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Genetic, Molecular, and Breeding Study of Coleus (Solenostemon scutellarioides (L.) Codd) during Growth and Development

Permanent Link: http://ufdc.ufl.edu/UFE0021516/00001

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Title: Genetic, Molecular, and Breeding Study of Coleus (Solenostemon scutellarioides (L.) Codd) during Growth and Development
Physical Description: 1 online resource (146 p.)
Language: english
Creator: Nguyen, Phuong Ngoc
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: anthocyanin, branching, breeding, coleus, flowering, genetics, habit, plant, scutellarioides, solenostemon, trailing, vigor
Horticultural Science -- Dissertations, Academic -- UF
Genre: Horticultural Science thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Solenostemon scutellarioides (syn. Coleus blumei Benth.), commonly known as coleus,is a member of the Lamiaceae family and is highly prized for its vibrant and colorful foliage. In the last decade, coleus has gained popularity in the floriculture industry with the introduction of a large number of vegetatively propagated varieties with novel foliage colors. Besides novel foliage colors, opportunities exist to increase their commercial value by manipulating plant architecture. A breeding program was initiated at the University of Florida with focus on two main objectives: (1) the development of new trailing types with bright foliage colors, and (2) the development of new varieties with improved vigor, foliage color quality and delayed flowering induction for production companies and consumers. At the completion of the breeding program, regarding new trailing types, several with bright orange colors were successfully produced and are currently being trialed in the greenhouse. Over a three year period, twenty-one new varieties were released from the breeding program with improved vigor, foliage color quality,and delayed flowering induction. During the development of new varieties with improved color quality, many varieties in the program were observed to become dark red when exposed to high light intensities. As a lack of information existed to explain color change in coleus, a focus on a third objective was pursued. A study was conducted on two related coleus varieties, 'Royal Glissade' and 'UF06-1-6', to determine the physiological and molecular mechanisms involved in this color change phenomenon. Light intensity was found to affect coloration by inducing the accumulation of anthocyanin pigments in the leaves. Expression of the structural genes PAL, CHS, F3H, DFR, ANS, and UFGT and regulatory genes AN1 and AN2 were analyzed. DFR and UFGT were found to play an important role in foliage coloration in coleus.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Phuong Ngoc Nguyen.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Clark, David G.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2008-06-30

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Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021516:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021516/00001

Material Information

Title: Genetic, Molecular, and Breeding Study of Coleus (Solenostemon scutellarioides (L.) Codd) during Growth and Development
Physical Description: 1 online resource (146 p.)
Language: english
Creator: Nguyen, Phuong Ngoc
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: anthocyanin, branching, breeding, coleus, flowering, genetics, habit, plant, scutellarioides, solenostemon, trailing, vigor
Horticultural Science -- Dissertations, Academic -- UF
Genre: Horticultural Science thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Solenostemon scutellarioides (syn. Coleus blumei Benth.), commonly known as coleus,is a member of the Lamiaceae family and is highly prized for its vibrant and colorful foliage. In the last decade, coleus has gained popularity in the floriculture industry with the introduction of a large number of vegetatively propagated varieties with novel foliage colors. Besides novel foliage colors, opportunities exist to increase their commercial value by manipulating plant architecture. A breeding program was initiated at the University of Florida with focus on two main objectives: (1) the development of new trailing types with bright foliage colors, and (2) the development of new varieties with improved vigor, foliage color quality and delayed flowering induction for production companies and consumers. At the completion of the breeding program, regarding new trailing types, several with bright orange colors were successfully produced and are currently being trialed in the greenhouse. Over a three year period, twenty-one new varieties were released from the breeding program with improved vigor, foliage color quality,and delayed flowering induction. During the development of new varieties with improved color quality, many varieties in the program were observed to become dark red when exposed to high light intensities. As a lack of information existed to explain color change in coleus, a focus on a third objective was pursued. A study was conducted on two related coleus varieties, 'Royal Glissade' and 'UF06-1-6', to determine the physiological and molecular mechanisms involved in this color change phenomenon. Light intensity was found to affect coloration by inducing the accumulation of anthocyanin pigments in the leaves. Expression of the structural genes PAL, CHS, F3H, DFR, ANS, and UFGT and regulatory genes AN1 and AN2 were analyzed. DFR and UFGT were found to play an important role in foliage coloration in coleus.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Phuong Ngoc Nguyen.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Clark, David G.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2008-06-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021516:00001


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1 GENETIC,MOLECULA RANDBREEDINGSTUDYOFCOLEUS ( S olenostemon scutellarioides (L.)CODD) DURINGGROWTHANDDEVELOPMENT By PHUONG NGOC NGUYEN ADISSERTATIONPRESENTEDTOTHEGRADUATESCHOOL OFTHEUNIVERSITYOFFLORIDAINPART IALFULFILLMENT OFTHEREQUIREMENTSFORTHEDEGREEOF DOCTOROFPHILOSOPHY UNIVERSITYOFFLORIDA 2007

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2 2007Phuong Ngoc Nguyen

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3 Tomyfamilyandfriendsfortheirloveandsupport.

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4 ACKNOWLEDGMENTS I thankDr.DavidClarkforbeingmya dvisorduringmyPh.D.researchandshowingme the politicsofsuccess.I thankmycommitteemembers,Dr.ZhanaoDeng,Dr.MariaGallo,Dr. KennethQuesenberryandDr.StevenSargentfortheirhelpandguidance.IamgratefultoDr. Quesenberryforhishel p andadvicewithquestionsregardingtopicsrelatedtoplantbreeding Ithankmylabmembers:Dr.JulianVerdonk,JooYoungKim,andThomas Colquhoun.I thankJulianforhis wonderful friendshipinsideandoutsideofthelabandforhelpful adviceon top icsrelatingtomolecularwork.IthankThomaswhomadetheofficeverylivelyand Joo Youngforherpatienceandkindness. Ithankeveryonewhohelpedwiththemassiveamountofgreenhouselaborthatwas requiredto makethecoleusbreedingprogramsucce ssful.IamsincerelygratefultoBecky HamiltonandGene Hannah.IthankGeneforhelpingwiththecoleuseventhoughretirement wasjustaroundthe cornerandBeckyforhercompanionshipandsupportthroughdifficulttimes.Iamgratefulfor thehelppro videdbythefollowingundergraduatestudentsfortheirhelp inthepotting, propagating,anddiscardingofthousandsofcoleusplants:CristinaTan,Holly Koorneef,Mark Jones,KyleKneel,andHeatherCampbell.IespeciallythankCristinafor herunceasin genergyin thegreenhouse,cart drivingability,andaboveallherfriendship. IacknowledgeallofmyfriendsfortheirfriendshipandsupportwhenIneeded themmost: AnaMariaBorda,ErnestoFonseco,CarmenValero,CamilaBrito,AnneFrances, KateSant os, DavidDobos,AlisonHeather,ElizabethNorris,KarunaRamachandran,and CatherineLangford.I thankmyfriendRosanneVernon,forwithoutherIwouldnotbeheretoday. Iamappreciativeofmyfamilyfortheirlove.IamgratefultomyMomandDadfor giving melifeandshelteringmealltheseyears.Ithankmyparentswhoinsistedthateducationwas

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5 importantandtopursueabetterlifethanwhattheyhad.Ithankmysiblings,Lan,Linda, Diane,and Sonnyfortheirjoysandcomfortandlatenightphone calls. LastbutnotleastIamsincerelygratefulfortheloveandsupportfrommypanino, Valeriano DalCin,whohasbeenmygreatestcompanionduringthelaststretchofmyPhD research.Toone whoismysoulmate,Ihopethecompanionshipwilllastal ifetime

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6TABLEOFCONTENTSpage ACKNOWLEDGMENTS...............................................................................................................4LISTOFTABLES...........................................................................................................................9LISTOFFIGURES.......................................................................................................................10ABSTRACT...................................................................................................................................13CHAPTER1LITERATUREREVIEW.......................................................................................................15IntroductionandRationale.....................................................................................................15LiteratureReview...................................................................................................................17ColeusPloidyandCytology............................................................................................17ColeusGenetics...............................................................................................................18TrailingGrowthHabit.....................................................................................................19Flowering.........................................................................................................................21FoliageColor...................................................................................................................23Summary.................................................................................................................................282THEGENETICSOFTRAILINGHABITANDTHEDEVELOPMENTOFNEWCOLEUS((Solenostemonscutellarioides(L.)CODD))VARIETIESWITHTRAILINGHABITANDBRIGHTERFOLIAGECOLOR.....................................................................30Introduction.............................................................................................................................30MaterialsandMethods...........................................................................................................33PlantMaterialsandGrowthConditionsforBreeding.....................................................33AnalysisforPloidyLevelDifferencesamongParentalGenotypesandHybrids...........34PollenViabilityandMicrosporogenesisAnalysis..........................................................34PollinationMethodology.................................................................................................35GrowthofSeedlings........................................................................................................36RatingScaleforGrowthHabitandFoliageColorBrightness........................................36RatingScaleforGenotypicandChi-squareAnalysis.....................................................36Chi-squareTestforHomogeneity...................................................................................37IntrogressionofColorUsingtheSelfingMethods..........................................................37Results.....................................................................................................................................38DeterminationofPloidyLevelDifferences....................................................................38PollenViabilityandMicrosporogenesisAnalysis..........................................................39GrowthHabitSegregation...............................................................................................40DeterminationofParentalGenotypes.............................................................................41FoliageColorBrightness.................................................................................................43IntrogressionofColor......................................................................................................44Discussion...............................................................................................................................45

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7 3 INFLUENCEOFLIGHTINTENSITYONCOLEUSFOLIAGECOLORCHANGES ..... 61 Introduction ................................ ................................ ................................ ............................. 61 MaterialsandMethods ................................ ................................ ................................ ........... 63 PlantMaterialsandGrowthConditions ................................ ................................ .......... 63 ExperimentalDesign ................................ ................................ ................................ ....... 64 Anthocyanin,c hlorophyllandpHdetermination ................................ ............................ 65 FreshWeightandLightMicroscopy ................................ ................................ ............... 66 RNAisolationandcDNAsynthesis ................................ ................................ ................ 66 IsolationofPartialClonesInvolvedinColoration ................................ .......................... 66 ExpressionAnalysisusingSemi QuantitativeRT PCR ................................ ................. 68 ResultsandDiscussion ................................ ................................ ................................ ........... 69 PhysiologicalMechanismsInvolvedinFoliageColorChange ................................ ...... 69 Lightintensitya ffectsfoliagecolorchangeinRGandUF ................................ ...... 69 RedcolorationinRGandUFfoliageisduetoanthocyaninaccumulation ............. 70 pHisnot afactoraffectingfoliagecolorationinRGandUF ................................ .. 71 Lightintensityaffectschlorophyllconcentrationandfoliagecoloration ................. 71 LightintensityaffectsplantgrowthinRGandUF ................................ .................. 72 MolecularMechanismsAffectingFoliageColorChangeinRGandUF ....................... 73 Isola tionandexpressionofclonesrelatedtoanthocyaninsynthesis ....................... 73 Enzymesinvolvedinanthocyaninsynthesisincoleus ................................ ............. 74 Lighti ntensityandanthocyaninaccumulationandtheireffectsongene expression ................................ ................................ ................................ ............. 75 F3HisresponsibleforimpartingthecolordifferencebetweenRGandUF ............ 77 Conclusions ................................ ................................ ................................ ............................. 78 4 DEVELOPMENTOFNEWNOVELCOLOREDCOLEUSCULTIVARSWITH IMPROVEDVIGOR,COLORQUALITY,ANDLATEFLOWERINGINDUCTION FORTHECOMMERCIALMARKET ................................ ................................ .................. 92 Introduction ................................ ................................ ................................ ............................. 92 MaterialsandMethods ................................ ................................ ................................ ........... 94 BreedingMethodology ................................ ................................ ................................ .... 94 CropCycleandYearlyTrials ................................ ................................ .......................... 95 ResultsandDiscussion ................................ ................................ ................................ ......... 100 Vigor ................................ ................................ ................................ .............................. 100 FoliageColorQuality ................................ ................................ ................................ .... 101 FloweringInduction ................................ ................................ ................................ ...... 103 CultivarRelea ses ................................ ................................ ................................ ........... 104 5 FUTUREWORK ................................ ................................ ................................ .................. 111 APPENDIX NEWCULTIVARRELEASESWITHIMPROVEDVIGOR,BRANCHING, COLORQUALITYANDLATEFLOWERING INDUCT ION 113 Introduction ................................ ................................ ................................ ........................... 113 2005Releases ................................ ................................ ................................ ....................... 113 2006Relea ses ................................ ................................ ................................ ....................... 119

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8 LISTOFREFERENCES ................................ ................................ ................................ ............. 133 BIOGRAPHICALSKETCH ................................ ................................ ................................ ....... 146

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9 LISTOFTABLES Table page 2 1 DNAcountsforRedQueen(RQ)andSedona(S)and14hybridsproducedfrom thecross(RQ)x(S). ................................ ................................ ................................ .......... 49 2 2 Comparativecellsi zeanalysisforRedQueen(RQ),Sedona(S),andfiveF 1 plants. ................................ ................................ ................................ ................................ 49 2 3 PollenviabilityestimatesforRedQueen(RQ),Sedona(S),andfiveF 1 plants. ......... 50 2 4 Chi squaretestforRedQueen(RQ)selfedpopulation. ................................ ................. 54 2 5 Chi squaretestforSedona(S)selfedpopulation. ................................ ........................... 55 2 6 Chi squaretestfortheF 1 population. ................................ ................................ ................ 55 2 7 Chi squaretestandtestforhomogeneityoftheF 1 andF 2 plants. ................................ ..... 56 3 1 Listofthedegenerateprimersandthecorrespondingconservedregions,andthe annealingtemperaturesusedduringamplification. ................................ ........................... 79 3 2 Listofthespecificprimersand oftheannealingtemperatureusedduringRT PCR expressionanalysisoncoleuscDNA. ................................ ................................ ................ 80 3 3. DataanalysisforanthocyanincontentforRGandUFindirectlightandshade conditions. ................................ ................................ ................................ .......................... 84 3 4 Dataanalysismeasuredforchlorophyllcontent(absorbancevalues649nmand 654nm)forRGandUFindirectlightandshadeconditions. ................................ ............ 87 3 5 Listofthepartialclonesinanthocyaninbiosynthesisincoleus. ................................ ....... 89

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10 LISTOFFIGURES Figure page 1 1 Generala nthocyaninsynthesispathway. ................................ ................................ ........... 29 2 1 Parentsusedinbreedingcrossestodevelopnewtrailingtypeswithbrightfoliage color. ................................ ................................ ................................ .............................. 48 2 2 PollengrainsofRedQueen(RQ)andSedona ................................ ............................. 50 2 3 MicrosporogenesisanalysisofRedQueenpollenmothercells. ................................ ..... 51 2 4 RedQueenselfedpopulation. ................................ ................................ ......................... 52 2 5 Sedonaselfedpopulation. ................................ ................................ ............................... 52 2 6 F1populationproducedfromthecrossRedQueenxSedona. ................................ .... 53 2 7 Distr ibutionoftheRedQueen(RQ)selfed,Sedona(S)selfed,andF1population forgrowthhabit. ................................ ................................ ................................ ................. 54 2 8 MeanratingvaluesforcolorbrightnessforRedQueen(RQ)selfed,Sedona(S) selfed ,F1,andtheF2/F3population. ................................ ................................ ................ 57 2 9 F1plantsproducedfromthecrossRedQueen(RQ)xSedona(S)withbrighter foliagecolors. ................................ ................................ ................................ ..................... 58 2 10 F2andF3plants(producedfromselfingF1andF2plants,respectively)with brighterfoliagecolorthanRQandasbrightasS. ................................ ............................. 59 2 11 DevelopmentofCopperPennywithtraili nghabitandorangecolor. ............................. 60 3 1 Visualfoliagecolorchangeofcoleusleavesthroughouttheexperiment. ........................ 81 3 2 Accumulation ofanthocyaninontheepidermalleaftissuesofRGandUFgrownin thedirectligh t(350mol 2s 1)andshade(150mol 2s 1). ................................ .......... 82 3 3 Anthocyanincontent(A535nm)forRG(solidsquare)andUF(solidtriangle)inthe sun(solidline)andintheshade(dashedline). ................................ ................................ .. 83 3 5 pHlevelforRG(solidsquare)andUF(solidtriangle)inthesun(solidline)andin theshade(dashedline). ................................ ................................ ................................ ...... 85 3 6 Totalc hlorophyllcontentforRG(solidsquare)andUF(solidtriangle)inthesun (solidline)andintheshade(dashedline). ................................ ................................ ........ 86 3 7 FreshweightforRG(solidsquare)andUF(solidtriangle)int hesun(solidline)and intheshade(dashedline). ................................ ................................ ................................ .. 88

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11 3 8 ExpressionanalysisofthegenesPAL1,CHS,F3H,DFR,ANS,UFGT,AN1,AN2, andcontrols18SandACTforRoyalGlissade(RG)andU F06 1 06(UF). ................ 90 4 1 Growthofseedlingsatthestartofthecropcycles. ................................ ......................... 106 4 2 30%shadeglassgreenhousetrial. ................................ ................................ ................... 107 4 3 FullsunoutdoorlocationatCitra,Florida. ................................ ................................ ...... 108 4 4 Percentagesofthepopulationreceivingratingscoresoffourorfivefor heightand branchingacrossthethreeyearlytrials. ................................ ................................ ........... 109 4 5 ColorqualityforthemonthsofJune,July,andAugustforyears2004,2005and 2006. ................................ ................................ ................................ ............................ 110 4 4 RoyalGlissade ................................ ................................ ................................ ............... 123 4 5 (A)TwistandTwirl(B)UF03 6 1B(C)UF03 6 1C ................................ ............... 123 4 6 UF03 8 2 4 ................................ ................................ ................................ ..................... 124 4 7 ElectricLime ................................ ................................ ................................ ................. 124 4 8 UF04 5 7 ................................ ................................ ................................ ....................... 125 4 9 UF04 1 8 3 ................................ ................................ ................................ ..................... 125 4 10 UF04 36 33 ................................ ................................ ................................ ................... 126 4 11 UF04 40 26 ................................ ................................ ................................ ................... 126 4 12 UF 04 40 125 ................................ ................................ ................................ ................. 127 4 13 UF04 51 33 ................................ ................................ ................................ ................... 127 4 14 UF04 51 41 ................................ ................................ ................................ ................... 128 4 1 5 UF04 51 42 ................................ ................................ ................................ ................... 128 4 16 UF04 47 64 ................................ ................................ ................................ ................... 129 4 17 SplishSplash ................................ ................................ ................................ ................. 129 4 18 PineappleSplash ................................ ................................ ................................ ........... 130 4 19 VelvetMocha ................................ ................................ ................................ ................ 130 4 20 UF06 4 140 ................................ ................................ ................................ ................... 131

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12 4 21 LemonSunsation ................................ ................................ ................................ ........... 131 4 22 BigRedJudy ................................ ................................ ................................ ................. 132

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13 AbstractofDissertationPresentedtotheGraduateSchool oftheUniversityofFlorid ainPartialFulfillmentofthe RequirementsfortheDegreeofDoctorofPhilosophy GENETIC,BREEDING, ANDMOLECULARSTUDYOFCOLEUS ( S olenostemon scutellarioides (L.)CODD) DURINGGROWTHANDDEVELOPMENT By Phuong Ngoc Nguyen December2007 Chair:DavidCl ark Major:HorticulturalScience Solenostem onscutellarioides (syn. Coleusblumei Benth .),commonlyknownascoleus,is amemberoftheLamiaceaefamilyandishighlyprizedforitsvibrantandcolorfulfoliage.Inthe lastdecade,coleushasgainedpopul arityinthefloricultureindustrywiththeintroductionofa largenumberofvegetativelypropagatedvarietieswithnovelfoliagecolors.Besidesnovel foliagecolors,opportunitiesexisttoincreasetheircommercialvaluebymanipulatingplant architectu re.AbreedingprogramwasinitiatedattheUniversityofFloridawithfocusontwo mainobjectives:(1)thedevelopmentofnewtrailingtypeswithbrightfoliagecolors,and(2)the developmentofnewvarietieswithimprovedvigor,foliagecolorqualitya nddelayedflowering inductionforproductioncompaniesandconsumers.Atthecompletionofthebreedingprogram, inregardstonewtrailingtypes,severalwithbrightorangecolorsweresuccessfullyproduced andarecurrentlybeingtrialedinthegreenhou se.Overathreeyearperiod,twenty onenew varietieswerereleasedfromthebreedingprogramwithimprovedvigor,foliagecolorquality, anddelayedfloweringinduction.Duringthedevelopmentofnewvarietieswithimprovedcolor quality,manyvarieties intheprogramwereobservedtobecomedarkredwhenexposedtohigh lightintensities.Asalackofinformationexistedtoexplaincolorchangeincoleus,afocusona thirdobjectivewaspursued.Astudywasconductedontworelatedcoleusvarieties,Ro yal

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14 GlissadeandUF06 1 6 todeterminethephysiologicalandmolecularmechanismsinvolvedin thiscolorchangephenomenon.Lightintensitywasfoundtoaffectcolorationbyinducingthe accumulationofanthocyaninpigmentsintheleaves.Expressionof thestructuralgenes PAL, CHS,F3H,DFR,ANS ,and UFGT andregulatorygenes AN1 and AN2 wereanalyzed. DFR and UFGT werefoundtoplayanimportantroleinfoliagecolorationincoleus.

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15 CHAPTER1 LITERATUREREVIEW IntroductionandRationale Thegenus Solenostemon isamemberoftheLamiaceaefamilyandcontainsmorethan500 species(Lebowitz,1985),oneofwhichiscoleus( ( Solenostemonscutellarioides (L.)Codd) ) ; ( syns Coleusblumei Benth.; Coleusscutellarioides (L.)Benth.).Coleusisaversatile annual plantthathasbeencommoninthecommercialbeddingplantindustryfordecadesandishighly prizedforitsvibrantandcolorfulfoliage.Originalspeciesutilizedforcultivationand hybridizationofcoleusintheearly1800swererelativelysimpl einregardstodiversityofcolor patterns,leafshape,andgrowthhabit(Stout,1916).Thefirstornamentalcoleuswasintroduced intoEuropefromJavabyDutchhorticulturistM.J.A.Willinkin1851,andduetoitsrapid growthrate,easypropagation,an dsuperiorperformanceinthelandscape,coleusquicklygained popularityasabeddingplant(Lebowitz,1985). In1877,WilliamBullintroduced150newvarietiesofcoleuswithcolorfulvariegations andofferedawideselectionofcoleusseedsforsaleat 0.43(U.S)perseed(PedleyandPedley, 1974).Withhighpricespossibleforagardenplant,thefevertobreednewvarietiesofcoleus ensued,withmaindevelopmentscenteringonbrightcolors,uniqueleafshapesanddistinctive plantforms(PedleyandP edley,1974).However,attheturnofthecentury,thequalityofcoleus declinedsincecoleuswasbeingdevelopedprimarilyasaseedcropandonlythoseplantsthat producedlargeamountsofseedswerefavorable.Thus,thetypesofcoleusthatsoonbecam e availablehadfoliageleavesthatweredullincolorandgrowthhabitsthatwerestunteddueto earlyflowering(PedleyandPedley,1974).Intheearly1940s,coleusbreedingwasrevivedagain duetoagrowinginterestindevelopingcoleusasacommercia lbeddingcrop.By1982,the BeddingPlants,Incseasonsalessummarylistedcoleusasthetenthmostimportantbedding

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16 plantgrownintheUnitedStates(Lebowitz,1985).Inthelastdecade,coleushasgainedfurther popularityinthefloricultureindustry withtheintroductionofalargenumberofvegetatively propagatedvarietieswithnovelfoliagecolors.Besidesfoliagecolor,opportunitiesexistto increasethecommercialvalueofcoleuswithregardtotwokeyareas:(1)themanipulationof plantarchi tecturebydevelopingnewtrailingvarietieswithbrightfoliagecolors,and(2)the developmentofnewvarietieswithimprovedvigor,colorquality,anddelayedfloweringfor productioncompani esandconsumers. Trailinggrowthhabitincoleusisdesirable asitincreasesitsutilityforuseinmixed containers,inhangingbaskets,andasgroundcoversinthelandscape.Fewtrailingvarietiesexist andthoseavailablearegenerallydarkgreenorredinfoliagecolor.Littleinformationisavailable regarding thegeneticsoftrailinggrowthhabitincoleusandtheoriginsofcurrenttrailingtypes remainunclear.Furthermore,noknownresearchtodatehasbeenpublishedonthedevelopment ofnewtrailingvarietieswithbrightfoliagecolors.Inordertodevelo pnewvarietieswiththese desirablecharacteristics,anunderstandingoftheinheritanceandthegeneticscontrollingtrailing growthhabitisrequired. Thesuccessofornamentalplantsforthecommercialmarketdependsoncertain characteristicsdesired bybothproductioncompaniesandconsumers.Inpastbreedingefforts, coleuswasprimarilybredfornovelfoliagecolorwithuprightgrowthhabitsforuseinthehome garden.Forcoleus,desirablecharacteristicsthatareincurrentdemandincludeimproved vigor, colorquality,andlatefloweringinduction.Plantswithgoodoverallplantvigorhavefastgrowth ratesforrapidturnoverandtheabilitytoproducemaximumcuttingsforproduction(Wilkins, 1988).Inadditiontovigor,colorqualityisdesirable incoleusbecauseconsumerspurchase varietiesbasedonaestheticappeal(Catanzaro,2005)andfadedc olorsdecreaseitsproductvalue

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17 (Oren shamir,2003).Coleuswithlateflowerinductionisdesirablebecausevarietiesthatflower earlyhavegreaterlan dscapemaintenanceandadecreaseinfoliagecolorsqualityduringseed production.Consideringthesetraits,acoleusbreedingprogramwasinitiatedin2003atthe UniversityofFloridatodevelopnewcultivarsthroughtraditionalbreedingmethodsfor imp rovedvigorforcommercialproduction,betterfoliagecolorqualityanddelayedflowering inductionforconsumers. Intheprocessofdevelopingnewcoleusvarietieswithimprovedcolorqualityforthe landscape,manyvarietieswereobservedtobecomedark redwhenexposedtohighlight intensities.TworelatedvarietiesRoyalGlissade(RG)andUF06 1 6(UF)(aseedlingproduced byself pollinatingRG)showedthisspecificcolorchangewhenexaminedunderdirectlightand shadeconditions.Asalackofinfor mationexiststoexplainthephysiologicalandmolecular mechanismsforcolorchangeincoleusfoliage,abroad basedapproachwaspursuedtoprovidea baselevelofinformationtoexplainthephysiologicalandmolecularmechanismsinvolvedin colorchange LiteratureReview ColeusPloidyandCytology Thegenus Solenostemon (syn. Coleus )containsseveralwell knownspeciesthataregrown assourcesformedicinalcompounds( C.ambionicus and C.forskohlii ),asfood( C.tuberosus ), andasfoliarornamentals ( C.frederici and C.blumei ).Themostpopularspeciesinthisgenus, S. scutellarioides (syn. C.blumei (Benth.),commonlyknownascoleus,hasbeenincultivation sincethe1800sforitsvibrantandcolorfulfoliage.Althoughmorethan500varietiesexis tonthe commercialmarket,littleisknownaboutitsgeneticsandbreeding(Lebowitz,1985).Diploids, triploids,andtetraploidsexistandchromosom ecountsforseveral Solenostem on speciessuggest basicchromosomenumbersofx=6,7,and 8(Darlington andWylie,1955;D ewet,1958).The

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18 majorityofcytologicalstudieson Solenostemon reporta2n=48chromosomenumber(Reddy, 1952).HakeemandRife(1966)foundtheappearanceofmultivalentsduringmeiosisincertain Solenostemoncultivarswith2n=48chro mosomesandsuggestedpolyploidyforthisspecies. Basedontheirfindingsandthefactthatgeneticexperimentsproducedsimplediploidratios,they furthersuggestedthatvarietiesbelongingto S.scutellarioides wereallotetraploids.Themost currentpl oidynumberforcoleus,asproposedbyGalbraith(1983),indicatedthatallcultivated hybridvarietiesunder S.scutellarioides aretetraploids,withn=24basedonfindingsfromrapid flowcytometry. ColeusGenetics Mostoftheliteratureoncoleusgene ticswasgeneratedintheearlytomid 1900s,with D.C.Rife(GeneticsLaboratory OhioStateUniversity)beingthemajorcontributorin providingtheinheritanceofseveraltraits(Rife,1939;Rife,1940;Rife,1944;Rife,1945).Most ofthetraitsstudi edincoleusaresimplyinheritedandcontrolledbyasinglegene(Lebowitz, 1985).Sincecoleusishighlyprizedforitsfoliage,manyofthegeneticstudiesintheearly1900s predominantlyfocusedonthegeneticsrelatingtoleafcolorandshape.Inreg ardstofoliage color,greencolorationproducedbychlorophyllpigmentsisdominanttodiluteyellow green (BoyeandRife,1938)andthelackofchlorophyll(albinism)iscontrolledbyrecessivegenes (Rife,1948).Solidpurpleonthelowerleafepidermis isdominanttogreen(unpigmented)lower epidermis(Rife1944),andredstemsandveinsaredominanttogreenones(Rife,1948).The brightredandpurplecolorationfoundincoleusisproducedprimarilybyanthocyanins,with purplecolorconferringdomin ance(Rife,1948).Theamountanddistributionofanthocyaninsin theleaves(leafcolor)iscontrolledbyanincompletedominantgene( N )(Rife,1948).In addition,adominantgene( S )isresponsiblefordarksplotchesintheuppersurfaceswhereasthe r ecessivealleleproducesnosplotches(Boye,1948).Inregardstoleafshape,traitsthatare

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19 dominantincludecrinkledleafsurfaces(Rife,1940)andnarrowedleaves(Rife,1972). TrailingGrowthHabit Plantarchitectureisthethree dimensionalorganiz ationoftheplantbody(Godin,2000). Forabovegroundpartsoftheplants,plantarchitectureincludesthebranchingpattern,aswellas size,shape,andpositionoftheleavesandflowerorgans(ReinhardtandKuhlemeier,2002).The progressivedevelopme ntofadditionalshoots,whichoccursbylateralbranching,establishesa particularpatternofbranchedgrowthhabit(GiffordandFoster,1989).Effortstomanipulate plantarchitecturebyincreasingbranchinginplantshaveimprovedtheirvalueascommer cial crops.Forseedcropssuchasguar [ Cyamopsistetragonoloba (L.)Taub.],varietieswithhigh amountofbranchinghavethepotentialforhighseedyieldandarethusconsideredmore desirablethanerectorbranchingvarieties(Liu,2006).Inredclover ( Trifoliumpratense L.),a lackofpersistencehasledtoahighsusceptibilitytorootrotandashortlifespan.Changingthe architecturetoimprovethepersistenceofexistingcommercialvarietieshasincreasedtheirasset valuetofarmers(Mirzaie No doushan,1999).Forcoleus,themanipulationofplantarchitecture todevelopnewvarietieswithamorebranchedhabit,i.e.trailingtypes,isdesirableasitincreases theirutilityforuseasgroundcovers,inhangingba skets,andinmixedcontainers. Th egeneticscontrollinggrowthhabithavebeeninvestigatedinalimitednumberofplants. Branchingtypesinguarisgroupedintofourbasicclassifications:erect(singleprimaryaxis), erectbranching(oneormorebranches),basalbranching(branchesat baseofprimaryaxis)or branching(branchingthroughouttheprimaryaxis).Erectbranchingandbranchingisdominant tobasalbranchinginguarandiscontrolledbyasinglegene(Liu,2006).Inthediploidred clover,prostratenessispartiallydominant toerectnesswithstrongevidenceforadominancex dominanceepistasiscontrollingdominanttoerectnessandprostratenesscontrolledbyafew genes(Mirzaie Nodoushan,1999).Intetraploid,cultivatedpeanut( Arachishypogaea L.),two

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20 typesofgrowthhab its,runner(trailing)andbunch(erect),existwithallelesHb1andHb2 affectingthegrowthhabit.Inonevarietyofpeanuts,atleastthreedominantalleles,inany combination[(Hb1,Hb1,Hb2,Hb2);(Hb1,Hb1,Hb2,hb2);and(Hb1,hb1,Hb2,Hb2)],are r equiredtoproducearunner.Two,one,ornodominantallelesarenecessarytoproducebunched (Ashri,1968).Inthenarrow leafedlupin( Lupinusangustifolia L.),threelociareinvolvedinthe determinationofrestrictedbranching(lackofbranching)(Ad hikari,2001).Branchinginpetunia iscontrolledbytheDECREASEDAPICALDOMINANCE(DAD)genesandmutationsofthese genesresultinplantswithanincreaseinbasalaxillarybranchesandadecreaseinheight (Simons,2007).Althoughhundredsofdifferen ttypesofcoleusexistwithanextensivevariation ingrowthhabit(BoyeandRife,1938),informationonthegeneticsfortrailinggrowthhabitis scarce. Contrastingviewshavebeenproposedastotheoriginsoftrailingtypessincemostmodern daycole usvarietiesweretheresultofinterspecifichybridizations(Lebowitz,1985).Duringthe early1920s,adifferentformofcoleuswasdiscoveredandclassifiedas Coleusrehneltianus .It wascharacterizedasagroundcoverwithalowandscramblinghabit, androotedwherethe trailingstemstouchedtheearth(Pedley,1974).AccordingtoBaileyHortorium(Lebowitz, 1985),currenttrailingvarietiesweredevelopedfrominterspecificcrossesbetween C. scutellarioides and Coleuspumila Blanco. C.pumila wasbe lievedtobeidenticalto C. rehneltianus becauseitshabitwastrailingascomparedto C.scutellarioides withanerectgrowth habit(Pedley,1974). Themostwell knowncommercialtrailingvarietycurrentlyonthemarketisRedTrailing Queen(RQ).RQi sadark purpleleavedcultivarwithlateralbranchesthatextendmorethan threefeetinlength.Althoughdesirableforitstrailinghabit,scarce informationonitsgeneticsis

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21 availableasonlytwoshortreferenceshavebeenfoundforthisvarietyinwo rksbyRife(1948) andReddy(1952).Rife(1948)observedthatplantsoftheTrailingQueenvarietywereusually highlysterile,whetherselfedorcrossed,andprogenywereonlyproducedwhenTrailing Queenwasusedasthematernalparent.Reddy(1952) studiedfourclonesofTrailingQueen andsuggestedthatTrailingQueenbelongedto C.rehneltianus becauseitwashighlybranched andhadflowersconspicuouslylargerthanothervarietiesassignedto C.blumei Flowering Floweringisnotpreferredin coleusbecauseflowersdecreaseitsfoliagequality.The productionofflowersoftenslowsplantgrowthduetotheremobilizationofstoredreservesfrom theleavestotheflowersandseeds,andincreaseslandscapemaintenance(PedleyandPedley, 1974).T hefloweringresponseinplantsisregulatedthroughacomplexinteractionofseveral definedoroverlappinggeneticpathways.Twogeneticpathwaysincludephotoperiod(Lin,2000) andthecircadianrhythm(SamachandCoupland,2000;Thomas,2006).Otherfa ctorsaffecting floweringresponsesinplantsarelightqualityandquantity(Thomas,2006). Floweringresponsesinplantsareoftensynchronizedbynightlengthandcanbedivided intofivemaingroups(ThomasandVince Prue,1997):short dayplants(SDP, flowerswhen nightlengthexceedsacriticallength);long dayplants(LDP,flowerswhennightlengthis shorterthanthecriticallength);day neutralplants(DNP,flowersirrespectiveofday/night length);intermediate dayplants(floweronlywhendayl engthisneithertooshortorlong);and ambiphotoperiodic dayplants(dualdaylengthrequiringplants,requiresbothSDandLDsina sequence).Mostcropsthataregrownforspringorsummerproductiondisplayaflowering responsetodaylengthandexhib itaphotoperiodicresponse(MattsonandErwin,2005). Variationsinfloweringtimesareapparentinmanyplantspecies. Lathryusodoratus has threefloweringresponseclasses:thosethatflowerinthewinter(DNP),inthespring(facultative

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22 LDP)andint hesummer(obligateLDP)(RossandMurfet,1985).Evaluationsforvariabilityin floweringtraitsfor66accessionsofroseclover( Trifoliumhirtum All)showedarangeof differencesintimetoreachmaturityfrom162to183days,withthreedifferentmat uringgroups observed:early,midseason,andlate(NunesandSmith,2003).Researchershavedescribedlong day,short day,andday neutralphotoperiodicresponsesinColeus(Lebowitz,1985).Coleus ( Coleusblumei Benth.)isaday neutralplant,while Coleu sfrederici and C.blumei x C.frederici (designatedas C.hybridus )flowerwhenthecriticaldaylengthisabove12hrandjustbelow13 hr(Halaban,1968).Forvarietiesof C.blumei developedinthebreedingprogramatUF,early, mid,andlateflowering varietieshavebeenobserved. Thehighamountofvariationinfloweringresponsesisattributedtoseveralinteracting factorsrelatingtoenvironmentalstimuliandthegeneticswithintheplant.Photoperiod,i.e.the synchronizationoffloweringresponse stodaylength,isamajorfactorinregulatingflowering responses(Thomas,2006).Photoperiodicchangesarealsocloselylinkedtoaninternaloscillator withintheplantknownasthecircadianclock(Halaban,1968)whichcontrolsthedailyrhythms ing eneexpressionandbehaviorandactasatimerinphotoperiodicresponse(Samachand Coupland,2000).Otherstimuliaffectingtheregulationoffloweringresponsesinplantsarelight qualityandquantity.Effectsoflightqualityhavebeenshownthroughs tudiesofsunversus shadeplants.Underashadecanopy,plantsshowarangeofresponsestothechangesinred(R) tofar red(FR)ratioofambientlight(Thomas,2006).Lightundershadedconditionsmimics shortend of dayFRtreatmentsandphytochromes actasphotoregulators(Thomas,2006).Light quantityresponsesarelinkedtophotosynthesisandtheavailabilityofassimilatesandis particularlyimportantduringearlydevelopmentofplants(Thomas,2006).

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23 C.blumei areday neutralplantsandthreema inclassesoffloweringresponses(early,mid, andlateflowering)arefoundwithinthespecies(Lebowitz,1985).Inabreedingprogramwhich selectslate floweringvarietiesbasedonphenotypiccharacteristics,itisimportanttoisolate thosecultivarst hatarelate floweringandusetheseasbreedinglinestogeneratenewpopulations withlatefloweringresponses. FoliageColor Anthocyaninsynthesisandproduction Colorpigmentationinplantshasbeenthe subjectofappliedresearchformorethanfourc enturies(Moletal.1998),andalthoughthe anthocyaninbiosyntheticpathwayhasbeenextensivelycharacterizedinflowers(Grotewold, 2006)onlyamoderateamountofinformationisavailableforleaves.Plantpigmentationisdueto theaccumulationoffl avonoids(includinganthocyanins),carotenoids,andbetalains(Moletal. 1998).Flavonoidsareconsideredtobethemostimportantplantpigmentsastheyprovidemost ofthecolorsinthevisiblespectrum(Koesetal.2005).Thebrightredandpurplecolo ration foundincoleusfoliageisproducedprimarilybyanthocyaninpigments,whereasgreenand yellowcolorationsareproducedbychlorophyllandcarotenoids,respectively(Lebowitz,1985). Theflavonoidpathwayispartofthelargerphenylpropanoidpathw ay,whichalsoproduces arangeofothersecondarymetabolites,suchaslignins,stilbenes,flavonoidsand hydroxycinnamicacids(Davies,2004).Thephenylpropanoidpathwaystartsfromphenylalanine, itselfderivedfromthe shikimate/chorismatepathways( D avies,2004).Thefirstgeneralenzyme oftheflavonoidbiosyntheticpathwayisphenylalanineammonialyase(PAL),whereasthe enzymechalconesynthase(CHS)providesthefirstcommittedsteptowardsthesynthesisof anthocyanins(Grotewold,2006) (Figure 1 1) .CHScatalyzesthecondensationofmalonyl CoA toyieldnaringeninchalcone(Schijlen,2004)andchalconeprovidestheprecursorsforallclasses offlavonoids,whichincludestheflavonolsandanthocyanins(Grotewold,2006).Naringenin

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24 chalconeisis omerizedbychalconeisomerase(CHI)toformflavanonenaringeninand subsequenthydroxylationofflavanonetodihydroflavonolsiscarriedoutbytheenzyme flavanone 3 hydroxylase(F3H)(Schijlen,2004).Thedihydroflavonolsrepresentonebranch pointin thepathwaywherefinecontrolofgeneexpressionoccurs,astheyarethesubstratesfor productionofboththecoloredanthocyaninsandthecolorlessflavonols(Davies,2003).Thenext enzymeinthepathway,dihydroflavonol4 reductase(DFR)providesone entrypointtothe biosynthesisofanthocyanins,anddependingontheplantspecies,DFRcanutilizeasasubstrate oneorallthreeofthedihydroflavonolsproducedbyF3Htoformthecorresponding leucoanthocyanidins(Grotewold,2006).Theconversionof leucoanthocyanidinsto anthocyanidinsiscarriedoutbythenextenzymeanthocyanidinsynthase(ANS).Thelast enzymeintheanthocyaninbiosyntheticpathwayisUFGT.UFGTcatalyzestheformationof anthocyanidin3 glucosidefromanthocyanidinundercytos olicneutralconditions(Saito,2002). Thepseudobaseformofanthocyanidin3 glucosideisthentransportedintothevacuolesfor storage(Saito,2002). Twogroupsofgenesinvolvedintheregulationofanthocyaninbiosynthesisinplantsare thestructura landregulatorygenes.Thestructuralgenesencodetheenzymesthatdirectly participateinthebiochemicalreactionsintheformationofanthocyaninsandtheregulatory genesencodetheproteinsthatregulateexpressionofthestructuralgenes(Gong,199 7).The regulatorygenesalsofunctionincontrollingthespatialandtemporalaccumulationofpigments (Gong,1997).Inmaize,theRandC1genefamiliesencodetranscriptionfactorsrelatedtothe MYCandMYBfamiliesthatregulatetheexpressionofthe structuralgenes(Gong,1997).In snapdragon,thepatternofredanthocyaninpigmentationisregulatedbytheproductofthe delila gene,whichexhibitshomologytotheproductsoftheRgeneinmaize(Gong,1997).Six

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25 anthocyaningenesareknowntoregula tetheexpressionof someofthestructuralgenesin Petuniahybrida (Quattrocchioetal.,1993).AN1encodesabasichelix loop helix(bHLH) proteinthatactivatestranscriptionofDFRanditsexpressionisregulatedbyAN2andAN4 (Speltetal.,2000).A N2isexpressedprimarilyinthepetallimbandencodesaMYBdomain transcriptionfactor(Quattrocchioetal.,1999)thatisfunctionallyinterchangeablewithC1from maize(Quattrocchioetal.,1998).AN4regulatestheexpressionofstructuralgenesprim arilyin theanthersanditalsoencodesaMYBdomaintranscriptionfactor(Speltetal.,2000).The activityofAN1,AN2,orAN4appearstoberegulatedpost transcriptionallybyacytosolic WD40repeatproteinencodedbyAN11inpetunia(Speltetal.,20 02).In Perillafrutescens ,a speciescloselyrelatedtocoleus,threeindependentgenes(A,B,andK)promotetheformationof anthocyaninindifferentpartsoftheplant.GeneAcontrolsanthocyaninproductioninleavesand stems,geneBpromotesonlyin thestem,andgeneKisessentialfortheexpressionofgeneAin theepidermisofleaves(Gong,1997).However,themolecularbasisofgeneticcontrolbygenes A,B,andKinanthocyaninproductioninPerillaremainsunclear(Yamazakietal.,2003). Phys iologicalfactorsaffectingplantcoloration:co pigmentationandpH. Once anthocyaninsaredepositedinthevacuoles,severalfactorsknowntoaffectplantcoloration includethephysiologicalfactorsofco pigmentationandvacuolarpH,andtheenvironmen tal factoroflightintensity.Co pigmentationandvacuolarpHhavebeenextensivelyinvestigatedin flowersbutlittleinformationexist s forthisphenomenoninleaves.Twotypesofco pigmentation thatexistsincludeintermolecularandintramolecularco pigmentation.Intermolecularco pigmentationinvolvestheinteractionofanthocyaninswithothernon coloredflavonoids (flavonols,carotenoids,ormetals)(Grotewold,2006).Non coloredflavonoidsprovidedepthto manywhiteorcreamflowers(Grotewold, 2006).Inintramolecularco pigmentation,

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26 anthocyaninchromatophoresarecovalentlymodifiedbyorganicacids,andtogetherwiththe formationofstackedcomplexes,resultinanincreaseinpigmentationandchangeinhuecolor (Grotewold,2006).Hereare afewspecialpointstokeepinmindwhenreviewingyourthesisor dissertation: VacuolarpHprovidestheproperhueforanthocyanincoloration,andcolorexpressionof anthocyaninsishighlyinfluencedbypH(TorskangerpollandAndersen,2005;Grotewold, 2006).Ingeneral,thevacuolarlumenineverycelltypeismoreacidicthanthesurrounding cytoplasm.TorskangerpollandAndersen(2005)examinedthecolorvariationofthree anthocyanins(cyanidin based)atfourteenpH valuesbetween1.1and10.5andfo undthathues changedwithpHlevels.ThethreepigmentshadreddishhuesatthelowestpHvaluesand changedtowardmorebluishtoneswithincreasingpHlevelsaround7.Inmorningglory( Ipomea tricolor )petals,thevacuolarpHisrelativelylowwhenthe flowerbudsopen,resultinginared color.Uponfurthermaturation,thevacuolarpHincreasesandthepetalsacquireastrongblue color(Yoshidaetal.1995).In P.hybrida ,wild typeflowersstayonthereddishsideofthecolor spectrumduetoanacid icvacuolarpH(Quattrocchioetal.,2006).Geneticscreensof P.hybrida mutantshaveidentifiedseveralgenes(PH1toPH7)involvedinvacuolaracidification.PH1to PH7genes,whenmutated,resultintheblueingofflowers(Moletal.1998;Grotewold, 2006; Quattrocchioetal.,2006)withoutlossoralterationofanthocyanincompositionandcontent (Moletal.1998).MutationsinthegenesANTHOCYANIN1(AN1),AN2,andAN11also causeanincreaseinpHofpetalextractsaswellaslossofanthocyaninp igments.AN1,encoding abasichelix loop helixprotein(bHLH),wasshowntoactivateanthocyaninbiosynthesisand vacuolaracidificationinpetalsthroughitsinteractionswithtwodistinctMYBproteinsencoded byAN2andPH4(Quattrocchioetal.,2006).

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27 Anenvironmentalfactoraffectingplant coloration:lightintensity. Lightintensityisan environmentalfactorthatisknownto affect colorationinplants. Forcoleusvarieties,foliage colorsusuallydarkenorfadeunderhighlightintensitiesdependin gonthevariety.The physiologicalandmolecularmechanismsforfoliagecolorchangehavenotbeeninvestigatedin coleus,buthavebeenforotherplantspecies. Lightexposurehasbeenshowntobeaprerequisiteforsignificantanthocyaninsynthesisin v egetativetissues(Steyn,2002).Generally,highlightintensityisrequiredtoinduceanthocyanin synthesiswhileanthocyaninlevelsinplantsmayvarywithrespecttolightexposurelevels (Steyn,2002).In Pennisetumsetaceum cvsRubrumandRedRidin gHood,pigmentationis highlydependentonlightforinduction,withcolorsofbothcultivarrangingfrompalegreenin lowlighttodarkpurpleinhigh lightenvironments(Beckwithetal.,2004).Light dependent anthocyaninproductionhasalsobeenobse rvedinyoung Brassicaoleracea L.seedlings(Rabino, 1986),inmaize(Pietrini,2002)andinapples(Merzlyak,2000).Insomecultivarsofapples,sun exposedsideshaveconsiderableamountsofaccumulatedanthocyanins,whereasshadedsides haveanabsenc eofpigments(Merzlyak,2000). Violacornuta L.cultivarYesterday,Todayand Tomorrow(YTT)haveflowersthatundergoadramaticanthocyanin basedontogeneticcolor fromwhitetolavendertodeeppurpleoverfivetoeightdays(Farzad,2002).Appearan ceof colorinYTTisregulatedbyanthocyaninsynthesisattheleveloftranscription.Lightirradiation leadstothetranscriptionalactivationofspecificanthocyaninbiosyntheticgenes,resultingin increasedamountsofmalvidins(Farzad,2002).Light isnecessaryfortheappearanceofdeep purplecolorinYTT,asevidencedbyalackofcolorchangeinflowersmaintainedinthedark (Farzad,2002).

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28 Theinductionofanthocyaninsynthesisinplantsexposedtohighlightintensitiesmayplay afunctional roleintheprotectionofthephotosyntheticapparatusagainstphotoinhibition. The firststepinphotosynthesisisthecaptureoflightphotonsbychlorophyll(Harpaz Saad,2007). Whentheabsorbedlightexceedsthecapacityoftheplanttousethetrappe denergythrough photosynthesis,damagetothephotosystemsoccurs(Huner,1998).Thisdamage,termed photoinhibition,canbecomeirreversibleandleadtosignificantdecreasesinplantproductivity (Huner,1998).Anthocyaninshavebeenshowntoabsorbthe excesslightenergythatwould otherwisebeinterceptedbychlorophyllsandprotectleavesfromphotoinhibitorydamageduring periodsofhighlightintensity(Neill,2003).Thephotoprotectiveroleofshieldingexcesslight fromchlorophyllbyanthocyanin shasbeenshownin Begoniapavonina (Gouldetal.,1995), Galaxurceolata (Hughes,2005),and Cornusstolonifera (Fieldetal.,2001). Summary Theeconomicsuccessofmanyornamentalplantsonthecommercialmarketrequiresnovel orimprovedcharacteristi csthatnotonlymakethemdistinctfromcurrentvarietiesbutalso increasetheirproductvalue.Withregardtocoleus,highgeneticvariabilityexiststoallowforthe manipulationofcertaintraitssuchasplantarchitecture(trailinggrowthhabit,vigo r,branching, andlateflowering)andfoliagecolorquality.Forthesuccessofthecoleusbreedingprogram,the developmentofnewvarietieswiththeseidealcharacteristicsandtheunderstandingofthe geneticsinvolvedinthecontrolofthesetraitsare thereforeessential.

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29Figure1-1.Generalanthocyaninbiosyntheticpathway.Phenylalanine 4-Coumaryl-CoA NaringeninchalconeCH S NaringeninCHIDihydrokaempferolF3H LeucopelargonidinDFR DihydromyricetinDihydroquercitinDFR DFRLeucocyanidinLeucodelphinidinPelargonidinDelphinidinCyanidinCyanidin-3-glucosideANS UFGTANS Delphinidin-3-glucosidePeonidin-3-glucosideMTPetunidin-3-glucosideMTMalvidin-3-glucosideMTF3’5’HF3’H UFGT ANS

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30 CHAPTER2 THEGENETICSOFTRAILINGHABITAND THEDEVELOPMENTOFNEWCOLEUS ( S olenostemon scutellarioides (L.)CO DD) VARIETIESWITHTRAILINGHABITAND BRIGHTERFOLIAGECOLOR Introduction Solenostemonscutellarioides (L.)Codd,commonlyknownascoleus,isamemberofthe Lamiaceaefamilyandisvaluedforitsvibrantandcolorfulfoliage(PedleyandPedley,1974).Its s tatusasacommonhouseholdbeddingplantdatesbacktoitsintroductioninEnglandfromits nativeoriginofIndonesiaintheearly1800s(Stout,1987).Morethan300speciesandseveral hundredvarietiesexistforthegenus Solenostemon andcommercialcu ltivarsarecharacterizedby theirleafcolors,leafshape,growthhabit,andfloweringcharacteristics(Lebowitz,1985).Most brightly coloredcoleusvarietiescommonlyfoundonthemarkethavevigorousgrowthhabits andaremainlyplantedinthelandscap eastallbeddingplants.Fewtrailinghabitvarietiesexist andthoseavailablehavefoliagethatisgenerallydarkgreenorred.Trailinggrowthhabitin coleusisdesirableasitincreasesitsutilityasanornamentalplantforuseinmixedcontainers, hangingbaskets,andasgroundcoverinthelandscape.Varietieswithtrailinghabitsare characterizedbyhavinganextensivelateralbranchingpatterninwhichthereisareductionin apicaldominanceandalargerquantityofaxillarymeristems(Davies,1 995).Themanipulation ofplantarchitecturehasbeenachievedduringtheimprovementofmanycropspecies.For example,inredclover,alackofpersistence,i.e.creepingthinstemsandmoreadventitiousroots, isamajorlimitationtoitswidespreadacc eptanceasaforagecrop(SmithandBishop,1993). Changingthearchitectureofexistingcommercialredclovercultivarsanddevelopingoneswith anincreaseinpersistencehaveincreasedtheirusebyfarmers(Mirzaie Nodoushan,1999). Thegenescontrollin ggrowthhabitvaryformanyplantspeciesbecausevariationsinplant architecturereflectdiversegrowthandreproductivestrategies(Goncharov,1999).Innarrow

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31 leafedlupin, Lupinusangustifolia L.,restrictedbranching(lackofbranching)isasimply inheritedrecessivetrait(Adhikari,2001).Inredclover, Trifoliumpratense L.,adominancex dominanceepistasiscontrolsprostrateness,withprostratenessbeingpartiallydominantto erectness(Mirzaie Nodoushan,1999).Althoughhundredsofdifferentt ypesofcoleusexistwith anextensivevariationingrowthhabit(BoyeandRife,1938),informationonthegeneticsfor growthhabitislimitingbecausemostofthegeneticstudiesincoleushavefocusedmainlyonits leafcolor(BoyeandRife,1938;Rife, 1938;Lebowitz,1985)andleafshape(Rife,1972;Rife andHakeem,1966).Typesofgrowthhabitobservedincoleusareuprightorerecthabitand trailinghabit,withvaryinglevelsforeachhabittype.Suchvariationsinhabitaretheresultofthe high levelofheterozygosityfoundincoleus. Contrastingviewshavebeenproposedastotheoriginsoftrailingtypessincemostmodern dayvarietiesweretheresultofinterspecifichybridizations(Lebowitz,1985).Duringtheearly 1920s,adifferentformof coleuswasdiscoveredandclassifiedas Coleusrehneltianus .Ithada low,scramblinghabit,seldomreachedmorethan25cminheight,androotedwherethetrailing stemstouchedtheearth(Pedley,1974).AccordingtoBaileyHortorium(1976),currenttraili ng varietiesweredevelopedfrominterspecificcrossesbetween Coleusscutellarioides and Coleus pumila Blanco. C.pumila hadatrailinghabitratherthanerectlike C.scutellarioides andwas believedtobeidenticalto C.rehneltianus (Pedley,1974). The majorityofcytologicalstudieson C.scutellarioides reporta2n=48chromosome number(Reddy,1952;Morton,1962;HakeemandRife,1966).Multivalentsappearduring meiosisincultivarswith2n=48chromosomesandindicatepolyploidy.Sincemanygenetic e xperimentsproducesimplediploidratios(Rife,1948and1952)thespeciesisthoughttobean allotetraploid(Lebowitz,1987).Allotetraploidsarisethroughthecombinationofchromosome

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32 setsfromtwoormorespecies(PoehlmanandSleper,1995).Contrasti ngviewsonchromosome numbershavebeenreportedfortrailingcoleusvarieties.Scheel(1931)reportedann=24for C. rehneltianus whileReddy(1947),fromobservationsofthreeclonesfromavarietynamed TrailingQueen( Coleusblumei ),reportedchromo somesnumbersofn=24+1andn=36.Rife (1948)observedthatplantsoftheTrailingQueenvarietywereusuallyhighlysterile,whether selfedorcrossedandonlyprogenywereproducedwhenTrailingQueenwasusedasthe maternalparent.Thecausefor highsterilityinTrailingQueenwasnotinvestigatedfurtherby Rifeorothers. Theaimofthisresearchwastoprovidefurtherinformationonthegeneticsfortrailing growthhabitincoleus.Understandingtheinheritanceoftrailinghabitwillprovide useful informationforthedevelopmentofnewcoleuscultivarswithdesirablehabitwhichcanbe combinedwithfoliagecolor.CommercialcultivarsRedTrailingQueen(RQ)andSedona(S) wereusedasparentallinesfortheproductionofF1hybridsint hebreedingprogram(Figure2 1).RQisadarkpurple leafedcultivarandwaschosenforuseasthegeneticsourcefortrailing habit.RQhasalowgrowinghabitandlateralbranchesthatextendmorethanthreefeetinlength, providingforgoodgroundcov erage.Sisastandarduprightcultivarandisoneofthefew commerciallyavailableplantsthathasbrightorangefoliage.Bothcultivarsflowerprofuselyand floweringinductionoccursyear round,facilitatinghand pollinationsinthegreenhouse. Seedlin gsresultingfromRQself pollinationweremostlyalltrailingbutdarkinfoliagecolor whileseedlingsresultingfromSself pollinationweremostlyuprightandhadbrightfoliage color.Thus,bothparentallinespossessedhighlevelsofhomozygosityfor useasbreeding materialsfortheanalysisoftrailinghabit(RQ)andfortheintrogressionoforangecolor(S)

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33 Littleinformationisavailableregardingthegeneticsof trailinggrowthhabitincoleus Furthermore,noknownstudytodatehasbeenpubli shedonthedevelopmentofnewtrailing varietieswithbrightfoliagecolorsthroughthemethodofcontrolledhand pollinations.Inorder todevelopnewvarietieswiththesedesirablecharacteristics,anunderstandingoftheinheritance fortrailinggrowth habitisrequired.Throughthisresearch,newrelevantinformationonthe knowntrailingvariety(RQ)isprovided.Themainobjectivesofthisstudywereto:(1)useRQ andSasbreedingparentstoproducehybrids,(2)determinethegeneticsfortrailing habitand isolatehybridswithtrailinghabit,(3)introgressorangecolorintotrailinghabittypes,and(4) developnewcoleusvarietieswithtrailinghabitandbrightfoliagecolors. MaterialsandMethods PlantMaterialsandGrowthConditionsforBreedi ng RQ,acommercialvarietywithdesirablebranchinggrowthhabitandS,acommercial varietywithdesirableorangecolor,wereobtainedfromProvenWinners,Inc.StockplantsofRQ andSweregrowninthree gallonpotsfilledwithsoillessmedium(Fafard lightweightmix#2; Apopka,Fl)inagreenhouseunderstandardconditions(21 25 o C).Cuttingswerepropagated fromeachvarietyandrootedinamisthouse,withmistingsetfor20secondsevery30min.After rooting,10clonalcuttingsfromeachparental lineweretransplantedto15.2 cmpotsandirrigated asneededwithliquidfertilizerwith150ppmnitrogen(15 5 15,N P 2 O 5 K 2 O Scotts,Apopka, Florida)inthegreenhouse.10clonalplantswereroutinelygrownfromeachparentallineto ensureanadequate numberforacontinualsupplyofflowersforpollinations.Theclonalplants weregrownuntilfloweringinductionoccurredatapproximatelysixweeksafterpotting.After flowering,fertilizationwasstoppedandtheplantswereirrigatedwithonlywatera sneeded.Not fertilizingwasthemosteffectivemeanstogenerateseedsbecauseplantsgrownunderlush conditionsdidnotproduceasmanyseedsasthosegrownunderasomewhatstressedcondition.

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34 AnalysisforPloidyLevelDifferencesamongParentalGenot ypesandHybrids Flowcytometryandcomparativecellsizewereusedtoassessdifferencesinploidylevels forRQ,S,andF1plants.Forflowcytometryanalysis,freshapicalmeristematictissues(~20mg) werechoppedandnucleiextractedin1mllysisbuff erandDAPI(4',6 diamidino 2 phenylindole).DAPIisaDNA specificstainwhichformsafluorescentcomplexbybindingto theminorgroveofA Trichsequences(Kapuscinski,1995).Largetissuedebriswasremovedby filtrationandtheextractionsolutions ubsequentlyusedforanalysis,withaminimumof2500 nucleicountedperextractionsolution.SampleswereanalyzedwithaPA1PloidyAnalyzer (Partec,Mnster,Germany)andDNAquantifiedusingaDPACsoftwareprovidedbythesame company.Aone parameter histogramwasobtainedandploidyleveldifferenceswereanalyzed bycomparingthem eanDNAcontentofeachsample. Comparativecellsizeanalysiswasperformedbymeasuringthelengthandwidthoffresh pollengrainsandstomates,andthelengthofgland ulartrichomes.Ithasbeenshownthatploidy leveldifferencescanbedeterminedbymeasuringtheseparametersinwhichcellsizeoften increaseswithincreasesinploidylevel(Ojiewo,2006;Kelly,2002).Guardcellsandglandular trichomeswereobtained fromepidermalpeelsaspreviouslydescribedbyReddy(1952). Measurements(inmicrons)weretakenatamagnificationof4Xforglandulartrichomesandat 40Xforpollengrainsandstomatesusingacompoundlightmicroscope(NikonLabophot 2, Japan).Data shownarethemeansof25pollengrainsandstomatalcellsandtwentyglandular trichomesforRQ,S,andfiveF1plants.At test(Excel)wasusedtodeterminesignificanceat the0.1level. PollenViabilityandMicrosporogenesisAnalysis Pollenviability estimateswerecalculatedbycountingthenumberofpollengrainsthat stainedfullyinaceto carmineoutofatotalof100.Pollenwascollectedfromanthersofthree

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35 flowersfromeachvarietyandthemeanvalue,generatedasapercentage,wasdetermined .Pollen estimatesandphotographsofpollengrainsweretakenatamagnificationof10Xusinga compoundlightmicroscope(NikonLabophot 2).Aceto orceinsmearswereusedforthestudyof microsporogenesisinRQtodeterminethecauseforlowpollenqual ity.Fourantherswere smearedinaceto orceinwithoutpriorfixation,coveredimmediatelywithacoverslip,andsealed withbees wax.Aftersealing,theslidewasgentlypressedbetweentwosheetsofblottingpaper andimmediatelyviewedunderadissecti ngscope.Approximately300pollenmothercellsfrom RQwereexamined.Photographsofcellsundergoingmeiosisweretakenatamagnificationof 100Xwithacameramagnificationof4000X.Methodsusedforpollenviabilityestimatesand microsporogenesisana lysiswereprevio uslydescribedbyReddy(1952). PollinationMethodology Allpollinations,selfsandreciprocalcrosses,wereperformedinthemorning(8 11am)ina greenhouseusingthefollowingtools:round tippaintbrushforpollencollection,tweezers for emasculation,permanentmarkerformarkingpollinatedflowersand95%ethanol.Self pollinationsweremadebyemasculatingflowers(pre anthesis)inthemorningandimmediately pollinatingthesameflowerwithpollencollectedfromthesameplant.Poll inatedflowerswere markedandseedscollectedapproximatelythreeweekslaterwhentheseedsandcalyx(seed capsule)weredarkbrownincolor.Cross pollinationswereperformedbyemasculatingflowers (pre anthesis)inthemorningwithtweezers.Topreve ntselfpollenfromcontaminatingthe flowers,thetweezersweredippedinalcoholafterallemasculations.Pollenfromthesecond parentallinewascollectedandimmediatelybrushedonthestigmaoftheemasculatedflowers fromthefirstparentalline.Co rollasofpollinatedflowersweremarkedwithafine tippedmarker andpollinationsweremadeonadailybasisuntil50 100seedswerecollectedfromeachparental line.SeedswerecollectedfromRQselfs,Sselfs,andthecrossRQ( ds

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36 werecollectedfromtheS( selfingF1andF2plants,respectively. GrowthofSeedlings Seedscollectedfromselfandcrosspollinationsweresprinkledonthetopsurfaceofflats filledwith soillessmedia(Fafard2 mix,Apopka,Florida)andgerminatedinamisthousewith mistcyclessetat20secondsforevery30minutes.Seedlingswereremovedfromthemisthouse after90%ofseedshadgerminated(10 14days),movedtoapolycarbonateshade house,and afterwardsweretransplantedonepercellintoa72cell packtray.Afterthreeadditionalweeks, theyweretransplantedto15.2 cmpotsandirrigatedasneededwithliquidfertilizerat150ppm nitrogen(15 5 15,N P 2 O 5 K 2 O Scotts,Apopka,Flor ida)ateveryotherirrigation.Theplants weregrownforanadditionalsixweeksandusedforsubsequentdatacollection. RatingScaleforGrowthHabitandFoliageColorBrightness Growthhabitwasratedonavisual1 5ratingscale,where1=upright,2 =semi upright,3 =prostrate,4=semi trailing,and5=trailing.The1 5ratingscalewasusedbecausethisscale hasbeenusedforotherplantsandallfivegrowthhabittypeshavebeenobservedincoleus (Pedley,1974).Growthhabitwascharacterized astheheightandbranchingpatternoftheplant, inwhichaplantwithuprightgrowthhabitwastallandhadpoorbranchingandaplantclassified astrailingwasshortandhadexcellentbranching.Foliagecolorwasratedonavisual1 5rating scale,whe re1=verydark(colorasdarkRQ),2=dark(colorslightlylighterthanRQ),3= medium(colorbetweenRQandS),4=bright(colorslightlydarkerthanS),and5=verybright (colorasbrightasS). RatingScaleforGenotypicandChi squareAnalysis T he1 5scalepreviouslyusedabovetorategrowthhabitsintheRQself,SselfandF1 populationswhere1=upright,2=semi upright,3=prostrate,4=semi trailing,and5=trailing

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37 wasmodifiedtoanalyzegenotypicratiosforthethreepopulations.U singatetraploidmodel,a plantobservedashavinganuprightgrowthhabit(ratingof1)hadadesignatedquadraplex genotypeofAAAA,assumingthatuprightgrowthhabitwasdominanttotrailing.Therefore,five genotypicclasseswereobserved.Basedont he1 5scale,1=upright(AAAA),2=semi upright (AAAa),3=prostrate(AAaa),4=semi trailing(Aaaa),and5=trailing(aaaa).Chi square(X 2 ) goodness of fitanalysisbasedonatetraploidmodelasillustratedforchromosomalsegregation byBurnham( 1962)wasusedtoanalyzethegrowthhabitsegregationforallpopulationsandto determinethegenotypeofRQandS. Chi squareTestforHomogeneity Afterthechi squaregoodness of fittestwasappliedtotheRQselfed,Sselfed,andF1 population(asd escribedabove),thegenotypesofRQandSweredetermined.Tofurthersupport thatthephenotypesmatchedthegivengenotypes,threeplantsfromtheF1(orF2)population fromeachgenotypicgroup(AAAA,AAAa,AAaa,Aaaa,andaaaa)wereselfed.Achi squa re testforhomogeneitybasedonanrxctable,aspreviouslydescribedbyCochran(1954),was performedtoobtainabetterestimateofthepopulationproportionandtodeterminewhether frequencycountsweredistributedidenticallyacrossthedifferentp opulations.Thevaluefrom thechi squaretestforhomogeneitywascalculatedaccordingto Equation2 1 X 2 = (observed expected) 2 ,(r 1)(c 1) df (2 1) expected wherer=rowsandc=columns.TheX 2 valuesforeachofthethreeplants ineachgenotypic groupweresummedtogiveanoveralltotalandaP valuewasobtained. IntrogressionofColorUsingtheSelfingMethods ThirtyF1plantscharacterizedwithsemi trailinghabitwereselectedforuseaslinesto introgressbrightcolor.Ab ackcrossmethodwasinitiallychosentointrogresscolorsusingSas

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38 thebackcrossparent.SinceprogenywithbrightcolorswereproducedbyjustselfingseveralF1 plants,selfingwasusedasthepreferredmethod.TheF1plantswereselfedoveraonemont h periodandseedscollectedtogenerateanF2population.F2plantsexhibitingbothtrailinghabit andbrightfoliagecolorwereselectedinthebreedingprogramaspotentialnewvarieties. Results DeterminationofPloidyLevelDifferences SinceRQandS wereusedforthefirsttimeasbreedingparents,flowcytometryand comparativecellsizeanalysiswasusedtodeterminetheircross compatibilitybyobservingif anyploidyleveldifferenceswerepresent.SeveralF1plantsproducedfromcrossesofRQand S werealsoanalyzedtodetermineiftheyhadthesameploidylevelastheparentallines.Itiswell knownthatasploidylevelsincreaseinaspecies,nuclearDNAcontentalsoincreases(Sugiyama, 2003) ADNAcontentvalueof112.4and110.4wereobtai nedforRQandS,respectively. RangesofDNAcontentvaluesfrom91.4to118.4wereobservedfortheF1plants.Resultsfrom flowcytometricanalysisindicatedthatnosignificantdifferencesinploidylevelswereobserved amongRQ,S,andtheF1plantst ested(Table2 1). Theapplicabilityofpollen,stomatalandglandulartrichomelengthasindirectmethodsto furthersupportthesimilarploidylevelsfoundforRG,S,andseveralF1plantswasinvestigated (Table2 2).Comparativecellsizeanalysishas beenshowntodifferentiateploidylevelsbetween species,whereincreasesinploidylevelscorrelatewithincreasesincellsize(Aryavand,2003; Reddy,1952).Pollengrainlengthsof37.6Mand38.7MweremeasuredforRQandS, respectively.Amongfive F1plantstested,arangeof36.9Mto40.8Mwasmeasured.In regardstostomatalcells,alengthof29.2MforRQand31.8MforSwasobserved,with rangesof30.4Mto33.6MmeasuredfortheF1plants.Nosignificantdifferenceswerefound whenglandu lartrichomesweremeasuredforallofthevarieties.RQandSdidnotdifferinthe

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39 sizeoftheirtrichomes,withalengthof50.8Mand49.7MmeasuredforRQandS, respectively.AmongtheF1plants,lengthsof48Mto52.3Mforglandulartrichomeswer e measured.Nosignificantdifferenceswerealsofoundwhenthewidthsofpollengrainsand stomatalcellswereinvestigated.Resultsfromthet testindicatednosignificantdifferencesin ploidylevelsbasedoncomparativecellsizeanalysisexistedbetw eenRQ,S,andtheF1plants (Table2 2),thusfurthersupportingtheresultsdeterminedfromflowcytometryanalysisthatRQ andShavethesameploidy.Furthersegregationanalysissuggeststhatbotharetetraploids (discussedlater). PollenViabilityan dMicrosporogenesisAnalysis RQandSwereshowntonotdifferinploidylevelsandthusweresexuallycompatible. However,differencesinseedsetandfertilitywereobservedwhenselfandreciprocalcross pollinationswereperformedonRQandS.Fromsel f pollinations,lowseedsetwasobservedin RQwhilehigherseedsetwasobservedinS.Ofamaximumoffourseedsthatcanbeproduced inaflower,onlyoneseedwasnormallyproducedinRQwhilethreetofourseedswereoften producedfromS(personalo bservation).Furthermore,self pollinationswereoftenunnecessary forSbecauseseedsetoccurredspontaneously.Manyspecieshavelowseedset,andpollen qualityand/orpollensterilityduetonon functionalpollenhavebeenreportedasfactorsaffectin g seedsetforsomeplantspecies(Byers,1995;McDadeandDavidar,1984).Inordertodetermine ifthelowseedsetobservedinRQwasduetopoorpollenquality,pollenviabilityestimatesand microsporogenesisanalysiswereinvestigated(Table2 3andF igures2 2and2 3). Alowpollenviabilityof14%wasobservedforRQwhilesignificantlyhigherpollen viabilityof98%wasobservedforS(Table2 3).WhenpollenviabilityoffiveF1plantswas compared,awiderangefromalowpollenviabilityof17% toahighof87%wasobserved. WhenRQpollengrainswereviewedunderalightmicroscope,manywerenotstainable(Figure

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40 2 2a),whereasSpollengrainswerefullystainable(Figure2 2b).Theshapesofpollengrainsof F1plantswithlowpollenviability werealsoconsistentwiththoseobservedinRQ(datanot shown).MicrosporogenesisanalysisshowedtheoccurrenceofmicronucleiinRQpollenmother cellsduetothefailureofunpairedchromosomes(enclosedincircle)tomigratetoeitherpole (Figure2 3a).Furthermore,theamountofirregularchromosomessegregatingtoeitherpole resultedintetradswithmicrosporesofdifferentsizes(Figure2 3b).Resultsfrompollenviability estimates,pollenshapeandmicrosporogenesisanalysissuggestthatthelo wseedsetobservedin RQislikelyduetopoorpollenviabilityandquality. GrowthHabitSegregation Inordertodeterminehowtrailinghabitwasinheritedincoleus,photographsshowingthe variousgrowthhabitformsobservedwhenselfingRQ(Figure2 4),S(Figure2 5),andintheF1 population(Figure2 6)weretaken.Furthermore,thedifferentgrowthhabittypesofall populationswereratedonavisual1 5scaleandtheirdistributionanalyzed(Figure2 7).A distributionoffivedifferentgrowthha bittypeswasobservedinRQselfedpopulation(Figure2 4).Of96selfsevaluated,1hadanuprightgrowthhabit,19weresemi upright,53wereprostrate, 21weresemi trailing,and2weretrailing.Thetwoprogenythatwerecharacterizedashaving traili nggrowthhabithadahabitevenmoretrailingthanRQ,suggestingthatRQwasnot completelyhomozygousfortrailinghabit.IntheSselfedpopulationthreedifferentgrowthhabit typeswereobserved(Figure2 5).Of100progenyevaluated,20wereupright ,57weresemi upright,and23wereprostrate.The20progenywithuprightgrowthhabitsweretallerandhad poorerbranchingthanS,suggestingthatSwasnotanuprightvarietyaspreviouslysuggested. FourgrowthhabittypeswereobservedintheF1popu lation(Figure2 6).Ofthe250F1plants evaluated,22wereupright,108weresemi upright,94wereprostrate,and26weresemi trailing. NoneoftheF1plantshadtrailinggrowthhabitsasobservedintheRQselfedpopulationwhile

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41 thosethatwereuprigh tweresimilartotheplantsintheSselfedpopulationcharacterizedas havinguprightgrowthhabits.ThecontinuousdistributionforgrowthhabittypesforRQself,S self,andF1populationisshowninFigure2 7. DeterminationofParentalGenotypes Ac hi squaretestforgoodness of fit(X 2 )wasusedtotesthypothesesofpossible genotypesforRQandS.ForRQ,alltypesofgrowthhabitswereobservedintheselfed population.Sinceboththequadraplex(AAAA)andnulliplex(aaaa)formswerepresent,the genotypeproposedforRQwastheduplexform(AAaa).InregardstoS,sinceonlyupright, semi upright,andprostrategrowthhabitswereobservedintheselfedpopulation,thetriplex form(AAAa)wasproposed.Achi squarevalueof2.02(P=0.73)wereca lculatedforRQ (Table2 4)andachi squarevalueof2.14andaP valueof0.34werecalculatedforS(Table2 5).IfRQ(AAaa)wascrossedtoS(AAAa),theF1populationshouldtheoreticallysegregateina 1:5:5:1ratio.Achi squa revalueof2.51(P=0 .47)was calculatedfortheF1population(Table 2 6).Resultsfromthechi squareindicatedagoodness of fitfortheobservedratiosintheRQ selfed,SselfedandF1populationsandthussupportedthegenotypesproposedforRQ(AAaa) andSedona(AAAa). Tofurthersupportthatthephenotypesmatchedtheirassignedgenotypes(e.g.aplant characterizedashavingasemi uprightgrowthhabithadthetriplex(AAAa)genotype),plants fromeachofthefivegenotypicclasseswereselected.Threeplantsfromeach ofthefive genotypicclasseswereselfedandsegregationforgrowthhabitintheF2populationwastested againstthechi squaretestforgoodness of fit.However,fortheupright(AAAA)andtrailing (aaaa)growthhabitclass,plantsfromtheF2generati onwereusedinstead,andtheF3population analyzed.

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42 Chi squaredatafortheplantsusedtomatchphenotypeswiththeirassignedgenotypesare shown inTable2 7.Forthefirstgenotypicclass,iftheplantswithuprightgrowthhabitshavea quadraplexg enotype(AAAA),thenalloftheprogenyselfedfromtheplantsshouldallsegregate foruprightgrowthhabits.TheF3populationsproducedfromeachofthreeF2plantsallhad uprightgrowthhabits.Forgenotypicclass2,iftheplantscharacterizedasbei ngsemi upright haveatriplexgenotype(AAAa),theprogenyshouldbeexpectedtosegregateina1:2:1ratio, withtheupright,semi upright,andprostrategrowthhabitobserved.Basedonthedatafromthe chi squaretest,theF2populationforeachplant segregatedaccordingtotheexpectedratio.X 2 valuesof1.84(P=0.34),0.34(P=0.18),and0.92(P=0.63)wereobserved.Forgenotypic class3,progenyfromplantswithaduplexgenotype(AAaa)shouldsegregateina1:8:18:8:1 ratiowithallgrowthha bitsobserved.X 2 valuesof4.04(P=0.40),6.41(P=0.17),and4.93(P= 0.29)wereobtainedforplantsingenotypicclass3.Plantstestedingenotypicclass4aresemi trailingwithasimplexgenotype(Aaaa).Progenypopulationresultingfromselfing plantswitha simplexgenotype(Aaaa)shouldsegregateina1:2:1ratioforprostrate,semi trailing,andtrailing growthhabits.Whenachi squaretestwastestedforgoodness of fit,X 2 valuesof1.22(P= 0.57),0.52(P=0.77),and1.69(P=0.43)were obtained.Lastly,forgenotypicclass5,when plantswithtrailinggrowthhabit(genotypeaaaa)areselfed,theprogenypopulationshouldbe expectedtosegregateonlyfortrailinggrowthhabits.Whentheprogenypopulationwas evaluated,allplantshadt railinggrowthhabit.DatafromtheX 2 andP valuesindicatedthata goodness of fitwasobservedforalloftheplantstestedineachofthefivegenotypicclasses. Datafromthechi squaretestforhomogeneity,listedasthetotalX 2 andP valueinTable 2 7, alsofurthersupportedtheresultsfromtheChi squaretest.

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43 SegregationfordifferentgrowthhabittypeswasobservedintheselfedpopulationofRG andS.FivedifferentgrowthhabittypeswerefoundinRQprogenybutonlythreegrowthhabits were foundinS. SinceallfivedifferentgrowthhabittypeswereobservedinRQselfedprogeny, aduplexgenotype(AAaa)wasproposed.ForS,sinceitsegregatedina1:2:1ratioforupright, semi upright,andprostrategrowthtypes,atriplexgenotype(AAAa) wasproposed.Foreachof theselfpopulationsofRQandS,thenumberofprogenycharacterizedwitheachgrowthhabit typeconstitutedtheobservedvalues,andtheseobservedvalueswerethencomparedtothe expectedvalues.Expectedvalueswerethen umbersofprogenyexpectedtobeseenifaduplex (AAaa)andatriplex(AAAa)wereselfed.IftheP valueobtainedwaslessthan1,thenthe genotypesproposedforRQandSwerecorrect.Thechi squaretestwasconductedfortheF1and F2populationsandb ecauseagoodness of fitwasalsoobserved,thedatafurthersupportedthe genotypesproposedforRQandS.Thedataforgrowthhabithadagoodness of fittoatetraploid model,andgeneticsforgrowthhabitincoleusappeartobecontrolledbyasinglet etrasomic locusgenewithcompletelyadditiveeffects. FoliageColorBrightness MeanratingvalueswerecalculatedforfoliagecolorbrightnessintheRQself,Sself,F1 andthecombinedF2/F3populations(Figure2 8).Ameanvalueof4.1wasobservedfor theS selfpopulationwhileameanvalueof1wasobservedintheRQselfedpopulation.Progeny producedfromselfingRQwerealldarkpurpleandgreenincolor,withthetwocolorsvarying slightlyindegreeofintensity.FortheSselfedpopulation,bri ghtercolorswereobserved,with colorsrangingfromyellow,orange,red,green,andmottledyellow/orange.IntheF1population, somecombinationsoffoliagecolorswereobservedwhichwerenotpresentineithertheRQorS selfedpopulation.Foliagecolo rsrangedfromdarkpurple(similartoRQ),purplewithlarge greenmargins,mottledlightpurple/green,mottledbrown/green,andmediumred(similarto

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44 someprogenyproducedfromselfingS)(Figure2 9).Theintrogressionofbrightfoliagecolor wasobser vedintheF1populationbecausesomeindividualshadbrightgreencolorintheir foliagewhichwasnotobservedintheRQselfedpopulation.ManyF1plantshadfoliagecolors thatweredarkerthanSbutstillbrighterthanthatofRQ.Inaddition,purple colorfromRQ appearedtobedominantandtheorangecolorfromStoberecessivebecausealloftheF1plants hadsomeformofpurpleintheirfoliage.Whenthemeansforcolorbrightnesswerecompared, colorbrightnessimprovedslightlyintheF1populat ion.Themeanratingvalueforcolor brightnessof1.3wasslightlyhigherthanthatoftheRQselfedpopulationbutwasstilllower thanthatoftheSselfedpopulation.InthecombinedF2/F3populationproducedfromselfingthe thirtyF1andF2plantsfo rgenotypicanalysis,themeanratingvalueforcolorbrightnesswas 2.65.Foliagecolorbrightnesswasfurtherimprovedinthenextgeneration,howeveralthoughthe valueof2.65washigherthanintheRQselfedandF1population,itstillremainedlower than thatoftheSselfedpopulation. IntrogressionofColor ThebackcrossmethodwasinitiallychosentointrogresscolorgenesfromSintoF1plants characterizedwithsemi trailinggrowthhabits.However,whenF1plantswereselfedfor genotypicanalysi s,brightcolorasseeninSwasobservedinindividualsintheF2populations. Thus,thebackcrossmethodwasnotneededtointrogresscolorintotrailingtypesandtheselfing methodwasusedinstead.ThirtyF1plantswithsemi trailinggrowthhabitand colorbrightness ratingsoftwoorhigherwereself pollinatedtoproduceanF2population.Foliagecolorobserved intheF2andF3populationsrangedfrompurple(asseeninRQ),green,mottledpinkwithgreen margins,mottledgreen/red,toorangecolor( asseeninS)(Figure2 10).Furthermore,manyF2 plantswithbrightfoliagecoloralsohadtrailinggrowthhabitsandoneF2seedlingwasproduced

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45 whichhadtrailinghabitequivalenttoRQandbrightorangecolorsimi lartothatofS(Figure2 11). Discus sion Theuseofflowcytometryandanalysisofcomparativecellsizeasmethodstodetermine ploidyleveldifferenceshavebeeninvestigatedformanyplantspecies(Dolezel,2005).IfSwas atetraploidandRQeitheradiploidortriploid,thenforasigni ficantdifferenceinploidylevelto occur,SwouldneedtohaveatminimumdoubledthechromosomecountofRQ(Reddy,1952). InastudyconductedbyReddy(1952),sizeofpollen,stomatalcells,andglandularhairswere measuredtodifferentiatediploids, triploids,andtetraploidsincoleus.Itwasobservedthat diploidspecieshadsmallerpollen,guardcellsandglandularhairscomparedtotriploids,which wereslightlylarger.Fortetraploids,thesizesofthecellsweremeasuredtobetwicethesizeof thosemeasuredinthediploids.Resultsfromflowcytometryandcomparativecellsizeanalysis inthisstudysuggestthatRQ,Sedona,andF1plantsderivedfromcrossingthetwoparentallines wereofthesameploidylevel. Adifferenceinploidyleveldi dnotappeartobethefactorcontributingtothelowseedset inRQandthecross incompatibilityfoundinS( initialobservationswhenself pollinationsweremadeinRQandS,lowseedsetwasfoundin RQwhile higherseedsetwasfoundinS(personalobservations).Similarlyinastudyconducted on Eupatoriumresinosum speciesbyByers(1995),seedset(numberofseedsandheadsineach cross)wasusedasameasureofcompatibility,withacrossconsideredcomp atibleiftherewasat leastoneseedproducedperhead.Lowseedsetforthisspecieswassuggestedtobeduetopoor pollenquality.Lowpollenqualityintermsofviabilityandmicronucleiinpollenmothercellsin RQappeartohavebeenfactorscontrib utingtoitslowseedsetandunsuccessfulseedsetwhen RQwasusedasthepollendonorinthecrossS(

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46 formationofmicronucleifromirregularmeiosishasbeenobservedinotherspeciessuchas Paspalumm aritimum (Adamowski,1999), Clarkiaexilis (Vasek,1961),and Pfaffiatuberosa (Taschetto,2004). Cross compatibilitybetweenRQandSwasexamined,butthelowseedsetin RQasaplausibleresultofself incompatibilitywasnotinvestigatedinthisstudy .Further examinationofself incompatibilityduetoincompatibilityallelesorfailureofproperpollentube growthmayofferabetterunderstandingo fthelowseedsetfoundinRQ. Thegeneticsoftrailinghabitincoleusappeartobecontrolledbyasin glegeneexpressed thoughadditivegeneaction.Ifoneassumesthatuprightgrowthhabitisdominanttotrailingwith additiveeffects,theadditionofoneuprightalleleshouldenhancetheexpressionforupright growthhabit.Completedominanceisnotsug gestedforthegenecontrollingtrailinghabitin coleusbecauseF1plantswithgenotypesAAAA,AAAa,AAaa,andAaaawouldallhaveupright growthhabits,andtheydidnot.Therefore,incompletedominanceisproposedwiththeeffectof thealleleA(forup rightgrowthhabit)beingadditive.Growthhabitscontrolledbytheinteraction ofseveralgeneshavebeenobservedforotherpolyploids.Inhexaploidwheat,one,two,or occasionallythreedominantgenesScontrolledspringgrowthhabitwhilerecessiveal lelesat thoselocicontrolledthewintergrowthhabit(AkermanandMacKey,1949).Pugsley(1971)later supportedthisclaimandconcludedthatanyofthreedominantgenesortheircombinations controlledthespringgrowthhabit.Forcoleus,trailinggrowt hhabit(aaaa)isqualitatively inheritedandcontrolledbyasinglegenewithadditiveef fectsandincompletedominance. Thegeneticsforfoliagecolorwasnotthemainfocusofthiswork,butsignificant observationswererecorded.Rife(1948)statedtha tsixmultiplealleles,designatedthepurple series,affectedtheamountanddistributionofanthocyanin.Theirinteractionsresultedinnine phenotypicvariationsrangingfromunevenpurple,brown,andtogreenwithpurplecolorbeing

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47 dominant(Rife,194 8).Insupportofhisresults,purplecolorfromRQwasobservedtobe dominanttotheorangecolorfromSasalloftheF1plantspredominantlyh adpurplecolorin theirfoliage.FoliagecolorbrightnesswasimprovedintheF1populationasthemeanratin g scoreforcolorwashigherthantheRQselfplantsbutlowerthanSselfplants.Colorbrightness wasfurtherimprovedintheF2populationwhencolorssimilartothoseseeninSwereobserved. Intheprocessofanalyzinggeneticsegregationfortrailing growthhabit,separate selectionsweremadeforthedevelopmentofvarietieswithbothtrailinghabitandbrightfoliage color.TheF1populationshowedaslightintrogressionofbrightercolorduetotheparentalline S.However,allF1plantswerestill relativelydarkincolorwithremnantsofpurplefoliagecolor andnoplantshadorangefoliagecolorsimilartoS.IntheF2generation,plantswithtrailing habitandbrighterfoliagecolorswereidentified(Figure2 11).Thegoalofdevelopingaplant withbothtrailinghabitandbrightorangefoliagecolorwasachieved,asoneseedlingin particular,CopperPenny,anF2seedlingselfedfromtheF1plantH05 66,possessedthesetwo desirablecharacteristics.WhenCopperPennywasfurtherselfed,F3 plantswithalltrailingand brightorangefoliagecolorswereproduced.Thus,inthreegenerations,nearhomozygosityfor trailinghabitandorangefoliagecolorwasreached(Figure2 11).Thesenewvarietiesare currentlybeingevaluatedoutdoorsforgar denandlandscapeperformanceforcolorstabilityand landscapeuseasgroundcovers.

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48 Figure2 1. Parentsusedinbreedingcrossestodevelopnewtrailingtypeswithbrightfoliage color. A)CommercialvarietyRedQueenwithdesirable trailinghabitanddark purplefoliage.B)Aclose upofRedQueenfoliage.C)Commercialvariety Sedonawithuprightgrowthhabitanddesirablebrightorangefoliage.D)A close upofSedonafoliage.

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49 Table2 1.DNAcountsforRedQueen(RQ)and Sedona(S)and14hybridsproducedfrom thecross(RQ)x(S). CultivarName DNAContent z RQ 112.39 S 110.39 H1 114.51 H2 105.38 H3 117.5 0 H4 114.03 H5 115.13 H6 102.05 H7 118.38 H8 97.32 H9 104.84 H10 115.14 H11 91.43 H12 101.63 H13 112.02 H14 116.06 Mean 109.26 StdDeviation 7.92 z DNAcountsgeneratedusingaploidyanalyzer(Partec).Nosignificantdifferencesinploidy levelswereobservedwhenallvarietieswerecomparedtoeachother. Table2 2. Comparativecellsizeanalysis forRedQueen(RQ),Sedona(S),andfiveF 1 plants. Pollensize(uM) x z Guardcellsize(uM) x z Glandulartrichomes (uM) yz Plant length width length width length RQ 37.6 + 7 24.7 + 4 29.2 + 3 20.5 + 2 50.8 + 5 S 38.7 + 6 25.3 + 4 31.8 + 2 22. 8 + 2 49.7 + 4 H1 38.2 + 6 25.4 + 4 30.4 + 3 21.6 + 2 53.6 + 5 H2 36.9 + 5 25.5 + 3 32.7 + 3 21.4 + 2 48.0 + 4 H3 39.0 + 6 25.8 + 5 31.3 + 3 20.3 + 2 52.3 + 5 H4 40.8 + 7 26.2 + 4 33.6 + 4 22.5 + 3 51.7 + 6 H5 39.6 + 4 25.2 + 4 32.6 + 4 21.5 + 2 50.9 + 4 x Measurements(u)weretakenatamagnificationof40Xusingacompoundlightmicroscope (NikonLabophot 2).Dataareshownasthemeanof25pollengrainsandstomatalguardcells. y Measurements(u)weretakenatamagnificationof4X.Dataareshow nasthemeanof20 glandulartrichomes. z NosignificantdifferenceswereobservedforthevarietiesandF 1 stestedwhenthemeansand standarddeviationswerecompared.

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50 Table2 3 PollenviabilityestimatesforRedQueen(RQ),Sedona(S),andfive F 1 plants. Genotype Pollenviability% y RQ 14% S 98% H1 87% H2 49% H3 20% H4 33% H5 17% y Estimateswerecalculatedbycountingthenumberofpollengrainsthatstainedfullyinaceto carmine outof100 .Awiderangeofpollenviabilitywasobserv ed forRQ(14%)andS( 98% ) Figure2 2. PollengrainsofRedQueen(RQ)andSedona (S)stainedwithaceto carmine. A)RQpollengrainsshowingmanynotstainable.B)Spollengrainsshowing manyfullystainable.Photographswer etakenatamagnificationof10xusinga compoundlightmicroscope(NikonLabophot 2).

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51 Figure2 3. MicrosporogenesisanalysisofRedQueenpollenmothercells. A)telophaseII withsomemicronuclei.B)tetradwithmicrospore sofdifferentsizes.Photographs weretakenatamagnificationof100Xwithacameramagnificationof4000X (NikonLabophot 2).

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52 Figure2 4. RedQueenselfedpopulation. Theplantsintheselfedpopulationwereratedon avisual1 5scale,where1=uprightand5=trailing.Fivedifferentgrowthhabit typeswereobserved.A)1=uprightgrowthhabit.B)2=semi uprightgrowth habit.C)3=prostrategrowthhabit.D)4=semi trailinggrowthhabit.E)5= trailinggrowthhabit. Figure2 5. Sedonaselfedpopulation. Theplantsintheselfedpopulationwereratedona1 5scale,where1=uprightand5=trailing.Threemaingrowthhabittypeswere observe d A)1=uprightgrowthhabit B)2=semi uprightgrowthhab it. C)3= prostrategrowthhabit. A B C D E A B C A B C A B C

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53 Figure2 6. F1populationproducedfromthecrossRedQueenxSedona. Theplantswere ratedona1 5ratingscale,where1=uprightand5=trailing.Fourmaingrowth habittypeswereobserved.A)1 =uprightgrowthhabit.B)2=semi upright growthhabit.C)3=prostrategrowthhabit.D)4=semi trailinggrowthhabit. A B C D

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54Figure2-7.Distributionofthe‘RedQueen’(RQ)selfed,‘Sedona’(S)selfed,andF1populationforgrowthhabit.Growthhabitwasratedona1-5scale,where1=upright,2=semi-upright,3=prostrate,4=semi-trailing,and5=trailing.ThedistributionofgrowthhabitintheF1populationwasbetweenthedistributionobservedintheselfpopulationsofRQandS.Table2-4.Chi-squaretestfor‘RedQueen’(RQ)selfedpopulation.GenotypexSegregationRatioObserved#Expected#X2P 1.AAAA112.7 2.AAAa81921.33.AAaa185348.04.Aaaa82121.35.aaaa122.72.020.73yTotal369696.0 xFivegenotypicclasseswereobservedforthefivedifferentgrowthhabittypesobserved.Genotypicclass1=upright(AAAAgenotype),class2=semi-upright(AAAagenotype),class3=prostrate(AAaagenotype),class4=semi-trailing(Aaaagenotype)and5=trailing(aaaagenotype).yTheP-valueof0.73supportsthegenotype(AAaa)proposedforRQ. Trailinghabitsegregation02040608010012012345Ratings Frequency RQself Sedonaself F1(RQxS) Frequency

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55 Table2 5. Chi squaretestfor S edona(S) self ed population. Genotype x SegregationRatio Observed# Expec ted# X 2 P 1.AAAA 1 20 25 2.AAAa 2 57 50 3.AAaa 1 23 25 2.14 0.34 y Total 4 100 100 x Threegenotypicclasseswereobservedforthethreedifferentgrowthhabittypesobserved. Genotypicclass1=upright(AAAAgenotype),class2=semi upr ight(AAAagenotype), andclass3=prostrate(AAaagenotype). y AP valueof0.34supportsthegenotype(AAAa)proposedforS. Table2 6. Chi squaretestfortheF 1 population. Genotype x SegregationRatio Observed# Expected# X 2 P 1.AAAA 1 22 20.8 2.AAAa 5 108 104.2 3.AAaa 5 94 104.2 4.Aaaa 1 26 20.8 2.51 0.47 y Total 12 250 250.0 x Fourgenotypicclasseswereobservedforthefourdifferentgrowthhabittypesobserved. Genotypicclass1=upright(AAAAgen otype),class2=semi upright(AAAagenotype), class3=prostrate(AAaagenotype),andclass4=semi trailing(Aaaagenotype). y AP valueof0.47supportsthegenotypesproposedforRedQueen(AAaa)andSedona (AAAa).

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56 Table2 7. Chi squaretesta ndtestforhomogeneityoftheF 1 andF 2 plants. Genotypic class Pedigree Upright Semi upright Prostrate Semi trailing Trailing X 2 y P y 1 H38 8 12 0 0 0 0 0.00 H65 4 14 0 0 0 0 0.00 H234 1 21 0 0 0 0 0.00 Total z N/A 2 H32 7 19 5 0 0 1.84 0.39 H34 7 10 11 0 0 3.43 0.18 H38 7 13 4 0 0 0.92 0.63 Total 5.19 0.20 3 H65 1 13 28 9 3 4.04 0.40 H85 0 2 22 13 2 6.41 0.17 H187 0 3 18 8 2 4.93 0.29 Total 15.38 0.08 4 H57 0 0 7 9 4 1.22 0.57 H66 0 0 5 9 3 0.52 0.77 H69 0 0 15 28 9 1.69 0.43 Total 3.43 0.50 5 H69 4 0 0 0 0 32 0.00 H69 18 0 0 0 0 23 0.00 H69 21 0 0 0 0 23 0.00 Total N/A x Threeplantsfromeachofthefivegenotypicclasswereselfedinordertosupportthe phenotypesassignedtoeac hgenotype..Thefivegenotypicclasseswere:1=upright(AAAA genotype),class2=semi upright(AAAagenotype),class3=prostrate(AAaagenotype),class4 =semi trailing(Aaaagenotype),andclass5=trailing(aaaagenotype). y Valuesforthechi s quaretestarelistedasX 2 withtheP valuesgivenforeachplantwithinthe genotypicclass. z Valuesforthechi squaretestforhomogeneityarelistedasthetotalX 2 andtheP valuesfor eachgenotypicclass.

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57Figure2-8.Meanratingvaluesforcolorbrightnessfor‘RedQueen’(RQ)selfed,‘Sedona’(S)selfed,F1,andtheF2/F3population.Colorbrightnesswasratedona1-5scalewhere1=verydark(colorasdarkasordarkerthanRQ),2=dark(colorslightlylighterthanRQ),3=medium(colorinbetweenRQandS,4=bright(colorslightlydarkerthanS),and5=verybright(colorasbrightasorbrighterthanS). ColorBrightness0.000.501.001.502.002.503.003.504.004.505.00Populations RQself Sedonaself F1 F2 AACB Rating

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58 Figure2 9. F1plantsproducedfromthecrossRedQueen(RQ)xSedona(S)withbrighter foliagecolors. (A D)FoliagecolorwasbrighterthanRQbutstilldarkerthanS andnewcolorcombinationswerealsoobservedwhichwerenotseenintheRQ andSselfpopulations. A B C D

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59 Figure2 10. F2andF3plants(produced fromselfingF1andF2plants,respectively)with brighterfoliagecolorthanRQandasbrightasS. A)plantinahangingbasket.B) varietywithorangefoliagecolor.C)close upofplantwithpinkfoliagecolor.D) close upofplantwithgreenfoliage color. A B C D A B C D A B C D

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60 Figure2 11. DevelopmentofCopperPennywithtrailinghabitandorangecolor. A)Red Queen.B)Sedona.C)F1varietyH66.D)F2varietyCopperPenny.E)F3 seedlingsfromselfingCopperPenny.F)Close uppictu reofoneseedlinginF3 population.

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61 CHAPTER3 INFLUENCEOFLIGHTINTENSITYONCOLEUSFOLIAGECOLORCHANGES Introduction Solenostemonscutellarioides (L.)Codd ,commonlyknownascoleus,isabeddingplant valuedprimarilyforitsvibrantcolorfulfoliage andnotforitsfloralcharacteristics(Lebowitz, 1985).Inplants,colorationofdifferentorganssuchasflower,fruitandleafisduetothe accumulationofbetalains,carotenoidsorflavonoids(anthocyanins)(Mol,1998).Anthocyanins arethemajorpig mentsthatimpartthewiderangeofredandpurplecolorsobservedincoleus leaves(Lebowitz,1985).Thesynthesisofanthocyaninsandcolorchangeinvegetativetissues duetolighthavebeeninvestigatedatthephysiologicalandmolecularlevelformany plant speciessuchasmaize(Singh,1999), Perillafrutescens (Gong,1997),andinbilberry(Jaakola, 2004)butlimitedworkhasbeendoneincoleus. Anthocyaninsarewater solublepigmentswhicharederivedfromabranchoftheflavonoid synthesispathw ayforwhichdiagrammaticrepresentationsareavailableinnumerous publications(Schijlen,2004;Moletal.,1998).Thethreepredominantanthocyaninpigments foundinplantsarepelargonidins,cyanidins,anddelphinidins(HoltonandCornish,1995).The g enesinvolvedinthecontrolofanthocyaninsynthesisandhencecolorationinplants (Quattrocchioetal.,1993)havebeendividedintotwotypes:structuralandregulatorygenes. Mostofthestructuralgeneshavebeenclonedfromseveralmodelplants,inc ludingmaize, Antirrhinum Petunia ,and Arabidopsis (Schijlen,2004).Theseencodephenylalanineammonia lyase(PAL),chalconesynthase(CHS),chalconeisomerase(CHI),dihydroflavonolreductase (DFR),flavanone 3 hydroxylase(F3H),flavonoid3 hydroxylas e(F3H),flavonoid3,5 hydroxylase(F3,5H),anthocyanidinsynthase(ANS),andUDP glucose:flavonoid3 O glucosyltransferase(UFGT)(HoltonandCornish,1995).Regulatorygeneswhichcontrol

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62 expressionofthesestructuralgenesincludetheR/BandC1/Pu rple(PI)familiesinmaize (Dooneretal.,1991),DELILAandELUTAgenesinsnapdragon(Goodrichetal.,1992),andthe ANTHOCYANINgenes(AN1,AN2,AN6,AN10,andAN11)inpetunia(Quattrocchioetal., 1993).CharacterizationoftheAN1,AN2,andAN11ge nesshowedthattheyaretranscription regulators(deVettenetal.,1997).AN1encodesthebasichelix loop helix(bHLH)familyof transcriptionfactors,AN2isaMYBtranscriptionfactor,andAN11encodesaWD 40repeat protein. Foliagecolormayalsobe dependentonotherfactorssuchasco pigmentationandvacuolar pH(Moletal.,1998).Co pigmentationinvolvestheinteractionofanthocyaninswithothernon coloredflavonols(e.g.flavonols),carotenoids,ormetals(e.g.Mg 2+ ),andthemodificationof anthocyaninsduetointeractionswithothermoleculesresultsinincreasedanthocyanin pigmentationandhuechanges(Grotewold,2006).Acidificationofthevacuoleresultsinred colorinPetuniaflowers;severalmutants(ph1 7)withdarkbluecolorshadh ighpHinthepetal extractswithoutachangeinanthocyanincompositionandquantity(vanHouwelingenetal., 1998). Thetypesofpigmentsinvolvedinthecolorationofcoleusfoliagehavebeeninvestigated (Lebowitz,1995)butlittleisknownabouttheir geneticcontrol.Thebrightredandpurplecolors observedintheleavesareproducedprimarilybyanthocyaninpigments,withmostofthe pigmentscomposedofacomplexofcyanidinandglucosecomponents(Lebowitz,1985).Green colorationismainlydueto chlorophyllpigments(Rife,1948).Asmottledcoleuscultivarsage, thecontentofcaroteneandxanthophyllinleavesincreases(Lebowitz,1985). Theregulationofanthocyaninsynthesisinvegetativetissuesbylighthasbeenwell documentedinmanyplant species(Meng,2004).Aproposedf unctionforanthocyaninsisthe

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63 photoprotectionofvegetativetissuesthatarepredisposedtophotoinhibition(Steyn,2002),as theycansignificantlymodifyboththequantityandqualityoflightincidentonchloroplasts (Krol etal.,1995).Underlowlightlevels,foliageof Pennisetumsetaceum cvsRubrumandRed Ridinghoodislight purpleorgreenwhileunderhighirradianceleavesbecomedarkpurple (Beckwith,2004).Mutantsdefectiveintheproductionofanthocya ninshavebeenshowntobe moresensitivetoUV Bdamageinwhichbleachingofthefoliageisobserved(Delpech,2000). InamajorityofplantsbeingdevelopedinthecoleusbreedingprogramatUF,foliagecolor becomesdarkredwhenplantsareexposedto highlightintensities.Inthisstudy,tworelated coleusvarieties RoyalGlissade (RG)and UF06 1 6 (UF)(aseedlingproducedbyself pollinatingRG)showingdifferentcolorchangeinresponsetolightintensitychangeswere examinedunderdirectligh tandshadeconditionsinthegreenhouse.Asalackofinformation existstoexplainthephysiologicalandmolecularmechanismsforcolorchangeincoleusfoliage, abroad basedapproachwaspursuedinthisstudytoprovideabaselevelofinformationfor this phenomenon.Themainobjectivesofthisstudyweretodetermine:(1)theeffectoflightintensity onfoliagecoloration,(2)theeffectofanthocyaninandchlorophyllaccumulationonfoliage coloration,and(3)theregulationofgenesinvolvedinant hocyaninsynthesisincoleus. MaterialsandMethods PlantMaterialsandGrowthConditions VegetativecuttingswithtwonodesweretakenfromstockplantsofRGandUF(72per variety)growninaglassgreenhouseunderstandardconditionsat23+2 o C.Cutti ngswere removedfromthemisthouseafterrooting(~tendays),transplantedinto12.7cmazaleapots,and grownunder25%shadeinthegreenhouseforanadditionalthreeweeks.Afterthreeweekswhen sixleafnodeswerepresentontheplants,64plantsth atwereuniformincolorandsizewere

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64 chosenfromeachvarietyandrandomlyassignedtotwolighttreatmentgroups:directlightand shadeinaglassgreenhouse.Ablackclothwasu sedtosettheshadetreatment. ExperimentalDesign Lightexperimentsfort hisstudywereconductedoveraperiodofthreeweeksbecauseRG andUFhadacompletefoliagecolorchangefromaninitialgreentoredwithinthisperiodin preliminaryexperiments.Twoindependentreplicatedlightexperimentswereconductedattwo diffe renttimes.Experiment1(E1)wasconductedfrom8March 29March,2007and Experiment2(E2)wasconductedfrom16May 6June,2007.PriortothestartofE1andE2, plantsofRGandUFwereinitiallygrownunder25%shade(moderatelight)andmovedt oeither 50%shade(lowlight)ordirectlight(highlight).InE1,them oderatelightintensitywas 250 mol 2 s 1 ,andplantswerethen transferredtoalowlight(150mol 2 s 1 )orhighlight (350 mol 2 s 1 ).In E2,themoderatelightwas340 mol 2 s 1 ,and plantswerethentr ansferredto thelowlight(225 mol 2 s 1 )orhighlight(450 mol 2 s 1 ).Thus,inthisstudy,RGandUFplants wereexposedtotwoinitialandfourbasiclightintensitylevels,fromth elowestlightintensityof 150 mol 2 s 1 inE1to the highestat450 mol 2 s 1 inE2. TheexperimentaldesignusedforE1andE2wasasplit splitplot,withthetwomain treatmentsbeingdirectsunandshadeandthesub treatmentsbeingtimeaftertheinitiationofthe experiment.Eachmaintreatmentwasdivid edintofourblocks,twoplantsperblock,withatotal ofeightplantssampledforeachvarietyperdatacollectiontime.Thedatacollectiontimewas dividedintofourweeklyintervals,designatedasweeks0,1,2,and3.Leaftissueatthefourth nodef romtheapexwascollectedinthemorning(~10am)ateachweeklyintervalforthe extractionofRNAandotheranalyses(anthocyaninandchlorophyllcontent,pH,andlight microscopy).Theseleaveswereactivelygrowingandstillexpandingbutnotoldenou ghtobe shadedbynewfoliage.

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65 Anthocyanin,chlorophyllandpHdetermination Foranthocyanindetermination,eightleafdiscs(1cm 2 )werecollectedfromfreshleaf tissuesandimmediatelyfrozeninliquidnitrogen.Samplesweregroundinliquidnitrogenan d anthocyaninswereextractedin1ml1%HCLiniso propanolandleftinthedarkat4 o Covernight (Singhetal.,1999).Sampleswerecentrifugedat10000Xgfor10min,diluted5Xwithdeionized water,and500mlofthesupernatantwereusedforthedetermina tionofabsorbanceby spectrophotometry(BioRadSmartSpec3000,California)at535nmand650nm.Anthocyanin contentwascalculatedaccordingtothefollowingformulaA=(A 535 (2*A 650 ))/leafareaused (8cm2)aspreviouslydescribedbyMurrayandHackett(1 991).Theabsorbanceat650nmwas deductedfromtheabsorbanceat535nmtocorrectthevalueforthechlorophyllcontent.Each meanisrepresentedastheabsorbancevalueofeightindependentdeterminationspertreatment. Forchlorophylldetermination,ei ghtleafdiscs(1cm 2 )werecollectedfromfreshleaf tissuesandimmediatelyfrozeninliquidnitrogen.Samplesweregroundinliquidnitrogenand chlorophyllwasextractedin1ml96%ethanolandleftinthedarkat4 o Covernight.Samples werecentrifuged at10000Xgfor10minand500mlofthesupernatantwereusedforthe determinationofabsorbanceat649nmand665nm.ThetotalchlorophyllA/Bconcentrationwas calculatedbysummingtheindividualcalculationsforchlorophyllAandBaccordingtothe formu laChlA=(9.78*A 665 ) (0.99*A 649 )/perleafareaused(8cm2)andChlB= (21.4*A 649 ) (4.65*A 665 )/perleafareaused(cm 2 )aspreviouslydescr ibed(Goncalves,etal. 2001). Eachmeanisrepresentedastheabsorbancevalueofeightindependent determ inationsper collection. ForpHdetermination,one3 cm 2 piecewascollectedfromfreshleaftissuesandgroundin 5000ldeionizedwaterwithamortarandpestleandmeasuredwithapHmeteraspreviously

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66 describedbySpelt(2002).ThepHmeasurementswer erecordedasthemeanofeightreplicate samplespercollectiontimepointinE2. SamplesforanthocyaninandchlorophyllanalysisandpHlevelwerecollectedforRGand UFforbothtreatmentconditionsatallweeklytimepointsthroughoutE1andE2.Anal ysisof variance(ANOVA)andaTukeystestwereperformedtodeterminetreatmenteffects(p=0.05). FreshWeightandLightMicroscopy Freshweightwasdeterminedbyexcisingtheentireplantatthesoillineandweighingiton abalance(PS7S01 S,Mettler Toledo).Freshweightwasrecordedasthetotalweightofeight plants(g)pertreatmentateachweeklycollection.Afterfreshweightwasdetermined,leaf tissueswereusedformicroscopy.Freshleaftissueswerehand sectionedalongthelongitudinal axis oftheleavesandimmediatelyviewedunderacompoundlightmicroscope(Nikon Labophot 2).Microscopyphotoswere takenatamagnificationof200X forplantsateach weeklytimepointinE1. RNAisolationandcDNAsynthesis TotalRNAwasextractedfrom coleusleavesusingtheTriZOL TM LSreagentmethod accordingtothemanufacturersmanual(GibcoBRL,Invitrogen,USA)withmodificationsas previouslydescribedbyVerdonk(2006).TotalRNAsampleswerefurtherpurifiedwitha phenol chloroformtreatme ntandprecipitatedwith3Msodiumacetateandiso propanol accordingtoSambrooketal.(1989).SamplesweretreatedwithDNase,withmodificationsas previouslydescribed(DalCinetal.,2005)andsinglestrandedcDNAwassynthesizedinafinal volumeo f20l,withmodificationsaspreviouslydescribed(DalCinetal.,2007). IsolationofPartialClonesInvolvedinColoration Atthebeginningoftheexperiments,nogenomicorEST(ExpressedSequenceTag) sequencesforcoleuswerepresentinthedatabase (http://www.ncbi.nlm.nih.gov/BLAST/).The

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67 identificationofspecificclonesencodingtheelementsinvolvedinanthocyaninbiosynthesisin coleuswaspursuedbytheamplificationofpartialclonesfromgenomicDNAorfromcDNA withdegenerateprimers.Thed esignofthedegenerateprimerswasperformedbythe identificationofconservedregionofthegenesofseveralspeciesinwhichtheelementsinvolved intheanthocyaninpathwayhavebeenextensivelycharacterized.Thedegenerateprimerswere specifically designedinthisstudy(ACT,PAL,CHS,DFR,UFGT,AN1,andAN2)orwere designedaspreviouslysequencedfrom Perillafrutescens (ANSandF3H)(Gong,1997)(Table 3 1).Theamplificationprofileforallcloneswasasfollows:aninitialholdat94 o Cfor5m in,40 cyclesandafinalholdat72 o Cfor5min.Eachcyclewascomposedofaninitialdenaturationfor 60sat94 o C,60satanannealingtemperature(Table3 1)specificfortheprimercoupleandan extensionat72 o Cfor60s.Reactionswerecarriedoutwit htheGeneAmpPCRSystem2700 (AppliedBiosystems,California)using0.025U/ lTaq polymerase(AmershamBiosciences, Piscataway,NJ),0.5lofcDNA,2Mprimers,1.5mMMgCl2,buffer1Xand1mMdNTPsina finalvolumeof25 l .Productswereelectrophores edina1.5%agarosegelandstainedwith ethidiumbromide.BandswerepurifiedwithaQiagen(USA)gelextractionkit,elutedfragments wereligatedintopGEMT easyvector(Promega,USA),andtransformedintochemically competentcells(XL1 Bluegenotype :recA1endA 1gyrA96thi 1hsdR17supE44relA1lac[F proABlacl9 colonyPCRusingSP6andT7plasmidspecificprimers.PositivecoloniesweregrowninLB mediaandplasmidsextractedwitht heTEG sodiumhydroxidemethod(Sambrooketal.1989) andplasmidsdigestedwithECORIrestrictionenzymetodetermineclonesize.Positiveplasmids weresequencedwithSP6andT7primersandsequenceswerealignedwiththeon lineblast x

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68 program(http:// www.ncbi.nlm.nih.gov/BLAST/)and(http://www.sgn.cornell.edu/tools/blast/)to confirmtheiridentity. ExpressionAnalysisusingSemi QuantitativeRT PCR RT PCRexpressionanalysisongenesisolatedinthisstudyandencodingPAL1,CHS, DFR,F3H,ANS,UFGT, AN1,andAN2wasperformedoncoleuscDNAusingspecificprimers (Table3 2)designedwiththeaidoftheon lineGeneFisherprogram (http://bibiserv.techfak.unibielefeld.de/genefisher/ ).PCRreactionconsistedof1lofcDNA, 0.75Mprimers,1.5mMMgCl2 ,buffer1Xand1mMdNTPsinafinalvolumeof20l.The amplificationwithspecific18Sandactinprimers(constitutivegenes)wasperformedtoverify similarquantitiesofcDNAusedforexpressionanalysis.Thechoiceoftwointernalcontrolswas usedto overcomethelimitationsduetotheuseofonlythe18Soronlythehousekeepinggene (Volkovetal.,2003).Theamplificationprofilewasasfollows:aninitialholdat94 o Cfor5min, avariablenumberofcycles(eachcyclecomposedofaninitialdenatur ationat94 o Cfor30s,30s atanannealingtemperaturespecificoftheprimercouple(Table3 2)andanextensionat72 o C for15s)andafinalholdat72 o Cfor5min.Productswereelectrophoresedina1.5%agarosegel stainedwithethidiumbromideandUVi mageswereacquiredwiththeGelLogic100Imaging System(Kodak,CT).Inordertoverifyprimerspecificity,amplificationproductsweregel purifiedandsequencedwiththespecificprimers.PCRreactionswererepeatedatadifferent numberofcyclestov erifyexpressionpatternandreproducibility. TheisolationofSsAN1andactin(SsACT)genomiccloneswasperformedon500ngof genomicDNAextractedfromcoleusleaveswiththeDNeasyPlantminikit(Qiagen,USA).The amplificationwasreportedasdescrib edabovefortheisolationofpartialclonesfromcDNA.The isolationofagenomicclonewasperformedinordertodesignacoupleofspecificprimers spanninganintron.TheseprimerswerealsousedtoverifythatgenomicDNAwasnotpresentin

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69thecDNA.ThepresenceofgenomicDNAmayinterferewiththeanalysisbyalteringthetemplateamountandthustheexpressionresults.ResultsandDiscussionPhysiologicalMechanismsInvolvedinFoliageColorChangeLightintensityaffectsfoliagecolorchangeinRGandUFRedcolorationincoleusisprimarilyproducedbyanthocyaninpigmentsandtheamountofanthocyaninpresentinleaftissueshasbeenshowntobeafunctionoflightintensity(Lebowitz,1985).TodetermineiflightaffectedfoliagecolorationinthecoleusvarietiesRGandUFinthisstudy,leavesrepresentativeofthecolorchangesforbothvarietiesateachweeklytimepointwerephotographed(Figure3-1).TheresultsindicatedthatleavesofbothRGandUFhadagradualtransitionfromgreentoredwithinthethreeweekperiodwhengrowninthedirectlighttreatments.Theobservationthatlightaffectscolorationincoleusfoliageissupportedbystudiesinotherplantspecies.InleavesofQuintiniaserrata,theoccurrencesofredareasonthelaminaweremoreprevalentinleavesexperiencinghighlightconditions(Gouldetal.,2000).SimilarlyinGalaxurceolata,leavesexposedtohighlightturnadeepshadeofredwhereasinleavesthatremainshadedstaygreen(Hughesetal.,2005).Furthermore,lightintensitywasobservedtoaffecthowfasttheplantstransitionedincolor.PlantsgrownunderdirectlightinbothE1andE2hadamorerapidchangefromgreentoredthanplantsgrownintheshade.WhenthefourlightintensitylevelsinE1andE2werecompared,RGandUFplantsgrownunderthelowestlightintensity(150mol-2s-1)hadtheslowestcolortransitionwhereasplantsgrownunderthehighestlightintensity(450mol-2s-1)hadthefastestcolortransition.Inaddition,RGandUFdifferedintheirrateofcolortransition.Atthestartoftheexperiment,UFleaveswerelessgreenincolorandthroughouttheexperiments,UFleavesreddenedearlierthanRGinbothdirectlightandshadetreatments.Ahighersensitivitytolight

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70 mayex plainthemorerapidtransitionfromgreentoredinUFthaninRG(Mancinelli,1990). SimilarresultswereobservedinastudyconductedbyMancinelli(1990)inwhichtomatoand cabbageseedlingsgrowingunderthesamelightconditionswerecompared.Itw asobservedthat maximumanthocyaninproductionintomatowasonly20%ofthatincabbage.Thus,plant differenceswereobservedinwhichcabbageproducedmoreanthocyaninsthantomatowhen grownunderthesamelightcondition. RedcolorationinRGandUF foliageisduetoanthocyaninaccumulation Ithasbeenreportedthatanthocyaninsarethemajorpigmentscontributingtothered colorationincoleusplants(Lebowitz,1985).Anthocyaninsarewatersolublepigments (Grotewold,2006)thataresynthesizedin thecytoplasm,activelytransportedacrossthe tonoplast,andaccumulatedinthevacuolesofepidermalormesophyllcells(Delpech,2000).To determinewheretheredcolorationoccurredinRGandUF,andifitwasduetotheaccumulation ofanthocyanins, lightmicroscopyphotosofcoleusleafepidermallayersweretakeninE1 (Figure3 2)andanthocyaninconcentrationmeasuredinabsorbance(A535)(Figure3 3)inE1 andE2.Anthocyaninswerefoundtoaccumulateonboththeupperandlowerepidermallayers throughouttheexperiments.Inaddition,colorationoftheupperepidermallayersforboth varietieswasdarkerincolorinplantsgrownunderdirectlightconditionscomparedtoshaded conditions.Similarobservationstothesehavebeenreportedformaize .Inseveralmaize varieties,exposuretolightinducedtheaccumulationofanthocyanininthevegetativetissuesand resultedinsun redphenotypes(Singhetal.,1999).Whentheupperepidermallayersofboth RGandUFwerecompared,UFwasobservedto accumulatemoreanthocyaninsintheupper epidermallayerthanRG.Thisobservationfurthersupportsthevisualobservationofthefoliage asseeninFigure3 1inwhichthereddeninginUFleavesoccurredearlierthaninRG.

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71 Whenanthocyaninconcentratio nsweremeasuredinRGandUF(Figure3 3),theresults furthersupportedthevisualobservationsandlightmicroscopyphotos.InbothE1andE2,RG andUFplantsgrownunderdirectlightatweek3hadhigherconcentrationsofanthocyaninsthan plantsgrow nintheshade.RGandUFhadthelowestanthocyaninconcentrationsatthelowest l ightintensity(150 mol 2 s 1 )andthehighestconcentrationsatt hehighestlightintensity (450 mol 2 s 1 ).Dependingonplantspecies,lightexposurehasbeenshowntobea prerequisite forsignificantanthocyaninsynthesisinvegetativetissuesandanthocyaninlevelsvaryinrelation tolightexposurelevels(Mancinelli,1983;Kroletal.,1995).ComparingRGandUF,atweek0 UFwasobservedtohavehigheranthocyaninconc entrationsthanRG.Atweek3forE1andE2, bothvarietieshadsimilaranthocyaninconcentrationsinthesun,whereasintheshade,UF accumulatedhigheramountsthanRG.Theseobservationsfurtherparalleltheresultsasobserved intheredderfoliageco lorofUFatw eek0andweek3inE1andE2. pHisnotafactoraffectingfoliagecolorationinRGandUF ChangesinpHlevelhavebeenproposedtoaffectplantcoloration(Moletal.,1998).In Petuniahybrida forexample,theacidificationofthevacuole resultsinaredcoloroftheflower andmutantshaveashiftofflowercolortowardsblue(Grotewold,2006).Inordertoobserve whethercolorchangesincoleusleavesweretheresultofchangesinleafpH,thepHwas measuredonaweeklybasisforE1an dE2(Figure3 4).Nosignificantchangeswereobserved forRGandUFplantsgrownunderdirectlightandshadetreatmentsandnodifferenceswere observedbetweenthevarieties.ItcanbeconcludedthatpHwasnotafactorcontributingtothe redcolorati oninRGandUF. Lightintensityaffectschlorophyllconcentrationandfoliagecoloration Todetermineiflightintensityaffectedchlorophyllconcentrationsandthuscoloration, pigmentcontentweremeasuredforRGandUFinbothexperiments(Figures3 6 and3 7).At

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72 week3,RGandUFplantsgrownintheshadeaccumulatedhigheramountsofchlorophyllthan plantsgrownindirectlight,withtheexceptionofUFintheshadeinE1.Bothvarietiesbehaved similarly,astheyhadthelowestchlorophyllconcentr ationsatthehighestlightintensity (450mol 2 s 1 )andthehighestchlorophyllconcentrationsatth emoderatelightintensity (225 mol 2 s 1 ).Similarresultshavebeenshowninstudiescomparingplantsgrowninthesun andshade.Underlowlight,plants producemorechlorophylltomaximizephotosynthesis whereasathighlightlesschlorophyllismadetoavoidphotodamage(ChenandXu,2005). AbalancebetweenchlorophyllandanthocyaninsappearedtoaffectplantcolorationinRG andUF.Foliageandlight microscopyphotosshowedthatthroughouttheexperimentUF accumulatedanthocyaninsontheupperepidermallayerearlierthanRG.Furthermore,green colorationdisappearedearlierinUFthaninRGinbothexperiments.Thissuggeststhatthe balancebetwee nchlorophyllandanthocyaninconcentrationsmaybemajorfactorsaffecting colorchangeincoleus.Theresultsregardingeitherchlorophylloranthocyaninconcentrations alonewerenotsufficienttoexplainthecolor differenceinUFandRG.At150 mol 2 s 1 (shade, E1),thedifferenceincolorbetweenUFandRGappearedtobepredominantlyduetochlo rophyll content,whereasat225 mol 2 s 1 and450 mol 2 s 1 (shadeanddirectlight,E2),colorwasmainly duetoanthocyaninaccumulationsinceonlyaslightd ifferenceinchlorophyl lquantitywas recorded.At350 mol 2 s 1 (directlight,E1),thebrighterredcolorobservedinUFwaslikelydue tobothalowchlorophyllcontentandhighlevelofanthocyaninaccumulation. LightintensityaffectsplantgrowthinR GandUF Lightisnecessaryforphotosynthesis(ChenandXu,2005)andtheamountoflightis criticalforplantgrowth(Lefsrud,etal.,2006).Lightintensityhadaneffectonplantgrowthfor bothRGandUF(Figure3 7).InE1,lightintensityfor shade anddirectlightwas150 mol 2 s 1 and350 mol 2 s 1 ,respectivelywhereasinE2,lightintensityfor shadeanddirectlightwas

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73 225 mol 2 s 1 and450 mol 2 s 1 ,respectively.Itmustbenotedherethattheday lengthinE1, whichwasconductedinMarch,wasalsotwohoursshorterona veragethaninE2conductedin May.Freshweightsofapproximatel y450gforRGand300gforUFwere recordedatweek3 whentheyweregrownat350 mol 2 s 1 (directlight,E1).Withaninc reaseinlightintensityto 450 mol 2 s 1 areductioninfreshweightt o232gforRGand271gforUFwasobservedatweek 3.ItcanbereasonedthatRGandUFshouldhavehigherfreshweightsunderhigherlight intensitiesduetohigherphotosyntheticactivity(Huneretal.,1998).Ho wever,alightintensityat 450 mol 2 s 1 an dtheadditionofalongerday lengthmayhaveresultedinphotoinhibition.Ithas beenshownthatphotoinhibitioncanleadtosignificantdecreasesinplantproductivi ty(Huner,et al.1998).At150 mol 2 s 1 lowestfreshweightwasrecordedforbothvarie ties,at150g(RG)and 87g(UF).Thelo wfreshweightinUFat150 mol 2 s 1 mayhelptoexplaintheirregularity observedinwhichUFhadthelowestchlorophyllconcentrationswhengrownintheshadein E1(Figure3 6) .Thuslightintensitiesat150 mol 2 s 1 and450 mol 2 s 1 weresuboptimalfor maximumplantg rowthwhilearangebetween350 mol 2 s 1 fordirectlightand225 mol 2 s 1 for shadeappearedtobeoptimalformaximumplantgrowth. MolecularMechanismsAffectingFoliageColorChangeinRGandUF Isolati onandexpressionofclonesrelatedtoanthocyaninsynthesis Thestructuralandregulatorygenesinvolvedintheanthocyaninbiosyntheticpathwayhave beenextensivelycharacterizedinothermodelspeciessuchasmaize,snapdragon,andpetunia (Winkel Shir ley,2001;Moletal.,1998;HoltonandCornish,1995).In Petuniahybrida ,atleast thirty fivegenesareknowntoinfluenceanthocyaninbiosynthesisinflowers(Wieringandde Vlaming,1984;Koes,1988).Inthisstudy,eightgenes(PAL1,CHS,F3H,DFR,A NS,UFGT, AN1andAN2)involvedintheanthocyaninsynthesispathwaywerechosenforexpression analysisincoleus.Thesixstructuralgeneschosenarelocatedatthebeginning(PALandCHS),

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74 middle(F3HandDFR),andend(ANSandUFGT)ofthepathway.At thestartofthestudy, thesegeneswerenotcharacterizedincoleus.Theuseofdegenerateprimersallowedforthe identificationofseveralpartialclonesencodingtheproteinsinvolvedinanthocyanin biosynthesisandtranscriptionfactorsthatareknown toregulatethosegenesincoleus.The fragmentsisolatedaftercloningandsequencingwerealignedwithothermajorplantspecies usingtheNCBIGenBankBLAST Xprogram.Alistoftheisolatedpartialclones,theirsequence identitiescomparedtootherp ublishedsequences,theGenBankaccessionnumbersandblaste values(measureofthesimilarityofthequeries)areshowninTable3 5. Enzymesinvolvedinanthocyaninsynthesisincoleus Thenumberofcyclesrequiredtogenerateanon saturatingoptimal levelofexpression indicatedthattranscriptamountsofDFRandUFGT(24cycles)wereexpressedinthemost abundance,followedbyPAL1andCHS(27cycles),F3H(29cycles)andANS(30cycles). TranscriptlevelsoftheregulatoryenzymesAN1andAN2weret heleastexpressedasatleast32 and37cycleswererequiredtoobserveanoptimallevelofexpression(Figure3 8).Alow numberof24cyclesrequiredtoseeexpressionofDFRandUFGTweretobeexpectedasboth enzymesareresponsibleforthesynthesis ofspecificanthocyaninsandthusplantcolorationin coleus.DFRcatalyzesthereductionofthreecolorlessdihydroflavonolstoleucoanthocyanidins (Schijlen,2004).Withthreedifferentsubstratestocatalyze,ahigherlevelofexpressionwas observedf orDFR.UFGThasbeenregardedasanindispensableenzymeoftheanthocyanin pathwayasitcatalyzesthefinalsteprequiredtostabilizeanthocyanidinstoaccumulateaswater solublepigmentsinthevacuoles(Schijlen,2004).Amoderatenumberof27cycle srequiredfor PAL1andCHSwerealsotobeexpectedastheybothbelongtomulti genefamilies(Durbinet al.,1995,Goto Yamamotoetal.,2002)andareinvolvedinnumerousotherprocessesbesides anthocyaninsynthesis(DixonandPalva,1995).Furthermor e,PALcatalyzesphenylalanine,the

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75 precursorforallflavonoidcompounds(Koes,1988)whereasCHSprovidesthefirstcommitted stepinanthocyaninsynthesis(Grotewold,2006).FollowingPALandCHS,F3HandANS required29cycles.TheactivityofF3Hmay bedependentontheproductsproducedbyCHS whileANScatalyzesonlyoneofthethreeproducts(leucoanthocyanidins)producedbyDFR (Schijlen,2004).HighernumberofcycleswasobservedfortheregulatoryelementsAN1and AN2andthismaybeduetothe factthattheyaretranscriptionfactorsandaretransientintheir regulation(Quattrocchioetal.,1999). Lightintensityandanthocyaninaccumulationandtheireffectsongeneexpression Theexpressionoftheseeightgenes(PAL1,CHS,F3H,DFR,ANS,UFG T,AN1.and AN2)wasanalyzedforRGandUFwhengrownindirectlightandshadeconditionsinE1and E2.Transcript levelsforallofthegeneswere observedtobehigherinUFthaninRGatweek0, thestartoftheexperiment.Higherexpressionlevelsob servedinUFsupportedtheresults previouslystatedinwhichUFwasdarkerincolorandhadhigheranthocyaninconcentrationsat week0thanRG.However,whenthetranscriptamountsforthestructuralgeneswerecompared betweentreatments(directlightv ersusshade)andinconjunctionwithanthocyanin concentrations,noclearpatternofexpressionwasobservedthroughouttheexperiments.Forboth varieties,transcriptamountsofPAL1,CHSandANShadminimalvariationinlevelsbetween directlightandsh adetreatmentsineitherE1orE2.DFRandUFGTexpressiondidnotcorrelate withtreatmentconditionsorwithanthocyaninconcentrations.Transcriptamountsofthesegenes inplantsgrownunderdirectlightwerenotalwayshigherthanplantsgrownunders hade. Furthermore,whereanincreaseinanthocyaninconcentrationswereobservedinbothvarieties, transcriptamountsforthegenesdidnotincrease.LightwasshowntoaffectcolorationinRGand UF,butnoevidencewasfoundforacorrelationbetweenge neexpressionandfoliagecolor ation.

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76 Althoughnocorrelationwasfoundbetweengeneexpressionandcoloration,acorrelationwas observedbetweentheexpressionpatternofUFGTandDFRandAN1. Regulatorygenescontrollingthetranscriptionofthestructu ralgeneshavebeenidentified bymutationinmaize,snapdragon,andpetunia(reviewedbyDooneretal.,1991).Inthisstudy, AN1mayplayaroleinregulatingtheexpressionofDFRandUFGT.InE2,expressionofAN1 followssimilarlytothatofUFGTand DFR,whileexpressionofAN2doesnotfollowthe expressionpatternsoftheotherbiosyntheticgenes.Twohypothesesthatmaybeproposedforthe similarexpressionpatternfoundbetweenAN1,UFGTandtoanextentDFRare:(1)AN1, UFGT,andDFRaresimila rlyregulated,or(2)AN1controlstheexpressionofUFGTand slightlyDFR.Thesecondhypothesisismorelikelytobefoundincoleusbecauseasimilar patternofcontrolofAN1onUFGTwasobservedinpetunia(Quattrocchioetal.,1993).The expressiono fAN2doesnotappeartobetightlycorrelatedwiththeexpressionoftheother biosyntheticgenesnordoesitappeartoregulatetheexpressionofAN1,aspreviouslydescribed bySpeltetal.(2000). Sincetranscriptamountsofthestructuralgenesdidno tcorrelatewithanthocyanin concentrations,regulationofthesegenesattheleveloftranscriptionisproposedtonotoccur. Thus,post transcriptionaland/orpost translationalregulationisthesuggestedreasonfor increasedanthocyanincontentinRGa ndUF.Evidenceforthesetwotypesofregulationhas beenshownin Petuniahybrida .Inpetuniacorollas,accumulationofanthocyaninsatthelater stagesofflowerdevelopmentwashypothesizedtoresultfromearlyaccumulationoftheCHS transcript.Since CHSexpressiondidnotalwayscoincidewithitseffectonanthocyanin accumulation,post translationaleffectswereproposed(Shvarts ,1997).TheactivityofAN2has

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77 alsobeenshowntoberegulatedpost transcriptionallybyacytosolicWD40repeatprotei n encodedbyAN11inpetunia(Shvarts,1997). F3HisresponsibleforimpartingthecolordifferencebetweenRGandUF Interestingly,onecleartrendobservedintheexperimentwasadifferenceintranscript amountsofF3HbetweenRGandUF.UFandRGdiffe redintheexpressionofF3Has consistentlyhighertranscriptamountswereobservedinUFthaninRGinalltreatmentsand experiments(Figure3 8).F3Hishighlyconservedamongwidelydivergentplantspeciesanda mutationresultinginalossofF3Hact ivity,bothin Petunia and Antirrhinum ,preventsthe progressionalongtheanthocyaninpathwayandresultsinwhiteflowers(Schijlenetal.,2004; Martinetal.,1991).TherapidanddarkercolorationofthefoliageobservedinUFis hypothesizedtobedu etotheoverexpressionofF3H.TheoverexpressionofF3Hmayleadtoa highformationofthesubstratedihydrokaempferol(DHK).Hydroxylationofthisproductatthe 3positionformsdihydroquercitin(DHQ)andultimatelytotheproductionofcyanidinsbas ed pigmentsandhencecolorformation(Scijlenetal.2004).Thus,theoverexpressionofF3Hmay haveresultedinthecolordifferencesobservedbetweenRGandUFthroughouttheexperiments. Furthermore,overexpressionofF3Hmayhelptoexplainthelowfr eshweightobservedin UFwhengrownunderth elowestlightintensityat150 mol 2 s 1 .Evenunderthelowestlight intensity,UFcontinuedtohaveacolorchangefromgreentored.Lightintensityhasbeenshown inthisstudytoaffecttheproductionofan thocyaninsinRGandUF.However,theaccumulation ofanthocyaninsandmaintenancecarriesanenergycost,asitmayreducelightcaptureand ultimatelycarbonassimilation(Steyn,2002).Therefore,thepoorgrowthofUFunderthelowest lightint ensityof 150 mol 2 s 1 maybeattributedtotheoverproductionofanthocyaninpigments andlowchlorophyllconcentrationsrequiredtosustainoptimalgrowth.

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78 Conclusions LightintensityaffectedfoliagecolorationinRGandUFinthisstudy.Asthelightintensity incr eased,thetransitionfromgreentoredcoloroccurredmorerapidlyinRGandUF.Foliage colorationwasduetotheaccumulationofanthocyaninpigmentsinbothvarieties.Anthocyanin synthesiswasnotobservedtoberegulatedattheleveloftranscription .AN1wasobservedto regulateUFGTandtoanextentDFR,butregulationofAN1,AN2,andtheotherstructuralgenes isproposedtooccurviapost transcriptionaland/orpost translationalregulation.Itisalso possiblethatothertranscriptionfactorsma ybeinvolvedintheregulationofthestructuralgenes andregulatorygenesAN1andAN2inthisstudy.

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79 Table3 1. Listofthedegenerateprimers andthecorrespondingconservedregions,andtheannealingtemperaturesusedduring amplification. Gene Forwa rdprimerandconservedregion Reverseprimerandconservedregion Tm PAL1 5 ARAGYGCNGAGCARCAYAACC 3 5 GGVAGAGGWGCACCRTTCCA 3 64 PAL1 SAEQHNQ WNGAPL CHS 5 CTGTCCTCCGCATGGCCAAG 3 5 GGCCCGAACCCAAACAGCA 3 64 CHS VLRMAKDL WGVLFGFG DFR 5 TYCA YGCWACYGTTCGTGATCC 3 5 CTTGYTTWATGATKSAGTARTGAG 3 60 DFR ATVRDPE HYSIIKQ F3H 5 SCARGAYTGGMGNGARATHGTVAC 3 5 GCYTGRTGRTCHGCRTTCTTGAABC 3 60 F3H QDWREIV FKNADHQ UFGT 5 ARAVKKTYCTCAAYRTHGGYCCWT 3 5 CCRAARAANGGCCTRCAAATCA 3 64 UFGT LNVGPS L ICRPFF AN1 5 CGGTTCAAACCGCTCAGTGGA 3 5 GGGTTTGGAGGATTAGAAGTGGAGT 3 64 AN1 SVQTAQW FSFPPGVG AN2 5 TATGGDGAAGGDAAGTGGCATC 3 5 GGAAKBTTCCGAGGTYGAGG 3 65 AN2 YGEGKWH VRPRPR ACT 5 CTCGGCAGTTGTGGTAAACATG 3 5 CCGTGTCGCTCCTGAAGAGC 3 64 ACT RVAPEEH GYMFTTTAE

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80 Table3 2. Listofthespecificprimers andoftheannealingtemperatureusedduringRT PCRexpressionanalysisoncoleuscDNA. Gene Forwardprimer Reverseprimer Tm( o C) PAL1 5 GCCAAGCCATTGATCTGAGGCAT 3 5 CGGAGCTTCTGCATCAAT GGGTA 3 65 CHS 5 GGAACTCAGTGTTCTGGATTGC 3 5 GGTGGATCTCAGCCCCTCCT 3 62 DFR 5 CTGGGACTTTGAATGTTGAGGA 3 5 GGAATGTGGGCATGAAGAATGG 3 64 F3H 5 TGATATGTCGGGTGGCAAGAAAGG 3 5 GCCTTCTCTCCTTCCCTCAACTTC 3 66 ANS 5 CGGCCTCCAGCTCTTCTACG 3 5 GGTCTCCGGTAGTGGCTGGA 3 66 UFGT 5 CTGCTTCCTGCTCTCTCTCTTCAC 3 5 CCGCAGTGAGTGACGAACACTC 3 65 AN1 5 GAGCGATGGATACTACAATGGA 3 5 GGAAACGAGAATGATACGCACA 3 62 AN2 5 GACCCGATATCAAAAGGGGCTT 3 5 CCGAGGTCGAGGCCTTACGAT 3 65 ACT 5 CTCGGCAG TTGTGGTAAACATG 3 5 CCGTGTCGCTCCTGAAGAGC 3 68 18S 5 TTAGCAGGCTGAGGTCTCGT 3 5 AGCGGATGTTGCTTTTAGGA 3 58

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81 Figure3 1 Visualfoliagecolorchangeofcoleusleavesthroughouttheexperiment. A) Experiment1(E1)conducte d8March 29March,2007andligh tlevelsfor directsunwas350 mol 2 s 1 andforshadewas150 mol 2 s 1 .B)Experiment2(E2) conducted16May 6June,2007withligh tlevelsfordirectsunwas450 mol 2 s 1 andforshadewas225 mol 2 s 1 .Photosweret akenateachweekinE1andE2 fromthestartoftheexperiments(W0). DirectLight Shade W 0 W 1 W 2 W 3 W 0 W 1 W 2 W 3 E1 RG RG UF UF E2

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82 Figure3 2 AccumulationofanthocyaninontheepidermalleaftissuesofRGandUFgrown inthedirectlight (350mol 2 s 1 )andshade(150 mol 2 s 1 ). Freshlea ftissueswere hand sectionedalongthelongitudinalaxisoftheleavesandviewedunderalight microscope(Nikon Labophot 2).Microscopyphotoswere takenata magnificationof200X forplantsateachweeklytimepoint(W1,W2,andW3) fromthestarto ftheexperiment(W0)inE1. Direct Light

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83Figure3-3.Anthocyanincontent(A535nm)forRG(solidsquare)andUF(solidtriangle)inthesun(solidline)andintheshade(dashedline).A)Experiment1(E1)conductedon8March–29March,2007andlightlevelsfordirectlightwas350mol-2s-1andforshadewas150mol-2s-1.B)Experiment2(E2)conductedon16May–6June,2007andthelightlevelsfordirectlightwas450mol-2s-1andforshadewas225mol-2s-1.Anthocyanincontentwasmeasuredateachweek(W1,W2,andW3)fromthestartoftheexperiments(W0).Dataaremeansofeightindependentdeterminationspertreatment. Anthocyanin0.000.050.100.150.200.250.300.350123Weeks RGlight RGshade UFlight UFshade Absorbance 535nm Anthocyanin0.000.100.200.300.400.500123Weeks RGlight RGshade UFlight UFshade Absorbance 535nm A B

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84 Table 3 3.DataanalysisforanthocyanincontentforRGandUF indirectlightandshade conditions. E 1 x Week Sun Shade E2 y Week Sun Shade RG 0 0.135cC z 0.158bC RG 0 0.094cD 0.112cD 1 0.137cC 0.089cD 1 0.250bB 0.144cD 2 0.199bB 0.110cC 2 0.366aA 0.131cD 3 0.280aA 0.101cD 3 0.385aA 0.222bC UF 0 0.219bB 0.228 aA UF 0 0.201bC 0.194bC 1 0.179bB 0.169bB 1 0.342aA 0.241bB 2 0.249aA 0.203aB 2 0.371aA 0.304aB 3 0.261aA 0.141bC 3 0.375aA 0.324aA x Experiment1(E1)wasconductedon8March 29March,2007andlightlevelsfo rdirect lig htwas350 mol 2s 1 andforshadewas150 mol 2s 1 y Experiment2(E2)wasconductedon16May 6June,2007andthelightlevelsfordirect lightwas450mol 2s 1 andforshadewas225 mol 2s 1 z Anthocyanincontent(Absorbance535nm)wasmeasuredateachweek( W1,W2,andW)from thestartoftheexperiments(W0).Dataaremeansofeightindependentdetermination.Within eachexperiment,significancewithincolumnsaredenotedinlowercaselettersandsignificance acrossrowsaredenotedinuppercaseletters.Va rianceanalysisandTukeystestwasperformed todeterminesignificanceatthe0.05level.

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85 Figure3 5. pHlevelforRG(solidsquare)andUF(solidtriangle)inthesun(solidline)and intheshade(dashedline) A)Experiment1(E1)wasconductedon8March 29 March,2007andligh tlevelsfordirectsunwas350 mol 2 s 1 andforshadewas 150 mol 2 s 1 .B)Experiment2(E2)wasconductedon16May 6June,2007and thelightlevelsfordirectsunwas 450mol 2 s 1 andforshadewas225 mol 2 s 1 pHdatawerecollectedateachweek(W1,W2,andW3)fromthestartofthe experiments(W0).Dataaremeansofeightindependentdeterminationsper treatment. pH 5.00 5.30 5.60 5.90 6.20 6.50 6.80 0 1 2 3 Weeks RGlight RGshade UFlight UFshade pH 5.00 5.30 5.60 5.90 6.20 6.50 6.80 0 1 2 3 Weeks RGlight RGshade UFlight UFshade l o g [ H + ] l o g [ H + ] A B

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86Figure3-6.TotalchlorophyllcontentforRG(solidsquare)andUF(solidtriangle)inthesun(solidline)andintheshade(dashedline).A)Experiment1(E1)wasconductedon8March-29March,2007andlightlevelsfordirectsunwas350mol-2s-1andforshadewas150mol-2s-1.B)Experiment2(E2)wasconductedon16May-6June,2007andthelightlevelsfordirectsunwas450mol-2s-1andforshadewas225mol-2s-1.Dataforchlorophyllcontent(Absorbance649nm+654nm)wastakenateachweek(W1,W2,andW3)fromthestartoftheexperiment(W0).Dataaremeansofeightindependentdeterminationspertreatment. Chlorophyll0.05.010.015.020.025.00123Weeks RGlight RGshade UFlight UFshade Chlorophyll0.05.010.015.020.025.00123Weeks RGlight RGshade UFlight UFshade Absorbance 649+654 nm Absorbance 649+654 nm A B

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87 Table3 4. Dataanalysismeasuredforchlorophyllcontent(absorbancevalues649nmand 654nm)forRGandUF indirectlightandshadeconditions. E1 x Week Sun Shade E2 y Week Sun Shade RG 0 10.60bC z 9.71cC RG 0 12.14aB 12.97bB 1 9.62bC 12.89bB 1 9.29aB 10.81bB 2 12.30aC 16.24aA 2 12.61aB 19.41aA 3 15.20aA 15.98aA 3 12.46aB 19.66aA UF 0 5.69cD 6.99cD UF 0 12.64 aB 13.14bB 1 6.80cD 7.58cD 1 8.24aC 13.69bB 2 12.12aC 4.82dD 2 9.61aB 18.86aA 3 12.22aC 7.57cD 3 10.61aB 17.75aA x Experiment1(E1)wasconductedon8March 29March,2007andlightlevelsfordirect light was350 mol 2 s 1 andforshadewas150 mol 2 s 1 y Experiment2(E2)wasconductedon16May 6June,2007andthelightlevelsfordirect lightwas450 mol 2 s 1 andforshadewas225 mol 2 s 1 z Chlorophyllcontent(Absorbance649nm+654nm)wasmeasuredatea chweek(W1,W2, andW)fromthestartoftheexperiments(W0).Dataaremeansofeightindependent determination.Withineachexperiment, significancewithincolumnsis denotedinlowercase lettersa ndsignificanceacrossrowsis denotedinuppercaselet ters.Varianceanalysisand Tukeystestwasperformedtodeterminesignificanceatthe0.05level.

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88Figure3-7.FreshweightforRG(solidsquare)andUF(solidtriangle)inthesun(solidline)andintheshade(dashedline).A)Experiment1(E1)wasconductedon8March–29March,2007andlightlevelsfordirectsunwas350mol-2s-1andforshadewas150mol-2s-1.B)Experiment2(E2)wasconductedon16May–6June,2007andthelightlevelsfordirectsunwas450mol-2s-1andforshadewas225mol-2s-1.Freshweightdatawascollectedateachweek(W1,W2,andW3)fromthestartoftheexperiments(W0).Dataweremeasuredasthetotalfreshweight(ingrams)ofeightplantspertreatment. Freshweight0501001502002503003504004505000123Weeks RGlight RGshade UFlight UFshade Grams Freshweight0501001502002503003504004505000123Weeks RGlight RGshade UFlight UFshade Grams A B

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89 Table3 5.Listofth epartialclonesinanthocyaninbiosynthesisincoleus. Acomparisonofthepartialclonestopublishedsequencesfromotherpl antspecies.In thetable,thelengthofthepartialclones,theaccessionnumbersandblaste values (similarityofsequences),andtheclosesthomologsto Vitisvinifera (Vv), Petuniahybrida (Ph), andanotherrelevantspeciesareshown[ Antirrhinumma jus (Am), Capsicumannuum (Ca), Ipomoeatricolor (It), Nicotianatabaccum (Nt), Pimpinellaanisum (Pa),and Silenelatifolia (Sl)] Gene Species Length Blaste values GenBankaccessionnumbers PAL1 Ss 698 0 Thisstudy(E U019242) Vv 5E 103 EF192469 Ph 1E 109 SGN E526590 Nt 8E 107 AB008199 CHS Ss 619 0 Thisstudy(E U01923 9) Vv 1E 99 AB066274 Ph 7E 100 X14599 Am 3E 102 X03710 DFR Ss 583 0 Thisstudy(E U019240) Vv 3E 78 X75964 Ph 5E 86 X79723 Am 2E 87 X15536 F3H Ss 459 0 Thisstudy(E U019241) Vv 2E 77 AM430949 Ph 1E 74 AF022142 Pa 1E 78 AY817674 UFGT Ss 412 0 Thisstudy(E U019243) Vv 3E 31 AB047099 Ph 2E 32 AF165148 Ac 3E 33 AB103471 AN1 Ss 558 0 Thisstudy(E U019237) Vv 1E 35 DQ886417 Ph 7E 36 EF423846 It 1E 31 AB154371 AN2 Ss 320 0 Thisstudy(E U019238) Vv 5E 41 DQ886417 Ph 7E 43 EF423846 Ca 3E 43 AJ608992 Actin Ss 422 0 Thisstudy(E U019236) Vv 2E 51 AM465189 Ph 6E 65 SGN U211816 Si 5E 52 AB094079

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90 Figure3 8. ExpressionanalysisofthegenesPAL1,CHS ,F3H,DFR,ANS,UFGT,AN1,AN2,andcontrols18SandACTfor RoyalGlissade(RG)andUF06 1 06(UF). A)Experiment1(E1)wasconductedon8March 29March,2007and ligh tlevelsfordirectsunwas350 mol 2 s 1 andforshadewas 150 mol 2 s 1 .B)Exp eriment2(E2)wasconductedon16 May 6June,2007andtheligh tlevelsfordirectsunwas450 mol 2 s 1 andforshadewas225 mol 2 s 1 .Samplesfor expressionanalysiswerecollectedfromleaftissuesateachweek(1W,2W,3W)fromthebeginningofthe experiment (T0).Representativeresultsofthesemi quantitativePCRaswellasthenumberofcyclesarereported. 27 27 29 24 30 24 32 37 32 34 RG UF PAL1 CHS Actin 18S AN2 AN1 UFGT ANS F3H DFR Number ofcycles A

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91Figure3-8.Continued.27 27 29 24 30 24 32 37 34 34 UF RG PAL1 CHS Actin 18S AN2 AN1 UFGT ANS F3H DFR Numberofcycles B

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92 CHAPTER4 DEVELOPMENTOFNEWNOVELCOLOREDCOLEU SCULTIVARSWITHIMPROVED VIGOR,COLORQUALITY,ANDLATEFLOWERINGINDUCTIONFORTHE COMMERCIALMARKET Introduction Solenoste monscutellarioides (L.)Codd,commonlyknownascoleus,isunlikemanyother popularornamentalplantsbecauseitishighlyprized foritsvibrantfoliageratherthanforits flowers(Lebowitz,1995).Althoughseedpropagatedcultivarsexistonthemarket,manynew varietiesofcoleusarenowpropagatedvegetatively.Thesuccessofornamentalplantsforthe commercialmarketdepends oncertaincharacteristicsdesiredbybothproductioncompaniesand consumers.Forcoleus,desirablecharacteristicscommontobothsidesarevigor,colorquality, andlatefloweringinduction.Plantswithgoodoverallplantvigorhavefastgrowthratesfo rrapid turnoverandtheabilitytoproducemaximumcuttingsforproduction(Wilkins,1988).Thebasis ofallvegetativeproductionrequiresplantswithgoodbranchingandthenumberofapical meristemsthatareproducedultimatelydeterminesthenumberof cuttingsproduced(Wilkins, 1988).Lateralbranchingisimportantfortheattractivenessofaplanttoconsumers(Hayashi, 2001)andthusvarietieswithgoodplantvigorintermsofexcellentbranchingpatt ernsareideal forthemarket. Theeconomicsucce ssofornamentalsusedforfoliagecharactersoftendependsonthe qualityofleafpigmentation.Flowerorfoliagecolorisoneofthemostimportantconsiderations whenconsumersmakeplantpurchases(Catanzaro,2005)andfadedcolorsdecreaseproduct val ue(Oren shamir,2003).Formanyornamentalcrops,thelossofcolorqualitythroughcolor fadingisaffectedbytwomainenvironmentalfactors:lightandtemperature.Lightquantityand qualityplayaroleinpigmentationchangesinplants.Smalllevelso fUVBcauseleavesof Dendranthemaxgrandiflorum cvSurftobecomebrighterinredcolor,whileinared green

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93 coleuscultivarWizardVelvetRed,UVBresultsintheemergenceofredpigments(Hoffman, 1999).UVAandUVBinducethesynthesisofanthocy aninsinleavesof Kalanchoexhybrida Colorado(Hoffman,1999).Theproductionofanthocyaninsinplantsmayserveasaprotective mechanismagainstUVradiationbyfunctioningintheprotectionofthephotosystems(Neilland Gould,2003).Damagestoth ephotosyntheticapparatus,whichresultsfromphotoinhibition,are oftenobservedinplantsasableachingoffoliagecolors.Thisbleachinginturnreducesfoliage colorquality(Krause,1999).Elevatedtemperaturesalsoaffectcolorqualitybydecreasin glevels ofanthocyaninproductioninplants(Shaked Sachray,2002;Dela,2003;Oren shamir,1999). Lowtemperatureat17 o Cinducesmoreaccumulationofredpigmentationinleavesof Cotinus coggygria cv.RoyalPurplecomparedtoplantsgrownatahigher temperatureof29 o C(Oren shamir,1997).In Photiniaxfraseri cv.RedRobinleaves,redpigmentationdecreaseswith increasedtemperatures(Oren shamir,1999).Inregardstocoleus,varietieswithbrightfoliage colorsthatdonotfadeunderhighlight intensityandhightemperatureconditionsarethus desirabletoconsumers. Thetimingoftransitionofaplantfromavegetativetoafloralstateisimportantforthe productionofornamentalplantsforthemarket(Chandler,2005).Earlyfloweringinorn amental cropsusedforfoliagecharacteristicsisoftenanundesirablecharacteristic.Earlyflowering increaseslandscapemaintenancecostsduetotheneedfordeadheadinganddecreasesfoliage colorqualityastheplantspendsmoremetabolicenergyto produceseeds. Gynuraaurantiaca hasthecommercialpotentialforuseasapottedplantforitsattractivefoliage,butflowering inductionthatleadstoproductionofmalodorousflowersisdifficulttoavoid(Pallez,2001). Manipulatingthetimingofflo raltransitionhasbeeninvestigatedthroughseveralmethodssuch aspruning(Gerber,2001),usingshadeclothstochangephotoperiodorlightintensity

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94 (Kobayashi,2006),andplantgrowthregulatorssuchasethephon(Hayashi,2001)butsuch methodsincrea seproductioncosts.Therefore,itisimportantforgrowerstomakeeffortsto produceinexpensiveattractiveplantsandforbreederstodevelopplantswithdelayedflowering toreducethecostofproduction(Hayashi,2001). Awiderangeofgeneticvariati onforplantvigor,foliagecolorqualityandflowering inductionisavailableincoleus.Wehaveobservedthatcoleusplantslackingvigoratthe seedlingstagedonotgrowfastenoughforcommercialproduction.Wehavealsoobservedthat plantswithstro ngapicaldominancedonotproduceenoughvegetativecuttingsforefficient production.Furthermore,earlyfloweringleadstoincreasedmaintenancerequirementsfor pruninginthelandscapeandlowerfoliagecolorqualityduetotranslocationofstoredres erves fromleavestoseeds. TheobjectiveoftheUFbreedingprogramistodevelopnewcultivarsthroughtraditional breedingmethodsforimprovedvigorforcommercialproduction,betterfoliagecolorqualityand delayedfloweringforconsumers.Throughou tthethreeyearsoftheprogram,improvements havebeenmadeinthecoleuspopulationforthesethreevariables,andnewcultivarshavebeen releasedwithallofthesecharacteristicsimprovedforthecommercialmarket. MaterialsandMethods BreedingMet hodology Attheinceptionofthebreedingprogramin2003,fifteencommercialcoleuscultivarswere grown,andopen pollinatedseedswerecollectedtogeneratethefirstseedlingpopulation.High levelsofvariabilityformanydifferenttraitssuchasfoli agecolor,leafshapeandsize,and growthhabitswereobserved,likelyduetothehighlyheterozygousnatureofcoleus(Lebowitz, 1985).Themethodofphenotypicrecurrentselectionwithhalf sibselectionwithprogenytest (PoehlmanandSleper,1995)was usedfortheimprovementofthequantitativecharacteristicsof

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95 plantvigor,colorquality,andlatefloweringinduction.Usingthehalf sibselectionmethod,a specificnumberofindividualswiththedesirablecharacteristicswereselectedfromanopen p ollinatedsourcepopulationandseedswereharvestedfromeachindividualseparately.A seedlingtestwasgrownfromeachcoleuslineattheendofthecropcycleinAugust October. Usinga72 cellpacktrayfilledwithsoillessmedia(Fafard2 mixmedia), 100seedsfromeach individualwereplantedandtheremainingseedswereretainedatroomtemperature.Basedon thetests,thenextcropcyclewasreconstitutedbyplantingtheremainingseedsfromthelines withseedlingsdisplayinghighlevelsofseedli ngvigorandvariabilityinfoliagecolors.Aseach cropcyclewasreconstitutedwiththeretainedseeds,individualswithsuperiorperformances fromeachyearlycropcyclewereisolatedfromthepopulationandwerere testedevery subsequentyearforrepl icatedperformance.Individualsreceivinghighratingscoresforvigor, colorquality,andlatefloweringinductioninadditiontonovelfoliagecolorhadthepotentialto bereleasedasnewcultivars. CropCycleandYearlyTrials Acropcycleconsistedof sixmajorparts:(1)sowingoftheseeds(2)threeselectionsof seedlingsateight,tenandtwelveweeksaftergerminationinagreenhouse(3)propagationof cuttingsinAprilforthesummeryeartrialsstartinginMay(4)summerfieldtrials,(5)seed c ollectionatendofthefieldtrialsinAugust,and(6)progenytestsinOctober November.The summeryeartrialsbeganwhencuttingspropagatedinAprilwereplantedforevaluation.Data werecollectedonceamonthattheendofJune,July,andAugustand endedwithselectionsmade forindividualsthatweresuperior. Sowingofseeds. Thestartofthecropcyclebeganwhentheseedsweresowninthefirst weekofJanuary.Aftersowingtheseedsusingahandseeder(SeedE ZSeederCompany),the flatsofso il(Fafard2 mixmedia)weremovedtoapolycarbonategreenhousewithtemperatures

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96 maintainedat21 27 o Cforthefirstweekofsowing.Duringthegerminationperiod(ten twenty) days,soiltemperatureswerekeptabove20 o C,aspreviouslydescribedbyLebow itz(1987).The flatsweremistedbyhandevery1hoursstartingat8amandendingat5pmtomaintain humidityuntilgerminationoccurred(fivetosevendays).Aftergermination,mistingcycleswere reducedtothreetimesperday,andoncetrueleavesfo rmed,theseedlingswerewatered overheadasneededandfertilizedwith50 100ppmnitrogenateveryotherirrigation.Seedlings werethentransplantedindividuallytoeachcellina72 cellpacktrayoncetheirfirstpairoftrue leaveswerepresent,appro ximatelythreeweeksaftersowing(Figure4 1). Selectionprocesses. Threeselectionsweremadeonseedlingsfromaninitialnumberof 30,000ateight,tenandtwelveweeksafterseedsowingforthevariablesofvigor,early floweringinduction,andnovel foliagecolor.Seedlingswereselectedvisuallyandthosewithlow vigororpoorbranchingwerediscardedfromtheprogram.Anyseedlingsthatinitiatedflowers duringthistimewerealsodiscarded.Remainingseedlings(~2,000)weretr ansplantedinto 12.7 c mazaleapots,pinchedoncetopromotebranching,andgrownuntilthe1stofMarch.In March,afinalselectionforseedlings(~500)withgoodvigor,branching,lackofflower inductionandnovelfoliagecolorwasconducted.Theremainingseedli ngsweret ransplantedinto 20.3 cmpotsandgrownunderthesameconditionsforsubsequentcuttingpropagationinApril. Propagationofcuttings. Afterthethreeselections,ninecuttingsweretakenfromeachof theremainingseedlingsandpropagatedinindividualce llsof72 cellpacktrayscontaining soillessmedia(Fafard2 mixmedia).Thelengthsof thecuttingswereatleast7.6 cmlongand hadatleasttwonodeswithfourpairsofleaves.Thecuttingswereplacedinamisthouse,with mistingcyclessetfortwent ysecondseverythirtyminutes.Afterthecuttingshadrooted(~tento fourteendays),theyweremovedtoa30%shadeglassgreenhouseandgrownunderstandard

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97 greenhouseconditions(temperature21 25 o C).Ifavarietydidnotrootwell,itwasdiscarded. A ftertwoweeksofgrowthinthegreenhouse,thecuttingsweresubsequentlyusedforevaluation inthegreenhouseandfieldtrialsstartinginAprilasdescribedbelow. Fieldtrials:2004,2005,and2006. YearlyfieldtrialsbeganinAprilaftercuttingswe re propagatedfromindividualsthatremainedintheprogramafterthethirdselectionprocess.These remainingindividualsexhibitedhighvigor,novelfoliagecolor,andhadnotinitiatedflowering. Twoevaluationlocations,aglassgreenhouseandfullsun outdoors,wereusedtoevaluatecoleus plantsforthecharacteristicsofvigor,colorqualityandfloweringinduction.Oftheninecuttings previouslypropagatedfromtheremainingindividuals,threewereusedinthegreenhousetrial, threewereplantedi nthefieldtrial,andtheremainderwasdiscarded. The30%shadegreenhousetriallocationconstitutedano pinchtrial,inwhichcuttings werenotpinchedsothattheirnaturalgrowthhabitcouldbeobserved(Figure4 2).Three replicateplantsofeach individualwereplantedin15.2cmazaleapots,spaced30 cmapart,and placedinnumericalorderbasedontheirexperimentallinenumbers.Afterdataforvigor(height +branching)werecollectedinthegreenhouse,thesameindividualswereevaluatedinth efield trialinJune,July,andAugust. TheoutdoorfullsunfieldtrialwasconductedatthePlantScienceResearchandEducation UnitinCitra,Florida.Individualswereplantedonraisedgroundbedscoveredwithsilver reflectivemulchtoincreasethe levelsoflightstressforevaluationofsuntolerance(Figure4 3). Thisfieldlocationwasusedonlyforthe2005and2006trials,whileanoutdoorlocationonblack tarpinGainesville,Floridawasusedforthe2004trial.Foreachyearlytrial,threer eplicate plantsperlinewerepinchedoncebeforep lantinginthefield,spaced30 cmapart,andirrigatedas needed.Slowreleasefertilizer(Osmocote8 16 12,N P 2 O 5 K 2 O)wasutilizedatplanting.The

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98 plantswereplantedinnumericalorderbasedontheir experimentallinenumberstofacilitatedata acquisition.Theindividualswereevaluatedona1 5scaleforcolorqualityandflowering induction.Datawerecollectedatthreepointsattheendofeachmonth(June,July,andAugust) foreachyearlytrial. Variablesevaluatedanddataanalysis. Va ria blesthatwereevaluatedforallcoleus individualsintheprogramwereplantvigor,foliagecolorqualityandfloweringinduction.Since vigorencompassedaplantsthree dimensionalformandtookintoaccount bothheightand width,avigorindexscorewasderivedbysummingtheindividualscoresforheightand branching.Foliagecolorqualitywasratedonthebrightnessofcolorpigmentation,with excellentratingsforplantsthatdidnotbleachorfadeincolo r.Floweringinductionwasratedon theappearanceoffloralinitiation. Forthegreenhousetriallocation,thevariablesofheightandbranchingwereratedona1 5 ratingscale.Forheight(measurementstakenfromsoilline),1=short(<10cm),2=mode rately short,3=average,4=moderatelytall,and5=tall(>30cm)andforbranching,1=poor,2=fair, 3=average,4=good,and5=excellent.Aftertheindividualswereratedonheightand branching,avigorindexscorewascalculatedbysummingth eheightandbranchingscoresfor eachindividual. Forthefieldtriallocation,colorqualityandfloweringinductionwerealsoratedona1 5 ratingscale.Forcolorquality,1=poor,2=fair,3=average,4=good,and5=excellentandfor flowering induction1=fullbloom,2=flowerbudsvisible,3=inflorescencevisible,4= inflorescencebudforming,and5=noflowering.Attheendofthecropcycle,thescores receivedinthegreenhouseandfieldweresummedandafinaloverallscorewascalcu latedfor eachindividual.Thus,anindividualcouldachieveamaximumscoreoftenpointsforvigor

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99 index(height+branching)inthegreenhouseandtenpointsforeachmonthofJune,July,and Augustinthefield(colorquality+floweringinduction),th usatotaloverallscoreoffortypoints wasachievable. Forthevariablesofplantvigor,colorquality,andfloweringinduction,apercentagewas calculatedforthenumberofindividualswithineachratingscore.Percentageswerecalculatedby dividingt hefrequencyofindividualsreceivingtheratingscorebythetotalpopulationnumber andmultiplyingby100.Bycalculatingthesepercentages,thenumberofindividualsthat receivedhighratingscoresoffourandfivescouldbecomparedacrossthethree yeartrialsto determineifimprovementsinthethreevariablescouldbeobservedacrossyearlypopulations. Seedcollectionandprogenytests. Seedswereharvestedattheendofeachyearlyfieldtrialin August Decemberfromindividualsthathadsuperio rperformancebasedontheirtotalrating scores.Matureflowerspikes(whencalyxweredarkbrownincolor)werecollectedandplacedin brownpaperbagsandair driedatroomtemperatureforoneweek.Afterair drying,theflower spikeswereplacedina roundandsmallwire meshsievecontainer,anddriedflowerpartswere separatedfromtheseeds.Theseedswereplacedinseedpacketsandstoredatroomtemperature becauseitwasobservedearlyonthatrefrigeratedstorageresultedinlowgermination per centages.Foreachsuperiorindividual,aprogenytestwasperformedtoevaluatetheseedling variability.Onehundredseedsofeachvarietyweresownonflatsofsoillessmedia(Fafard2 mix media),placedinamisthousewithmistingsetforthirtysecon dseverytwentyminutes,and transferredtoagreenhouseaftergermination.Theseedlingsweregrownandevaluatedbetween October Novemberandlineswithhighvigorandvariabilityinfoliagecolorswerenoted(data notshown).Onlyseedsfromtheselines wereusedtoreconstitutethenextcropcyclestartingthe followingJanuary.

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100 ResultsandDiscussion Attheinceptionofthecoleusbreedingprogram,fourteencommercialcultivarswereused asparentallinestostartthebreedingprogram.Thesefourteenc ultivarsdifferedinfoliagecolors, growthhabits,leafshapes,andfloweringinductiontimes.Thefourteencultivarsusedwere StainedGlassworks,Elfers,Micanopy,EmbersGatorMint,Astatula,HurricaneLouise,Kiwi Fern,Tilt a Whirl,Yalaha,Freckles, IndianFrills,OrangeGatorMint,TrailingNova,andRed Queen.Unknowntousatthetime,parentallinesElfersandStainedGlassworksbecamethe predominantsourcesforexcellentgeneticsinsubsequentcropcyclesandproducedmanyofthe varietieswith thedesiredcharacteristicsofimprovedvigor,colorquality,anddelayedflowering induction. Vigor Avigorindexwasusedtoevaluateindividualsfortheirthree dimensionalform.Since vigorcomprisesboththeheightandwidthofaplant,heightandbr anchingwasratedforeach individualandavigorindex(heightrating+branchingrating)wascalculatedtoevaluatethe individualsforbothcharacteristics.Vigorwasratedinthegreenhousebecauseitwasa characteristicthatwaseasilyscreenedfora ttheseedlingstage.Seedlingsthatweretallandhad goodbranchingatanearlystagedisplayedthesamecharacteristicsatmaturitywhenevaluatedin thefield.Slightimprovementsweremadeforvigorinthegreenhouseacrossthethreeyeartrials byre ducingheightandincreasingbranchinginthepopulations(Figure4 4).In2004,92%ofthe populationwasmoderatelytallortallbutonly45%ofthepopulationhadgoodtoexcellent branching.Manyindividualshadstrongapicaldominance,didnotproduce enoughcuttings neededformassproduction,andoftengrewleggy.Theseindividualswithstrongapical dominancemayhavehadgenotypesAAAAorAAAainregardstogrowthhabit.Thusselections weremadeforindividualswithgenotypesAAaabecauseindividu alswithgenotypesAAaa,

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101 Aaaa,oraaaaweredesirabletodecreaseapicaldominanceandtoincreasebranching.In2005, 90%ofthepopulationwasmoderatelytallortallbutanimprovementwasmadeforbranchingin which65%ofthepopulationreceivedscore soffourorfive.Thepopulationin2005hada minimalreductioninapicaldominancebutbetterbranchingthanthepopulationin2004.In 2006,manyoftheindividualswereidealas59%ofthepopulationwasmoderatelytallortallbut 74%ofthepopulati onhadgoodtoexcellentbranching.Inregardstodataforvigorindex, individualswithscoresoffourtofivepointsforheightandfourtofivepointsforbranchingwith anoverallvigorindexscoreofeighttotenpointsdesirable.In2004,66%ofthe population achievedscoresofeighttotenpointsforthevigorindexbuttheindividualsinthiscategorywere mostlytallasseenintheheightdata.In2005,anincreaseinthevigorindexwasobservedas 74%ofthepopulationreceivedscoresofeightt otenpoints;however,individualsinthis percentagegroupweresimilarinheightbuthadhigherbranchingratingsthanin2004.In2006, althoughthevigorindexratingdecreasedto65%,moreoftheindividualswithinthispercentage groupwereidealas theyhadexcellentbranchingscoresandreducedapicaldominance. Improvementsweremadeforvigorbyselectingforplantswithdecreasedapicaldominanceand higherlateralbranches.Itappearedthatequallevelsofvigorweremaintainedeachsubsequent y ear,butindividualsthatwerebeingdevelopedwereshorterandbetterbranchedin2006. Furthermore,2005/2006mayappeartobeabreakingpointintermsoftheallocationofbiomass betweenheightandbranching.Aplantcanonlyproduceacertainlevelo fbiomass when dedicatedtobranching,thereisonlysomuchbiomassitcanallocatetoheight. FoliageColorQuality Afterthevarietieswereratedinthegreenhouseforheight,branching,andvigorindex, theyweretransplantedtothefieldlocationfo revaluationofcolorqualityandflowering induction.Colorqualitywasnotafeasiblecharacteristictorateinthegreenhousebecausemost

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102 plantsmaintainedexcellentcolorqualitywhengrownunderoptimalglassgreenhouseconditions withlittleUV.For manyplants,highlightintensity(withUVradiation)(Krause,1999)and elevatedtemperaturesresultsincolorfading(Shaked Sachray,2002;Dela,2003;Oren shamir, 1999)andthusplantswithhighratingsinthefieldwerethosethatmaintainedtheirvi brant foliagecolor.Improvementincolorqualitywasmadeintheprogramattheendofthreeyearly trials,asdemonstratedbyahigherpercentageofthepopulationreceivingscoresoffourandfive points(Figure4 5).InthemonthofJuneforallyearly trials,over90%ofthepopulation receivedscoresoffourorfiveforcolorqualityandminimalcolorfadingandbleachingofthe foliagewereobserved.Theseplantshadbeenplantedinthefieldforonemonthandwerenotyet affectedbyhigherirradian celevelsandtemperatures.ForthemonthofJuly,animprovement wasmadeinboth2005and2006comparedto2004.Over90%ofthepopulationmaintained theirratingscoresoffourorfiveforcolorqualityin2005 2006comparedto64%in2004.The montho fAugustwasanimportantmonthastheplantshadbeenplantedinthefieldforthree monthsandplantsthathadexcellentcolorqualitydistinguishedthemselvesfromtheplantsthat hadpoorcolorquality.In2004,only35%ofthepopulationmaintainedco lorqualityrating scoresoffourorfive.Aslightdecreaseinfoliagecolorqualitywasobservedin2005withonly 29%ofthepopulationreceivingratingscoresoffourorfive.However,in2006,afterthree yearlytrials,significantimprovementswerem adeforcolorqualityas53%ofthepopulation maintainedgoodtoexcellentcolorquality.Augustalsoservedasanimportantmonthtoscreen plantsforexcellentfoliagecolorqualitybecausethismonthmayhavehadthehighest accumulatedirradianceand temperaturelevelswhencomparedtothemonthsofJune andJuly (personalobservation).

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103 Selectionsofnewvarietiesbasedonvisualcolorqualityratingsasconductedinthis breedingprogramhavebeenperformedforotherplantspeciessuchasdaylilies (Owings,2005) and Abeliaxgrandiflora (Robacker,2006).Thereis stillroomforimprovementsforcolor qualityincoleusbecausehalfofthepopulationin2006stilldemonstratedcolor fading/bleaching.Bycollectingseedsfromplantswithhighcolorrat ings,thepercentageof plantsreceivingratingscoresoffourorfivecanbegraduallyincreasedinsubsequent populations. FloweringInduction Afterthethreeselectionprocessesweremadeattheseedlingstageinthegreenhouse,all seedlingsthathade arlyfloweringinductionwerediscardedfromtheprogram.Flowering inductionwaseasilyscreenedforatthejuvenilitystage,however,floweringatthematuritystage requiredevaluationofthevarietiesinthefield.Improvementsfordelayedfloweringin duction weremadeinthebreedingprogramasthepercentageofvarietiesthathadjustinitiatedflowering orhadnotfloweredincreasedsignificantlyin2006from2004forthemonthsofJulyandAugust (Figure4 6).ForthemonthofJuly,33%ofthepopul ationin2004receivedscoresoffourorfive forfloweringwhile67%ofthepopulationhadalreadyflowered.Animprovementwasmadein 2005as56%receivedscoresoffourorfivewithonly44%inflower.Anotherimprovementwas madein2006as63%receiv edscoresoffourorfiveandonly37%ofthepopulationwasin flower.Withinathreeyearperiod,areductionof30%forearlyfloweringwasachievedforJuly. Augustwasanimportantmonthfordatacollectiononfloweringinductionbecausebythistime a largerpercentageofthepopulationhadalreadyflowered.In2004,only20%ofthepopulation hadjustinitiatedfloweringorhadnotfloweredasofAugust.In200537%ofthepopulationhad justinitiatedfloweringorhadnotfloweredasofAugust,and in2006,thatfigureincreasedto 48%.Takentogether,thesefiguresrepresenta28%reductioninearlyfloweringoverthreeyears

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104 ofselection.Furtherimprovementsfordelayedfloweringinductioncanlikelybeachievedsince halfofthepopulationin200 6wasalreadyinflowerinAugust.Bycollectingseedsfromlater floweringvarieties,thepercentageofvarietiesthatflowerlatermaybeincreasedinsubsequent populations.Asimilarprograminwhichcoleusvarietieswereratedforlatefloweringwas c onductedinthespringof2003andfallof2004inBatonRouge,Louisiana.Forty five commercialcultivarswereevaluatedinthelandscapeforslownesstoflowerandthecoleus cultivarAuroraBlackCherryreceivedhighestratingforlatefloweringinduc tion(Owings, 2005). Augustwasdesignatedasthelastdatacollectionmonthbecauseultimatelyseedshadtobe collectedoffofvarietiesthatreceivedhighratingscoresforallthreecharacteristics.InFlorida, hurricaneseasonrunsfromJune1sttoNo vember30thwiththemajoroccurrencesofstormsin September.Seedcollectionwasdelayedaslongaspossibletocollectseedsonlyfromvarieties thatwerelateflowering.IfseedcollectionwasdelayeduntiltheendofSeptember,theriskof losingplan tstoenvironmentalconditionswasincreased.Itwasobserved,however,thatvarieties withexcellentcolorqualitymaintainedvibrantfoliagecolorsafterthelastdatacollectionperiod inAugustuntilthefirstfrost.In2005severalmajorstormsaffect edthecoleuspopulationin September2004and2005,butin2006therewerenostormsandseedswerecollecteduntillate November. CultivarReleases Attheendoftheyearlytrials,themaximumscoreavarietycouldattainwasfortypoints (tenpointsin thegreenhouseandthirtypointsinthefield).Varietieswithhighoverallrating scoreswereretainedaftereachyearlytrialandhadthepotentialtobecomereleasesfromtheUF breedingprogram.Fourteencultivarswerereleasedin2005andsevenwerer eleasedin2006.All

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105 ofthereleaseshadimprovedplantvigor,colorqualityanddelayedfloweringinduction.Cultivar releaseinformationforthetwenty onenewreleasesisshowninAppendix1. Itisinterestingtonotethatmanyofthecultivarsrelease dfromtheprogramweretraced backtotwopredominantparentalcultivarsusedattheinceptionofthebreedingprogram.Elfers andStainedGlassworksproducedmanyvarietieswithexcellentvigor,colorqualityandlate floweringinduction.Elfersisatal lvarietywithbrightred/greenfoliagecolor,excellent branching,andlatefloweringinduction.StainedGlassworksisavarietywithlowvigor,buthas brightorange/redfoliagecolorandexceptionallylatefloweringinduction.Itisalsointerestingto notethatexcellentgeneticscanbetracedbacktothematernalparentinanopen pollinated situation.SeedscollectedfromElfersandStainedGlassworkshaveconsistentlyproduced severalcultivarsthathavebeenselectedassuperioreveryyear.Ofthet wenty onevarieties releasedfromtheprogram,thegeneticsforthirteenofthevarietiesweretracedbacktoeither ElfersorStainedGlassworks(seeAppendix1).Vigordoesnotappeartobeaproblematic characteristictobreedforincoleus,howevercol orqualityandlatefloweringinductionmay requirefurtherimprovements.

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106 Figure4 1. Growthofseedlingsatthestartofthecropcycles. Seedlingsweregerminatedina polycarbonategreenhouseunder standardconditions(temperatureat21 27 o C) withoverheadmist.Threeweeksaftergermination,seedlingsweretransplanted individuallytoacell.

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107 Figure4 2. 30%shadeglassgreenhousetrial. Threereplicatecuttingsofeachindiv idualwere placedinnumericalorderaccordingtotheirexperimentallinenumbers.The individualswereratedforheightandbranchingona1 5scale.Forheight (measurementstakenfromthesoilline),1=short(<10cm),2=moderatelyshort, 3=average ,3=moderatelytall,and5=tall(>30cm)andforbranching,1= poor,2=fair,3=average,4=good,and5=excellent.Aftertheindividualswere ratedonheightandbranching,avigorindexscorewascalculatedbysummingthe heightandbranchingsc oresforeachindividual.

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108 Figure4 3. FullsunoutdoorlocationatCitra,Florida. Threereplicatecuttingsofeach individualwereplantedonraisedgroundbedscoveredwithsilvermulch. Individualswereratedf orthevariablesofsuntoleranceandfloweringinduction ona1 5scale.Suntolerancewasratedona1 5scale,with1=poor,2=fair,3= average,4=good,and5=excellent.Floweringinductionwasratedona1 5 scalewhere1=flowersvisibleand plantswereinfullbloom,2=flowerbuds visible/fullblooms,3=inflorescencevisible,4=formationofinflorescencebud, and5=nofloweringinduction.ThefieldtrialstartedinMayandendedin August.

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109Figure4-4.Percentagesofthepopulationreceivingratingscoresoffourorfiveforheightandbranchingacrossthethreeyearlytrials.A)Heightandbranchingwerescoredona1-5ratingscale.Forheight,1=short,2=moderatelyshort,3=average,4=moderatelytall,and5=tall.Forbranching,1=poor,2=fair,3=average,4=good,and5=excellent.B)Avigorindexwasderivedbymultiplyingtheheightratingscorebythebranchingratingscore. VigorIndex0102030405060708090100200420052006Year Height Branching VigorIndex020406080100200420052006Year HeightandBranching Percentagewithratingsof4-5 Percentagewithratingsof4-5 A B

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110Figure4-5.ColorqualityforthemonthsofJune,July,andAugustforyears2004,2005and2006.Percentagesofthepopulationreceivingratingscoresoffourorfiveforcolorqualityacrossthethreeyearlytrials.Colorqualitywasratedona1-5ratingscale,where1=poor(colorfading/bleaching),2=fair,3=average,4=good,and5=excellent(nocolorfading/bleaching). ColorQuality0102030405060708090100JuneJulyAugustMonths 2004 2005 2006 Percentagewithratingsof4-5

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111 CHAPTER5 FUTUREWORK Thedevelopmentofseveraldozennewcoleusvarietieswithmoretrailinggrowthhabit thanRQandbrightorangecolorsimilartoSwasac hieved.Inthreegenerations,trailinghabit andorangecolorwascombinedinoneF2individual(H05 66 5).However,itappearedthatall F2plantswithbothtrailinghabitandorangecolorhadearlyfloweringinductionasobservedin bothparentalvarieti es.Itappearsthatearlyfloweringinductionandbrightorangecolorare linked;asflowersareundesirableincoleus,improvementsarestillneededtodevelopnew varietieswithtrailinggrowthhabit,brightfoliagecolors,andlatefloweringinduction. Inregardstothecoleusbreedingprogram,improvementscanstillbemadeforthe selectionofnewvarietieswithimprovedcolorqualityandlatefloweringinduction.Asthecrop cycledidnotproduceapopulationreceivingratingscoresof5forallthree variables,thereisstill roomforimprovements.Suchimprovementscanbemadebycontinuallyselectingvarietieswith thedesiredcharacteristicsandcollectingseedsfromthoseselectedvarieties.Mostofthetraitsin coleusaresimplyinherited(Rife, 1946);thusafocusontheimprovementofcoleusforother desirabletraitsmaybeofinterest.Twotraitsofinterestarecold toleranceandlargerflowers.The developmentofvarietiesthatcanbegrowninlowerhardinesszoneswillimprovetheir landsc ape rangeandmarketvalue.Likewise,developingcoleusforlargeranduniqueflower formswill openanewmarketniche.Intergenericcrossesbetweencoleusandarelatedgenus, Plectranthus, maybefeasibleasPlectranthusexhibitsgoodcoldhardinessand larger,tubular flowerforms.As bothgenusbelongtothesamefamily(Lamiaceae)suchintergenericcrosses maybeperformed. Inregardstothemolecularstudyofanthocyaninsynthesisincoleus,ithasbeenshowntha t lightinfluencesfoliagecoloration byincreasingproductionofredpigmentation.However,as onlyafewgeneswereidentifiedinthisstudy,theidentificationofadditionalgenesmaybe necessarytofurtherunderstandthephysiologicalandmolecularmechanismsinvolvedinthe

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112 colorationof coleusfoliage.Inaddition,thebiochemistryofanthocyaninandcarotenoid accumulationisessentialtounderstandthedifferentpigmentsthatcontributetofoliage coloration. Besidestheeffectoflightinfluencingcolorationincoleus,colorationlea dingtosplotches andvariegationshasbeenproposedtobeduetotheactivityoftransposons(LebowitzandKloth, 1986).Astransposonsaffectthenucleargenome,Mendelianinheritancewillbeobserved for foliarvariegation.Coleuswillbeanidealcandi datetostudytransposonactivityduetoits high levelofgeneticvariabilityforcolor.

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113 APPENDIXA NEWCULTIVARRELEASESWITHIMPROVEDVIGOR,BRANCHING,COLOR QUALITYANDLATEFLOWERINGINDUCTION Introduction ThecoleusbreedingprogramattheUnive rsityofFlorida,inGainesvilleFlorida,was initiatedin2003withaprimaryemphasisondevelopingnewvegetativelypropagatedcultivars withimprovedcolorqualityandlateflowering.Thesecondaryemphasisofourprogramfocused ondevelopingnewcult ivarswithnovelcolor,increasedvigor,andbranchingforoptimal commercialmassproduction.ThefollowingUFvarietieswereselectedbecausetheyhaveafew orallofthesedesirabletraits.Listedbelowarethecultivarreleases,theirorigins,foliar descriptionandgeneralperformancecharacteristics.Foliagecolorwasdeterminedusingthe RoyalHorticulturalSocietyColourChart(RoyalHorticulturalSociety,1939)inaglass greenhouse underfullsunconditions. 2005Releases RoyalGlissade Royal Glissade(Figure4 4)isanopen pollinatedvarietyofElfers(FloridaCitySeries, LakeBrantleyPlantCorp).Elferswaschosenforitslate flowering,excellentvigorandoptimal branching.Foliageisaspeckledpeagreen(61/1)onadelftrose(o20/ 1)backgroundonthe adaxialleafsurface.Youngleavesstartasspeckledpeagreen(61/1).Foliageisagarnetlake (828)ontheabaxialleafsurface.Leafshapeiscuneate acutewithcrenatemargins.Mature leavesaverage14cmlongand7cmwideonplan tsgrown15weeksafterplanting.Royal Glissadeisintendedforuseasadecorativefoliageplantforthelandscape.Thiscultivaris exceptionalbecausefoliagecolorremainsconsistentunderalltestinglocationsandhasaunique foliagecolorthatrefl ectsgold/bronzeinthesunlightandprovidesabrightcontrastamongother

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114 ornamentals.WithitsfloweringoccurringnearOctober,thisvarietyhasallofthedesirabletraits ofsun tolerance,lateflowering,vigor,branching,andcolorretention. Twist andTwirlanditstwosports,UF03 6 1BandUF03 6 1C Allthreecultivars(Figure4 5)areopen pollinatedvarietiesofStainedGlassworks Copper(Miracle groPlants/Metrolina).StainedGlassworksCopperwaschosenforitsbright redfoliageco lorandlatefloweringcharacteristics.TwistandTwirlfoliageispolychromatic canaryyellow(2/1),scheelesgreen(860),jasperred(o18),andaconiteviolet(937).Young leavesstartasavariegatedspinachgreen(o960)andivygreen(ooo1060/2)and othercolors appearwhenleavesmature.UF03 6 1Bfoliageisavermilion(18)andivygreen(ooo1060/1). UF03 6 1Cfoliageisacanaryyellow(2/1)andscheelesgreen(860).TwistandTwirl, UF03 6 1B,andUF03 6 1Chavedifferentfoliagecolorb utidenticalgrowthcharacteristics. Leafshapeislanceolateandmarginsarehighlylobed.Matureleavesaverage12cmlongand8 cmwideonplantsgrown15weeksafterplanting.TwistandTwirl,UF03 6 1B,andUF03 6 1Careintendedforuseasade corativefoliageplantinthelandscapeandareuniquedueto theirmulti colored,highlylobed,cut flowerfoliageshapeandexceptionalhighvigor.Being nearlyisogenic,thesecultivarsperformwelltogetherwhileprovidingdifferencesinfoliage color. Withitsmulti colored,redandgreenfoliage,thiscultivarseriescanbeeasilycombined withotherornamentalplants. UF03 8 24 Anopen pollinatedvarietyofElfers,its foliageisaspeckledpurplemadder(1028)ona lettucegreen(861)backgroun dontheadaxialsurface.Marginedgeandabaxialsurfacearea purplemadder(1028).Leafshapeiscuneate acuteandmarginslightlyserrate.Matureleaves average12cmlongand11cmwideonplantsgrown15weeksafterplanting.UF03 8 24

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115 (Figure4 6) isintendedforuseasadecorativefoliageplantinthelandscape.Thiscultivarworks wellforcommercialcuttingproduction,havingexcellentvigorandbranching.Withitsgreenand darkerpurplemarkings,thiscultivarworksexceptionallywellincomb inationwithother ornamentalsandasaserieswithRoyalGlissade. ElectricLime ElectricLime (Figure4 7)isanopen pollinatedvarietyofUF03 1 6.UF03 1 6isan open pollinatedvarietyofAstatula.Astatula(FloridaCitySeries,LakeB rantleyPlantCorp) waschosenforitslate flowering,excellentvigor,optimalbranching,andsun tolerance.Foliage isavariegatedcitrongreen(763)onascheelesgreen(860)backgroundontheadaxialleaf surface.Asleavesmature,foliageisavarieg atedchartreusegreen(663/1)onascheelesgreen (860)backgroundontheadaxialleafsurface.Leafshapeisovateandcuneate acutewithcrenate margins.Matureleavesaverage14cmlongand7cmwideonplantsgrown15weeksafter planting.ElectricLi meisexceptionalbecausefoliagecolorremainedbrightgreeninalltesting locations.WithitsfloweringoccurringnearSeptember,thiscultivarhasallofthedesirable traits.Thiscultivarisintendedforuseasadecorativefoliageplantforthela ndscapeandin pottedcontainersandwillprovideasharpcontrastamongotherornamentals.ElectricLime currently hasaplantpatentappliedfor. UF04 5 7 UF04 5 7 (Figure4 8) isanopen pollinatedvarietyofUF03 6 1.UF03 6 1isaselection of StainedGlassworksCopper. Foliageisamagentarose(o27/1)diffusingtoanivygreen (ooo1060/2),withsmallspotsofscheelesgreen(860/2)nearthemargin.Abaxialsurfaceisa magentarose(o27/1).Leafshapeisrounded acutewithcrenatemargins. Matureleavesaverage 6.5cmlongand5cmwideonplantsgrown15weeksafterplanting.UF04 5 7isdifferent

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116 fromothercommercialcultivarsbecauseitsbrightpinkfoliageremainsconsistentunderpartial tofullshadeconditionsandthereforecanbe usedinmixedcontainersneedingabrightcolor contrast.Withitsconsistentslowgrowinghabit,thiscultivarisintendedforuseasalow maintenancecultivarforpottedcontainersinthelandscape. UF04 18 3 UF04 18 3 (Figure4 9) isanopen polli natedvarietyofUF03 13 9.UF03 13 9isa selectionofMicanopy(FloridaCitySeries,LakeBrantleyPlantCorp).UF03 13 9waschosen foritsnovelorangefoliagecolor.Micanopywaschosenforitsexcellentvigorandbranching. Foliagehasinterveina lareasandareofacanaryyellow(2/ 3 )intheleafcenteranddiffusinginto acarmine(21/ 1 )/purplemadder( 10 28)towardtheleafmargin.Theabaxialsurfaceisjasperred (o18/ 3 ).Leafshapeiscordate acutewithhighlycrenatemargins.Matureleavesa verage11cm longand10cmwideonplantsgrown15weeksafterplanting. UF04 18 3 isintendedforuseas adecorativeplantforbothsunandshadeconditions.Althoughthiscultivarhasfairratingsfor vigorandbranching,itscombinationofhighlylo bedandcrinklytexturedfoliage,redfoliage color,semi dwarfgrowthhabit,andlatefloweringmakesthiscultivarhighlyuniqueand differentfromothercommercialcultivars. UF04 36 33 UF04 36 33(Figure4 10)isanopen pollinatedvarietyofUF0 3 6 18.UF03 6 18isa selectionofStainedGlassworksCopper.UF03 6 18waschosenforitsvariegatedcolor pattern,excellentvigorandbranching.Foliageisachinarose(o24)withivygreen(ooo1060/2) spotsthroughoutleafblade.Marginisedged withscheelesgreen(860/2).Leafshapeiscuneate acutewithmildserratemargins.Matureleavesaverage10cmlongand8cmwideonplants grown15weeksafterplanting.UF04 36 33isuniquewithitscombinationofdark purple

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117 stemsandbrightredfoli age.Thiscultivarisintendedforuseasadecorativefoliageplantfor bothsunnyandshadylandscapes,withredfoliagecolorbecomingmoreintenseunderpartial shade.Withitshighvigorandbranching,thiscultivarisexcellentformasspropagationa nd commercialcuttingproduction. UF04 40 26 UF04 40 26 (Figure4 11) isanopen pollinatedvarietyofUF03 13 9.UF03 13 9isa selectionofMicanopy. Foliageissulphuryellow(1/3)inthecenter,interveinalareasan aureolin(3),andaschee lesgreen(860).Leafshapeiscordate acutewithmarginhighlycrenate. Matureleavesaverage11.5cmlongand9cmwideonplantsgrown15weeksafterplanting. UF04 40 26isdistinctivewithitsfoliagecolorpatternandcrinklytexture,highvigor,a nd branching.Despiteitshighvigorrating,thiscultivarisintendedforuseasapottedplantdueto itshighlycompactandmoundedgrowthhabitandwouldworkextremelywellasalandscape borderplant. UF04 40 125 UF04 40 125 (Figure4 12) isan open pollinatedvarietyofUF03 13 9.UF03 13 9is aselectionofMicanopy. Foliageisasulphuryellow(1/3)initscenterdiffusingtoascheeles green(860)nearthemargin,andthebladespeckledwithIndianlake(826).Leafshapeis cordate acu teandleafmarginismildlyserrate.Matureleavesaverage10cmlongand8cmwide onplantsgrown15weeksafterplanting.UF04 40 125isintendedforuseasadecorative foliageplantinthelandscapeandasapottedplantincontainers.UF04 40 125 isanexcellent sun tolerantcultivar,withfoliagecolorsremainingconsistentlybrightinalllocationstested.It alsoworkswellformasspropagationandcommercialcuttingproduction.Thisvarietyreceived

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118 anawardforbestperformanceattheFloric ultureFieldDayevaluatedatGainesville,Floridain 2006. UF04 51 33 UF04 51 33(Figure4 13)isanopen pollinatedvarietyofAuroraOrange(Ball FloraPlant).AuroraOrangewaschosenforitsbrightorangefoliagecolor,leafshapeand growthh abit.Foliageisacamelliarose(622/1)diffusingtoadarkcamelliarose(622)nearthe margin,themarginspeckledwithivygreen(ooo1060/3),andthemarginedgealettucegreen (861/3)ontheadaxialsurface.Abaxialsurfaceisporcelainrose(620/3 ).Leafshapeisoblique acuteandtheleafmarginishighlycrenate.Matureleavesaverage10cmlongand5cmwideon plantsgrown15weeksafterplanting.UF04 51 33waschosenforitsnovelrose colored foliageandlatefloweringcharacteristics.Wit halowervigorandbranchingrating,thiscultivar isintendedforuseinsmallmixedcontainersinpatiogardensinshadylocations. UF04 51 41 UF04 51 41(Figure4 14)isanopen pollinatedvarietyofAuroraOrange.Foliageisa speckledchinaros e(o24/1)onapurplemadder(1028)background.Leafshapeisoblique acute andleafmarginlobed.Matureleavesaverage13cmlongand5cmwideonplantsgrown15 weeksafterplanting.UF04 51 41isintendedforuseasadecorativefoliageplantinsun nyor shadylandscapes.UF04 51 41isuniqueforitscombinationoflobedleaves,roseanddark purplefoliage,lobedleafshape,moundedgrowthhabit,andlateflowering.UF04 51 41will provideasharpcontrastofcolorandtextureagainstgreensin thelandscape.Withahigh branchingrating,UF04 51 41isexceptionalforrapidcommercialcuttingproduction.

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119 UF04 51 42 UF04 51 42(Figure4 15)isanopen pollinatedvarietyofAuroraOrange.Foliageisa roseopal(o22/3)darkeningtoadark roseopal(o22/3)nearthemarginontheadaxialsurface. Theabaxialsurfaceismagentarose(o27/1).Youngleavesbeginasjasperred(o18/1).Leaf shapeisoblique acuteandmarginmediumcrenate.Matureleavesaverage6.5cmlongand3cm wideonplan tsgrown15weeksafterplanting.UF04 51 42growthhabitandlowvigormake thiscultivaridealfor4containerproduction.UF04 51 42waschosenforitsexceptionalrose opalfoliagecolor.Despitehavingafairratingonvigorandbranching,thef oliagecolorwasthe selectionfactorandthecolorisconsiderednovelwhencomparedtoothercommercialcultivars. 2006Releases UF04 47 64 UF04 47 64(Figure4 16)isanopen pollinatedvarietyofElfers(FloridaCitySeries, LakeBrantleyPlant Corp.).Elferswaschosenforitslate flowering,excellentvigorandoptimal branching.Foliageisatyrianrose(o25)ontheadaxialandmagenta(27)ontheabaxialleaf surface.Leafshapeiscuneate acutewithcrenatemargins.Matureleavesaverage10 cmlongand 6cmwideonplantsgrown15weeksafterplanting.UF04 47 64isintendedforuseasa decorativefoliageplantforthelandscape.Thiscultivarwaschosenforitslargeleafsize, exceptionalvigor,andrichdeeprose coloredfoliage.Witha latefloweringinduction,this cultivarremainsbrightthroughoutthesummerseason. SplishSplash SplishSplash (Figure4 17) isanopen pollinatedvarietyofElfers. Foliageismottled sapgreen(62/1),maroon(1030),anddutchvermillion(717/2) onboththeadaxialandabaxial leafsurface.Leafshapeisrounded acutewithcrenatemargins.Matureleavesaverage11am longand8cmwideonplantsgrown15weeksafterplanting. SplishSplashisuniqueforits

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12 0 spectacularmottledfoliagecolorand worksgreataloneorasanadditionformixedcontainers. Withafullgrowthhabit,highvigor,andexcellentbranching,itisintendedforuseadecorative foliageplantandwillworkwellas ahedgeplantinthelandscape. PineappleSplash Pineapple Splash(Figure4 18)isanopen pollinatedvarietyofUF03 8 21.UF03 8 21isaselectionofElfers.UF03 8 21waschosenforitsbrightcolorfoliageandlate flowering.Foliageiscarmine(21/1)alongthemid veindiffusingtoafuchsiapurple (28)ona uraniumgreen(63/2)backgroundwithsplotchesofuraniumgreen(63/3)throughouttheadaxial leafsurface.Foliagecolorisauraniumgreen(63/3)ontheabaxialleafsurface.Leafshapeis acute acuminatewithcrenatemargins.Matureleavesav erage8cmlongand3cmwideonplants grown15weeksafterplanting.PineappleSplashwaschosenforitsuniquebrightyellowand marooncenterfoliage.Althoughthiscultivarhasafairtoaveragevigorrating,itbecameoneof thefewcultivarsthat lookedfantasticandremainedexceptionalattheendofthetrial.Withan excellentsuntoleranceandlatefloweringrating,thiscultivarworksbestinsmallplantings and/orinmixed containersforsunnylocations. VelvetMocha VelvetMocha (Figure 4 19) isanopen pollinatedvarietyofElfers. Foliageismaroon (1030)alongthemid veinonajasperred(o18)backgroundontheadaxialleafsurfaceanda maroon(1030/1)ontheabaxialleafsurface.Leafshapeisacuminate acuminatewithentireor l obedmargins.Matureleavesaverage9cmlongand2.5cmwideonplantsgrown15weeks afterplanting. VelvetMochahasbothexceptionalvigorandbranchingandlateflowering.This cultivarisintendedforuseasafoliageplantinshadylandscapes.Its uniquejasperredfoliage coloronalanceolateleafshapeandmoundedhabitmakesthiscultivarstandoutfromothers.

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121 UF06 04 140 UF06 04 140 (Figure4 20) isanopen pollinatedvarietyofElfers. Foliageispeagreen (61)speckledonarosered (724)backgroundontheadaxialleafsurface.Youngleavesstartas speckledpeagreen(61).Foliagecolorisamaroon(1030/3)ontheabaxialleafsurface.Leaf shapeisrounded acutewithcrenatemargins.Matureleavesaverage12cmlongand7cmwide on plantsgrown15weeksafterplanting. UF06 04 140isintendedforuseasadecorative foliageplantinthelandscape.Thiscultivarhasexcellentcolorforbothsunnyandshady locations,butappearsmorerose redunderhighlightintensities.Withflow eringinduction occurringinlateOctoberandexceptionalbranchingandgrowthhabit,thiscultivarisidealfor gardenperformance. LemonSunsation LemonSunsation(Figure4 21)isanopen pollinatedvarietyofUF04 18 3.UF04 18 3isaselection ofUF03 13 9.UF03 13 9isaselectionofMicanopy(FloridaCitySeries, LakeBrantleyPlantCorp).UF04 18 3waschosenforitsnovelandbrightfoliagecolor. UF03 13 9waschosenforitsnovelorangefoliagecolor.Micanopywaschosenfori ts excellentvigorandbranching.Foliageisacanaryyellow(2/1)andsapgreen(62)ontheadaxial leafsurface.Foliagecolorisaspiraearose(o25)ontheleafmarginsandasapgreen(62)onthe abaxialleafsurface.Leafshapeiscuneate acutewith laceratemargins.Matureleavesaverage8 cmlongand5cmwideonplantsgrown15weeksafterplanting.LemonSunsationhasoneof thebrightestyellowfoliageseenonacoleusplantforfullsunconditions.Thiscultivarhas excellentbranchinganduni queleafshapes.Withamaroonborderalongtheleafmarginssetin contrastwiththeyellowfoliagecolor,thiscultivarworkswellinmixedcontainersorasa decorativefoliageplantinthelandscapeforgardenerswantingtoaddadynamiccontrast.

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122 Big RedJudy BigRedJudy (Figure4 22) isanopen pollinatedvarietyofElfers.F oliageisacherry (722)ontheadaxialleafsurfaceandamaroon(1030/1)ontheabaxialleafsurface.Leafshapeis rounded acuteandcrenatemargins.Matureleavesaver age12cmlongand7cmwideonplants grown15weeksafterplanting. BigRedJudywasnamedforitslargeredleavesandrobust growthhabit.Thiscultivarworkswellinlargepottedcontainersorinthelandscape.Itsexcellent vigor,brightfoliageco lor,lateflowering,andoverallappearancedistinguishesitf romotherred coleusvarieties.

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123 Figure4 4. RoyalGlissade Fi gure4 5. (A) TwistandTwirl(B)UF03 6 1B(C) UF03 6 1C A B C

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124 Figure4 6. UF03 8 24 Figure4 7. ElectricLime

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125 Figure4 8. UF04 5 7 Figure4 9. UF04 18 3

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126 Figure4 10. UF04 36 33 Figure4 11. UF04 40 26

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127 Figure4 12. UF04 40 125 Figure4 13. UF04 51 33

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128 Figure4 14. UF04 51 41 Figure4 15. UF04 51 42

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129 Figure4 16. UF04 47 64 Figure4 17. SplishSplash

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130 Figure4 18. PineappleSplash F igure4 19. VelvetMocha

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131 Figure4 20. UF06 4 140 Figure4 21. LemonSunsation

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132 Figure4 22. BigRedJudy

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144 TaschettoO,PagliariniM(2004)Meioticbehaviorintetraploidpopulationsof Pfaffiatuberosa (Amaranthaceae).ActaBotCroat 63(1):17 24 ThomasB(2006)Lightsignalsandflowering.JofExpBot57(13):3387 3393 ThomasB,Vince PrueD(1997)Photoperiodisminp lants,2nded.AcademicPress,NewYork, NY,pp.1 26. TorskangerpollK,AndersenOM(2005)Colourstabilityofant hocyaninsinaqueoussolutionsat variouspHvalues.FoodChem89:427 440 VakninH,Bar AkivaA,OvadiaR,Nissim LeviA,ForerI,WeissD,Oren ShamirM(2005) Activeanthocyanindegradationin Brunfelsiacalycina (yesterday today tomorrow)flowers. Pla nta222:19 26 ValladaresF,ChicoJ,ArandaI,BalaguerL,DizengremelP,ManriqueE,DreyerE(2002)The greaterseedlinghigh lighttoleranceof Quercusrobur over Fagussylvatica islinkedtoagreater physiologicalplasticity.Trees16:395 403 V anH ouwelingenA,SouerE,SpeltK,KloosD,MolJ,KoesR(1998)Analysisofflower pigmentationmutantsgeneratedbyrandomtransposonmutagenesisin Petuniahybrida PlantJ 13(1):39 50 VasekF(1961)Multiplespindle Ameioticirregularityin Clarkia exilis .AmJ Bot49:536 539 VergerM,PaguesLE(1993)Bulkvegetativepropagationofhybridlarch( Larixxeurolepis Henry).AnnalesdesSciForestieres50(2):205 215 VijayraghavanU(2001)Howplantspatternflowers:lessonsfrommoleculargeneticst udiesof floweringin Arabidopsisthaliana amodelplant.CurrSci80(2):233 243 WadeH,BibikovaT,ValentineW,JenkinsG(2001)Interactionswithinanetworkof phytochromes,crytochromeandUV Bphototransductionpathwaysregulatechalconesynthase g eneexpressionin Arabidopsis leaftissue.PlantJ25(6):675 685 WeissD,HalevyAH(1991)Theroleoflightreactionsintheregulationofanthocyaninsynthesis inpetuniacorollas.PlantPhysiol81(1):127 133 WhitelamGC,DevlinPF(1997)Rolesofdi fferentphytochromesin Arabidopsis photomorphogenesis.PlantCellEnvir20:752 758 WieringH,deVlamingP(1984) Geneticsofflowerandpollencoloursinpe tunia .K.C.Sink,ed (Berlin:Springer Verlag):49 75 WilkinsHF(1988)Techniquestomaximize cuttingproduction.ActaHort226:137 144

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145 Winkel ShirleyB(2001)Flavonoidbiosynthesis:Acolorfulmodelforgenetics,biochemistry, cellbiology,andbiotechnology.PlantPhysiol126:485 493 Winkel ShirleyB(2002)Biosynthesisofflavonoidsandeff ectsofstress.CurrOpinPlantBiol5: 218 223 YabuyaT,SaitoM,IwashinaT,YamaguchiM(2000)Stabilityofflowercolorsdueto anthocyanin flavonecopigmentationinJapanesegardeniris, Irisensata Thunb.Euphytica115: 1 5 YakovlevaI,TitlyanovE (2001)EffectofhighvisibleandUVirradianceonsubtidal Chondrus crispus :stress,photoinhibitionandprotectivemechanisms.AquaticBotany71:47 61 YamaguchiT,Fukada TanakaS,InagakiY,SaitoN,Yonekura SakakibaraK,TanakaY, KusumiT,IidaS (2001)GenesencodingtheVvacuolarNa+/H+exchangerandflower coloration.PlantCellPhysiol42(5):451 461 YamazakiM,MakitaY,SpringbobK,SaitoK(2003)Regulatorymechanismsforanthocyanin biosynthesisinchemotypesof Perillafrutescens var.cri spa.BiochemEngineeringJ14:191 197 YoshidaK,KondoT,OkazakiY,KatouK(1995)Causeofbluepetalcolour.Nature373:291 ZufallRA,RausherMD(2003)Thegeneticbasisofaflowercolorpolymorphisminthe commonmorningglory( Ipomeapurpurea ). JHered 94(6):442 448

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146 BIOGRAPHICALSKETCH PennyNguyenwasborninGoGong,Vietnam andimmigratedwithherfamilytothe UnitedStatesattheageof5.AftergraduatingfromPensacolaHighSchool ,shecompletedher Bachelor ofScience degree atJackson villeUniversity,Jacksonville,Floridawithamajorin biologyandminorconcentrationsinchemistryandmarinescience.Whileworkingonamanatee researchprograminthesummerof2003,shewasacceptedintothedoctoralprogramatthe UniversityofFlo rida.PennywasarecipientoftheAlumniFellowshipandbeganhergraduate studiesinpla ntbreeding.Throughoutthe4 yearsofherresearch,shefocusedonthegeneticsof trailinghabit,howlightinfluencedfoliagecolorchanges,anddevelopingnewcol euscultivars forthecommercialmarket.Sheandhermajoradvisorsuccessfullyimplementedthefirstcoleus breedingprogramattheUniversityofFlorida.Afterhercompletion,21newcoleuscultivars havebeenreleased,with8soldonthecommercialmark etandgarneringroyalties.Herlistof achievementsincludeapatentappliedforonecoleuscultivar(ElectricLime)andthe developmentofseveraldozennewcoleusvarietieswithtrailinghabitandbrightorangefoliage colors.Sheiscurrentlyaplant breederatCarolinaNurseries,MoncksCorner,SouthCarolina.