (whethe r hot or cold) then leaf wax would be worthy of study Howeve r, Pensa cola bahiag rass ha d the least solute lea kag e (14.6% -15.4%) of 10 Paspalum specie s exposed to heat stress of 54C y et the third lowest lea f epic uticular wax content (1.35 to 1.79 mg wax dm (Tischler and B urson, 1995; Tischler e t al., -2 1990). Ther efor e, lea f epic uticular wa x content did not appea r worthy of investig ation. Ic e h a s b e e n s h o w n t o f o r m t r a v e l a n d s p r e a d t h r o u g h o u t b a r l e y ( Hordeum sp.) throug h the leaf midrib first, then throug hout the leaf (Wisniewski et al., 1997; Pearce and F uller, 2001). This indicate s the leaf midrib x y lem is the reg ion of initial leaf ice g ro wt h w hic h e ve ntu a lly inv a de s th e e nti re le a f. Xy le m di a me te r o f t e mpe ra te pla nts is signific antly less than for tr opical plants (Ha berla ndt, 1914). I nterna l leaf a natomy may be re lated to diffe renc es in fre eze-tolera nce. Everg ree n leave s with narrow interce llular space s and/or small mesophy ll cells were repor ted to have ic e nucle ation tempera tures down to betwe en -10 to -12C (Saka i and L arc her, 1987) L arg e xy lem diameter s have be en shown to be r elated to c avitations and a ir e mbo lis ms c a us e d b y fr e e zing a nd su bs e qu e nt t ha wi ng in w oo dy pla nt s pe c ie s ( Da vis et al., 1999). X y lem cell wa ll porosity and per meability has bee n rela ted to deep supe r c o o l i n g i n s o m e w o o d y p l a n t s p e c i e s ( W i s n i e w s k i e t a l 1 9 9 1 ; W i s n i e w s k i 1 9 9 5 ) In woody tempera te plants a re lationship has been shown be tween xy lem diameter and

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19 c a vit a tio ns c a us e d b y fr e e zing (D a vis e t a l., 19 99 ). Whe n xy le m so lut ion fr e e zes dissolved ga ses in the liquid phase ar e re lease d as bubbles in the ice phase. When thawing if air bubbles a re small they may dissolve back into the liquid phase. I f bubbles c oa le sc e a nd be c ome la rg e e no ug h to blo c k th e xy le m c on du it f low the y c a n c a us e cavitation of the liquid columnar flow. Cavitation has bee n confirme d with cry oscanning elec tron microscopy to occur during thawing in Fraxinus mandshurica Rupr. (U tsu mi e t a l., 19 99 ). On c e c a vit a tio n o c c ur s e mbo lis m f oll ow s. A xy le m e mbo lis m become s filled with atmospheric a ir and wa ter va por and disrupts tra nslocation (Da vis et a l., 19 99 ). I n s ome ha rd wo od tr e e s ( Be tul a p lat y ph y lla Sukatschev. a nd Sa lix sa c ha lin e ns is F r. Sc hm. ), ve sse ls r e c ov e r b e fo re sp ri ng g ro wt h w ith wa te r r e fi ll, pr e su ma bly fr om r oo t pr e ssu re (U tsu mi e t a l., 19 99 ). I n s ome de c idu ou s h a rd wo od s, embolism is permanent and ne w spring g rowth provides f unctional and f illed x y lem vessels (Da vis et al., 1999). Xy lem transport a nd the neg ative pre ssures sustained f or cavitation of w ater was re viewed a nd expanded with an experiment that compa red approximations of x y lem sap with Z -tube mate rials var y ing in we tting surf ace and incre asing pressure (Smith, 1994). Approx imations of biologica l pressure s, where x y lem cavitation would occ ur, ra ng ed betwe en -0.1 a nd -0.6 MPa. Xy lem structure and susce ptibili ty to cavitation by fre ezing e x periments have been done under c ontrolled pressur e that mimics droug ht-stress (Da vis et al., 1999). Cavitation would theoretica lly occur depending on x y le m p re ssu re (P x ) af ter a thaw. The rela tionship of P x the vapor pressure of wa ter (P wv ) in the embolism air bubble, the surfa ce te nsion of the xy lem sap (t), a nd the air bubble radius (r ) is shown in the fo llo wi ng e qu a tio n: P x # P wv(2t/r)

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20 E m p i ri ca l l y t h i s re l at i o n s h i p ca n b e q u an t i fi ed b y co n t ro l l i n g P x Mean xy lem diameter and per cent loss of xy lem conductivity in 12 tempera te har dwood spec ies was quantified with a f ree ze-thaw e x periment with a c onstant x y lem pressure of -0.5 MPa. Davis et a l. (1999) found tha t upon thawing a 30 m mean diamete r threshold for 12 wo od y sp e c ie s e xiste d a bo ve wh ic h c a vit a tio n w ou ld o c c ur T his c a vit a tio n r e su lte d in 7.2% loss of conduc tivity upon thawing Species with diamete rs g rea ter than 40 m had as much a s 95% loss of conduc tivity afte r a f ree ze-thaw c y cle. Perce nt loss of conductivity was shown to be a Weibull function of mean spec ies vessel diame ter. F rom the da ta of 12 sp e c ie s a 50 % l os s o f c on du c tiv ity fr om a be stfi t c ur ve wa s u se d to approximate the mean c ritical ca vitation diameter (45 m). Predicted pe rce nt loss of conductivity (Da vis et al., 1999) wa s calc ulated as c 100(1 (d $ d) / d) 4 4 c I n the formula, the sum of diameter s g rea ter than or equal to the c avitation diameter (d ) c raised to the f ourth power ( (d $ d ) ) and d was the summation of a ll the diameters 44 c raised to the f ourth power The be st fit d was 44 m ( r =0.96). The se value s can be 2 calc ulated in woody specie s using the published pr ocedur e (D avis, et al., 1999). Woody plant twigs a re c ut to standard leng ths and faste ned to a Z -tube without constricting or defor ming ve ssels. Woody plant twig se ctions can ha ve their ve ssels evac uated with a pu mp t o me a su re c on du c tiv ity Su c c ule nt g ra ss l e a ve s c a nn ot b e ha nd le d in the sa me manner as woody twig se ctions. Grass lea ves ca nnot be studied with the same a ppara tus that was used to study woody twig pie ces xy lem conducta nce a nd cavitation. I n bahiag rass, obser vations and microsc opic visualizations from controlled fre ezes might provide some information as to how smaller x y lem diameter s might be

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21 he lpf ul i n to le ra tin g a le a f f re e ze e ve nt. Th us a n in ve sti g a tio n o f l e a f a na tom y differ ence s in bahiag rass may be wa rra nted. P hys iol og ic al I f we assume that plants deve loped fre eze-tolera nce ba sed on desicc ation tol e ra nc e me c ha nis ms, the n c old -t ole ra nt l ine s o f a su btr op ic a l pl a nt c ou ld h a ve a t le a st two major phy siologica l mechanisms involved 1) hig her pe rce ntag e of unsa turated F As (C 16 :1, C18 :1, C18 :2, C18 :3) (w hic h w ou ld r e su lt i n in c re a se d me mbr a ne pe rm e a bil ity and fluidity ), 2) hig her c oncentr ation of osmotica (w hich would re sult in reduced w ater loss and reduc ed fr eezing point-depre ssion). 4 F A c omp os iti on of so me tr op ic a l a nd su btr op ic a l C g ra sse s h a s b e e n r e po rt e d in c old te mpe ra tur e a c c lim a tio n e xpe ri me nts Se a sh or e Pa sp a lum ( Paspalum vaginatum S w a r t z ) a s u b t r o p i c a l g r a s s h a s b e e n u s e d a s a t u r f g r a s s b e c a u s e o f i t s s a l t t o l e r a n c e In an experiment whe re a cold-tolera nt, intermediately -tolera nt, and cold-se nsitive line wer e subjec ted to controlled c old stress (8/4 day /night for three wee ks) the coldtolerant line initially had a hig her double bond FA inde x than the other lines as well as a t the end of the cold stress pe riod (Cy ril et al., 2002). L inolenic ac id (C18:3), content incre ased the most of all the FAs in the c old-tolerant line a nd not in the intermediate a nd se ns iti ve lin e s. B e rm ud a g ra ss ( Cynodon dactylon (L .) Pers) mode rate ly cold-tolera nt and c old -s e ns iti ve lin e s w e re c omp a re d u nd e r a sim ila r c old te mpe ra tur e str e ss r e g ime in which lea ves, cr owns and roots we re a naly zed for F A composition (Samala et a l., 1998). L inolenic ac id increa sed in the modera tely cold-tolera nt line as a re sult of cold trea tment. G e ne ti c A r e vie w o f t he lit e ra tur e did no t sp e c if ic a lly ide nti fy fr e e zetol e ra nc e g e ne tic reg ulation. Howeve r, infer ence s and possible ana logie s may be deduc ed fr om plant cold-

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22 toleranc e litera ture. Cold-tolera nce ( the ability of the plant to withstand above -fr eezing tempera tures without damag e) w ork in g enetics is ava ilable and c ould be used to provide insight or pe rhaps illuminate the complexity of fr eezetoleranc e at the g enetic le vel. Cold-toleranc e is a hig hly complex and inter-re lated tra it under g enetic c ontrol (Revilla et al., 2005) I n Arabidopsis thal iana ( L.) Hey nh. the numbers of g enes responsive to c old stress rang e fr om 1,000 to an extrapolated 7,000 g enes, de pending on the experiment and the micr oarr ay results (Guy 2003). Ge nes that ar e downreg ulated may be as important a s those up-re g ulated and e x presse d in microarr ay studies. Her itability of plant lea f-tissue fr eezetoleranc e wa s not found in the literature Cold-toleranc e her itability may provide insig ht as to possible heritability rang es for leaf tissue free ze-toleranc e. He ritability estimates for c old-toleranc e ac ross diffe rent c rop specie s has ra ng ed fr om moderate to high ( Revilla et al., 2005), with sig nificant g enoty pe x environment (G E) intera ction. Reported ( Revilla et al., 2005) maize see dling vig or, ma ize pu rp lin g of le a ve s, ma ize y e llo wi ng of le a ve s, ma ize dr y ing of le a ve s; l e nti l seedling fre eze-tolera nce, popla r fr eezetoleranc e, whe at fre eze-tolera nce, Brassica sp. Fr eezetoleranc e etc ., have be en tra its that depend on the sea son (Fa ll or Spring) acc limation or non-acc limation and highintensity or low-intensity fre ezing. Additive eff ects ha ve bee n repor ted (Revilla et a l., 2005) as the most important eff ects in many plants. Nonadditive g ene e ffe cts (dominant and e pistatic eff ects) a re important in many sp e c ie s. Ma te rn a l g e ne e ff e c ts a pp e a r t o a ff e c t c old -t ole ra nc e of e a rl y a nd la te g ro wt h stag es. I nterpopulation re curr ent selec tion or mass selection has be en re commended since a dditive ge ne ef fec ts are the most im portant. Yield unde r cold c onditions has been rec ommended as a method to evaluate cold-tolera nce ( Revilla et al., 2005).

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23 Fieldfre eze stress experiments have been r eporte d to have hig h experimental err or, hig h environmenta l interac tion and low corr elation with controlled fr eeze c hamber experiments (Revilla et al., 2005). Repor ted limited success in bre eding for f ree zing toleranc e using field selec tion has been a ttributed to major limit ing f actor s: lim ited g enetic dive rsity ineffe ctive sele ction criter ia, and limited knowledg e of f ree ze-toleranc e g enetic c ontrol. A p o mi xis ( a p o s p o r y a n d p s e u d o g a my ) e xis ts in b a h ia g r a s s a t t h e te tr a p lo id ( 4 x) chromosome leve l (Chen et al., 2001) Apomictic mode of r eproduc tion has restricte d tr a ns fe r o f d e sir e d tr a its be tw e e n s e xua l di plo id a nd a po mic tic te tr a plo id g e rm pla sm (F orbes a nd Bur ton, 1961). Attempts to manipulate apomoxi s in bahiag rass ha ve ra ng ed from fa ilure (Ha y war d, 1999) to limit ed, but imprac tical re sults (Burton, 1982; B urton, 19 86 ; B ur ton 1 99 2; B ur ton 1 99 9) A po mixis h a s h a mpe re d b re e din g c old -t ole ra nc e in tetraploid bahia g rass in the southea st US. Bre eding and sele ction in sex ual diploid (2 x ) chromosome leve l in bahiag rass lines for cold-tolera nce tr ait in addition to day leng thinsensitivit y trait has bee n a multi-state ef fort to extend the g razing season throug h the cool short-da y g rowing season ( Blount et a l., 2001). Outlin e of the Rese arch P lan Th e pu rp os e of the re se a rc h w a s to de te rm ine wh ic h me c ha nis ms c a us e d L TF T i n dip loi d b a hia g ra ss: Quantify the ra ng e of L TFT e x pression in diploid bahiag rass g enoty pes Ve ri fy wh e the r L TF T w a s a le a f o r r oo t f re e zetol e ra nc e tr a it Deter mine whether there wer e ana tomical differ ence s betwee n fre eze-tolera nt and fr e e zese ns iti ve g e no ty pe s De te rm ine wh e the r t he re we re ph y sio log ic a l di ff e re nc e s ( os mol a lit y a nd fa tty a c id composition) betwee n fre eze-sensitive a nd fre eze-tolera nt ge noty pes

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24 Quantify L TFT he redity (broa d-sense -and na rrowsense) De te rm ine the mod e of L TF T g e ne a c tio n ( a dd iti ve a nd do min a nt) Ch apt er 2 d escr ib es t he ex peri men ts us ed t o q uan ti fy th e ran ge of L TFT expression and the conf irmation of leaf -tissue fre eze-da mag e as a leaf eff ect, instea d of a root eff ect. I n order to quantify the extremes of the r ang e of the L TFT e x pression, fre eze-tolera nt and fre eze-sensitive plants would be c ollected a nd tested under controlled fre eze eve nts. Canopy damag e ra tings would be used to quantify the L TFT tr ait. The limit s of L TFT e x pression would be de termined by repe atedly colder fre eze trea tments. Additions of new plant mater ial to the experiment would require confirma tion of L TFT, with a sing le fre eze trea tment of the entire g enoty pe collec tion. To determine whe ther canopy damag e fr om free ze treatments wa s the result of e ither lea f dama g e, or of root damag e, fr eezesensitive g enoty pes identified in the initial experiments would be frozen in a manner that only the leave s would be subjecte d to the fre eze trea tments. Chapter 3 de scribes the experiments conducted to qua ntify anatomica l differ ence s betwee n fre eze-tolera nt and fre eze-sensitive g enoty pes. Diff ere nces in c ontrasting leaf anatomy might provide information about lea f-tissue fr eezetoleranc e. The reg ion of anatomica l investiga tion was based on the observa tion of leaf da mag e immediately afte r a fr eeze tre atment. The midrib re g ion of the lea f blade of a f ree ze-sensitive g enoty pe (F L 9) wa s discolored a nd damag ed within hours af ter plac ing pla nts in full sunl ight, a fter a fr eeze c hamber trea tment. I n contra st, the midrib reg ion of the lea f blade of a fre eze-tolera nt ge noty pe (F L 67), subjecte d to the same fr eeze tre atment, was not damag ed. Diff ere nces be tween the midrib vascular bundle ana tomy betwee n these two g e no ty pe s ( F L 9 a nd F L 67 ) w ou ld b e qu a nti fi e d. I f d if fe re nc e s w e re sig nif ic a nt f or the se two contra sting lines, additional fr eezesensitive and fr eezetolerant g enoty pes would be

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25 added to the inve stiga tion. Anatomical diffe renc es by fre eze-tolera nce c lass neede d to be confirme d acr oss multi ple lines, in order to support the hy pothesis that leaf a natomy was associate d with L TFT. Chapter 4 de scribes the experiments used to investiga te the two main theor ies of leaf fre eze tolera nce: 1) fre eze temper ature depre ssion by the mecha nism of increa sed mol a lit y a nd 2) me mbr a ne int e g ri ty by the me c ha nis m of inc re a se d me mbr a ne fl uid ity mediated by an incr ease in unsaturated f atty acid c ompositi on. Fr eezetolerant g enoty pes should have hig her molality than fre eze-sensitive g enoty pes, if the L TFT me chanism wa s incre ased molality Comparing the molality of the midrib re g ion of the lea f blade wher e damag e first a ppear ed in fre eze-sensitive lines, to fre eze-tolera nt lines, should determine whether molality was the L TFT me chanism. F ree ze-tolerant g enoty pes should have a hig he r p or tio n o f l e a f u ns a tur a te d f a tty a c ids tha n f re e zese ns iti ve lin e s, if fa tty a c id composition is t he L TFT me chanism. Chapter 5 de scribes the experiments used to quantify the L TFT inhe ritance (broa dand na rrowsense) and the mode of g ene a ction (additive a nd nonadditive portions). Plant breede rs nee d to know the nar row-se nse her itability (h ) of a trait. The 2 narr ow-sense heritability is the additive mode of g ene a ction, and it can be manipulated b y t h e p l a n t b r e e d e r t h r o u g h s e l e c t i o n T h e h i g h e r t h e h o f a t r a i t t h e m o r e r a p i d l y a 2 plant bree der c an make prog ress sele cting improved populations. One method of ob ta ini ng the inf or ma tio n n e e de d to c a lc ula te the h is t he dia lle l ma tin g de sig n o f p a re nts 2 that repr esent a wide ra ng e of tra it ex pression. A diallel mating desig n would be used wher e F 1 prog eny would be g rown fr om ever y possible combination of crosse s of a g roup of f ree ze-tolerant a nd fre eze-sensitive pa rents. B eca use informa tion was lacking on the number of car y opses nee ded to sow, g erminate and g row suff icient F1 pr og eny for

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26 the diallel study a ser ies of experiments would have to be conduc ted to support the diallel study

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27 CH APT ER 2 L EA F -T I SSU E F REE Z ETO L ERA NC E T RA I T D I VE RSI TY I N B AH I AG RA SS Introduction 4 B a hia g ra ss i s a pla nt w ith the C fi xati on pa thw a y su bty pe tha t us e s N AD Pma lic e nzy me (N AD PME ) i n th e le a f b un dle sh e a th c hlo ro pla st c e ll o f t he Kr a nz a na tom y to 2 provide c arbon dioxide (CO ) for r ibulose bisphophate ca rboxy lase/oxy g enase (Rubisco) 4 (B rown, 1999). O ne of the limit ations of the C g rass-g rowing season a nd rang e is the lack of or limited leaftissue free ze-toleranc e (Rowley et al., 1975). N o consistent photosy nthetic pathwa y explanation could be ar rived a t to ex plain leaf lesions cause d by cold temper ature s, other than to attribute othe r unknown fa ctors der iving f rom the 44 tr op ic a l or ig in o f t he C sp e c ie s b e ing e xami ne d ( L on g 1 99 9) T he C sp e c ie s d e c lin e in 4 perc entag e of f lora c ompositi on with increa sing la titude. The C specie s g rowing season has bee n noted to approximate a temper ature limit This tempera ture limit is reache d wh e n th e a ve ra g e da ily min imu m te mpe ra tur e fo r t he wa rm e st m on th o f t he y e a r i s le ss 4 than 18C (L ong 1983). This approximated limit ation of the C specie s rang e had be en 2 postulated to be a r esult of impaired ne t CO uptake throug h slow or irre versible photo4 inhibiti on of photosy nthesis. L ow temper ature toleranc e has be en found in the C g enus Zea Genoty pes of Zea mays L and Ze a p e re nn is ( A. S. Hi tc hc .) Re e v e s & Ma ng le sd or f wer e found to be tolerant to low temper ature photo-inhibiti on. These g enoty pes showed 4 source s of orig in effe cts when c ompare d to modern Zea mays L cultivars. The C g ener a are not excluded from hig h altitude and low temper ature sites. Four Paspalum three

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28 Muhlenbe rgia and one Er ag ro sti s specie s were found g rowing betwee n 4,000 and 4,500 m in altitude in the Peruvian Andes, whe re me an annua l tempera ture wa s 3 to 6C. South American ba hiag rass a cce ssions in t he U.S. Depa rtment of Ag riculture, Ag riculture Re sear ch Servic e, Ge rmplasm Resource s I nformation Ne twork S9-g rasse s (U SDA AR S GR I N S9 ) d a ta ba se we re se a rc he d f or c oll e c tio n s ite la tit ud e in S ou th Americ a noting their winter injury rating at Griff in, Georg ia (USDA, A RS, 2001). Cou ntr ie s o f o ri g in i nc lud e d A rg e nti na B oli via B ra zil, Pa ra g ua y a nd Ur ug ua y So uth Am e ri c a n b a hia g ra ss a c c e ssi on sit e s r a ng e d r ou g hly fr om 2 1 to 3 4 S la tit ud e (U .S. O f f i c e o f G e o g r a p h y, 1 9 6 3 ; U S O f f i c e o f G e o g r a p h y, 1 9 5 6 ; U S O f f i c e o f G e o g r a p h y, 1957; U.S. Office of Ge og raphy 1968; USDA, ARS, 2001). When acce ssions were g ro wn a t G ri ff in, Ge or g ia th e y va ri e d f ro m li ttl e to n o w int e r i nju ry thr ou g h c omp le te wi nte r i nju ry A lth ou g h th e USD A A RS G RI N S9 sy ste m r e po rt e d w int e rinj ur y str e ss an d s u rv i v al i n s t ea d o f l ea ft i s s u e d am age fr o m fr ee z e e v en t s t h e w i n t er i n j u ry ra t i n gs provide a n indication of diverse g enoty pe tolera nce to be low-fr eezing tempera tures. Leaf-tissue Fr eez e-t olerance Sc re e nin g g e no ty pe s f or le a ftis su e fr e e zetol e ra nc e c a n b e do ne in t he fi e ld o r i n g rowth cha mbers by assessing leaf -tissue fre eze-injury Fr eezetolerant g enoty pes sh ou ld h a ve low le a ftis su e inj ur y sy mpt oms F ie lddis c ri min a tio n o f f or a g e g e rm pla sm for low leve ls of leaf -tissue fre eze-injury would be desira ble. Fie ld-discriminated g ermplasm should be a pplicable to commer cial for ag e produc tion. Disadvantag es of field-f ree ze events include limitations of unpredicta ble and diff icult-to-re peat a nnual fre eze eve nts. Field-discr imination is espec ially difficult when a ttempting to scre en plant m a t e r i a l t o b e g i n a s t u d y. Convenient, replica ble, controlled e x periments in environme ntal chambe rs allow c on tr ol o f c oo lin g ra te s a nd fr e e zestr e ss e xpos ur e du ra tio n b y e xpos ing a ll t he pla nts to

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29 the same tre atment. The disadva ntag e of the environmenta l chamber s is that plants are co o l ed b y ad v ec t i o n wh i ch i s n o t t h e s am e c o o l i n g co n d i t i o n p l an t s ex p er i en ce d u ri n g a radia tive frost (Ashw orth and Kie ft. 1995). Howe ver, the advanta g e of c ontrolled and replica ble environme ntal conditions favors its use for initial screening s, as well as descr iptive experiments. 4 C g ra sse s ( Paspalum dilatatum Poir., Eragrostis curvula Schrad Nees hemarthria altissi ma Poi r Stapf CE Hubbard, Pennisetum c landestinum Hochst. ex Chiov., Acroce ras macrum Stapf., Cynodon dactylon L ., Setaria anceps Stapf., Di git ar ia sp. ) have been sc ree ned using a temper ature -g radie nt bar, whic h froze excised lea f piec es on a temper ature continuum. Fre ezing tre atment was f ollowed by deter mination of lethal 50 tempera ture (L T ), base d on elec troly tic leaka g e of e x cised lea f piec es (Rowley et al., 50 1975). Elec troly tic leaka g e has be en used w ith linear re g ression to deter mine L T of Setaria anceps Stapf., Chloris gayana Kunth. and Ce nc hr us c ili ar is L (I vory and Whit eman, 1978) Electroly tic leaka g e, as a measure of lea f fr eezetoleranc e diffe renc es among tropical g rasse s, identified the temper ature s at which the g enoty pes we re f ree zetolerant. 4 Pe rc e nt f oli a r f re e zeda ma g e of C g ra sse s ( Setaria anceps Stapf, Se tar ia t rin e rv ia Stapf, Di git ar ia m ac ro glo ssa Henr ., Digitaria setivalva Stent., Di git ar ia s mu tsi Stent., Paspalum plicatulum Michx ., Paspalum guenoarum Are chav., Pa sp alu m r oja sii Hac k., 3 Paspalum dilatatum Poir., Panicum max imum Jac q. ) a nd C g ra sse s ( Lolium perenne L ., Lolium x hybridum) was c alculate d on a dry matter (D M) basis on har vested, sepa rate d, and drie d leave s (Hac ker e t al., 1974; I vory and Whiteman, 1978). I n addition to being time-consuming the DM method of a ssessing f oliar fr eezedamag e wa s destructive, requiring the har vesting of top g rowth. Ha rvesting top gr owth interfe res with plant

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30 rec overy measure ments, cumulative stress a s a re sult of free ze-injury and quantific ation of g e no ty pe wi nte r s ur viv a l. This study conce ntrates on investig ating varia bility among bahiag rass lines lea ftis su e fo r f re e zetol e ra nc e T he le a ftis su e fr e e zetol e ra nc e tr a it ( L TF T) wa s id e nti fi e d in this study as an a bility to maintain gr een, a ppare ntly undamag ed lea ves af ter experiencing a fr eeze ( tempera tures below 0C) e vent. Th e re wa s a ne e d to sc re e n b a hia g ra ss g e no ty pe s th a t mi g ht r e pr e se nt a div e rs e ra ng e of L TF T. Re pr e se nta tiv e fr e e zese ns iti ve a nd fr e e zetol e ra nt ( L TF T) tr a it e xpre ssi on g e no ty pe s w e re ne e de d to inv e sti g a te po ssi ble me c ha nis ms c on tr ibu tin g to this trait. Screening of diploid (2 x ) plant mater ial additionally might provide information helpful in a bre eding prog ram a imed at extending g razing during the winter se ason in the southeaster n United States. M at e r ial s a nd M e th ods Leaf-tissue Fr eez e-t olerance Trait Scre ening E xperim ent 1 An initial ex periment wa s conducte d in which 26 bahiag rass lines we re subjec ted to prog ressively lower be low-fr eezing tempera tures. The purpose wa s to quantify div e rs ity of L TF T t ra it i n e xistin g c omm e rc ia l di plo id a nd te tr a plo id cultivars, as w ell as diploid selections from a ba hiag rass bre eding prog ram initiated by Dr. Ann B lount at the University of F lorida North F lorida Resea rch a nd Educa tion Center in Marianna Florida Fr om this i nitial scree ning e x periment, lines we re ide ntified as hig hor lowL TFT. A rg e nti ne , a te tr a plo id a po mic t, w a s u se d a s th e sta nd a rd lin e kn ow n to ha ve the le a st L TFT of commerc ial cultivars. This standar d was used to c ompare the L TFT of sexual diploid bahiag rass lines in the study A selec tion of the commerc ial diploid cultivar Pensac ola obtaine d from Dr Paul Mislevy University of F lorida Resea rch a nd

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31 Ex tension Center a t Ona, F lorida, wa s used bec ause the larg est ac rea g e in Flor ida pasture land is planted to that varie ty The line Sand Mounta in was include d beca use it had been r eporte d to have improve d cold-tolera nce c ompare d to Pensacola a nd Arg entine (B lount, personal communica tion, 2001; Ball and B lount, 2003). Sand Mountain seed wa s o bta ine d f ro m th e Al a ba ma Se e d Co mmi ssi on T he re ma ini ng re pr e se nta tiv e dip loi d c lon e s in the e xpe ri me nt w e re se le c te d b y Dr B lou nt f ro m a pp ro xima te ly 24 ,0 00 pla nts that wer e obser ved af ter fr eeze e vents in north Florida and included in a bree ding prog ram for cold-tolera nce. Plants we re pr opag ated ve g etatively from rhizome/stolon piece s orig inating f rom the Univer sity of F lorida North F lorida Resea rch a nd Educa tion Center a t Marianna Florida Twenty -three clones we re pr opag ated ve g etatively Plantlets from three c ultivars or lines wer e see d-g rown (A rg entine, Pensac ola, Sand Mountain). Plants were maintained in the same media, and g iven nutrient mana g ement and sche duled drip irrig ation sy stem in the Ag ronomy teac hing g ree nhouse under ambient day leng th, during the fa ll of 2001. The g ree nhouse temper ature was set f or supplemental steam he at to circ ulate whe n air tempe rature dropped be low 22C. The g ree nhouse maxim um tempera tures ra rely exceede d 50C, as rec orded w ith a minimum /max imum thermocouple (Ac urite). Round pot size was 12.5 cm diamete r x 12.5 cm heig ht. The potting me dia was Scotts Ter ralite Ag ricultural Mix (Scotts -Sierra Horticultural Produc tion Com pany 14111 Scotts L ane Rd., Mar y sville, OH 43041). 25 2 Nutrient mana g ement wa s 1 g of a 164-8-1 NP O -K O-F e ana ly sis gr anular fer tiliz er pr e -p la nt i nc or po ra te d in to t he me dia of e a c h p ot. I ro nit e 100 ( I ro nit e Pr od uc ts Company Scottsdale, AZ 85258) wa s sprinkled at the ra te of 1 g per pot on the top of the media to pre vent iron chlorosis. An ove rhea d spray irrig ation sy stem was set to run f or five min four times during the day

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32 Temper ature trea tments were single -nig ht expos ure to -1C, -3C, -5C or 7C. The same sets of plants wer e prog ressively exposed to colder temper ature trea tments. After a temper ature trea tment, plants were rate d then re turned to the g ree nhouse for 7 d befor e imposing the next coldest temperature trea tment. Fre ezing c hamber was a blood cooler (Pharmac y GEM Refr ige rator Company Philadelphia, PA) with sty rofoa m ins ula tio n a dd e d to c ov e r t he sli din g do or s. Te mpe ra tur e s w e re se t by a dju sti ng c on tr ols me a su ri ng a ve ra g e te mpe ra tur e s w ith the rm oc ou ple s ( F ish e r S c ie nti fi c I nte rn a tio na l) during a compr essor c y cle a nd adjusting c ompressor r un-time cy cles. Tr eatment tempera ture dura tion was a total of 10 h, be g inning a t 22:00 and ending at 08:00 the following morning The USDA A RS GRI N S9 winter-surviva l descriptor r ating (1 to 9) was used a s the basis for r ating plant canopy leaf -dama g e (1 = 0% plant ca nopy leaf damag e, 9 = 100% plant leaf canopy damag e) no la ter than 48 hour s after trea tment (Table 2-1). Canopy leave s were considere d damag ed if they appea red w ater -soake d, brown, or de sicca ted and c urled. Rating s were a visual estimate of the portion of the entire pla nt canopy that was da mag ed (Ta ble 2-1). T ab l e 2 -1 C an o p y l ea fd am age ra t i n g s y s t em s h o ws p er ce n t age o f c an o p y l ea fd am age and ca nopy g ree n leaf Canopy leaf -dama g e Canopy leaf -dama g e Canopy g ree n leaf Rating % % 1 0 100 2 12 88 3 25 75 4 38 62 5 50 50 6 62 38 7 75 25 8 88 12 9 100 0

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33 Ba hiag rass lines we re r eplicate d four times and r andomized as to which shelf the y wer e assig ned in the fr eezing trial. To ac count for position eff ects, two re plications per line wer e included in the uppe r shelf a nd two in the lower she lf during eac h fre ezing trea tment. L ine position by replica tion was identified during eac h succe ssive fre ezing trea tment by having plants in plastic tray s so that positi ons were identical in ea ch replica tion. Data log g ers ( Optic StowAway Temp Onset Computer, 470 Mac Arthur Blvd., B ourne, MA 02537) rec orded a ctual temper ature s in eac h shelf. B eca use of limit ed coole r spac e, lines we re subjec ted to trea tments in spli t replica tions (replica tions 1 and 2 subjec ted to tempera ture tre atment, then re plications 3 and 4). Plants wer e g iven 1 8 d to r e c o v e r in th e U F I F A S A g r o n o my te a c h in g g r e e n h o u s e a f te r th e la s t f r e e ze tempera ture then r ated f or cold stre ss damag e. Leaf-tissue Fr eez e-t olerance Trait Scre ening E xperim ent 2 An experiment was c onducted in an e nvironmental g rowth cha mber (Environmenta l Growth Chamber s, 510 East Washington Stree t Chag rin Fa lls, OH 44022) that could a ccommodate all of the lines with their re plications, blocked for position effec ts, in a single c ontrolled fre eze-e vent. A sing le fre eze trea tment which acc ommodated all of the ba hiag rass lines in one sing le trea tment was done to e nsure that results obtained with the modified blood coole r we re c onsistent. To ensure a ir flow su rr ou nd e d th e e nti re po tte d p la nts un if or mly w oo de n p a lle ts w ith e xpa nd e d w ir e me sh we re us e d to ra ise po tte d li ne s o ff the fl oo r. B a ff le s a nd a re c ir c ula tio n f a n w e re us e d to redire ct air f low from the c ooling unit fa n in an attempt to reduc e foliar desicc ation from cold, dry air flow. B aff les wer e moved a nd adjusted in a ser ies of experiments using Arg entine plants, five pla nts per block, e ight positions in the gr owth chambe r, and prog rammed to run a t -3C from 2200 till 0800. The -3C treatment tempe rature was

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34 us e d b e c a us e pr e vio us tr ia ls i n th e mod if ie d b loo d c oo le r s ho we d mo re da ma g e to Arg entine plants at that tempe rature than at -1C. When statistical analy sis of canopy leaf -dama g e ra tings we re not sig nificant for block (g rowth cha mber position) eff ects, a single controlled-f ree ze event wa s imposed on all the test lines. The EGC wa s programmed to maintain a -6 that temperature treatment from 2200 till 0800. Thirty bahiagrass lines were used to screen for LTFT. Vegetatively propagated lines were grown as previously described for the first LTFT screening experiment. Potted plants were enclosed in plastic bags to maintain humidity at a constant level. Relative humidity has been shown to shift the percent of leaf freeze-damage (Ivory and Whiteman, 1978) in freeze chamber trials with tropical grasses. The higher the relative humidity the more leaf freeze-damage was recorded at treatment temperatures ranging from -1 to -5C. Since potted plants came directly from a controlled greenhouse irrigation system, plastic bags were used to seal moisture and prevent desiccation of plants during the 10-h freeze-temperature treatment. Additionally, bagging individual pots prevented the compressor from failing due to ice formation when the large number of potted plants released moisture from the potting media as the fans recirculated the -6C air. Leaf vs. Root Effects Experiment 3 A third experiment was conducted using the modified blood cooler to determine if the observed freeze-damage was due to the entire plant (potted roots and leaves) being subject to low temperature or if the damage was only from the leaves being frozen. Potted plants of lines selected to represent a range of LTFT traits were immersed in controlled recirculation water baths (Polyscience Model 71, Niles IL) set at 5C. Potted plants were weighted down with river rock so the warmed recirculation water covered the

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35 entire r oot sy stem with only the leave s exposed to the below-fr eezing air tempe rature trea tment (Fig ure 21). F ig ur e 21. Wat e rba th e xpe ri me nt s ho wi ng tub s, he a te rs a nd we ig hte d p ots Actual f ree zing stress tre atments wer e -2.7 a nd -3.2 C for 10 h, star ting f rom 2 2 0 0 a n d e n d in g a t 0 8 0 0 th e f o ll o w in g mo r n in g to e n s u r e th a t l e a f ti s s u e F r e e ze trea tment durations wer e similar to whole-pla nt 10-h fre eze trea tments. Tempera ture tr e a tme nts be low -3 .2 C w e re c on str a ine d b y the the rm a l ma ss o f t he wa te r i n th e pa ns the incre ased r elative humidity in the cooler as a r esult of the wa ter surf ace and the condensa tion of ice on the f ree zer compre ssor. Results and Discus sion Leaf-tissue Fr eez e-t reat m ent Scre ening E xperim ent 1 Observa tions showed initial leaf-tissue fr eezeinjury sy mptoms on t hawing included wa ter-soa ked lesions, followed by leaf wilting and c urling afte r being exposed to full sunlight. Fur ther fr eezeinjury sy mptoms under full sunlight included tissue browning followed by leaf desicc ation. L ight f ree ze-injury sy mptoms i n bahiag rass we re ty pically leaf -tip browning and dry ing. Mor e seve re f ree ze sy mptoms were br owning and

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36 desicc ation of entire leave s or tillers. Canopy leaf -dama g e ra tings of the 26 lines combined ar e pre sented in Table 2-2. Table 22. Mean simple ef fec ts across 26 ba hiag rass lines subjec ted to targ et tem pera tu re t reat men ts of p rogres si vel y col der f reez in g event s, L TFT scre ening experiment 1. Temper ature Canopy leaf -dama g e Canopy g ree n leaf C rating % -1 1.0a* 100 -3 4.2b 60 -5 6.2c 35 -7 8.3d 9 *M e a ns wi th t he sa me le tte r a re no t si g nif ic a ntl y dif fe re nt a t P = 0. 05 c on fi de nc e le ve l. L ine /c lon e tr e a tme nt s imp le e ff e c ts w e re sig nif ic a nt a t P < 0. 00 01 le ve l ( Ta ble 2-2). Simple eff ects, a vera g ed ac ross all lines, showed the te ndency as trea tment tempera ture de cre ased f or lea f ca nopy damag e ra tings to incre ase. A t -3C mean bahiag rass mea n canopy damag e had a rating of 4.2 (e quivalent to an ave rag e 60% g ree n undamag ed lea f ca nopy ). At -5C, bahia g rass mea n canopy had a r ating of 6.2 50 ( e q u i v a l e n t t o a n a v e r a g e 3 5 % g r e e n u n d a m a g e d l e a v e s i n t h e p l a n t c a n o p y ) T h e LT values ha ve bee n used to establish fre eze-tolera nce sta ndards in experiments which ha ve div e rs e pla nt l ine s ( I vo ry a nd Whit e ma n, 19 78 ). F or thi s g ro up of se le c te d b a hia g ra ss 50 lines, a lethal tempe rature (L T ), whe re 50% of the lea ves of the c anopy leave s were g ree n and 50% w ere damag ed, could be interpolated from a linear regression from -3 to -7C where 50 12.813x + 98.656 = L T = -3.8C Replication eff ects we re sig nificant a t the targ et trea tment tempera tures of 3C and -5C, ( P < 0.001 and P < 0.01, re spectively ). Examination of the rec orded te mpe ra tur e s d ur ing the se pa ra te ru ns pe r r e pli c a tio n s ho we d th a t te mpe ra tur e s w ith in targ eted tempe rature trea tments were differ ent (Ta ble 2-3). T emper ature differ ence s wer e suff icient to cause differ ence s in canopy leaf -dama g e ra tings in the same lines from

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37 tempera tures ra ng ing f rom -2.7 throug h -6.0 C in the targ eted tre atments of -3 a nd 5C. The 0.2C differ ence betwee n replica tions at the targ et trea tment tempera ture of 1C and the 0.1C differ ence betwee n actua l tempera tures of 6.5 and -6.6C we re insufficient to ca use diffe renc es in leaf canopy rating s betwee n runs. Results showed that bahiag rass c an be se nsitive enoug h to be visually rate d differ ently as a r esult of experiencing fre eze temper ature differ ence s as small as 0.8C. Results also showed the need to subjec t all the plant materia l at one time to the temper ature trea tment. Table 23. Mean simple ef fec ts across a ll bahiag rass lines with ac tual tempera ture trea tments of prog ressively colder fre ezing e vents, ca nopy leaf -dama g e, L TFT sc ree ning e x periment 1. T ar get t em p er at u re Ac t u al t em p er at u re C an o p y l ea fd am age C C rating -1 -0.5 1.1f* -1 -0.7 1.1f -3 -2.7 3.6e -3 -3.5 4.8d -5 -4.0 5.7c -5 -6.0 6.8b -7 -6.5 8.6a -7 -6.6 7.9a *M e a ns wi th t he sa me le tte r a re no t si g nif ic a ntl y dif fe re nt a t P = 0. 05 c on fi de nc e le ve l. I n spite of all ef forts to reduc e position and replica tion effe cts, splitti ng the materia ls into separate runs produc ed additional var iance which may have r educe d the str e ng th o f l ine e ff e c ts. L ine e ff e c ts w e re sig nif ic a nt ( P < 0.05) ( Table 24) and identified lines that had c onsistently high r ating s as trea tment tempera tures dec rea sed. Arg entine had the highe st canopy leaf -dama g e ra ting a t -1 and -3C, whic h was consistent with the ear liest reporte d frost ra tings (B urton, 1946). L ines FL 67 and C4-36 ha d th e low e st a nd se c on d lo we st, re sp e c tiv e ly o f c a no py le a fda ma g e ra tin g s a c ro ss t r e a t m e n t t e m p e r a t u r e s w h i c h d e m o n s t r a t e d c o n s i s t e n t LT F T a s q u a n t i f i e d b y r a t i n g s In contra st, severa l lines (i.e., FL 17, FL 19) appe are d to have L TFT a t -3C, but not at colder

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38 te mpe ra tur e s. Sa nd Mo un ta in e xhibi te d s o mu c h v a ri a bil ity be tw e e n r e pli c a tio ns tha t it wa s n ot s ig nif ic a ntl y dif fe re nt f ro m ot he r l ine s. Th is e xpe ri me nt c on fi rm e d v a ri a bil ity in bahiag rass L TFT. F ree ze treatments we re c older a nd more than twic e the 4 h ty pical du ra tio n o f n a tur a l f re e ze e ve nts in p e nin su la r F lor ida T he e nti re pla nt ( le a ve s, sto lon s, roots) wa s frozen, which w as a f ree ze stress never experience d in Florida. Ta ble 2-4 shows that there wer e some lines at the ta rg eted 7C treatment, which a ppare ntly had only modera te fre eze-tolera nce a t these extreme fr eeze stre ss conditions. Table 24. Prog ressively lower f ree zing temper ature events e ffe cts on ca nopy fre ezeda ma g e of se le c te d s e xua l di plo id a nd a po mic tic te tr a plo id li nes L TFT scre ening experiment 1. Targ et temper ature C Ploidy L ine/cultivar -1 -3 -5 -7 Rating 2x FL 11 1b* 7. 0a b 9. 0a 9. 0a 2 x Sand Mt. 1b 4. 7a bc de 7. 7a 8. 7a 2 x FL 9 1b 6. 5a bc 7. 5a 8. 7a 2 x FL 38 1b 4. 0a bc de 7. 5a 8. 7a 2 x FL 66 1b 4. 0a bc de 7. 5a 8. 7a 2 x Pensacola 1b 6. 5a bc 7. 2a 8. 5a 2 x FL 2 1b 4. 2a bc de 7. 2a 9. 0a 2 x FL 30 1b 5. 5a bc d 7. 2a 8. 5a 2 x FL 31 1b 5. 5a bc d 7. 0a b 9. 0a 2 x FL 35 1b 3. 7a bc de 6. 7a b 8. 7a 2 x FL 45 1b 3. 5bc de 6. 7a b 8. 7a 2 x FL 82 1b 6. 0a bc 6. 7a b 8. 0a 4 x Arg entine 2a 7. 2a 6. 5a b 8. 7a 2 x FL 19 1b 2. 2de 6. 5a b 8. 7a 2 x CO175 1b 5. 0a bc de 6. 2a b 8. 5a 2 x FL 17 1b 1. 7e 6. 2a b 8. 7a 2 x FL 41 1b 3. 2c de 6. 2a b 9. 0a 2 x FL 69 1b 3. 0c de 6. 0a b 8. 7a 2 x FL 52 1b 3. 5bc de 5. 7a b 8. 7a 2 x FL 56 1b 4. 5a bc de 5. 7a b 8. 2a 2 x C4-36 1b 2. 2de 5. 5a b 6. 0bc 2 x FL 54 1b 3 5 b cde 5 2 ab 7 2 ab 2 x FL 86 1b 3. 7a bc de 5. 2a b 8. 0a 2 x FL 58 1b 4. 0a bc de 5. 0a b 8. 2a 2 x FL 24 1b 3 5 b cde 4 2 ab 7 2 ab 2 x FL 67 1b 2. 2de 2. 7b 5. 0c *M e a ns wi th t he sa me le tte r a re no t si g nif ic a ntl y dif fe re nt a t P = 0. 05 c on fi de nc e le ve l.

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39 B e c a us e of the se ve ri ty of the pr og re ssi ve fr e e zes (1 0 h du ra tio n, e nti re pla ntfre ezing, r epea ted and c older fr eezes) it wa s important to also view and ra te amount of whole-pla nt damag e of a ll 26 lines after 18 d r ecove ry in the g ree nhouse (Ta ble 2-5). Th e a bil ity to r e c ov e r f ro m f re e ze s tr e ss e ve nts is i mpo rt a nt f ro m a se le c tio n a s w e ll a s a prac tical standpoint. Florida f ree zes are f ollowed by war m periods fa vorable for bahiag rass re g rowth or c ontinued g rowth during periods of shor ter da y leng th. FL 67 had the most reg rowth, the lea st damag e, and w as ra ted as 2.5. A r ating of 2.5 mea nt that FL 67 frozen clone s, after an 18d re cover y period, ha d 81% of the c anopy of control EL 67 clones maintaine d in the g ree nhouse (Ta ble 2-5). As fre eze temper ature trea tments were lowere d, the trend w as for bahiag rass line canopy -dama g e ra tings to incre ase ( Table 24). At -1C Arg entine wa s the only line that wa s d a ma g e d. At -3 C c a no py -d a ma g e ra tin g s r a ng e d f ro m 1. 7 to 7. 2 ( e qu iva le nt t o gre en l ea f c an o p i es ra n gi n g fr o m 9 1 t o 2 2 %, re s p ec t i v el y ). At -5 C ca n o p y -d am age rating s rang ed fr om 2.7 to 9.0 (equivalent to g ree n leaf canopie s rang ing f rom 21 to 0%, re sp e c tiv e ly A t 7 C lin e c a no py -d a ma g e ra tin g s r a ng e d f ro m 5. 0 to 9. 0 ( e qu iva le nt t o g ree n leaf canopie s rang ing f rom 50 to 0%, re spectively ). At -7C the only line that was signific antly differ ent from the 26 lines w as F L 67, which also ha d the lowest ca nopy fre eze-da mag e ra ting ( 5.0). I t was evide nt that the limit of the L TFT tr ait had bee n approa ched by the -7C trea tment. The hig hest fre eze-stre ss toleranc e of a ll the lines tested (Ta ble 2-4) w as F L 67, as shown by the ra tings: 2.2, 2.7, and 5.0 a t tempera tures -3, -5, a nd -7C, respe ctively I n order to contrast mec hanisms within a plant population, cold-sensitive clones wer e nee ded. Te traploid Arg entine could not be inc luded in further mecha nism studi es using diploid lines beca use of the c onfounding of ploidy and ce ll siz e with other

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40 Table 25. Whole plant recove ry rating s as a pe rce nt of control plants, 18 d af ter trea tment of selec ted sexual diploi d and apomictic te traploid lines by pr og re ssi ve ly low e r f re e zing te mpe ra tur e e ve nts Som a tic c hr omo so me number L ine/cultivar Fr ee z e s t re s s d am age rating 2 x FL 11 8.5a* 2 x Sa nd Mt. 7 0 ab 2 x FL 9 8.5a 2 x FL 38 8.7a 2 x FL 66 8.0ab 2 x Pensacola 5.0a 2 x FL 2 8.5a 2 x FL 30 8.0a 2 x FL 31 9.0a 2 x FL 35 8.2a 2 x FL 45 8.5a 2 x FL 82 7.7ab 4 x Arg entine 8.1a 2 x FL 19 7.5ab 2 x CO175 7.0ab 2 x FL 17 7.7ab 2 x FL 41 8.5a 2 x FL 69 8.0a 2 x FL 52 9.0a 2 x FL 56 7.0ab 2 x C4-36 5.2b 2 x FL 54 6.7ab 2 x FL 86 7.5ab 2 x FL 58 7.7ab 2 x FL 24 6.7ab 2 x FL 67 2.5c *M e a ns wi th t he sa me le tte r a re no t si g nif ic a ntl y dif fe re nt a t P = 0.05 conf idence level. dif fe re nc e s b e tw e e n th e dip loi d a nd te tr a plo id l ine s. L ine F L 11 wa s a s se ns iti ve to c a n o p y f r e e z e d a m a g e ( T a b l e 2 4 ) a s A r g e n t i n e ( r a t i n g s o f 7 0 a n d 7 2 r e s p e c t i v e l y) at -3 C At t em p er at u re s o f 5 an d -7 C l i n e F L 1 1 s h o we d m o re ca n o p y fr ee z ed am age than Arg entine. F L 11 was not a vig orous line and wa s propag ated with diffic ulty for the se c on d L TF T s c re e nin g e xpe ri me nt. L ine s F L 31 a nd F L 52 we re no t us e d a s c lon e s to contra st L TFT be cause they had died ( fre eze-stre ss damag e ra ting = 9.0) and ha d not shown ca nopy leaf -dama g e as se vere ly as other lines a t -3C.

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41 A commer cial standa rd ser ves as a benchma rk to eva luate new g ermplasm for potential cultivar improveme nt. Be cause Pensacola was a population (g rown fr om seed, which mea nt that plant-to-plant var iability was inher ent), a veg etatively propag ated c lone was ne eded tha t would behave simil arly to Pensacola. L ine FL 9 had ca nopy fre ezeda ma g e ra tin g s th a t w e re sim ila r t o Pe ns a c ola (T a ble 24) a nd wa s th e re fo re inc lud e d in anatomica l (Chapter 3) and osmolality and fa tty acid pr ofile (Chapte r 4) studies instead of Pensac ola. Leaf-tissue Fr eez e-t olerance Scree nin g Experim ent 2 I n the first scr eening experiment, one fr eezetolerant (F L 67) and one intermediate ly fre eze-tolera nt (C4-36) line had be en identified to re prese nt the tolerant levels of the tr ait expression. There was a need to f ind more than two c lones with hightrait expression under fr eezestress. Additional plant materia l was found a nd pr op a g a te d. Tw o c lon e s w e re ob ta ine d f ro m so uth e a ste rn Ok la ho ma D r. B lou nt a lso added supe rior clone s from the bre eding prog ram. The re a lso was a ne ed to expand the sensitive end of the L TFT tr ait expression, simi lar to Arg entine, in orde r to compar e the rang e of tra it ex pression within one ploidy level (diploid). No a dditional free ze-sensitive lin e s w e re a dd e d to the se c on d s c re e nin g e xpe ri me nt b e c a us e dip loi d b a hia g ra ss w ith those traits wer e not ava ilable. The se cond scr eening experiment included 30 lines (Table 2-6). All lines were included in one sing le temper ature trea tment. Each potted line w as enclose d in a sea led plastic bag to maintain high va por pre ssure a round the lea ves. The single fre ezing tre atment re sulted in canopy fre eze-da mag e ra ting diff ere nces tha t varied among clones (T able 26).

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42 Ta ble 26. Ca no py fr e e zeda ma g e of se le c te d s e xua l di plo id a nd a po mic tic te tr a plo id lin e s, a s a ff e c te d b y a sin g le fr e e ze t re a tme nt ( -6 C) in a n e nv ir on me nta lly controlled c hamber Som a tic c hr omo so me number L ine/cultivar C an o p y fr ee z ed am age rating 2 x 2 x FL9 6.2a* 2 x Sand Mountain 5.6ab 2 x FL 31 5.2abc 2 x FL 52 5.2abc 2 x FL 17 5.2abc 4x Arg entine 5.0abcd 2 x FL 45 5.0abcd 2 x FL 58 4.8abcd 2 x CT18 4.7abcd 2 x FL 11 4.6abcde 2 x C436 4.4abcde f 2 x FL 30 4.2abcde f 2 x FL 82 4.2abcde f 2 x FL 2 4.0abcde f 2 x Pensacola 4.0abcde f 2 x FL 38 3.8abcde f 2 x FL 56 3.8abcde f 2 x FL 54 3.6bcdef 2 x FL 41 3.6bcdef 2 x FL 82 3.6bcdef 2 x FL 24 3.6bcdef 2 x FL 19 3.4bcdef 2 x FL 66 3.4bcdef 2 x CO5 3.4bcdef 2 x CO175 3.0cdef 2 x FL 69 2.8cdef 2 x OK1 2.6def 2 x OK2 2.2ef 2 x FL 67 2.0f 2 x CO6 2.0f *M e a ns wi th t he sa me le tte r a re no t si g nif ic a ntl y dif fe re nt a t P = 0. 05 c on fi de nc e le ve l. Ca no py fr e e zeda ma g e ra tin g s ( Ta ble 26) fo r e a c h li ne we re low e r i n th is experiment than in the first L TFT e x periment. The bag g ed plants may not have be en as desicc ated f rom the cold, dry air fr om the fre ezer fa n, as whe n they wer e uncove red. L ines cha ng ed ra nking in a mount of canopy fre eze-da mag e, but that was e x pecte d under a uniform f ree ze trial compare d to split runs in the first scr eening experiment. For

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43 example, line FL 9 was e ven less fr eezetolerant (r ating = 6.2) tha n fre eze-sensitive standard A rg entine (r ating = 5.0). Sand Mountain, the pur ported fr eezetolerant line, develope d from Pensac ola, had a rating that was not sig nificantly differ ent from the fre eze-sensitive lines. Rating differ ence s betwee n FL 9 and Ar g entine mea ns were not signific antly differ ent. Al tho ug h r a w r a nk of lin e ra tin g s w a s c ha ng e d in thi s e xpe ri me nt, so me ne wl y introduced c lones showed supe rior L TFT. A new c lone from Dr Blounts bre eding prog ram, CO6, had a s low a ca nopy fre eze-da mag e ra ting ( 2.0) as F L 67. I n addition, the ne wl y inc lud e d O kla ho ma lin e s, OK 1 a nd OK 2 ( ob ta ine d f ro m D Re df e a rn ) h a d n e a rl y as low ra tings (2.6 a nd 2.2, respe ctively ). L ow ca nopy damag e ra tings c ould be expected since Okla homa experienc es much long er a nd colder winter pe riods than north F lorida and south Geor g ia, wher e most of the plant mater ial orig inated. This second L TFT e x periment supporte d the selec tion of FL 9 as a diploid clone to r e pr e se nt t he se ns iti ve e nd of tr a it e xpre ssi on T his e xpe ri me nt a lso su pp or te d th e us e of c lon e s F L 67 O K1 O K2 a nd CO 6 to re pr e se nt a hig h e xpre ssi on of the L TF T t ra it i n further mecha nism (Chapter 3 and Cha pter 4) a nd g enetics studies (Cha pter 5). Leaf vs. Root Ef fects Experim ent 3 Simpl e tre atment ef fec ts across a ll lines used in the water -bath e x periment we re signific ant at P < 0.03. Simple effe cts showed that a s canopy tempera ture de cre ased, the canopy fre eze-da mag e ra ting incr ease d signific antly Root temperature was ke pt constant. I f ca nopy fre eze-da mag e ra tings we re a result of g enoty pe diffe renc es in root fre eze-da mag e, then ther e should have been no dif fer ence in rating s when tre atment te mpe ra tur e s w e re de c re a se d. Me a n s imp le e ff e c ts s ho we d th a t L TF T w a s a dir e c t r e su lt o f l e a f d a m a g e f r o m b e l o w f r e e z i n g t e m p e r a t u r e s n o t a r e s u l t o f r o o t i n j u r y.

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44 The wa ter-ba th experiment (which kept r oots at 5C while subjecting leaf tissue to below-fr eezing tempera tures) w ith 6 clones having differ ing c anopy fre eze-da mag e, sh ow e d th e mod if ie d c oo le r w a s a ble to i mpo se te mpe ra tur e tr e a tme nts (2. 7 3. 2 C) that resulted in ra tings that sepa rate d the lines. L ine or c lone diffe renc es (Ta ble 2-7) we re sig nif ic a nt f or the -2 .7 C t re a tme nt ( P < 0.0144) a nd the -3.2C trea tment ( P < 0.043). L ine FL 9 was c onfirmed a s a fr eezesensitive line simil ar to Ar g entine at -3.2C and a ppear ed to be e ven less tolera nt than Arg entine at 2.7C. L ines FL 11 and FL 82 wer e not sig nificantly differ ent from the A rg entine fr eezesensitive standard at -3.2C. Sand Mountain was not diffe rent than Pensa cola, f rom which it was se lected, a t e ith e r t e mpe ra tur e tr e a tme nt. Th e se ra nk ing s a nd c la ssi fi c a tio ns we re sim ila r t o th os e obtained in the first two e x periments whe re the entire pla nt was fr ozen (Table 26). No lines were included in the tub experiments that had fr eezetoleranc e bec ause w hat was being tested wa s whether leaf injury was a result of true leaf -dama g e or a result of root injury during fre ezing stre ss. There fore lines that were fre eze-sensitive we re use d for that experiment. I f ca nopy injury was the r esult of fre ezing r oots, then fre eze-sensitive lines should have shown no da mag e in the wa ter-ba th experiment, since roots wer e protec ted. Ta ble 27. Ca no py fr e e zeda ma g e of se le c te d s e xua l di plo id a nd a po mic tic te tr a plo id lines while root sy stem was kept a bove fr eezing (5C) F ebruary 2002. Som a tic c hr omo so me number L ine/cultivar Temper ature trea tment -2.7C -3.2C Rating 4 x Arg entine 1.5b* 7.0a 2 x FL 9 3.2a 7.0a 2 x FL 82 1.2b 5.2ab 2 x FL 11 3.2a 5.2ab 2 x Sand Mountain 1.2b 4.0b 2 x Pensacola 1.0b 3.7b *M e a ns wi th t he sa me le tte r a re no t si g nif ic a ntl y dif fe re nt a t P = 0. 05 c on fi de nc e le ve l.

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45 Who le -p la nt f re e ze r e c ov e ry ra tin g s o f t he lin e s/c lon e s te ste d in the wa te rba th experiment after an 18-d r ecove ry period in the g ree nhouse (Ta ble 2-8) w ere not signific antly differ ent among lines. This might be e x pecte d since the lines se lected f or this ex periment we re c hosen for their hig h sensitivity to free ze-stress fr om the initial L TFT sc ree ning e x periment. The levels of whole-plant freeze-damage ratings (6.0 to 7.2) of control clones/lines were greater than expected, and may be a result of being submerg ed in wate r (a pox ia) in addition to having leave s frozen. Also, these lines we re succe ssively frozen, re sted for 7 d, the n frozen ag ain. Succe ssive fre eze eve nts were apoxic, stressing r oots with a lack of oxy g en, in addition to having foliag e fr ozen. Cumulative stress may have a ccounte d for une x pecte dly sensitive whole-pla nt free zedamag e ra tings. Table 28. Whole-plant rec overy rating s as a pe rce nt of control plants af ter 18 da y rec overy in gr eenhouse afte r fr eeze tre atment (-3.2C) w hile root sy stem was ke pt above f ree zing (5C) F ebrua ry 2002. Som a tic c hr omo so me number L ine/cultivar Fr ee z ed am age rating 2 x FL 11 7.3a* 4 x Arg entine 7.2a 2 x Pensacola 7.0a 2 x FL 9 6.7a 2 x FL 82 6.5a 2 x Sand Mountain 6.0a *M e a ns wi th t he sa me le tte r a re no t si g nif ic a ntl y dif fe re nt a t P = 0. 05 c on fi de nc e le ve l. Over all, results confir med the hy pothesis that L TFT w as truly a re sult of leafda ma g e a nd no t of ro ot d a ma g e Co ntr oll e d f re e ze t re a tme nts c ou ld b e us e d to se pa ra te and ca teg orize bahiag rass c lones and lines for the L TFT tr ait expression. Summ ary Plants were se lected f or fr eezetoleranc e as w ell as for fre eze-sensitive re sponse, and quantifie d in plant-ca nopy fre eze-da mag e ra tings. Experiments included

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46 prog ressively lower f ree zing temper ature s, uniform fre ezing tempe rature trial of all lines at a sing le eve nt, and wate r-ba th experiments where the root temper ature was ke pt abovefre ezing while the leaf canopy was e x posed to various be low-fr eezing tempera tures. Prog ressively lower f ree zing temper ature s to -7C identified a super ior (F L 67), intermediate (C4-36) a nd low-L TFT ( FL 9) line. A unifor m free zing temper ature trial (6 C) c on fi rm e d r e su lts of the ini tia l L TF T s c re e nin g tr ia l a nd ide nti fi e d n e w l ine s w ith highL TFT ( CO6, OK1, and O K2). Wat e rba th e xpe ri me nts c on fi rm e d th a t c a no py le a ftis su e fr e e zeda ma g e wa s a re s u l t o f LTF T an d n o t o f r o o t d am age re s u l t i n g fr o m fr ee z i n g. L i n es wh i ch s h o we d h i gh levels of c anopy leaf -tissue fre eze-da mag e in wholeplant fre ezing tria ls showed similar damag e eve n when r oots were kept at 5C while the c anopy was e x posed to two prog ressively colder below-f ree zing temper ature s (-2.7C and 3.2C, respec tively ). L ines which showe d low levels of c anopy leaf -tissue fre eze-da mag e in wholeplant fre ezing tria ls showed similar damag e whe n roots wer e kept a bove-f ree zing.

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47 CH APT ER 3 AN AT OM Y R EL AT ED TO L EA F -T I SSU E F REE Z ETO L ERA NC E Introduction Tropica l savanna g rass lea f fr ost resistance has bee n repor ted to be less than te mpe ra te c lim a te g ra ss a nd fo rb fr os t r e sis ta nc e (S a ka i a nd L a rc he r, 19 87 ). Som e tropical g rasse s can toler ate tempe rature s as low as -4C. A f ew tropic al g rass spec ies can toler ate tempe rature s as low as -8 to 10C. I n contra st, temperate g rasse s can tolerate tempera tures within a ra ng e of 10 to -25C without showing lea f-da mag e. Some tempera te g rass spec ies ca n tolerate tempera tures as low a s -30C. The majority of 4 3 tropical g rasse s have the C phy siology and the major ity of temper ate g rasse s have the C ph y sio log y T he fi rs t a ssu mpt ion mig ht b e tha t le a ftis su e fr e e zeinj ur y ma y be the re su lt 4 of the C phy siology. 4 Not all tropical g rasse s have the C phy siologica l pathway (Kna pp and Medina 1999). Ther efor e, fr eezetoleranc e and intoler ance may not be a dire ct re sult of differ ing 3, c a rb on -f ixat ion ph y sio log y T he Pa nic oid e a e su bf a mil y of g ra sse s h a s C int e rm e dia te 3 4 4 (betwe en C and C ), and all thre e C subty pes of phy siology as we ll as the unique lea f anatomy that is associated w ith each ty pe and subty pe of c arbonfix ation phy siology Another a rg ument ag ainst cate g orizing tropica l gr ass fre eze-tolera nce by car bon-fixation phy siology would be the e x istence of a wide va riation in the trait shown in Chapter 2. A def inition of the leaftissue free ze-toleranc e (L TFT) is the ability of a g enoty pe to ma in ta in g r e e n a p p a r e n tl y u n d a ma g e d le a v e s a f te r e xp e r ie n c in g a f r e e ze

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48 (temper ature s below 0C) eve nt. Anatomical par ameter s, other than c ell arr ang ement a ro un d th e va sc ula r t iss ue ma y be re la te d to dif fe re nc e s in fr e e zetol e ra nc e Sm a ll c e ll siz e mig ht be re lated to L TFT. E verg ree n leave s with narrow interce llular space s and/or sma ll m e so ph y ll c e lls we re re po rt e d to low e r i c e nu c le a tio n te mpe ra tur e s d ow n to -1 0 to -1 2 C ( Sa ka i a nd L a rc he r, 19 87 ). Ot he r r e po rt e d o bs e rv a tio ns sh ow e d f ro st h a rd ine ss corr elated w ith small cell siz e (Sutcliffe 1977). Small cells had g rea ter spec ific ar ea a nd less volume strain per unit surfac e ar ea pr oduced dur ing ic e for mation than larg e ce lls. In w o o d y p l a n t s d e e p s u p e r c o o l i n g h a s b e e n r e l a t e d t o c e l l w a l l p o r o s i t y, p e r m e a b i l i t y, x y lem pit membrane, a nd cell wa ll tensile streng th (Wisni ewski, 1995). Whe n p la nt t iss ue fr e e zes (i f n o r a pid su pe rc oo lin g oc c ur s in iti a lly to approximately -10C) ice forms outside the living c ell in the apoplast (Saka i and L arc her. 1987). Tempe rature s experience d in Florida f ree ze events ar e usually above 10C. Pr ob a bil iti e s o f a -9 C f re e ze o c c ur ri ng in F lor ida ra ng e d f ro m P = 0.26 at Milton in the northern pa nhandle, to P = 0.000 at Home stead in the souther n peninsula (B radle y 1975). Ther efor e, we can a ssume ice f ormation in plants in Florida fr eeze e vents form outside the living c ell, in the apoplast. Sakai a nd L arc her ( 1987) desc ribe ice formation occur ring in the plant vessels first, followe d by ice spre ading throug hout the plant body in the interce llular air spa ce a nd film of water on the ce ll walls. This im plies vessel water is directly linked to the apoplast. Sy mplast free zing occ urs af ter a poplast fre ezing. Apoplastic ice formation may be an importa nt proce ss to relate to lea f ana tomy I ce ha s been shown to f orm, trave l, and sprea d throug hout barley leave s ( Hordeum sp.) throug h the leaf midrib first, then throug hout the leaf (Wisniewski et al., 1997). This supports ear lier work tha t showed the fr eezing sequenc e in a plant or g an,

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49 which conta ined wate r-f illed vessels. The ve ssels froze first, followe d by differ ent tissues in the plant org an (Saka i and L arc her, 1987) Th e xy le m di a me te r o f t e mpe ra te tr e e s is le ss t ha n f or tr op ic a l c lim bin g pla nts (T a ble 31) L ime wa s th e on ly fr e e zese ns iti ve tr op ic a l tr e e lis te d th a t ha d a sma ll diameter (Ha berla ndt, 1914). L arg e xy lem diameter s in branche s have be en shown to be r elated to c avitations and air embolisms, caused by fre ezing a nd subsequent thawing in 12 woody plant specie s (Da vis et al., 1999). At a xy lem pressure of -0.5 MPa, spec ies with branc h diameter vessels g rea ter than 40 m had nea rly complete c avitation. Species with xy lem diameter s rang ing f rom 30 m to 40 m ha d p a rt ia l c a vit a tio n a t 0. 5 M Pa Sp e c ie s w ith sma ll x y lem diameter s (less than 30 m) showed no f ree ze-induced c avitation at -0.5 MPa. Ta ble 31. Com pa ri so n o f v e sse l di a me te rs of tr op ic a l c lim bin g pla nts c omp a re d to tree s (Vesse l diameters obtaine d from Ha berla ndt, 1914). Clim bers Diameter ( m) Tree s Diameter ( m) Hypanther guape va 650 Oak 250 Calamus rotang L 350 Elm 158 An iso sp e rm a p as sif lor a Manso 300 Ash 140 Pa ssi flo ra lau rif oli a L 200 Birc h 85 Pa ssi flo ra e du lis Sims 200 Alder 76 Gl y c ine sin e ns is Sims 200 L ime 60 Aristolochia sp. 140 Pear 40 Se rja nia sp. 120 Box 28 These r eports stimulated an intere st in investigating leaf anatomica l differ ence s be tw e e n id e nti fi e d f re e zetol e ra nt a nd fr e e zese ns iti ve ba hia g ra ss l ine s. Th e nu ll hy po the sis te ste d w a s th a t th e re we re no a na tom ic a l di ff e re nc e s b e tw e e n th e mid ri b vascula r bundles of diploid (2 x ) bahia g rass lines diffe ring in L TFT.

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50 M at e r ial s a nd M e th ods Init ial Twoline Expe r im e nt An initial ex periment wa s conducte d 1 J anuar y 2003 to determine if the re w ere vis ibl e a na tom ic a l di ff e re nc e s b e tw e e n b a hia g ra ss c lon e s sh ow ing e xtre me dif fe re nc e s in L TFT. I nitial investigation used two lines re prese nting e x tremes in L TFT e x pression d u ri n g co n t ro l l ed fr ee z e e x p er i m en t s L i n e F L 9 wa s ch o s en as a ge n o t y p e r ep re s en t i n g a fre eze-sensitive line during controlled, succ essively colder fre eze-e vent trea tments (6.5 at C, 7. 5 a t C a nd 8. 7 a t C) L ine F L 9 w a s a lso c ho se n b e c a us e it r e sp on de d w ith the lowest ca nopy fre eze-da mag e ra ting ( 6.2) fr om a sing le, unac climated fr eeze e vent of C. L ine F L 67 wa s c ho se n to re pr e se nt t he mos t f re e zetol e ra nt b e c a us e of its consistent behavior in the experiments in which FL 9 was include d (Chapter 2). Timing of a natomical compa risons during short day s, when fr eeze e vents occ ur na tur a lly in n or th c e ntr a l F lor ida w a s im po rt a nt b e c a us e ba hia g ra ss h a s b e e n s ho wn to respond to day leng th (Sinclair et al., 2001; Sinclair e t al., 2003). On 1 January 2003, leaf lamina portions wer e cut f rom four pla nts of eac h line at three fully expanded leaf positions. Standardizing the sa mpled leaf reg ion was important to minimi ze variability Reported a natomical wor k with Pensacola ba hiag rass used the midpoint of the leaf blade a s a re fe re nc e to m a ke a fr e sh sa mpl e c ut o f 5 -c m lo ng (F lor e s e t a l., 19 93 .) A we a kn e ss in using the F lores tec hnique was tha t the leaf tip and leng th may vary with water rela tions ex perie nced dur ing le af e x pansion so that the sampled re g ion may vary from plant-to-plant. The leaf -blade collar w as used a s a re fer ence point from which to cut lamina samples. The leaf lamina sample fr om which sec tions were to be made wa s sta nd a rd ize d a s a 3c m lo ng po rt ion se ve re d w ith sc iss or s. Th e sta nd a rd ize d s a mpl e

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51 reg ion beg an 2 cm f rom the collar (mea suring towards the la mina tip/dis tal end). The total sample portion, 3 cm long which continued towa rds the lamina tip/distal end, was sever ed with scissors 5 cm fr om the collar. The sample included the widest part of the lamina, and pr ovided a standa rdized reg ion of tissue to ex amine re g ardle ss of leaf po sit ion a nd pla nt s a mpl e d. Cut sa mpl e s w e re pr e se rv e d in 4 mL /10 0 mL g luteralde hy de/wate r. Th re e le a f p os iti on s w e re de fi ne d in the ini tia l tw olin e ba hia g ra ss s tud y : f ir st fully expanded leaf second f ully expanded leaf and third fully expanded leaf from the top of the stem. Sampling at these three different positions allowed for observation of differ ence s in leaf da mag e, depe nding on le af position, during fre eze cha mber wor k. Ol de r l e a ve s ( se c on d, thi rd a nd old e r l e a ve s e me rg e d f ro m th e wh or l) a pp e a re d to express more fr eezeinjury than the first e merg ing a nd first fully expanded leaf of a whorl. Sampling f rom four diff ere nt plants of the same ve g etative pr opag ated g enoty pe would quantify plant-to-plant var iance leaving g enoty pe and le af position anatomica l eff ects to be a naly zed statisti cally I n a se pa ra te inv e sti g a tio n o f p ott e d p la nts of F L 9 a nd F L 67 le a ve s e xpos e d to ambient temper ature s after a contr olled fre eze eve nt showed prog ressive discolor ed damag e deve lopment in the midrib vascular r eg ion of the lea f, bef ore the ty pical wa terso a ke d f re e zeda ma g e d s y mpt oms a pp e a re d. I n th is p re lim ina ry e xpe ri me nt, fr e sh sections showed r elative diff ere nces in va scular bundle siz e in the midrib are a. The midrib area was discolore d and appa rently damag ed first be fore the traditional wa tersoaked le aftissue free ze-injury sy mptoms developed. I t was dec ided to measure the x y lem diameter of clone s FL 9 and F L 67.

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52 L eaf samples wer e stored individually in vials filled with 40 g kg -1 g lut e ra lde hy de /w a te r f or a min imu m of 24 ho ur s to fi x ce lls a nd pr e se rv e sa mpl e s u nti l they wer e sec tioned (F unk, persona l communication, 2003). An I nterna tional Minot Custom M ichrotome (I EC CTF Microtome) (Cry ostat I nterna tional Equipment Company Nee dham Heig hts, MA 31883) was use d with a setting of -20C and a cutting thi c kn e ss o f 1 5 mi c ro ns T he tis su e e mbe dd ing me diu m w a s T iss ue -T e k O CT compound #4583 (102.4 g kg poly viny l alcohol, 42.6 g kg poly ethy lene g ly col, 855.0 -1 -1 g kg non-re active ing redie nts) (Miles I nc. Diag nostic Division, Elkhart, I N 46515). -1 Embedding wells on brass micr otome discs wer e fa brica ted from timing ta pe, which fac ilitated filling with embedding medium and prompt fre ezing with the lea f sample a s near vertica l as possible, so sections would be per pendicular to the lamina and make anatomica l measure ments consistent from sample to sample. Slides wer e kept c lean in a slide well, filled with a solution made of 100 ml of 95 g kg ethy l alcohol in which 10 -1 drops of g lacial a scetic a cid had be en adde d. Slides to be used wer e re moved from the well, airdried, then c oated with fr eshly made Hoppe s adhe sive (1 g g elatin, 2 g phenol cry stal, 15 mL g ly cer in, 100 mL deionized water ) and dr ied in cover ed tra y s for 24 hours prior to being used to rec eive a nd hold microtomed sections. F resh Cry stal violet stain, which wa s desig ned to show lig nified ce ll walls as violet, was made as a 10 g kg -1 deionized water solution. S lides with sections were stained for 15 min in sl ide wells. S l i d e s w e r e t h e n c l e a n e d i n s l i d e w e l l s w i t h p r o g r e s s i v e l y h i g h e r c o n c e n t r a t i o n s o f e t h yl a lc oh ol i n s olu tio ns wi th d e ion ize d w a te r ( 50 mL 7 5 mL 8 0 mL 9 5 mL a nd 10 0 mL ethy l alcohol /100 mL ) to remove excess cry stal violet stain and excess tissue water A final rinse of rea g ent-g rade x y lene r emoved the la st trace s of wate r. Slides were then d r ie d in c o v e r e d tr a y s f o r 2 4 h p r io r to ma k in g p e r ma n e n t m o u n ts w it h F lo te xx Mountant (F isher Scientific I nterna tional). An Oly mpus BH2 microscope (2

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53 Corporate Center Dr ., Melville, NY 11747) wa s used with a stag e microme ter to mea sure va sc ula r p a ra me te rs O c ula r u nit s w e re c a lc ula te d f ro m oc ula r a nd le ns po we rs wi th micrometer units converted to micr ons at 400X powe r. Eigh t-line Experim ent Results of the first experiment wer e used to c onduct a se cond experiment. The second experiment included additional diploid clones to confirm initial results using additional lines repr esenting the two extremes in LTFT and an intermediate line. On 3 Dec ember 2002, Dr. Ke n Quese nberr y identified eig ht diploid clones that were se ve re ly da ma g e d b y a lig ht f ro st a t th e Ag ro no my F or a g e Un it, Ha g ue F lor ida U p to this ti me, FL 9 had bee n the most free ze-sensitive diploid ( 2x ) l ine w ith a pp ro xima te ly the same r esponse a s the standar d cultivar Pensac ola. Fr eezesensitive lines (1-30-3, 1-30-4, 222-1, and F L 9) and f ree ze-tolerant lines (F L 67, OK1, OK2, CO6) wer e incre ased f rom piece s of rhizomes/stol ons. Be fore planting in plastic pots (12.5 c m diameter x 12.5 cm depth, model 500, B etter Plastics, Kissimmee, F L ), veg etative rhizomatous/s toloniferous piec es we re dippe d in Hormodin 2 (E.C. Geig er I nc. Rt. 63 Box 285, Harley sville, PA 19438) to induce rooting Plants were ma intained in the same me dia, g iven nutrient mana g ement, and schedule d drip irrig ation sy stem in the Ag ronomy Tea ching Gre enhouse unde r ambient day leng th. The potting me dia was Scotts Ter ralite Ag ricultural Mix (Scotts-Sierra Horticultural Produc tion Com pany 14111 Scotts L ane Rd., Mar y sville, OH 43041). 25 2 Nutrient mana g ement wa s 1 g of a 164-8-1 NP O -K O-F e ana ly sis gr anular fer tiliz er pr e -p la nt i nc or po ra te d in to t he me dia of e a c h p ot. I ro nit e 100 (I ro nit e Pr od uc ts Com pa ny Sc ott sd a le A Z 85 25 8) wa s sp ri nk le d ( 1 g pe r p ot) on the top of the me dia to preve nt iron chlorosis. An over head spr ay irrig ation sy stem was set to run f or 5 min four times during the day

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54 When leaves w ere mature e noug h to sample by leaf positions (1 Februa ry 2003), leaf samples wer e collec ted as done previously Classes of L TFT w ere based on pr evious co n t ro l fr ee z e c h am b er ex p er i m en t s (C h ap t er 1 ) a n d fi el d ra t i n g. B e c a us e the da ma g e d r e g ion a pp e a re d to be the mid ri b r e g ion th e e nti re mid ri b va sc ula r b un dle re g ion wa s o bs e rv e d. Me a su re me nts of the mid ri b v a sc ula r b un dle diameter wer e made using the outer suber ized walls of the bundle-she ath ce lls as the limit Where ova l config urations wer e enc ountere d, two values we re r ecor ded: majoraxis diameter minor-axis di ameter Mean dia meter va lues wer e ca lculated a s (majorax i s d i am et er + m i n o r a x i s d i am et er / 2 ). Ar ea ca l cu l at i o n s we re d o n e b y m u l t i p l y i n g x min or a xis r a diu s x ma jor a xis r a diu s. Tw o ma jor xy le m ve sse ls p e r v a sc ula r b un dle occur red in the ba hiag rass lines mea sured. The x y lem vessel diamete r and c ross-sec tion a re a we re c a lc ula te d in the sa me ma nn e r a s f or va sc ula r b un dle s. Xy le m c e ll w a ll thickness was me asure d acr oss the visually appar ent ave rag e ce ll wall thickness, thus eliminating e x tremes. All mea surements we re ma de using a microsc ope at 400X w ith an ocular micrometer with units calibrated with a sta g e microme ter a s stated above The SAS (SAS I nstitut e I nc., 1987) g ener al linear model was used to a naly ze data. Results and Discus sion Init ial Twoline Expe r im e nt Sin c e le a f i nte rn a l a na tom y wa s a ne w a re a of inv e sti g a tio n, it w a s im po rt a nt t o test the null hy pothesis that there w ere no differ ence s betwee n the bahiag rass line xy lem diameter s with the same somatic c hromosome number ( 2 n = 2 x = 20 ). Me a n s imp le eff ects a cross a ll leaf positions showed that the diffe renc es we re sig nificant (T able 32). Th e 1 Jan ua ry sa mpl ing of lin e s F L 9 ( fr e e zese ns iti ve ) a nd F L 67 (f re e zetol e ra nt) showed that the hig h-L TFT line ha d a lar g er midrib diamete r (201 m vs. 149 m, respe ctively ).

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55 Table 32. Mean midrib xy lem diameter acr oss three le af positions of two lines sampled 1 January 2003. L eaf -tissue fre eze-tolera nce c lass Genoty pe line Xy lem diameter Number of me a su re me nts mn Sensitive FL 9 201a* 164 Tolera nt FL 67 149b 120 *M e a ns wi th t he sa me le tte r a re no t si g nif ic a ntl y dif fe re nt a t P = 0. 05 c on fi de nc e le ve l. Absolute vessel diame ter va lues of F L 9 and F L 67 wer e hig her tha n those repor ted (Table 3-1) f or all temper ate woody plants except for oa k (250 m ) a n d wi t h i n t h e r an ge of five of the tropica l vining spec ies (120 to 200 m). Simpl e mea n leaf position effec ts were signific ant ac ross both lines (Table 33). The xy lem diameter of the fir st fully expanded leaf was sig nificantly smaller than the second a nd third fully expanded leave s. The re sults show that what had bee n define d as the first fully expanded leaf was still ex perie ncing anatomica l chang es whe n sampled. Table 33. Mean midrib xy lem diameter of three leaf positions across two lines (F L 9, FL 67) var y ing in L TFT, sa mpled 1 J anuar y 2003. L e a f p os iti on Xy le m di a me te r Nu mbe r o f m e a su re me nts m n First 158c* 120 Second 190a 80 Third 184b 80 *M e a ns wi th t he sa me le tte r a re no t si g nif ic a ntl y dif fe re nt a t P = 0. 05 c on fi de nc e le ve l. Observa tion in the various controlled fr eezer trials (Chapter 2) indicate d that fre eze-injury sy mptoms appeare d more re adily on older lea ves, such a s the second a nd third fully expanded leave s, rather than on the first fully expanded leaf and the ne w leaf emer g ing f rom the whorl. L eaf position (thus l eaf ag e) f ree zing seque nce ma y be similar in bahiag rass a s in Hordeum sp. (bar ley ) (Pea rce and F uller, 2001b), de pending on the sever ity of the fr eezing trea tment. When org ans of upr ooted bar ley wer e tested unde r controlled fr eeze c onditions i n the labora tory fre ezing or der oc curr ed first fr om nuclea ted leave s, roots, older lea ves, and y oung er le aves w ith secondar y tillers being the

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56 last to freeze (Pearce and Fuller, 2001b). If the xylem diameter is one of the mechanisms that is related to LTFT it might explain why older leaves freeze before younger leaves. Visualization of the third emerged fully expanded leaf cross-section of the freezetolerant (Figure 3-1) and freeze-sensitive (Figure 3-2) lines showed clear differences in xylem diameter as well as parenchyma cells within the midrib region. This was the region that was apparently damaged within hours after exposure to sunlight after a 10hour freeze. Figure 3-1.FL67 bahiagrass (freeze-tolerant) section showing bundle sheath, girder system of sclerenchymous tissue supporting the vascular bundle. Adaxial (bottom) midrib vascular bundle towards the left of center. Third emerged fully expanded leaf position at 100X. Figure 3-2.FL9 (freeze-sensitive) section showing larger vascular bundle area than FL67. Third emerged fully expanded leaf position at 100X.

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57 Eigh t-line Experim ent Th e F e br ua ry sa mpl ing da te sh ow e d th a t me a su re d p a ra me te rs in t he fi rs t experiment using two c ontrasting lines, held consistently when c ontrasting four f ree zetolerant to four fre eze-sensitive lines. Mea n L TFT c lass leaf x y lem diameter trends acr oss 3 leaf positions were simil ar f or the two-line a nd eig ht-line experiments. L eaf tis su e fr e e zetol e ra nt l ine s te nd e d to ha ve low e r m e a n xy le m di a me te r v a lue s ( a c ro ss a ll three leaf positions) than free ze-sensitive lines. The fr eezetolerant line, F L 67, had a mean xy lem diameter of 149 m in the two-line experiment (Table 3-4), a nd the mean of 4 fre eze-tolera nt lines in the eig ht-line experiment was 169m in the 8-line experiment (Table 3-5). Me an lea f xy lem diameter trends ac ross 3 leaf positions were similar for both the two-line and e ightline L TFT e x periments. The low-L TFT line, F L 9, had a me an x y lem diameter of 201 m in the two-line experiment (Table 3-4), a nd the mean of 4 low-L TFT lines in the e ightline experiment was 221m (Table 35). Mean simple ef fec ts for the midrib aba x ial vascula r bundle diame ters we re sig nif ic a nt. F re e zetol e ra nt l ine s h a d la rg e r v e sse l di a me te rs a nd va sc ula r b un dle diameter s than fre eze-sensitive lines (Ta ble 3-4). T ab l e 3 -4 Mean simple ef fec ts of vessel and va scular bundle diameter s of four f ree zetolerant vs. four fre eze-sensitive ba hiag rass c lones sampled 1 F ebrua ry 2003. Parame ter L TFT c lass Number of Sensitive Tolera nt P > F me a su re me nts mn Vessel diame ter 221 169 0.0001 445 Vasc ular bundle dia meter 1170 921 0.0001 445 Mean simple ef fec ts for the midrib aba x ial vascula r bundle diame ter we re sig nificant. Freez e-s ens it iv e li nes had lar ger ves sel areas and vas cul ar bu nd le a reas th an h ighL TFT lin e s ( Ta ble 34) M e a n s imp le a re a e ff e c ts w e re hig hly sig nif ic a nt a nd c le a rl y dis tin g uis he d b e tw e e n f re e zese ns iti ve a nd fr e e zetol e ra nt c lon e s ( Ta ble 35) T he e ig ht-

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58 line experiment corr oborate d and supported the initial ex ploratory two-line experiment. Th e re la tio ns hip fo r s ma lle r xy le ms t o b e a sso c ia te d w ith fr e e zetol e ra nc e wa s c on sis te nt. Ei the r xy le m or va sc ula r b un dle dia me te r o r a re a me a su re me nts c ou ld b e us e d to ide nti fy ba hia g ra ss c lon e s w ith the L TF T t ra it ( Ta ble 36) X y le m c e ll w a ll t hic kn e ss was a para meter tha t did not dis tinguish betwe en clone L TFT c lasses. L ine FL 67, one of the g e no ty pe s w ith fr e e zetol e ra nc e h a d th e low e st a ve ra g e xy le m c e ll w a ll t hic kn e ss (20 m) O K2 a no the r l ine wi th f re e zetol e ra nc e h a d th e hig he st xy le m c e ll w a ll thickness of a ll the lines tested (32 m). Table 35. Analy sis of varianc e compa ring mean ve ssel and vasc ular bundle a rea simple effe cts of four highvs. four lowle aftissue free ze-tolerant c lones sampled 1 F ebrua ry 2003. Parame ter L TF T c la ss P > F Number of Se ns iti ve To le ra nt me a su re me nts mm n 2 Vessel a rea 0.039 0.023 0.0001 445 Vasc ular bundle a rea 1.064 0.677 0.0001 445 The c ritical xy lem diameter that distinguished betwe en fr eezetolerant a nd fre ezesensitive g enoty pes appe are d to be betwe en 209 and 187 m Table 3-6) The c ritical x y lem are a distinguishing betwee n fre eze-tolera nt and fre eze-sensitive L TFT g enoty pes was be tween 0.035 a nd 0.028 mm The va scular bundle diameter and ar ea f ollowed 2 trends similar to the xy lem diameter compar ed with the ar ea in spite of the oval shape of x y lem vessels. The c ritical vasc ular bundle dia meter w as betwe en 1160 and 980 m. Critical vascula r bundle a rea appea red to be betwee n 1.039 and 0.759 mm 2 The que stion is t hen ra ised how smaller xy lem diameter s might provide a n advanta g e to bahiag rass plants that ar e complete ly frozen, then thaw ed in ambient te mpe ra tur e s in fu ll s un lig ht? Ob se rv a tio n o f t he ba hia g ra ss l ine s, du ri ng the ve ry fi rs t da y of re c ov e ry in f ull su nli g ht a ft e r a c on tr oll e d f re e ze e ve nt, sh ow e d d a ma g e ini tia lly

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59 Table 36. Mean midrib aba x ial (fa cing the bottom side of the leaf ) xy lem and vasc ular bundle par ameter s from a F ebrua ry 2003 sampling da te. L TFT c la ss Canopy d am age ra t i n g @ -6 C Genoty pe Xy lem diameter Xy lem are a Xy lem c e ll w a ll thi c kn e ss Vasc ular bu nd le diameter Vasc ular bundle ar ea m mm mm mm m 22 2 Sensitive ---1-30-4 242a* 0.046a 22bc 1230a 1.178a Sensitive ---1-30-3 223b 0.039b 22d 1170b 1.069b Sensitive 6.2b FL 9 209c 0.036c 24 1120b 0.963c Sensitive ---2-22-1 209c 0.035c 21d 1160b 1.039b Tolera nt 2.6a OK1 187d 0.028d 22bc 980d 0.759d Tolera nt 2.2a OK2 170e 0.023e 32cd 950d 0.716d Tolera nt 2.0a FL 67 157f 0.022e 20e 900e 0.654e Tolera nt 2.0a CO6 158f 0.020e 21cd 850f 0.579f *M e a ns wi th t he sa me le tte r a re no t si g nif ic a ntl y dif fe re nt a t P = 0. 05 c on fi de nc e le ve l. Selected a fter a fre eze eve nt 3 Dece mber 2002 a t Hag ue, F lorida, fr om a visual rating compar ed to Arg entine (c anopy damag e ra ting of 2, slightly damag ed) vs. number ed s en s i t i v e l i n es t h at we re ex t re m el y s en s i t i v e, b ei n g fr o z en t o t h e gr o u n d (c an o p y d am age rating of 9, all foliag e dama g ed). a lon g the le a f m idr ib ( F ig ur e 33) f oll ow e d b y the la min a pr op e r. Th e le a f l a min a wo uld have tha wed f irst in full sunli g ht. Transpira tion demand on the lamina in full sunlight would have ma de lea f xy lem pressure (P x ) more ne g ative. At pre ssures of 0.5 MPa, fre eze-thaw cy cles ha ve bee n under shown to c ause e mbolism s which re duced transpira tion in plant species with small x y lem diameter s (Davis e t al., 1999). I t could be possible that under full tra nspiration stress on initially thawing x y lem pressure s experience d in the leaf x y lem could be more neg ative than the 0.5 MPa in the controlled experiment conducte d by Davis et a l. (1999) bec ause the roots would still be fr ozen in the pots. Following that rea soning, r oot pressure would not alleviate the tr anspiration demand a s might be possible in fieldg rown ba hiag rass plants. Without pos itive root pr e ssu re th e mid ri b d a ma g e wo uld be vis ua lize d w ith in a sh or t pe ri od of tim e T his sequenc e of e vents could have been the case for f ree ze-sensitive bahiag rass c lones re mov e d f ro m c on tr oll e d f re e ze t ri a ls. B a hia g ra ss m idr ib l e a f d a ma g e wa s se e n w ith in

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60 hours of plac ing f rozen, potted plants in full sunlight. I n the work r eporte d by Davis et a l. ( 19 99 ) c a vit a tio n o c c ur re d in xy le m ve sse ls u po n th a wi ng f oll ow e d b y a ir e mbo lis ms, which re stricted vesse l conductanc e in plants with vessels larg er tha n the cr itical embolism diameter value of 45 m for the 12 woody specie s investiga ted. Fur ther detailed discussion and r efe renc es conc erning cavitation, embolism and vesse l conducta nce a re inc luded in Chapter 1. I n bahiag rass, the two la rg est x y lem vessels of a bahiag rass lea f occ ur in the single midrib vascular bundle, on the ada x ial reg ion of the lea f cr oss-section (F igur es 3-1 a nd 32) T his is t he re g ion tha t vi su a lly a pp e a re d d a ma g e d in su sc e pti ble lin e s w ith in ho ur s o f c on tr oll e d f re e ze t ri a ls. Fig ure 33. Fr eezesensitive line within hours of being place d in full sunlight afte r a c ontrolled fre eze eve nt, 10 h at -6C, shows damag ed midrib reg ions init ially The same midrib reg ion appea red da mag ed first in har d fre ezes experience d in a ba hia g ra ss e xpe ri me nt c on ta ini ng fr e e zese ns iti ve lin e s p la nte d in the fi e ld a t th e No rt h

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61 Florida Ag ricultural Rese arc h and Educ ation Center ( NFREC-Suwanne e Va lley ) af ter fre eze eve nts in Dece mber 2004 a nd J anuar y 2005. Fieldg rown plant roots we re not frozen, y et leaf midribs showed similar damag e sy mptoms t he af ternoon of the day of the fr ee z e w h en ai r t em p er at u re s wa rm ed ab o v e f re ez i n g. Vi s u al s y m p t o m s o f l ea f d am age in the mid-rib leaf reg ion of fre eze-sensitive plants we re similar to those fr om controlled fr e e ze t ri a ls. Ev e n w ith po sit ive ro ot p re ssu re fr om u nfr oze n r oo ts, ve sse l a ir e mbo lis ms would have r estricted w ater supply under tra nspiration demand unde r full af ternoon sunlight in the field. Ob se rv a tio n o f f re e zese ns iti ve ba hia g ra ss c lon e da ma g e su g g e sts tha t th e sma ll leaf x y lem diameter in lines with free ze-toleranc e (O K1, OK2, F L 67, CO6) may be a mecha nism to survive a fre eze eve nt. For f ree ze-sensitive bahiag rass lines (F L 9, 1-30-3, 130 -4 2 -2 21) th a wi ng wo uld c a us e la rg e dia me te r xy le m ve sse l a ir e mbo lis m fo rm a tio n, wh ic h w ou ld p re ve nt l e a f b la de c e ll r e hy dr a tio n. Th e a ir e mbo lis m w ou ld lead to lea f ce ll plasmoly sis and leaf -dama g e, which w as obser ved and r ated in fr eezese ns iti ve lin e s. Summ ary A two-line investig ation of bahia g rass wa s conducte d initially to quantify anatomica l differ ence s betwee n a fr eezetolerant a nd a fr eezesensitive clone. Statist ically signific ant diffe renc es we re f ound in the mid-rib x y lem diameter and ar ea, a s well as the va scular bundle diameter and ar ea be tween F L 9 (fr eezesensitive) and F L 67 (fr eezetolerant). T he xy lem cell wa ll thickness was not a pa rame ter that wa s associate d with L TFT. To ve ri fy wh e the r e nc ou nte re d a na tom ic a l di ff e re nc e s w e re c on sis te nt a c ro ss bahiag rass c lones exhibi ting these L TFT tr aits, an eig ht-line investiga tion was conduc ted.

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62 Smaller midrib x y lem diameter s, x y lem are as, vasc ular bundle dia meters, a nd vascula r bundle ar eas w ere associate d with L TFT c lones. Critical values of L TFT c lones wer e estimated, base d on the eig ht-line data. Estimated c ritical value s (values a bove being fre eze-sensitive a nd values be low being fre eze-tolera nt) wer e ca lculated f or the xy lem diameter (198 m), x y lem area (0.031 mm, vascular bundle diameter (1070 m), and 2) vascula r bundle a rea (0.899 mm ). 2

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63 CH APT ER 4 P HYS I OL OGI CA L ME CH ANI S MS AS S OC I ATE D W I TH L EAFTI SSU E F REE Z ETO L ERA NC E Introduction A def inition of leaf-tissue fr eezetoleranc e (L TFT) would be the a bility of a g e n o ty p e to ma in ta in g r e e n a p p a r e n tl y u n d a ma g e d le a v e s a f te r e xp e r ie n c in g a f r e e ze (t e mpe ra tur e s b e low 0 C) e ve nt. To a ssu me tha t th e L TF T t ra it i n b a hia g ra ss i s th e re su lt of one sing le mecha nism may be too simplist ic. Sever al mecha nisms may be broug ht into action, including both the a poplast and the sy mplast. Xy lem vessels ar e conside red to be par t of the plant apopla st beca use vesse ls are c omposed of dea d cell wa lls. B a hia g ra ss xy le m ( a pa rt of the le a f a po pla st) a na tom y wa s in ve sti g a te d a nd re la te d to L TFT thr oug h a propose d embolism mechanism (Chapter 3). Sy mplast mechanisms may be involved in bahiag rass a s well and should there fore be investig ated. A r eview of the major sy mplast mechanisms involved, the major bioche mical and phy siologica l mecha nisms with a proposed sche me that integ rate d plant fre eze-tolera nce w as thoroug hly discussed (Chapte r 1). Plant phy siologica l and metabolic r esponse to coldand fr eezestress has be en sh ow n to ov e rl a p b ioc he mic a l pa thw a y s in vo lve d in os mot ic h ig h s a lin ity a nd dr ou g htstress profiles in g ene e x pression studies (Guy 2003). This biochemica l pathway overla p ha s in dic a te d th e hy po the sis tha t tr op ic a l a nd su btr op ic a l pl a nts a dju sti ng to g e olo g ic a lly c ha ng ing c lim a te s ma y ha ve uti lize d s imi la r p hy sio log ic a l pa thw a y s a nd me c ha nis ms t o wi ths ta nd the de sic c a tio n e ff e c ts o f f re e zing (G uy e t a l., 19 92 ). Pla nt t iss ue sa lt-

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64 toleranc e has be en shown to include a ccumulation of or g anic, a s well as inorg anic, so lut e s in or de r t o r e g ula te c e ll o smo tic po te nti a l ( Or c utt a nd Ni lse n, 20 00 ). B oth org anic ( sug ars, a mino acids, and poly ols) sy nthesis and ac cumulation as we ll as inorg anic ions (Na K Ca ) ac cumulate in orde r to reduc e the osmotic potential ++ + + sufficie ntly to maintain cell turg idity throug h water uptake in halophy tes. Plant membrane stability and its permea bility under sa lt-stress have also bee n shown to be rela ted to membrane phospholipid saturation. Further discussion is covere d in Chapter 1. Two major phy siologica l mechanisms have been a ssociated with fr eezetolerant plants (Chapter 1) : 1) fre eze point-depre ssion through inc rea sed osmolality ; and 2) incre ased me mbrane fluidity at lower tempera tures throug h incre ased poly unsaturate d fatty acid c ontent. Since no studies in bahiag rass we re f ound rela ting osmolality and me mbr a ne fl uid ity a t be low -f re e zing te mpe ra tur e s, the re wa s a ne e d to inv e sti g a te the se two potential mecha nisms. A short re view of how osmolality aff ects the f ree zing point of a solution may be he lpf ul. Va po r p re ssu re a nd fr e e zing te mpe ra tur e of a so lut e de pe nd s o n th e os mol a lit y (moles of solute dissolved in a g iven volume of solution). Osmolality is defined a s the moles solute per kilog ram of solution. (Holtzclaw et a l., 1984). A solution will freeze at a lower tempera ture than pur e wa ter. F ree zing point-depre ssion temperature can be calc ulated with the following formula: f f = i K m f Def inition of the formula terms include s the following : K is the fre ezing constant for water (-1.86C m ), m = the molality of the solute, and i = the Van t Hoff 1 dissociation constant. A per fec t solute would have i = 1.0. A n aqueous solution of 1 molal (1000 milli osmoles kg ) with a pe rfe ct solute would produce an osmotic potential -1

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65 of -2.27 MPa. This relationship ([(-2.27 MPa/-1.86C mol kg-1)/-2.27 MPa mol kg-1] = s/ f ) can be simplified, relating osmotic potential ( s) with freezing point-depression f : s = 0.537 f Figure 4-1 shows the relation between osmotic pressure (Pa), freezing temperature (C), osmolality (osmole kg-1), and relative humidity (%) of a solution. Figure 4-1.Relationship of osmolality, freezing temperature, osmotic pressure, and relative humidity of an aqueous solution with a pure solute (Source of figure from Wolf and Bryant, 2001). These relationships could be used to test the hypothesis that the mechanism of the LTFT trait is a result of freezing point-depression from solute increase in leaf symplast cells. Cells that would especially be important in the bahiagrass lamina would be those arranged radially in the bundle sheath adjacent to the xylem. This single layer of bundlesheath cells, which are immediately adjacent to the xylem vessel, is where the second photosynthetic carbon reduction occurs. The second layer of cells removed from the xylem vessels are mesophyll cells, where the primary photosynthetic carbon reduction reaction occurs. If ice forms initially in the xylem vessels, the single layer of bundle-

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66 sheath would be the ce lls which would experience osmotic stress first. Cells with lower fr e e zing te mpe ra tur e s r e su lti ng fr om i nc re a se d o smo ly te c on c e ntr a tio ns wo uld be a ble to resist desicc ation (loss of wate r fr om the cell as it cr ossed the membra ne to ac hieve osmotic equilibrium). Cell desiccation during fre eze-stre ss is caused f rom the lower os mot ic po te nti a l ( ) of the g rowing apoplastic ice cry stals (assuming ice c ry stals formed in the vessels). L et us assume a hy pothetical fr eeze e vent to -10C is slow enoug h so super cooling does not occ ur (supe r cooling is an unstable condition when ic e, liquid and g as phases a re not in equilibrium; as discussed thoroug hly in Chapter1). A ssume that leaf tis su e fr e e zeda ma g e is d e fi ne d b y : w a te rso a ke d s y mpt oms fr om w hic h me so ph y ll tis su e da ma g e c ou ld b e inf e rr e d f ro m f re e zeind uc e d o smo tic str e ss, c e ll v olu me reduc tion to a critical volume a nd irreve rsible plasmoly sis upon thawing a nd attempted re h y d ra t i o n I n t h e 1 0 C h y p o t h et i ca l fr ee z eev en t as s u m e l ea ft i s s u e f re ez ed am age was not see n in a bahiag rass line, upon be ing pla ced in f ull solar radia tion in the g ree nhouse af ter tre atment. I n this hy pothetical example, if lower osmolality within the mid-rib reg ion protecte d the line to -10 C by depre ssing the f ree zing temper ature os mol a lit y wo uld ha ve to b e 5 o smo le kg (1 0 M Pa ) f ro m in c re a se d s olu te -1 c o n c e n tr a ti o n L e a f ti s s u e f r e e ze p r o te c ti o n v ia th e me c h a n is m o f d e p r e s s e d f r e e ze tempera ture a s a func tion of osmotic potential could be tested in the c ells of intere st by measuring the osmotic potential of bahiag rass lea ves, within the reg ion of visual damag e. F atty Ac id Com position Membrane bi-lipid lay er f luidity is thought to be influenc ed by the composition of fatty acids (F As). Unsa turated F As have a lower melting point. The la rg er the pr op or tio n o f u ns a tur a te d F As th e low e r t he te mpe ra tur e a me mbr a ne ph a se sh if ts f ro m a

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67 fluid liquid cry stalline phase to a g el phase (B uchana n, et al., 2000). Cell re cover y from fre eze-thaw stress cy cle, whe re c ell volume initially is decre ased a s a re sult of de hy dr a tio n s tr e ss f oll ow e d b y re hy dr a tio n, re qu ir e s me mbr a ne fl uid ity in o rd e r t o maintain functional integ rity The F A composition in plants appear s to be a dy namic, c omp le x pla nt m e ta bo lic pr oc e ss i nv olv ing me mbr a ne a nd or g a ne lle compar tmentalization, t ranspor t, and control. L ipid sy nthesis is mainly located in the chloroplast with export to the endoplasmic re ticulum. High leve ls of interac tive communication and tra nsport occur betwee n these two org anelles. Exported F As ca n be modified in the ce ll after the y are exported and integ rate d into their cell loca tion (i.e. g alac tolipids phospholipids, s phering olipids, etc.). Membra ne-bound de saturase s, as well as a soluble chloroplast desa turase c an be a ctivated to add or chang e the loca tion of a c is -double bond. De saturase s introduces a phy sical kink into a fatty acid c hain, which maintains membra ne mobility and per meability and re duces the tempera ture a t wh ic h th e lip id m e lti ng te mpe ra tur e oc c ur s. Th e te mpe ra tur e a t w hic h th e imp e rm e a ble and inflexible phy sical membra ne g el phase occur s under this model depe nds on the unsaturate d FA c ompositi on. A continuum of membra ne fle x ibilit y is impl ied from abovefre ezing to belowfre ezing de pending on FA c ompositi on. Artificial membr ane, F A m i x t u r e b e h a v i o r a n d t h e o r y in v itr o may be d if feren t t han who lepl ant FA composition in t he fie ld present some c halleng es. Ag e of tissue, org an sampled, se asonal chang es (pr esumably as a r esult of war ming or cooling tempera tures), a bove-f ree zing tempera ture tre atment, and plant g enoty pe may aff ect F A composition. Assumpti ons made in v itr o may be diffic ult to translate to the field. Stag e of pla nt development has a n e ff e c t on F A c omp os iti on (N ish ida a nd Mu ra ta 1 99 6) G ra sse s v a ry in p re do min a nt, unsaturate d FA c ompositi on by org an sampled ( Massard e t al., 2000). Seasona l chang es

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68 in FA c ompositi on can oc cur in g razed g rass a nd clover fora g es during the sea son from spring throug h fall (L oor et a l., 2002). Cold treatment (a bove-f ree zing temper ature s) of g rasse s cause d chang es in tissue FA c ompositi on (Samala e t al.,1998). By st ud y in g FA com po si ti on ov er t im e in si ght ca n b e gain ed o n t he re lat iv e FA c omp os iti on a s a fr a c tio n to ta l e xtra c te d f a tty a c ids (g kg TE F A) G ra zed or c ha rd g ra ss -1 ( Dactylis glomerata L .) F A composition, quantified as g kg TEFA chang ed fr om May -1 to J uly (Table 4-1). Satura ted FA composition increase d from May to J uly sampling dates. The trend for saturate d FA c ompositi on to increa se with incre asing tempera ture from May to J uly would be expected f rom the F A theory Small chang es in the fr action incre ase of the fra ction of TEF A ar e see n in the data ( Table 41) for 14:0 my ristic, and 18:0 stearic. T he 16:0 palmitic FA f rac tion increa sed the most of all the satur ated F As (fr om 192 to 224 g kg during the May to J uly sampling pe riod). The tri-unsatura ted FA -1 18 :3 li no len ic, decr ease d fr om 55 8 t o 5 01 g kg-1 du ri ng th e sea so n. Th e mo del of FA saturation fr action incr easing during war mer tempe rature s and dec rea sing dur ing c ooler te mpe ra tur e s w ou ld e xpla in t he pla nt r e sp on se to t e mpe ra tur e by inc re a sin g the lin ole nic fr a c tio n o f T EF A. Ho we ve r, on ly sma ll o r n o in c re a se s in oth e r u ns a tur a te d F As (1 6:1 p a l m i t o l e i c 1 8 : 1 o l e i c a n d 1 8 : 3 l i n o l e i c ) o c c u r r e d d u r i n g t h e s e a s o n ( T a b l e 4 1 ) In orcha rd g rass, lea f linolenic ac counted f or the lar g est portion (> 500 g kg ) of the total -1 extracted F As (Ta ble 4-1). D epending on the month of the sea son, di-unsaturate d linoleic or satura ted palmitic FA a ccounte d for the se cond or third hig hest g kg TEFA -1 The da ta would have been mor e helpf ul in providing fa ll pre-f ree ze event ana logie s for ba hia g ra ss i f F A a na ly sis c ou ld h a ve be e n d on e thr ou g ho ut t he g ro wi ng se a so n in to October

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69 Table 41. Fa tty acid c ompositi on of g razed orc hard g rass (T able f rom L oor et a l., 2002). Total extracted f atty acids F a tty a c id Ma y June July P > F statistic of season e ffe ct g kg -1 14:0 0.05 0.07 0.06 0.01 16:0 19.20 21.10 22.40 0.01 16:1 0.02 0.04 0.03 0.93 18:0 1.60 1.80 1.90 0.01 18:1 2.20 1.80 2.20 0.01 18:2 20.40 20.50 21.10 0.01 18:3 55.80 53.40 50.10 0.01 14:0 = My ristic, 16:0 = palmitic, 16:1 = palmitoleic, 18:0 = stea ric, 18:1 = ole ic, 18:2 = lin ole ic 1 8:3 = lin ole nic G r a s s e s v a r y i n p r e d o m i n a n t u n s a t u r a t e d F A c o m p o s i t i o n b y o r g a n s a m p l e d In be rm ud a g ra ss ( Cynodon dactylon L .) cr owns linoleic and linolenic a ccounte d for 706g kg TEFA in a cold-toler ant cultivar Midiron a nd 693 g kg in a cold-se nsitive line U3 -1 -1 b e f o r e i m p o s i n g a b o v e f r e e z i n g c o l d t e m p e r a t u r e t r e a t m e n t ( S a m a l e t a l 1 9 9 8 ) In se a sh or e Pa sp a lum ( Paspalum vaginatum Swartz.) crow ns, the predominant F As wer e linoleic and linolenic, which a ccounte d for 690 to 726 g kg TEFA depending on the -1 g enoty pe, bef ore c old tempera ture tre atment (Cy ril et al., 2002). Per ennial ry eg rass total unsaturate d FA g kg TEFA and an unide ntified g roup of pa sture g rass lea ves wa s -1 re po rt e d to be 89 9 g kg a nd 80 4 g kg r e sp e c tiv e ly (H a wk e 1 97 3) Pe re nn ia l r y e g ra ss -1 -1 leaf was pre dominately composed of two unsa turated F As (682 g kg linolenic, -1 146 g kg linoleic) and one saturate d FA ( 119 g kg stearic ). -1 -1 C o l d ( a b o v e f r e e z i n g ) t r e a t m e n t o f g r a s s e s c h a n g e s t i s s u e F A c o m p o s i t i o n In bermuda g rass, c old-tolerant Midiron and c old-sensitive U3 cr own tissue FA c ompositi on res po nd ed t o ab ov e-free z in g cold accl im ati on tr eat men t b y chan ging t hei r FA c omp os iti on (S a ma la e t a l., 19 98 ). Un sa tur a te d F A: sa tur a te d F A r a tio (U SF A: SF A) is a c on ve nie nt m e tho d o f e xpre ssi ng F A c omp os iti on A t th e be g inn ing of the e xpe ri me nt,

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70 bo th cul ti vars had a US FA: S FA rat io of 2 .6 5. At th e end of t he ex peri men t, US FA: S FA ratios for Midiron a nd U3 wer e 2.94 and 2.71, r espec tively L inolenic (C18:3) incre ased by 13 8 g kg TE F A i n M idi ro n, a c c ou nti ng fo r t he ma jor ity of the inc re a se in -1 USFA:SFA. I n seashor e Paspalum (Cy ril et al., 2002) linolenic ( C18:3) was the F A that incre ased ( from 219 g kg to 234 g kg TEFA ) as a result of c old acc limation treatment. -1 -1 Such a small incre ase in USF A minimally chang ed the USF A:SFA fr om 2.33 to 2.36. Reported F A cha ng es in seashor e Paspalum ar e so small that FA c ompositi on may not explain differe nces in g enoty pe coldtoleranc e. I f F A composition of membrane inf lue nc e s c e ll f re e zetol e ra nc e thr ou g h in c re a se d me mbr a ne fl uid ity F A c ha ng e s w ou ld have to be much larg er to a pproxi mate ar tificial membrane and mix ture be havior a t below-f ree zing temper ature s. The double bond inde x (DB I ) has be en used a s a method to compar e plant tissue FA c ompositi on. DB I is calcula ted as a weig hted portion of the poly -unsatura ted FA s [(2 x C18:2+3 x C:18:3)/100] I n bermuda g rass DB I incre ased most for c old-tolerant Midiron crown tissue, rising from an initial 1.67 prior to cold tre atment to 1.80 afte r c old -a c c lim a tio n ( Sa ma la e t a l., 19 98 ). Col dse ns iti ve U3 be rm ud a g ra ss D B I a pp e a re d to chang e ver y littl e as a result of c old-ac climation treatment, fr om 1.62 to 1.65. I n seashor e Paspalum, cold-tolera nt SeasI sle1 DB I incre ased the most, from 1.77 to 1.95 (Cy ril et al., 2002). I ntermedia te cold-toler ant cultivar A daly d had an inter mediate DB I (1.65) initially but at the end of the cold ac climation treatment ha d the same D BI (1.65) a s coldsensitive line PI 299042. Currently there is a lack of information on bahiag rass lea f fa tty acid c ompositi on. Membrane fluidity may be an importa nt mechanism in fre eze-tolera nce f or fr eezetol e ra nt l ine s. Th e re g ion of ini tia l da ma g e in f re e zese ns itv e ba hia g ra ss l ine s a pp e a re d to

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71 tol e ra nt l ine s. Th e re g ion of ini tia l da ma g e in f re e zese ns itv e ba hia g ra ss l ine s a pp e a re d to be in the midrib reg ion, initi ally befor e the e ntire lea f blade was da mag ed upon thawing afte r a f ree ze event. The bundle shea th cells and mesophy ll cells surrounding the vessel reg ion would be the re g ion that would experience membrane stress from de hy dration and c e ll v olu me re du c tio n d ur ing ic e fo rm a tio n a nd re hy dr a tio n a nd c e ll v olu me inc re a se during a fr eeze/thaw c y cle. B uchana n et al. (2000) have show n that the majority of lea f lipids i n Arabidopsis thal iana L (564 g kg leaf DM) is involved with membrane -1 g ly cer olipids, another 27 g kg leaf DM leaf membrane sphingolipids and sterols, a nd -1 259 g kg leaf DM lipids associated with chlorophy ll. Thus, the majority 591 g kg leaf -1 -1 DM of lea f total lipid contents, is invol ved with the lea f membra ne. The discussion of the literature and theor y of unsatura ted FA composition of the plasmalemma c ontributing to mem bran e fl ui di ty un der f reez e st res s s uggest s t hat a la rger po rt io n o f un sat urat ed FA ma y c on tr ibu te to m e mbr a ne fl uid ity M e mbr a ne fl uid ity is n e e de d u nd e r f re e zestr e ss freez e/t haw cy cle s. I f t he m echan is m o f t he L TFT t rai t i n b ahi agrass is a res ul t o f FA composition, then it i s hy pothesized that the lines with free ze-toleranc e should have hig he r u ns a tur a te d g kg TE F A, hig he r U SF A: SF A a nd hig he r D B I tha n li ne s w ith -1 fre eze-sensitive lines. M at e r ial and M e th ods Osm olality Nine lines re prese nting va ry ing L TFT w ere studied (Chapter 2) L ines that wer e fre eze-sensitive include d 1-30-3, 130-4, 2-221, and F L 9. An intermedia te line was C436 L ine s th a t e xpre sse d f re e zetol e ra nc e we re OK -1 O K2, CO 6, a nd F L 67 Pl a nts wer e veg etatively propag ated 11 Nove mber 2004. Roots of veg etative por tions were

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72 dipped in Hormodin-2 (0.3% indolebuty ric a cid, E.C. Geig er, I nc. Ha rley sville, PA). Pot tin g me dia us e d a s th e so il f or thi s st ud y wa s Sc ott s T e rr a lit e Ag ri c ult ur a l Mi x (Scotts-Sierra Horticultural Produc tion Com pany 14111 Scotts L ane Rd., Mar y sville, OH 43 04 1) T he ro un d p la sti c po ts w e re 18 c m in dia me te r x 18 c m in he ig ht, Cla ssi c 400 Nurser y Supplies, Farle ss Hills, P A. Nutrient mana g ement wa s 1 g of a 164-8-1 N-P2O5-K2O -F e ana ly sis gr anular fer tiliz er pr e-pla nt incorpora ted into the media of eac h pot. One g I ronite 1-00 (I ronite Products Company Scottsdale, AZ 85258) wa s a pp lie d to the me dia in e a c h p ot t o p re ve nt i ro n c hlo ro sis I rr ig a tio n w a s c on du c te d d a ily by hand to kee p moisture as uniform a s possible. On 23 November 2004, plants were place d in a g ree nhouse with a he ater set to come on whe never the temper ature fell below 20C. An air conditioning unit was set to cool the g ree nhouse whe never tempera tures exceede d 30C. Pl ant location wa s randomized within the gr eenhouse to acc ount for tempera ture va riability A minimum/ maxi mum thermometer wa s place d in two locations to r e c or d te mpe ra tur e va ri a bil ity B a hia g ra ss h a s b e e n s ho wn to b e se ns iti ve to d a y le ng th (Sinclair et a l., 2001; Si nclair e t al., 2003), and the se lines wer e g rown during short day s. We could assume that ac climation from shortene d day leng th would have oc curr ed. A We sc or 55 00 Va pr o Pr e ssu re Os mom e te r (We sc or 4 59 S. M a in S tr e e t, L og an, Utah 84321) was used to r ecor d osmolality Potted plants were r emoved f rom the g ree nhouse 26 De cembe r 2004 and c arr ied to the labora tory Fr esh lea f blade s were cut at the collar of the third fully expanded leaf in a whorl. A 5mm plug of lamina of third leaf was c ut with a No. 2 cor k cutter c overing the midrib section betwe en 2 and 2.5 c m o u t o n t h e l ea f b l ad e f ro m t h e l ea f c o l l ar T h i s re gi o n wa s t ar get ed b ec au s e o f d am age visualiz ed in the midrib are a as pr eviously discussed (Chapte r 3). I mmediately the leaf disc was c ut in half along the midrib vessel with a disposable micr otome blade. This wa s

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73 done to pre vent contamination of the osmometer cha mber by protruding midrib or curle d la min a dis c T he tw o h a lve s o f t he le a f d isc we re he ld w ith tw e e zer s a nd imm e dia te ly spray ed with Cry okwik (Shield Chemical Co. Damon/I EC Division, 300 2 Ave., n d Nee dham, MA 02194). Minimum temperatur es produc ed with Cry okwik wer e re corde d to -46C. The intended pur pose of f ree zing samples wa s to ly se ce lls. I mmediately on thawing the two halves of the leaf disc wer e plac ed in the osmometer c hamber Values w e r e o n l y u t i l i z e d o n l y i f t h e o s m o m e t e r c a l i b r a t e d c o n s i s t e n t l y b e t w e e n r u n s If calibra tion was not achie ved, conta mination had occur red in the osmometer chambe r, and the entire unit was disassembled, cle aned, w arme d to opera ting tempe rature and calibra ted bef ore r unning a nother fr eshly core d lamina sample. F atty Ac id Com position Tifton 9 bahiag rass wa s harve sted 25 Octobe r 2005 fr om a field that ha d been c ut for ha y This field was se lected be cause it was isolated from a ny bahiag rass by planted pine tree s, thereby ensuring population purity having been pla nted from c ertified se ed. Sufficient mater ial was ha rvested ( 41 g fre sh weig ht) to develop e x trac tion methodology Clone leaf ma terial wa s limi ted in the diallel experiment (Chapter 5) plot at University of F lor ida No rt h F lor ida Re se a rc h a nd Ed uc a tio n Ce nte r Suw a nn e e Va lle y (N F REC Suwannee Valley ). Extraction methodology was re fined on Tifton 9 ba hiag rass be fore using c lone leaf materia l from the diallel experiment plots. L eaf whorls wer e cut a t the collar of the third fully expanded leaf and then plac ed on ice in a cooler Samples were transporte d to the labora tory Ba g g ed samples we re dippe d in liquid ni trog en for 5 seconds, a nd then stored in a f ree zer at -20C. To preve nt losing fr esh g ree n leaf tissue to an ear ly frost, on 29 Oc tober 2005 veg etative c lone leave s were harve sted at the diallel plot at Univer sity of F lorida

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74 NFRECSuwanne e Va lley Random leaf sa mples were selec ted from e ach 10plant plot. L eaf whorls wer e cut a t the collar of the third fully expanded leaf A minimum of 30 g fre sh plant material wa s harve sted from e ach plot, sea led in a plastic ba g and kept in an iced c ooler. F our blocks of ha rvested pla nt material fr om pare nt clone row s in the diallel study at NF REC-Suwannee V alley wer e sampled. Tw o replica tions per block for a total of eig ht samples per L TFT c lone wer e used. When a ll plots wer e har vested, samples wer e tra nsported to the Univer sity of F lorida at Ga inesville, Florida, w here samples wer e dipped in liquid nit rog en for 5 seconds, then store d in a fre ezer a t -20C. Grinding of sa mpl e s w a s d on e to o bta in u nif or m le a f p a rt ic le s f or F A e xtra c tio n. Un if or m pa rt ic le siz e should reduc e F A extraction varia bility L eaf samples wer e re moved frozen f rom -20C storag e and c ut into roughly 2 cm leng ths with stainless st eel sc issors. Samples wer e loade d into the stainless steel g rinding cup (Wiley ), cove red in liquid nitroge n, mace rate d and pushed dow n to the cutting bla des with a c era mic pestle (Coors ), then c ov e re d in liq uid nit ro g e n a nd g ro un d. Th e g ri nd ing se qu e nc e wa s w ith the low e st setting, g round for 10 seconds, then inspe cted a nd mix ed with a stainless stee l spatula. L iquid nitroge n was a dded, g round at the se cond lowest setting for 10 se conds, mix ed and inspec ted, with a fina l addition of liquid ni trog en and g rinding for a n additional 15 seconds a t that setting. The g round Tifton 9 wa s kept in a sea led 150 ml beake r in a fr e e zer to d e ve lop fa tty a c id e xtra c tio n me tho do log y a nd to u se the tis su e a s a sta nd a rd in a more complete e x periment. Fa tty acid c ontent of bahia g rass wa s unknown so a pre liminary trial was c on du c te d to de te rm ine wh ic h s a mpl e size a nd e xtra c tio n me tho d w ou ld b e the mos t eff icient to proce ss samples. Three differ ent tissue sample sizes were used bec ause of the conce rn of insuff icient fa tty acid qua ntities: 0.5, 1, and 2 g. Thr ee e x trac tion procedur es

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75 wer e compa red: 1) Cold extraction using a 50:50 hexane/tert-B uty l ether, 2) petroleum ether and 3) dire ct ester ification with methanoloic HCl with 2, 2-dimethox y porpane (DMP). A pe anut oil standard wa s used with blanks. All methods of extraction produce d simil ar pe rce ntag es of f atty acids. The half g sample produc ed ar eas on the integ rator f or unique FA s that had to be ma g nified on the g as chr omatog raph ( GC) integ rator output. I t w a s d e c ide d to us e the 1 g tis su e sa mpl e size be c a us e it p ro du c e d c le a r a nd re pli c a ble are as that could be smoothly and consistently integ rate d. Ex trac tion method samples were analy zed on a Hew lett Packar d 5890 g as chroma togr aph (G C). Procedur es we re c ompare d for time involved in FA extraction as well as c onsistent results. Ex trac tion Method 2, using petr oleum ether was sele cted be cause of the e ase of extraction in addition to y ielding F A profile r esults consistent with all other methods used. Method 2 wa s modified to ensure tha t gr ound leaf materia l was cove red by rea g ents. The modified Extraction Method 2 in its entirety was a s follows: A 1 g g round leaf sample wa s place d in a Py rex (Corning ) 16 x 125 m m disposable scre w ca p culture tube via a f unnel, 2 ml of a 1M methanoloicHCl with 5% (v/v) 2,2-dimethoxy propane (DMP) wa s added to e ach tube and tubes we re se aled with Te flon-lined ca ps. Tubes wer e plac ed in an 80C wa ter ba th and hea ted for 1 h. T ubes we re c ooled in ambient te mpe ra tur e wa te r b a th f or 10 min T wo ml 9 0 g kg Na Cl w e re a dd e d to e a c h tu be to -1 sto p th e re a c tio n. He xan e (1 .5 ml) wa s th e n a dd e d to e a c h tu be a nd tub e s th or ou g hly mixe d b y vo rt e xing T ub e s w e re c e ntr if ug e d a t 3, 00 0 G fo r f ive min to a llo w p ha se s e p a r a t i o n A p p r o x i m a t e l y 1 m l o f t h e u p p e r p h a s e c o n t a i n i n g t h e f a t t y a c i d m e t h yl est ers (FAM ES ) was pi pet ted and seal ed i nt o au to sam pl er vi als for GC anal y si s o f FA

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76 profile. Samples we re store d at -20C immediately until GC analy sis could be conducte d. GC analy sis included hexane blank standa rds for e very 10 bahiag rass tissue sam pl es. Th e As so cia ti on of O ffi cia l A gricu lt ural Ch emi st s (A OAC ) st and ards of FA (F AME Mix RM-6, Supelco, 595 North Ha rrison Rd., Be lefonte, PA) w ere included at the beg inning, middle and e nd of the GC ana ly sis. Analy sis of varianc e wa s conducte d with SAS (S AS I nstitut e I nc., 1987). Results and Discus sion Osm olality Th e re we re sig nif ic a nt d if fe re nc e s a mon g g e no ty pe s in re g a rd to l e a f d isc o s m o l a l i t y ( P < 0.019). Me an lea f disc osmolality by g enoty pe is listed in Table 4-2 separ ated by Dunca ns multiple rang e test at the 5% level of c onfidenc e. L e a f d isc os mol a lit y did no t c or re la te wi th b a hia g ra ss l ine L TF T c la ss ( P < 0.261). D iffer ence s betwee n the g rand me an of the se two cla sses wer e not signific ant: mean f ree ze-sensitive = 623 mmol kg and fr eezetolerant = 649 mmol kg -1 -1 L ines that wer e fr eezetolerant ra ng ed fr om 545 to 817 mmol kg L ines that wer e -1 freez e-s ens it iv e ran ged fro m 4 89 to 85 9 m mo l k g T here for e th e me chan is m o f L TFT -1 do e s n ot a pp e a r t o th a t of inc re a se d s olu te sy nth e sis a nd sto ra g e in c e ll v a c uo le s in c e lls around the midrib vascular bundle, nor in hig her solute c oncentr ations in the x y lem ve sse ls. L eaf disc osmolality can be used to ca lculate f ree ze tempera ture de pression. The require d osmolality can be calc ulated for the fre ezing point depr essed be low 0C. An ff example follows using Va nt Hoff s equa tion ( = iK m) for the solute conce ntration f neede d to depre ss the fre ezing tempe rature to a = -6C. Assume that the disa ssociation

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77 constant of plant solutes (suc h as simple sug ars) approximates a value of I = 1. The f fr ee z e co n s t an t fo r wa t er = K = -1 8 6 C m o l k g We can solve f or m (in units of mol kg ) -1 -1 ff by rea rra ng ing V ant Hof fs e quation: m = / K = -6C/-1.86C mol kg = 3.2 mol kg -1 -1 F re e zetol e ra nt l ine s F L 67 O K1 O K2 a nd CO 6 w ou ld n e e d to ha ve ha d o smo la lit y values a pproac hing 3 moles kg I nstead, osmolality values ra ng ed fr om 545 to 817 -1 mmol kg Another w ay of looking at the data is that an osmolality /molarity of 545 -1 mmol kg would g ive a f = (1. 86 C m ) x ( 0. 54 5 m k g ) = 1 .0 C. B y the sa me -1 -1 -1 method of ca lculation 817 mmol kg would g ive a f = 1 5 C. A c tu a l c o n tr o ll e d f r e e ze -1 chambe r data showed c anopy injury occur red a t -6C, thus showing solute conc entra tion was not the mec hanism protecting bahiag rass lines with fre eze-tolera nce. Table 42. Osmolality of bahia g rass sexual diploid li nes re prese ntative of fr eezet o l e r a n c e a n d f r e e z e s e n s i t i v i t y. L TFT Genoty pe C an o p y fr ee z ed am age rating L e a f d isc os mol a lit y Class L ine (1 to 9) mmol kg -1 Sensitive 1-30-3 --859a* Tolera nt FL 67 2.0 817a Tolera nt OK-1 2.6 679abc Sensitive 2-22-1 --631abc Sensitive 1-30-4 --610abc Tolera nt CO6 2.0 556bc Tolera nt OK-2 2.2 545bc Sensitive FL -9 6.2 489c I ntermedia te C4-36 4.4 463c *M e a ns wi th t he sa me le tte r a re no t si g nif ic a ntl y dif fe re nt a t P = 0. 05 c on fi de nc e le ve l. S el ec t ed af t er a f re ez e e v en t De ce m b er 3 2 0 0 2 at Ha gu e, Fl o ri d a f ro m v i s u al ra t i n g compar ed to Arg entine (c anopy fre eze ra ting of 2, slightly damag ed) vs. number ed sensitive lines that were extremely sensitive, being frozen to the g round (c anopy fre ezedamag e ra ting of 9, all foliag e dama g ed). Ca no py fr e e zeda ma g e ra tin g fr om c on tr oll e d f re e ze t o 6 C in e nv ir on me nta l g ro wt h chambe r. F atty Ac id Com position Seven F As ac counted f or the major c ompositi on in the bahiag rass lines tested (Table 4-3). Othe r outputs were small and not identified, which is a similar re sult found

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78 by other investig ators (Cy ril et al., 1998; Samala e t al., 1998). F inding a nd char acte rizing seve n major F As for ba hiag rass c ontrasts with only four major FAs char acte rized in bermudag rass a nd seashor e Paspalum (C16:0, C18:0, C18: 2, C18:3). My ristic (C14:0), a satur ated F A, was not sig nificantly differ ent ac ross lines g rown in the diallel plot (rang ing f rom 9 to 11 g kg TEFA ). Tifton 9 wa s signific antly differ ent, but -1 only slightly (8 g kg TEFA ). Tifton 9 value s were used as standa rds to ensure that the -1 GC wa s o pe ra tin g c on sis te ntl y wh ile a na ly zing the lin e s th a t w e re ve g e ta tiv e ly propag ated in the diallel c ross plot. All other FAs ( C16:0, C16: 1, C18:0, C18: 1, C18:2, a nd C18 :3) we re sig nif ic a ntl y dif fe re nt ( P < 0.0001) a mong g enoty pes. Palmiti c (C16:0) c omprised the lar g est portion of all satura ted FA s, rang ing f rom 175 to 206 g kg TEFA depending on the bahiag rass line. Palmitic, a satura ted FA was -1 the only FA in be rmudag rass a nd seashor e Paspalum that did not chang e in composition under c old treatment. Table 43. Ba hiag rass lea f blade fatty acids, a s a fr action of total e x trac ted fa tty acids (g kg TEFA ). -1 L TFT L ine Saturated f atty acids Unsatura ted fa tty acids C14 :0 C16 :0 C18 :0 C16 :1 C18 :1 C18 :2 C18 :3 Sensitive Arg entine 11a* 175e 17c 35bc 15b 134d 544a Sensitive 1-30-4 9ab 183cde 17c 29d 16b 174a 516ab Sensitive 1-30-3 11a 185cd 17c 31cd 15b 173a 497ab Sensitive 2-22-1 11a 195b 20b 38b 18b 167ab 474b Sensitive FL 9 10a 206a 20b 31cd 18b 170a 478b I ntermedia te C4-36 10a 182de 20b 37bc 16b 158bc 502ab Tolera nt F67 9ab 192bc 19b 38b 14b 154c 494ab Tolera nt OK-1 9ab 189bcd 19b 33bcd 16b 158c 512ab Tolera nt OK-2 11a 195b 18bc 37bc 15b 154c 499ab --Tifton9 8b 213a 24a 51a 28a 140d 364c Assigne d L TFT c lass based on c ontrolled fre ezer trials or f ield behavior A ll l ine s v e g e ta tiv e ly pr op a g a te d a nd ma na g e d th e sa me e xce pt T if ton 9, wh ic h w a s a population that was har vested fr om an adjac ent plot to standardize protocol bef ore a na ly zing lim ite d le a f m a te ri a l f ro m th e dia lle l c ro ss. *M e a ns fo llo we d b y the sa me le tte r a re no t si g nif ic a ntl y dif fe re nt a t th e 5% le ve l. **C14:0 = my ristic, C16:0 = palmitic, C16:1 = palmitol eic, C18:0 = stea ric, C18:1 = oleic, C18:2 = linoleic, C18:3 = linolenic.

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79 Ste a ri c (C 18 :0) is a sa tur a te d F A, wh ic h d id n ot d ist ing uis h a mon g L TF T l ine s in bahiag rass a nd constituted a minor portion of the total prof ile. I n bermuda g rass a nd seas ho re P asp alu m t he s tea ri c mi ni mal ly chan ged bet ween genot y pes in g kg TEFA -1 afte r cold-tr eatment a cclimation. L inolenic (C18:3) comprise d the larg est portion of the unsatur ated F As, rang ing in the diallel cross plot lines from 499 to 544 g kg TEFA Arg entine, the most fre eze-1 sensitive of all the ba hiag rasse s, had the hig hest g kg TEFA (544), whic h was -1 signific antly differ ent than all the other lines, reg ardle ss if they wer e fr eezetolerant or fr e e zese ns iti ve T his is t he re ve rs e of wh a t w ou ld b e e xpe c te d f ro m th e lit e ra tur e in ma ny sp e c ie s ( Cha pte r 1 ). Th e se re su lts a re the op po sit e of wh a t w a s f ou nd in bermuda g rass (Sama la et al., 1998) and sea shore Paspa lum (Cy ril et al., 2002) c rowns. I n Berm ud agrass co ld -t ol eran t l in e Mi di ron had app rox im ate ly 85 hi gher g kg TEFA -1 than cold-intolera nt line U3. I n seashor e Paspalum, cold-toler ant line SeaI sle1 had highe r (282 g kg TEFA ) poly unsaturate d linolenic (C18:3) than cold-intoler ant line -1 PI 299042 (231 g kg TEFA ). These chang es in linolenic g kg TEFA betwee n the two -1 -1 lin e s o c c ur re d a ft e r a 21 d c old tr e a tme nt o f 8 C d a y /4 C ni g ht t e mpe ra tur e s w ith a da ily 10-h photoperiod. L inoleic (C18:2) wa s unusually and sig nificantly low in Arg entine (134 g kg -1 TEFA ) y et sig nificantly high in the r emaining low-L TFT g roup of lines (130-4, 1-303, 2-22-1, a nd FL 9) with values r ang ing f rom 167 to 173 g kg TEFA Fr eezetolerant a nd -1 int e rm e dia te -L TF T b a hia g ra ss l ine s ( F L 67 O K1 O K2 a nd C436 ) w e re a sso c ia te d w ith low and sig nificant va lues that wer e diffe rent fr om the fre eze-sensitive lines (154 to 158 g kg TEFA ). I f Arg entine linoleic g kg TEFA had not bee n so low, bahiag rass lines -1 -1 may have be en sepa rate d by class of f ree ze-toleranc e base d on the diffe renc es in this FA.

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80 Perhaps this was c oincidental in this ex periment, but lower C18:2 values have be en a sso c ia te d w ith c old -t ole ra nt l ine s th a t ha ve be e n s ub je c te d to a c c lim a tio n tr e a tme nt i n 4 other C g rasse s. I n seashor e Paspalum, cold-toler ant SeaI sle1 C18:2 was reduc ed by 56 g kg TEFA and only by 26 g kg TEFA for c old-intolerant PI 299042. -1 -1 I n bahiag rass lines g rown unde r the same condition, oleic g kg TEFA did not -1 chang e betwe en lines and c onstitut ed only a minor portion of the F A profile. T he differ ence in Tifton 9 oleic g kg TEFA could be a ttributed to differe nt gr owing -1 condition para meters in an a djace nt plot. P almitoleic (C16:1) constituted another minor portion of the F A profile a nd did not dist inguish be tween c lasses of ba hiag rass lines differ ing in L TFT. This char acte rization of the FA pr ofile of ba hiag rass ve g etative c lones g rown during short day leng ths and cool we ather (har vested 29 Oc tober 2005) show ed ther e wa s no signific ant FA profile that c ould be used to distinguish whe ther a line had hig hor lo wL TFT. I n fac t, Argent in e, t he l owe st L TFT l in e had th e hi ghest C1 8: 3 g kg TEFA -1 4 of all the lines. The se re sults were in c ontrast to two other C specie s which had the re ve rs e da ta fo r C 18 :3 g kg TE F A, wh e re the mos t c old -t ole ra nt l ine s h a d th e hig he st -1 C18:3 and the lowest C18:2 g kg TEFA (Cy ril et al., 2002; Samala e t al., 1998). -1 Ano th er way to com pare po ly un sat urat ed FA p rof il es i s t o u se t he D BI (T a ble 44) T he DB I wa s si g nif ic a nt f or g e no ty pe s ( P < 0. 00 01 ) b ut n ot f or L TF T c la ss. Th e DB I re fl e c te d g e no ty pe dif fe re nc e s. Th e DB I wa s n ot u se fu l in se le c tin g lin e s w ith fre eze-tolera nce. B ahiag rass DB I rang ed fr om 1.76 to 1.90. I n Be rmudag rass, the c oldto ler ant li ne D BI was 1. 81 an d t he co ld -i nt ol eran t l in e DBI was 1. 65 Bah iagras s D BI values we re c lose to the rang e of published be rmudag rass va lues. DB I did not identify which lines had f ree ze-toleranc e.

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81 Table 44. L eaf blade double bond inde x (DB I ) and unsa turated f atty acid: satura ted fatty acid r atio (USFA :SFA) of lines r epre sentative of f ree ze-toleranc e and f r e e z e s e n s i t i v i t y. L TFT L ine DB I USFA:SFA Sensitive Arg entine 1.90a* 3.59a Sensitive 1-30-4 1.89a 3.51ab Sensitive 1-30-3 1.84abc 3.36ab Sensitive 2-22-1 1.76c 3.08de Sensitive FL -9 1.77bc 2.94e I ntermedia te C4-36 1.82bc 3.37ab Tolera nt FL -67 1.80bc 3.13de Tolera nt OK-1 1.85ab 3.30bc Tolera nt OK-2 1.80bc 3.14cd --Tifton 9 1.64d 2.73f *M e a ns wi th t he sa me le tte r a re no t si g nif ic a ntl y dif fe re nt a t P = 0. 05 c on fi de nc e le ve l. The DB I values may be helpf ul in chara cter izi ng profile r elationships but perhaps should be used with ca ution until a cle are r under standing of the role of F A satura tion and membrane fre eze-tolera nce is a chieve d. The ba hiag rass da ta showed tha t having a lar g er pr op or tio n o f C 18 :3 d id n ot k e e p A rg e nti ne fr om s til l be ing the mos t se ns iti ve lin e to canopy damag e during a fr eeze e vent. Obviously in bahiag rass, the F A profile doe s not explain why some lines are fre eze-tolera nt or fre eze-intolera nt. Another me thod of indexi ng FA pr ofiles is calc ulating the unsaturate d F A: sa tur a te d F A r a tio (U SF A: SF A) w hic h is sh ow n in Ta ble 44. Ag a in, the sa me limit ing r easons, that the D BI did not help identify bahiag rass lines diffe ring in L TFT, apply to the USFA:SFA. V alues for bahiag rass lines at this sampling time a ppear to be sli g htl y hig he r ( 2. 73 to 3 .5 9) tha n th os e fo r b e rm ud a g ra ss a c c lim a te d w ith c old tempera ture (2.65 to 2.94) I n bermuda g rass, the hig her U SFA:SFA identifie d the coldtol e ra nt l ine V a lue s f or ba hia g ra ss r a tio s w e re muc h h ig he r t ha n f or se a sh or e Pa sp a lum I n seashor e Paspalum, the line with the hig her U SFA:SFA r atio identified the c oldtolerant line (2.65) and the c old-intolerant line (2.31) Perhaps USF A:SFA ra tios of g kg -1 TEFA may be too simplist ic to explain why g enoty pes var y in free ze-toleranc e. Ne ither

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82 leaf osmolality nor lea f F A profile e x periments identified me chanisms which mig ht distinguish one line of ba hiag rass fr om the other in L TFT, a s rated by canopy damag e. Summ ary More than one mecha nism could be involved in mediating L TFT. A poplastic and sy mplastic mecha nisms mi g ht be involved. Fr eezepoint depression throug h incre ased osmolality is a mecha nism some plants use to prevent fr eezedamag e at tempe rature s below 0C. Results from the osmolality experiment showed that diffe renc es in clone L TFT w ere not a re sult of leaf osmolality I ncre ased me mbrane fluidity at lower tempera tures throug h incre ased poly unsaturate d fatty acid c ontent has bee n associate d wi th freez e-t ol eran t p lan ts R esu lt s o f t he FA e x peri men t s ho wed th at u ns atu rat ed FA c on te nts wa s n ot t he me c ha nis m by wh ic h f re e zetol e ra nt c lon e s su rv ive fr e e ze e ve nts wi th s ma ll a mou nts of c a no py da ma g e I n f a c t, r e su lts we re the re ve rs e of wh a t w ou ld h a v e b e e n p r e d i c t e d b y t h e u n s a t u r a t e d F A m o d e l f o r m e m b r a n e f l u i d i t y.

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83 CH APT ER 5 G E N E T IC B E H A V IO R O F T H E LE A F T IS S U E F R E E Z E T O LE R A N C E T R A IT Introduction Ba hiag rass re production is controlled by the somatic chr omosome number (Qua rin et al., 2001). T etra ploid (2 n= 4 x= 40 ) b a hia g ra ss r e pr od uc e s b y se e d th a t is formed a pomictically identical to the mother pla nt. Diploid (2 n =2 x = 20 ) b a hia g ra ss re pr od uc e s b y se e d th a t is fo rm e d b y se xua l un ion of ma le a nd fe ma le g a me te s. Di plo id ba hia g ra ss w ill be dis c us se d in de ta il l a te r. B oth so ma tic ba hia g ra ss c hr omo so me numbers, diploid and tetra ploid, are he terozy g ous. I t is in t he diploid, sex ual condition, that a ra ng e of he terozy g ous g enetic inf ormation ca n be expressed a nd selec ted for in the pr og e ny I n th e dip loi d c on dit ion pr og e ny tr a it e xpre ssi on c a n b e us e d to qu a nti fy tr a it heritability mode of g ene a ction, g ener al combining ability and spec ific combining a bil ity T he dip loi d b a hia g ra ss s oma tic c on dit ion wa s u se d in thi s st ud y of L TF T t ra it t o q u a n t i f y: T r a i t h e r i t a b i l i t y Mode of g ene a ction Ge ne ra l c omb ini ng a bil ity Spe c if ic c omb ini ng a bil ity Re su lts wi ll b e dis c us se d in te rm s o f p ra c tic a l a pp lic a tio ns in a n e ff or t to de ve lop dip loi d bahig rass populations with improved L TFT. Tetraploid Reproduction Tetra ploid (4x ) bahia g rass re produce s apomictically (without mix ing of the g a me tic g e ne tic inf or ma tio n th ro ug h n or ma l do ub le fe rt ili zat ion ). Te tr a plo id b a hia g ra ss

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84 has bee n considere d an oblig ative apomictic ty pe of r eproduc tion (where unreduc ed embry os produce seed tha t are g enetica lly identical to the mater nal plant). F acultative te tr a plo id apomictic plants occ ur in extremely small numbers natura lly and have been studied for their me chanism of poly embry onic see d set (Qua rin, 1999), and de g ree of se xua lit y (C he n e t a l., 20 01 ), or us e d in c ro sse s ( B ur ton a nd Ha nn a h, 19 86 ). Te tr a plo id bahiag rass c ultivars, lines or bioty pes have been c ited as having less frost re sistance a nd more winter -killing than diploid bahiag rasse s (B urton, 1946; Bur ton, 1955). Due to the c omp le x for ms o f a po mixis a nd dif fi c ult y in i de nti fy ing se xua l or fa c ult a tiv e a po mic ts i n tetraploid bahia g rass fr ost sensitive cultivars wer e not used in this resea rch to de termine the g enetics of leaf -tissue fre eze-tolera nce tr ait. Diploid Reproduction Diploid (2 x ) bahia g rass re produce s sexually (B urton, 1948; Bur ton, 1982). A highdeg ree of cr oss-pollination occurs in diploid bahiag rass (Wer ner a nd Bur ton, 1991). S e l f p o l l i n a t i o n o f d i p l o i d b a h i a g r a s s s h o w e d a h i g h d e g r e e o f s e l f i n c o m p a t i b i l i t y, po stu la te d to be of the S ty pe (B ur ton 1 95 5) Wh e n 5 7 c lon e s o f P e ns a c ola ba hia g ra ss wer e selfpollinated, seed se t aver ag ed 6% c ompare d to the same c lones which a vera g ed 89.5% see d when ope n-pollinated (F igur e 5-1) A small number of pla nts from a sexual population may not be as self -incompatible a s the majority of the population and ma y be able to set a s high a s 25% to 30% see d (B urton, 1955). The ref ore, the re ma y be a limited number of c lones within a population which may contribute less tra it ex pression in a cross due to the deg ree of selfing I dentifica tion of the pare nt clone ability to self may be imp or ta nt w he n c on du c tin g c ro sse s in ba hia g ra ss t ha t in ve sti g a te the he ri ta bil ity of tr a it e x p r e s s i o n i n p r o g e n y.

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85 A re view of the literature did not revea l work that quantified f ree ze-toleranc e g ene a ction, whether the trait wa s additive or dominant in plants. I nformation on g ener al c omb ini ng a bil ity s pe c if ic c omb ini ng a bil ity a nd ho w h e ri ta ble L TF T t ra it m ig ht b e in other g ener a would be he lpful in study ing the trait in bahiag rass. No spe cific infor mation on L TFT tr ait g ener al combining ability specific combining a bility and her itability was found. Ge nera l summary information was f ound for c old-toleranc e and f ree ze-toleranc e b re ed i n g. Br eeding for plant toler ance of low temper ature stress (a bove-f ree zing and be lowfr e e zing ) c a n b e a pp ro a c he d c on c e ptu a lly u sin g the c on tin uu m of inc re a sin g pla nt s tr e ss as temper ature decr ease s (Fig ures 11 and 1-2) Challeng es and suc cesse s in coldtoleranc e bre eding could provide insig ht into results of bree ding f or L TFT in ba hiag rass. Br eeding for c old-toleranc e (the ability for a plant to withstand temperatur es betwe en 20C and 0C) was r eporte d to be complex, depending on the mode of g ene a ction, the specie s, ploidy level, the mec hanisms involved and the bre eding method used and f urther complicated in the f ield by g enoty pe x environment interac tions (Revilla et al., 2005). Br eeding for f ree ze-toleranc e is eve n more c omplex and diffic ult because much of the e va lua tio n w or k mu st b e do ne in u nc on tr oll e d f ie ld p lot s. I n f ie ld p lot s, va ri a tio ns in s oil moisture, tempera ture, soil ty pe, microniches, ther mody namics of plant c over insulation versus ba re soil, plant size, plant heig ht, radiation, conve ction and many more va riables that cannot be controlled c ontribute to high e rror terms, CVs, phenoty pe var iance s and g e no ty pe x en vir on me nt i nte ra c tio ns tha t a re dif fi c ult to c on tr ol a nd re mov e sta tis tic a lly from phenoty pe eva luations. Additional challeng es include limited and inconsistent knowledg e of the trait g enetic c ontrol. Difficulty obtaining c onsistent plant responses incre ases the f arthe r re moved the fie ld trials are from the a dapted e nvironment of the

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86 plant. Traditional bre eding techniques f or improving low tempera ture tolera nce r ang ing from mass sele ction to molecular g enetics, ha ve bee n attempted in maize, Brassica sp., tomato, and other c rops (Revilla et a l., 2005). Abra ham (1988) r eviewe d basic fr eezestress par ameter s, g enetic va riation and inh e ri ta nc e a nd br e e din g me tho do log y Po or c or re la tio n o f c on tr oll e d f re e ze t e st r e su lts with field eva luation trials was re ported. Spatial and te mporal var iability in the field wa s po int e d o ut a s a ma jor pr ob le m to ov e rc ome wh e n s c re e nin g la rg e nu mbe rs of pla nts in c on tr oll e d e nv ir on me nta l c ha mbe rs F re e ze t ri a ls w e re lim ite d to the nu mbe r o f p la nts that could be teste d. Mode of g ene a ction (additive vs. nonadditive) varie d, depending on the cr op. Fr eezetoleranc e tra its were dif ficult to select f or and to qua ntify Transg ressive se g reg ation for f ree ze-toleranc e wa s difficult to show in prog eny of bree ding tr ials. Attempts to quantify ing g enetic va riability in trials did not sol ve problems with lack of prog eny expression of transg ressive se g reg ation for f ree zetoleranc e tra it. Crosses of hardy pare nts rare ly showed improveme nt bey ond the level of the most tolerant par ent. Abra ham (1988) sug g ests one re ason (that pr og eny of cr osses rar ely showed improveme nt bey ond the har dy pare nts) might have been tha t the crop g enetic ba se wa s narr ow. The la ck of improve ment in free ze-toleranc e throug h crop bree ding w as a r esult of the lac k of the g enetic r esourc es. Concentr ation on bree ding f or improved fr eezetoleranc e mec hanisms and collec ting g ermplasm at the f ring e ra ng es of crop a daptation might be helpful (Abr aham, 1988) The L TFT tr ait has bee n selec ted in diploid bahiag rass. Rec urre nt restricte d phenoty pic selec tion (RRP S) of superior clone poly cross prog eny is curre ntly used in a F lor ida br e e din g pr og ra m ( B lou nt e t a l., 20 01 ). RRPS h a s b e e n u se d to su c c e ssf ull y improve bahia g rass for ag e y ield with the rele ase of an improved c ultivar, Tifton 9, a fter

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87 nin e c y c le s o f s e le c tio n ( B ur ton 1 99 1) Po ly c ro ss m a tin g sy ste ms h a ve be e n u se d to develop sy nthetic lines with superior tra its in heterozy g ous, self-infe rtile crops in for ag es (P oe hlm a n a nd Sle pe r, 19 95 ). Un de rs ta nd ing the L TF T t ra it g e ne tic c on tr ol c ou ld a ssi st plant bree ders. The L TFT mode of g ene a ction could be e x ploited in order to shorten the tim e re qu ir e d to re le a se imp ro ve d b a hia g ra ss c ult iva rs Sh or te nin g c ult iva r r e le a se tim e would benef it the forag e and f orag e-f ed livestock industries in Flor ida and the southeaster n United States Coastal Plains reg ion (Chapter 1) Diallel cr ossing de signs ha ve bee n used to provide infor mation on trait g ener al a nd sp e c if ic c omb ini ng a bil ity a mon g a se le c t g ro up of pa re nt l ine s ( Gr if fi ng 1 95 6) A ll po ssi ble ma tin g c omb ina tio ns of a g ro up of pa re nts ( p ) ar e made resulting in p ( p -1) c ro sse s if se lf -p oll ina tio ns a re no t in c lud e d. Th re e g ro up s o f p la nts a re c omp a re d in 1 ref ere nce to the studied tra it: 1) the pare nt lines; 2) one set of F prog eny and 3) the 1 rec iproca l set of F prog eny A full diallel study with self-fe rtile plants would include a 1 fo ur th g ro up of pla nts : in br e d p ro g e ny (S ) f ro m se lf po lli na te d p la nts F or thi s a na ly sis to have r eliable r esults, it is important that plants do not self-pollinate during the cr ossing proce ss, hence the discussion on self-pollination. Two re asons for de veloping a cr ossing scheme that included selfpollinations: 1) diallel cr ossing sc hemes whic h include the 1 pa re nts F of fs pr ing a nd se lf -p oll ina tio ns a re mor e po we rf ul a t se pa ra tin g g e ne tic e ff e c ts from envir onmental ef fec ts than simpli fied diallel studies; 2) ther e wa s a nee d to know the per cent of self-pollination of the pa rent c lones, which mig ht confound lea f fr eezetol e ra nc e tr a it. The initial literature revie w and discussion above was important and r elates to the method of appr oach c ross technique in bahiag rass discussed in mate rial and me thods.I n a he te ro zy g ou s p la nt, su c h a s b a hia g ra ss, the g e ne ra l c omb ini ng a bil ity fo r a tr a it i s

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88 important when c onducting poly crosse s. Parent clone s with high-g ener al combining ability for the tr ait would be desire d. Hig h-g ener al combining ability would provide 1 ma te rn a l pl a nts the op po rt un ity to d e ve lop F se e d th a t w ou ld e xpre ss t he L TF T t ra it. Hig h-g ener al combining ability for L TFT w ould be desira ble. When the additive mode of g e ne a c tio n f or a tr a it i s a la rg e c omp on e nt o f t ra it h e ri ta bil ity s e le c tio n f or tha t tr a it can be come e fficie nt. Recurr ent re stricted phenoty pic selec tion would be one method of conce ntrating the desire d trait in a population using the additive mode of g ene a ction for a tra it. Fr eezetoleranc e of pla nts is a complex t rait which mig ht be mediated by more than 1000 g enes ( Guy 2003). The poly g enic tra it ex pression may be more of an a dditive mode of g ene a ction. Nar row-se nse her itability (h ) is calc ulated as the additive portion 2 of the g enetic va rianc e divided by the total g enetic va rianc e (Poehlman a nd Sleper, 1995; Allard, R.W., 1966). Narr ow-sense heritability calc ulations of L TFT the ref ore w ould be heavily influence d by the g ener al combining ability Additive g ene a ction should be imp or ta nt w he n u sin g a RRPS b re e din g sy ste m. T hu s, the imp or ta nc e of a tte mpt ing to quantify h for L TFT is e vident. 2 Th e br oa dse ns e he ri ta bil ity (H ) i s c a lc ula te d a s th e g e ne tic va ri a nc e fo r a tr a it 2 divided by the sum of the phenoty pic var iance (Poehlman and Slepe r, 1995; Allard, R.W ., 1966). The H often c an over estimate her itability beca use of g enoty pe x 2 phenoty pe intera ction trait eff ects. Additionally H includes dominant and e pistatic g ene 2 action ef fec ts. Dominant and epistatic g ene a ctions occur in the hy brid condition and ar e difficult to manipulate in a he terozy g ous, self-incompa tible plant such as bahia g rass. Do min a nt a nd e pis ta tic g e ne a c tio ns ma y no t be inv olv e d w ith L TF T i f t he tr a it i s poly g enica lly controlled by a lar g e number of g enes a s Guy (2003) sug g ested. The

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89 diallel mating de sign c an be use d to estimate H I n a diallel mating desig n phenoty pe 2 va ri a nc e s c a n b e pa rt iti on e d in to g e ne ra l c omb ini ng s pe c if ic c omb ini ng ma te rn a l, pa te rn a l a nd a ll o f t he po ssi ble int e ra c tio n e ff e c ts ( L y nc h a nd Wal sh 1 98 8) Ph e no ty pic va ri a nc e c a n b e pa rt iti on e d in to 1 2 c omp on e nts w hic h c a n b e e xtra c te d f ro m th e a na ly sis of var iance (Z hang et al., 2005). A diallel mating sc heme c an be use ful as a pr eliminary t o o l i n es t i m at i n g t ra i t h er i t ab i l i t y d ev el o p i n g an i n i t i al b re ed i n g s y s t em an d i n i t i at i n g a se le c tio n s c he me H ig hL TF T h e ri ta bil ity w he the r h or H w ou ld a llo w m or e ra pid 22 development of improved populations than low her itability As a res ul t o f t he p revi ou s d is cus si on a need occu rred to in ves ti gate t he L TFT trait g ene a ction and her itability in bahiag rass a t the diploid level. The hy pothesis that ba hia g ra ss w a s ma inl y a se lf -i nc omp a tib le pla nt n e e de d to be te ste d s inc e se lf -f e rt ili ty c ou ld s ke w c omb ini ng a bil ity re su lts T he hy po the sis tha t L TF T w a s a he ri ta ble tr a it neede d to be tested. I f L TFT is he ritable, ther e would be a need to e stimate the level of heritability I f L TFT is a heritable trait, then the qua ntify ing of the mode of g ene a ction would be bene ficial to bahia g rass bre eder s. M at e r ial and M e th ods Se e d P r oduc ti on F r om Cl one s I n the summer of 2003 f our fr eezesensitive low-L TFT c lones (2-221, 1-30-4, 130-3, and F L 9), four fre eze-tolera nt highL TFT c lones (F L 67, CO6, OK1, and O K2), a nd one interme diate-L TFT c lone (C4-36) w ere incre ased ve g etatively A total of nine diploid parent clone s were used in this study Fr eezesensitive clones 2-221, 1-30-3, a nd 1-30-4 we re se lected f rom UF -I FAS Da iry Resear ch Center Hag ue, F lorida af ter a light frost eve nt which produc ed 100% le afdamag e. F ree ze-sensitive clone F L 9 was sele cted from contr olled fre eze cha mber trials. F ree ze-tolerant c lones FL 67, C4-36, and CO6

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90 orig inated fr om a bre eding prog ram for fre eze-tolera nce a t University of F lorida-I FAS North F lorida Resea rch a nd Educa tion Center (NF REC) near Marianna Florida Fr eezetolerant c lones OK1 a nd OK2 orig inated fr om naturalized bahiag rass c ollected a nd supplied by Dar ren Redf ear n, from Oklahoma Rhi zome pie c e s w e re dip pe d in Ho rm od in 2 (E .C. Ge ig e r, Ha rl e y sv ill e PA ) t o induce r ooting. Potting media was Scotts Terr alite Ag ricultural Mix (Scotts-Sierra Horticultural Produc tion Com pany 14111 Scotts L ane Rd., Mar y sville, OH 43041). Pot siz e wa s 18 cm x 19 cm, Classic 400 (Nurse ry Service s, Fa irless Hills, PA) to provide su ff ic ie nt r oo t vo lum e T he po tti ng me dia wa s Sc ott s T e rr a lit e Ag ri c ult ur a l Mi x (Scotts-Sierra Horticultural Produc tion Com pany 14111 Scotts L ane Rd., Mar y sville, 25 2 OH 43 04 1) N utr ie nt m a na g e me nt w a s 1 g of a 16 -4 -8 -1 NP O -K OF e a na ly sis g ranula r fe rtiliz er pr e-pla nt incorpora ted into the media of e ach pot. I ronite 1-00 (I ronite Products Company Scottsdale, AZ 85258) wa s applied (1g per pot) on the top of the media to pre vent iron chlorosis. An ove rhea d spray irrig ation sy stem was set to run for f ive min four times during the day Additional fertilization after e ach f lowering and 25 2 c ro ssi ng c y c le inc lud e d a pp lic a tio n o f 1 g pe r p ot o f a 16 -4 -8 -1 NP O -K OF e a na ly sis g ranula r fe rtiliz er, a nd I ronite 1-00 After eac h flush of inflore scense s, crossing and seed ha rvest, plants we re c lipped to a 10-c m heig ht, irrig ation withdrawn f or 48 hours and fe rtiliz ed with the same amount of nutrients to induce a nother c y cle of infloresc ence emer g ence A drip emitter sy stem was installed to deliver 3.78 L min per pot four times -1 per da y to maintain optimum plant g rowth. Da y leng th was maintained a t 20 h of light with a timed bank of inc andesc ent lights to promote ve g etative g rowth and f lower pr od uc tio n. Pla nts we re g ro wn a t th e Un ive rs ity of F lor ida s Ag ro no my te a c hin g g la ss g ree nhouse whe re te mpera tures we re se t for stea m heat to circ ulate whe n tempera tures

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91 wer e below 22C. Gr eenhouse maxi mum temperature s rar ely exceede d 50C, as rec orded w ith a minimum /max imum thermometer (A curite) Crosses wer e made using the technique de scribed a nd ref err ed to as a pproac h crosse s by Bur ton (1955). I n approa ch cr osses, one inflore scenc e fr om eac h of the pa re nts is p la c e d in a ba g to e xclu de fo re ig n p oll e n, a nd no a nth e r e ma sc ula tio n is conducte d. Approa ch cr osses are made with plants that ar e assumed to be mainly selfinfertile. The ref ore, a pproac h crosse s are mutual crosses. Crosses w ere made the evening (betwe en 5pm and midnig ht) befor e flore ts opened. Potted plants wer e ph y sic a lly a pp ro a c he d, wi th t he fl ow e r s ta lk a nd e xclu sio n b a g su pp or te d b y a wi re to make the c ross. Where potted plants we re not possible to put toge ther for crossing a rh izom e pie c e wi th s e ve ra l no de s w hic h in c lud e d th e fl ow e r a nd le a ve s ( re fe rr e d to here afte r as r amets) we re br oken off from ea ch clone inserted into a c ontainer of water with their flore ts enclosed within a ba g and supported by wire. Selfpollinations were made by slipping a g lassine bag over a single infloresc ence I nitially dialy sis tubing was u s ed t o ex cl u d e f o re i gn p o l l en i n cr o s s es / s el fs b u t i t wa s re p l ac ed b y gl as s i n e b ags beca use of the a ppare nt highe r humidity and static holding of pollen in dialy sis tubing when visually inspecting bag g ed cr osses. Daily tapping of bag g ed cr osses or selfs w as conducte d to ensure pollen dispe rsal. Anther emer g ence and pollen dehisc ence appea red to occur first in the middle, followed by the base and fina lly by the apica l portion of the infloresc ence over a period of a pproxi mately three day s. Daily tapping was c onsidered nece ssary to assure unifor m seed set. See d heads w ere allowed to ripe n for a minimum of 14 d af ter c rossing /selfing to ensure a dequate maturity No later than 21 d af ter c ro ssi ng /se lf -p oll ina tio n s e e d h e a ds we re ha rv e ste d to pr e ve nt s e e d s ha tte r a nd ke pt i n marke d envelope s and kept in airconditioned storag e until hand-thre shing c ould be

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92 acc omplished. Seed per he ad we re c ounted if a c ary opsis was forme d. No g rading of seed by weig ht was conduc ted, only total seed pe r hea d wer e counte d. A minimum of 10 poll inations were attempted on all possible cr osses or selfpo lli na tio ns Si nc e the re we re nin e pa re nts ( p ) 36 initial crosses a nd 36 rec iproca l c ro sse s w e re ma de fo r a tot a l of 72 dif fe re nt p os sib le c omb ina tio ns of c ro sse s [ p ( p -1) = [9(9-1) =72]. All nine pare nts were self-pollinated ( p = 9) in o rd e r t o te st t he hy po the sis that bahiag rass c lones wer e mainly self-infe rtile. A minimum of 10 self-pollinations wer e attempted. I f see d g erminate d sufficie ntly from the se lf-pollinations of pare nt 1 clo nes an at tem pt wou ld be m ade t o i ncl ud e S pro geny in a ful l d ial lel st ud y of t he L TFT trait. I nformation on the number of see d produce d from a ba hiag rass c ross and self pollination was nece ssary to ensure tha t sufficient plants could be g rown for a diallel study Bur ton (1955) re ported pe rce nt of flore ts that set seed in openpollinations (8 9. 5% ) a nd se lf -p oll ina tio ns (6 .0 %) bu t no t se e d n umb e r p e r i nf lor e sc e nc e T he re wa s a need to know how the see d number that would be produce d from 1) a cross a nd 2) fr om a selfpollination. This i nformation wa s neede d befor e the diallel study could proc eed beca use suffic ient prog eny seedling s had to be g rown for the diallel study Em erge nce from Seed B a hia g ra ss s e e d g e rm ina tio n h a s b e e n a c ha lle ng e c omm e rc ia lly a s w e ll a s in bree ding due to its prolonged g ermination per iod and wea k seedling s (Gate s et al., 2004) Dormanc y cause d by unscar ified see d coat a nd adher ing le mma and pale a on the car y opsis was overc ome par tially by acid sc arific ation (West and Marousky 1985; Marousky and West, 1988). A small trial was c onducted with four replica tions of two trea tments on two lots of bahiag rass see d. Commercially available mecha nically hulled

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93 a nd fu ng ic ide -t re a te d Pe ns a c ola ba hia g ra ss w a s th e fi rs t lo t of se e d u se d. Th e Pe ns a c ola seed w as har vested two y ear s prior (2002) Two-y ear old seed wa s used due to better g ermination of se ed that had pa ssed an a fterripene d dormanc y period, ra ther than sa me1 season se ed (West and Ma rousky 1985). The se cond lot of see d was F bree der se ed (not threshe d, g rade d, scar ified, or tre ated with fung icide) c rea ted from a cross made 17 Aug ust 2003. The purpose w as to deter mine whether bree der se ed g ermination (quantified a s emerg ence ) wa s enhanc ed or pr evente d by acid sc arific ation. The Pensacola seed lot wa s the optimum seed ag e and tr eatment f or g ermination and emer g ence Pots were f illed with the same media a s in previously discussed trials. Trea tment, with concentr ated sulphuric a cid, occ urre d 6 J une 2004 followe d by sowing and plac ing pots unde r a timed mist bed in the Ag ronomy Tea ching Gre enhouse. Emerg ence counts of 100 see d wer e conduc ted 17 d af ter tre atment and sowing (Table 5-1). Table 51. Ger mination test via emerg ence of 100 see d scar ified with conc entra ted sulphuric ac id for 15 minutes, 23 J une 2004. L ine/cultivar Acid sca rified Control % Pensacola 0.0b 15.5a C4-36 x FL 9 13.2a 0.0b *M e a ns wi th t he sa me le tte r a re no t si g nif ic a ntl y dif fe re nt a t P = 0. 05 c on fi de nc e le ve l. 1 Acid sca rifica tion of bree der F seed ( C4-36 x FL 9) wa s compara ble to the control trea tment of mecha nically hulled Pensacola seed. A cid sca rifica tion may have been le thal to mechanic ally hulled scar ified Pensac ola see d. Concern tha t excessive trea tment with concentr ated sulphuric a cid might da mag e fr esh, un-hulled se ed as w ell as hulled seed, r esulted in reduc ing sc arific ation time to 5 min for the diallel study All seed fr om a c ro ss o r s e lf we re mixe d u nif or mly 9 July 20 04 Se e d w e re c ov e re d in conce ntrated sulphuric acid ( 16 M) for 5 min in a plastic c entrifug e tube. See d and

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94 sulphuric ac id mix ture we re stirre d with a g lass rod af ter initially cover ing the mix ture with acid. At the e nd of 5 min the mix ture wa s deca nted into a plastic straine r to ca tch seed in a sink with running wa ter for a minimum of 15 seconds. Washed see d wer e air dried during the nig ht on coffe e filters. See d wer e counte d into four 100-see d lots, or evenly divided into four lots in order to have four r eplications per cross/self. On 10 J uly 2004, four 100seed r ows of see d wer e sown (or seed lots less than 40 0 w e re e ve nly div ide d in to f ou r r ow s) a nd ma rk e d w ith pla sti c la be ls i n e a c h p la sti c tray in which Scotts Terra lite Ag ricultural Mix was used f or media. The rema inder of the se e d lo t, i f t he re wa s a ny w a s sp re a d a c ro ss t he tr a y a nd g e ntl y pr e sse d b y ha nd to ensure seedto-media c ontact. Tra y s were place d on propag ation tables in a scr eene d g ree nhouse at the U niversity of F lorida I FAS Nor th Florida Rese arc h and Educ ation CenterSuwannee Valley An automated mist sy stem cover ing a ll tray s was timed for 15 min at 0800, 0100, 1200, 1400, and 1600 daily Seed moisture has be en re ported to be critica l for bahia g rass g ermination (Williams and W ebb, 1958). Thr ee da y s later, g ermination wa s visible. The automated mist sy stem was adjusted a fter se edling s had sever al leave s to 5-minute durations during the set times and alter nate da y s for irr iga tion. 25 2 Nutrient mana g ement wa s throug h timed irrig ation with a 1-0-1 ( N-P O -K O) h yd r o p o n i c c u c u m b e r f e r t i l i z e r t h a t d e l i v e r e d 1 2 0 m g k g N i n s o l u t i o n 2 2 J u l y, 2 4 J u l y, -1 and 27 Jul y 2004. On 30 J uly N was inc rea sed to 180 mg kg to promote plant g rowth. -1 Seedling emer g ence was c ounted 25 J uly 2004 for a ll crosses/selfpollinations. Di all e l J uly 31, 2004, wher e possible, a minimum of 40 seedling s per c ross wer e tr a ns pla nte d to Spe e dli ng 8 x 16 c e ll ( 12 8 c e ll) a ir -p ru nin g sty ro fo a m tr a y s in Sc ott s Terr alite Ag ricultural Mix Plastic tray s were maintained in the scr eene d g ree nhouse at

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95 the Univer sity of F lorida I FAS Nor th Florida Rese arc h and Educ ation CenterSuwannee Valley under a timed mist sy stem, as desc ribed for g ermination. Ther e we re not sufficie nt self-pollination seedling s acr oss the nine par ent clones to c onduct a f ull diallel study due to their low g ermination and e merg ence rate s (1.0% to 14.0%) There fore a 1 Gr if fi ng Mo de l 3 d ia lle l de sig n w ith pa re nt c lon e s a nd F pr og e ny (i nc lud ing re c ipr oc a ls) was used in a randomized complete bloc k desig n with four re plications. Plot s were composed of 10 pla nts spaced 30 c m apar t for a total plot leng th of 3.5 m. Rows were space d 76 cm apa rt. The diallel plot was pla nted 27 Aug ust 2004. Parent clones w ere planted as ve g etative r hizom e cutting s dipped in Hormodin 2 to encour ag e root development. Par ent clones w ere composed of f our diploid free ze-sensitive clones (2 -2 21, 130 -4 1 -3 03, a nd F L 9) a nd fo ur dip loi d fre eze-tolera nt clones (F L 67, C4-36, 1 CO6, OK1, and O K2). The F prog eny seedling s from 70 cr osses wer e tra nsplanted fr om Speedling tray s. I n addition to the formal diallel study a Pensac ola population was inc lud e d a s a sta nd a rd c omm e rc ia l di plo id c ult iva r a s w e ll a s a n A rg e nti ne c lon e a s a standard f or lea f fr eezesensitivity Wil mington-ty pe tetra ploid clones (identified a s 31 57 32 3 15 73 3, a nd 31 57 34 ) a s w e ll a s a te tr a plo id P a ra g ua y 22 c lon e we re a lso inc lud e d to c omp a re le a ftis su e fr e e zetol e ra nc e in t e tr a plo ids to d ipl oid s. Th e se additional tetraploid clone s were propag ated a s veg etative c uttings in the same ma nner a s the par ent clones. B order rows and bor der pla nts were planted on all four sides of the plot are a. F ield maintenanc e of the plot included a tilled border alley 1.8 m wide; handwee ding of rows wa s done whe n nece ssary Spot application of herbic ides wer e used, a s nece ssary during the g rowing season to contr ol weeds in row alley s or broa dcast ove r the entire plot. De pending on the wee ds and their loca tion the label ra tes of the f ollowing herbic ides wer e used: 10 g kg v/v Diquat (Z enec a Ag Products, Wil mington, DE) 10 g -1

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96 kg v/v Roundup Ultra Max (Monsanto Co., St. L ouis, MO) or 1.16 L ha Ba nvel -1 -1 (Micro F lo Company L L C, Memphis, TN). I rrig ation was a pplied in the fall of 2004 a s ne c e ssa ry to e sta bli sh the so wi ng N o f e rt ili zer wa s a pp lie d a ft e r s ow ing in 2 00 4. Pla nts appea red g ree n and in a ve g etative state as the winter fre ezes approa ched. A fre eze eve nt 15 Dece mber 2004 re ache d -2.5C at 0715 and 2.8C at 2215 as rec orded a t the 60-cm he ight a bove the soil at Univer sity of F lorida-I FAS Nor th Florida Resear ch and E ducation Center -L ive Oak. Ca nopy damag e ra tings we re ma de 17 Dec ember 2004. Statist ical ana ly sis of both parent a nd prog eny was c onducted with the same model, whe re r ating = g enoty pe + bloc k + re plication within blocks + g enoty pe x block. A fre eze eve nt rea ched 3.7 C at 0730 at the 60-c m heig ht on 20 Dece mber 2004. o This was followed by a fr eeze e vent on Dec ember 21, 2004 that rema ined at -4.1 C from o 0715 throug h 0730 at the 60 cm he ight. Cumulative ca nopy damag e ra tings we re ma de 22 De c e mbe r 2 00 4 o n a 1 to 9 s c a le wh e re 1 = no c a no py da ma g e a nd 9 = c omp le te ca n o p y d am age W i n t er i n j u ry ra t i n gs we re m ad e b as ed o n ac cu m u l at ed ca n o p y d am age 12 Marc h 2005, befor e spring canopy reg rowth, on a 1 to 9 sca le wher e 1 = no w inter injury and 9 = c omplete plant dea th. Fe rtiliz er w as applied 15 Ma rch 2005, w hich supplied the following nutrients on a 22 kg ha basis: 89.6 N, 22.4 P O5, 44.8 K 0, 56.0 S, 11.2 Ca, 0.39 Zn, 0.17 B 0.06 Cu, -1 1.06 Fe and 0.78 Mn. The e ntire plot are a wa s clipped 7 June 2005 to a 10 cm heig ht wi th a ro ta ry mow e r a nd top g ro wt h r e mov e d b y ra kin g A ll p la nts we re un if or mly define d by vertica lly cutting into squar es 15 cm x 15 cm with a Sti hl edg er 14 June throug h 16 J une 2005. Pruned pla nt material wa s removed ma nually from the plot ar ea. I ndividual turf piece s were left uniformly square 30 cm from turf cente r to turf c enter,

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97 within the row. The pur pose of squa ring eac h of the 10 individual plants within a plot row wa s to stage all plant materia l to uniform siz e. Squaring turf piec es also enha nced individual origina l plant identity in the winter for canopy fre eze-injury rating Squaring turf piec es additionally was done to equalize bare soil that mi g ht radiate heat dur ing the winter fr eezes a nd confound c anopy fre eze-injury Plots wer e mowed 8 Septe mber 2005 to a 10 cm heig ht with a rotary mower a nd top gr owth remove d by raking Mowing was c on du c te d a t th is t ime to p re ve nt p la nts fr om b e c omi ng do rm a nt. Ne w v e g e ta tiv e g ro wt h wa s st imu la te d w ith a n a pp lic a tio n o f f e rt ili zer ide nti c a l to the 15 Ma rc h a pp lic a tio n r a te and nutrients. A sec ond season of canopy fre eze-da mag e ra tings we re ta ken in orde r to compar e rating s taken on 4-month old see dlings in 2004 to mature pla nts in 2005. Canopy fre ezeda ma g e ra tin g s in 20 05 we re ma de on a pe rc e nta g e of c a no py le a fda ma g e (0 % t o 10 0% ). A f re e ze e ve nt o c c ur re d 2 2 D e c e mbe r 2 00 5 w hic h r e a c he d 3. 7 C a t 07 00 a t a o 60 cm heig ht above the soil. This eve nt was followed by a fr eeze on 23 De cembe r 2005 wh i ch re m ai n ed b et we en 5 9 an d -3 2 C fo r 4 h an d b el o wfr ee z i n g fo r 8 5 h R at i n gs of pe rc e nt c a no py le a fda ma g e we re ma de the a ft e rn oo n o f 2 3 D e c e mbe r 2 00 5. Ma in g enoty pe ef fec ts were analy zed statisti cally using the g ener al linear model with SAS (SAS I nstitut e I nc., 1987). Combining a bility was a naly zed with DI AL L EL -SAS05 (Z hang et al., 2005). V aria nces w ere used of the phe noty pe compone nts derived f rom DI AL L EL -SAS output t o calc ulate H and h by published proce dure ( L y nch and Walch, 22 1998; Wu, personal communication, 2006). The broadsense he ritability (H ) wa s 2 c a lc ula te d f ro m th e g e ne tic va ri a nc e div ide d b y the ph e no ty pic va ri a nc e : g p H = V / V 2

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98 g A The g enetic va rianc e (V ) was c alculate d as the sum of the a dditive varianc e (V ) and D dominance va rianc e (V ): g = A D V V + V A The a dditive varianc e (V ) wa s calc ulated as 4 x ge nera l combining a bility varia nce V(g ): A V = 4 x V( g) D The dominanc e var iance (V ) wa s calc ulated as 4 x specific c ombining ability V(s): D V = 4 x V( s) p The total phenoty pic var iance (V )wa s calc ulated as the summation of all 12 partitioned 1 varia nces ( g ener al combining ability specific combining a bility F prog eny rec iproca l eff ects, mate rnal e ffe cts, non-mater nal ef fec ts and all their intera ctions derived f rom the DI AL L EL -SAS output). Narrow -sense heritability was c alculate d as Ap h = V/ V 2 Results and Discus sion Se e d P r oduc ti on fr om Cl one s A cr ossing sc heme of nine clones w ith their rec iproca ls would equal the ne eded 72 possible crosses. Of the possible 72 crosse s, 70 were succe ssfully completed (Table 5-2). Crosses of OK1 x OK2 (H x H) were not possible (Table 52) due to lac k of flower emer g ence sy nchrony so other cr osses wer e made when those c lones flower ed. When flowering sy nchrony did not favor c rosses, selfpollinations were made Flowe ring sy nc hr on y of so me c lon e s ( OK 1, OK 2, 222 -1 1 -3 03, a nd 130 -4 ) w a s d if fi c ult to achie ve during Aug ust 2003 through D ece mber 2003 in spite of maintaining day leng th at 2 0 h w i t h a r t i f i c i a l l i g h t i n g T h e s e c l o n e s r e p r e s e n t e d t h e e x t r e m e s o f t h e LT F T r a n g e It ma y be po ssi ble tha t op tim um d a y le ng th f or fl ow e ri ng c ou ld b e dif fe re nt f or the se

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99 difficult-to-c ross clones. All of the nine par ent clone se lf-pollinations were made (Table 5-2). Table 52. Crosses and self -pollinations made Aug ust 2003 through D ece mber 2003 of fr e e zetol e ra nt, int e rm e dia te a nd fr e e zese ns iti ve ba hia g ra ss c lon e s. M al e ( % ) H H H H I L LLL Femal e ( & ) F L 6 7 C O 6 O k 1 O K 2 C 4 -3 6 F L 9 1 -3 0 -3 1 -3 0 -4 2 -2 2 -1 T FL67 12 9 6 5 19 12 12 8 14 T C O 6 9 1 8 1 2 7 1 1 1 1 91 2 7 T O K 1 6 1 2 1 1 8 6 9 91 0 T O K 2 5 6 1 9 1 0 6 7 8 6 I C436 18 13 8 11 14 10 10 14 11 S F L 9 1 3 1 1 6 6 1 0 1 3 8 91 0 S 1303 12 9 9 7 9 8 17 15 10 S 1304 8 12 9 8 14 8 15 21 7 S 2221 14 7 10 6 11 10 10 7 20 T = tolera nt, I = interme diate, and S = se nsitive Em erge nce from Seed 1 R es u l t s o f e m er gen ce fr o m s ee d d et er m i n ed wh et h er o r n o t s u ff i ci en t F s ee d l i n gs from cr osses would be a vailable f or the diallel study Emerg ence of selfpollinations would deter mine whether or not self-pollinations could be inc luded in the diallel study Perce nt emerg ence among the prog eny g enoty pes (Ta ble 5-4), w hether develope d from 11 se lf -p oll ina tio ns (S ) o r c ro sse s o f p a re nts (F ), wa s si g nif ic a nt ( P < 0.0001). I t was obvious that obtaining suff icient selfpollinated seedling s for a diallel study would not be 1 possible, based on the r ate of emer g ed see dlings (S s are marke d in bold ty pe on the dia g on oa l, T a ble 53) Se lf -p oll ina te d p ro g e ny pe rc e nt e me rg e nc e ra ng e d f ro m 1. 0% to 14.5%. Self-pollination prog eny had a me an 5.4% e merg ence rate Table 53 shows low self-pollination prog eny emer g ence (1% to 7%) for c lones other than F L 9 (14.5%) ref lected the low rate of selfpollination leading to viable seed. The se data (Table 5-3) confirm ba hiag rass a s quantified by seedling emer g ence Mean e merg ence results are simil ar to the r eporte d mean ba hiag rass self -pollination seed set of 6.0% (B urton, 1955).

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100 Rece nt work has shown se lf-pollinations of clones of a Pensacola bahiag rass population aver ag ed a me an of 11% seed se t, with a rang e fr om 0.2% to 44% (Ac una, per sonal c o m m u n i c a t i o n 2 0 0 6 ) S e l f p o l l i n a t i o n o c c u r s i n b a h i a g r a s s p o p u l a t i o n s a n d m a y p l a y a part in trait expression. L ine FL 9 was the only clone whe re suf ficient self -fe rtility might influence the eva luation of L TFT tr ait expression. Self-fer tilit y in bahiag rass, c ould be 1 assumed to be a neg ligible influe nce in the L TFT tr ait expression of F prog eny of par ent clones used in the study except for line F L 9. Table 53. Perce nt emerg ence of cr osses and self -pollinations made Aug ust 2003 throug h Dec ember 2003 of fr eezetolerant, interme diate, and f ree zese ns iti ve ba hia g ra ss c lon e s. Male ( % ) HHHHI L L L L Fe male ( & ) FL 67 CO6 Ok1 OK2 C4-36 FL 9 1-30-3 1-30-4 2-22-1 T FL 67 1.0 23.3 6.5 13.5 19.5 29.0 54.3 45.3 22.7 T CO6 11.7 1.7 30.0 12.3 29.3 32.3 9.3 44.5 12.3 T OK1 36.3 22.3 6.3 20.7 --20.5 2.5 44.0 9.0 T OK2 12.3 15.7 --6.3 22.7 16.3 9.5 9.3 6.5 I C4-36 65.0 28.7 16.7 13.0 2.3 33.3 65.7 25.0 38.5 S FL 9 22.7 21.7 6.3 23.3 28.7 14.5 30.3 8.5 58.0 S 1-30-3 32.3 19.0 8.5 15.3 13.3 26.5 5.7 1.0 0.5 S 1-30-4 22.7 9.0 12.5 21.0 22.7 39.3 1.0 3.5 1.7 S 2-22-1 31.7 20.7 26.7 11.3 42.3 42.0 2.0 2.0 6.5 *T = tolera nt, I = interme diate, and S = se nsitive. Pensacola open pollinated standa rd see d lot not acid-sca rified. ** Se lf -p oll ina tio ns bo lde d o n th e dia g on a l. F e ma le a nd ma le dif fe re nc e s in se e dli ng e me rg e nc e c ou ld a ff e c t L TF T t ra it expression, through the materna l or pater nal contributions to the trait. I f there wer e dif fe re nc e s in se e dli ng e me rg e nc e d e pe nd ing on ini tia l or re c ipr oc a l c ro sse s, L TF T t ra it expression would be biased by differ ence s in perce nt emerg ence by the ty pe of pollination. Theref ore, it wa s important to know if the ty pe of pollination was 1 signific ant. Statisti cal a naly sis of F populations showed ty pe of pollination (initial ve rs us re c ipr oc a l c ro ss) wa s n ot s ig nif ic a nt ( P = 0.05). Uniform emergence of

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101 seedling s, reg ardle ss whether the par ent clone w as the fe male or ma le par ent, was 1 confirme d (Table 5-4). Comparison of F emer g ence with the Pensacola seed standa rd showed no sig nificant diff ere nce ( Table 54). Self-pollination emer g ence was sig nif ic a ntl y le ss t ha n e me rg e nc e of se e d d e ve lop e d f ro m c ro sse s. 1 Table 54. Mean e merg ence of cr oss F s from nine c lones vary ing in the le af f ree zetoleranc e tra it and self-pollination prog eny of nine c lones compar ed to an open-pollinated standa rd. Polli nation Genoty pe Emerg ence % Open Pensacola 27.0a* 1 I nitial cross F populations 24.0a 1 Reciproc al cr oss F populations 20.0a 1 Self S populations 5.4b *M e a ns wi th t he sa me le tte r a re no t si g nif ic a ntl y dif fe re nt a t P = 0. 05 c on fi de nc e le ve l. Diallel 2004 Th e re we re sig nif ic a nt g e no ty pe e ff e c ts ( P < 0.0001) f or par ent clone c anopy fr e e zeda ma g e ra tin g s a ft e r t he 17 De c e mbe r 2 00 4 f re e ze e ve nt ( Ta ble 55) F ie ld r e su lts of par ent clone c anopy damag e ra tings we re not c onsistent with L TFT c ateg ories assig ned to clones in pre vious controlled fre eze experiments (Chapter 2). Clones that ha d p re vio us ly be e n e xtre me ly se ns iti ve to f re e ze e ve nts (1 -3 03 a nd 130 -4 ) w e re le ss damag ed than c lones that had pre viously been toler ant of fr eeze e vents (F L 67, CO6, and OK2). The skewing of clone behavior in the field, compa red to c are fully controlled environmenta lly controlled g rowth cha mber (E GC) trials, may have be en the r esult of newly established veg etative planting s from re cently -divided plant mater ial. I n spite of ir ri g a tio n to e ns ur e a de qu a te fi e ld m ois tur e b loc k e ff e c ts w e re sig nif ic a nt ( P < 0.0001). Table 56 shows canopy damag e ra ting by cross a nd rec iproca l cross. The 1 seedling s of F s resulting from OK1 ( fema le) x CO6 (male) had the lowest ca nopy dam age rati ng of al l t he p rogeny (1. 6). Th is was a cro ss of a h ighL TFT x hi gh-L TFT

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102 c lon e a nd mor e ty pic a l of sp e c if ic c omb ini ng a bil ity w he re sp e c if ic c ro sse s w ou ld transfe r the tra it. The rec iproca l cross, CO6 x OK1, had signific antly highe r ca nopy dam age (2. 9). Cr os ses of h ighL TFT l in e OK1 x in ter med iat e-L TFT C 4-3 6, lo w-L TFT 130 -3 lo wL TF T 1 -3 04, a nd low -L TF T 2 -2 21 r e su lte d in pr og e ny wi th s ig nif ic a ntl y lower c anopy damag e ra tings than the rec iproca l crosses ( Table 56). The r ecipr ocal c ro ss d a ta su g g e sts tha t ma te rn a l e ff e c ts a s w e ll a s p oll e n s ou rc e ma y be imp or ta nt t o consider, w hen making specific crosse s with parent c lones. Ta ble 55. Pa re nt c lon e c a no py fr e e zeda ma g e ra tin g s u se d to de ve lop a dia lle l c ro ss rate d afte r a f ree ze event 17 De cembe r 2004. Parent c lone L TFT Canopy damag e ra ting Categ ory (1 to 9) FL 67 T 3.1a* CO6 T 3.2a OK1 T 2.8b OK2 T 4.0a C4-36 I 3.7a FL 9 S 3.6a 1-30-3 S 2.7b 1-30-4 S 2.6b 2-22-1 S 3.6a *M e a ns wi th t he sa me le tte r a re no t si g nif ic a ntl y dif fe re nt a t P = 0. 05 c on fi de nc e le ve l. Ba sed on controlled f ree ze experiments (1 = 0% a nd 9 = 100% da mag e). T = tolera nt, I = interme diate, S = sensitive. Table 56. Mean c anopy leaf fre eze-da mag e ra tings (1 to 9) of prog eny rate d afte r a fre eze eve nt (17 Dec ember 2004) as a result of fe male par ent and pollen source F e ma le pare nt ( & ) Ma le pa re nt ( % ) FL 67 CO6 OK1 OK2 C4-36 FL 9 1-30-3 1-30-4 2-22-1 FL 67 3.2abc 2.8bc 2.8bc 2.5c 3.2ab 3.4ac 3.1abc 3.1abc CO6 3.5a* 2.9abc 2.9abc 3.1abc 3.2ab 3.3ab 2.6bc 3.4b OK1 2.8bc 1.6d --3.6a 3.2ab 3.0abc 2.9abc 2.6c OK2 2.9abc 3.1abc --3.2abc 2.9abc 3.3ab 3.6a 3.7a C4-36 3.3ab 3.5a 2.7bc 3.3ab 2.8bc 3.0abc 3.3ab 3.2abc FL 9 3.0abc 3.3ab 3.0abc 2.3c 3.1abc 2.8bc 3.5a 3.0abc 1-30-3 3.3ab 3.3ab 2.7bc 2.9abc 3.1abc 2.8bc --2.4c 1-30-4 3.3ab 3.4ab 2.7bc 3.2abc 2.9abc 3.2ab --3.0abc 2-22-1 3.3ab 2.9abc 2.4c 3.0abc 3.4ab 3.7a 3.3ab 3.4b *M e a ns wi th t he sa me le tte r a re no t si g nif ic a ntl y dif fe re nt a t P = 0. 05 c on fi de nc e le ve l.

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103 I t was nec essar y to conduct fur ther statistical ana ly sis using a f ix ed model of Griff ing s Model 3 (Z hang and Kha ng 1997). B eca use the softwa re ( Z hang et al., 2005) c ou ld n ot o pe ra te wi th m iss ing c ro sse s, no t a ll o f t he pa re nts c ou ld b e us e d to te st combining a bility Prog eny of cr osses 1-30-4 x 1-30-3 and 130-3 x 1-30-4 had bee n c omp ro mis e d a nd re mov e d f ro m th e fi e ld p lot s. Th e re fo re o nly pa re nts wi th f ull complements of pr og eny could be use d. This eliminated one hig h-L TFT ( OK2) a nd one low-L TFT ( 1-30-4) pare nt and their prog eny from the diallel test for combining a bility L ines FL 67, CO6, C4-36, FL 9, 1-30-3 a nd 2-22-1 we re use d in the model afte r per sonal communication with the author of the softwar e (Z hang persona l communication, 2005). A total of 6 par ents and 24 prog eny wer e used f or a total of 30 e ntries with 4 replica tions per e ntry using D I AL L EL -SAS05 (Z hang et al., 2005). Th e re we re no sig nif ic a nt e ff e c ts ( P < 0.2156) f or combining ability (Table 5-7). B loc k e ff e c ts ( P< 0.001 for Ty pe I err or sums of square s) as we ll as a hig h CV (15%), contributed to the lac k of statistical signif icanc e to entrie s, and spec ific and g ener al c omb ini ng a bil iti e s ( P < 0. 21 5 for type I error). The young age of the plants (4 months) may have c ontributed to the lack of signific ance for g ener al and spe cific c ombining abilities. I t appea red tha t the new planting of the plot was c ontributing to the hig h rating CV, as well as to block e ffe cts (Ta ble 5-7). T here fore it was dec ided to allow another g ro wi ng se a so n to oc c ur in o rd e r f or pla nts to s ta bil ize a s ma tur e pla nts A n a tte mpt to captur e the L TFT tr ait eff ects a fter a n adequa te fre eze in 2005 would be made Diallel 2005 Th e c a no py da ma g e ra tin g in 2 00 5 w a s a pe rc e nt c a no py da ma g e e sti ma te (0 = 0 % c an o p y d am age t o 1 0 0 = 1 0 0 % c an o p y fr ee z ed am age ). Fr ee z e d am age ra t i n gs we re sig nif ic a nt f or g e no ty pe e ff e c ts ( P < 0. 00 01 ) a lth ou g h b loc k e ff e c ts w e re sti ll

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104 Table 57. Analy sis of varianc e of c anopy damag e ra tings made afte r 17 De cembe r 20 04 fr e e ze u sin g the fi xed mod e l G ri ff ing s Me tho d 3 a nd 6 b a hia g ra ss c l o n e s v a r yi n g i n L T F T t r a i t a n d t h e i r p r o g e n y. Source Df Sum of square s Mean square F value P > F Model 32 12.269 0.383 1.68 0.0311 Error 85 19.412 0.228 Total 117 31.681 Ty pe I rep 3 4.004 1.335 5.84 0.0011 Ty pe I entry 29 8.264 0.285 1.25 0.2156 sig nif ic a nt ( P < 0. 00 01 ). Pa re nt c a no py da ma g e ra tin g s ( Ta ble 58) f or the mos t pa rt wer e consistent with the L TFT c ateg ories deve loped from pr evious controlled EG C fre eze trials. Clones CO6, OK1, OK2, a nd C4-36 had low ca nopy damag e ra tings (15% to 2 8% da ma g e ). Un e xpe c te dly F L 67 ha d a c a no py da ma g e ra tin g of 51 %, wh e re a s in controlled EGC trials this clone ha d been one of the fir st high-L TFT lines ide ntified and studied as a r esult of consistent tolera nce to f ree zing temper ature s. L ine FL 9 was a nother clone whic h chang ed in fre eze-tolera nce be havior (T able 58). F L 9 had 29% c anopy da ma g e w hic h w a s n ot d if fe re nt f ro m th e c lon e s w ith the hig he st L TF T t ra it. Th is w a s a complete r ever sal of beha vior, when c ompare d to controlled fr eeze e vents, which identified F L 9 as a f ree ze-sensitive clone. Clones 1-303, 1-30-4 a nd 2-22-1 r ang ed fr om 60 to 81% ca nopy damag e and w ere signific antly highe r (a pproxi mately twofold) than the majority of the c lones which ha d previously been c lassified hig h in L TFT ( CO6, OK1, OK2, a nd C4-36). E ff o rt s t o re d u ce fi el d v ar i ab i l i t y h ad b ee n t ak en i n t h e d i al l el m at i n g d es i gn study The e ntire field ha d been c lipped, fer tiliz ed, plants individually trimmed, and wee ded as unifor mly as possible. Yet the r esults of L TFT, a s quantified by perc ent cano py dam age after th e 22 Decem ber 2 00 5 fr eez e even t, sh owe d d if feren t L TFT toleranc e for pare nt clones in the field, c ompare d to their previous be havior unde r

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105 controlled fr eeze tria ls. The phenomenon of controlled fr eeze tria l results which do not corr elate w ell with field trials is a problem that re peate dly arose in the plant fre ezetoleranc e litera ture (A braha m, 1988). Soil and micro-climate va riability among many uncontrolled pa rame ters, ar e the c halleng es of a ttempting fie ld evaluations of plant materia l. Bre eder s have f elt that evalua tions of whether or not plant materia ls are f ree zetolerant unde r field pr oduction sy stems have to be the ultimate test of perf ormanc e. Ta ble 58. Pa re nt c lon e c a no py fr e e zeda ma g e ra tin g s u se d to de ve lop a dia lle l c ro ss rate d afte r a 22 D ece mber 2005 fr eeze. Parent c lone L TFT Canopy damag e ra ting cate g ory % FL 67 T 51b* CO6 T 28c OK1 T 25c OK2 T 15c C4-36 I 15c FL 9 S 29c 1-30-3 S 76a 1-30-4 S 81a 2-22-1 S 60b *M e a ns wi th t he sa me le tte r a re no t si g nif ic a ntl y dif fe re nt a t P = 0. 05 c on fi de nc e le ve l. Ba se d o n c on tr oll e d f re e ze e xpe ri me nts T = tolera nt, I = interme diate, S = sensitive. T a b le 5 9 s h o w s th e p r o g e n y c a n o p y r a ti n g s a f te r 2 2 D e c e mb e r 2 0 0 5 f r e e ze e v e n t T h e F L9 p r o g e n y w i t h t h e e x c e p t i o n o f o n e c r o s s ( F L9 & x OK2 % = 67% c anopy damag e) ha d low canopy damag e ra tings, re g ardle ss whether FL 9 was the f emale or the p o l l e n s o u r c e o f t h e c r o s s W i t h t h e e x c e p t i o n o f F L9 & x OK2 % t h e c r o s s e s w h e r e F L9 was the f emale ha d canopy damag e ra tings ra ng ing f rom 24% to 43%. I n the 22 Dec ember 2005 fre eze eve nt the FL 9 pare nt clones cha ng ed beha vior, to fre eze-tolera nt (c a no py da ma g e ra tin g = 29 %, Ta ble 58) in c omp a ri so n to fr e e zese ns iti ve in controlled fr eezing trials. The fr eezetoleranc e beha vior in 22 Dec ember 2005 was evident in all of the c rosses whe re F L 9 was the pollen sour ce, a nd had low ca nopy

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106 damag e ra tings. Prog eny canopy damag e of c rosses fr om all female pare nt clones (F L 67, CO 6, OK 1, OK 2, C436 1 -3 03, 130 -4 a nd 222 -1 ) w ith F L 9 ma le pa re nt c lon e s, rang ed fr om 25% to 43%. I f all of the c rosses in which F L 9 had bee n a fe male par ent had shown low c anopy damag e ra tings, the hig h rate of selfing of F L 9 (14.5%) might be suspected a s the ca use of low pr og eny canopy fre eze-da mag e ra tings. The one excepted c r o s s ( F L9 & x OK2 % ), with hig h canopy damag e (67% in Table 5-9) shows that selfing of F L 9 did not ex plain prog eny behavior from that cr oss. The hig her r ate of selfing of FL 9, compar ed to the other pare nt clones (F L 67, CO6, OK1, OK2, C4-36, 130-3, 1-304, and 2-221) may not expl ain the sig nificantly highe r ca nopy damag e ra ting of 67% ( T a b l e 5 9 ) f o r t h e F L9 & x OK2 % cross. I n fac t, the canopy damag e ra ting of prog eny of FL 9 crosse s, states a c ase f or g ener al combining ability for the L TFT tr ait. Table 59. Prog eny canopy fre eze-da mag e ra tings (0 to 100% ) ra ted af ter 22 De cembe r 2005 as a r esult of fe male par ent and pollen sourc e. F e ma le Parent ( & ) Ma le pa re nt ( % ) FL 67 CO6 OK1 OK2 C4-36 FL 9 1-30-3 1-30-4 2-22-1 FL 67 56abc 53bc 54fbc 51bc 33cd 66ab 57bc 65ab CO6 63ab* 37cd 49c 39cd 35cd 59abc 59abc 52bc OK1 65ab 52bc --35cd 43cd 53bc 57bc 59bc OK2 65ab 52bc --52bc 67ab 59abc 61abc 68ab C4-36 45c 47c 51bc 43cd 24d 39cd 39cd 53bc FL 9 32d 35cd 35cd 34cd 25d 25d 30d 43cd 1-30-3 60ab 40cd 55bc 59abc 38cd 37cd --29d 1-30-4 58bc 49c 53bc 66ab 52bc 28d --33cd 2-22-1 68ab 62abc 45cd 60abc 48c 35cd 72a 46cd *M e a ns wi th t he sa me le tte r a re no t si g nif ic a ntl y dif fe re nt a t P = 0. 05 c on fi de nc e le ve l. Statist ical ana ly sis for combining ability was c onducted using Griff ing s fix ed model Method 3 with the same c lones and prog eny as in 2004. Results were signific ant ( P > 0. 03 2) fo r t he a na ly sis of va ri a nc e T he L TF T p he no ty pe tr a it w a s p a rt iti on e d in to its so ur c e s o f v a ri a nc e to c a lc ula te he ri ta bil ity (T a ble 510 ). B ro a dse ns e he ri ta bil ity

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107 (H ) of L TFT w as ca lculated to be 25.8. N arr ow-sense heritability (h of L TFT w as 22 ) calc ulated to be 7.6% The L TFT tr ait in this ex periment, ha s a low broa d-sense heritability and nar rowsense he ritability L ow fre eze-tolera nce he ritability is consistent with ge nera l trends re po rt e d b y Ab ra ha m ( 19 88 ). Ta ble 510 sh ow s th e la rg e va ri a nc e c on tr ibu tio ns to phenoty pe expression of L TFT f rom the var ious sources. Phenoty pe var iance contributions are espec ially substantial from the non-ma terna l interac tions, reciproc al 1 c ro ss e ff e c ts, re c ipr oc a l c ro ss x e nv ir on me nt i nte ra c tio ns a nd F int e ra c tio ns Ph e no ty pic e xpre ssi on of the L TF T t ra it d e pe nd s h e a vil y on the e nv ir on me nt, a s sh ow n in Table 510. Table 510. L TFT phe noty pe ef fec ts partitioned into their varianc e sourc es fr om a fixed six pare nt diallel mating de sign a naly zed with Griffing s Method Three Var iance source Diallel-SAS notation Var iance L SD 0.05 Gene ral c ombining ability Vg 9.297 6.060 gi -gj Specific c ombining ability V 22.313 9.389 m Materna l contribution V 6.198 4.948 mi mj Materna l interac tion V 14.876 7.666 nij Non-mate rnal c ontribution V 29.751 10.841 nijnkj Non-mate rnal intera ction V 74.378 10.841 nijnkjl Non-mate rnal x environment V 59.502 15.332 r Reciproc al cr oss effe cts V 44.627 13.278 rijrkl Reciproc al cr oss x environment V 89.253 18.778 1 sij F eff ects V 26.776 10.285 1 sijsik F interac tion V 66.940 16.262 1 sijskl F x environment V 44.627 13.278 The a dditive ge ne ac tion portion of the L TFT tr ait heritability is a small portion of total heritability (7.6%) compar ed to total heritability (H = 25.9%) The dominant 2 g ene a ction portion of L TFT tr ait heritability is 18.3%, as ca lculated by differ ence [18.3% = (0.259 0.076) x 100]. Broadsense he ritability of L TFT ma y be conside red larg ely composed of dominant g ene a ction (70.5%) with a minor component of a dditive

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108 g ene a ction (29.5%) as ca lculated a s perc entag es of br oad-se nse her itability Selection for the L TFT tr ait must manag e the low he ritability of 25.9% ( H = 0.259) by 2 maxi miz ing se lection prog ress throug h bree ding sc hemes that utiliz e this mode of g ene action. The dominant g ene a ction of the L TFT tr ait could be e x ploited by making controlled c rosses of se lected pa rents. Ta ble 5-9 shows c rosses with FL 9 as either pare nt had sig nificantly lower c anopy damag e. The conce rn of se lfing interfe ring with crossing f o r F L9 c a n b e n e g a t e d d u e t o t h e 6 7 % c a n o p y d a m a g e o f p r o g e n y f r o m t h e O K 2 x F L9 cross. Crosses whic h produce d prog eny with significa ntly low canopy damag e we re rec iproca l FL 9 X C4-36 cr osses. Appar ently there is high spec ific combining ability for L TFT be tween those two lines. T h e a d d i t i v e c o m p o n en t o f t h e LTFT t ra i t gen e a ct i o n co u l d b e u t i l i z ed t h ro u gh 1 RRPS t o e ith e r i mpr ov e su pe ri or F pr og e ny of se le c te d li ne s o f c ro sse s o r s y nth e tic populations made fr om poly crosse s of superior pare nt clones. Afte r seve ral c y cles of RRP S, either improved sy nthetic populations or prog eny lines could be e valuated f or e ve ntu a l c ult iva r d e ve lop me nt. Es se nti a lly th e a dd iti ve c omp on e nt c ou ld b e us e d to conduct a family and line bre eding prog ram a s an alter native or in c onjunction with the traditional poly cross bre eding scheme The diallel study data supports using family bree ding a s a strate g y for de veloping su pe ri or L TF T b a hia g ra ss f a mil ie s. F ro m th e mos t f re e zetol e ra nt f a mil ie s, the mos t fre eze-tolera nt individual plants could be selecte d and used a s pare nts in advance d po ly c ro sse s. Br eeding strateg y in L TFT in ba hiag rass c ould move bey ond identification of superior individual plants to be used in tra ditional poly crosse s. Poly crosse s do not

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109 ensure cross-pollination of super ior lines. Polli nation sy nchrony is assumed. Wit hout prog eny -testing of clone s used in poly crosse s, combining a bility is not identified. Specific c rosses of pa rent c lones assure g enetic inf ormation exchang e. Pare nt clone com bi ni ng abi li ty may be an im po rt ant para met er i n b reed in g for s up eri or L TFT populations. Proge ny of F L 9 had sig nificantly less canopy damag e in Table 5-9, reg ardle ss of pollen source Prog eny -testing assure s superior pa rent c lones as we ll as identifies combining ability for the de sired tra it. Cl assic sy nthetic population 1 de ve lop me nt u se s th e e va lua tio n o f p a re nt c lon e F pr og e ny to i de nti fy pa re nts wi th su pe ri or c omb ini ng a bil ity Su pe ri or pr og e ny -t e ste d p a re nt c lon e s c ou ld b e us e d in succe ssive poly cross c y cles fr om which see d would be incr ease d for e ventual super ior cultivar re lease Summ ary Re pr od uc tiv e be ha vio r i n b a hia g ra ss i s c omp lic a te d. Chr omo so me nu mbe r i s often linked with the ty pe of se x ual re production in bahiag rass. Diploid (2 x ) b a hia g ra ss plants are commonly sexual. Tetraploid (4 x ) bahia g rass plants ar e ty pically apomictic. Ma nip ula tio n o f t ra it e xpre ssi on mus t c ur re ntl y be br e d a nd se le c te d f or a t th e dip loi d somatic chromosome le vel. Se lf -p oll ina tio n in po pu la tio ns tha t a re ma inl y c ro sspo lli na te d ma y sk e w t ra it e xpre ssi on Se lf -p oll ina tio n w a s sh ow n to oc c ur fo r o ne of the nin e pa re nt c lon e s a t a 14.5% leve l. The other e ight c lones used in the study had low ra tes of selfpollination. The a vera g e selfpollination rate of 5.4% among clones used a s pare nts in thi s study was s i m i l ar t o t h e p u b l i s h ed ra t e o f 6 % ( Bu rt o n 1 9 5 5 ). P ro gen y t es t i n g s h o we d t h at al t h o u gh FL 9 had a hig h rate of selfing there was a cross whe re c anopy damag e ra ting wa s e xtre me ly dif fe re nt f ro m a ll o f t he oth e r c ro sse s. I f s e lf ing ha d d omi na te d tr a it

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110 expression in FL 9 crosse s, prog eny from all of the crosse s from F L 9 would have expressed the same trait. Data sug g est that crossing actua lly took place in a t least one cross of F L 9, and led to prog eny with differ ent levels of c anopy damag e. Ge nera l combining a bility for L TFT tr ait in FL 9 could also be involved in prog eny fre ezetoleranc e beha vior. Deter mining the he ritability of a tra it is i mportant in bree ding f or improved populations. Hightrait heritability allows rapid population improveme nt per c y cle of selec tion. L ow-tra it heritability not only reduc es the ra te of population improvement but im pact s t he b reed in g str ate gy and sel ect io n o f br eedi ng met ho d. Heri tab il it y of L TFT was found to be rela tively low. Br oad-se nse her itability was c alculate d as H = 26%, 2 wh ic h in c lud e d b oth a dd iti ve a nd do min a nt m od e s o f g e ne a c tio n o n L TF T t ra it expression. Narr ow-sense heritability was c alculate d as h = 8%, w hich expresses the 2 additive g ene a ction as a por tion of L TFT phe noty pe expression. Mode of g ene a ction for a trait is important in developing an ef ficient bre eding prog ram. I f additive g ene a ction for a trait such as L TFT w ere the pre dominant method of he ri ta bil ity th e n ma ss a nd re c ur re nt s e le c tio n b re e din g sc he me s ( su c h a s RRP S) would shift the conce ntration of the de sired tra it in the population. I f dominant g ene action for a tra it were the pre dominant mode of her itability then isolated cr osses of superior individual plants would be more eff icient. Ba sed on the re sults of this s tudy modified mating scheme s were sug g ested. I dentifica tion and rete ntion of superior c lones with high c ombining ability for L TFT w as rec ommended. Prog eny testing of clones wa s rec ommended as a method of identify ing superior c lones with high c ombining ability for the L TFT tr ait. A sy nthetic population develope d from poly crosse s of superior pare nts was re commended.

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111 The dominant mode of g ene a ction might be use d in making individual cr osses resulting in superior prog eny The a dditive mode of g ene a ction for L TFT tr ait could be utiliz ed in either improving sy nthetic populations or in developing improved L TFT lines fr om i nd ivi du a l c ro sse s.

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112 CH APT ER 6 CONCL USI ONS Introduction Florida livestock (dair y horses, be ef c attle, shee p, and g oats) that depe nd on fora g es ac counted f or $1.009 billion in sales and 18,076 jobs in 1997. Bahia g rass is the fora g e base upon which that livestock industry depends. Shortag e of ba hiag rass during the months of Dec ember throug h Marc h occur s as a r esult of sporadic fre eze eve nts. L eaf -tissue cold-toler ance (the a bility of a g enoty pe to maintain appa rently g ree n and undamag ed lea ves af ter a fre eze eve nt) would bene fit the Florida livestock industry The bahiag rass lea f-tissue fr eezetoleranc e (L TFT) trait could extend fora g e supply for liv e sto c k d ur ing the wi nte r s e a so n if the tr a it w e re inc or po ra te d in to a c omm e rc ia lly available cultivar. Curre ntly bahiag rass bre eding is confined to diploid sex ual lines due to the apomictic block in tetra ploid lines. A re view of the cold-tolera nce a nd fre eze-tolera nce liter ature resulted in a theore tical sche me on which to view va rious toleranc e mec hanisms on a continuum of plant response to gr adually lower a nd ultimately below-f ree zing temper ature stress. B e c a us e L TF T h a s b e e n f ou nd in d ipl oid bahiag rass a need oc curr ed to initially quantify the ra ng e of tha t trait with repre sentative lines. Major a rea s of investig ation which mig ht e l u c i d a t e m e c h a n i s m s t h a t m a y b e i n v o l v e d i n t h e L T F T t r a i t i n c l u d e d : a n a t o m y, phy siology and g enetics ( heritability of the tra it, not mol ecula r g ene a ctivation or silencing ). Unde rstanding which mec hanism or mecha nisms mi g ht be involved in the

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113 L TF T t ra it m ig ht h e lp e xpe dit e the de ve lop me nt a nd re le a se of su pe ri or dip loi d bahiag rass c ultivars in the near future tha t might extend the fora g e base in Florida dur ing Dec ember throug h Marc h. An additional potential would be the e x pansion of the ba hia g ra ss f or a g e pr od uc tio n a re a fa rt he r n or th i f L TF T c ou ld b e inc or po ra te d in to bahiag rass. Summ ary of Ob jec tives Range of Leaf -tissue F ree zetoler ance Trait Expression The fir st objective in this study was to quantify the ra ng e of f ree ze-toleranc e e xpre ssi on in d ipl oid ba hia g ra ss g e no ty pe s. A r a ng e of L TF T e xpre ssi on wa s f ou nd in the field fir st. Diploid clone bahiag rass c lones wer e sele cted f rom Dr. B lounts bree ding prog ram, a collection of a pproxi mately 24,000 plants at Maria nna, F lorida, collec tion from natur alized plants in southeastern Oklahoma and obser vation in prog eny at Hag ue, Florida The ra ng e of L TFT w as quantified using a ca nopy damag e ra ting of 1 to 9 (1= no c anopy fre eze damag e, 9 = c omplete ca nopy fre eze damag e). A n initial, 26-line stu dy us e d c on tr oll e d f re e ze t ri a ls o f p ro g re ssi ve ly c old e r t e mpe ra tur e s f ro m 1 C to -7C. Additions of ge rmplasm wer e conf irmed with a 30-line study conducte d in a larg er, e nvironmentally controlled fr eeze c hamber which could hold all of the replica tions at one single fre eze eve nt. The line trends f or L TFT e x pression we re confirme d in the 30-line study A contribution to science of the ra ng e of L TFT e x pression study was that this was indeed a trait. L ines with fre eze-tolera nce or fre eze-sensitivity could be identifie d throug h repe ated c ontrolled fre ezing tria ls. The tolera nce to lea f-f ree zing was de mon str a te d to be a tr a it, no t a ra nd om o c c ur re nc e Co nf ir ma tio n o f L TF T a s a tr a it o p en ed t h e p o s s i b i l i t i es o f u s i n g t h e t o o l s av ai l ab l e t o m an i p u l at e t h e t ra i t t h ro u gh

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114 traditional g enetics, a s well as to furthe r investig ate the tr ait on a molecula r leve l. C o n f i r m a t i o n o f LT F T a s a t r a i t m a d e f u r t h e r e x p l o r a t i o n o f m e c h a n i s m s m e a n i n g f u l If L TF T m e c ha nis ms c ou ld b e ide nti fi e d, those mechanisms could be exploited to manipulate the trait. Another contribution for further scientific investigations is the observation that relative humidity needs to be controlled in freeze trials. The use of a plastic bag to enclose potted plants in a freeze trial may be beneficial in future experiments. Baffles and test plants, blocked by location, were used in the environmentally controlled chamber, where 30-lines were tested. A contribution to further investigations is the need to run small tests to reduce the CV and position effects in controlled freeze chamber experiments. A practical contribution of quantifying and confirming the LTFT trait in bahiagrass is the potential for plant breeders to use identified freeze-tolerant genotypes to extend the grazing season into the cool, fall period. Genotypes were identified and confirmed through controlled freezing trials, which withstood freeze events more severe than what is normally experienced in Florida. What was not achieved in the studies of LTFT trait range was solving the question of why an entire leaf would look damaged in a whorl of leaves, while the leaves adjacent to the completely damaged leaf appeared green. Canopy freeze-damage ratings were for the entire canopy of a potted plant. Undamaged leaves in a whorl tended to be those which had just emerged from the whorl through the first fully-expanded leaf. Canopy freeze-damage ratings were an average for the canopy leaves, and did not take into account the individual leaf freeze-damage behavior. Further investigations of bahiagrass leaf-tissue freeze-damage could record leaf damage by leaf position (and thus leaf age). Infrared thermography could be used to

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115 rec ord the a ctual fr eeze pr ocess in the lea f. F urther inve stiga tions need to be c onducted to answer why an entire leaf in a leaf whorl, is damag ed ra ther than a portion of the lea f. Conf irm ation of Leafor Roottissue F ree zeTolerance The sec ond objective in this study was to dete rmine whe ther the L TFT tr ait might be root fr eezetoleranc e ra ther than le af f ree ze-toleranc e. B eca use potted plants wer e completely frozen during trials (roots and le aves) there was the possibility that root eff ects, ra ther than le af e ffe cts, wer e showing as L TFT. Water -bath e x periments we re conducte d wher e the r oot tempera ture wa s kept above fre ezing while the leaf canopy was e xpos e d to va ri ou s b e low -f re e zing te mpe ra tur e s to c on fi rm c a no py da ma g e wa s a re su lt of lea f-tissue dama g e and not of root fre eze-stre ss. Water-bath e x periments conf irmed that canopy leaf fre eze-da mag e wa s a re sult of L TFT. L ines which showe d high le vels of canopy leaf fre eze-da mag e in wholeplant fre ezing tria ls showed similar damag e eve n when r oots were kept at 5C while the c anopy was e x posed to two prog ressively colder below-f ree zing temper ature s (-2.7C and 3.2C, respec tively ). L ines which showe d low levels of c anopy leaf fre eze-da mag e in wholeplant fre ezing tria ls also showed similar d am age wh en ro o t s we re k ep t ab o v e f re ez i n g. A c on tr ibu tio n to sc ie nc e is t he c on fi rm a tio n th a t L TF T i s a re su lt o f l e a f e ff e c ts, not root eff ects. I nvestig ators ca n fre eze entire bahiag rass plants and qua ntify leaf damag e. A pr actica l contribution of the L TFT le afeff ect c onfirmation is that produce rs c a n b e a ssu re d th a t if a fr e e ze e ve nt o c c ur s th a t da ma g e s th e g re e n b a hia g ra ss f oli a g e it is the canopy that is damag ed, not the roots. W h a t w a s n o t a c h i e v e d i n t h i s o b j e c t i v e w a s a m e a s u r e o f r o o t d a m a g e In retrospe ct, viable r oot weig ht of control clone s versus fr eezetrea tment clones bef ore a nd

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116 afte r the fr eeze tre atment could have been done Howeve r, the limitations of plant materia l and fre ezer spac e constra ined destruc tive measure ment techniques. Anatom ical Diff ere nces Between F ree zeSensitive and F ree zeTolerant Lines The third objec tive in this st udy was to dete rmine whe ther ther e we re a natomical dif fe re nc e s b e tw e e n f re e zese ns iti ve a nd fr e e zetol e ra nt l ine s th a t mi g ht b e re la te d to fr e e zetol e ra nc e A tw olin e inv e sti g a tio n o f b a hia g ra ss w a s c on du c te d in iti a lly to quantify anatomica l differ ence s betwee n a fr eezetolerant a nd a fr eezesensitive clone. Statist ically signific ant diffe renc es we re f ound in the leaf midrib x y lem diameter An eig ht-line investiga tion was conduc ted to confirm a natomical diffe renc es in 4 fre ezetol e ra nt a nd a 4 f re e zese ns iti ve c lon e s. Sma lle r m idr ib xy le m di a me te rs xy le m a re a s, vascula r bundle, diame ters and va scular bundle are as we re a ssociated with fr eezetolerant clones. What was not ac hieved in this study was a confirma tion in the field of vessel diameter differ ence s of prog eny of fr eezetolerant x free ze-sensitive pare nt lines. The limit ation of the fir st y ear in the field (2004) was the e ffe ct of see dlings e x perie ncing field fr eeze c onditions not providing canopy fre eze-da mag e ra tings that a mature pla nt would (in 2005). Onc e fr eeze e vents identified fr eezetolerant a nd fre eze-sensitive prog eny from a toler ant and se nsitive cross, fre eze-sensitive line lea ves would have been damag ed, pre venting x y lem diameter measure ments. The ea rliest an ana tomical verific ation could have been done of prog eny from a c ross would have been la te fa ll of 2006. A c on tr ibu tio n to sc ie nc e wa s th e ide nti fi c a tio n o f a n a na tom ic a l f e a tur e in bahiag rass lea ves that may provide a possible expl anation of w hy entire le aves a re damag ed, instead of leaf portions, after a fr eeze e vent. Fur ther investig ation may

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117 confirm whe ther or not ve ssel air e mbolism may preve nt water flow whe n fre ezese ns iti ve lin e s a re re c ov e ri ng un de r f ull tr a ns pir a tio n s tr e ss. A pra ctical c ontribution of anatomical diff ere nces ma y be a he ighte ned conc ern by plant bree ders that improve d L TFT ba hiag rass populations have as hig h an in v itr o org anic matter dige stibil ity (I VOMD) a s possible. Care must be take n that selec tion for L TF T, wi th i ts a sso c ia te d a na tom y of sma lle r, le ss d ig e sti ble ve sse ls, no t r e su lt i n le ss dige stible forag e. F ur the r r e se a rc h n e e ds to b e c on du c te d in le a f a na tom y to c on fi rm wh e the r s ma ll x y lem diameter is a L TFT me chanism in bahiag rass. I n the late f all, field-g rown, mature prog eny (which ha ve bee n identified as f ree ze-tolerant a nd fre eze-sensitive) of tolerant and sensitive cr osses, nee d to be sec tioned and mea sured f or lea f xy lem diameter P hysiol ogical Diff erences Between Freeze-sensitive and Freeze-tolerant Lines The four th objective wa s to determine whe ther phy siologica l mechanisms might acc ount for diffe renc es betwe en fr eezetolerant a nd fre eze-sensitive lines. An experiment was initiated to test the hy pothesis that differ ence s betwee n fre eze-tolera nt and fr eezesensitive bahiag rass c lones wer e a r esult of diffe renc es in osmolality L eaf samples wer e take n at the midrib reg ion, where damag e had be en visualized as occ urring afte r a f reeze event. The samples were then frozen, to burst cells, then measured for osmolality. Results from that experiment showed that the differences in highand low-LTFT were not a result of leaf osmolality. An experiment was c onducted to test the hy pothesis that differ ence s betwee n fr e e zetol e ra nt a nd fr e e zese ns iti ve ba hia g ra ss c lon e s w a s a re su lt o f d if fe re nc e s in c e ll unsaturate d fatty acid ( FA) content. I n late fa ll, prior to a fre eze eve nt, leaves of clones repr esenting fre eze-tolera nce a nd fre eze-sensitivity wer e sampled. Tota l fatty acids of

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118 leaf samples wer e extracte d, and the f atty acids mea sured a s a portion of total fa tty acids e xtra c te d. Re su lts of tha t e xpe ri me nt s ho we d th a t un sa tur a te d f a tty a c id c on te nt, a s a portion of the total fa tty acids e x trac ted from the le af, w as not the mec hanism by which clones with fr eezetoleranc e could survive fre eze eve nts with small amounts of ca nopy damag e. I n fac t, results were the re verse of wha t would have be en pre dicted by unsaturate d FA the ory The most fre eze-sensitive (A rg entine) ha d the hig hest proportion of tri-unsatur ated C18:3 FA (linolenic) e x presse d as g kg TEFA highe st DBI and -1 hig he st U SF A: SF A. Wha t w a s n ot a c hie ve d in thi s e xpe ri me nt i s th e e xtra c tio n o f f a tty acids fr om only cell membra nes. Contributions to science from the osmolality experiment were that there wer e g enoty pe diffe renc es in bahiag rass c lones. The diff ere nces in osmolality depending on g enoty pe, sug g ests that bahiag rass ha s a ra ng e in osmoreg ulation. This information may be helpf ul in designing water -use a nd water -stress e x periments with bahiag rass. Ge no ty pe dif fe re nc e s in os mol a lit y imp ly tha t po pu la tio ns of int e rb re e din g dip loi d p la nts sh ou ld e xpre ss a ra ng e of os mor e g ula tio n. F ur the r c on tr ibu tio ns of os mol a lit y differ ence s in ge noty pes sug g est droug ht-toleranc e re sear ch, bre eding and per haps se le c tio n to imp ro ve ba hia g ra ss p op ula tio ns A dir e c t c on tr ibu tio n to sc ie nc e fr om t his curr ent study is the finding tha t osmolalit y did not predict whe ther a bahiag rass g enoty pe had hig hor lowL TFT. O bserva tion in the field of the 22 De cembe r 2005 fr eeze e vent at L ive Oak, F lorida, showed tha t highL TFT lines w ere frozen and c overe d with frost. Visualized frost confirme d that the mecha nism of free ze point-depression via incr ease d osmolality was not the mec hanism that preve nted fre eze-tolera nt lines from experiencing leaf -tissue fre eze-da mag e. The contribution to science is that osmolalit y may not be

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119 inv olv e d in the L TF T t ra it i n b a hia g ra ss, a nd tha t r e se a rc h e ff or ts s ho uld be fo c us e d in other a rea s of investig ation. Contributions to science from the f atty acid e x periment include the quantifica tion of the sa tur a te d a nd un sa tur a te d f a tty a c ids in b a hia g ra ss l e a ve s for use by animal scientists and food science investigators. Scientists in the areas of livestock nutrition, livestock products, and consumer health are working collaboratively to improve the health of meat-product consumers. Fatty acid composition, metabolism, and profiles by species is an active research area at this time. A p ra c tic a l c on tr ibu tio n c a n b e the dir e c t us e of the inf or ma tio n o f b a hia g ra ss fatty acid c ompositi on. Ruminant nutriti on investiga tion is usi ng the fa tty acid pr ofile of fee ds to manipulate the fa tty acid pr ofile of mea t and milk products, so that a hea lthier consumer pr oduct ca n be deve loped. Fur ther re sear ch in fa tty acids is re commended in the le af midrib ar ea of lowL TF T l ine s. Th e re g ion of da ma g e su g g e ste d th a t th e ph oto sy nth e tic c e lls of the Kr a ntz anatomy circ ling the la rg e midrib vessel c ould be the first c ells to be damag ed in the fr e e zetha w p ro c e ss. I n o rd e r t o s tud y the se c e lls e xtra c tio n me tho do log y of jus t th os e cells would have to be deve loped. The bundle sheath c ells are wher e the se cond photosy nthetic ca rbon re duction step occ urs. The la y er of cells immediately contig uous to the bundle shea th cells are the mesophy ll cells, high in c hloroplasts, which conta in the primary photosy nthetic re action. I n Arabidopsis thal iana L ., g ly cer olipids associated wi th m e mbr a ne str uc tur e a c c ou nt f or 56 4 g kg of the le a f l ipi d D M p or tio n. Chl or op la st -1 lipids account for 259 g kg of the lea f lipid DM portion (Buc hanan e t al., 2000). -1 Chang es in non-membra ne fa tty acid spe cies in the total fa tty acid e x trac tion method could be a ccounte d for by chang es in the chloropla st lipi d portion or the re maining 177 g

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120 kg po rt ion T he re fo re fu tur e re se a rc h o n f a tty a c id c omp os iti on sh ou ld f oc us on le a f c e ll -1 membrane s. Furthe rmore, r esea rch ne eds to focus on the midrib bundle sheath ce lls, not the entire lamina. The r eason f or this rec ommendation was that the midrib wa s where leaf injury was fir st visibl e af ter a fre eze eve nt, and full sunlight. Methodolog y for bahiag rass midrib bundle shea th cell membra ne sepa ration, and f atty acid e x trac tion, and qu a nti fi c a tio n, ma y be tte r d e te rm ine wh e the r m e mbr a ne fa tty a c id c omp os iti on is a me c ha nis m f or the L TF T t ra it. Here dity and Mode of Ge ne Act ion of Leaf-tissue Fr eez e-t olerance The fina l objectives in the bahia g rass L TFT study wer e to quantify here dity and mod e of g e ne a c tio n a t th e dip loi d c hr omo so me le ve l. N ume ro us c ro sse s a nd se lf po lli na tio ns we re ma de to a ssu re su ff ic ie nt v ia ble se e d w ou ld b e so wn Pr og e ny a nd se lf pollination seedlings we re g rown. The diallel mating study was modified be cause of insufficient see dlings fr om self pollinations. S elf-pollination was shown to oc cur f or one of the nine se lected L TFT pa rent c lones (F L 9) at a 14.5% leve l as measur ed by seedling emer g ence The other eig ht clones used in the study had low ra tes of selfpollination (1.0 to 6.5%). One of the 13 c rosses with FL 9 was sig nificantly highe r in ca nopy damag e than the re mainder of the cr osses. I f selfing had bee n the major sourc e of pr og eny canopy damag e ra ting would ha ve bee n consistent acr oss all crosses. The c ontribution to science wa s quantify ing the heritability (H = 26%, h = 8%) 22 a nd the a dd iti ve (8 %) a nd the do min a nt ( 18 %) mod e s o f g e ne a c tio n f or the L TF T t ra it. The low he ritability shows bree ders that pr og ress unde r selec tion will be slow, and depends on the additive portion of the mode of g ene a ction. Be cause of the he terozy g ous nature of bahia g rass the dominant mode of g ene a ction has not bee n traditionally utiliz ed. I n bahiag rass, hy brid deve lopment is not practical. Per haps the dominant mode of a ction

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121 could be use d in future in attempts to fix t he L TFT tr ait using c hromosome doubling a nd crossing with a fa cultative tetra ploid apomict, should that possi bility become a re ality The pra ctical c ontribution of mode of g ene a ction is the confirma tion that re c ur re nt s e le c tio n ( RRPS) of dip loi d ty pe s, wi th r e te nti on of su pe ri or L TF T c lon e s, sh ou ld r e su lt i n im pr ov e d L TF T i n la te r s e le c tio n c y c le s. Sugg e st e d F ur th e r Re se ar c h Molecular g enetics w ork contra sting fr eezetolerant a nd fre eze-sensitive c lones could be a naly zed for enzy mes, cry o-protec tants, and stress g ene se quence s that are upre g ula te d o r d ow nre g ula te d, du ri ng sh or t da y s a nd c oo l f a ll t e mpe ra tur e s. I t w ou ld a pp e a r t ha t sa mpl e s ta ke n in the fi e ld d ur ing the fa ll, pr ior to a fr e e ze e ve nt, a nd sto re d in liq uid nit ro g e n f or la te r a na ly sis ma y pr ov ide inf or ma tio n o n a dd iti on a l me c ha nis ms inv olv e d in the L TF T t ra it. Mo re tha n o ne me c ha nis m ma y be inv olv e d in the L TF T t ra it. Observa tion showed that approximately two wee ks after a fr eeze e vent, the hig hL TFT lines w ould not gr ow new le aves. A ll plants in t he fie ld appea red to be dormant. Since hig h-L TFT lines c ould gr ow new le aves w ithin the 18 d gr eenhouse rec overy period, ne w leaf emer g ence may depend on w arm tempe rature s. I n the field, fr eezetolerant lines did not g row ne w leave s after a fr eeze e vent. Ther e is a ne ed to find bahiag rass plants that have the trait of ne w leaf -eme rg ence during cool temper ature s and short day s. L eaf -eme rg ence was found in a plant introduction from Arg entina, USDA AR S GR I N S9 sy ste m pl a nt i ntr od uc tio n ( PI ) n umb e r 5 08 85 2. L e a fe me rg e nc e in addition to high-L TFT tr aits were observe d for plant introduc tion number 508852 for three y ear s at University of F lorida I FAS Nor th Florida Rese arc h and Educ ation Center L ive Oak, F lorida. The PI 508852 clone is a c onserva tively -g rowing small-leaf ed plant with undesirable f orag e g rowth habit.

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122 Crosses should be made w ith forag e-ty pe diploid bahiag rass c ultivars such as Ti ft on 9 in or de r t o e va lua te a nd se le c t pr og e ny wi th b oth le a fe me rg e nc e a nd L TF T t ra it i n fo ra get y p e p ro gen y C ro s s es o f P I 5 0 8 8 5 2 x FL9 s h o u l d b e m ad e b ec au s e o f t h e h i gh combining a bility of F L 9 for L TFT. Prog eny of cr osses of PI 508852 x FL 9 may have a highe r proba bility of expressing both leafemer g ence and L TFT. Dr. Ann B lount has develope d photoperiod-insensitive coldadapte d (PCA) diploid bahiag rass populations throug h cy cles of RRPS. Advanced c y cles ha ve improved L TF T t ra it a nd le a fe me rg e nc e (B lou nt, pe rs on a l c omm un ic a tio n, 20 05 ). Cr os se s o f P I 50 88 52 x ad va nc e d RR PS c y c le pla nts ma y pr od uc e imp ro ve d le a fe me rg e nc e in prog eny during the winter months. Using a nar row g ene ba se selec ted for L TFT in diploid bahiag rass, may be a strateg y to more quickly introg ress the tra it into a population. The testing of prog eny of superior c lones should help deter mine combining a bility in final deve lopment of sy nth e tic po pu la tio ns fr om p oly c ro sse s. F or e xamp le F L 9 w ou ld b e a c lon e tha t c ou ld b e i n c l u d e d i n a p o l yc r o s s b e c a u s e o f i t s a b i l i t y t o t r a n s m i t t h e L T F T t r a i t t o i t s p r o g e n y. Diallel mating could be use d to determine mode of g ene a ction for g ree n leaf emer g ence trait of bahia g rass during cool wea ther a nd short winter da y s. Mode of g ene action for g ree n leaf -eme rg ence may be diffe rent than f or L TFT. D iffer ent bre eding strateg ies may have to be used to maxi miz e lea f-e merg ence trait expression in an imp ro ve d c ult iva r p op ula tio n o f a fo ra g e or uti lit y tur f b a hia g ra ss.

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127 Ma ro us ky F .J., a nd S.H We st. 19 88 G e rm ina tio n o f b a hia g ra ss i n r e sp on se to tempera ture a nd scar ification. J. Am. S oc. Hor tic. Sci. 113:845-849. Ma ssa rd o, F ., L Co rc ue ra a nd M. Al be rd i. 2 00 0. Em br y o p hy sio log ic a l r e sp on se s to cold by two cultivars of oa t during germination. Crop Sci. 40:1694-1701. Mc Ke rs ie B .D ., a nd Y. L Y a Ac ov 1 99 4. Str e ss a nd str e ss c op ing in c ult iva te d p la nts Kl uw e r A c a de mic Pub lis he rs D or dr e c ht, Ne the rl a nd s. N i l s e n E T a n d D M O r c u t t 1 9 9 6 P h ys i o l o g y o f p l a n t s u n d e r s t r e s s J o h n W i l e y & Sons, New York, N Y. Orc utt, D.M., and E.T. Nilsen. 2000. The phy siology of plants under stress: Soil and biotic fac tors. J ohn Wil ey & Sons, Ne w York, N Y. Palta, J .P., and L .S. W eiss. 1993. I ce f ormation and fr eezing injury : An overvie w on the su rv iva l me c ha nis ms a nd mol e c ula r a sp e c ts o f i nju ry a nd c old a c c lim a tio n in herba ceous plants. p. 143-176. In P.H. Li, and L. Christersson (eds.) Advances in plant cold hardiness. CRC Press, Boca Raton, FL. Pearc e, R.S. 2001. Pl ant fre ezing a nd damag e. Ann. B ot. 87:417-424. Pearc e, R.S., and M.P. Fuller. 2001. F ree zing of ba rley studied by infrar ed video thermog raphy Plant Phy siol. 125:227-240. Poe hlm a n, J.M., a nd D. A. Sle pe r. 19 95 B re e din g hy br id c ult iva rs 4 e d. I ow a Sta te th University Press, Ames, I O. Quamme, H.A 1995. Dee p superc ooling in buds of woody plants. p. 183-199. In R E J. L e e G .J. Wa rr e n, a nd L .V G us ta ( e ds .) B iol og ic a l ic e nu c le a tio n a nd its applications. Am. Phy to. Soc., St. P aul, MN. Quar in, C.L 1999. Effe ct of pollen sourc e and pollen ploidy on endosper m formation and see d set in pseudog amous apomictic Paspalum notatum Sex ual Plant Reproduction. 11:331-335. Qu a ri n, C.L ., F Espinoza, E.J. Martinez, S.C. Pessino, and O.A. Bovo. 2001. A rise in ploidy level induce s the expression of apomix is in Paspalum notatum Sex ual Plant Reproduction. 13:243-249. Rajasheka r, C.B. 2000. Cold re sponse and f ree zing tolera nce in plants. p. 321-342. In R.E. Wi lkinson (ed.) Sec ond edition. Plant environment interac tion. Marce l Dekke r, Ne w York, N Y.

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128 Re vil la P. A B utr on M .E Ca rt e a R. A. Ma lva r, a nd A. Or da s. 20 05 B re e din g fo r c old toleranc e. p. 301-607. In M. As hr a f, a nd P.J. C. Ha rris (eds.) Abiotic stresses: Pla nt r e sis ta nc e thr ou g h b re e din g a nd mol e c ula r a pp ro a c he s. F oo d Pr od uc ts Press, New York, NY Rowley J A., C.G. Tunnicliffe and A.O. T ay lor. 1975. Fr eezing sensitivity of lea f tissue 4 of C g rasse s. Aust. J Plant Phy siol. 2:447-451. Sakai, S., and W. L arc her. 1987. F rost survival of plants. Spring erVer lag Be rlin. Samala, S., J Yan, a nd W.V. Baird. 1998. Cha ng es in polar lipid fatty acid c ompositi on during cold ac climation in 'Midiron' and U3' Be rmudag rass. Crop Sci. 38:188-195. Saupe, S.G. 2004. Dete rmining osmotic potential by the fre ezing point depr ession method. [Onli ne]. Available a t htt p:/ /e mpl oy e e s. c sb sju .e du /SSA UPE /bi ol3 27 /L a b/w a te r/ wa te rla bfr e e z.h tm (ver ified 28 April 2004). Sinclair, T.R., J .D. Ray P. Misl evy and L .M. Premazzi 2003. Growth of subtropic al fora g e g rasse s under e x tended photoper iod during short-day leng th months. C rop Sci. 43:618-623. Sin c la ir T .R. P. Mis le vy a nd J.D. Ra y 2 00 1. Sho rt ph oto pe ri od inh ibi ts w int e r g ro wt h of subtropica l gr asses. Planta 213:488-491. Smit h, A.M. 1994. Xy lem transport a nd the neg ative pre ssures sustainable by water Ann. B ot. 74:647-651. St. J ohn, J B. 1979. Chemica l modification of lipids in chill ing se nsitive species. p. 405430. In J.M. L y on s, D. Gr a ha m, a nd J.K. Ra iso n ( e ds .) L ow te mpe ra tur e str e ss i n crop pla nts. Acade mic Press, New York, NY S t ep o n k u s P L M F. Do wge rt R Y. E v an s an d W Go rd o n -K am m 1 9 8 2 C ry o b i o l o gy of isolated protoplasts. p. 459-474. In P.H. Li, and A. Sakai (eds.) Plant cold hardiness and freezing stressmechanisms and crop implications. Vol. 2. Academic Press, New York, NY. Stier, J.C., D.L. Filiault, and J.P. Palta. 2003. Visualization of freezing progression in turfgrasses using infrared video thermography. Crop Sci. 43:415-420. Sutcliffe, J. 1977. Plants and temperature. Edward Arnold, London, England. Taiz, L., and E. Geiger. 2002. Ice formation in higher-plant cells. Plant physiology online: A companion to plant physiology. [Online]. Available at http://www.plantphys.net/article.php?ch=25&id=254 (verified 10 February 2005).

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129 Tischler, C.R., and B .L Bur son. 1995. Evaluating differ ent bahiag rass c y toty pes for heat toleranc e and le af e picuticular w ax content. Euphy tica 84:229-235. Tischler, C.R., P.W Voig ht, and B .L Bur son. 1990. Evaluation of Paspalum g e rm pla sm for va riation in leaf w ax and heat tolera nce. E uphy tica 50:73-79. Tomashow, M.F 1999. Plant cold acc limation: Free zing tolera nce g enes a nd reg ulatory mecha nisms. Ann. Rev. Pl ant Phy siol. Molec. Bio. 50:571-599. USDA, ARS, National Gene tic Resource s Progr am. Germplasm Re sources Information Ne two rk ( GR IN ) [ O n l i n e D a t a b a s e ] N a t i o n a l G e r m p l a s m R e s o u r c e s L a b o r a t o r y, B e lts vil le M a ry la nd A va ila ble a t htt p:/ /w ww .a rs -g ri n. g ov /c g ibin/npgs/html/tax _site_acc .pl? S9%20Paspalum%20notatum (ver ified 22 Fe bruar y 2006) U.S. Office of Ge og raphy 1963. Arg entina off icial standar d names g azetteer no. 103. US Governme nt Printi ng Offic e, Washing ton. U.S. Office of Ge og raphy 1956. Urug uay g azetteer no. 21. US Governme nt Printi ng Of fi c e Wa sh ing ton D C. U.S. Office of Ge og raphy 1957. Parag uay g azetteer no. 35. US Governme nt Printi ng Of fi c e Wa sh ing ton D C. U.S. Office of Ge og raphy 1963. Br azil; official standard na mes approve d by the united states boar d on g eog raphic names. US Gove rnment Printing Of fice Washington, DC Vali, G. 1995. Principles of ic e nucle ation. p. 1-28 In R.E.J L ee, G .J Warren, a nd L .V. Gusta. (e ds.) B iologica l ice nucle ation and its applications. Am. Phy to. Soc., St. P aul, MN. Wanner, L A., and O. Juntti la. 1999. Cold-induced f ree zing tolera nce in Ar ab ido ps is Plant Phy siol. 120:391-400. Werner, B K., and G.W. B urton. 1991. Recur rent re stricted phenoty pic selec tion for y ield alters morpholog y and y ield of Pensac ola bahiag rass. Crop Sci. 31:48-50. Wes t. S .H ., a nd F .J. Ma ro us ky 1 98 5. Me c ha nis m of do rm a nc y in P e ns a c ola ba hia g ra ss. Crop Sci. 29:787-791. Wil liams, R.C and B .C. W ebb. 1958. Seed moisture r elationships and g ermination behavior of ac id-scar ified baha igr ass see d. Ag ron. J. 50: 235-237. Wisn ie ws ki, M. 19 95 D e e p s up e rc oo lin g in w oo dy pla nts a nd the ro le of c e ll w a ll structure p. 163-182. In R.E.J L ee, G .J Warren, a nd L .V. Gusta. (e ds.) Biolog ical ice nuclea tion and its applications. Am. Phy to. Soc., St. P aul, MN.

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130 Wis niewski, M., S.E. L indow, and E.N. A shworth. 1997. Obser vation of ice nuclea tion and propa g ation in plants using infra red the rmog raphy Plant Phy siol. 113:327334. Wolfe, J ., and G. B ry ant. 2001. Cellular cr y obiology : Thermody namic and me chanic al eff ects. I nt. J Refrig era tion 24:438-450. Wright, D.L ., A.R. Blount, R.D. Ba rnett, and R.O. My er. 2005. Tillag e and ove rsee ding pa stu re s f or wi nte r f or a g e pr od uc tio n in no rt h F lor ida SSAG R14 3, Ag ro no my depar tment, Florida c oopera tive extension service, I nstitut e of F ood and Ag ri c ult ur a l Sc ie nc e s, Un ive rs ity of F lor ida G a ine sv ill e F L [O nli ne ]. A va ila ble a t http:// edis.ifas.ufl.e du/AG146 (Ve rified 15 Apr il 2005). Utsumi, Y., Y. Sano, R. Funada S. Fujikawa, a nd J un Ohtani. 1999. The pr og ression of cavitation in ea rly wood vessels of F raxinus mandshurica var japonica dur ing fre ezing a nd thawing Plant Phy siol. 121:897. Z hang Y., and M.S. Kang 1997. DI AL L EL -SAS: A SAS prog ram for Griff ing s diallel analy ses. Ag ron. J. 89: 176-182. Z hang Y., M.S. Kang and K.R. L amkey 2005. DI AL L EL -SAS05: A comprehe nsive prog ram for Griff ing' s and Ga rdner -Eber hart a naly sis. Agr on. J 97:1097-1106.

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131 B I OG RA PHI CA L SKE TCH The c andidate s formal e ducation cove rs diverse fields of study A B ache lor of Ar ts i n p sy c ho log y wa s a wa rd e d b y F lor ida I nte rn a tio na l U niv e rs ity M ia mi, F lor ida in 1975 with a ce rtificate of Hig h Distinction. A chang e in intere sts toward ag riculture le d to ear ning a Ba chelor of Scienc e in Ag ronomy from the Unive rsity of F lorida in 1978. I ntere st in tropical fora g es and r uminant livestock led to work that re sulted in a Master of Sc ie nc e the sis (f or a g e g ro wt h a nd qu a lit y of F lor ig ra ze p e re nn ia l pe a nu t Ar ac his gla br ata Be nth. under six clippi ng reg imes) with a minor in animal scienc e in 1980 from the Univer sity of F lorida. Employ ment with the University of F lorida I FAS F lorida Coopera tive Ex tension Service commence d in 1980. Five y ear s of work in Madison County Florida wer e followed by a ra nk incre ase a nd promotion to Count y Ex tension Direc tor in Union County Ex tension educa tion prog rams in ag riculture a nd 4-H y outh development be ne fi te d c lie nte le to t he e xten t th a t th e c a nd ida te wa s p ro mot e d to the hig he st r a nk in Ex tension, and wa s one of the f ew e x tension ag ents to be aw arde d an additional 9% salary incre ase f or produc tivity At the time of this writing, the candida te had 26 y ear s of service with the Florida Cooperative E x tension Service. I nterna tional experience cover ed a c hildhood spent in S outh America Five y ear s wer e spent in the Santa Cruz reg ion of Bolivia. Eig ht y ear s were spent in the Guajira peninsula of Colombia. I n later y ear s development wor k in the Caribbea n on short-term projec ts with various ag encie s was par t of independe ntly planned pr ofessional

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132 development. F lorida Associa tion of Voluntary Ag encie s for Car ibbean Ac tion sponsored 4-H y outh developments work in the Commonwea lth of Dominica. The c andidate w as involved in a Philip M orris-f unded tobac co-budg et projec t in Belize, and c ollaborate d with Partners of A merica on two peanutdevelopment pr ojects with far mer c oopera tives in Haiti. The ca ndidate wr ote a g rant that the U .S. Age ncy for I nterna tional Deve lopment (USAI D) c oopera tively funded w ith Partners of Ame rica that trained f ive Haitian ag ricultural c oopera tive leade rs and se cure d incre ased pe anut proc essing e qu ipm e nt i mpa c tin g the liv e s o f m or e tha n 2 ,0 00 ind ivi du a ls. T h e c an d i d at e h as t wo ch i l d re n an d fo u r gr an d ch i l d re n at t h e t i m e o f t h i s wr i t i n g.


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Permanent Link: http://ufdc.ufl.edu/UFE0013608/00001

Material Information

Title: Leaf-Tissue Freeze-Tolerance Mechanisms in Bahiagrass (Paspalum notatum Fluegge)
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0013608:00001

Permanent Link: http://ufdc.ufl.edu/UFE0013608/00001

Material Information

Title: Leaf-Tissue Freeze-Tolerance Mechanisms in Bahiagrass (Paspalum notatum Fluegge)
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0013608:00001


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Full Text











LEAF-TISSUE FREEZE-TOLERANCE MECHANISMS IN BAHIAGRASS
(Paspalum notatum Fluegge)















By

JACQUE WILLIAM BREMAN


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2006
































Copyright 2006

by

Jacque William Breman



































To my parents, Janet and John Breman















ACKNOWLEDGMENTS

We all stand on the shoulders of those who precede us. We travel trails blazed by

others. I hope that this five-year effort provides support and information for others who

travel this trail of investigation.

Acknowledgments are important in recognizing those key people who made this

effort possible: The supervisory committee members, the graduate coordinator, family,

friends and staff.

The supervisory committee guides the student's learning and research project.

Each committee member leaves a legacy in the way the student is molded. A student's

standing in the academic world reflects the quality of the committee. I'd like to thank

Dr. Quesenberry, who taught me skills in cytology and plant breeding and shared his love

of teaching. Dr. Blount renewed my enthusiasm for field work and plant exploration.

Dr. Sinclair challenged me to think critically about results, as well as the need to quantify

and record all data for reflective thinking. Dr. Barnett taught me plant breeding

strategies to shorten the time needed to provide improved cultivars. Dr. Coleman taught

me the joy of research, the excitement of questing after new knowledge, and the deep

satisfaction of obtaining results. Dr. Coleman and I were truly a team on our projects

with animal- science applications.

A good graduate coordinator also provides guidance and mentorship. I was

fortunate to have an excellent graduate coordinator. Dr. Wofford taught me the

importance of understanding and quantifying the mode of trait inheritance to develop









efficient breeding programs. Additionally, Dr. Wofford opened my vision to larger

horizons during our visits. I hope we can continue those chats in the future.

Family is crucial for emotional support. My study was perhaps more taxing since

it was undertaken while working full time for the Florida Cooperative Extension Service.

Graduate school and independent research taxes the endurance of the soul when

experiments go awry, equipment fails, and examination and other deadlines loom

ominously. Continuing the effort unaided at night and on weekends was made possible

by family support. My brothers Jim and Jeff Breman have been there for me.

I want to especially thank and recognize my parents, John and Janet Breman.

Despite their age, they at times worked physically beside me when experiments had to be

prepared or completed. My parents were always available by phone when I needed an

emotional lifeline. Their emotional support and prayers were unwavering in spite of the

experienced darkness of the times. My parents deserve the highest respect and honor for

their dedication.

I thank my daughter, Laurel Schaafsma, and my son, Jonathan Breman, for their

respect, love, and emotional support. Their spouses, Keith Schaafsma and Jennifer

Breman, understood my need to find respite by spending time with my grandchildren

(Nathan Breman, Gavin Breman, and Alec Schaafsma).

Someone who has walked with me throughout the last portion of this project is

my fiance, Alicia. My best friend and my life companion-to-be has celebrated the

project's successes, grieved the failures, and steadfastly been there each step of the long

way regardless of the challenges. Without Alicia, it would have been difficult to gather

myself up from setbacks to continue toward completion. We both look forward to the









day when we can finally be together as husband and wife instead of 1,500 miles apart.

Alicia is my joy and happiness.

I have to recognize a friend who was also a staff member in the Animal Science

department. John Funk, biological scientist, is one of those special people who went

above and beyond his job to make sure the student's research was successful. John made

sure that equipment was working, materials were available, technique was proper, and

results were satisfactory before letting me work on my own. During the long night and

weekend hours, I knew I could count on John to check on me sometimes to make sure my

research was going well. John taught me the proper microtome technique which made

the anatomical study successful. In the process of the research work, I gained a friend for

whom I have so much respect.

Getting equipment to operate and developing a consistent technique are crucial to

obtaining consistent results. Two special people who helped me in the fatty acid analysis

work were Robert "Bob" Querns, Biological Scientist, and George Person, laboratory

technician. Bob was helpful with suggesting various procedures. George was a true

partner in reviewing current literature with me and comparing different procedures. He

made sure that the Hewlett-Packard gas chromatograph was working properly, which

produced data that had the lowest possible coefficient of variability. The three of us

celebrated the completion of the fatty acid experiments as happily as if we had been a

team working for years.

Richard Fethiere, coordinator of Research Programs in the Agronomy Forage

Evaluation Laboratory, was instrumental in letting me take priority so I could analyze

two years' worth of nitrogen and in vitro organic matter digestibility results. This









expedited the animal-science research projects with Dr. Coleman in time to present an

abstract and poster for the International Grasslands Congress in Ireland. Edwin "Ed"

Bowers, biologist at Brooksville Subtropical Agricultural Research Station, helped me

get through some of the near infrared scanning quirks of the software and equipment in a

most patient and supportive manner. This helped us achieve results in time for the

imposed deadlines.

Acknowledgments of graduate committee, family, friends and staff have only

covered the high points of this project. This is in no way meant to minimize or exclude

faculty, family members, friends, and staff who have been supportive and helpful in so

many ways. We are not islands. We do not stand alone. As we travel the course of our

days, we travel trails formed by others and hopefully leave signs along the way to make

the journey easier for others. I hope that this project helps others along the way. May I

help others as kindly as those who have graciously offered their assistance.















TABLE OF CONTENTS

page

ACKNOWLEDGMENTS .............................................. iv

LIST OF TABLES ............ ......................................... xi

LIST OF FIGURES ....................................... ........ xiv

ABSTRACT ......... ............................... .. .......... xv

CHAPTER

1 INTRODUCTION .......................................... 1

Plant Cold-injury and Cold-tolerance ......................... .......... 1
Chill-injury .................... ............................ 2
Freeze Stress ....................................... ............ 5
Freeze Process ............. ...................... ........... 5
Suggested Scheme ............................................. 7
Plant Freeze-tolerance Summary ................ .................. 15
Bahiagrass in the Southeast US ........................................ 16
Importance and Use ................ ................ ............ 16
Adaptation and Geographic Distribution ............................ 16
Calendar Forage Dry Matter Production ............................ 17
Commercial Cultivar Freeze-tolerance Variability ....................... 17
Potential Bahiagrass Leaf Freeze-tolerance Trait Mechanisms ................. 18
Anatomical ................... ............................. 18
Physiological ............. ...................... ........... 21
Genetic ............ .......................................... .21
Outline of the Research Plan .......................................... 23

2 LEAF-TISSUE FREEZE-TOLERANCE TRAIT DIVERSITY IN
BAHIAGRASS ............ ..................................27

Introduction ............ ........................................... 27
Leaf-tissue Freeze-tolerance ................ ........................ 28
Materials and Methods ................ ............................. 30
Leaf-tissue Freeze-tolerance Trait Screening Experiment 1 ................ 30
Leaf-tissue Freeze-tolerance Trait Screening Experiment 2 ................ 33










Leaf vs. Root Effects Experiment 3 ............... ................ 34
Results and Discussion .............. .................... ......... 35
Leaf-tissue Freeze-treatment Screening Experiment 1 .................... 35
Leaf-tissue Freeze-tolerance Screening Experiment 2 ..................... 41
Leaf vs. Root Effects Experiment 3 ............... ................ 43
Summary ............ ............................................ 45

3 ANATOMY RELATED TO LEAF-TISSUE FREEZE-TOLERANCE .......... 47

Introduction ............ ........................................... 47
Materials and Methods ................................................ 50
Initial Two-line Experiment ............... ...................... 50
Eight-line Experiment ..................................... ....... 53
Results and Discussion .............. .................... ......... 54
Initial Two-line Experiment ............... ...................... 54
Eight-line Experiment ..................................... ....... 57
Summary ............ ............................................ 61

4 PHYSIOLOGICAL MECHANISMS ASSOCIATED WITH LEAF-TISSUE
FREEZE-TOLERANCE .............................. ..... ........ 63

Introduction ............ ........................................... 63
Fatty Acid Composition ........................................... 66
Material and Methods .............. .................... .......... 71
Osmolality ............. ............................ .71
Fatty Acid Composition ........................................... 73
Results and Discussion .............. .................... ......... 76
Osmolality ........... .........................................76
Fatty Acid Composition ........................................... 77
Summary ............ ............................................. 82

5 GENETIC BEHAVIOR OF THE LEAF-TISSUE FREEZE-TOLERANCE
TRAIT ............ ...............................................83


Introduction ....................
Tetraploid Reproduction ..........
Diploid Reproduction ............
Material and Methods ...........
Seed Production From Clones ..
Emergence from Seed .........
D iallel ...................
Results and Discussion ...........
Seed Production from Clones ...
Emergence from Seed .........
Diallel 2004 ...............
Diallel 2005 ...............
Sum m ary ...................


. . . .. . . . 8 3
. . . . . . . . . 8 3
. . . .. . . . 8 4
. . . .. . . . 8 9
. . . . . . . . . 8 9
................................. 92
................................. 94
................................. 98
. . . . . . . . 9 8
. . . .. . . . 9 9
....................... ........ 101
.............................. 103
.............................. 109









6 CONCLUSIONS ...................................................112

Introduction ............ .......................................... 112
Summary of Objectives ................. ........................... 113
Range of Leaf-tissue Freeze-tolerance Trait Expression .................. 113
Confirmation of Leaf- or Root-tissue Freeze-Tolerance .................. 115
Anatomical Differences Between Freeze-Sensitive and Freeze-Tolerant
Lines ............ ........................................ 116
Physiological Differences Between Freeze-sensitive and Freeze-tolerant
Lines ..................................................... 117
Heredity and Mode of Gene Action of Leaf-tissue Freeze-tolerance ........ 120
Suggested Further Research ................ ....................... 121

REFERENCES ............. .......................................... 123

BIOGRAPHICAL SKETCH ............................................ 131















LIST OF TABLES

Table page

2-1 Canopy leaf-damage rating system shows percentage of canopy leaf-damage
and canopy green leaf ................. ............... ............ 32

2-2 Mean simple effects across 26 bahiagrass lines subjected to target temperature
treatments of progressively colder freezing events, LTFT screening
experiment 1 ............. ........................ ........... 36

2-3 Mean simple effects across all bahiagrass lines with actual temperature
treatments of progressively colder freezing events, canopy leaf-damage,
LTFT screening experiment 1 ................ ...................... 37

2-4 Progressively lower freezing temperature events effects on canopy freeze-
damage of selected sexual diploid and apomictic tetraploid lines, LTFT
screening experim ent 1 .............................. ..... ........ 38

2-5 Whole plant recovery ratings as a percent of control plants, 18 d after
treatment of selected sexual diploid and apomictic tetraploid lines by
progressively lower freezing temperature events .......................... 40

2-6 Canopy freeze-damage of selected sexual diploid and apomictic tetraploid
lines, as affected by a single freeze treatment (-60C) in an environmentally
controlled chamber .................................. ........... 42

2-7 Canopy freeze-damage of selected sexual diploid and apomictic tetraploid
lines while root system was kept above freezing (50C) February, 2002 ......... 44

2-8 Whole-plant recovery ratings as a percent of control plants after 18 day
recovery in greenhouse after freeze treatment (-3.20C) while root system was
kept above freezing (50C) February 2002 .............................. 45

3-1 Comparison of vessel diameters of tropical climbing plants compared to trees ... 49

3-2 Mean midrib xylem diameter across three leaf positions of two lines sampled
1 January 2003 .................................... .. ........... 55

3-3 Mean midrib xylem diameter of three leaf positions across two lines (FL9,
FL67) varying in LTFT, sampled 1 January 2003 ......................... 55









3-4 Mean simple effects of vessel and vascular bundle diameters of four freeze-
tolerant vs. four freeze-sensitive bahiagrass clones sampled 1 February 2003 .... 57

3-5 Analysis of variance comparing mean vessel and vascular bundle area simple
effects of four high- vs. four low- leaf-tissue freeze-tolerant clones sampled 1
February 2003 ............. ....................... ........... 58

3-6 Mean midrib abaxial (facing the bottom side of the leaf) xylem and vascular
bundle parameters from a February 2003 sampling date .................... 59

4-1 Fatty acid composition of grazed orchard grass (Table from Loor et al., 2002) ... 69

4-2 Osmolality of bahiagrass sexual diploid lines representative of freeze-
tolerance and freeze sensitivity ................. ................. ... 77

4-3 Bahiagrass leaf blade fatty acids, as a fraction of total extracted fatty acids ..... 78

4-4 Leaf blade double bond index (DBI) and unsaturated fatty acid: saturated fatty
acid ratio ............... .............................81

5-1 Germination test via emergence of 100 seed scarified with concentrated
sulphuric acid for 15 minutes, 23 June 2004 ......................... 93

5-2 Crosses and self-pollinations made August 2003 through December 2003 of
freeze-tolerant, intermediate, and freeze-sensitive bahiagrass clones ........... 99

5-3 Percent emergence of crosses and self-pollinations made August 2003 through
December 2003 of freeze-tolerant, intermediate, and freeze-sensitive
bahiagrass clones .................................. .......... 100

5-4 Mean emergence of cross F,'s from nine clones varying in the leaf freeze-
tolerance trait and self-pollination progeny of nine clones compared to an
open-pollinated standard ............... ......................... 101

5-5 Parent clone canopy freeze-damage ratings used to develop a diallel cross
rated after a freeze event 17 December 2004 ............................ 102

5-6 Mean canopy leaf freeze-damage ratings (1 to 9) of progeny rated after a
freeze event (17 December 2004) as a result of female parent and pollen
source ............ ............................................. 102

5-7 Analysis of variance of canopy damage ratings made after 17 December 2004
freeze using the fixed model Griffing's Method 3, and 6 bahiagrass clones
varying in LTFT trait, and their progeny ............................. 104









5-8 Parent clone canopy freeze-damage ratings used to develop a diallel cross
rated after a 22 December 2005 freeze ................................. 105

5-9 Progeny canopy freeze-damage ratings (0 to 100%) rated after 22 December
2005 as a result of female parent and pollen source ....................... 106

5-10 LTFT phenotype effects partitioned into their variance sources from a fixed
six parent diallel mating design analyzed with Griffings Method Three ....... 107















LIST OF FIGURES


Figure page

1-1 Chill-injury symptom response of chill-sensitive plants leading to reversible
or irreversible cell and plant injury ................ ................... 3

1-2 Freeze-injury symptom response of plants leading to reversible or irreversible
cell and plant injury ................. ............................ 8

2-1 Water-bath experiment showing tubs, heaters, and weighted pots ............. 35

3-1 FL67 bahiagrass (freeze-tolerant) section showing bundle sheath, girder
system of sclerenchymous tissue supporting the vascular bundle ............. 56

3-2 FL9 (freeze-sensitive) section showing larger vascular bundle area than FL67 ... 56

3-3 Freeze-sensitive line within hours of being placed in full sunlight after a
controlled freeze event, 10 h at -60C, shows damaged midrib regions initially ... 60

4-1 Relationship of osmolality, freezing temperature, osmotic pressure, and
relative humidity of an aqueous solution with a pure solute .................. 65















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

LEAF-TISSUE FREEZE-TOLERANCE MECHANISM IN BAHIAGRASS
(Paspalum notatum Fluegge)

By

JACQUE WILLIAM BREMAN

May 2006

Chair: Ann R. Blount
Cochair: Kenneth H. Quesenberry
Major Department: Agronomy

Bahiagrass (Paspalum notatum Fluegge) is the forage base that Florida livestock

producers depend on. Leaf-tissue freeze-tolerance (LTFT) is a trait found in some

bahiagrass plants after a freeze event. Understanding the LTFT mechanism could help

expand Florida's forage base into the winter months to financially benefit a $1 billion-

plus industry that sustains an estimated 18,076 jobs. A need arose to find the mechanism

that allowed plants with LTFT to survive freeze events.

The range of the LTFT trait expression was quantified using controlled freezing

trials. Eight clones were selected for anatomical study. Four clones that were freeze-

tolerant and four clones that were freeze-sensitive were compared and contrasted for

anatomical and physiological differences.

Leaf xylem diameter was an indicator of LTFT. The lines with freeze-tolerance

had xylem diameters ranging from 157(Jm to 187 jam. The lines that were freeze-

sensitive had xylem diameters ranging from 209 jam to 242 jam.









Leaf osmolality was not a mechanism involved in high-LTFT clones. Clones

with freeze-tolerance ranged from 545 to 817 mmol kg-'. Clones that were freeze-

sensitive ranged from 489 to 859 mmol kg'.

Leaf fatty acid (FA) composition was not a mechanism involved in LTFT. Seven

FAs (C14:0 myristic, C16:0 palmitic, C16:1 palmitoleic, C18:0 stearic, C18:1 oleic,

C18:2 linoleic, C18:3 linolenic) accounted for the major composition in the bahiagrass

lines tested. Leaf fatty acid composition was not a mechanism involved in high-LTFT

clones. Unsaturated FA profile, unsaturated FA:saturated FA, and double bond index

were contrasted for nine clones that varied in LTFT. None of the FA parameters could

predict bahiagrass LTFT.

The LTFT heritability was calculated from a diallel mating design using

Griffing's fixed model for Method 3. The LTFT heritability was low (H2 = 25%, h2 =

8%). Dominant gene action accounted for most LTFT trait-expression heritability.















CHAPTER 1
INTRODUCTION

Plant Cold-injury and Cold-tolerance

Florida agriculture and natural-resource industries in 1997 accounted for $31.4

billion in sales, $18.2 billion in exports, $12.3 billion in value-added revenue, and

314,000 jobs (Hodges et al., 2000). Erratic and unpredictable freeze and above-freezing

events have historically damaged crops as far south on peninsular Florida as Miami-Dade

County. Millions of dollars of crop losses have resulted from these freeze events. Entire

agricultural industries have been changed or moved as a result of severe freeze events.

The Florida citrus industry permanently moved to the southern peninsula to escape freeze

events that could not be managed using traditional wind machines, grove heaters or

sprinkler irrigation (Crocker, personal communication, 2000). The ornamental,

transplant, and hydroponics industry in Florida has used overhead sprinkler irrigation, as

well as covered or heated greenhouses, to protect their high-value crops (Hochmuth,

personal communication, 2005). Anything that puts agriculture of this economic scale at

risk is something that needs to be studied and managed. Freeze-temperature tolerance in

crop plants needs to be studied so crop damage and loss risk can be reduced.

The Florida forage-based livestock industry (dairy, horses, beef cattle, sheep and

goats) accounted for $1.009 billion in sales and 18,076 jobs in 1997 (Hodges et al.,

2000). For the Florida livestock industry to remain economically viable, it must grow the

bulk of the required forages within the state. Forages in Florida have economic value in







2

addition to forming the feed foundation for the forage-fed livestock industry. Florida hay

and pasture outputs were estimated at an additional $55.9 million, creating an additional

2,910 jobs in 1997.

Bahiagrass (Paspalum notatum Fluegge) is the pasture base livestock producers

depend on during the warmer months, on an estimated 2.5 million acres in Florida

(Chambliss, 2002). Livestock producers supplement, during the winter months, with hay

from north-central Florida northwards as a direct result of frost injury to bahiagrass

pastures. Frost injury has occurred on bahiagrass as far south as Lake Okeechobee

(Mislevy, 2005, personal communication). Alternative small-grain winter forages

planted from north-central Florida throughout the panhandle have been historically

damaged from severe freeze events. Small grain winter forages are costly to sow. They

are also at risk from freeze and drought (Wright et al., 2005) because they produce forage

December through April, while bahiagrass is not available for grazing. Conserved

forages are an important and costly requirement for Florida livestock producers from

Lake Okeechobee through peninsular Florida and encompassing the panhandle region.

Freeze-injury during cool winter months puts bahiagrass at risk in Florida. Because of

this risk factor it is important to understand plant-leaf freeze-injury as it pertains to

forage production.

Chill-injury

Because some plant leaf chill-injury symptoms physically resemble leaf freeze-

injury symptoms, understanding the main mechanisms involved in plant-leaf chill-injury

may provide insight into plant freeze-injury and its avoidance or tolerance. Chill-injury

and plant tolerance of that injury can be a subject of confusion in the literature. There is








3

a need to define some terms used in the literature and review basic mechanisms that

apply to cold injury and plant cold-tolerance.

Chill-injury is foliar, and whole-plant damage, as a result of temperatures above

0C and below some threshold temperature unique for that species and even for a specific

genotype. Freeze-injury is plant damage at temperatures below 0C or when radiative

frosts occur with ice formation (defined later).

Chill-injury can be conceptualized as the plant responding with increasing

damage to cold stress as ambient temperature approaches 0C, and depending on the

duration of that stress (Figure 1-1). Damage to bahiagrass foliage from chill stress has

not been documented. However, there may be mechanism continuums that may apply to

further our understanding of freeze-injury in bahiagrass.


200C




Chill
stress
Critical
.......................................................................................................................................................... ............................................................................... ........... te m p e r a tu r e
temperature


Reversible Not
reversible



0oC
0oc ----------------------------

Time



Figure 1-1. Chill-injury symptom response of chill-sensitive plants leading to reversible
or irreversible cell and plant injury (Adapted from Nilsen and Orcutt, 1996)

Temperature ranges where damage to chill-sensitive plants occurs vary. Values

such as from 12 to 0C (Buchanan et al., 2000.) and from to 200 to 0C (Lyons et al.,







4

1979; Hudak and Salaj, 1999) have been reported. In contrast, chill-resistant plants can

tolerate chilling temperatures without irreversible injury. Chill-sensitive plants tend to be

of tropical or subtropical origin or have the vegetative portion of their life cycle only

during the warmest portion of the year (Guy, 2003). Chill-sensitive plants comprise

many major field crops, such as cotton, soybean, maize, and rice. Chill-sensitive plants

rarely survive a freeze event in which ice forms in the tissue. Symptoms of chilling injury

depend on chill-stress severity (proximity of temperature to 0C) and duration of the chill

stress temperature (Nilsen and Orcutt, 1996). Chill stress severity and duration (Figure

1-1) may determine plant damage. Chill stress may determine whether plant damage is

reversible. Chill stress symptoms may include

* Changes in membrane structure and composition
* Decreased protoplasmic streaming
* Electrolytic leakage
* Plasmolysis
* Increased or reduced respiration (depending on severity of chill stress)
* Production of abnormal metabolites
* Reduced plant growth
* Surface lesions on leaves and fruit
* Abnormal curling, lobbing, and crinkling of leaves
* Water soaking of tissues
* Cracking, splitting, and dieback of terminal growth
* Rapid leaf wilting followed by water-soaked patches that develop into sunken pits
of collapsed tissue, that usually dry up on warming, leaving necrotic areas of leaf
tissue
* Plant death

Perturbation of the membrane may be the first sign of chill-injury (Lyons et al.,

1979). Whether or not the plant has sufficient time to become acclimated to chilling

temperature affects the severity of the damage (Buchanan et al., 2000). Acclimation

resembles lowering the critical temperature for a plant to experience injury symptoms.







5

Acclimation signals can include shortening day-length, cooler temperatures (Tomashow,

1999), changes in nutrition, water relation, and stage of growth (Buchanan et al., 2000).

Freeze Stress

Freeze stress is similar to chill stress, but the temperatures are below 0C and may

include the presence of ice (Nilsen and Orcutt, 1996). Ice formation may not always

occur at temperatures below 0C, but if formed, ice is formed outside the cell membrane,

in the apoplast.

Freeze Process

The freeze process includes dehydration stress to the living cells in the symplast.

Because the osmotic potential of ice is lower than that of water, cell water exits the cell

toward the growing ice crystal. As the freeze process continues, the cell dehydrates, cell

volume is reduced, and the osmotic concentration in the cell increases (Rajashekar,

2000). The extra cellular and intracellular freezing process and injury to plant cells have

been largely described and visualized by microscopy (Asachina, 1978).

Infrared thermography has been used to visualize and record the freezing process.

When water freezes, heat is given off from the phase change from water to ice. Infrared

photography (thermography) have been used to obtain real-time data. From data

gathered from thermography, supercooling as well as the freezing temperature of the

plant tissue can be identified (Taiz and Geiger, 2002).

The actual process of ice nucleation on leaf blades of various species has been

visualized and recorded with infrared thermography (Wisniewski et al., 1997). Infrared

thermography recorded freezing temperatures of -1.5 to -2.10C for three species of

barley (Hordeum sp.) (Pearce and Fuller, 2001) under natural freeze conditions. The

order of freezing, for up-rooted plants placed in a controlled environment chamber,







6

occurred first for a droplet of water placed on the barley leaf blade, which froze and acted

as the ice nucleation site. Sequential and successive infrared thermography of the

nucleated leaf blade showed the midrib xylem vessel region froze from the site of

external ice nucleation. Freezing of the barley leaf progressed down the entire midrib

xylem vessel region. Once the entire midrib vessel region was frozen the remainder of

the leaf blade froze. The next organ that froze was the exposed roots, followed by older

leaves, then younger leaves, and finally the secondary tillers. The freezing order thus

followed ice nucleation outside of living plant, followed by leaf invasion of the apoplast

through the xylem, then the mesophyll of the lamina. In potted Lolium perenne L. and

Poa supine Schrad. in controlled environmental chamber, the order of freezing recorded

by infrared thermography was from the roots to crown through connective tissue,

followed by freezing of the shoots and leaves (Stier et al., 2003).

Nucleation is one of the variables in determining at what temperature plants

freeze. Plant ice nucleators can be divided into two classes (Pearce, 2001a): extrinsic

and intrinsic. Extrinsic nucleators are outside of the plant which aid ice formation and

are also referred to as heterogeneous nucleators. Heterogeneous nucleators are

substances that catalyze the formation of a stable ice nucleus. When water molecules

come together spontaneously to form a stable ice nucleus the term homogeneous

nucleation is used. Extrinsic heterogeneous nucleators (Lal and Lal, 1990) can be water

droplets, dust particles, ice nucleation-active (INA) bacteria, or wind agitation. Intrinsic

nucleators are substances within the cell which catalyze ice formation. An example of

intrinsic nucleators might be those found in rye (Secale cereale L.) cells (Brush et al.,

1994). In rye, intrinsic nucleators were found to be complexes of proteins,







7

carbohydrates, and phospholipids in which both disulfide bonds and free sulfhydrol

groups were important for nucleating activity.

Suggested Scheme

Freeze-injury can occur to freeze-sensitive plants at or just below 0C when ice

nucleation occurs. Freeze-tolerant plants can withstand freezing at temperatures -3C

lower than freeze-sensitive plants. Freeze-tolerant plants will thaw, rehydrate and

function normally by various mechanisms. There is a need to organize plant response

and published mechanisms of freeze-tolerance.

Many schemes have been used in attempts, to integrate the various mechanisms

of freeze-avoidance and freeze-tolerance with observed plant responses and biochemical

adjustments. Earlier attempts such as those referenced (Levitt, 1978) did not have the

current body of knowledge available from plant molecular and biochemical science (Guy,

2003). A scheme for classifying whether plants are freeze-sensitive or freeze-tolerant

was proposed by Sakai and Larcher (1987). The concept of a continuum in plant injury

in response to stress duration and intensity was proposed by Nilsen and Orcutt (1996) and

illustrated previously (Figure 1-1). These concepts can be integrated with established

freeze-stress mechanisms as shown in Figure 1-2. The proposed scheme of plant

tolerance to freeze-stress can be useful in organizing the literature. Figure 1-2 can

helpful in classifying plants based on freeze intensity and duration. helpful in classifying

plants based on freeze intensity and duration.

This conceptual scheme would classify plants as freeze-sensitive if they were

damaged by shallow freezes down to -30C. Freeze-tolerant plants would be those which

could withstand freezing temperatures below -3C. Both shallow and deep freezes occur







8

in peninsular Florida. Bahiagrass genotypes with freeze-tolerance may have more than

one protective mechanism.


0 C Freeze- Heterogeneous nucleation
sensitive

-30 C ..................................................................................................................
-3C
Freeze- Heterogeneous nucleation after
-10o C tolerant supercooling due to solute
accumulation

Homogenous nucleation


Dehydration tolerance +
-50oC Membrane fatty acids +
Cryoprotectants



Time

Figure 1-2. Freeze-injury symptom response of plants leading to reversible or
irreversible cell and plant injury (Conceptualized by the author based on
data from Sakai and Larcher, 1987; Nilsen and Orcutt, 1996; Guy, 2003)

One protective mechanism postulated (Levitt, 1978; Pearce, 2001) for shallow

freezes (-1 to -30C) has been freezing-point depression of cell sap by heterogeneous ice

nucleation after transient supercooling. Therefore, freeze-tolerant bahiagrass genotypes

may be transiently protected by supercooling during short freeze events. Supercooling is

simply defined as water or a solution below the equilibrium freezing point of water

(00 to -10C) and above the homogeneous nucleation temperature of water (-40 to -410C)

(Chen et al., 1995). Supercooling is a mechanism that allows plants to avoid what could

be lethal intracellular freezing by reducing the freezing point of the cell solution (Hudak,

J. and J. Salaj, 1999). Supercooling is an unstable thermodynamic situation when a

liquid solution is not in phase equilibrium with the solid ice phase. The process of







9

conversion from an unstable state, such as supercooled liquid, to a stable state such as a

frozen phase, is initiated by nucleation (Vali, 1995). Nucleation occurs when a small

volume of the new ice phase occurs. Nucleation is followed by growth of the new

thermodynamically stable ice phase. Growth of the stable ice phase is controlled by the

latent heat of water. The system experiences equilibrium when the rate of ice formation

equals the rate of ice melting, and the rate of water vaporization equals the rate of water

vapor condensation. Palta and Weiss (1993) suggest that herbaceous plants nucleate as

a result of bacterial flora on the surface of their leaves at temperatures between -5

and 0C. Bacterial flora could confound supercooling effects.

Factors affecting ice formation include the presence of intrinsic ice nucleators and

extrinsic ice nucleators (dust, bacteria, fungi, wind agitation, dew or ice crystals) (Lal and

Lal, 1990; Palta and Weiss, 1993; Vali, 1995). Factors affecting ice growth rate include

cooling rate, cell wall porosity and membrane porosity. Freeze-tolerant plants can

prevent protoplast injury by using extrinsice nucleators to form extra-cellular ice in the

apoplast (cell wall space, intercellular space, and xylem). Protoplast injury at

temperatures between -5 and 0C, can be prevented by forming ice crystals with

intrinsic ice nucleators. Freeze-tolerant plants lower their freezing temperature by

accumulating solutes during deeper freezes (-3 to -100C). Freeze-tolerant plants may

experience transient supercooling after heterogeneous ice nucleation even though their

freezing point has been depressed by accumulating solutes.

Under shallow freezing temperature conditions (to -30C), supercooling may have

an adaptive advantage. Even though eventual ice nucleation may occur with shallow

freezing, the speed of ice crystal growth may not be rapid enough to cause protoplast

damage. Under colder temperature regimes, supercooling can cause more plant tissue







10

damage once heterogeneous ice nucleation occurs due to the high rate of freezing (Wolfe

and Bryant, 2001). This may be the case for herbaceous tissue freeze-injury from rapid

tissue freezing, but the opposite case may be made for woody tissue where deep

supercooling is a mechanism of deep freeze-tolerance (Wisniewski, 1995; Quamme,

1995).

Plant osmoregulation can be done by two methods. Metabolic synthesis of

solutes such as sugars and other osmotica can occur during the acclimation process.

Water can be moved from one tissue to another in a process that dehydrates the tissue,

increases the osmotic concentration and depresses the freezing point. The mechanism of

osmotically reducing the freezing point of cell sap by increasing solute concentration has

been summarized by Sutcliffe (1977). The temperature at which a solution of cell sap

freezes (T,), has been related to osmotic potential (y, in kPa) (Sutcliffe, 1977).

Tf= y/12.2

If plant sap osmotic potential ranges between -3000 to -4000 kPa then freezing

depression as a strict result of osmotic potential would range from -2.5 to -3.3C. This

is not much protection. The actual osmotic potential of the tissue can be determined

experimentally by the freezing point-depression method using thermocouples and

extracted plant sap using the Van't Hoff formula, then correcting for supercooling and

room temperature (Saupe, 2004). An even more accurate calculation can be determined

if the solute composition can be determined and tabular solute values could be used.

Thermodynamic and mechanical effects have been integrated into a scale which relates

the hydraulic pressure of water in Pascals, the equilibrium freezing temperature,

osmolality and the relative humidity of water at that pressure, temperature and osmolality

(Wolfe and Bryant, 2001). However, freezing point-depression from increased osmotic







11

concentration and subsequently more negative osmotic potential is only for a few

degrees, as calculated above.

Freeze stress-induced cell dehydration happens once ice nucleation occurs at

colder temperatures than can be protected by osmotic concentration and temporary

supercooling. Other plant mechanisms must protect the plant from desiccation. Cell

dehydration progresses when water leaves the cell through the cell membrane to the

apoplast where the ice crystal continues to grow. The ice crystal will continue to grow

until the ice phase comes to thermodynamic equilibrium with the liquid and gas water

phases within the symplast + apoplast system. At progressively colder below-freezing

temperatures, mechanisms that prevent cell dehydration become increasingly important.

Membrane integrity is important in preventing cell dehydration. Membrane

integrity is damaged when freezing-induced dehydration occurs (Pearce, 2001).

Membrane flexibility becomes an important factor in determining whether or not

membrane integrity will occur as the cell volume contracts through loss of water, and

then re-hydrates on thawing to full turgor pressure (Buchanan et al., 2000). Research in

rye protoplast showed freezing to below -10C without acclimation caused membrane

failure and ice nucleation that invaded the protoplast (Steponkus et al., 1981). It was

membrane failure that preceded invasion of the protoplast with ice. In non-acclimated

protoplasts, intracellular ice formation was strongly dependent on the cooling rate within

the range of -5 to -30C. The greater the cooling rate the more intracellular ice was

formed. Under slow-cooling rate experiments, rye protoplast injury resulted from

contraction during cold-stress treatment and lyses on warming. Microscopic

investigations of the plasma lemma have correlated deletion of the membrane into







12

vesicles as the cell volume is reduced through dehydration during slow freezing (Hudak

and Salaj, 1999).

Plasmalemma inflexibility and deletion may be related to lower flexibility of

membranes with higher concentrations of saturated fatty acids. Seedling wheat cultivars

acclimated at low, above-freezing temperatures, showed an increase in a- and y-linolenic

fatty acid as a percent of the membrane composition. These cultivars had lower lethal

freeze temperatures (as much as 100C lower for some cultivars) than unacclimated

seedlings (St. John, 1979). Cold acclimation duration increased the membrane ratio of

polyunsaturated fatty acids (C18:3/C18:2) from 0.37 to 0.86. Species in which cold-

acclimated plasma membranes have the highest ratio of di-unsaturated phospholipids and

lowest proportion of glucocerebrosides tend to be the most cold-hardy (Buchanan et al.,

2000).

Membrane fluidity may be the result of the membrane fatty acid (FA)

composition. Fatty acid composition can change the temperature that membrane phase

changes occur (Buchanan et al., 2000). The more unsaturated the FA content the lower

the temperature the membrane remains in the liquid-crystal phase. Shifting to the gel

stage has been associated with more saturated FA content and membrane damage at

warmer temperatures. Phase transition temperatures of gel to liquid of

phosphatidylcholine (PC) species which differed by the FA group ranged from 55C for

disteroyl-PC (16:0-PC) to -190C for dioleoyl-PC (18:1-PC) (Palta and Weiss, 1993).

Cool-temperature acclimated potato (Solanum commersonii Dun.) plasma membrane

extraction showed a 28.5% increase in 18:2 FA and a 10.1% decrease in 16:0 FA. An

increase in plasmalemma ATPase activity and an additional 50C freeze tolerance

(from -4 to -90C) was the result of cool-temperature acclimation.







13

As long as the membrane remains in the liquid-crystal stage, the cell can function

normally, albeit at a slower rate as temperatures decrease. Once the membrane gel phase

occurs, membrane functions, such as osmoregulation and translocation, become impaired

when crystal patches of membrane form, and membrane permeability increases

(Buchanan et al., 2000). Plants have desaturase enzymes, which can change the position

of the double bonds on a fatty acid chain. Desaturases are activated for normal

membrane FA turnover. After acclimation signals (cool temperatures, short days, etc.)

are experienced, desaturases are up-regulated with the net effect of an increase in

unsaturated fatty acid composition of the membrane PC. The actual mechanism can

simply be moving the location of the double bond on the FA carbon chain. For example,

moving the double bond from the second and third carbon position (melting

point = 400C) on an 18-carbon chain FA (C,,) to between the ninth and the tenth carbon

position (melting point = -200C) can change the melting temperature or phase shift 600C.

This mechanism would impact freeze-tolerance. The longer the membrane would remain

functional and in the liquid-crystalline phase, the longer it could remain freeze-tolerant.

Once the phase shift occurred from liquid-crystalline to a gel at a temperature

below -0C, if apoplastic ice existed, membranes would allow rapid ice crystal growth

leading to increased dehydration stress and eventual cell damage. Gel phase membranes

may also be more prone to shearing and forming endocytic vesicles, which decrease

membrane material during cell volume reduction during freeze events, as mentioned

previously. Upon thawing and attempting to rehydrate the integrity of the cell, the

membrane is compromised and cell death occurs.

The role of acclimation in preparing the plasmalemma for freeze-stress has been

mentioned. Chemical and structural changes in the plasmalemma which help resist







14

freeze dehydration, mechanical stress, molecular packing, and other freeze events, can be

induced by temperatures as low as -30C in Robinia pseudacacia L. Duration of

acclimation temperature tends to increase freeze-tolerance (Hudak and Salaj, 1999).

Hardened (chemical and structural changes that occur through acclimation) plasma

membrane becomes highly folded in some species, invaginated in others, and associated

with polyphenolic anti-freeze compounds in still other species. Changes in acclimated

plasma membrane are assumed to aid in plasma membrane integrity under severe freeze-

stress.

Protein metabolism is important in cold acclimation and freeze-tolerance, but

perhaps not for the historical reason Levitt had postulated (Guy and Carter, 1982).

Glutathione was thought to protect membrane protein sulfhydrol groups during freeze

stress thereby preventing protein denaturing, which was believed to lead to cell freeze-

damage. High levels of glutathione had been found to be correlated with acclimated

freeze hardiness in several plant species. The protein metabolism that may facilitate

understanding plant cold acclimation and acclimated plant ability to withstand more

severe freeze stress is the genetic up-regulation and down-regulation of enzymatic

proteins controlling cell processes (Guy, 1990). A partial listing of low temperature

responsive genes affected processes which included respiration, carbohydrate

metabolism, lipid metabolism, phenylpropanoid metabolism, antioxidant metabolism,

regulatory enzymes in ten different plants, and ten functionally unknown proteins (Guy et

al., 1994). Spinach plants acclimated to low temperature compared to control and

drought-stressed plants at low temperature showed gene products that were expressed

either to low temperature acclimation or to drought-stress. Freezing tolerance was

enhanced when these proteins accumulated in the leaf. Since part of freeze-stress







15

tolerance is tolerance to desiccation these results support previous literature reviewed.

The overlapping and quantitative nature of genes involved in freeze-stress tolerance has

begun to be substantiated with microarray results using Arabidopsis thaliana (L.) Heynh

(Thomashow, 1999; Wanner and Junttila, 1999). Perhaps as many as 1,000 to 7,000

genes in the entire Arabidopsis genome of 28,000 to 30,000 genes may be involved in

freezing stress tolerance (Guy, 2003).

Freeze-tolerance acclimation may be induced by short days, cool temperatures,

application of abscisic acid (ABA) drought-stress, potassium fertilization and

occasionally phosphorous fertilization (McKersie and Leshem, 1994). Acclimated plant

generalizations include increased osmotic concentration, decreased tissue water content,

increased starch and protein concentration, lipid accumulation, fatty acid unsaturation,

increased soluble protein content, increased mRNA, increased polysomes, increased

tRNA and increased ABA decreased respiration.

Some freeze-tolerant plants can also limit the growth of apoplastic ice crystals

with proteins and polysaccharides, limiting the extent of protoplast dehydration from ice

crystal growth. In rye the development of freeze-tolerance to -300C by acclimation was

accompanied by a tenfold increase in protein extracted from the extracytoplasmic regions

of the leaves (Griffith et al., 1993). Photomicrography of protein extracts subjected to

low temperatures showed ice crystals were nucleated at temperatures approaching 0C

and then the ice crystal growth was restricted.

Plant Freeze-tolerance Summary

Understanding cold temperature tolerance mechanisms can be helpful in

developing freeze resistant crop plants. Genotype differences in plant chill or freeze-

tolerance have been shown to be related to many factors. Changes in water relations,







16

solute accumulations, membrane fatty acid composition and numerous metabolic events

and path ways are under genetic control. Thus, breeding and selection for chill or freeze

resistance appears to be a realistic objective. Plant breeders and molecular geneticists

could utilize this increasing body of information to develop chill and freeze-tolerant crop

plants.

Bahiagrass in the Southeast US

Importance and Use

Bahiagrass is the major pasture grass used by the livestock industry in Florida as

well as the Southern Coastal Plain soils of Georgia and Alabama (Blount et al., 2001). In

Florida alone, bahiagrass is estimated to cover over 1 million hectares (Chambliss, 2000).

Bahiagrass uses include pasture, hay, sod and seed.

Adaptation and Geographic Distribution

Bahiagrass is adapted to sandy soils in warm, humid tropical and warm temperate

regions. This grass tolerates soils with low fertility, low pH and persists under

continuous stocking (Gates et al., 2004). Bahiagrass can survive on drought soils and

soils with intermittent flooding. It was introduced in Florida as an improved forage and

has become naturalized (established as part of the local flora) throughout the Southern

Coastal Plain and the Gulf Coast of the Southern USA. 'Pensacola', a more cold-hardy

diploid cultivar has been found growing as far north as southern Oklahoma and in

Virginia and grows from Texas to North Carolina, extending into Arkansas and

Tennessee. Freeze-tolerance apparently limits the range of bahiagrass to those regions

where brief and shallow below-freezing temperatures are experienced.









Calendar Forage Dry Matter Production

The bahiagrass growing season in the southeastern United States is from April to

October. The growing season becomes shorter moving from the Coastal Plain to the

Piedmont (Gates et al., 2004). Forage growth distribution throughout the year appears to

be influenced by temperature and photoperiod with more dry matter (DM) production

during the warmer season, longer daylengths and at lower latitudes. Twice as much

forage was clipped from Pensacola bahiagrass plots in July compared to October at 320 N

in the southeastern United States. In southeast Brazil (220 45'S) the general trend was

for 90% of the total annual DM accumulation to occur during the warm "summer" half of

the year and 10% during the "winter" half. Shortening daylengths have been shown to

reduce bahiagrass growth even when temperatures were warm. Extending daylength

with artificial lighting resulted in increased bahiagrass growth (Sinclair et al., 2001;

Sinclair et al., 2003).

Commercial Cultivar Freeze-tolerance Variability

As early as 1942 leaf tissue tolerance to frost and freezing was recognized as an

important trait ofbahiagrass cultivars (Burton, 1946). A scale of 1 to 5 with 1 = most

frost tolerant and 5 = most frost sensitive was used to rate four bahiagrass cultivars after

a freeze event of -5C. 'Common' tetraploid (4x) was rated 5, 'Wallace' (4x) was rated

3, 'Paraguay' (4x) was rated 2, 'Wilmington' (4x) was rated 2 and 'Pensacola' diploid

(2x) was rated 1. 'Tifton 9' is a diploid cultivar, that is considered to be cold-tolerant.

Tifton 9 is a Georgia release developed from a collection of Pensacola seed collected

from 16 farms and subjected to nine years of restricted recurrent phenotypic selection

(RRPS) (Gates et al., 2004).







18

Potential Bahiagrass Leaf Freeze-tolerance Trait Mechanisms

Anatomical

Freeze-stress produces osmotic stress. Severe osmotic stress during freeze events

followed by immediately high-transpiration demands in Florida field conditions might be

minimized by leaf desiccation protection. Leaf desiccation protection could be enhanced

by a thicker wax layer. If leaf wax would prevent leaf desiccation under severe

temperature stress (whether hot or cold) then leaf wax would be worthy of study.

However, 'Pensacola' bahiagrass had the least solute leakage (14.6% -15.4%) of 10

Paspalum species exposed to heat stress of 540C yet the third lowest leaf epicuticular

wax content (1.35 to 1.79 mg wax dm2 (Tischler and Burson, 1995; Tischler et al.,

1990). Therefore, leaf epicuticular wax content did not appear worthy of investigation.

Ice has been shown to form, travel and spread throughout barley (Hordeum sp.)

through the leaf midrib first, then throughout the leaf (Wisniewski et al., 1997; Pearce

and Fuller, 2001). This indicates the leaf midrib xylem is the region of initial leaf ice

growth which eventually invades the entire leaf. Xylem diameter of temperate plants is

significantly less than for tropical plants (Haberlandt, 1914).

Internal leaf anatomy may be related to differences in freeze-tolerance.

Evergreen leaves with narrow intercellular spaces and/or small mesophyll cells were

reported to have ice nucleation temperatures down to between -10 to -120C (Sakai and

Larcher, 1987). Large xylem diameters have been shown to be related to cavitations and

air embolisms caused by freezing and subsequent thawing in woody plant species (Davis

et al., 1999). Xylem cell wall porosity and permeability has been related to deep super

cooling in some woody plant species (Wisniewski et al., 1991; Wisniewski, 1995). In

woody temperate plants a relationship has been shown between xylem diameter and







19

cavitations caused by freezing (Davis et al., 1999). When xylem solution freezes,

dissolved gases in the liquid phase are released as bubbles in the ice phase. When

thawing, if air bubbles are small they may dissolve back into the liquid phase. If bubbles

coalesce and become large enough to block the xylem conduit flow they can cause

cavitation of the liquid columnar flow. Cavitation has been confirmed with cryo-

scanning electron microscopy to occur during thawing in Fraxinus mandshurica Rupr.

(Utsumi et al., 1999). Once cavitation occurs embolism follows. A xylem embolism

becomes filled with atmospheric air and water vapor and disrupts translocation (Davis et

al., 1999). In some hardwood trees (Betula platyphylla Sukatschev. and Salix

sachalinensis Fr. Schm.), vessels recover before spring growth with water refill,

presumably from root pressure (Utsumi et al., 1999). In some deciduous hardwoods,

embolism is permanent and new spring growth provides functional and filled xylem

vessels (Davis et al., 1999). Xylem transport and the negative pressures sustained for

cavitation of water was reviewed and expanded with an experiment that compared

approximations of xylem sap with Z-tube materials varying in wetting surface and

increasing pressure (Smith, 1994). Approximations of biological pressures, where xylem

cavitation would occur, ranged between -0.1 and -0.6 MPa.

Xylem structure and susceptibility to cavitation by freezing experiments have

been done under controlled pressure that mimics drought-stress (Davis et al., 1999).

Cavitation would theoretically occur depending on xylem pressure (Px) after a thaw. The

relationship of Px, the vapor pressure of water (Pwv) in the embolism air bubble, the

surface tension of the xylem sap (t), and the air bubble radius (r) is shown in the

following equation:


Px < Pwv-(2t/r)







20

Empirically, this relationship can be quantified by controlling Px. Mean xylem

diameter and percent loss of xylem conductivity in 12 temperate hardwood species was

quantified with a freeze-thaw experiment with a constant xylem pressure of -0.5 MPa.

Davis et al. (1999) found that upon thawing, a 30 (im mean diameter threshold for 12

woody species existed above which cavitation would occur. This cavitation resulted in

7.2% loss of conductivity upon thawing. Species with diameters greater than 40 (im had

as much as 95% loss of conductivity after a freeze-thaw cycle. Percent loss of

conductivity was shown to be a Weibull function of mean species vessel diameter. From

the data of 12 species a 50% loss of conductivity from a best-fit curve was used to

approximate the mean critical cavitation diameter (45 jam). Predicted percent loss of

conductivity (Davis et al., 1999) was calculated as

100(1- (d > d,)4/Ed4)

In the formula, the sum of diameters greater than or equal to the cavitation diameter (d,)

raised to the fourth power (1 (d > d )4) and Ed4 was the summation of all the diameters

raised to the fourth power. The best fit d was 44 jm (r2 =0.96). These values can be

calculated in woody species using the published procedure (Davis, et al., 1999). Woody

plant twigs are cut to standard lengths and fastened to a Z-tube without constricting or

deforming vessels. Woody plant twig sections can have their vessels evacuated with a

pump to measure conductivity. Succulent grass leaves cannot be handled in the same

manner as woody twig sections. Grass leaves cannot be studied with the same apparatus

that was used to study woody twig pieces xylem conductance and cavitation.

In bahiagrass, observations and microscopic visualizations from controlled

freezes might provide some information as to how smaller xylem diameters might be







21

helpful in tolerating a leaf freeze event. Thus an investigation of leaf anatomy

differences in bahiagrass may be warranted.

Physiological

If we assume that plants developed freeze-tolerance based on desiccation

tolerance mechanisms, then cold-tolerant lines of a subtropical plant could have at least

two major physiological mechanisms involved 1) higher percentage of unsaturated FAs

(C16:1, C18:1, C18:2, C18:3) (which would result in increased membrane permeability

and fluidity), 2) higher concentration of osmotica (which would result in reduced water

loss and reduced freezing point-depression).

FA composition of some tropical and subtropical C4 grasses has been reported in

cold temperature acclimation experiments. Seashore Paspalum (Paspalum vaginatum

Swartz), a subtropical grass, has been used as a turfgrass because of its salt tolerance. In

an experiment where a cold-tolerant, intermediately-tolerant, and cold-sensitive line

were subjected to controlled cold stress (80/40 day/night for three weeks) the cold-

tolerant line initially had a higher double bond FA index than the other lines as well as at

the end of the cold stress period (Cyril et al., 2002). Linolenic acid (C18:3), content

increased the most of all the FAs in the cold-tolerant line and not in the intermediate and

sensitive lines. Bermudagrass (Cynodon dactylon (L.) Pers) moderately cold-tolerant and

cold-sensitive lines were compared under a similar cold temperature stress regime in

which leaves, crowns and roots were analyzed for FA composition (Samala et al., 1998).

Linolenic acid increased in the moderately cold-tolerant line as a result of cold treatment.

Genetic

A review of the literature did not specifically identify freeze-tolerance genetic

regulation. However, inferences and possible analogies may be deduced from plant cold-







22

tolerance literature. Cold-tolerance (the ability of the plant to withstand above-freezing

temperatures without damage) work in genetics is available and could be used to provide

insight or perhaps illuminate the complexity of freeze-tolerance at the genetic level.

Cold-tolerance is a highly complex and inter-related trait under genetic control

(Revilla et al., 2005). In Arabidopsis thaliana (L.) Heynh. the numbers of genes

responsive to cold stress range from 1,000 to an extrapolated 7,000 genes, depending on

the experiment and the microarray results (Guy, 2003). Genes that are down-regulated

may be as important as those up-regulated and expressed in microarray studies.

Heritability of plant leaf-tissue freeze-tolerance was not found in the literature.

Cold-tolerance heritability may provide insight as to possible heritability ranges for leaf-

tissue freeze-tolerance. Heritability estimates for cold-tolerance across different crop

species has ranged from moderate to high (Revilla et al., 2005), with significant genotype

x environment (GE) interaction. Reported (Revilla et al., 2005) maize seedling vigor,

maize purpling of leaves, maize yellowing of leaves, maize drying of leaves; lentil

seedling freeze-tolerance, poplar freeze-tolerance, wheat freeze-tolerance, Brassica sp.

Freeze-tolerance etc., have been traits that depend on the season (Fall or Spring),

acclimation or non-acclimation and high-intensity or low-intensity freezing. Additive

effects have been reported (Revilla et al., 2005) as the most important effects in many

plants. Nonadditive gene effects (dominant and epistatic effects) are important in many

species. Maternal gene effects appear to affect cold-tolerance of early and late growth

stages. Interpopulation recurrent selection or mass selection has been recommended

since additive gene effects are the most important. Yield under cold conditions has been

recommended as a method to evaluate cold-tolerance (Revilla et al., 2005).







23

Field-freeze stress experiments have been reported to have high experimental

error, high environmental interaction and low correlation with controlled freeze chamber

experiments (Revilla et al., 2005). Reported limited success in breeding for freezing

tolerance using field selection has been attributed to major limiting factors: limited

genetic diversity, ineffective selection criteria, and limited knowledge of freeze-tolerance

genetic control.

Apomixis (apospory and pseudogamy) exists in bahiagrass at the tetraploid (4x)

chromosome level (Chen et al., 2001). Apomictic mode of reproduction has restricted

transfer of desired traits between sexual diploid and apomictic tetraploid germplasm

(Forbes and Burton, 1961). Attempts to manipulate apomoxis in bahiagrass have ranged

from failure (Hayward, 1999) to limited, but impractical results (Burton, 1982; Burton,

1986; Burton, 1992; Burton, 1999). Apomixis has hampered breeding cold-tolerance in

tetraploid bahiagrass in the southeast US. Breeding and selection in sexual diploid (2x)

chromosome level in bahiagrass lines for cold-tolerance trait in addition to daylength-

insensitivity trait has been a multi-state effort to extend the grazing season through the

cool short-day growing season (Blount et al., 2001).

Outline of the Research Plan

The purpose of the research was to determine which mechanisms caused LTFT in

diploid bahiagrass:

* Quantify the range of LTFT expression in diploid bahiagrass genotypes

* Verify whether LTFT was a leaf or root freeze-tolerance trait

* Determine whether there were anatomical differences between freeze-tolerant and
freeze-sensitive genotypes

* Determine whether there were physiological differences (osmolality and fatty acid
composition) between freeze-sensitive and freeze-tolerant genotypes







24

* Quantify LTFT heredity (broad-sense-and narrow-sense)

* Determine the mode of LTFT gene action (additive and dominant)

Chapter 2 describes the experiments used to quantify the range of LTFT

expression and the confirmation of leaf-tissue freeze-damage as a leaf effect, instead of a

root effect. In order to quantify the extremes of the range of the LTFT expression,

freeze-tolerant and freeze-sensitive plants would be collected and tested under controlled

freeze events. Canopy damage ratings would be used to quantify the LTFT trait. The

limits of LTFT expression would be determined by repeatedly colder freeze treatments.

Additions of new plant material to the experiment would require confirmation of LTFT,

with a single freeze treatment of the entire genotype collection. To determine whether

canopy damage from freeze treatments was the result of either leaf damage, or of root

damage, freeze-sensitive genotypes identified in the initial experiments would be frozen

in a manner that only the leaves would be subjected to the freeze treatments.

Chapter 3 describes the experiments conducted to quantify anatomical differences

between freeze-tolerant and freeze-sensitive genotypes. Differences in contrasting leaf

anatomy might provide information about leaf-tissue freeze-tolerance. The region of

anatomical investigation was based on the observation of leaf damage immediately after

a freeze treatment. The midrib region of the leaf blade of a freeze-sensitive genotype

(FL9) was discolored and damaged within hours after placing plants in full sunlight, after

a freeze chamber treatment. In contrast, the midrib region of the leaf blade of a

freeze-tolerant genotype (FL67), subjected to the same freeze treatment, was not

damaged. Differences between the midrib vascular bundle anatomy between these two

genotypes (FL9 and FL67) would be quantified. If differences were significant for these

two contrasting lines, additional freeze-sensitive and freeze-tolerant genotypes would be







25

added to the investigation. Anatomical differences by freeze-tolerance class needed to be

confirmed across multiple lines, in order to support the hypothesis that leaf anatomy was

associated with LTFT.

Chapter 4 describes the experiments used to investigate the two main theories of

leaf freeze tolerance: 1) freeze temperature depression by the mechanism of increased

molality, and 2) membrane integrity by the mechanism of increased membrane fluidity

mediated by an increase in unsaturated fatty acid composition. Freeze-tolerant genotypes

should have higher molality than freeze-sensitive genotypes, if the LTFT mechanism was

increased molality. Comparing the molality of the midrib region of the leaf blade, where

damage first appeared in freeze-sensitive lines, to freeze-tolerant lines, should determine

whether molality was the LTFT mechanism. Freeze-tolerant genotypes should have a

higher portion of leaf unsaturated fatty acids than freeze-sensitive lines, if fatty acid

composition is the LTFT mechanism.

Chapter 5 describes the experiments used to quantify the LTFT inheritance

(broad- and narrow-sense) and the mode of gene action (additive and nonadditive

portions). Plant breeders need to know the narrow-sense heritability (h2) of a trait. The

narrow-sense heritability is the additive mode of gene action, and it can be manipulated

by the plant breeder through selection. The higher the h2 of a trait, the more rapidly a

plant breeder can make progress selecting improved populations. One method of

obtaining the information needed to calculate the h2 is the diallel mating design of parents

that represent a wide range of trait expression. A diallel mating design would be used

where Fl progeny would be grown from every possible combination of crosses of a

group of freeze-tolerant and freeze-sensitive parents. Because information was lacking

on the number of caryopses needed to sow, germinate and grow sufficient Fl progeny for







26

the diallel study, a series of experiments would have to be conducted to support the

diallel study.















CHAPTER 2
LEAF-TISSUE FREEZE-TOLERANCE TRAIT DIVERSITY IN BAHIAGRASS

Introduction

Bahiagrass is a plant with the C4 fixation pathway subtype that uses NADP-malic

enzyme (NADP-ME) in the leaf bundle sheath chloroplast cell of the Kranz anatomy to

provide carbon dioxide (CO2) for ribulose bisphophate carboxylase/oxygenase (Rubisco)

(Brown, 1999). One of the limitations of the C4 grass-growing season and range is the

lack of or limited leaf-tissue freeze-tolerance (Rowley et al., 1975). No consistent

photosynthetic pathway explanation could be arrived at to explain leaf lesions caused by

cold temperatures, other than to attribute other unknown factors deriving from the

tropical origin of the C4 species being examined (Long, 1999). The C4 species decline in

percentage of flora composition with increasing latitude. The C4 species growing season

has been noted to approximate a temperature limit. This temperature limit is reached

when the average daily minimum temperature for the warmest month of the year is less

than 180C (Long, 1983). This approximated limitation of the C4 species range had been

postulated to be a result of impaired net CO2 uptake through slow or irreversible photo-

inhibition of photosynthesis. Low temperature tolerance has been found in the C4 genus

Zea. Genotypes of Zea mays L. and Zeaperennis (A.S. Hitchc.) Reeves & Manglesdorf

were found to be tolerant to low temperature photo-inhibition. These genotypes showed

sources of origin effects when compared to modem Zea mays L. cultivars. The C4 genera

are not excluded from high altitude and low temperature sites. Four Paspalum, three







28

Muhlenbergia and one Eragrostis species were found growing between 4,000 and 4,500

m in altitude in the Peruvian Andes, where mean annual temperature was 30 to 60C.

South American bahiagrass accessions in the U.S. Department of Agriculture,

Agriculture Research Service, Germplasm Resources Information Network S9-grasses

(USDA ARS GRIN S9) database were searched for collection site latitude in South

America noting their winter injury rating at Griffin, Georgia (USDA, ARS, 2001).

Countries of origin included Argentina, Bolivia, Brazil, Paraguay, and Uruguay. South

American bahiagrass accession sites ranged roughly from 210 to 340 S latitude (U.S.

Office of Geography, 1963; U.S. Office of Geography, 1956; U.S. Office of Geography,

1957; U.S. Office of Geography, 1968; USDA, ARS, 2001). When accessions were

grown at Griffin, Georgia, they varied from little to no winter injury through complete

winter injury. Although the USDA ARS GRIN S9 system reported winter-injury stress

and survival instead of leaf-tissue damage from freeze events, the winter injury ratings

provide an indication of diverse genotype tolerance to below-freezing temperatures.

Leaf-tissue Freeze-tolerance

Screening genotypes for leaf-tissue freeze-tolerance can be done in the field or in

growth chambers by assessing leaf-tissue freeze-injury. Freeze-tolerant genotypes

should have low leaf-tissue injury symptoms. Field-discrimination of forage germplasm

for low levels of leaf-tissue freeze-injury would be desirable. Field-discriminated

germplasm should be applicable to commercial forage production. Disadvantages of

field-freeze events include limitations of unpredictable and difficult-to-repeat annual

freeze events. Field-discrimination is especially difficult when attempting to screen plant

material to begin a study.

Convenient, replicable, controlled experiments in environmental chambers allow

control of cooling rates and freeze-stress exposure duration by exposing all the plants to







29

the same treatment. The disadvantage of the environmental chambers is that plants are

cooled by advection, which is not the same cooling condition plants experience during a

radiative frost (Ashworth and Kieft. 1995). However, the advantage of controlled and

replicable environmental conditions favors its use for initial screenings, as well as

descriptive experiments.

C4 grasses (Paspalum dilatatum Poir., Eragrostis curvula Schrad Nees,

hemarthria altissima Poir Stapf CE Hubbard, Pennisetum clandestinum Hochst. ex

Chiov., Acroceras macrum Stapf., Cynodon dactylon L., Setaria anceps Stapf., Digitaria

sp.) have been screened using a temperature-gradient bar, which froze excised leaf pieces

on a temperature continuum. Freezing treatment was followed by determination of lethal

temperature (LT50), based on electrolytic leakage of excised leaf pieces (Rowley et al.,

1975). Electrolytic leakage has been used with linear regression to determine LT50 of

Setaria anceps Stapf., Chloris gayana Kunth. and Cenchrus ciliaris L. (Ivory and

Whiteman, 1978). Electrolytic leakage, as a measure of leaf freeze-tolerance differences

among tropical grasses, identified the temperatures at which the genotypes were freeze-

tolerant.

Percent foliar freeze-damage of C4 grasses (Setaria anceps Stapf, Setaria trinervia

Stapf, Digitaria macroglossa Henr., Digitaria setivalva Stent., Digitaria smutsi Stent.,

Paspalum plicatulum Michx., Paspalum guenoarum Arechav., Paspalum rojasii Hack.,

Paspalum dilatatum Poir., Panicum maximum Jacq.) and C3 grasses (Lolium perenne L.,

Lolium x hybridum) was calculated on a dry matter (DM) basis on harvested, separated,

and dried leaves (Hacker et al., 1974; Ivory and Whiteman, 1978). In addition to being

time-consuming, the DM method of assessing foliar freeze-damage was destructive,

requiring the harvesting of top growth. Harvesting top growth interferes with plant







30

recovery measurements, cumulative stress as a result of freeze-injury and quantification

of genotype winter survival.

This study concentrates on investigating variability among bahiagrass lines leaf-

tissue for freeze-tolerance. The leaf-tissue freeze-tolerance trait (LTFT) was identified in

this study as an ability to maintain green, apparently undamaged leaves after

experiencing a freeze (temperatures below 0C) event.

There was a need to screen bahiagrass genotypes that might represent a diverse

range of LTFT. Representative freeze-sensitive and freeze-tolerant (LTFT) trait

expression genotypes were needed to investigate possible mechanisms contributing to

this trait. Screening of diploid (2x) plant material additionally might provide information

helpful in a breeding program aimed at extending grazing during the winter season in the

southeastern United States.

Materials and Methods

Leaf-tissue Freeze-tolerance Trait Screening Experiment 1

An initial experiment was conducted in which 26 bahiagrass lines were subjected

to progressively lower below-freezing temperatures. The purpose was to quantify

diversity of LTFT trait in existing commercial diploid and tetraploid cultivars, as well as

diploid selections from a bahiagrass breeding program initiated by Dr. Ann Blount at the

University of Florida North Florida Research and Education Center in Marianna, Florida.

From this initial screening experiment, lines were identified as high- or low-LTFT.

'Argentine,' a tetraploid apomict, was used as the standard line known to have the least

LTFT of commercial cultivars. This standard was used to compare the LTFT of sexual

diploid bahiagrass lines in the study. A selection of the commercial diploid cultivar

'Pensacola' obtained from Dr. Paul Mislevy, University of Florida Research and







31

Extension Center at Ona, Florida, was used because the largest acreage in Florida pasture

land is planted to that variety. The line 'Sand Mountain' was included because it had

been reported to have improved cold-tolerance compared to Pensacola and Argentine

(Blount, personal communication, 2001; Ball and Blount, 2003). Sand Mountain seed

was obtained from the Alabama Seed Commission. The remaining representative diploid

clones in the experiment were selected by Dr. Blount from approximately 24,000 plants

that were observed after freeze events in north Florida and included in a breeding

program for cold-tolerance. Plants were propagated vegetatively from rhizome/stolon

pieces originating from the University of Florida North Florida Research and Education

Center at Marianna, Florida. Twenty-three clones were propagated vegetatively.

Plantlets from three cultivars or lines were seed-grown (Argentine, Pensacola, Sand

Mountain). Plants were maintained in the same media, and given nutrient management

and scheduled drip irrigation system in the Agronomy teaching greenhouse under

ambient daylength, during the fall of 2001. The greenhouse temperature was set for

supplemental steam heat to circulate when air temperature dropped below 220C. The

greenhouse maximum temperatures rarely exceeded 50C, as recorded with a

minimum/maximum thermocouple (Acurite). Round pot size was 12.5 cm diameter x

12.5 cm height. The potting media was Scotts Terralite Agricultural Mix (Scotts-Sierra

Horticultural Production Company, 14111 Scotts Lane Rd., Marysville, OH 43041).

Nutrient management was 1 g of a 16-4-8-1 N-P205-K20-Fe analysis granular fertilizer

pre-plant incorporated into the media of each pot. Ironite 1-0-0 (Ironite Products

Company, Scottsdale, AZ 85258) was sprinkled at the rate of 1 g per pot on the top of the

media to prevent iron chlorosis. An overhead spray irrigation system was set to run for

five min four times during the day.







32

Temperature treatments were single-night exposure to -10C, -30C, -50C or -70C.

The same sets of plants were progressively exposed to colder temperature treatments.

After a temperature treatment, plants were rated then returned to the greenhouse for 7 d

before imposing the next coldest temperature treatment. Freezing chamber was a blood

cooler (Pharmacy GEM Refrigerator Company, Philadelphia, PA) with styrofoam

insulation added to cover the sliding doors. Temperatures were set by adjusting controls,

measuring average temperatures with thermocouples (Fisher Scientific International)

during a compressor cycle and adjusting compressor run-time cycles. Treatment

temperature duration was a total of 10 h, beginning at 22:00 and ending at 08:00 the

following morning. The USDA ARS GRIN S9 winter-survival descriptor rating (1 to 9)

was used as the basis for rating plant canopy leaf-damage (1 = 0% plant canopy leaf-

damage, 9 = 100% plant leaf canopy damage) no later than 48 hours after treatment

(Table 2-1). Canopy leaves were considered damaged if they appeared water-soaked,

brown, or desiccated and curled. Ratings were a visual estimate of the portion of the

entire plant canopy that was damaged (Table 2-1).

Table 2-1. Canopy leaf-damage rating system shows percentage of canopy leaf-damage
and canopy green leaf.
Canopy leaf-damage Canopy leaf-damage Canopy green leaf
Rating % %
1 0 100
2 12 88
3 25 75
4 38 62
5 50 50
6 62 38
7 75 25
8 88 12
9 100 0







33

Bahiagrass lines were replicated four times and randomized as to which shelf they

were assigned in the freezing trial. To account for position effects, two replications per

line were included in the upper shelf and two in the lower shelf during each freezing

treatment. Line position by replication was identified during each successive freezing

treatment by having plants in plastic trays so that positions were identical in each

replication. Data loggers (Optic StowAway Temp, Onset Computer, 470 MacArthur

Blvd., Bourne, MA 02537) recorded actual temperatures in each shelf. Because of

limited cooler space, lines were subjected to treatments in split replications replicationss

1 and 2 subjected to temperature treatment, then replications 3 and 4). Plants were given

18 d to recover in the UF-IFAS Agronomy teaching greenhouse after the last freeze

temperature then rated for cold stress damage.

Leaf-tissue Freeze-tolerance Trait Screening Experiment 2

An experiment was conducted in an environmental growth chamber

(Environmental Growth Chambers, 510 East Washington Street Chagrin Falls, OH

44022) that could accommodate all of the lines with their replications, blocked for

position effects, in a single controlled freeze-event. A single freeze treatment which

accommodated all of the bahiagrass lines in one single treatment was done to ensure that

results obtained with the modified blood cooler were consistent. To ensure air flow

surrounded the entire potted plants uniformly, wooden pallets with expanded wire mesh

were used to raise potted lines off the floor. Baffles and a recirculation fan were used to

redirect air flow from the cooling unit fan in an attempt to reduce foliar desiccation from

cold, dry air flow. Baffles were moved and adjusted in a series of experiments using

Argentine plants, five plants per block, eight positions in the growth chamber, and

programmed to run at -30C from 2200 till 0800. The -30C treatment temperature was







34

used because previous trials in the modified blood cooler showed more damage to

Argentine plants at that temperature than at -1 C. When statistical analysis of canopy

leaf-damage ratings were not significant for block (growth chamber position) effects, a

single, controlled-freeze event was imposed on all the test lines. The EGC was

programmed to maintain a -60 that temperature treatment from 2200 till 0800.

Thirty bahiagrass lines were used to screen for LTFT. Vegetatively propagated

lines were grown as previously described for the first LTFT screening experiment.

Potted plants were enclosed in plastic bags to maintain humidity at a constant level.

Relative humidity has been shown to shift the percent of leaf freeze-damage (Ivory and

Whiteman, 1978) in freeze chamber trials with tropical grasses. The higher the relative

humidity the more leaf freeze-damage was recorded at treatment temperatures ranging

from -1 to -50C. Since potted plants came directly from a controlled greenhouse

irrigation system, plastic bags were used to seal moisture and prevent desiccation of

plants during the 10-h freeze-temperature treatment. Additionally, bagging individual

pots prevented the compressor from failing due to ice formation when the large number

of potted plants released moisture from the potting media as the fans recirculated

the -60C air.

Leaf vs. Root Effects Experiment 3

A third experiment was conducted using the modified blood cooler to determine if

the observed freeze-damage was due to the entire plant (potted roots and leaves) being

subject to low temperature or if the damage was only from the leaves being frozen.

Potted plants of lines selected to represent a range of LTFT traits were immersed in

controlled recirculation water baths (Polyscience Model 71, Niles IL) set at 50C. Potted

plants were weighted down with river rock so the warmed recirculation water covered the







35

entire root system with only the leaves exposed to the below-freezing air temperature

treatment (Figure 2-1).




















Figure 2-1. Water-bath experiment showing tubs, heaters, and weighted pots

Actual freezing stress treatments were -2.7 and -3.2 C for 10 h, starting from

2200 and ending at 0800 the following morning to ensure that leaf-tissue Freeze

treatment durations were similar to whole-plant 10-h freeze treatments. Temperature

treatments below -3.20C were constrained by the thermal mass of the water in the pans,

the increased relative humidity in the cooler, as a result of the water surface, and the

condensation of ice on the freezer compressor.

Results and Discussion

Leaf-tissue Freeze-treatment Screening Experiment 1

Observations showed initial leaf-tissue freeze-injury symptoms on thawing

included water-soaked lesions, followed by leaf wilting and curling after being exposed

to full sunlight. Further freeze-injury symptoms under full sunlight included tissue

browning followed by leaf desiccation. Light freeze-injury symptoms in bahiagrass were

typically leaf-tip browning and drying. More severe freeze symptoms were browning and







36

desiccation of entire leaves or tillers. Canopy leaf-damage ratings of the 26 lines

combined are presented in Table 2-2.

Table 2-2. Mean simple effects across 26 bahiagrass lines subjected to target
temperature treatments of progressively colder freezing events, LTFT
screening experiment 1.
Temperature Canopy leaf-damage Canopy green leaf
C rating %
-1 1.0a* 100
-3 4.2b 60
-5 6.2c 35
-7 8.3d 9
*Means with the same letter are not significantly different at P = 0.05 confidence level.

Line/clone treatment simple effects were significant at P < 0.0001 level (Table

2-2). Simple effects, averaged across all lines, showed the tendency as treatment

temperature decreased for leaf canopy damage ratings to increase. At -30C mean

bahiagrass mean canopy damage had a rating of 4.2 (equivalent to an average 60% green

undamaged leaf canopy). At -50C, bahiagrass mean canopy had a rating of 6.2

(equivalent to an average 35% green undamaged leaves in the plant canopy). The LT50

values have been used to establish freeze-tolerance standards in experiments which have

diverse plant lines (Ivory and Whiteman, 1978). For this group of selected bahiagrass

lines, a lethal temperature (LTs5), where 50% of the leaves of the canopy leaves were

green and 50% were damaged, could be interpolated from a linear regression from -30 to

-70C where

12.813x + 98.656 = LT50=-3.80C

Replication effects were significant at the target treatment temperatures of-30C

and -50C, (P < 0.001 and P < 0.01, respectively). Examination of the recorded

temperatures during the separate runs per replication showed that temperatures within

targeted temperature treatments were different (Table 2-3). Temperature differences

were sufficient to cause differences in canopy leaf-damage ratings in the same lines from







37

temperatures ranging from -2.7 through -6.0 C in the targeted treatments of -3 and -

5C. The 0.20C difference between replications at the target treatment temperature of-

1C and the 0.1 0C difference between actual temperatures of -6.5 and -6.6C were

insufficient to cause differences in leaf canopy ratings between runs. Results showed

that bahiagrass can be sensitive enough to be visually rated differently, as a result of

experiencing freeze temperature differences as small as 0.80C. Results also showed the

need to subject all the plant material at one time to the temperature treatment.

Table 2-3. Mean simple effects across all bahiagrass lines with actual temperature
treatments of progressively colder freezing events, canopy leaf-damage,
LTFT screening experiment 1.
Target temperature Actual temperature Canopy leaf-damage
C 0C rating
-1 -0.5 1.1f*
-1 -0.7 1.lf
-3 -2.7 3.6e
-3 -3.5 4.8d
-5 -4.0 5.7c
-5 -6.0 6.8b
-7 -6.5 8.6a
-7 -6.6 7.9a
*Means with the same letter are not significantly different at P = 0.05 confidence level.

In spite of all efforts to reduce position and replication effects, splitting the

materials into separate runs produced additional variance, which may have reduced the

strength of line effects. Line effects were significant (P < 0.05) (Table 2-4) and

identified lines that had consistently high ratings as treatment temperatures decreased.

Argentine had the highest canopy leaf-damage rating at -1 and -3C, which was

consistent with the earliest reported frost ratings (Burton, 1946). Lines FL67 and C4-36

had the lowest and second lowest, respectively, of canopy leaf-damage ratings across

treatment temperatures, which demonstrated consistent LTFT as quantified by ratings. In

contrast, several lines (i.e., FL17, FL19) appeared to have LTFT at -30C, but not at colder








38

temperatures. Sand Mountain exhibited so much variability between replications that it

was not significantly different from other lines. This experiment confirmed variability in

bahiagrass LTFT. Freeze treatments were colder and more than twice the 4 h typical

duration of natural freeze events in peninsular Florida. The entire plant (leaves, stolons,

roots) was frozen, which was a freeze stress never experienced in Florida. Table 2-4

shows that there were some lines at the targeted -70C treatment, which apparently had

only moderate freeze-tolerance at these extreme freeze stress conditions.

Table 2-4. Progressively lower freezing temperature events effects on canopy freeze-


Ploidy


damage of selected sexual diploid and apomictic tetraploid lines, LTFT
screening experiment 1.
Target temperature, C
-1 -3 -5 -7
Line/cultivar Rating


2x FL11 lb* 7.0ab 9.0a 9.0a


Sand Mt.


4.7abcde


2x FL9 lb 6.5abc
2x FL38 lb 4.0abcde
2x FL66 lb 4.0abcde
2x Pensacola lb 6.5abc
2x FL2 lb 4.2abcde
2x FL30 lb 5.5abcd
2x FL31 lb 5.5abcd
2x FL35 lb 3.7abcde
2x FL45 lb 3.5bcde
2x FL82 lb 6.0abc
4x Argentine 2a 7.2a
2x FL19 lb 2.2de
2x CO175 lb 5.0abcde
2x FL17 lb 1.7e
2x FL41 lb 3.2cde
2x FL69 lb 3.0cde
2x FL52 lb 3.5bcde
2x FL56 lb 4.5abcde
2x C4-36 lb 2.2de
2x FL54 lb 3.5bcde
2x FL86 lb 3.7abcde
2x FL58 lb 4.0abcde
2x FL24 lb 3.5bcde
2x FL67 lb 2.2de
*Means with the same letter are not significantly different at


7.7a


8.7a


7.5a 8.7a
7.5a 8.7a
7.5a 8.7a
7.2a 8.5a
7.2a 9.0a
7.2a 8.5a
7.0ab 9.0a
6.7ab 8.7a
6.7ab 8.7a
6.7ab 8.0a
6.5ab 8.7a
6.5ab 8.7a
6.2ab 8.5a
6.2ab 8.7a
6.2ab 9.0a
6.0ab 8.7a
5.7ab 8.7a
5.7ab 8.2a
5.5ab 6.0bc
5.2ab 7.2ab
5.2ab 8.0a
5.0ab 8.2a
4.2ab 7.2ab
2.7b 5.0c
P = 0.05 confidence level.







39

Because of the severity of the progressive freezes (10 h duration, entire plant-

freezing, repeated and colder freezes) it was important to also view and rate amount of

whole-plant damage of all 26 lines after 18 d recovery in the greenhouse (Table 2-5).

The ability to recover from freeze stress events is important from a selection as well as a

practical standpoint. Florida freezes are followed by warm periods favorable for

bahiagrass regrowth or continued growth during periods of shorter daylength. FL67 had

the most regrowth, the least damage, and was rated as 2.5. A rating of 2.5 meant that

FL67 frozen clones, after an 18d recovery period, had 81% of the canopy of control

EL67 clones maintained in the greenhouse (Table 2-5).

As freeze temperature treatments were lowered, the trend was for bahiagrass line

canopy-damage ratings to increase (Table 2-4). At -1C Argentine was the only line that

was damaged. At -30C canopy-damage ratings ranged from 1.7 to 7.2 (equivalent to

green leaf canopies ranging from 91 to 22%, respectively). At -50C canopy-damage

ratings ranged from 2.7 to 9.0 (equivalent to green leaf canopies ranging from 21 to 0%,

respectively. At -70C line canopy-damage ratings ranged from 5.0 to 9.0 (equivalent to

green leaf canopies ranging from 50 to 0%, respectively). At -7C the only line that was

significantly different from the 26 lines was FL67, which also had the lowest canopy

freeze-damage rating (5.0). It was evident that the limit of the LTFT trait had been

approached by the -70C treatment. The highest freeze-stress tolerance of all the lines

tested (Table 2-4) was FL67, as shown by the ratings: 2.2, 2.7, and 5.0 at temperatures

-3, -5, and -70C, respectively.

In order to contrast mechanisms within a plant population, cold-sensitive clones

were needed. Tetraploid Argentine could not be included in further mechanism studies

using diploid lines because of the confounding of ploidy and cell size with other







40

Table 2-5. Whole plant recovery ratings as a percent of control plants, 18 d after
treatment of selected sexual diploid and apomictic tetraploid lines by
progressively lower freezing temperature events.
Somatic chromosome Freeze stress damage
number Line/cultivar rating
2x FLU1 8.5a*
2x Sand Mt. 7.0ab
2x FL9 8.5a
2x FL38 8.7a
2x FL66 8.0ab
2x Pensacola 5.0a
2x FL2 8.5a
2x FL30 8.0a
2x FL31 9.0a
2x FL35 8.2a
2x FL45 8.5a
2x FL82 7.7ab
4x Argentine 8.1a
2x FL19 7.5ab
2x CO175 7.0ab
2x FL17 7.7ab
2x FL41 8.5a
2x FL69 8.0a
2x FL52 9.0a
2x FL56 7.0ab
2x C4-36 5.2b
2x FL54 6.7ab
2x FL86 7.5ab
2x FL58 7.7ab
2x FL24 6.7ab
2x FL67 2.5c
*Means with the same letter are not significantly different at P = 0.05 confidence level.

differences between the diploid and tetraploid lines. Line FL11 was as sensitive to

canopy freeze-damage (Table 2-4) as Argentine (ratings of 7.0 and 7.2, respectively)

at -30C. At temperatures of -5 and -70C line FL11 showed more canopy freeze-damage

than Argentine. FL11 was not a vigorous line and was propagated with difficulty for the

second LTFT screening experiment. Lines FL31 and FL52 were not used as clones to

contrast LTFT because they had died (freeze-stress damage rating = 9.0) and had not

shown canopy leaf-damage as severely as other lines at -30C.







41

A commercial standard serves as a benchmark to evaluate new germplasm for

potential cultivar improvement. Because Pensacola was a population (grown from seed,

which meant that plant-to-plant variability was inherent), a vegetatively propagated clone

was needed that would behave similarly to Pensacola. Line FL9 had canopy freeze-

damage ratings that were similar to Pensacola (Table 2-4) and was therefore included in

anatomical (Chapter 3) and osmolality and fatty acid profile (Chapter 4) studies instead

of Pensacola.

Leaf-tissue Freeze-tolerance Screening Experiment 2

In the first screening experiment, one freeze-tolerant (FL67) and one

intermediately freeze-tolerant (C4-36) line had been identified to represent the tolerant

levels of the trait expression. There was a need to find more than two clones with high-

trait expression under freeze-stress. Additional plant material was found and

propagated. Two clones were obtained from southeastern Oklahoma. Dr. Blount also

added superior clones from the breeding program. There also was a need to expand the

sensitive end of the LTFT trait expression, similar to Argentine, in order to compare the

range of trait expression within one ploidy level diploidd). No additional freeze-sensitive

lines were added to the second screening experiment because diploid bahiagrass with

those traits were not available. The second screening experiment included 30 lines

(Table 2-6).

All lines were included in one single temperature treatment. Each potted line was

enclosed in a sealed plastic bag to maintain high vapor pressure around the leaves. The

single freezing treatment resulted in canopy freeze-damage rating differences that varied

among clones (Table 2-6).







42

Table 2-6. Canopy freeze-damage of selected sexual diploid and apomictic tetraploid
lines, as affected by a single freeze treatment (-60C) in an environmentally
controlled chamber.
Somatic chromosome Canopy freeze-damage
number Line/cultivar rating
2x
2x FL9 6.2a*
2x Sand Mountain 5.6ab
2x FL31 5.2abc
2x FL52 5.2abc
2x FL17 5.2abc
4x Argentine 5.0abcd
2x FL45 5.0abcd
2x FL58 4.8abcd
2x CT18 4.7abcd
2x FL11 4.6abcde
2x C436 4.4abcdef
2x FL30 4.2abcdef
2x FL82 4.2abcdef
2x FL2 4.0abcdef
2x Pensacola 4.0abcdef
2x FL38 3.8abcdef
2x FL56 3.8abcdef
2x FL54 3.6bcdef
2x FL41 3.6bcdef
2x FL82 3.6bcdef
2x FL24 3.6bcdef
2x FL19 3.4bcdef
2x FL66 3.4bcdef
2x C05 3.4bcdef
2x CO175 3.0cdef
2x FL69 2.8cdef
2x OK1 2.6def
2x OK2 2.2ef
2x FL67 2.0f
2x C06 2.0f
*Means with the same letter are not significantly different at P = 0.05 confidence level.

Canopy freeze-damage ratings (Table 2-6) for each line were lower in this

experiment than in the first LTFT experiment. The bagged plants may not have been as

desiccated from the cold, dry air from the freezer fan, as when they were uncovered.

Lines changed ranking in amount of canopy freeze-damage, but that was expected under

a uniform freeze trial compared to split runs in the first screening experiment. For







43

example, line FL9 was even less freeze-tolerant (rating = 6.2) than freeze-sensitive

standard Argentine (rating = 5.0). Sand Mountain, the purported freeze-tolerant line,

developed from Pensacola, had a rating that was not significantly different from the

freeze-sensitive lines. Rating differences between FL9 and Argentine means were not

significantly different.

Although raw rank of line ratings was changed in this experiment, some newly

introduced clones showed superior LTFT. A new clone from Dr. Blount's breeding

program, CO6, had as low a canopy freeze-damage rating (2.0) as FL67. In addition, the

newly included Oklahoma lines, OK1 and OK2 (obtained from D. Redfearn) had nearly

as low ratings (2.6 and 2.2, respectively). Low canopy damage ratings could be expected

since Oklahoma experiences much longer and colder winter periods than north Florida

and south Georgia, where most of the plant material originated.

This second LTFT experiment supported the selection of FL9 as a diploid clone

to represent the sensitive end of trait expression. This experiment also supported the use

of clones FL67, OK1, OK2, and CO6 to represent a high expression of the LTFT trait in

further mechanism (Chapter 3 and Chapter 4) and genetics studies (Chapter 5).

Leaf vs. Root Effects Experiment 3

Simple treatment effects across all lines used in the water-bath experiment were

significant at P < 0.03. Simple effects showed that as canopy temperature decreased, the

canopy freeze-damage rating increased significantly. Root temperature was kept

constant. If canopy freeze-damage ratings were a result of genotype differences in root

freeze-damage, then there should have been no difference in ratings when treatment

temperatures were decreased. Mean simple effects showed that LTFT was a direct result

of leaf-damage from below-freezing temperatures, not a result of root injury.







44

The water-bath experiment (which kept roots at 50C while subjecting leaf tissue

to below-freezing temperatures) with 6 clones having differing canopy freeze-damage,

showed the modified cooler was able to impose temperature treatments (-2.7, -3.20C)

that resulted in ratings that separated the lines. Line or clone differences (Table 2-7)

were significant for the -2.70C treatment (P < 0.0144) and the -3.20C treatment

(P < 0.043). Line FL9 was confirmed as a freeze-sensitive line similar to Argentine

at -3.20C and appeared to be even less tolerant than Argentine at -2.70C. Lines FL11 and

FL82 were not significantly different from the Argentine freeze-sensitive standard

at -3.20C. Sand Mountain was not different than Pensacola, from which it was selected,

at either temperature treatment. These rankings and classifications were similar to those

obtained in the first two experiments where the entire plant was frozen (Table 2-6). No

lines were included in the tub experiments that had freeze-tolerance because what was

being tested was whether leaf injury was a result of true leaf-damage or a result of root

injury during freezing stress. Therefore, lines that were freeze-sensitive were used for

that experiment. If canopy injury was the result of freezing roots, then freeze-sensitive

lines should have shown no damage in the water-bath experiment, since roots were

protected.

Table 2-7. Canopy freeze-damage of selected sexual diploid and apomictic tetraploid
lines while root system was kept above freezing (50C) February 2002.
Somatic chromosome Temperature treatment
number Line/cultivar -2.70C -3.20C
Rating
4x Argentine 1.5b* 7.0a
2x FL9 3.2a 7.0a
2x FL82 1.2b 5.2ab
2x FL11 3.2a 5.2ab
2x Sand Mountain 1.2b 4.0b
2x Pensacola 1.0b 3.7b
*Means with the same letter are not significantly different at P = 0.05 confidence level.







45

Whole-plant freeze recovery ratings of the lines/clones tested in the water-bath

experiment after an 18-d recovery period in the greenhouse (Table 2-8) were not

significantly different among lines. This might be expected since the lines selected for

this experiment were chosen for their high sensitivity to freeze-stress from the initial

LTFT screening experiment. The levels of whole-plant freeze-damage ratings (6.0

to 7.2) of control clones/lines were greater than expected, and may be a result of being

submerged in water (apoxia) in addition to having leaves frozen. Also, these lines were

successively frozen, rested for 7 d, then frozen again. Successive freeze events were

apoxic, stressing roots with a lack of oxygen, in addition to having foliage frozen.

Cumulative stress may have accounted for unexpectedly sensitive whole-plant freeze-

damage ratings.

Table 2-8. Whole-plant recovery ratings as a percent of control plants after 18 day
recovery in greenhouse after freeze treatment (-3.20C) while root system
was kept above freezing (50C) February 2002.

Somatic chromosome Freeze-damage
number Line/cultivar rating
2x FL11 7.3a*
4x Argentine 7.2a
2x Pensacola 7.0a
2x FL9 6.7a
2x FL82 6.5a
2x Sand Mountain 6.0a
*Means with the same letter are not significantly different at P = 0.05 confidence level.

Overall, results confirmed the hypothesis that LTFT was truly a result of leaf-

damage and not of root damage. Controlled freeze treatments could be used to separate

and categorize bahiagrass clones and lines for the LTFT trait expression.

Summary

Plants were selected for freeze-tolerance as well as for freeze-sensitive response,

and quantified in plant-canopy freeze-damage ratings. Experiments included







46

progressively lower freezing temperatures, uniform freezing temperature trial of all lines

at a single event, and water-bath experiments where the root temperature was kept above-

freezing while the leaf canopy was exposed to various below-freezing temperatures.

Progressively lower freezing temperatures to -70C identified a superior (FL67),

intermediate (C4-36) and low-LTFT (FL9) line. A uniform freezing temperature trial (-

6C) confirmed results of the initial LTFT screening trial and identified new lines with

high-LTFT (C06, OKI, and OK2).

Water-bath experiments confirmed that canopy leaf-tissue freeze-damage was a

result of LTFT and not of root damage resulting from freezing. Lines which showed high

levels of canopy leaf-tissue freeze-damage in whole-plant freezing trials showed similar

damage even when roots were kept at 50C while the canopy was exposed to two

progressively colder below-freezing temperatures (-2.70C and -3.20C, respectively).

Lines which showed low levels of canopy leaf-tissue freeze-damage in whole-plant

freezing trials showed similar damage when roots were kept above-freezing.















CHAPTER 3
ANATOMY RELATED TO LEAF-TISSUE FREEZE-TOLERANCE

Introduction

Tropical savanna grass leaf frost resistance has been reported to be less than

temperate climate grass and forb frost resistance (Sakai and Larcher, 1987). Some

tropical grasses can tolerate temperatures as low as -40C. A few tropical grass species

can tolerate temperatures as low as -8 to -100C. In contrast, temperate grasses can

tolerate temperatures within a range of -10 to -250C without showing leaf-damage.

Some temperate grass species can tolerate temperatures as low as -300C. The majority of

tropical grasses have the C4 physiology and the majority of temperate grasses have the C3

physiology. The first assumption might be that leaf-tissue freeze-injury may be the result

of the C4 physiology.

Not all tropical grasses have the C4 physiological pathway (Knapp and Medina,

1999). Therefore, freeze-tolerance and intolerance may not be a direct result of differing

carbon-fixation physiology. The Panicoideae subfamily of grasses has C3, intermediate

(between C3 and C4), and all three C4 subtypes of physiology, as well as the unique leaf

anatomy that is associated with each type and sub-type of carbon-fixation physiology.

Another argument against categorizing tropical grass freeze-tolerance by carbon-fixation

physiology would be the existence of a wide variation in the trait shown in Chapter 2.

A definition of the leaf-tissue freeze-tolerance (LTFT) is the ability of a genotype

to maintain green, apparently undamaged leaves after experiencing a freeze







48

(temperatures below 0C) event. Anatomical parameters, other than cell arrangement

around the vascular tissue, may be related to differences in freeze-tolerance. Small cell

size might be related to LTFT. Evergreen leaves with narrow intercellular spaces and/or

small mesophyll cells were reported to lower ice nucleation temperatures down to -100 to

-120C (Sakai and Larcher, 1987). Other reported observations showed frost hardiness

correlated with small cell size (Sutcliffe, 1977). Small cells had greater specific area and

less volume strain per unit surface area produced during ice formation than large cells.

In woody plants, deep supercooling has been related to cell wall porosity, permeability,

xylem pit membrane, and cell wall tensile strength (Wisniewski, 1995).

When plant tissue freezes (if no rapid supercooling occurs initially to

approximately -100C) ice forms outside the living cell in the apoplast (Sakai and Larcher.

1987). Temperatures experienced in Florida freeze events are usually above -100C.

Probabilities of a -90C freeze occurring in Florida ranged from P = 0.26 at Milton in the

northern panhandle, to P = 0.000 at Homestead in the southern peninsula (Bradley,

1975). Therefore, we can assume ice formation in plants in Florida freeze events form

outside the living cell, in the apoplast. Sakai and Larcher (1987) describe ice formation

occurring in the plant vessels first, followed by ice spreading throughout the plant body

in the intercellular air space and film of water on the cell walls. This implies vessel

water is directly linked to the apoplast. Symplast freezing occurs after apoplast freezing.

Apoplastic ice formation may be an important process to relate to leaf anatomy.

Ice has been shown to form, travel, and spread throughout barley leaves

(Hordeum sp.) through the leaf midrib first, then throughout the leaf (Wisniewski et al.,

1997). This supports earlier work that showed the freezing sequence in a plant organ,







49

which contained water-filled vessels. The vessels froze first, followed by different

tissues in the plant organ (Sakai and Larcher, 1987).

The xylem diameter of temperate trees is less than for tropical climbing plants

(Table 3-1). Lime was the only freeze-sensitive tropical tree listed that had a small

diameter (Haberlandt, 1914).

Large xylem diameters in branches have been shown to be related to cavitations

and air embolisms, caused by freezing and subsequent thawing in 12 woody plant species

(Davis et al., 1999). At a xylem pressure of-0.5 MPa, species with branch diameter

vessels greater than 40 jam had nearly complete cavitation. Species with xylem diameters

ranging from 30 jam to 40 jam had partial cavitation at -0.5 MPa. Species with small

xylem diameters (less than 30 jam) showed no freeze-induced cavitation at -0.5 MPa.

Table 3-1. Comparison of vessel diameters of tropical climbing plants compared to
trees (Vessel diameters obtained from Haberlandt, 1914).
Diameter Diameter
Climbers ((Im) Trees ((jm)
Hypanther guapeva 650 Oak 250
Calamus rotang L. 350 Elm 158
Anisosperma passiflora Manso 300 Ash 140
Passiflora laurifolia L. 200 Birch 85
Passiflora edulis Sims 200 Alder 76
Glycine sinensis Sims 200 Lime 60
Aristolochia sp. 140 Pear 40
Serjania sp. 120 Box 28

These reports stimulated an interest in investigating leaf anatomical differences

between identified freeze-tolerant and freeze-sensitive bahiagrass lines. The null

hypothesis tested was that there were no anatomical differences between the midrib

vascular bundles of diploid (2x) bahiagrass lines differing in LTFT.







50

Materials and Methods

Initial Two-line Experiment

An initial experiment was conducted 1 January 2003 to determine if there were

visible anatomical differences between bahiagrass clones showing extreme differences in

LTFT. Initial investigation used two lines representing extremes in LTFT expression

during controlled freeze experiments. Line FL9 was chosen as a genotype representing a

freeze-sensitive line during controlled, successively colder freeze-event treatments (6.5 at

-30C, 7.5 at -50C and 8.7 at -70C). Line FL9 was also chosen because it responded with

the lowest canopy freeze-damage rating (6.2) from a single, unacclimated freeze event of

-60 C. Line FL67 was chosen to represent the most freeze-tolerant because of its

consistent behavior in the experiments in which FL9 was included (Chapter 2).

Timing of anatomical comparisons during short days, when freeze events occur

naturally in north central Florida, was important because bahiagrass has been shown to

respond to daylength (Sinclair et al., 2001; Sinclair et al., 2003). On 1 January 2003, leaf

lamina portions were cut from four plants of each line at three fully expanded leaf

positions.

Standardizing the sampled leaf region was important to minimize variability.

Reported anatomical work with Pensacola bahiagrass used the midpoint of the leaf blade

as a reference to make a fresh sample cut of 5-cm long (Flores et al., 1993.). A weakness

in using the Flores technique was that the leaf tip and length may vary with water

relations experienced during leaf expansion so that the sampled region may vary from

plant-to-plant. The leaf-blade collar was used as a reference point from which to cut

lamina samples. The leaf lamina sample from which sections were to be made was

standardized as a 3-cm long portion severed with scissors. The standardized sample







51

region began 2 cm from the collar (measuring towards the lamina tip/distal end). The

total sample portion, 3 cm long, which continued towards the lamina tip/distal end, was

severed with scissors 5 cm from the collar. The sample included the widest part of the

lamina, and provided a standardized region of tissue to examine regardless of leaf

position and plant sampled. Cut samples were preserved in 4 mL/100 mL

gluteraldehyde/water.

Three leaf positions were defined in the initial two-line bahiagrass study: first

fully expanded leaf, second fully expanded leaf, and third fully expanded leaf from the

top of the stem. Sampling at these three different positions allowed for observation of

differences in leaf damage, depending on leaf position, during freeze chamber work.

Older leaves (second, third, and older leaves emerged from the whorl) appeared to

express more freeze-injury than the first emerging and first fully expanded leaf of a

whorl. Sampling from four different plants of the same vegetative propagated genotype

would quantify plant-to-plant variance, leaving genotype and leaf position anatomical

effects to be analyzed statistically.

In a separate investigation of potted plants of FL9 and FL67, leaves exposed to

ambient temperatures after a controlled freeze event showed progressive discolored

damage development in the midrib vascular region of the leaf, before the typical water-

soaked freeze-damaged symptoms appeared. In this preliminary experiment, fresh

sections showed relative differences in vascular bundle size in the midrib area. The

midrib area was discolored and apparently damaged first before the traditional water-

soaked leaf-tissue freeze-injury symptoms developed. It was decided to measure the

xylem diameter of clones FL9 and FL67.







52

Leaf samples were stored individually, in vials filled with 40 g kg'

gluteraldehyde/water for a minimum of 24 hours to fix cells and preserve samples until

they were sectioned (Funk, personal communication, 2003). An International Minot

Custom Michrotome (IEC CTF Microtome) (Cryostat International Equipment

Company, Needham Heights, MA 31883) was used with a setting of-200C and a cutting

thickness of 15 microns. The tissue embedding medium was Tissue-Tek OCT

compound #4583 (102.4 g kg- polyvinyl alcohol, 42.6 g kg- polyethylene glycol, 855.0

g kg-' non-reactive ingredients) (Miles Inc. Diagnostic Division, Elkhart, IN 46515).

Embedding wells on brass microtome discs were fabricated from timing tape, which

facilitated filling with embedding medium and prompt freezing with the leaf sample as

near vertical as possible, so sections would be perpendicular to the lamina and make

anatomical measurements consistent from sample to sample. Slides were kept clean in a

slide well, filled with a solution made of 100 ml of 95 g kg-' ethyl alcohol in which 10

drops of glacial ascetic acid had been added. Slides to be used were removed from the

well, air-dried, then coated with freshly made Hoppe's adhesive (1 g gelatin, 2 g phenol

crystal, 15 mL glycerin, 100 mL deionized water) and dried in covered trays for 24 hours

prior to being used to receive and hold microtomed sections. Fresh Crystal violet stain,

which was designed to show lignified cell walls as violet, was made as a 10 g kg'

deionized water solution. Slides with sections were stained for 15 min in slide wells.

Slides were then cleaned in slide wells with progressively higher concentrations of ethyl

alcohol in solutions with deionized water (50mL, 75 mL, 80 mL, 95 mL, and 100 mL

ethyl alcohol /100 mL) to remove excess crystal violet stain and excess tissue water. A

final rinse of reagent-grade xylene removed the last traces of water. Slides were then

dried in covered trays for 24 h prior to making permanent mounts with Flo-texx

Mountant (Fisher Scientific International). An Olympus BH-2 microscope (2







53

Corporate Center Dr., Melville, NY 11747) was used with a stage micrometer to measure

vascular parameters. Ocular units were calculated from ocular and lens powers with

micrometer units converted to microns at 400X power.

Eight-line Experiment

Results of the first experiment were used to conduct a second experiment. The

second experiment included additional diploid clones to confirm initial results using

additional lines representing the two extremes in LTFT and an intermediate line.

On 3 December 2002, Dr. Ken Quesenberry identified eight diploid clones that were

severely damaged by a light frost at the Agronomy Forage Unit, Hague, Florida. Up to

this time, FL9 had been the most freeze-sensitive diploid (2x) line, with approximately

the same response as the standard cultivar Pensacola. Freeze-sensitive lines (1-30-3,

1-30-4, 2-22-1, and FL9) and freeze-tolerant lines (FL67, OKI, OK2, CO6) were

increased from pieces ofrhizomes/stolons. Before planting in plastic pots (12.5 cm

diameter x 12.5 cm depth, model 500, Better Plastics, Kissimmee, FL), vegetative

rhizomatous/stoloniferous pieces were dipped in Hormodin 2 (E.C. Geiger Inc. Rt. 63

Box 285, Harleysville, PA 19438) to induce rooting.

Plants were maintained in the same media, given nutrient management, and

scheduled drip irrigation system in the Agronomy Teaching Greenhouse under ambient

daylength. The potting media was Scotts Terralite Agricultural Mix (Scotts-Sierra

Horticultural Production Company, 14111 Scotts Lane Rd., Marysville, OH 43041).

Nutrient management was 1 g of a 16-4-8-1 N-P205-K20-Fe analysis granular fertilizer

pre-plant incorporated into the media of each pot. Ironite 1-0-0 (Ironite Products

Company, Scottsdale, AZ 85258) was sprinkled (1 g per pot) on the top of the media to

prevent iron chlorosis. An overhead spray irrigation system was set to run for 5 min four

times during the day.







54

When leaves were mature enough to sample by leaf positions (1 February 2003),

leaf samples were collected as done previously. Classes of LTFT were based on previous

control freeze chamber experiments (Chapter 1) and field rating.

Because the damaged region appeared to be the midrib region, the entire midrib

vascular bundle region was observed. Measurements of the midrib vascular bundle

diameter were made using the outer suberized walls of the bundle-sheath cells as the

limit. Where oval configurations were encountered, two values were recorded: major-

axis diameter, minor-axis diameter. Mean diameter values were calculated as (major-

axis diameter + minor axis diameter/2). Area calculations were done by multiplying 7u x

minor axis radius x major axis radius. Two major xylem vessels per vascular bundle

occurred in the bahiagrass lines measured. The xylem vessel diameter and cross-section

area were calculated in the same manner as for vascular bundles. Xylem cell wall

thickness was measured across the visually apparent average cell wall thickness, thus

eliminating extremes. All measurements were made using a microscope at 400X with an

ocular micrometer with units calibrated with a stage micrometer as stated above. The

SAS (SAS Institute Inc., 1987) general linear model was used to analyze data.

Results and Discussion

Initial Two-line Experiment

Since leaf internal anatomy was a new area of investigation, it was important to

test the null hypothesis that there were no differences between the bahiagrass line xylem

diameters with the same somatic chromosome number (2n = 2x = 20). Mean simple

effects across all leaf positions showed that the differences were significant (Table 3-2).

The 1 January sampling of lines FL9 (freeze-sensitive) and FL67 (freeze-tolerant)

showed that the high-LTFT line had a larger midrib diameter (201 (im vs. 149 jam,

respectively).







55

Table 3-2. Mean midrib xylem diameter across three leaf positions of two lines
sampled 1 January 2003.

Leaf-tissue Number of
freeze-tolerance class Genotype line Xylem diameter measurements
p.m n
Sensitive FL9 201a* 164
Tolerant FL67 149b 120
*Means with the same letter are not significantly different at P = 0.05 confidence level.

Absolute vessel diameter values of FL9 and FL67 were higher than those reported

(Table 3-1) for all temperate woody plants except for oak (250 rim) and within the range

of five of the tropical vining species (120 to 200 rim).

Simple mean leaf position effects were significant across both lines (Table 3-3).

The xylem diameter of the first fully expanded leaf was significantly smaller than the

second and third fully expanded leaves. The results show that what had been defined as

the first fully expanded leaf was still experiencing anatomical changes when sampled.

Table 3-3. Mean midrib xylem diameter of three leaf positions across two lines (FL9,
FL67) varying in LTFT, sampled 1 January 2003.
Leaf position Xylem diameter Number of measurements
gIm n
First 158c* 120
Second 190a 80
Third 184b 80
*Means with the same letter are not significantly different at P = 0.05 confidence level.

Observation in the various controlled freezer trials (Chapter 2) indicated that

freeze-injury symptoms appeared more readily on older leaves, such as the second and

third fully expanded leaves, rather than on the first fully expanded leaf and the new leaf

emerging from the whorl. Leaf position (thus leaf age) freezing sequence may be similar

in bahiagrass as in Hordeum sp. (barley) (Pearce and Fuller, 2001b), depending on the

severity of the freezing treatment. When organs of uprooted barley were tested under

controlled freeze conditions in the laboratory, freezing order occurred first from

nucleated leaves, roots, older leaves, and younger leaves with secondary tillers being the







56

last to freeze (Pearce and Fuller, 2001b). If the xylem diameter is one of the mechanisms

that is related to LTFT it might explain why older leaves freeze before younger leaves.

Visualization of the third emerged fully expanded leaf cross-section of the freeze-

tolerant (Figure 3-1) and freeze-sensitive (Figure 3-2) lines showed clear differences in

xylem diameter as well as parenchyma cells within the midrib region. This was the

region that was apparently damaged within hours after exposure to sunlight after a 10-

hour freeze.


Figure 3-1. FL67 bahiagrass (freeze-tolerant) section showing bundle sheath, girder
system of sclerenchymous tissue supporting the vascular bundle. Adaxial
(bottom) midrib vascular bundle towards the left of center. Third emerged
fully expanded leaf position at 100X.


Figure 3-2. FL9 (freeze-sensitive) section showing larger vascular bundle area than
FL67. Third emerged fully expanded leaf position at 100X.









Eight-line Experiment

The February sampling date showed that measured parameters in the first

experiment using two contrasting lines, held consistently, when contrasting four freeze-

tolerant to four freeze-sensitive lines. Mean LTFT class leaf xylem diameter trends

across 3 leaf positions were similar for the two-line and eight-line experiments. Leaf-

tissue freeze-tolerant lines tended to have lower mean xylem diameter values (across all

three leaf positions) than freeze-sensitive lines. The freeze-tolerant line, FL67, had a

mean xylem diameter of 149 pm in the two-line experiment (Table 3-4), and the mean of

4 freeze-tolerant lines in the eight-line experiment was 169pm in the 8-line experiment

(Table 3-5). Mean leaf xylem diameter trends across 3 leaf positions were similar for

both the two-line and eight-line LTFT experiments. The low-LTFT line, FL9, had a mean

xylem diameter of 201 pm in the two-line experiment (Table 3-4), and the mean of 4

low-LTFT lines in the eight-line experiment was 221 pm (Table 3-5).

Mean simple effects for the midrib abaxial vascular bundle diameters were

significant. Freeze-tolerant lines had larger vessel diameters and vascular bundle

diameters than freeze-sensitive lines (Table 3-4).

Table 3-4. Mean simple effects of vessel and vascular bundle diameters of four freeze-
tolerant vs. four freeze-sensitive bahiagrass clones sampled 1 February 2003.
LTFT class Number of
Parameter Sensitive Tolerant P> F measurements
Mum n
Vessel diameter 221 169 0.0001 445
Vascular bundle diameter 1170 921 0.0001 445

Mean simple effects for the midrib abaxial vascular bundle diameter were significant.

Freeze-sensitive lines had larger vessel areas and vascular bundle areas than high-LTFT

lines (Table 3-4). Mean simple area effects were highly significant and clearly

distinguished between freeze-sensitive and freeze-tolerant clones (Table 3-5). The eight-







58

line experiment corroborated and supported the initial exploratory two-line experiment.

The relationship for smaller xylems to be associated with freeze-tolerance was consistent.

Either xylem or vascular bundle diameter or area measurements could be used to

identify bahiagrass clones with the LTFT trait (Table 3-6). Xylem cell wall thickness

was a parameter that did not distinguish between clone LTFT classes. Line FL67, one of

the genotypes with freeze-tolerance, had the lowest average xylem cell wall thickness

(20 jam). OK2, another line with freeze-tolerance, had the highest xylem cell wall

thickness of all the lines tested (32 (Jm).

Table 3-5. Analysis of variance comparing mean vessel and vascular bundle area
simple effects of four high- vs. four low- leaf-tissue freeze-tolerant clones
sampled 1 February 2003.
LTFT class Number of
Parameter Sensitive Tolerant P> F measurements
mm2 n
Vessel area 0.039 0.023 0.0001 445
Vascular bundle area 1.064 0.677 0.0001 445

The critical xylem diameter that distinguished between freeze-tolerant and freeze-

sensitive genotypes appeared to be between 209 and 187 jtm Table 3-6). The critical

xylem area distinguishing between freeze-tolerant and freeze-sensitive LTFT genotypes

was between 0.035 and 0.028 mm2. The vascular bundle diameter and area followed

trends similar to the xylem diameter, compared with the area in spite of the oval shape of

xylem vessels. The critical vascular bundle diameter was between 1160 and 980 jtm.

Critical vascular bundle area appeared to be between 1.039 and 0.759 mm2.

The question is then raised how smaller xylem diameters might provide an

advantage to bahiagrass plants that are completely frozen, then thawed in ambient

temperatures in full sunlight? Observation of the bahiagrass lines, during the very first

day of recovery in full sunlight after a controlled freeze event, showed damage initially







59

Table 3-6. Mean midrib abaxial (facing the bottom side of the leaf) xylem and vascular
bundle parameters from a February 2003 sampling date.
Canopy
damage Xylem Vascular
LTFT rating Xylem cell wall bundle Vascular
class -6 C Genotype diameter Xylem area thickness diameter bundle area
[1m mm2 mm 1[m2 mm2
Sensitive ----- 1-30-4 242a* 0.046a 22bc 1230a 1.178a
Sensitive ----- 1-30-3 223b 0.039b 22d 1170b 1.069b
Sensitive 6.2b FL9 209c 0.036c 24 1120b 0.963c
Sensitive -----t 2-22-1 209c 0.035c 21d 1160b 1.039b
Tolerant 2.6a OK1 187d 0.028d 22bc 980d 0.759d
Tolerant 2.2a OK2 170e 0.023e 32cd 950d 0.716d
Tolerant 2.0a FL67 157f 0.022e 20e 900e 0.654e
Tolerant 2.0a C06 158f 0.020e 21cd 850f 0.579f
*Means with the same letter are not significantly different at P = 0.05 confidence level.
TfSelected after a freeze event 3 December 2002 at Hague, Florida, from a visual rating
compared to Argentine (canopy damage rating of 2, slightly damaged) vs. numbered
sensitive lines that were extremely sensitive, being frozen to the ground (canopy damage
rating of 9, all foliage damaged).

along the leaf midrib (Figure 3-3), followed by the lamina proper. The leaf lamina would

have thawed first in full sunlight. Transpiration demand on the lamina in full sunlight

would have made leaf xylem pressure (Px) more negative. At pressures of-0.5 MPa,

freeze-thaw cycles have been under shown to cause embolisms which reduced

transpiration in plant species with small xylem diameters (Davis et al., 1999). It could be

possible that under full transpiration stress on initially thawing, xylem pressures

experienced in the leaf xylem could be more negative than the -0.5 MPa in the controlled

experiment conducted by Davis et al. (1999) because the roots would still be frozen in the

pots. Following that reasoning, root pressure would not alleviate the transpiration

demand as might be possible in field-grown bahiagrass plants. Without positive root

pressure, the midrib damage would be visualized within a short period of time. This

sequence of events could have been the case for freeze-sensitive bahiagrass clones

removed from controlled freeze trials. Bahiagrass midrib leaf damage was seen within







60

hours of placing frozen, potted plants in full sunlight. In the work reported by Davis et

al. (1999) cavitation occurred in xylem vessels upon thawing, followed by air embolisms,

which restricted vessel conductance in plants with vessels larger than the critical

embolism diameter value of 45 (im for the 12 woody species investigated. Further

detailed discussion and references concerning cavitation, embolism and vessel

conductance are included in Chapter 1.

In bahiagrass, the two largest xylem vessels of a bahiagrass leaf occur in the

single midrib vascular bundle, on the adaxial region of the leaf cross-section (Figures 3-1

and 3-2). This is the region that visually appeared damaged in susceptible lines within

hours of controlled freeze trials.
























midrib regions initially.

The same midrib region appeared damaged first in hard freezes experienced in a

bahiagrass experiment containing freeze-sensitive lines planted in the field at the North
afe acntole rez eet,1 ha -, hwsdmae







61

Florida Agricultural Research and Education Center (NFREC-Suwannee Valley) after

freeze events in December 2004 and January 2005. Field-grown plant roots were not

frozen, yet leaf midribs showed similar damage symptoms the afternoon of the day of the

freeze when air temperatures warmed above freezing. Visual symptoms of leaf damage

in the mid-rib leaf region of freeze-sensitive plants were similar to those from controlled

freeze trials. Even with positive root pressure from un-frozen roots, vessel air embolisms

would have restricted water supply under transpiration demand under full afternoon

sunlight in the field.

Observation of freeze-sensitive bahiagrass clone damage suggests that the small

leaf xylem diameter in lines with freeze-tolerance (OKI, OK2, FL67, C06) may be a

mechanism to survive a freeze event. For freeze-sensitive bahiagrass lines (FL9, 1-30-3,

1-30-4, 2-22-1), thawing would cause large diameter xylem vessel air embolism

formation, which would prevent leaf blade cell rehydration. The air embolism would

lead to leaf cell plasmolysis and leaf-damage, which was observed and rated in freeze-

sensitive lines.

Summary

A two-line investigation of bahiagrass was conducted initially to quantify

anatomical differences between a freeze-tolerant and a freeze-sensitive clone.

Statistically significant differences were found in the mid-rib xylem diameter and area, as

well as the vascular bundle diameter and area between FL9 (freeze-sensitive) and FL67

(freeze-tolerant). The xylem cell wall thickness was not a parameter that was associated

with LTFT.

To verify whether encountered anatomical differences were consistent across

bahiagrass clones exhibiting these LTFT traits, an eight-line investigation was conducted.







62

Smaller midrib xylem diameters, xylem areas, vascular bundle diameters, and vascular

bundle areas were associated with LTFT clones. Critical values of LTFT clones were

estimated, based on the eight-line data. Estimated critical values (values above being

freeze-sensitive and values below being freeze-tolerant) were calculated for the xylem

diameter (198 jam), xylem area (0.031 mm2), vascular bundle diameter (1070 jam), and

vascular bundle area (0.899 mm2).















CHAPTER 4
PHYSIOLOGICAL MECHANISMS ASSOCIATED WITH LEAF-
TISSUE FREEZE-TOLERANCE

Introduction

A definition of leaf-tissue freeze-tolerance (LTFT) would be the ability of a

genotype to maintain green, apparently undamaged leaves after experiencing a freeze

(temperatures below 0C) event. To assume that the LTFT trait in bahiagrass is the result

of one single mechanism may be too simplistic. Several mechanisms may be brought

into action, including both the apoplast and the symplast. Xylem vessels are considered

to be part of the plant apoplast because vessels are composed of dead cell walls.

Bahiagrass xylem (a part of the leaf apoplast) anatomy was investigated and related to

LTFT through a proposed embolism mechanism (Chapter 3). Symplast mechanisms may

be involved in bahiagrass as well and should therefore be investigated. A review of the

major symplast mechanisms involved, the major biochemical and physiological

mechanisms with a proposed scheme that integrated plant freeze-tolerance was

thoroughly discussed (Chapter 1).

Plant physiological and metabolic response to cold- and freeze-stress has been

shown to overlap biochemical pathways involved in osmotic, high salinity, and drought-

stress profiles in gene expression studies (Guy, 2003). This biochemical pathway overlap

has indicated the hypothesis that tropical and subtropical plants adjusting to geologically

changing climates may have utilized similar physiological pathways and mechanisms to

withstand the desiccation effects of freezing (Guy et al., 1992). Plant tissue salt-







64

tolerance has been shown to include accumulation of organic, as well as inorganic,

solutes in order to regulate cell osmotic potential (Orcutt and Nilsen, 2000). Both

organic (sugars, amino acids, and polyols) synthesis and accumulation as well as

inorganic ions (Na K Ca ) accumulate in order to reduce the osmotic potential

sufficiently to maintain cell turgidity through water uptake in halophytes. Plant

membrane stability and its permeability under salt-stress have also been shown to be

related to membrane phospholipid saturation. Further discussion is covered in Chapter 1.

Two major physiological mechanisms have been associated with freeze-tolerant

plants (Chapter 1): 1) freeze point-depression through increased osmolality; and 2)

increased membrane fluidity at lower temperatures through increased polyunsaturated

fatty acid content. Since no studies in bahiagrass were found relating osmolality and

membrane fluidity at below-freezing temperatures, there was a need to investigate these

two potential mechanisms.

A short review of how osmolality affects the freezing point of a solution may be

helpful. Vapor pressure and freezing temperature of a solute depends on the osmolality

(moles of solute dissolved in a given volume of solution). Osmolality is defined as the

moles solute per kilogram of solution. (Holtzclaw et al., 1984). A solution will freeze at

a lower temperature than pure water. Freezing point-depression temperature can be

calculated with the following formula:

Af = i Km

Definition of the formula terms includes the following: Kf is the freezing

constant for water (-1.860C m'), m = the molality of the solute, and i = the Van't Hoff

dissociation constant. A perfect solute would have i = 1.0. An aqueous solution of 1

molal (1000 milliosmoles kg -1) with a perfect solute would produce an osmotic potential







65

of -2.27 MPa. This relationship ([(-2.27 MPa/-1.860C mol kg-')/-2.27 MPa mol kg-] =

Ys/Af) can be simplified, relating osmotic potential (Is) with freezing point-depression

Af:

Y, = 0.537Af

Figure 4-1 shows the relation between osmotic pressure (Pa), freezing

temperature (C), osmolality (osmole kg-1), and relative humidity (%) of a solution.

(Pa) I e... I iii, I I ,m I i a.. I I ,I I 1 Iu I
v "0o "b o "o o o


Equilibrium freezing I i -I c i
temperature (0 C) 6d i

I I I I I I I I I I
(Os lkg )
(Osmolality k, ;

Relative 0 oo 0oo 0; 0 0 0 o-
humidity () 0 M gO V


Figure 4-1. Relationship of osmolality, freezing temperature, osmotic pressure,
and relative humidity of an aqueous solution with a pure solute
(Source of figure from Wolf and Bryant, 2001).

These relationships could be used to test the hypothesis that the mechanism of the LTFT

trait is a result of freezing point-depression from solute increase in leaf symplast cells.

Cells that would especially be important in the bahiagrass lamina would be those

arranged radially in the bundle sheath adjacent to the xylem. This single layer of bundle-

sheath cells, which are immediately adjacent to the xylem vessel, is where the second

photosynthetic carbon reduction occurs. The second layer of cells removed from the

xylem vessels are mesophyll cells, where the primary photosynthetic carbon reduction

reaction occurs. If ice forms initially in the xylem vessels, the single layer of bundle-







66

sheath would be the cells which would experience osmotic stress first. Cells with lower

freezing temperatures resulting from increased osmolyte concentrations would be able to

resist desiccation (loss of water from the cell as it crossed the membrane to achieve

osmotic equilibrium). Cell desiccation during freeze-stress is caused from the lower

osmotic potential (W) of the growing apoplastic ice crystals (assuming ice crystals formed

in the vessels).

Let us assume a hypothetical freeze event to -100C is slow enough so super

cooling does not occur (super cooling is an unstable condition when ice, liquid and gas

phases are not in equilibrium; as discussed thoroughly in Chapterl). Assume that leaf

tissue freeze-damage is defined by: water-soaked symptoms from which mesophyll

tissue damage could be inferred from freeze-induced osmotic stress, cell volume

reduction to a critical volume and irreversible plasmolysis upon thawing and attempted

rehydration. In the -100C hypothetical freeze-event assume leaf-tissue freeze-damage

was not seen in a bahiagrass line, upon being placed in full solar radiation in the

greenhouse after treatment. In this hypothetical example, if lower osmolality within the

mid-rib region protected the line to -10 C by depressing the freezing temperature,

osmolality would have to be 5 osmole kg -' (10 MPa) from increased solute

concentration. Leaf-tissue freeze protection via the mechanism of depressed freeze

temperature as a function of osmotic potential could be tested in the cells of interest by

measuring the osmotic potential of bahiagrass leaves, within the region of visual damage.

Fatty Acid Composition

Membrane bi-lipid layer fluidity is thought to be influenced by the composition of

fatty acids (FAs). Unsaturated FAs have a lower melting point. The larger the

proportion of unsaturated FAs, the lower the temperature a membrane phase shifts from a







67

fluid liquid crystalline phase to a gel phase (Buchanan, et al., 2000). Cell recovery from

freeze-thaw stress cycle, where cell volume initially is decreased as a result of

dehydration stress followed by rehydration, requires membrane fluidity in order to

maintain functional integrity. The FA composition in plants appears to be a dynamic,

complex plant metabolic process involving membrane and organelle

compartmentalization, transport, and control. Lipid synthesis is mainly located in the

chloroplast with export to the endoplasmic reticulum. High levels of interactive

communication and transport occur between these two organelles. Exported FAs can be

modified in the cell after they are exported and integrated into their cell location (i.e.

galactolipids, phospholipids, spheringolipids, etc.). Membrane-bound desaturases, as

well as a soluble chloroplast desaturase can be activated to add or change the location of

a cis-double bond. Desaturases introduces a physical "kink" into a fatty acid chain,

which maintains membrane mobility and permeability, and reduces the temperature at

which the lipid melting temperature occurs. The temperature at which the impermeable

and inflexible physical membrane gel phase occurs under this model depends on the

unsaturated FA composition. A continuum of membrane flexibility is implied from

above-freezing to below-freezing depending on FA composition. Artificial membrane,

FA mixture behavior, and theory in vitro may be different than whole-plant FA

composition in the field present some challenges. Age of tissue, organ sampled, seasonal

changes (presumably as a result of warming or cooling temperatures), above-freezing

temperature treatment, and plant genotype may affect FA composition. Assumptions

made in vitro may be difficult to translate to the field. Stage of plant development has an

effect on FA composition (Nishida and Murata, 1996). Grasses vary in predominant,

unsaturated FA composition by organ sampled (Massard et al., 2000). Seasonal changes







68

in FA composition can occur in grazed grass and clover forages during the season from

spring through fall (Loor et al., 2002). Cold treatment (above-freezing temperatures) of

grasses caused changes in tissue FA composition (Samala et al.,1998).

By studying FA composition over time insight can be gained on the relative FA

composition as a fraction total extracted fatty acids (g kg-' TEFA). Grazed orchard grass

(Dactylis glomerata L.) FA composition, quantified as g kg-' TEFA, changed from May

to July (Table 4-1). Saturated FA composition increased from May to July sampling

dates. The trend for saturated FA composition to increase with increasing temperature

from May to July would be expected from the FA theory. Small changes in the fraction

increase of the fraction of TEFA are seen in the data (Table 4-1) for 14:0 myristic, and

18:0 stearic. The 16:0 palmitic FA fraction increased the most of all the saturated FAs

(from 192 to 224 g kg-' during the May to July sampling period). The tri-unsaturated FA,

18:3 linolenic, decreased from 558 to 501 g kg-1 during the season. The model of FA

saturation fraction increasing during warmer temperatures and decreasing during cooler

temperatures would explain the plant response to temperature by increasing the linolenic

fraction of TEFA. However, only small or no increases in other unsaturated FAs (16:1

palmitoleic, 18:1 oleic, and 18:3 linoleic) occurred during the season (Table 4-1). In

orchard grass, leaf linolenic accounted for the largest portion (> 500 g kg-') of the total

extracted FAs (Table 4-1). Depending on the month of the season, di-unsaturated

linoleic or saturated palmitic FA accounted for the second or third highest g kg-' TEFA.

The data would have been more helpful in providing fall pre-freeze event analogies for

bahiagrass if FA analysis could have been done throughout the growing season into

October.







69

Table 4-1. Fatty acid composition of grazed orchard grass (Table from Loor et al.,
2002).
Total extracted fatty acids
P > F statistic of
Fatty acidt May June July season effect
g kg1
14:0 0.05 0.07 0.06 0.01
16:0 19.20 21.10 22.40 0.01
16:1 0.02 0.04 0.03 0.93
18:0 1.60 1.80 1.90 0.01
18:1 2.20 1.80 2.20 0.01
18:2 20.40 20.50 21.10 0.01
18:3 55.80 53.40 50.10 0.01
?14:0 = Myristic, 16:0 = palmitic, 16:1 = palmitoleic, 18:0 = stearic, 18:1 = oleic, 18:2=
linoleic, 18:3 = linolenic

Grasses vary in predominant, unsaturated FA composition by organ sampled. In

bermudagrass (Cynodon dactylon L.) crowns linoleic and linolenic accounted for 706g

kg-' TEFA in a cold-tolerant cultivar Midiron and 693 g kg-' in a cold-sensitive line U3

before imposing above-freezing cold temperature treatment (Samal, et al., 1998). In

seashore Paspalum (Paspalum vaginatum Swartz.) crowns, the predominant FAs were

linoleic and linolenic, which accounted for 690 to 726 g kg-' TEFA, depending on the

genotype, before cold temperature treatment (Cyril et al., 2002). Perennial ryegrass total

unsaturated FA g kg-' TEFA and an unidentified group of pasture grass leaves was

reported to be 899 g kg-' and 804 g kg-', respectively (Hawke, 1973). Perennial ryegrass

leaf was predominately composed of two unsaturated FAs (682 g kg-' linolenic,

146 g kg-' linoleic) and one saturated FA (119 g kg-' stearic).

Cold (above-freezing) treatment of grasses changes tissue FA composition. In

bermudagrass, cold-tolerant Midiron and cold-sensitive U3 crown tissue FA composition

responded to above-freezing cold acclimation treatment by changing their FA

composition (Samala et al., 1998). Unsaturated FA: saturated FA ratio (USFA:SFA) is a

convenient method of expressing FA composition. At the beginning of the experiment,







70

both cultivars had a USFA:SFA ratio of 2.65. At the end of the experiment, USFA:SFA

ratios for Midiron and U3 were 2.94 and 2.71, respectively. Linolenic (C18:3) increased

by 138 g kg-' TEFA in Midiron, accounting for the majority of the increase in

USFA:SFA. In seashore Paspalum (Cyril et al., 2002) linolenic (C18:3) was the FA that

increased (from 219 g kg-' to 234 g kg-' TEFA) as a result of cold acclimation treatment.

Such a small increase in USFA minimally changed the USFA:SFA from 2.33 to 2.36.

Reported FA changes in seashore Paspalum are so small that FA composition may not

explain differences in genotype cold-tolerance. If FA composition of membrane

influences cell freeze-tolerance through increased membrane fluidity, FA changes would

have to be much larger to approximate artificial membrane and mixture behavior at

below-freezing temperatures.

The double bond index (DBI) has been used as a method to compare plant tissue

FA composition. DBI is calculated as a weighted portion of the poly-unsaturated FAs [(2

x C18:2+3 x C:18:3)/100]. In bermudagrass DBI increased most for cold-tolerant

Midiron crown tissue, rising from an initial 1.67 prior to cold treatment to 1.80 after

cold-acclimation (Samala et al., 1998). Cold-sensitive U3 bermudagrass DBI appeared to

change very little as a result of cold-acclimation treatment, from 1.62 to 1.65. In seashore

Paspalum, cold-tolerant SeasIslel DBI increased the most, from 1.77 to 1.95 (Cyril et al.,

2002). Intermediate cold-tolerant cultivar Adalyd had an intermediate DBI (1.65)

initially but at the end of the cold acclimation treatment had the same DBI (1.65) as cold-

sensitive line PI299042.

Currently, there is a lack of information on bahiagrass leaf fatty acid composition.

Membrane fluidity may be an important mechanism in freeze-tolerance for freeze-

tolerant lines. The region of initial damage in freeze-sensitve bahiagrass lines appeared to







71

tolerant lines. The region of initial damage in freeze-sensitve bahiagrass lines appeared to

be in the midrib region, initially, before the entire leaf blade was damaged upon thawing

after a freeze event. The bundle sheath cells and mesophyll cells surrounding the vessel

region would be the region that would experience membrane stress from dehydration and

cell volume reduction during ice formation and rehydration and cell volume increase

during a freeze/thaw cycle. Buchanan et al. (2000) have shown that the majority of leaf

lipids in Arabidopsis thaliana L. (564 g kg-' leaf DM) is involved with membrane

glycerolipids, another 27 g kg-' leaf DM leaf membrane sphingolipids and sterols, and

259 g kg-' leaf DM lipids associated with chlorophyll. Thus, the majority, 591 g kg-' leaf

DM of leaf total lipid contents, is involved with the leaf membrane. The discussion of

the literature and theory of unsaturated FA composition of the plasmalemma contributing

to membrane fluidity under freeze stress suggests that a larger portion of unsaturated FA

may contribute to membrane fluidity. Membrane fluidity is needed under freeze-stress

freeze/thaw cycles. If the mechanism of the LTFT trait in bahiagrass is a result of FA

composition, then it is hypothesized that the lines with freeze-tolerance should have

higher unsaturated g kg-' TEFA, higher USFA:SFA and higher DBI than lines with

freeze-sensitive lines.

Material and Methods

Osmolality

Nine lines representing varying LTFT were studied (Chapter 2). Lines that were

freeze-sensitive included 1-30-3, 1-30-4, 2-22-1, and FL9. An intermediate line was C4-

36. Lines that expressed freeze-tolerance were OK-1, OK-2, C06, and FL67. Plants

were vegetatively propagated 11 November 2004. Roots of vegetative portions were







72

dipped in Hormodin-2 (0.3% indole-butyric acid, E.C. Geiger, Inc. Harleysville, PA).

Potting media used as the soil for this study was Scotts Terralite Agricultural Mix

(Scotts-Sierra Horticultural Production Company, 14111 Scotts Lane Rd., Marysville,

OH 43041). The round plastic pots were 18 cm in diameter x 18 cm in height, Classic

400 Nursery Supplies, Farless Hills, PA. Nutrient management was 1 g of a 16-4-8-1

N-P205-K20-Fe analysis granular fertilizer pre-plant incorporated into the media of

each pot. One g Ironite 1-0-0 (Ironite Products Company, Scottsdale, AZ 85258) was

applied to the media in each pot to prevent iron chlorosis. Irrigation was conducted daily

by hand to keep moisture as uniform as possible. On 23 November 2004, plants were

placed in a greenhouse with a heater set to come on whenever the temperature fell below

200C. An air conditioning unit was set to cool the greenhouse whenever temperatures

exceeded 300C. Plant location was randomized within the greenhouse to account for

temperature variability. A minimum/maximum thermometer was placed in two locations

to record temperature variability. Bahiagrass has been shown to be sensitive to daylength

(Sinclair et al., 2001; Sinclair et al., 2003), and these lines were grown during short days.

We could assume that acclimation from shortened daylength would have occurred.

A Wescor 5500 Vapro Pressure Osmometer (Wescor, 459 S. Main Street,

Logan, Utah 84321) was used to record osmolality. Potted plants were removed from the

greenhouse 26 December 2004 and carried to the laboratory. Fresh leaf blades were cut

at the collar of the third fully expanded leaf in a whorl. A 5mm plug of lamina of third

leaf was cut with a No. 2 cork cutter covering the midrib section between 2 and 2.5 cm

out on the leaf blade from the leaf collar. This region was targeted because of damage

visualized in the midrib area as previously discussed (Chapter 3). Immediately the leaf

disc was cut in half along the midrib vessel with a disposable microtome blade. This was







73

done to prevent contamination of the osmometer chamber by protruding midrib or curled

lamina disc. The two halves of the leaf disc were held with tweezers and immediately

sprayed with Cr ok\\ ik' (Shield Chemical Co. Damon/IEC Division, 300 2nd Ave.,

Needham, MA 02194). Minimum temperatures produced with Cryokwik were recorded

to -460C. The intended purpose of freezing samples was to lyse cells. Immediately on

thawing, the two halves of the leaf disc were placed in the osmometer chamber. Values

were only utilized only if the osmometer calibrated consistently between runs. If

calibration was not achieved, contamination had occurred in the osmometer chamber, and

the entire unit was disassembled, cleaned, warmed to operating temperature, and

calibrated before running another freshly cored lamina sample.

Fatty Acid Composition

Tifton 9 bahiagrass was harvested 25 October 2005 from a field that had been cut

for hay. This field was selected because it was isolated from any bahiagrass by planted

pine trees, thereby ensuring population purity, having been planted from certified seed.

Sufficient material was harvested (41 g fresh weight) to develop extraction methodology.

Clone leaf material was limited in the diallel experiment (Chapter 5) plot at University of

Florida North Florida Research and Education Center-Suwannee Valley (NFREC-

Suwannee Valley). Extraction methodology was refined on Tifton 9 bahiagrass before

using clone leaf material from the diallel experiment plots. Leaf whorls were cut at the

collar of the third fully expanded leaf, and then placed on ice in a cooler. Samples were

transported to the laboratory. Bagged samples were dipped in liquid nitrogen for 5

seconds, and then stored in a freezer at -200C.

To prevent losing fresh green leaf tissue to an early frost, on 29 October 2005

vegetative clone leaves were harvested at the diallel plot at University of Florida







74

NFREC-Suwannee Valley. Random leaf samples were selected from each 10-plant plot.

Leaf whorls were cut at the collar of the third fully expanded leaf. A minimum of 30 g

fresh plant material was harvested from each plot, sealed in a plastic bag and kept in an

iced cooler. Four blocks of harvested plant material from parent clone rows in the diallel

study at NFREC-Suwannee Valley were sampled. Two replications per block for a total

of eight samples per LTFT clone were used. When all plots were harvested, samples

were transported to the University of Florida at Gainesville, Florida, where samples were

dipped in liquid nitrogen for 5 seconds, then stored in a freezer at -200C. Grinding of

samples was done to obtain uniform leaf particles for FA extraction. Uniform particle

size should reduce FA extraction variability. Leaf samples were removed frozen from

-200C storage and cut into roughly 2 cm lengths with stainless steel scissors. Samples

were loaded into the stainless steel grinding cup (W\Il ) '), covered in liquid nitrogen,

macerated and pushed down to the cutting blades with a ceramic pestle (Coors), then

covered in liquid nitrogen and ground. The grinding sequence was with the lowest

setting, ground for 10 seconds, then inspected and mixed with a stainless steel spatula.

Liquid nitrogen was added, ground at the second lowest setting for 10 seconds, mixed

and inspected, with a final addition of liquid nitrogen and grinding for an additional 15

seconds at that setting. The ground Tifton 9 was kept in a sealed 150 ml beaker in a

freezer to develop fatty acid extraction methodology and to use the tissue as a standard in

a more complete experiment.

Fatty acid content ofbahiagrass was unknown so a preliminary trial was

conducted to determine which sample size and extraction method would be the most

efficient to process samples. Three different tissue sample sizes were used because of the

concern of insufficient fatty acid quantities: 0.5, 1, and 2 g. Three extraction procedures







75

were compared: 1) Cold extraction using a 50:50 hexane/tert-Butyl ether, 2) petroleum

ether and 3) direct esterification with methanoloic HC1 with 2, 2-dimethoxyporpane

(DMP). A peanut oil standard was used with blanks. All methods of extraction produced

similar percentages of fatty acids. The half g sample produced areas on the integrator for

unique FA's that had to be magnified on the gas chromatograph (GC) integrator output.

It was decided to use the 1 g tissue sample size because it produced clear and replicable

areas that could be smoothly and consistently integrated.

Extraction method samples were analyzed on a Hewlett Packard 5890 gas

chromatograph (GC). Procedures were compared for time involved in FA extraction as

well as consistent results.

Extraction Method 2, using petroleum ether was selected because of the ease of

extraction in addition to yielding FA profile results consistent with all other methods

used. Method 2 was modified to ensure that ground leaf material was covered by

reagents. The modified Extraction Method 2 in its entirety was as follows: A 1 g ground

leaf sample was placed in a P) ri\' (Coming) 16 x 125 mm disposable screw cap culture

tube via a funnel, 2 ml of a 1M methanoloic-HCl with 5% (v/v) 2,2-dimethoxypropane

(DMP) was added to each tube and tubes were sealed with Teflon-lined caps. Tubes

were placed in an 800C water bath and heated for 1 h. Tubes were cooled in ambient

temperature water bath for 10 min. Two ml 90 g kg-' NaCl were added to each tube to

stop the reaction. Hexane (1.5 ml) was then added to each tube and tubes thoroughly

mixed by vortexing. Tubes were centrifuged at 3,000 G for five min to allow phase

separation. Approximately 1 ml of the upper phase containing the fatty acid methyl

esters (FAMES) was pipetted and sealed into auto sampler vials for GC analysis of FA







76

profile. Samples were stored at -200C immediately until GC analysis could be

conducted.

GC analysis included hexane blank standards for every 10 bahiagrass tissue

samples. The Association of Official Agricultural Chemists (AOAC) standards of FA

(FAME Mix RM-6, Supelco, 595 North Harrison Rd., Belefonte, PA) were included at

the beginning, middle and end of the GC analysis. Analysis of variance was conducted

with SAS (SAS Institute Inc., 1987).

Results and Discussion

Osmolality

There were significant differences among genotypes in regard to leaf disc

osmolality (P < 0.019). Mean leaf disc osmolality by genotype is listed in Table 4-2

separated by Duncan's multiple range test at the 5% level of confidence.

Leaf disc osmolality did not correlate with bahiagrass line LTFT class

(P < 0.261). Differences between the grand mean of these two classes were not

significant: mean freeze-sensitive = 623 mmol kg-' and freeze-tolerant = 649 mmol kg-.

Lines that were freeze-tolerant ranged from 545 to 817 mmol kg-'. Lines that were

freeze-sensitive ranged from 489 to 859 mmol kg-'. Therefore the mechanism of LTFT

does not appear to that of increased solute synthesis and storage in cell vacuoles in cells

around the midrib vascular bundle, nor in higher solute concentrations in the xylem

vessels.

Leaf disc osmolality can be used to calculate freeze temperature depression. The

required osmolality can be calculated for the freezing point depressed below 0C. An

example follows using Van't Hoff's equation (Af = iKm) for the solute concentration

needed to depress the freezing temperature to a Af = -60C. Assume that the disassociation







77

constant of plant solutes (such as simple sugars) approximates a value of I = 1. The

freeze constant for water = Kf= -1.86C mol kg-'. We can solve for m (in units of mol kg-')

by rearranging Van't Hoff's equation: m = Af / Kf= -6C/-1.86C mol kg' = 3.2 mol kg1.

Freeze-tolerant lines FL67, OKI, OK2 and CO6 would need to have had osmolality

values approaching 3 moles kg-'. Instead, osmolality values ranged from 545 to 817

mmol kg-'. Another way of looking at the data is that an osmolality/molarity of 545

mmol kg' would give a Af = (-1.860C m-') x (0.545 m kg') = 1.0C. By the same

method of calculation 817 mmol kg-' would give a Af = -1.50C. Actual controlled freeze

chamber data showed canopy injury occurred at -60C, thus showing solute concentration

was not the mechanism protecting bahiagrass lines with freeze-tolerance.

Table 4-2. Osmolality of bahiagrass sexual diploid lines representative of freeze-
tolerance and freeze sensitivity.
Canopy freeze-damage Leaf disc
LTFT Genotype rating osmolality
Class Line (1 to 9) mmol kg-1
Sensitive 1-30-3 ---t 859a*
Tolerant FL67 2.01 817a
Tolerant OK-1 2.6 679abc
Sensitive 2-22-1 --- 631abc
Sensitive 1-30-4 --- 610abc
Tolerant CO6 2.0 556bc
Tolerant OK-2 2.2 545bc
Sensitive FL-9 6.2 489c
Intermediate C4-36 4.4 463c
*Means with the same letter are not significantly different at P = 0.05 confidence level.
TSelected after a freeze event December 3, 2002 at Hague, Florida from visual rating
compared to Argentine (canopy freeze rating of 2, slightly damaged) vs. numbered
sensitive lines that were extremely sensitive, being frozen to the ground (canopy freeze-
damage rating of 9, all foliage damaged).
$Canopy freeze-damage rating from controlled freeze to -60C in environmental growth
chamber.

Fatty Acid Composition

Seven FAs accounted for the major composition in the bahiagrass lines tested

(Table 4-3). Other outputs were small and not identified, which is a similar result found









by other investigators (Cyril et al., 1998; Samala et al., 1998). Finding and

characterizing seven major FAs for bahiagrass contrasts with only four major FAs

characterized in bermudagrass and seashore Paspalum (C16:0, C18:0, C18:2, C18:3).

Myristic (C14:0), a saturated FA, was not significantly different across lines grown in the

diallel plot (ranging from 9 to 11 g kg-' TEFA). Tifton 9 was significantly different, but

only slightly (8 g kg-' TEFA). Tifton 9 values were used as standards to ensure that the

GC was operating consistently while analyzing the lines that were vegetatively

propagated in the diallel cross plot. All other FAs (C16:0, C16:1, C18:0, C18:1, C18:2,

and C18:3) were significantly different (P < 0.0001) among genotypes.

Palmitic (C16:0) comprised the largest portion of all saturated FAs, ranging from

175 to 206 g kg-' TEFA, depending on the bahiagrass line. Palmitic, a saturated FA, was

the only FA in bermudagrass and seashore Paspalum that did not change in composition

under cold treatment.

Table 4-3. Bahiagrass leaf blade fatty acids, as a fraction of total extracted fatty acids
(g kg-' TEFA).
Saturated fatty acids Unsaturated fatty acids
LTFTf Linel C14:0 C16:0 C18:0 C16:1 C18:1 C18:2 C18:3
Sensitive Argentine 1la* 175e 17c 35bc 15b 134d 544a
Sensitive 1-30-4 9ab 183cde 17c 29d 16b 174a 516ab
Sensitive 1-30-3 11a 185cd 17c 31cd 15b 173a 497ab
Sensitive 2-22-1 Ila 195b 20b 38b 18b 167ab 474b
Sensitive FL9 10a 206a 20b 31cd 18b 170a 478b
Intermediate C4-36 10a 182de 20b 37bc 16b 158bc 502ab
Tolerant F67 9ab 192bc 19b 38b 14b 154c 494ab
Tolerant OK-1 9ab 189bcd 19b 33bcd 16b 158c 512ab
Tolerant OK-2 11a 195b 18bc 37bc 15b 154c 499ab
--- Tifton9 8b 213a 24a 51a 28a 140d 364c
"Assigned LTFT class based on controlled freezer trials or field behavior.
}All lines vegetatively propagated and managed the same, except Tifton 9, which was a
population that was harvested from an adjacent plot to standardize protocol before
analyzing limited leaf material from the diallel cross.
*Means followed by the same letter are not significantly different at the 5% level.
**C14:0 = myristic, C16:0 = palmitic, C16:1 = palmitoleic, C18:0 = stearic, C18:1 =
oleic, C18:2 = linoleic, C18:3 = linolenic.







79

Stearic (C18:0) is a saturated FA, which did not distinguish among LTFT lines in

bahiagrass and constituted a minor portion of the total profile. In bermudagrass and

seashore Paspalum the stearic minimally changed between genotypes in g kg-' TEFA

after cold-treatment acclimation.

Linolenic (C18:3) comprised the largest portion of the unsaturated FAs, ranging

in the diallel cross plot lines from 499 to 544 g kg-' TEFA. Argentine, the most freeze-

sensitive of all the bahiagrasses, had the highest g kg-' TEFA (544), which was

significantly different than all the other lines, regardless if they were freeze-tolerant or

freeze-sensitive. This is the reverse of what would be expected from the literature in

many species (Chapter 1). These results are the opposite of what was found in

bermudagrass (Samala et al., 1998) and seashore Paspalum (Cyril et al., 2002) crowns.

In Bermudagrass, cold-tolerant line Midiron had approximately 85 higher g kg-' TEFA

than cold-intolerant line U3. In seashore Paspalum, cold-tolerant line SeaIslel had

higher (282 g kg-' TEFA) polyunsaturated linolenic (C18:3) than cold-intolerant line

PI299042 (231 g kg' TEFA). These changes in linolenic g kg- TEFA between the two

lines occurred after a 21 d cold treatment of 8C day/4C night temperatures with a daily

10-h photoperiod.

Linoleic (C18:2) was unusually and significantly low in Argentine (134 g kg'

TEFA) yet significantly high in the remaining low-LTFT group of lines (1-30-4, 1-30-3,

2-22-1, and FL9) with values ranging from 167 to 173 g kg-' TEFA Freeze-tolerant and

intermediate-LTFT bahiagrass lines (FL67, OKI, OK2, and C4-36) were associated with

low and significant values that were different from the freeze-sensitive lines (154 to 158

g kg-' TEFA). If Argentine linoleic g kg-' TEFA had not been so low, bahiagrass lines

may have been separated by class of freeze-tolerance based on the differences in this FA.







80

Perhaps this was coincidental in this experiment, but lower C18:2 values have been

associated with cold-tolerant lines that have been subjected to acclimation treatment in

other C4 grasses. In seashore Paspalum, cold-tolerant SeaIslel C18:2 was reduced by 56

g kg' TEFA and only by 26 g kg-' TEFA for cold-intolerant PI299042.

In bahiagrass lines grown under the same condition, oleic g kg-' TEFA did not

change between lines and constituted only a minor portion of the FA profile. The

difference in Tifton 9 oleic g kg-' TEFA could be attributed to different growing

condition parameters in an adjacent plot. Palmitoleic (C16:1) constituted another minor

portion of the FA profile and did not distinguish between classes of bahiagrass lines

differing in LTFT.

This characterization of the FA profile of bahiagrass vegetative clones grown

during short daylengths and cool weather (harvested 29 October 2005) showed there was

no significant FA profile that could be used to distinguish whether a line had high- or

low- LTFT. In fact, Argentine, the lowest LTFT line had the highest C18:3 g kg-' TEFA

of all the lines. These results were in contrast to two other C4 species which had the

reverse data for C18:3 g kg-' TEFA, where the most cold-tolerant lines had the highest

C18:3 and the lowest C18:2 g kg-' TEFA (Cyril et al., 2002; Samala et al., 1998).

Another way to compare polyunsaturated FA profiles is to use the DBI

(Table 4-4). The DBI was significant for genotypes (P < 0.0001) but not for LTFT class.

The DBI reflected genotype differences. The DBI was not useful in selecting lines with

freeze-tolerance. Bahiagrass DBI ranged from 1.76 to 1.90. In Bermudagrass, the cold-

tolerant line DBI was 1.81, and the cold-intolerant line DBI was 1.65. Bahiagrass DBI

values were close to the range of published bermudagrass values. DBI did not identify

which lines had freeze-tolerance.







81

Table 4-4. Leaf blade double bond index (DBI) and unsaturated fatty acid: saturated
fatty acid ratio (USFA:SFA) of lines representative of freeze-tolerance and
freeze sensitivity.
LTFT Line DBI USFA:SFA
Sensitive Argentine 1.90a* 3.59a
Sensitive 1-30-4 1.89a 3.5 ab
Sensitive 1-30-3 1.84abc 3.36ab
Sensitive 2-22-1 1.76c 3.08de
Sensitive FL-9 1.77bc 2.94e
Intermediate C4-36 1.82bc 3.37ab
Tolerant FL-67 1.80bc 3.13de
Tolerant OK-1 1.85ab 3.30bc
Tolerant OK-2 1.80bc 3.14cd
--- Tifton 9 1.64d 2.73f
*Means with the same letter are not significantly different at P = 0.05 confidence level.

The DBI values may be helpful in characterizing profile relationships but perhaps

should be used with caution until a clearer understanding of the role of FA saturation and

membrane freeze-tolerance is achieved. The bahiagrass data showed that having a larger

proportion of C18:3 did not keep Argentine from still being the most sensitive line to

canopy damage during a freeze event. Obviously, in bahiagrass, the FA profile does not

explain why some lines are freeze-tolerant or freeze-intolerant.

Another method of indexing FA profiles is calculating the unsaturated

FA:saturated FA ratio (USFA:SFA), which is shown in Table 4-4. Again, the same

limiting reasons, that the DBI did not help identify bahiagrass lines differing in LTFT,

apply to the USFA:SFA. Values for bahiagrass lines at this sampling time appear to be

slightly higher (2.73 to 3.59) than those for bermudagrass acclimated with cold

temperature (2.65 to 2.94). In bermudagrass, the higher USFA:SFA identified the cold-

tolerant line. Values for bahiagrass ratios were much higher than for seashore Paspalum.

In seashore Paspalum, the line with the higher USFA:SFA ratio identified the cold-

tolerant line (2.65) and the cold-intolerant line (2.31). Perhaps USFA:SFA ratios of g kg'

TEFA may be too simplistic to explain why genotypes vary in freeze-tolerance. Neither







82

leaf osmolality nor leaf FA profile experiments identified mechanisms which might

distinguish one line ofbahiagrass from the other in LTFT, as rated by canopy damage.

Summary

More than one mechanism could be involved in mediating LTFT. Apoplastic and

symplastic mechanisms might be involved. Freeze-point depression through increased

osmolality is a mechanism some plants use to prevent freeze-damage at temperatures

below 0C. Results from the osmolality experiment showed that differences in clone

LTFT were not a result of leaf osmolality. Increased membrane fluidity at lower

temperatures through increased polyunsaturated fatty acid content has been associated

with freeze-tolerant plants. Results of the FA experiment showed that unsaturated FA

contents was not the mechanism by which freeze-tolerant clones survive freeze events

with small amounts of canopy damage. In fact, results were the reverse of what would

have been predicted by the unsaturated FA model for membrane fluidity.















CHAPTER 5
GENETIC BEHAVIOR OF THE LEAF-TISSUE FREEZE-TOLERANCE TRAIT

Introduction

Bahiagrass reproduction is controlled by the somatic chromosome number

(Quarin et al., 2001). Tetraploid (2n=4x=40) bahiagrass reproduces by seed that is

formed apomictically, identical to the mother plant. Diploid (2n=2x=20) bahiagrass

reproduces by seed that is formed by sexual union of male and female gametes. Diploid

bahiagrass will be discussed in detail later. Both somatic bahiagrass chromosome

numbers, diploid and tetraploid, are heterozygous. It is in the diploid, sexual condition,

that a range of heterozygous genetic information can be expressed and selected for in the

progeny. In the diploid condition progeny trait expression can be used to quantify trait

heritability, mode of gene action, general combining ability, and specific combining

ability. The diploid bahiagrass somatic condition was used in this study of LTFT trait to

quantify:

* Trait heritability
* Mode of gene action
* General combining ability
* Specific combining ability

Results will be discussed in terms of practical applications in an effort to develop diploid

bahigrass populations with improved LTFT.

Tetraploid Reproduction

Tetraploid (4x) bahiagrass reproduces apomictically (without mixing of the

gametic genetic information through normal double fertilization). Tetraploid bahiagrass







84

has been considered an obligative apomictic type of reproduction (where unreduced

embryos produce seed that are genetically identical to the maternal plant). Facultative

tetraploid apomictic plants occur in extremely small numbers naturally and have been

studied for their mechanism of polyembryonic seed set (Quarin, 1999), and degree of

sexuality (Chen et al., 2001), or used in crosses (Burton and Hannah, 1986). Tetraploid

bahiagrass cultivars, lines or biotypes have been cited as having less frost resistance and

more winter-killing than diploid bahiagrasses (Burton, 1946; Burton, 1955). Due to the

complex forms of apomixis and difficulty in identifying sexual or facultative apomicts in

tetraploid bahiagrass frost sensitive cultivars were not used in this research to determine

the genetics of leaf-tissue freeze-tolerance trait.

Diploid Reproduction

Diploid (2x) bahiagrass reproduces sexually (Burton, 1948; Burton, 1982). A

high-degree of cross-pollination occurs in diploid bahiagrass (Werner and Burton, 1991).

Self-pollination of diploid bahiagrass showed a high degree of self-incompatibility,

postulated to be of the S type (Burton, 1955). When 57 clones of Pensacola bahiagrass

were self-pollinated, seed set averaged 6% compared to the same clones which averaged

89.5% seed when open-pollinated (Figure 5-1). A small number of plants from a sexual

population may not be as self-incompatible as the majority of the population and may be

able to set as high as 25% to 30% seed (Burton, 1955). Therefore, there may be a limited

number of clones within a population which may contribute less trait expression in a

cross due to the degree of selfing. Identification of the parent clone ability to self may be

important when conducting crosses in bahiagrass that investigate the heritability of trait

expression in progeny.