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1 DORMANCY IN PRE-VARIETY GERMPLASM OF NATIVE COREOPSIS SPECIES By DZINGAI RUKUNI A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008
2 2008 Dzingai Rukuni
3 To my wife Miriam and my daughters Shingira i Tadziripa and Tanyaradzwa Nyasha who gave me all the support that I needed to accomplish this research and dissertation. This is a special dedication to my son Dzingai Ju nior who arrived on October 28th 2008. Also to my mother and late father who inspired me to achieve all that I have to this day.
4 ACKNOWLEDGMENTS I would like to express gratitude to my chair, Dr. Jeffrey G. Norcini and co-chair Dr. Daniel J. Cantliffe for stirring m e through these studies. Besides his busy schedule, Dr. Cantliffe always spared some time to exchange ideas and pass fruitful comment s and suggestions. Dr. Bernard E. Hauser contributed valuable info rmation and experience on seed anatomy studies, work that was new to me, but his advice made every aspect so enjoyable. I thank my committee members, Dr. Michael E. Kane and Dr. David G. Clark for their valuable contribution toward this work in committee meetings and outside. I would like to thank Nicole Shaw for the assistance in acquiring laboratory and greenhouse supplies and her valuable experien ce in greenhouse management during seed production in the Citra greenhouse. Social and academic interact ion was important in members of the Seed Physiology Lab (Building 710) and I express my appreciation to Jean Marie Mitchell, Elizabeth Thomas, Shubin Saha and Elio Jovicich. I would al so like to thank Karen Kelly at the Interdisciplinary Centre for Biotechnology Research (ICBR) at the University of Florida for assistance with the operation procedures of the SEM equipment. My studies would not have gone so well without patience, support and encouragement from my wife Miriam. During this period we welcomed our first son, Dzingai Rukuni, Jr. on 28th October 2008. I also thank my daughters Tanya radzwa and Shingirai for their support and comfort. I would also like to thank members of my family in Zimbabwe, particularly my mother for the encouragement and my late father for the in spiration. Last but not least, I owe my friends some appreciation, most of whom are in touch and provided support through cyberspace.
5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........8 LIST OF FIGURES.........................................................................................................................9 ABSTRACT...................................................................................................................................11 CHAPTER 1 INTRODUCTION..................................................................................................................13 1.1 Overview....................................................................................................................... 13 1.2 Taxonomy a nd Distribution..........................................................................................14 1.3 Problem Statem ent and Research Objectives................................................................ 15 2 LITERATURE REVIEW.......................................................................................................19 2.1 Seed Dormancy in Coreopsis Species........................................................................... 19 2.2 Seed Morphology and Anatomy ...................................................................................20 2.3 Germination.................................................................................................................. 21 2.3.1 Temperature and Light......................................................................................21 2.3.2 Gibberellic Acid................................................................................................ 24 2.3.3 Ethylene 25 2.3.4 Cytokinins......................................................................................................... 25 2.4 Dormancy......................................................................................................................25 2.4.1 Abscisic Acid (ABA)........................................................................................ 26 2.4.2 Afterripening..................................................................................................... 29 2.4.3 Coat Imposed Dor mancy................................................................................... 31 2.5 Overcomi ng Dormancy................................................................................................. 31 2.5.1 Nitrat es 31 2.5.2 Moist Cold and W arm Stratification................................................................. 32 2.5.3 Seed Priming ..................................................................................................... 32 2.5.4 Other Dormancy Breaking Substances............................................................. 33 3 ACHENE MORPHOLOGY AND ANATOMY AFFECT DORMANCY IN Coreopsis ( Asteraceae) SPECIES........................................................................................................... 35 3.1 Introduction................................................................................................................... 35 3.2 Materials and Methods.................................................................................................. 38 3.2.1 Plant Material.................................................................................................... 38 220.127.116.11 Anatomy and accom panying germination studies.............................. 38 18.104.22.168 Endo-mannanase analysis and related germ ination studies............ 38
6 3.2.2 Microscopy........................................................................................................ 39 22.214.171.124 External seed morphology .................................................................. 39 126.96.36.199 Scanning electron mi croscope (SEM)................................................ 39 188.8.131.52 Light mi croscopy................................................................................ 39 184.108.40.206 Determination of seed tissue ploidy ................................................... 40 3.2.3 Image Capture and Editing................................................................................ 41 3.2.4 Germination Tests ............................................................................................. 41 3.2.5 Influence of Seed Envelopes on Germination................................................... 41 3.2.6 Statistical Analysis ............................................................................................42 3.2.7 Endo-Mannanase Activity in Coreopsis Endosperms ...................................42 3.2.8 Influence of ABA, Tetcyclacis and Supraoptim al Temperature (30oC) on Germination and EBM activity......................................................................... 44 3.3 Results and Discussion..................................................................................................44 3.4 Conclusions................................................................................................................... 49 3.5 Summary .......................................................................................................................50 4 OPTIMIZING GERMINATION IN Coreopsis SEEDS ........................................................ 63 4.1 Introduction................................................................................................................... 63 4.2 Materials and Methods.................................................................................................. 65 4.2.1 Seed Material.................................................................................................... 65 4.2.2 Germination Tests ............................................................................................. 66 4.2.3 Plant Growth Regulators...................................................................................67 4.2.4 Cold Stratification............................................................................................. 67 4.2.5 Statistical Analysis ............................................................................................67 4.3 Results........................................................................................................................ ...67 4.3.1 Temperature and Light......................................................................................67 4.3.2 Gibberellic Acid, AB A and Tetcyclacis............................................................ 68 4.3.3 Cold Stratification............................................................................................. 68 4.4 Discussion..................................................................................................................... 68 4.5 Summary .......................................................................................................................70 5 AFTERRIPENING ALLEVIATES DORMANCY IN FRESH Coreopsis SEEDS..............86 5.1 Introduction................................................................................................................... 86 5.2 Materials and Methods.................................................................................................. 88 5.2.1 Plant Material.................................................................................................... 88 5.2.2 Germination Tests ............................................................................................. 88 5.2.3 Gibberellic Acid and Potassium Nitrate ............................................................ 89 5.2.4 Cold Stratification............................................................................................. 89 5.2.5 Removal of Seed Coverings.............................................................................. 89 5.2.6 Data Analysis.................................................................................................... 90 5.3 Results........................................................................................................................ ...90 5.3.1 Temperature and Light......................................................................................90 5.3.2 Gibberellic Acid, Potassium Nitr ate and Cold Stratification............................ 90 5.3.3 Germination After Re moval of Embryo Coverings.......................................... 91
7 5.4 Discussion..................................................................................................................... 91 5.5 Summary .......................................................................................................................96 6 SEED PRIMING ALLEVIATES DORMANCY IN Coreopsis floridana AND PERMITS GERMINATION AT HIGH TEMPERAT URES IN C. floridana AND C. lanceolata .............................................................................................................................107 6.1 Introduction................................................................................................................. 107 6.2 Materials and Methods................................................................................................ 109 6.2.1 Plant Material.................................................................................................. 109 6.2.2 Osmotic Priming Protocol...............................................................................109 6.2.3 Solid Matrix Priming (SMP) Protocol ............................................................ 109 6.2.4 Germination Tests ...........................................................................................110 6.2.5 Statistical Analysis ..........................................................................................110 6.3 Results and Discussion................................................................................................ 110 6.3.1 Coreopsis floridana .........................................................................................110 6.3.2 Coreopsis lanceolata .......................................................................................112 6.4 Summary .....................................................................................................................113 7 CONCLUSION..................................................................................................................... 120 LIST OF REFERENCES.............................................................................................................125 BIOGRAPHICAL SKETCH.......................................................................................................150
8 LIST OF TABLES Table page 1-1 Distribution and characteristics of Coreopsis species used in the study........................... 17
9 LIST OF FIGURES Figure page 3-1 External appearance of Coreopsis seeds............................................................................ 52 3-2 Light microscopy image of internal seed anatomy............................................................ 53 3-3 Lateral and micropylar endosperm of Coreopsis seeds..................................................... 54 3-4 Cotyledons (embryo) and the single cell-layer endosperm tissue showing DAPI stain ed fluorescent nuclei in C. lanceolata seeds..............................................................55 3-5 Influence of removal of pericarp and/or endosperm on germination in light or dark........ 56 3-6 Influence of the removal of pericarp and testa or endosperm on germ ination in light at 10, 20 or 30oC................................................................................................................57 3-7 Relationship between germination per centage and percentage seeds with EB M activity................................................................................................................................60 3-8 Correlation between C. lanceolata germination, and EBM activity levels ....................... 61 3-9 Percentage of endosperms exhibiting EB M activity, white bars and Y1 ax is, and EBM activity levels, diamond marks and Y2 axis, in C. lanceolata endosperms............. 62 4-1 Effect of temperature on germination of Coreopsis seeds in light and dark..................... 72 4-2 Influence of 100 M GA or ABA on germination of Coreopsis species at constant 20oC in light and dark.......................................................................................................76 4-3 Germination of various Coreopsis sp ecies at constant 20oC in light and dark after dark, cold (5oC) stratification.............................................................................................80 4-4 Response of Coreopsis sp ecies seeds to GA3 or GA4+7.....................................................84 4-5 Response of Coreopsis seeds to ABA............................................................................... 84 4-6 Response of Coreopsis seeds to tetcy clacis, a GA bios ynthesis inhibitor, (inhibits the oxidation of ent -kaurene to ent -kaurenol in the GA biosynthesis pathway).....................85 5-1 Effects of five incubation temperatur es at two dry afterripening periods on germination in C. floridana seeds in light and dark ........................................................... 97
10 5-2 Progressive dormancy loss with in creasing dry afterripening duration in C. lanceolata seeds. ................................................................................................................98 5-3 Effect of dry afterripening duration and imbibition temperature on germination in C. lanceolata seeds in light or dark. ....................................................................................... 99 5-4 Influence of various incubation temper atures on dorma ncy at two dry afterripening periods in C. lanceolata seeds imbibed in light or dark.................................................. 102 5-5 Effect of increasing concentrations of KNO3 on dormancy relief in 60-day dry afterripened C. lanceolata seeds in light and dark........................................................... 103 5-6 Germination response of 60-day dry afterripened C. floridana seeds after various cold stratifi cation (5oC) durations in dark........................................................................ 103 5-7 Cold stratification (5oC) effects on 60-day dry afterripened C. lanceolata seeds........... 104 5-8 Influence of increasing dry afterr ipening duration on germination (at 20oC) of intact C. floridana seeds in dark; or when pericarp was removed in dark; or when naked embryos were imbibed in dark......................................................................................... 105 5-9 Influence of embryo envelopes on C. lanceola ta seed germination at increasing dry afterripening periods of inta ct seeds imbibed in light or dark; or when pericarp was removed in light or dark; and in naked embryos in light or dark.................................... 106 6-1 Germination of C. floridana seeds in dark after priming in PEG for 3, 4, 5, 6 or 7 days in light ......................................................................................................................115 6-2 Germination of C. floridana seeds in dark at 20oC (LSD = 20) or 30oC (LSD = 9) after priming in 100 mg GA4+7/L PEG for 3, 4 or 5 days in light...................................115 6-3 Germination of C. floridana seeds in dark at 20 or 30oC after priming in 100 mg BA/L PEG for 3, 4 or 5 days in light...............................................................................116 6-4 Mean germination time (MGT) in C. floridana seeds in dark after osmoprim ing in PEG plus various BA concentrations for 4 days in light.................................................116 6-5 Germination of C. floridana seeds in dark after 4 days of SMP in various volumes of solutions in light.. ........................................................................................................... ..117 6-6 Mean germination time (MGT) of C. floridana seeds in dark after SMP in clay (0.5 g) with various volumes of benzyladenine (BA, 100 mg/L water) or gibberellic acid (GA, 100 mg/L water) or wa ter control (C) in light........................................................ 118 6-7 Mean germination time (MGT) of C. lanceolata seeds in dark at 30oC after osmopriming in PEG plus BA (100 mg/L PEG) for various durations in light............... 119
11 Abstrac of Dissertation Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy DORMANCY IN PRE-VARIETY GERMPLASM OF NATIVE Coreopsis SPECIES By Dzingai Rukuni December 2008 Cochair: Jeffrey G. Norcini Cochair: Daniel J. Cantliffe Major: Horticultural Sciences Coreopsis is Floridas state native wildflow er, and the Florida Department of Transportation adopted it for roadside revegetation. However, C. floridana and C. lanceolata seeds have dormancy that can impede economic establishment of sustainable populations. Coreopsis seeds (achenes) consis t of a pericarp, testa and endosperm surrounding a dicotyledonous embryo. In C. lanceolata, lateral endosperm was a single cell-layer, but 1 to 3 cell-layers at the micropylar end, while in C. floridana a single cell-layer persisted throughout. Endosperm ploidy was 1.5 times that of embryo, consistent with angiosperms. Endosperm removal alleviated dormancy in dark-imbibed C. floridana seeds, while in bot h species, excision of the same tissue permitted germination at supraoptimal temperatures. Optimum germination temperatures in C oreopsis were 15 and 20oC, and germination declined above 25oC. In C. lanceolata, endo-mannanase (EBM) activity was de tected at 90 hours imbibition but C. floridana seeds did not exhibit enzyme activity anytime during germination. There was an association between EBM activity and endosperm rupture. Ab scisic acid, tetcyclacis or supraoptimal temperatures inhib ited EBM activity and germination.
12 In 4-week-old C. floridana seeds, pericarp, testa and endosperm imposed dormancy in dark but all naked embryos germinated. Exoge nous gibberellic acid (GA) overcame dormancy in dark. Cold (5oC) stratification partially overcame dormancy in dark-imbibed C. floridana seeds, but potassium nitrate was not effective. Four-week-old C. lanceolata seeds required 150 days dry afterripening to overcome dormancy; e ndosperm enforced dormancy for 90 days, while naked embryos were non-dormant. Potassium nitrate, light and GA did not alleviate dormancy in C. lanceolata and cold stratificati on reduced germination. Osmopriming C. floridana seeds in PEG or PEG+GA improved germination rate and uniformity at 30oC but these treatments were less effective than priming in PEG+6benzyladenine (BA), which led to 100% germination at 20 or 30oC in dark. When solid matrix priming (SMP) was used, BA gave similar resu lts as in osmopriming, but GA effectiveness improved. In C. lanceolata osmopriming in PEG+BA increased germination at supraoptimal temperatures. Dormancy alleviation in Coreopsis was modulated by embryo e nvelopes. There is great potential for using SMP to enhance germination and improve field establishment in Floridas native Coreopsis sowing programs.
13 CHAPTER 1 INTRODUCTION 1.1 Overview The genus Coreopsis is Fl oridas state wildflower The Florida Department of Transportation (FDOT) deems use of this native wildflower appropriate and consistent with roadside ecosystem management goals (FDOT, 2008). Roads impact the ecosystem in several ways such as altered vegetation patterns, hydrology, biodiversity and habi tat, decrease water quality via runoff, and affect a larger area than they occupy (Forman and Alexander, 1998; Trombulak and Frissel, 2000; Gelbard and Beln ap, 2003; Hansen and Clevenger, 2005; Flory and Clay, 2006; Watts et al. 2007). Planting and rest oration of locally adap ted (ecotypes) native wildflowers besides their aesthetic value, has the potential for lowering roadside maintenance costs by reducing mowing and re-seeding frequenc y (McCully, 1987; Bryant and Harper-Lore, 1997; Dana et al., 1996; Norcini and Al drich, 1998; Norcini et al. 2001a, 2001b; FDOT, 2008). For example, use of native wildflowers in Texa s roadside plantings increased aesthetic value, reduced maintenance costs, enhanced wildlife ha bitat and biodiversity, augmented soil erosion control and suppressed noxious weeds (Bryant and Harper-Lore, 1997). Othe r states using local ecotypes for revegetation programs include, Idaho, Michigan, Ohio and Wisconsin. Native wildflower populations established w ith locally or regionally adapted ecotype germplasm are likely to be se lf-sustaining with proper manage ment (Norcini and Aldrich, 1998; Norcini et al. 2001b; FDOT, 2008), because the environm ent favors flowering, seed production (Norcini et al. 2001b; Norcini and Aldrich, 1998) and the po ssible establishment of a persistent seed bank (Kabat et al., 2007; Frances, 2008), coupled with o pportune conditions for subsequent seed germination and recruitment (Frances, 2008).
14 Because of adaptive attributes and their aest hetic value, Florida ecotypes are especially valued for ecological restorati on, roadside revegetation, mine s ite reclamation, and enhancement of natural areas. Seeds of Florida ecotypes of native Coreopsis species are costly ($110$220/kg) because of limited supply (Norcini a nd Aldrich, 2008), and ar e well adapted, low maintenance plants (Norcini and Aldrich, 1998; Norcini et al. 2001b; Kabat et al. 2007; Norcini, 2008). However, native Florida ecotypes of Coreopsis can have germination problems that hinder establishment of sustainable populations. Additionally, some species require light for germination. 1.2 Taxonomy and Distribution Coreopsis is a genus of about 132 species and is distributed throughout the Americas, the near Pacific and Africa. Form erly known as Calliopsis, Coreopsis belongs to the family Asteraceae ( Compositae ), the daisy or aster family. The aster family has some of the most important agricultural and horticultural species such as lettuce ( Lactuca sativa ) and sunflower ( Helianthus annuus ). Coreopsis species in the current study belong to the Division Magnoliophyta; Class, Magnoliopsida; Order, Asterales; Family, Asteraceae; Tribe, Coreopsideae; Genus, Coreopsis : C. basalis, C. floridana, C. lanceolata, C. leavenworthii and C. pubescens (USDA-NRCS, 2007; Crawford and Mort, 2005). According to Crawford and Mort (2005) the species basalis, lanceolata and pubescens belong to the section Coreopsis, leavenworthii to section Calliopsis, and floridana to section Eublepharis. Important botanical and reproductive information on the study spec ies was documented by Smith (1976) and is summarized in Table 1-1. Furt her hybridization work in Coreopsis is reported by Smith (1983) and Smith and Deng (2008), and Czarnecki et al. (2008) presents information on genetic variation in C. leavenworthii. Coreopsis species have sporophytic self-incompatibility (Ferrer
15 and Good-Avila, 2007) to maintain genetic variabili ty, and cross-pollination is necessary for seed production. The genus Coreopsis is commonly referred to as tick seed because the flat, small, indehiscent fruit (achene) is oval to round, and has two short spines or awns at one end that give it a bug-like appearance. The achene (hereafter referred to as seed) is a cypsela because it develops from an inferior ovary (Esau, 1977). Ti ckseed flowers generally have eight showy ray flowers (petals) that usually have toothed ends. There are 14 tickseed species in Florida; C. floridana is endemic while C. leavenworthii is nearly so (USDA NRCS, 2007; Wunderlin and Hansen 2008). 1.3 Problem Statement and Research Objectives Poor seed germination due to dorm ancy is an obstacle to propagating and establishing non-domesticated species. Successive sowing cycl es of seeds without applying germination improving treatments leads to reduced dor mancy in cultivat ed populations of Helianthus annuus (Gandhi et al. 2005) and Echinaceae purpurea (Qu et al. 2005) due to sustained passive selection against dormancy. In C. leavenworthii and C. lanceolata prevariety germplasm, seeds harvested in different seasons had va riable germination levels (Norcini et al. 2004, Kabat, 2004; Norcini et al. 2006, Kabat et al. 2007). Using higher seeding rates to compensate for low germination can be expensive because of high seed prices (Kabat et al. 2007; Norcini 2008; FDOT, 2008) which range from $110 and $220 pe r kilogram (Norcini and Aldrich, 2008). Dry afterripening incr eases germination of C. lanceolata and C. leavenworthii (Norcini et al. 2004, 2006; Norcini and Aldrich 2007a). However, afterripening under cold moist conditions induces dormancy in C. lanceolata (Banovetz and Scheiner (1994a 1994b). Gibberellic acid stimulates germination in non-afterripened C. basalis seeds (Carpenter and Ostmark, 1992). However, there is a lack of informa tion on germination behavior in native Coreopsis seeds.
16 Optimizing germination continues to present a cha llenge to both seed pro ducers and end users of pre-variety Coreopsis germplasm, and techniques to improve germination are presently being explored. The objective of this study was to investigate methods of alleviating dormancy, and enhance germination to attain acceptable field establishment under a wide variety of environmental conditions, and promote efficient and cost effective native Coreopsis sowing programs in roadside revegetation and ecological restoration projec ts. Several approaches were used to accomplish the objective, and these were: 1) studying Coreopsis seed anatomy, and examining effects of embryo envelopes on dormancy regulation, and assessing the relationship between endo-mannanase (EBM) enzyme activity in endosperms and need for during germination; 2) appraisal of temperature and light effects on germination, and GA to promote germination; 3) evaluation of influence of dry afterripening on germination; and 4) exploration of seed priming techniques to improve germination.
17Table 1-1. Distribution and characteristics of Coreopsis species used in the study. Species Distribution Main blooming time Chromosome number Habitat Hybridizations C. basalis (A. Diert.) Blake. Goldenmane tickseed It was apparently introduced into the USA, but is now widespread on the coastal plain from Florida to North Carolina. Southern states; North Carolina, South Carolina, Georgia, Florida, Alabama, Mississippi, Arkansas, Louisiana and Texas. April-July n=13 low sandy areas Successful with: C. wrightii only when it was the male parent (pollen donor) Failed with: C. grandiflora var. grandiflora and saxicola, C. auriculata, C. rosea and C. tinctoria .: C. floridana E.B. Smith. Florida tickseed Endemic to Florida. Thought to be hybrid of C. linifolia and C. gladiata (or possibly C. falcata). Distribution Florida mid OctoberDecember n=ca.78 Mid October-December Individuals blooming late in the season (December-March) are nearly scapose. This probably represents a high polyploidy (duodecaploid) derived from hybridization. low sandy bogs, ditches, pine barrens, everglades Successful with: none Failed with: C. linifolia (n=13 and n=26) and C. gladiata C. lanceolata L. Lanceleaf tickseed. Varieties: glabella and villosa Michx. Connecticut, New Jersey, Delaware, Pennsylvania, Maryland, West Virginia, New York, North Carolina, South Carolina, Georgia, Florida, Alabama, Tennessee, Kent, Mississippi, Louisiana, Texas, Arkansas, Oklahoma, Missouri, Kansas, Illinois, Indiana, Iowa, Michigan, Wisconsin. mid MarchMay, in the south to June-July in the north. n=13 (+0-2B). Exhibits variability and has intergrading phases. The most prevalent variation is a subglabrous and pubescent phase. Four phases have been drawn up: typical entire-leaved, robust and dwarf hairy. All intergrade and it does not appear useful to segregate any of them. In Florida, all dwarf glabrous or villosa. prairies, glades, sandy slopes and roadsides. Successful with: C. auriculata, C. grandiflora var grandiflora, harveyana and saxicola; C. pubescens var. pubescens. Failed with: C. intermedia, C. nuecensoides, C. basalis, C. rosea, C. gladiata, C. tinctoria var. tinctoria and C. palmata.
18Table 1-1. Continued Species Distribution Main Blooming time Chromosome number Habitat Hybridizations C. leavenworthii Torr. & Gray. Leavenworths tickseed. Varieties: garberi Gray curtissii Sherff and lewtonii (Small) Sherff. Nearly endemic to Florida. Alabama 2 counties. AprilOctober (sporadically all year in southern Florida) n=12 low wet sandy flatwoods and savannas. Successful with: C. tinctoria var. tinctoria and Failed with: C. basalis, C. wrightii, C. nuecensoides, C. linifolia (n=26), C. rosea, C. gladiata, and C.grandiflora var. longipes. C. pubescens Ell. Star tickseed. Varieties: debilis, pubescens and robusta Virginia, North Carolina, South Carolina, Georgia, Florida, Alabama, Tennessee, Kent, Mississippi, Louisiana, Arkansas, Oklahoma, Missouri, Illinois. June-August. n=13 (+0-2B). alluvial banks, pan woods, sandy fields. Successful with: C. pubescens var pubescens X var debilis, C. auriculata, C. intermedia, C. grandiflora var. saxicola and C. lanceolata. Failed with: C. basalis, C. nuecensoides, C. palmata, and C. tripteris. Based on information from Smith (1976), Wunderlin (2008) and Norcini (Pers. Comm.).
19 CHAPTER 2 LITERATURE REVIEW In seed biology, there are four chronol ogical developmental program s, namely, morphogenesis, maturation, dormancy and germina tion, in that order (B ewley and Black, 1994). Integration of the phases is le ss strong than previously though t since some stages can be bypassed without detriment to the seed (Golovina et al., 2001; Gutierrez et al. 2007). A brown seeded tomato mutant does not exit the developmental program and germinates after physiological maturity without ente ring dormancy or quiescence (Downie et al. 2004); vivipary also occurs in corn kernels (White et al. 2000). Many dormancy features are determined by genetics and mediated by the environment, a nd the balance between the phytohormones abscisic acid (ABA) and GA plays a crucial role in seed dormancy (White et al. 2000; Finch-Savage and Leubner-Metzger, 2006; Leubner-Metzger 2003; Groot and Karsen, 1992). 2.1 Seed Dormancy in Coreopsis Species Coreopsis lanceolata seed dorm ancy is influenced by afterripening (Banovetz and Scheiner, 1994a, 1994b), and the phytohormones GA3 and ethylene govern germination in Coreopsis basalis seeds collected from natural stands (Carpenter and Ostmark, 1992). In C. leavenworthii seeds, warm stratification alleviated dormancy (Kabat, 2004; Kabat et al. 2007), while in C. lanceolata seeds dry afterripening overcame dormancy (Norcini et al., 2004, 2006; Norcini and Aldrich, 2007a). Fresh C. basalis, C. floridana and C. leavenworthii seeds require slight afterripening (Nor cini and Aldrich, 2007a). Coreopsis leavenworthii and C. lanceolata prevariety seeds harvested in different seasons had variab le dormancy levels (Norcini et al. 2004, 2006; Kabat, 2004; Kabat et al. 2007). Cold stratification induces dormancy in C. lanceolata seeds (Banovetz and Scheiner (1994a, 1994b).
20 2.2 Seed Morphology and Anatomy The typical fruit of Asteraceae the achene or cypsela (hereaft er referred to as seed), is comprised of a dicotyledonous embryo surrounded by a thin endosperm, testa, and pericarp (Borthwick and Robbins, 1928; Esau, 1977; Puttock, 1994; Sancho et al., 2006). Seeds of Asteraceae are classified as cypselas because they develop from an inferior ovary (Esau, 1977). Like other angiosperms, the diploid pericarp a nd testa are of maternal origin. The triploid endosperm has one set of genes from the paternal parent and two sets fro m maternal parent, and the diploid bi-parental embryo is derived equally from both male and female parental gametes (Friedman 1998; Herr 1999; Floyd and Friedman 2001; Friedman and Williams 2003). In lettuce ( Lactuca sativa ), the embryo is enveloped by a two-cell la yer of endosperm which could be three or more cell layers thick at the micropylar end, a testa and pericarp (Borthwick and Robbins, 1928; Jones, 1974; Psaras et al. 1981; Nijsse et al. 1998). In seeds of Arabidopsis thaliana and Lepidium sativum (both Brassicaceae ), endosperm is single cell-layered (Muller et al. 2006). Pericarp and testa protect the dispersal unit while endosperm nourishes the embryo during seed development and germination (Floyd and Fr iedman 2000; Friedman and Floyd, 2001; Williams and Friedman 2002; Feurtado et al. 2004). Costa et al. (2004) reported that endosperm contains proteins that repress precocious germin ation and promote desiccation tolerance. The developmental origin of the endosperm from a second fertilization event was independently discovered by Nawaschin of Russia in 1898 and Guignard of France in 1899 (Friedman, 1997; Friedman and Floyd, 2001). Before then, the endosperm had been regarded as a fission product of polar nuclei of the female gametophyte (embryo sac). There are three types of endosperm development, viz : cellular, helobial and nuclear (sync ytial) of which the last is the most common in angiosperms (Carmichael and Friedman, 1995; 1996; Friedman 1998; 1992a; 1992b; 1990a; 1990b; Friedman, 1994; 1995; 1998; Friedman and Carmichael, 1996; Floyd et
21 al., 1999; Floyd and Friedman 2000; Friedman and Floyd, 2001; Williams and Friedman 2002; Costa et al. 2004). The endosperm could be regarded as an organism homologous (but not in function) to the embryo rather than as a tissue because it is gene tically identical to embryo except for gene dosage (Friedman, 1998; 1995; Friedman, 1998; 1991; 1990a; 1990b). The three defining characteristics of an endosperm are a ltruism, determinate growth and programmed death (Friedman, 1998; 1995). 2.3 Germination Germination is the sum of all the processes th at occur in a seed and results in exit from dormancy or quiescence to produce a seedling. This occurs when sufficient moisture is provided under optimal temperatures, adequate oxygen leve ls and may sometimes require exacting light quality and/or quantity (Baskin et al. 2003; Bewley and Black, 1994; Finch-Savage and Leubner-Metzger, 2006; Tamura et al. 2006; Bradford et al. 2007). Water uptake in seeds occurs in a triphasic pattern, star ting with an initial stage of ra pid water uptake, followed by a lag phase, and finally, a period of resumed water up take as a result of embryonic axis growth (Bewley and Black, 1994). Failure of seeds to enter the third stage of mass increase is a sign of dormancy (Loercher, 1974; Bewley and Black, 1994; Baskin and Bask in, 1998; Jayasuriya et al. 2007). 2.3.1 Temperature and Light Temp erature influences germination via its i nherent control of resp iration and metabolic rates in seeds (Bewley and Black, 1994; Baskin and Baskin, 1998). Germination and respiration rates vary with temperature and water potential (Dahal et al. 1996; Marshall and Squire, 1996; Steinmaus et al. 2000; Cheng and Bradfor d, 1999; Grundy, 2002; Larsen et al., 2004). Seeds have an optimum, maximum and minimum temper ature at which germination occurs, and nondormant seeds germinate in a wide temperat ure range (Bewley and Black, 1994; Baskin and
22 Baskin, 1998; Pritchard et al. 1999; Kabat et al. 2007). Fluctuating temperatures promote germination in Cynara cardunculus and Sisymbrium altissimum seeds under high osmotic potential (Huarte and Benech-Ar nold, 2005), and alternating temper atures in the natural habitat means that seeds germinate under wider environmen tal conditions (del Monte and Tarquis, 1997; Chachalis and Reddy, 2000; Steinmaus et al. 2000; Steadman, 2004; Baskin et al. 2006; Tarasoff et al., 2007; Merritt et al. 2007; Leon et al. 2006; 2007; Cristaudo et al., 2007; Merritt et al. 2007). Temperature and light interaction cause variable germination responses (Taylorson and Hendricks, 1972; Steadman, 2004). For example, light responses vary with temperature in germinating Abutilon theophrasti and Setaria faberi seeds (Leon and Owen, 2003). Light is a critical environmental factor regulating germination, and acts through the phytochrome system (Hendricks et al. 1968; Wooley and Stoller, 1978; van der Woude and Toole, 1980; Ensminger and Ikuma, 1988; Ensminger and Ikuma, 1988; Derkx et al. 1994; Poppe and Schafer, 1997; Casal et al. 1997; 1998; Toyomasu et al. 1998; Milberg et al., 2000; Yamaguchi and Kamiya, 2002; Oh et al. 2006; Appenroth et al. 2006; Donohue et al., 2007; 2008). Phytochrome photoreceptors are located in the embryonic axis (Bewley and Black, 1994). The phytochrome system is widely believed to control germination through two forms of the inter-convertible phytochrome pigment that exists as Pfr or Pr, with Pfr being the biologically active form (Bewley and Black, 1994; Baskin and Baskin, 1998). Exposure of seeds to red light (660 nm) converts the pigment to the Pfr form and irradiation with far-red light (730 nm) causes it to revert (Poppe and Schafer, 1997; Casal et al. 1997; 1998). Three types of phytochrome re sponses operate in seeds: Low Fluence Response (LFR), Very Low Fluence Response (VLFR) and Hi gh Irradiance Response (HIR). Very low proportions of Pfr are sufficient to induce VLFR under low fluence red light, whereas higher
23 levels are required to induce LFR. Under far-r ed light, VLFR would occur but not LFR. The HIR occurs under continuous far-red light. The LFR response is under control of the more stable phytochrome B while the labile phytochrome A mediates VLFR and HIR. Reciprocity experiments are utilized to disse ct these light responses since LF R and VLFR exhibit reciprocity but HIR does not (Yanovsky et al. 1997; Casal et al. 1997; 1998). The best-c haracterized light response is controlled by phytochrome B, and lettuce seeds are a good example for LFR (Grubisic et al., 1985; Shinomura et al., 1994; Yanovsky et al. 1997; Leon and Owen, 2003; Yamauchi et al. 2004; Appenroth et al. 2006;). In six annual Asteraceae weeds ( Bidens pilosa, Galinsoga parviflora, Guizotia scabra, Parthenium hysterophorus, Tagetes minuta and Verbesina encelioides ), germination was promoted by light (Karlson et al. 2008). Lettuce seed s responded to light quality at 4 to 32% seed moisture content (Vertucci et al., 1987; Lewak and Khan, 1977). In lettuce, far-red irradiance can not reverse the effects of red lig ht exposure when applie d 14 hours after red light induction of germination (Loercher, 1974). The li ght requirement in some lettuce seeds could be substituted for by cold stratification (Lewak and Khan, 1977; van der Woude and Toole, 1980). Light and cold stratification promoted germination in Amaranthus tuberculatus (Leon and Owen, 2003; Leon et al. 2006; 2007). Nitric oxide was found to promote light-mediated events in germinating light-requiring lettuce seeds (Beligni and Lamattina, 2000). Germination is less dependent on light in larger seeds (Milberg et al., 2000; Schutz et al., 2002; Jankowska-Blaszczuk and Daws, 2007). Small seeds of C. lanceolata germinated frequently at shallow depths and large ones at deeper positions (Banovetz and Scheiner, 1994b). In Tragopogon pratensis ( Asteraceae ) seeds, dormancy is controlled by seed size (van Molken et al. 2005). In buried weed seeds with VLFR, sensitiv ity to light is high, and 70 to 400% more
24 seeds germinated when tillage was carried out in daylight than at night (Scopel et al. 1994). Many seeds such as those of Polygonum aviculare lose the light-requirement for germination as dormancy wanes (Batlla and Benech-Arnold, 2005). In many species, activation of phytochrome is linked to subsequent de novo GA production (Grubisic et al., 1985; Yanovsky et al. 1997; Toyomasu et al. 1998; Kucera et al., 2005; Oh et al. 2006; Yamauchi et al. 2007; Donohue et al. 2007; 2008), and enhanced sensitivity of embryos to synthesized GA (Hil horst and Karsen, 1988; Yamaguchi and Kamiya, 2002). 2.3.2 Gibberellic Acid Gibberellic acid promotes germ ination (Hole et al. 1989; Yamaguchi et al. 1998; Debeaujon and Koornneef, 2000; Olszewski et al., 2002; Feurtado et al. 2004; Ali-Rachedi et al. 2004; Kucera et al., 2005; Finch-Savage and Le ubner-Metzger, 2006; Muller et al., 2006; Yamauchi et al. 2007). Exogenous application of GA i nduces germination in many seeds by substituting for the light require ment (Braun and Khan, 1975; Derkx et al. 1994; Yoshioka et al., 1998; Yamaguchi et al. 1998; Yamaguchi and Kamiya, 2002; Hedden, 2002; Yamauchi et al. 2007). Gibberellins stimulate embryo grow th potential by promo ting cell elongation and division of embryo hypocotyls (Taiz and Zieger, 2002), and at times, induces de novo biosynthesis of hydrolases that di gest and weaken endosperm (Wu et al. 2001; Yamaguchi and Kamiya, 2002; Kucera et al., 2005; Oh et al., 2006). The importance of GA in germination is demonstrated in Arabidopsis thaliana and tomato GA-deficient mutants that require exog enous GA for germination (Leon-Kloosterziel et al., 1996; Debeaujon and Koorneef, 2000; Olszewski et al., 2002; Hedden, 2002; Mo and Bewley, 2003). Gibberellin requirement for germ ination is determined by testa integrity in
25 Arabidopsis testa mutants (Debeaujon and Koornneef, 2000). Ethylen e action substitutes for GA requirement in Arabidopsis seed germination (Leon-Kloosterziel et al., 1996). 2.3.3 Ethylene The role of ethylene in germination is widely acknowledged (W ang and Ecker, 2002; Kepczynski and Kepczynka, 1997) Ethylene promotes germination in lettuce seeds by stimulating radial cell expansion in the embr yonic hypocotyl (Abeles, 1986). The phytohormone promotes germination in seeds of lettuce (Nascimento et al., 2000; 2001), peanut ( Arachis hypogeae ) (Ketring and Morgan, 1972), Avena fatua (Cranston et al. 1996), barley (Locke et al., 2000), tomato (Siriwitayawan et al. 2003), beet ( Beta vulgaris ) (Hermann et al. 2007), Lannea microcarpa (Neya et al. 2008) and in Stylosanthes humilis (Pinheiro et al. 2008). Ethylene also overcomes thermoinhibition in lettuc e seeds (Abeles, 1986; Nascimento et al., 2000; 2001). Seeds of some species do not respond to ethylene, or promotive effects are minimal (Kucera et al., 2005). 2.3.4 Cytokinins Cytokinins can either inhibit or promot e germination, and response is dependent on environmental conditions such as temperatur e (Hutchinson and Kieb er, 2002). Cytokinins promote germination through regulation of ce ll division (Khan, 1971; Braun and Khan, 1975; Hutchinson and Kieber, 2002). They also alleviat e thermoinhibition in lettuce seeds (Abeles, 1986; Kucera et al., 2005). 2.4 Dormancy Seed dorma ncy is an innate property that prevents seeds from germinating under any environmental conditions that are otherwise favo rable for germination in a particular species (Baskin and Baskin, 1998; 2004; Baskin et al. 2003; Bewley and Black, 1994; Finch-Savage and Leubner-Metzger, 2006). Primary dormancy is acqui red at seed maturation (Bewley and Black,
26 1994; Baskin and Baskin, 1998; Mollard et al. 2007). Dormancy is a quantitative character though its inheritance is not we ll understood (Foley and Fennimo re, 1998). Finch-Savage and Leubner-Metzger (2006) proposed a broad classi fication of five seed dormancy classes: physiological dormancy, morphological dormanc y, morphophysiological dormancy, physical dormancy and combinational dormancy. Non-deep physiological dormancy is the most common and occurs in both gymnosperms and a ngiosperms (Baskin and Baskin, 1998; 2004). In the natural environment, seed dormancy plays an important role by stretching the germination window by several s easons or years, an important survival strategy (Paterson et al. 1976; Cavieres and Arroyo, 2000; Foley, 2001; Traba et al., 2004; Masin et al. 2006; Karlsson et al. 2006). Based on 2-year buria l results, the longevity of C. lanceolata seeds were 2, 6 and 13 years for small, medium and larg e seeds respectively (Banovetz and Scheiner, 1994a). Seed age-related changes in dormancy leve ls in a soil seed bank is adaptive rather than spontaneous, and maintains genetic divers ity (Valleriani and Tielborger, 2006). Growth and maturation environment of ma ternal plants influence dormancy in Arabidopsis thaliana (Blodner et al. 2007; Donohue et al., 2007; 2008), Sicyos deppei (OrozcoSegovia et al. 2000), and Avena fatua and A. barbata (Paterson et al., 1976). In a particular species, there are variations in seed dormancy among populations, maternal parents and years of seed production (Andersson a nd Milberg, 1998; Pritchard et al. 1999; El-Keblawy and AlAnsari, 2000). The shift in seed population sensitivity to envi ronmental cues together with individual seed variation in sensitivity accounts for the wide array of dormancy phenotypes (Silverton, 1999; Mo and Bewley, 2003; Bradford, 2005). 2.4.1 Abscisic Acid (ABA) Abscisic acid is a putative dorma ncy e nhancer (Schopfer and Plachy, 1984; Hole et al. 1989; Page-Degivry and Garello, 1992; Leon-Kloosterziel et al., 1996; Leung and Giraudat,
27 1998; Toorop et al. 2000; Feurtado et al. 2004; Benech-Arnold et al., 1999, 2006; Ali-Rachedi et al. 2004; Kucera et al. 2005; Finch-Savage and Leubner-Metzger, 2006; Muller et al. 2006; Leon et al. 2006; 2007; Toh et al. 2008). In dormant imbibed seeds, ABA is produced in the embryo and regulates germination by arresting embryo growth (Taiz and Zeiger, 2002). The nature of ABA receptors and its signal transduction are still not well understood (Leung and Giraudat, 1998). Abscisic acid is synthesized in plants in response to abiotic stress such as drought, and this phytohormone plays a major ro le during seed development, maturation and dormancy (White et al. 2000; Suzuki et al. 2000; Frey et al. 2004). Abscisic acid is a sesquiterpenoid synthesized from xanthophylls (Nambara and MarionPoll, 2003; Taylor et al., 2000). Carotenoids are the main precursors of ABA and inhibition of their biosynthesis would preven t ABA biosynthesis. Studies in which carotenoid biosynthesis inhibitors are used to study ABA effects in seed germination could however be misleading, because carotenoids are channeled to ma ny other physiological processes (Yoshioka et al. 1998). Abscisic acid in seeds is inactivated by metabolism into phaseic and dihydrophaseic acids (Jacobsen et al., 2002; Feurtado et al. 2004). Abscisic acid is required during late seed maturation to induce dormancy (Hole et al. 1989), and in Arabidopsis thaliana, this is controlled by four master regulator genes, viz ABSCISIC ACID INSENSITIVE 3 (ABI3), FUSC A3 (FUS3), LEAFY COTYLEDON 1 (LEC1) and LEC2 (Gutierrez et al. 2007). Production of ABA during seed development is of dual origin, arising from developing embryo and ma ternal tissues, but only embryonic ABA induces lasting dormancy (Hole et al. 1989; Page-Degivry and Garell o, 1992; Groot and Karssen, 1992; Nambara; Suzuki et al., 2000 and Marion-Poll, 2003; Kucera et al., 2005). Developing seeds of Cucumis sativus were more sensitive to ABA than mature seeds (Amritphale et al. 2005). This
28 disparity in embryo ABA sensitivity prevents vivipary. Pre-harvest sprouting in Sorghum bicolor was caused by low embryonic sens itivity to ABA (Steinbach et al. 1997). However, Berry and Black (1992) proposed that the osmo tic environment within the tomato fruit contributed more to preventing precocious germination of deve loping seeds than did endogenous seed ABA. Embryo axes excised from seeds afte r radicle protrusion were insensitive to ABA in Aesculus hippocastanum (Gumulevskaya and Azarkovich, 2004). In ABA-deficient tomato seeds, exogenous ABA restricted embryo growth potential (Groot and Karsen, 1992) Embryonic ABA in Arabidopsis seeds influences dormancy (Debeaujon and Koornneef, 2000; Debeaujon et al., 2000). Plant mutants impaired in ABA biosynthesis and/or sensitivity produce viviparous seeds that germinate on the mother plant. Such mutants have been isolated in tomato, Zea mays, Sorghum bicolor Triticum aestivum and Arabidopsis thaliana ; and these genotypes are invaluable in the study of ABA and its role in seed dormancy (Leon-Kloosterziel et al. 1996; Steinbach et al. 1997; Kawakami et al. 1997; Donohue, 2005; Gualano et al., 2007; Gianinetti and Vernieri, 2007). Seed sensitivity to ABA is associated with physiological status (Ni and Bradford, 1992; 1993). Lettuce seeds imbibed in conditions favoring germination had rapid decline in endogenous ABA content (Braun and Khan, 1975). Dry dormant seeds of the Cvi ecotype of Arabidopsis thaliana were found to contain higher amounts of ABA than dry, non-dormant seeds, and when imbibed, ABA content declined in both dormant and non-dormant seeds until 3 days after sowing when ABA content increased and remained high only in dormant seeds (AliRachedi et al. 2004). The mechanisms blocking germination when exogenous ABA is used were found to be different from those that blocked germination in dormant Arabidopsis thaliana seeds (Chibani et al. 2006).
29 Abscisic acid breakdown permits seeds to exit dormancy (Jacobsen et al. 2002; Feurtado et al. 2004). Fluridone, an ABA biosynthesis in hibitor overcame dormancy in lettuce seeds (Yoshioka et al., 1998; Gonai et al. 2004), and in a light-requiri ng lettuce cultivar, the ABAinhibitor norflurazone promoted germination in dark-imbibed seeds (Roth-Bejerano et al., 1999). The capacity of embryos to metabolize (de-ac tivate) ABA and low ABA sensitivity are two factors associated with dormancy termination in Chamaecyparis nootkatensis seeds (Schmitz et al., 2000, 2002), and similar results were found with Pinus monticola (Feurtado et al. 2004) and in barley (Jacobsen et al. 2002). This corroborates that ABA is involved in dormancy imposition (Gonai et al. 2004; Tamura et al. 2006; Toh et al. 2008). Abscisic acid inhibits endosperm rupture in Lepidium sativum, Arabidopsis thaliana and Nicotiana tabacum seeds (Leubner-Metzger, 2002; Muller et al., 2006), and inhibits water uptake in Brassica napus seeds by preventing cell wall loosening (Schopfer and Plachy, 1985, 1984). Abscisic acid action is asso ciated with arrested embryo grow th as confirmed by studies in which micropylar endosperm is removed (Finch-Savage and Leubner-Metzger, 2006). 2.4.2 Afterripening Afterripening is a natural process that occurs in dry seeds under a set of environmental conditions. This process allows them to ge rm inate after a certain period from physiological maturity, and may take a few weeks to years de pending on species, storage relative humidity and temperature, and oxygen levels (Widrlechner, 2006) Afterripening occurs below a certain seed moisture content and is delayed when seeds are too dry; it is accelerat ed by high temperatures and elevated oxygen levels (Bewle y and Black, 1994). It is a co mmon property of many dicot and monocot seeds (Baskin and Baskin, 1976; Fennimore and Foley, 1998; Steadman et al., 2003; Merritt et al. 2007; Tarasoff et al. 2007; Cristaudo et al. 2007), but is a phenomenon whose physiology is not well understood (We ttlaufer and Leopold, 1991; Berma-Lugo and
30 Leopold 1992; Bewley and Black, 1994; Baskin a nd Baskin 1998). The process is associated with non-enzymatic biochemical reactions betw een carbohydrates (sugars) and proteins (amino acids) called Maillard and Amadori reactions (Wettlaufer and Leopold, 1991; Berma-Lugo and Leopold 1992; El-Keblawy and Al-Ansari, 2000; Murthy and Sun, 2000; Murthy et al. 2003). These reactions occur best at warm temperatures Changes associated with afterripening are linked to seed aging, implying that these two ar e part of a continuum, with full afterripening being the peak of seed vigor after whic h seed deterioration occurs (Foley, 2001). Dry afterripening is not an ab rupt change from dormancy to full germinability, rather seeds become more responsive to a wider rang e of conditions favoring germination and less responsive to those which do not (Bewley and Black, 1994; Baskin and Baskin, 1998; Foley, 2001). The equilibrium seed moisture content, which is influenced by se ed storage temperature and relative humidity, is an im portant factor in afterripening (Baskin and Baskin, 1979; LeubnerMetzger, 2005; Paterson et al., 1976; Fennimore and Foley, 1998; Steadman et al., 2003). Afterripening can occur at temperatures as low as -20oC (Baskin et al. 2006). In Asteraceae, dormancy is overcome by afterripening (Leubner-Metzger, 2003). Possible mechanisms for dormancy alleviation through afterripening are postulated to act via the capacity of seeds to degrade ABA and synthesize GA during imbibition (Ramagosa et al. 2001). Afterripened barley seeds have increased ABA degradation during imbibition, compared to non-afterripened ones (Jacobsen et al. 2002; Chono et al. 2006). Afterripening involves both a decline in ABA levels a nd sensitivity (Steinbach et al. 1997; Ramagosa et al., 2001). Response of Arabidopsis seeds to dormancy breaking treatme nts such as cold stratification, nitrate and light were dependent on afterri pening (Leubner-Metzge r, 2002; Ali-Rachedi et al. 2004; Finch-Savage et al., 2007). Afterripening is not only important in making decisions on
31 sowing certain seed lots, but is a critical factor in weed mana gement in cropping systems (Foley, 2001). 2.4.3 Coat Imposed Dormancy The two mo st common ways by which primary dormancy is conferred are coat-imposed dormancy conferred by restrictive embryo cove rings, and embryo dormancy in which dormancy is regulated by the embryos physiological status (Foley and Fennimore, 1998; Li et al., 1999; Ren and Kermode, 1999; Baskin et al., 2003; Leubner-Metzger, 2003; 2005; Baskin et al. 2006; Chen et al. 2007). Seed coats may influence dormanc y through interference with water uptake and gaseous exchange, leaching endogenous inhibito rs to embryo, or mechanical restriction of embryo growth (Shull 1913; Borthwic k and Robbins 1928; Jones 1974; Koller et al. 1963; Watkins and Cantliffe, 1983; Abeles, 1986; Soltani, 2003; Leubner-Mezger, 2003; BenechArnold et al., 1999, 2006; Chen et al. 2007; Bradford et al. 2007). In lettuce, presence of endosperm regulated dormancy (Leubner-Metzger, 2003). In Arabidopsis thaliana, testa removal or mutational defects in testa struct ure, led to reduced dormancy (Debeaujon and Koorneef, 2000). 2.5 Overcoming Dormancy 2.5.1 Nitrates Compounds that donate the nitric oxide (NO) m olecule stimulate germination via the nitric oxide pathway (Jovanovic et al., 2005). Cyanide, nitric oxide, sodium nitroprusside, nitrates and nitrites have dormancy breaking properties in seeds of Arabidopsis (Alboresi et al., 2005; Bethke et al., 2006a, 2007) and Zoysia japonica (Xu et al., 2005), because they donate nitric oxide, a signaling molecule that plays an important role in seed dormancy loss (Bethke et al., 2006a, 2006b). Potassium nitrate is recommende d for dormancy alleviation in many species (ISTA, 1985; AOSA, 1998).
32 2.5.2 Moist Cold and Warm Stratification Cold (0-10oC) or warm (>15oC) stratification are pre-sowing treatments in which seeds are imbibed for various durations, normally in dark, before they are exposed to requisite germination conditions; stratification requirements vary by species. Cold stratification or moist chilling alleviates dormancy in many species (ISTA, 1985; del Monte and Tarquis, 1997; Pritchard et al. 1999; Yang et al. 1999; AOSA, 1998; Cavieres and Arroyo, 2000; Soltani, 2003). Cold stratification reduces em bryo sensitivity to ABA (Jacobsen et al. 2002; Feurtado et al. 2004; Chono et al. 2006) and promotes ABA catabolism (Sondheimer et al. 1968), thereby breaking dormancy. Another mechanism by whic h cold stratification breaks dormancy is by enhancing embryo sensitivity to GA (Leon et al. 2006, 2007). Moist chilling replaces the light requirement in some species (Noronha et al., 1997; Leon et al., 2006, 2007). In the Asteraceae species Guizotia scabra Parthenium hysterophorus and Verbesina encelioides cold stratification overcomes dormancy, whereas wa rm stratification relieves dormancy in Bidens pilosa and Galinsoga parviflora (Karlson et al. 2008), and in C. leavenworthii (Kabat et al ., 2007). Twelve weeks of warm stratification followe d by 20 weeks cold stratification alleviated dormancy in Empetrum hermaphroditum seeds (Baskin et al. 2002). Lettuce seeds imbibed at low temperatures germinate better when transferred to supraoptimal temperatures compared to those initially imbibed at warmer temperatures (Nascimento, 2003). 2.5.3 Seed Priming Priming is a pre-sowing imbibition treatm ent that improves seed germination and field establishment through advancing th e biological pre-germination processes, but does not permit radicle protrusion or loss of desiccation toleranc e (Cantliffe, 1981). Seeds are primed in various liquids (osmopriming or osmocond itioning) or in hydrated inert so lid-matrix materials (solid
33 matrix priming [SMP] or matriconditioning) (C antliffe, 2003). Various specific terms are used to describe priming, for example, priming seeds in salt solutions is calle d halopriming. When priming in water, it is called hydropriming or drum priming, using heat/cold, thermopriming, and seed hydration with biological agents biopriming (Ashraf and Foolad, 2005). Seed priming can also be used to improve germination under stress conditions such as high soil temperatures which inhibit germinatio n, as for example in le ttuce seeds (Guedes and Cantliffe, 1980; Cantliffe et al. 1984; Cantliffe, 1991; Sung et al. 1998; Nascimento and Cantliffe, 1998; Nascimento et al 2000, 2001; Nascimento, 2003). 2.5.4 Other Dormancy Breaking Substances Chem icals with phytohormone-like action such as N6-benzyladenine (BA) and fusicoccin overcome dormancy in lettuce seeds by prom oting embryonic hypocotyl elongation (Abeles, 1986). Recently discovered brassinosteroids relieve seed dormancy (Bishop and Koncz, 2002; Kucera et al. 2005). Indole-3-acetic acid (IAA), a naturally occurring auxin overcomes dormancy in many species (Kepinski and Leyser, 2002; Kucera et al., 2005). Of late, polypeptide hormones were discovered in plants and could be involved in dormancy alleviation (Ryan et al., 2002). Organic chemicals like ethanol, methanol, but anol, propanol, iso-propanol, pentanol, aldehydes, nitriles and ketones can break dormancy in red rice (Cohn et al., 1989; Cohn, 2002). Seed anatomy influences permeability of alc ohols through the pericarp, testa, endosperm and into embryo. However, these chemicals may not directly break dormancy. For example, in red rice, alcohols are converted to carboxylic acids which then overcome dormancy (Taylorson, 1988; Abeles, 1986; Taylorson and Hendricks, 1980; Priestly and Leopold, 1980; Reynolds, 1977, 1987).
34 Non-organic chemicals like sele nium compounds break dormancy in Stylosanthes humilis seeds (Pinheiro et al., 2008). Sodium hypochlorite overc omes dormancy in lettuce through endosperm weakening (Drew and Brocklehurst, 1984) Various salts alleviated seed dormancy in Haloxylon ammodendron (Tobe et al., 2004), and potassium hydroxide reduced dormancy in Zoysia japonica (Xu et al. 2005). Seeds of parasitic species such as Orobanche Cumana and Striga hermonthica exit dormancy when they get exposed to root exudates from their hosts (Matusova et al., 2004).
35 CHAPTER 3 ACHENE MORPHOLOGY AND ANATOMY AFFECT DORMANCY IN COREOPSIS ( ASTERACEAE ) SPECIES 3.1 Introduction Seed dorma ncy impedes propagation and esta blishment of many nondomesticated native species such as Coreopsis (Norcini and Aldrich, 2007a; Kabat et al., 2007). The state of Florida is encouraging planting of native wildflowers in roadside revegetation projects (FDOT, 2008), but germination can be poor due to dormancy (Norcini and Aldrich, 1998; Norcini et al., 2001b, 2004, 2006). In native C. leavenworthii and C. lanceolata harvest season of prevariety germplasm influenced the proportion of dormant seeds (Norcini et al. 2004, 2006; Kabat et al. 2007). Prevariety germplasm of native Coreopsis seeds are in high demand because they are well adapted, low maintenance plants (Norcini and Aldrich, 1998; Kabat et al. 2007; Norcini, 2008), with potential to be self -sustaining given proper manageme nt (Norcini and Aldrich, 1998; Norcini et al. 2001b; FDOT, 2008), making them highly su itable for roadside revegetation. Increasing seeding rates to compensate for low ge rmination can be expensive because seeds cost between $110 and $220 per kilogram (Norcini and Aldrich, 2008), and supply is limited. Information on dormancy and germination physiology in C. floridana and C. lanceolata is limited. In Coreopsis species, seed anatomy is not well documented. Information on morphology and anatomy of seeds is important to unders tanding their biology (Bewley and Black, 1994). Many species have coat-imposed dorma ncy (Groot and Karsen, 1992; Welbaum et al., 1998; Sung et al., 1998; Debeaujon and Koornneef, 2000; Toorop et al., 2000; Yamaguchi and Kamiya, 2002; Leubner-Metzger et al., 2003; Kucera et al., 2005; Muller et al., 2006). In Asteraceae of which Coreopsis belongs, the typical achene or cyps ela (seed) develops from an inferior ovary (Esau, 1977), and is comprised of a dicotyledonous em bryo enclosed in an
36 endosperm, testa, a nd pericarp (Sancho et al., 2006; Puttock, 1994; Borthwick and Robbins, 1928). In angiosperms, pericarp and testa are di ploid and of maternal origin, and the embryo and endosperm are derived from both male and female parent gametes but the former is diploid, while the later is triploid (Friedman, 1998; He rr, 1999; Floyd and Friedm an, 2001; Friedman and Williams, 2003). In lettuce ( Lactuca sativa ) the embryo is enveloped by a two cell-layer of endosperm which could be three or more cell-laye rs thick at the micropylar end (Borthwick and Robbins, 1928; Jones, 1974; Psaras et al., 1981; Nijsse et al., 1998). The selective permeability of endosperm, te sta and pericarp controls dormancy and germination by regulating diffusion of solute s (Shull, 1913; Borthwick and Robbins, 1928; Jones, 1974) and oxygen (Bradford et al., 2007) to the embryo. Some seeds have high concentrations of abscisic acid (ABA) in embr yo envelopes, and leachi ng of ABA to the embryo during imbibition maintained dormancy (Chen et al., 2007). Pericarp and testa protect the dispersal unit while endosperm is important to embryo growth during seed development, and also germination in endospermic seeds. In lettuce, the endosperm regulates dormancy by physically restricting embryo gr owth (Leubner-Metzger, 2003). Gibberellic acid (GA) is implicated in induc tion of embryo growth pot ential and initiation of hydrolases that weaken structures su rrounding the embryo (Groot and Karsen, 1992; Welbaum et al., 1998; Toorop et al., 2000; Morohashi and Matsushima, 2000; Yamaguchi and Kamiya, 2002; Leubner-Metzger et al., 2003; Kucera et al., 2005; Muller et al., 2006; Chen et al., 2007). Gibberellins stimulated germination in non-afterripened C. basalis seeds (Carpenter and Ostmark, 1992). Removal of seed tissues enveloping the embryo in many species had similar effects as GA (Leon-Kloosterziel et al., 1996; Debeaujon and Koorneef, 2000; LeubnerMezger, 2003). Light is impor tant in dormancy regulation an d is presumed to initiate de novo
37 GA biosynthesis (Hilhorst a nd Karsen, 1988; Shinomura et al., 1994; Poppe and Schafer, 1997; Milberg et al., 2000; Yamaguchi and Kamiya, 2002; Yamaguchi et al., 2004; Leon and Owen, 2003; Leon and Knapp, 2004). The color or transm ittance of the embryo envelopes determined the quality and quantity of light reaching the embryo (Bewley and Black, 1994; Xu et al., 2005). Light and/or GA are important in alleviating coat-imposed dormanc y in several species (Watkins and Cantliffe, 1983; Groot and Karsen, 1992; Leon-Kloosterziel et al., 1996; Sung et al., 1998; Debeaujon and Koornneef, 2000; Leubner-Metzger et al., 2003; Kucera et al., 2005; Muller et al., 2006). Endo-mannanase (EBM) activity is associat ed (through endosperm digestion) with germination of several species including lettuce (Dutta et al., 1997; Nonogaki and Morohashi, 1999) and tomato (Nonogaki and Morohashi, 1996; 1999; Nonogaki et al. 1998; Toorop et al., 2000; Wu et al. 2001; Mo and Bewley, 2003; Downie et al., 1999; 2004). Activity of this enzyme varies in individual tomato seeds in a population, and single-se ed assay techniques are appropriate to measure activity levels (Still et al. 1997; Still and Bradford, 1997; Mo and Bewley, 2003). Lettuce seed germination at supr aoptimal temperatures in some cultivars is linked to greater EBM activity (Abeles, 1986; Dutta et al., 1997; Nascimento et al., 2000; 2001). Thermoinhibition in imbibed lettuce (Yoshioka et al., 1998; Gonai et al. 2004) and Arabidopsis thaliana (Tamura et al. 2006; Toh et al. 2008) seeds is associated wi th endosperm resistance to rupture and elevated de novo ABA biosynthesis. Abscisic acid biosynthesis inhibitors permitted germination at elevated te mperatures in lettuce (Gonai et al. 2004) and A. thaliana (Yoshioka et al., 1998; Tamura et al. 2006; Toh et al. 2008) seeds. There is strong evidence of an association between EBM hydr olase activity in endosperm and dormancy termination.
38 Dormancy in Coreopsis continues to present a challeng e to revegetation efforts, and dormancy regulation needs to be understood in order to develop techniques to alleviate it. The overall objectives were to 1) identify seed tissu es (components), 2) test whether these tissues could be excised to diminish or terminate dor mancy, and 3) establish whether EBM activity was involved in release from dormanc y. Morphology and anatomy of C. floridana and C. lanceolata seeds were examined, and their potential roles in dormancy investigated in li ght or dark. Various tissues surrounding the embryo were excised, and seeds or embryos were imbibed at various temperatures in light or dark. The EBM activ ity in endosperms of imbibed seeds was also quantified to determine if this enzyme might be linked to dormancy. To address this, a singleseed assay technique was used to measure enzyme activity in C. floridana and C. lanceolata seeds, and to ascertain effects of ABA ( putative dormancy enhancer), tetcyclacis (GA biosynthesis inhibitor) and high temperature (30oC) on enzyme activity. 3.2 Materials and Methods 3.2.1 Plant Material 220.127.116.11 Anatomy and accompanying germination studies Seeds of C. floridana E.B. Sm ith (Ex. Polk County, FL) and C. lanceolata L (composite population, Ex. Leon, Wakulla, Gadsden and Jeffe rson Counties, FL) were harvested in 2005 from cultivated populations in Flor ida (Norcini and Aldrich, 2007a). 18.104.22.168 Endo-mannanase analysis and related germination studies Coreopsis floridana and C. lanceolata seed lots used in anatomical studies (above) were sown to raise seedlings in a growth cham ber in mid August 2006. These were transplanted at the end of October 2006 into 11 liter pots containing pine bark and drip fert igated in a high roof, plastic, passively ventilated greenhouse under na tural photoperiods. Fertigation was set to supply 2.5 liters/plant/day with a nutrient solution containing (ppm): nitrogen (140), phosphorus
39 (50), potassium (200), calcium (120), magnesium ( 50), sulfur (65), iron (2.8), boron (0.7), copper (0.2), manganese (0.8), zinc (0.2) and molybdenum (0.05). The EC of the solution was 1.8 dSm1 and pH was 5.5. Bumble bees ( Bombus impatiens; Kopert Biological Systems, Romulus, MI) were used to ensure cross pollination since Coreopsis is self-incompatib le (Ferrer and GoodAvila, 2007). Seeds of C. lanceolata were collected in May 2007 and those of C. floridana in November 2007. Seeds were bulked over 4 weeks in each species to provide sufficient quantities. Coreopsis floridana seeds were dried to 8.7% seed moisture content and C. lanceolata 8.1%, sealed in mois ture-proof plastic bags, and stored at 10oC and 50% relative humidity in dark. 3.2.2 Microscopy 22.214.171.124 External seed morphology A LEICA MZFLIII Stereomi croscope (Leica Microsystems Inc., Bannockburn, IL) and a Kodak MDS290 camera (Eastman Kodak Co., Rocheste r, NY) were used to record seed images. 126.96.36.199 Scanning electron microscope (SEM) Seed anatomy and morphology were studied usi ng SEM. Seeds were exam ined whole, or cut longitudinally or transversely to view internal anatomy. Specimens from the two Coreopsis species were sputter coated with platinum and examined using a HITACHI S-4000 FE-Scanning Electron Microscope (Hitachi High Technol ogies America Inc., Pleasanton, CA). 188.8.131.52 Light microscopy Individual seeds of each species were bisect ed transversely or longitudinally and fixed overnight in a solution of 500 mM phosphate buffer (pH 7.0), 20 mM EDTA, 20 mM MgSO4, 0.5% paraformaldehyde and 1.5% (v:v) glutaralde hyde. A light vacuum was applied until the seeds sunk to the bottom of the tubes. Samples were washed twice in phosphate buffered saline (PBS) to remove the fixative. The specime ns were post-fixed overnight in 2% OsO4 (osmium) in
40 100 mM phosphate buffer (pH 7). They were washed twice in PBS to remove the osmium, dehydrated in a graded ethanol seri es starting from 10% to 100%. The ethanol was replaced with increasing concentrations of Spurrs resin (Elect ron Microscopy Sciences, Ft. Washington, PA). To speed up infiltration of the resin, samples we re heated for 3 minutes in a bio-microwave (PELCO BioWave 34700 + Coldspot, Ted Pella Inc., Redding, CA) at a power setting of 250 W, vacuum pressure 575 mm Hg, and a temperature of 37oC in each of the Spurrs series from 10% to 90%. At 100% Spurrs, the microwave heatin g duration was increased to 4.5 minutes. The seed specimens were positioned lengthwise or cr oss-sectional in molds with 100% Spurrs resin and incubated at 65oC until polymerization was complete. Sections 1.0 m thick were made using a Leica Reichert Ultracut R microtome (Leica AG, Reichert Division, Wien, Aust ria). Sections were mounted on glass slides and stained with 1% toluidine blue (T-blue) for 5 minutes on a wa rm hot-plate, and excess dye rinsed away using deionized water. The slides were mounted us ing Permount SP15-100, (Fisher Scientific, Atlanta, GA) and cover glasses. The st ained sections were observed us ing a Zeiss Axioskop microscope (Carl Zeiss MicroImaging Inc., Thornwood, NY). 184.108.40.206 Determination of seed tissue ploidy Ploidy levels were determined to verify the iden tity of seed tissues. Seeds were fixed and em bedded similarly to that for light microscopy. Sections (5 m) were cut and stained with 0.1 g/ml 4,6-diamidino-2-phenylindole (DAPI) (S igma-Aldrich, St. Louis, MO) to stain deoxyribonucleic acid (DNA). Methylene blue (Polysciences Inc., Warrinton, PA) was used to quench auto-fluorescence from cell walls. Using a Zeiss Axioskop microscope, DNA fluorescence was quantified with a DAPI filter (excitation: 350/50 nm; emission LD 420). DAPI stained DNA fluorescence techniques were used to quantify relative ploidy levels in seed tissue nuclei (Williams and Friedman 2002; Friedman 1992b). Fluorescence measurements were made
41 in white pixels on 10 endosperm and 10 embryo nuclei per image (section). Twenty seed sections were evaluated for each species ( C. floridana or C. lanceolata ). Background fluorescence was quantified and adjustments made on the nuclei fluoresce nce readings. Mean fluorescence was calculated for each tissue type to obtain the ra tio of average endosperm to embryo measurements. 3.2.3 Image Capture and Editing Digital images were recorded with a Kodak MDS290 cam era and stored in Tagged Image File Format (TIFF). Images were edited (a djusting color) and labe led using Adobe Photoshop version 7.0 (Adobe Systems Inc., San Jose, CA). 3.2.4 Germination Tests Constant temperatures of 20 and 30oC were provided in an Isotemp incubator model 304R (Fisher Scientific, Fair Lawn, NJ) in continuous cool white fluorescent light (~25 molm2s-1) or dark. Darkness was achieved by covering Petri dishes with aluminum foil. Four replications of 25 seeds were placed on double blue blotter pa per (Anchor Paper Company, St. Paul, MN) moistened with de-ionize d water in 5 cm glass Petri dishes. Germination, defined as visible radicle protrusion of at least 2 mm, was recorded every 2 days up to 14 days. Interim germination counts for dark incubated seeds were made under a dimgreen light (25W, A19, Specialty 90912, General Electric Company, Cleveland, OH) in a dark room. 3.2.5 Influence of Seed Envelopes on Germination Experiments with C. floridana and C. lanceolata seeds harvested in 2005 were conducted in spring/summer 2006 (effects of embryo covers at 20oC) and spring/summer 2007 (effects of embryo covers at various temperatures). Pericarp plus testa and endosperm of Coreopsis floridana and C. lanceolata seeds were removed under a dissecting microscope (Model, ASZ37L3, Bausch and Lomb, Roches ter, NY). Ten to twenty embr yos per replicate and three or
42 four replicates per treatment were tested for germination. Germination tests were conducted on a thermo-gradient table, Type db 5000 (Van dok and de Boer Machinefabriek BV, Enkhuizen, The Netherlands) at 10, 20 or 30oC in continuous dark or light with intact seeds (S), intact seeds minus the pericarp and testa (S-P), and naked embryos (E). Germination test duration was 14 days. Intact seeds were used as controls. 3.2.6 Statistical Analysis Digital images recorded in fluorescence m icroscopy were analyzed using NIH Image Version 1.61/ppc software (Nationa l Institute of Health, Bethesda, MD). Germination data for each species were analyzed separately. Data were adjusted for viability as determined by a TZ test (ISTA 2003), arcsine-square root transformed, if necessary, and analyzed by general linear model methods (SAS Version 9.1; SAS Institu te, Cary, NC). Pearson product-moment correlation coefficients (r) were also computed in SAS Version 9.1. Treatment means were separated by least significant difference (LSD; =0.05). 3.2.7 Endo-Mannanase Activity in Coreopsis Endosperms Studies on EB M activity and germination we re conducted after 6 months storage for C. floridana and 12 months for C. lanceolata seeds. Endo-mannanase activity during imbibition of C. floridana and C lanceolata seeds was detected using a gel-diffusion assay (Bonina, 2005; Nascimento et al., 2001; Downie et al. 1994; Still et al., 1997). Seeds were incubated at 20oC in light as described for germination tests. To prepare gel plates, 0.05% (w/v) galactom annan (Locust Bean Gum, Sigma Chemical Co., St. Louis, MO) was dissolved in incuba tion buffer (0.1 M citric acid, 0.2M sodium phosphate, pH 5.0) by stirring and heating for 30 minutes. The solution was then clarified by centrifugation at 15,000 g for 15 minutes at 4C. Agar (Plant TC/Micropropagation grade, PhytoTechnology Laboratories, Shawnee Missio n, KS) was dissolved at 0.7% (w/v) in the
43 clarified solution and stirred wh ile heating to the boiling point. Thirty ml of solution was dispensed into 150x25 mm plastic Petri dishes (Falc on, Franklin Lakes, NJ). After solidification, 2-mm wells were made using a disposable plasti c pipette, and excised gel removed by aspiration. Endosperms were separated from the embryos under a dissecting microscope as described previously. In some experiments, endosperms were divided into lateral a nd micropylar sections. Each endosperm (whole, micropylar tip or lateral) was placed into an individual microtiter plate (Nalge Nunc, Naperville, IL) well containing 20 L of sterile incubation buffer (0.1 M citric acid, 0.2 M sodium phosphate, pH 5.0) and incubate d in the dark for 2 hours at 25C. After incubation, 10 L of buffer from each microtiter well was transferred to gel-diffusion plate wells. Endo-mannanase (Megazyme International Ireland Ltd., Wicklow, Ireland) was diluted to make a standard (control) EBM solution of 1 U (1 U = enzyme activity converting 1 mol of galactomannan per minute under standard conditions). The standard (10 L) was transferred into wells in the gel-diffusion plates alongside endosperm extracts. Pe tri dishes were sealed with Parafilm M (Pechiney Plastic Packaging, Chicago, IL), covered with aluminum foil, and incubated at 25oC for 24 hours. Gels were stained with 10 mL Congo Red (Sigma Chemical Co., St. Louis, MO) in distilled water (0.4% w/v) added to each plate. Plates were ag itated on an orbital shaker for 20 minutes at 60 rpm during staining. The stain solution was decanted and the gel was gently rinsed with distilled water for 1 min after which 10 mL of citrate-phos phate buffer pH 7.0 was added and plates shaken again for 3 minutes on the orbi tal shaker at 60 rpm. The buffer was decanted and plates were scanned within 5-10 minutes usi ng a Scan Jet 3c/T (Hew lett Packard, Palto Alto, CA). Scanned images were captured using WinRhizotm (Regent Instruments Inc., Quebec, Canada) software. The diameter of the cleare d areas on the plates was proportional to EBM
44 activity. Germination tests (as described earlier) were conducted and counts recorded daily up to 14 days. 3.2.8 Influence of ABA, Tetcyclaci s and Supraoptimal Temperature (30oC) on Germination and EBM activity Seeds were incubated at 20 or 30oC with 100 M ABA or 100 M tetcyclacis (a GA biosynthesis inhibitor) in light, as described in germination test s above. Light conditions were chosen because C. floridana and C. lanceolata seeds had highest germin ation in in light. Ten endosperms were excised as described previous ly after incubating seeds for 24 hours on each substrate, and endosperms were incubated for an additional 120 hours on respective media. Endosperms from control intact seeds were excised after 144 hours. In another control (incubated in de-ionized water), endosperms were excised after 24 hours but re-incubated in deionized water for a further 120 hours. Enzyme activity was evaluated using procedures described above. Accompanying germination test s were conducted on respective substrate and temperature combinations. 3.3 Results and Discussion Seeds of C. floridana and C. lanceolata were dark brown to black with pale brown or beige wings (Figure 31). Only seeds of C. lanceolata had cavernous callus tissue (knob-like structures) at both longitudinal ends. Additionally, seeds of bot h species had hollow spikes on the pericarp (Rose, 1889; Tadesse et al., 1995). W ings are a possible adaptation for dispersal in wind, whereas callus and spikes could facilitate water floatation duri ng dispersal. Light microscopy and SEM images of Coreopsis seeds showed an outer w oody pericarp (fruit wall), a testa (seed coat) and a single cell-layered e ndosperm that enveloped a dicotyledonous embryo (Figures 3-2 and 3-3). The inner part of the woody pericarp was composed of polygonal parenchyma cells. Measurements of imbibiti on rate and equilibrium moisture content
45 demonstrated that embryo envelops in both spec ies were freely permeable to water. In C. lanceolata the radicle (micropylar) end of the endospe rm consisted of one to three-cell layers, while in C. floridana the micropylar endosperm remained a single cell-layer throughout (Figure 3-3). In lettuce seeds the endosperm is composed of two cell layers but the micropylar end could have more than two cell-layers (Borthwick and Robbins, 1928; Psaras et al., 1981; Bewley and Black, 1994; Nijsse et al., 1998). Muller et al. (2006) reported a single cell-layer endosperm in Lepidium sativum and Arabidopsis thaliana ; however, they found th at in both species the micropylar end had one to two-cell layers. The embryo had distinctive smaller epidermal cells and larger inner cells (Figure 3-2) resembling lettuce seeds (Borthwick and Robbi ns, 1928). Placement of the dicotyledonous spatulate embryo in both species was symmetric al and longitudinal. Seed morphology and anatomy of the two Coreopsis species was similar and typical of Asteraceae (Esau, 1977; Borthwick and Robbins, 1928; Psaras et al., 1981; Nijsse et al., 1998). The mean ratio ( standard error) of endosperm to embryo DNA fluorescence (Figure 34) for C. floridana was 1.54 0.03 and 1.52 0.02 for C. lanceolata The 3:2 DNA ratio indicated that the single-cell layer tissue enclosing the embryo was endosperm. Triploid endosperms and diploid embryos have been report ed in many angiosperm seeds (Friedman and Williams, 2003; Williams and Friedman, 2002; Carmichael and Friedman, 1996, 1995; Friedman 1998, 1992a, 1992b, 1990a, 1990b; Floyd and Friedman, 2001, 2000; Floyd et al., 1999). In light at 20oC, germination of C. floridana naked embryos, seeds without pericarp and testa, and intact seeds was 100%; however, in dark germinati on declined to 77% in naked embryos, 43% in seeds without pe ricarp and testa, and 15% in in tact seeds (Figure 3-5). In C. lanceolata, germination was nearly 100% when na ked embryos, seeds without pericarp and
46 intact seeds were incubated in light or dark. However, co mpared to intact seeds of C. lanceolata germination was rapid in naked embryos and s eeds without pericarp a nd testa (Figure 3-5), indicating some level of dormancy imposition by the pericarp (Bewley and Black, 1994). Tissues enveloping the embryo in both spec ies imposed dormancy. In dark-imbibed C. floridana seeds, removal of pericarp plus testa and endosperm nearly alleviated dormancy. In light-requiring lettuce cultivars, removal of e ndosperm relieved dormancy in dark (Leubner, 2003; Gonai et al., 2004). Debeaujon and Koornneef ( 2000) reported that testa-structural mutants of Arabidopsis thaliana had varying dormancy levels as dictated by testa integrity. Intact seeds of C. floridana had an obligate light requirement but not C. lanceolata (Milberg et al., 2000; Yamaguchi et al., 2004; Leon and Owen, 2003; Leon and Knapp, 2004). In light, excised embryos of C. floridana germinated 100% in 4 days at 30oC while at 20oC it took 8 days, and more than 14 days at 10oC. Naked embryos of C. lanceolata germinated 100% at 20oC and 30oC in about 4 days but 10 days at 10oC. Greater germination rates by naked embryos indicated that pericarp, testa and endosperm had some restriction on germination of C. floridana and C. lanceolata seeds. In both species, intact seeds and those without pericarp did not attain 100% germination at 30oC, because this was near the upper limit for germination. Pericarp restricts germin ation in thermoinhibited Tagetes minuta achenes (Taylor et al. 2005). Lettuce (Cantliffe et al., 1984; Yoshioka et al., 1998; Nascimento et al., 2000, 2001; Gonai et al., 2004) and; Arabidopsis (Toh et al., 2008) seeds have reduced germination at supraoptimal temperatures. When pericarp, testa and endosperm were removed, both C. floridana and C. lanceolata seeds germinated normally at elevated temperatures. Endo-mannanase activity was assayed in imbibed C. floridana and C. lanceolata endosperms to determine if enzyme activity was linked to dormancy. Coreopsis lanceolata
47 seeds exhibited EBM activity from 90 hours of imbibition onwards; visible radicle protrusion was observed after 96 hour s. Non-germinated C. lanceolata seeds had non-de tectable EBM activity. Coreopsis floridana seeds did not exhibit EBM activity even in the post germination phase. Coreopsis floridana seeds did not exhibit EBM activity possibly because of the absence of galactomannans in the endosperm, which imp lies that a different enzyme system could be involved in endosperm digestion. In lettuce seeds, hydrolytic enzymes other than mannanase might be involved in endosperm weakening (Dutta et al. 1994); cellulolytic, hemicellulolytic or pectolytic enzymes promoted germination in dormant dark-imbibed lettuce seeds (Ikuma and Thimann, 1963). Endosperm composition of bot h species is not known and no literature was available detailing the identity of deposits in these endosperms. Galactomannans constitute the bulk of deposits in lettuce endosperms (Dutta et al., 1997; Nonogaki and Morohashi, 1999) and enzyme activity has been used to detect germina tion patterns in that sp ecies (Abeles, 1986; Dutta et al., 1997; Nascimento et al., 2000, 2001; Bonina, 2005). Endo-mannanase activity has also been reported in endosperms of germinati ng tomato seeds (Nonogaki and Morohashi, 1996; Nonogaki et al. 1998; Mo and Bewley, 2003). Endo-mannanase activity levels were significantly correlated with C. lanceolata seed germination (r= 0.88, p = 0.0199), incubation du ration (r=0.88, p = 0.0196), and proportion of seeds with enzyme activity (r=0.93, p = 0.0077). Germination percentage was well correlated with proportion of seeds expressing EBM activ ity (r=0.97, p = 0.0012), and incubation duration (r=0.99, p = 0.0001). The proportion of seeds e xhibiting EBM activity was highly correlated with incubation duration (r = 0.99, p = 0.0002) (Figure 3-7). Endo-mannanase activity increased as germination time progressed, togeth er with the proportion of seeds expressing activity of this enzyme. The pe rcentage of seeds exhibiting EBM activity reached 100% before
48 maximum germination was attained, implying that enzyme activity preceded germination (Figure 3-7). In seeds of lettuce (Abeles, 1986; Dutta et al., 1997; Nascimento et al., 2000, 2001; Bonina, 2005, 2007), tomato (Nonogaki and Mor ohashi, 1996, 1999; Still and Bradford, 1997; Nonogaki et al., 1998), and in this case C. lanceolata EBM activity is associated with germination. Conditions unfavorable for germin ation inhibited EBM activity in C. lanceolata seeds (Halmer et al., 1976; Toorop et al. 2000). Germination percenta ge and EBM activity levels were highly correlated (0.87, p = 0.0001). When s eeds were imbibed in ABA at optimal (20oC) temperatures, germination percentage was 28% after 14 days, and only germinated seeds expressed normal levels of EBM activity (~0.45 U) while EBM activity levels were low (>0.10 U) in ungerminated seeds (Figure 3-8). At 30oC (control) germination was 9% and nongerminated seeds exhibited low levels of enzyme activity. Seeds incubate d in a combination of ABA and 30oC had 4% germination and no enzyme activity was detected from ungerminated seeds (Figure 3-8). The correlation coefficient between the proportion of endosperms expressing EBM activity and enzyme activity levels was 0.9 8 (p=0.0025) (Figure 3-9). Failure of some C. lanceolata seeds to germinate on ABA substrate or at 30oC was linked to low or no EBM activity. In lettuce, thermo-tolerant cultivars had greater EBM activity during germination at 35oC compared to thermo-sensitiv e ones (Abeles, 1986; Dutta et al., 1997; Nascimento et al., 2000, 2001, 2005; Bonina, 2005, 2007). Thermoinhibi tion in lettuce was circumvented by removal of the endosperm (Cantliffe et al. 1984; Sung et al. 1998; Nascimento et al 200, 2001, 2005). In Arabidopsis thaliana seeds, thermoinhibition was associated with endosperm resistance to rupture and elevated de novo ABA biosynthesis in embryos (Tamura et al. 2006; Toh et al. 2008), and fluridone (ABA biosynthesis inhibitor) relieved thermoinhibition
49 (Yoshioka et al., 1998; Gonai et al., 2004). In seeds such as those of Datura ferox conditions that maintained dormancy inhibited EBM activity (Bewley, 1997). The mechanism of thermoinhibition at 30oC and ABA-suppressed germination in C. lanceolata seeds was associated with low EBM activity in endosperms. Seeds incubated in 100 M tetcyclacis (GA biosynthesis i nhibitor) failed to germinate and endosperms had no detectable EBM activity (dat a not shown). Gibberel lin-deficient tomato mutants do not germinate or e xhibit EBM activity without ex ogenous GA application (Mo and Bewley, 2003). When seeds were imbibed on de-ionized water for 24 hours and endosperm separated from embryo, EBM activity was detect ed in excised endosperms incubated for a further 120 hours on same substrate (Figure 3-9, End. Ctrl). In tomato seeds, EBM activity occurred only if endosperm was separated from embryo after at leas t 6 hours of initial imbibition, indicating that a signal was requi red from embryo (Mo and Bewley, 2003). Moreover in Arabidopsis thaliana and Lepidium sativum endosperm weakening processes, GA could substitute for this embryo signal (Muller et al. 2006). Gibberellic aci d induced enzymatic endosperm weakening in lettuce (Halmer et al. 1976) and tomato (Groot et al., 1988) seeds. It seems de novo GA biosynthesis may be required to i nduce EBM activity and germination in C. lanceolata seeds. 3.4 Conclusions Coreopsis floridana and C. lanceolata seeds have an outer pericarp, a testa, endosperm and dicotyledonous embryo, and this is the first study to report the anatomy of Coreopsis seed s, paving way for further seed physiology research of this species. The pericarp, testa and endosperm are involved in imposing dormancy in C. floridana and C. lanceolata Correlative and diagnostic evidence suggests that EBM activity is a prerequisite for germination in C. lanceolata seeds, but not in C. floridana In C. floridana, a different enzyme system could be
50 involved in endosperm digestion during germinati on, but this was not explored further. This EBM activity in C. lanceolata seeds is reported here for the first time and may provide an answer to circumventing dormancy in non-afterripened C. lanceolata seeds. Seed enhancement technologies aimed at breaking dormancy a nd/or circumventing thermoinhibition for germination improvement and field uniformity s hould focus on techniques that weaken pericarp, testa and endosperm. 3.5 Summary Morphology and anatomy of C. floridana and C. lanceolata achenes (seeds) were studied in order to ascertain possible tissue functions in dormancy modul ation. Seeds were examined using light microscopy, scanning electron micr oscopy and fluorescence microscopy. Seeds of both species had a woody pericarp, testa a nd an endosperm surrounding a dicotyledonous embryo. The lateral endosperm in C. lanceolata was a single cell-layer but was one to three celllayers thick at the micropylar end, while in C. floridana there was a single cell-layer throughout. Florescence microscopy confirmed endosperm pl oidy was 1.5 times that of embryo, concurring with double fertilization outcomes in angiosperms. Coreopsis floridana seeds were dormant in dark; however, C. lanceolata seeds germinated in light and dark, but are normally dormant unless dry afterripened at 10oC for 150 days. Dormancy in dark-imbibed C. floridana seeds at 20oC was nearly alleviated by removal of perica rp, testa and endosperm; removal of the same tissues in light-imbibed seeds entirely overcame inhibition in both specie s at temperatures as high as 30oC. In other Asteraceae lack of endo-mannanase (EBM) activity restricts germination. A single-seed assay technique was used to evaluate EBM activity in C. floridana and C. lanceolata Coreopsis floridana seeds did not exhibit EBM activity, but in C. lanceolata, EBM activity occurred (at 90 hours) before radicle protrusion. Th ere was a significant correlation (r = 0.97) between proportion of s eeds exhibiting EBM activity and endosperm
51 rupture. Inhibited ra dicle protrusion in 100 M abscisic acid substrate and/or at 30oC was associated (r = 0.87) with reduced EBM activity. Tetcyclacis (100 M) prevented EBM activity and induced dormancy. Thus, dormancy in C. floridana is overcome by imbibing seeds in light while uniform and rapid germination of C. lanceolata is consistent with endosperm weakening. Potentially, this mechanism may be important to circumventing dormancy in non-afterripened seeds of C. lanceolata
52 Figure 3-1. External appearance of Coreopsis seeds. A) C. floridana convex (i) and concave (ii) side. B) C. lanceolata convex (iii) and concave (iv) side. Bar scale = 1 mm. C) SEM longitudinal section of C. lanceolata seed (v), and whole seed (vi).
53 Figure 3-2. Light microscopy image of internal seed anatomy A) C. floridana. B) C. lanceolata Bar scale = 25 m.
54 Figure 3-3. Lateral and micropylar endosperm of Coreopsis seeds A) C. floridana lateral endosperm is a single cell-layer. B) C. lanceolata lateral endosperm is also a single cell-layer. C) Micr opylar endosperm of C. floridana seeds are single cell-layered. D) C. lanceolata seeds have micropylar endosperm with one or more cell-layers. Short white arrows on C. lanceolata image indicate the radicle/endosperm interface.
55 Figure 3-4. Photograph of the cotyledons (embr yo) and the single cell-layer endosperm tissue showing DAPI stained fluorescent nuclei in C. lanceolata seeds.
56 A Treatment S-P+LS-P+DE+LE+DS+LS+D Germination % 0 20 40 60 80 100 B Treament S-P+LS-P+DE+LE+DS+LS+D Germination % 0 20 40 60 80 100 Figure 3-5. The influence of removal of pericarp and/or endosperm on germination in light or dark A) C. floridana seeds, at 7 days (white bars, LSD=18) and at 14 days (stipuled bars, LSD=11). B) Coreopsis lanceolata seeds at 7 days (white bars, LSD=33) and at 14 days (stipuled bars, LSD=16) in light or dark at 20oC. S, Intact seed; P, pericarp; E, naked embryo; L, light and D, da rk. Experiments were conducted in spring/summer 2006.
57 A Incubation period (days) 024681 0 Germination % 0 20 40 60 80 100 E+10 E+20 E+30 B Incubation period (days) 024681 0 Germination % 0 20 40 60 80 100 S-P+10 S-P+20 S-P+30 Figure 3-6. The influence of the removal of peri carp and testa or endos perm on germination in light at 10, 20 or 30oC. A), B) and C) C. floridana. D), E), and F) C. lanceolata Seeds. LSDs = 13, 19, 16, 21 and 21 at 2, 4, 6, 8 and 10 days respectively for C. floridana (across all embryo envelope treatmen ts). LSDs = 18, 12, 18, 14 and 18 at 2, 4, 6, 8 and 10 days respectively for C. lanceolata (across all embryo envelope treatments). S, Intact seed; P, peri carp; E, naked embryo. Experiments were conducted in spring/summer 2007.
58 C Incubation period (days) 024681 0 Germination % 0 20 40 60 80 100 S+10 S+20 S+30 D Incubation period (days) 024681 0 Germination % 0 20 40 60 80 100 E+10 E+20 E+30 Figure 3-6. Continued
59 E Incubation period (days) 024681 0 Germination % 0 20 40 60 80 100 S-P+10 S-P+20 S-P+30 F Incubation period (days) 024681 0 Germination % 0 20 40 60 80 100 S+10 S+20 S+30 Figure 3-6. Continued
60 Incubation time (hours) 080100120140160180200 Germination % or % seeds with EBM 0 20 40 60 80 100 EBM activity (U) 0.00 0.05 0.10 0.15 0.20 % seeds with EBM Germination % EBM activity Figure 3-7. Relationship between germination percentage and percentage seeds with EBM activity (Y1 axis) and EB M activity (Y2 axis) in C. lanceolata seeds. EBM activity assessments were made every 6 hours and act ivity was first detect ed after 90 hours. Standard EBM activity was 1U, (1 U = EBM activity converting 1 mol of galactomannan/minute under standard conditions).
61 r=0.87 Treatment Control30 deg. CABA+20ABA+30 Germination % 0 20 40 60 80 100 EBM activity (U) 0.00 0.05 0.10 0.15 0.20 Figure 3-8. Correlation between C. lanceolata germination (white bars and Y1 axis), and EBM activity levels (diamond marks and Y2 axis ). Enzyme activity was measured from endosperms of non-germinated seeds at the end of a 14-day incubation period under the respective treatments. Germinated s eeds from non-control treatments had normal enzyme activity similar to control. Standard EBM activity was 1U, (1 U = EBM activity converting 1 mol of galactomannan/minute under standard conditions).
62 r=0.98 Treatment Int. CtrlEnd. Ctrl30 deg.CABA+20ABA+30 % endosperms with EBM activity 0 20 40 60 80 100 EBM activity (U) 0.0 0.1 0.2 0.3 0.4 0.5 Figure 3-9. Percentage of endosperms exhibitin g EBM activity, white bars and Y1 axis, and EBM activity levels, diamond marks and Y2 axis, in C. lanceolata endosperms. In 30 deg C, ABA+20 and ABA+30 treatments, endosperms were separated from embryos after 24 hours imbibition on respective media and were incubated for an additional 120 hours on same substrate. Intact cont rol: endosperms were excised after 144 hours incubation at 20oC in de-ionized water. End cont rol: endosperms were excised after 24 hours at 20oC in de-ionized water and re-incubat ed for another 120 hours in same substrate. Enzyme activity was measured after incubating endospe rms for a total of 144 hours under each treatment. Standard EBM activity was 1U, (1 U = EBM activity converting 1 mol of galactomannan/minute under standard conditions).
63 CHAPTER 4 OPTIMIZING GERMINATION IN COREOPSIS SEEDS 4.1 Introduction Native Coreopsis wildflowers are useful in Florida ecosy stem restoration and aesth etic plantings on roadsides and public parks. Widespread use can be hindered by low germination due to seed dormancy. In order to study dormancy in Coreopsis, various factors to alleviate dormancy need to be elucidated. Normally, germination o ccurs when seeds are provided with adequate moisture, optimal temperature, oxygen and sometimes light. Failure to germinate signifies dormancy (Bewley and Black, 1994; Baskin and Baskin, 1998, 2004). Various studies suggest that most seeds posses some level of dormancy at maturity (Finch-Savage and Leubner-Metzger, 2006; Steinbach et al., 1995, 1997) and both dormancy release and subsequent germination depend on environmental cues (Baskin and Baskin, 1978, 2004). The balance between germination and dormancy is determined by genetics, physiological status (Finch-Savage and Leubne r-Metzger, 2006; Bradford et al. 2007), and environmental factors including seed maturation environment (Sung et al. 1998; Orozco-Segovia et al., 2000; Blodner et al. 2007). Norcini et al. (2004) reported that environmental conditions during seed maturity affects seed viability and dormancy in Coreopsis lanceolata In some seeds, nitrates and cold stratification (moi st chilling) break dormancy (ISTA, 1985; AOSA, 1998), while alternating temperatures overcome dormancy in other species (Leon and Knapp, 2004). Temperature controls germination in non-dormant seeds by its inherent influence on metabolic and physiological processes (Bewley and Black, 1994; Baskin and Baskin, 1998). Information on effects of environmental factors (moisture, temperature, oxygen, light) and seed properties (genotype, physiological and physical status) is important in dormancy studies because these factors regulate germination, and germination is a method of assessing dormancy.
64 There is strong evidence that ABA imposes and maintains dormancy (Finch-Savage and Leubner-Metzger, 2006). In many species th e phytohormone ABA inhibits germination (Schopfer and Plachy, 1 984; Leon-Kloosterziel et al., 1996; Leung and Giraudat, 1998; Toorop et al. 2000; Benech-Arnold et al., 2006; Kucera et al. 2005; Finch-Savage and LeubnerMetzger, 2006; Taylor et al. 2000). In dormant seeds of barley (Jacobsen et al. 2002; Chono et al. 2006), and Chamaecyparis nootkatensis (Schmitz et al., 2000, 2002), ABA is synthesized de novo during imbibition and accumulates due to the inability of the embryo to metabolize it. Arabidopsis thaliana ABA-mutants (insensitive or defi cient) lack seed dormancy (LeonKloosterziel et al. 1996). In species where coat-imposed dormancy is involved, there is an indirect interaction between ABA an d GA in dormancy regulation (Jacobson et al. 2002; Leubner-Metzger, 2003). Gibberellic acid promotes germination (Debeaujon and Koornneef, 2000; Olszewski et al., 2002; Yamaguchi and Kamiya, 2002; Kucera et al., 2005) by stimulating cell elongation and division of embryo hypocotyls (Taiz and Zieger, 2002), and in some species, induces de novo biosynthesis of hydrolases that digest and weaken endosperm (Yamaguchi and Kamiya, 2002; Kucera et al., 2005). Exogenous application of GA can substitute for light in positively photoblastic seeds (Braun and Khan, 1975; Yoshioka et al., 1998; Yamaguchi and Kamiya, 2002). The control of light response in germination is medi ated by phytochrome photoreceptors (Loercher, 1974; Bewley and Black, 1994). P hytochrome controls germination through two inter-convertible protein conformations, termed Pfr or Pr, with Pfr being the biologically active form. Exposing seeds to red light (6 60 nm) converts the pigment to the Pfr form and irradiation with far-red light (730 nm) causes it to revert (Poppe and Schafer, 1997; Casal et al. 1997;
65 1998). There are three known types of phytochrome responses in seeds; Low Fluence Response (LFR), Very Low Fluence Response (VLFR) and High Irradiance Response (HIR) (Shinomura et al. 1994; Poppe and Schafer, 1997; Milberg et al., 2000; Casal et al., 1998; Yamaguchi and Kamiya, 2002; Leon and Owen, 2003; Yamauchi et al. 2004). Very low amounts of Pfr are sufficient to induce VLFR, whereas higher levels are required to induce LFR. The VLFR occurs under far-red light, but LFR does not. The HIR occurs under continuous high-irradiance far-red light (~715 nm). The LFR response is under contro l of the more stable phytochrome B while the labile phytochrome A mediates VLFR and HIR. Light is postulated to induce de novo GA biosynthesis in imbibed light-requiring seeds, and to enhance sensitivity of embryos to GAs (Shinomura et al., 1994; Hilhorst and Karsen, 1988; P oppe and Schafer, 1997; Yamaguchi and Kamiya, 2002; Yamauchi et al. 2004). Light is an important el ement in dormancy alleviation and germination in many seeds (Goggin et al. 2008). This study sought to identify important fact ors that might influence germination of C. basalis, C. floridana, C. lanceolata, C. leavenworthii and C. pubescens seeds. The effects of temperature, light, nitrate and cold stratificati on were examined. Additionally, effects of ABA, GA, and tetcyclacis (a GA biosynthesis inhibitor) were investigated to ga in insight into possible roles of GA and ABA in germination of Coreopsis seeds. 4.2 Materials and Methods 4.2.1 Seed Material Five Coreopsis species were studied: C. basalis (A. Dietr.) S.F. Blake, C floridana E.B. Smith, C. lanceolata L ., C. leavenworthii Torr. & A. Gray, and C. pubescens Elliot. Coreopsis basalis, C. floridana, C. leavenworthii and C. lanceolata (North Florida ecot ype) were prevariety germplasm. Coreopsis floridana and C. leavenworthii are wetland species, with C. floridana being endemic to Florida and C. leavenworthii nearly so (USDA-NRCS, 2007). Coreopsis
66 basalis and C. lanceolata are upland species but C. pubescens is a facultative wetland species, and all three are more widely distributed than the wetland species (USDA-NRCS, 2007). Except for C. basalis which was harvested from a natural stand in northern Florida, seeds of C. floridana, C. lanceolata (North Florida ecotype NF) and C. leavenworthii were harvested from cultivated populations in Florida (Norcini a nd Aldrich, 2007a). A North Carolina ecotype of C. lanceolata (NC) and a West Virginia ecotype of C. pubescens (WV) were purchased from Ernst Conservation Seeds (Meadville, PA). Two other accessions of C. lanceolata a dwarf form (DW) and a typical form (Lance Leaf type LL), were purchased from Applewood Seed Company (Arvada, CO). 4.2.2 Germination Tests A thermo-gradient table, Type db 5000 (Van dok and de Boer Machinefabriek BV, Enkhuizen, The Netherlands) was used (m inimi zes non-treatment variation) to provide temperatures of 15, 20, 25 and 30oC in continuous light or da rk. When a single constant temperature was required, an Isotemp incubator model 304R (Fisher Scien tific, Fair Lawn, NJ) was set at 20oC in continuous light or dark. Four replications of 25 seeds each were placed on double blue blotter paper (Anchor Paper Company, St. Paul, MN) moistened with de-ionized water in 5 cm glass Petri dishes. Dark was achieved by wrapping Petri dishes with alum inum foil and maintaining a dark environment in an incubator. Germination counts were ma de at 7 and 14 days. The 7-day counts for seeds germinated in dark were made under a dim-green light (25W, A19, Specialty 90912; General Electric Company, Cleveland, OH) in a dark ro om. Germination was defined as visible protrusion of radicle to at leas t 2 mm. A tetrazolium test (ISTA, 2003) was used to assess seed viability of non-germinated seeds.
67 4.2.3 Plant Growth Regulators Germination tests were conducted at 20oC, except that the blotter papers were moistened with 0 to 100 M of tetcyclacis (BASF, Florham Park, NJ), GA3 (Fluka Chemie AG, Buchs, Switzerland), GA4+7 (Plant Protection Limited, Yalding, United Kingdom), or ABA (Sigma Chemical Company, St. Louis, MO). At intermed iate counts, blotter pape rs were re-moistened with the appropriate concentration of pl ant growth regulator or tetcyclacis. 4.2.4 Cold Stratification Seeds were imbibed in the dark at 5oC (Precision incubato r model 816, Precision Scientific Group, Chicago, IL) for 7 or 14 days. Af ter the prescribed peri od, four replicates (25 seeds each) of Petri dishes were transferred to an incubator at 20oC for germination tests in light or dark. 4.2.5 Statistical Analysis Data were analyzed separately. Germination percentages were adjusted for percentage of viable seeds, arcsine-squre root transformed if necessary (to normalize data), and then analyzed; however, non-transformed means ar e presented. Analysis of variance was performed using PROC GLM (SAS Version 9.1; SAS Institute, Ca ry, NC). Treatment means were separated using the least significant difference (LSD ) test at the 5% significance level. 4.3 Results 4.3.1 Temperature and Light In all specie s, the greatest germ ination was attained at 15 and 20oC. Depending on species, germination improved in light, (Figure 4-1). In C. lanceolata the NC ecotype germination was promoted by light comp ared to the NF ecotype. However, C. floridana seeds were dormant in the dark (Figure 4-1). Wh en all 5 species were imbibed at 25 and 30oC
68 germination percentage fell and th is effect was more pronounced in dark. However, germination was maximized if seeds were returned to optimal temperatures. 4.3.2 Gibberellic Acid, ABA and Tetcyclacis Except C. floridana and C. leavenworthii where dark germination was prom oted gibberellic acid did not affect germination of any species; interestingly, GA reduced germination in C. leavenworthii seeds imbibed in light. Abscisic acid reduced germination in all species, but the reduction was more dras tic in dark (Figure 4-2). When exogenous GA concentrations were increased in dark-imbibed C. floridana seeds, germination also increased; GA3 and GA4+7 had similar efficacy in stimulating germination (Figure 4-4). In C. lanceolata (NF), neither GA3 or GA4+7 affected germination. Germination in C. floridana and C. lanceolata (NF) seeds declined as concen trations of ABA or tetcyclacis increased (Figures 4-5 and 4-6). 4.3.3 Cold Stratification Stratification at 5oC for 7 or 14 days did not affect germination of C. basalis, C. lanceolata, C. pubescens or C. lanceolata seeds in light, but there wa s a reduction in germination in dark (Figure 4-3). In C. lanceolata dark germination reduction was pronounced in the NC ecotype compared to the NF ecotype. However, cold stratification of C. floridana and C. leavenworthii seeds promoted germinati on in dark, but in light C. leavenworthii seeds had reduced germination (Figure 4-3). 4.4 Discussion Coreopsis seeds had greatest germination percentage at constant 15 or 20oC which concurs with other reports (ISTA, 1985; Carpenter and Ostmark, 1992; Banovetz and Scheiner, 1994; AOSA, 1998). Seeds that did not germ inate at elevated temperatures (30oC) germinated normally when returned to optimal temperatures, which indicates that higher temperatures
69 inhibited germination but did not kill the seeds. Coreopsis basalis, C. pubescens and four accessions of C. lanceolata did not require light for germ ination but light was vital in C. floridana and C. leavenworthii Coreopsis floridana seeds tolerated higher imbibitional temperatures in light compared to other Coreopsis species. Light requirement in C. floridana and C. leavenworthii seeds may be an ecological adaptation. Gibberellic acid improved germination in dark-imbibed C. floridana and C. leavenworthii but not other species. Both GA3 or GA4+7 had similar efficacy in promoting germination in C. floridana. In light-requiring lettuce seed s (Braun and Khan, 1975; Psaras et al. 1981; Vertucci et al., 1987; Bewley and Black, 1994; Nijsse et al. 1998; Yoshioka et al., 1998), in Bidens pilosa (Forsyth and Brown, 1982), and in othe r light-requiring seeds (Olszewski et al. 2002; Yamaguchi and Kamiya, 2002) exogenous GA promot ed germination. In endospermic seeds, such as those of Coreopsis gibberellins may stim ulate embryo growth and induce hydrolases that digest and weaken endosperm, allowing ra dicle protrusion (Yamaguchi and Kamiya, 2002; Kucera et al. 2005). Cold stratification promoted germination in dark-imbibed C. floridana and C. leavenworthii seeds but reduced germination in C. basalis, C. lanceolata and C. pubescens imbibed in dark. Cold stratificat ion reduced seed germination in C. lanceolata (Banovetz and Scheiner, 1994), Phacelia dubia (Baskin and Baskin, 1978) and Asparagus acutifolius (Conversa and Elia, 2008). However, in light-requiring lettuce seeds, cold stratification improved germination (van der Woude and Toole, 1980; Psaras, 1981; Bewley and Black, 1994; Nijsse et al. 1998). Differences in cold stratification requi rement between species might be an ecological adaptation.
70 Abscisic acid probably prevents the germination of Coreopsis seeds by antagonizing GA action, as has been shown for other species (Leubner-Metzger, 2003; Finch-Savage and LeubnerMetzger, 2006). Abscisic acid biosynthesis i nhibitors alleviated dor mancy in lettuce seeds (Roth-Bejerano et al. 1999). Tetcyclacis inhibited germination by impairing de novo GA biosynthesis (Debeaujon and Koornneef, 2000). Data show that factors impeding GA activity in Coreopsis seeds inhibit germinati on (Figures 4-5 and 4-6). This study identified factors influencing germination in Coreopsis species, and there is apparent inter-specific variation related to natural ecological habitats (Paterson et al. 1976; Leon et al. 2006). In addition to si milar temperature optimum for germination (15 or 20oC) in all Coreopsis species, light, GA and cold stratification promoted germination only in the wetland species C. floridana and C. leavenworthii. This information is important to Coreopsis seeds end users (for example, nursery personnel) because it enables them to optimize germination, or use these methods as pre-germination seed trea tments to promote germination and improve subsequent seedling recovery. 4.5 Summary This study sought to optimize fact ors influencing germ ination in C. basalis, C. floridana, C. lanceolata, C. leavenworthii and C. pubescens to facilitate dormancy studies. Germination effects of temperatures (15, 20, 25 and 30oC), differing light regimes (continuous white light or dark), GA and two pre-germination cold stratification treatments (5oC for 7 or 14 days) were examined. Additionally, effects of ABA and tetcyc lacis were examined to evaluate germination response. Optimum germination temperature range in light or dark in all species was 15 to 20oC. Coreopsis basalis, C. pubescens and four accessions of C. lanceolata did not require light for germination but for C. floridana and C. leavenworthii, light was essential. Cold stratification reduced total germination in C. basalis, C. pubescens and four accessions of C. lanceolata but
71 improved germination in dark-imbibed C. floridana and C. leavenworthii seeds. Gibberellic acid (100 M GA3) did not promote germination in C. basalis, C. pubescens and C. lanceolata but improved germination in dark-imbibed C. floridana and C. leavenworthii seeds. Abscisic acid and tetcyclacis inhibited germination in all spec ies. Factors important in germination of the wetland species C. floridana and C. leavenworthii were similar, and likewise in the upland species C. basalis and C. lanceolata, and the facultative species C. pubescens.
72 A LSD=17 Temperature ( o C) 0 1 52 02 53 0 Germination % 0 20 40 60 80 100 B LSD=16 Temperature ( o C) 015 20 25 30 Germination % 0 20 40 60 80 100 Figure 4-1. Effect of temp erature on germination of Coreopsis seeds in light (broken lines) and dark (solid lines). A) C basalis. B) C. floridana C) C. leavenworthii D) C. pubescens. E) C. lanceolata North Florida ecotype. F) C. lanceolata North Carolina ecotype. G) C. lanceolata dwarf type. H) C. lanceolata lanceleaf type.
73 C LSD=16 Temperature ( o C) 0 1 52 02 53 0 Germination % 0 20 40 60 80 100 D LSD=17 Temperature ( o C) 0 1 52 02 53 0 Germination % 0 20 40 60 80 100 Figure 4-1. Continued
74 E LSD=12 Temperature ( o C) 0 1 52 02 53 0 Germination % 0 20 40 60 80 100 F LSD=9 Temperature ( o C) 0 1 52 02 53 0 Germination % 0 20 40 60 80 100 Figure 4-1. Continued
75 G LSD=13 Temperature ( o C) 0 1 52 02 53 0 Germination % 0 20 40 60 80 100 H LSD=10 Temperature ( o C) 0 1 52 02 53 0 Germination % 0 20 40 60 80 100 Figure 4-1. Continued
76 A LSD=12 Germination conditions Control GA ABA Germination % 0 20 40 60 80 100 B LSD=5 Germination conditions Control GA ABA Germination % 0 20 40 60 80 100 Figure 4-2. Influence of 100 M GA or ABA on germination of Coreopsis species at constant 20oC in light (white bars) and dark (black bars). A) C basalis. B) C. floridana. C) C. leavenworthii. D) C. pubescens E) C. lanceolata North Florida ecotype. F) C. lanceolata North Carolina ecotype. G) C. lanceolata dwarf type. H) C. lanceolata lanceleaf type.
77 C LSD=16 Germination conditions Control GA ABA Germination % 0 20 40 60 80 100 D LSD=22 Germination conditions Control GA ABA Germination % 0 20 40 60 80 100 Figure 4-2. Continued
78 E LSD=8 Germination conditions Control GA ABA Germination % 0 20 40 60 80 100 F LSD=8 Germination conditions Control GA ABA Germination % 0 20 40 60 80 100 Figure 4-2. Continued
79 G LSD=10 Germination conditions Control GA ABA Germination % 0 20 40 60 80 100 H LSD=6 Germination conditions Control GA ABA Germination % 0 20 40 60 80 100 Figure 4-2. Continued
80 A LSD=8 Cold stratification duration (days) 071 4 Germination % 0 20 40 60 80 100 B LSD=6 Cold stratification duration (days) 071 4 Germination % 0 20 40 60 80 100 Figure 4-3. Germination of various Coreopsis species at constant 20oC in light (white bars) and dark (black bars) after dark, cold (5oC) stratification. A) C basalis. B) C. floridana C) C. leavenworthii D) C. pubescens E) C. lanceolata North Florida ecotype. F) C. lanceolata North Carolina ecotype. G) C. lanceolata dwarf type. H) C. lanceolata lanceleaf type.
81 C LSD=16 Cold stratification duration (days) 071 4 Germination % 0 20 40 60 80 100 D LSD=16 Cold stratification duration (days) 071 4 Germination % 0 20 40 60 80 100 Figure 4-3. Continued
82 E LSD=5 Cold stratification duration (days) 071 4 Germination % 0 20 40 60 80 100 F LSD=10 Cold stratification duration (days) 071 4 Germination % 0 20 40 60 80 100 Figure 4-3. Continued
83 G LSD=9 Cold stratification duration (days) 071 4 Germination % 0 20 40 60 80 100 H LSD=9 Cold stratification duration (days) 071 4 Germination % 0 20 40 60 80 100 Figure 4-3. Continued
84 GA Concentration ( M) 020406080100 Germination % 0 20 40 60 80 100 CF GA3 CL GA3 CF GA4+7 CL GA4+7 Figure 4-4. Response of Coreopsis species seeds to GA3 or GA4+7. Germination tests were conducted in dark at 20oC. CF, C. floridana (LSD=14); CL, C. lanceolata (LSD=11). Means comparison was within species. ABA Concentration ( M) 020406080100 Germination % 0 20 40 60 80 100 CF CL Figure 4-5. Response of Coreopsis seeds to ABA. Germination tests were conducted under white fluorescent light at 20oC. CF, C. floridana (LSD=13); CL, C. lanceolata (LSD=6). Means were compared within each species.
85 Tetcyclacis concentration ( M) 020406080100 Germination % 0 20 40 60 80 100 CF Dark CF Light CL Dark CL Light Figure 4-6. Response of Coreopsis seeds to tetcyclacis, a GA bios ynthesis inhibitor, (inhibits the oxidation of ent -kaurene to ent -kaurenol in the GA biosynthesis pathway). Germination tests were conducted unde r white light or in dark at 20oC. CF, C. floridana (LSD=8); CL, C. lanceolata (LSD=9). Means comparison was within species.
86 CHAPTER 5 AFTERRIPENING ALLEVIATES DORMANCY IN FRESH COREOPSIS SEEDS 5.1 Introduction Indigenous Coreopsis sp ecies are important in Florid a for ecological restoration and aesthetic reasons. However, large sowing ac tivities can be hampered by poor germination because of dormant seeds. Past research identifie d afterripening as one of the factors influencing dormancy (Kabat, 2004; Norcini et al., 2006; Kabat et al. 2007; Norcini and Aldrich, 2007a), but other factors such as embryo envelopes were not examined. Afterripening (dry) is a natural process that occurs in dry seeds, which allows seeds to germinate after a certain period from physiological maturity (Widrlechner, 2006). The process, which can take a few weeks to years depending on species, occurs below a certain seed moisture content and is delayed when seeds are too dry; it is accelerated by high temperat ures and elevated oxygen levels (Baskin and Baskin, 1976, 1998; Bewley and Black, 1994; Steadman et al. 2003). Dry afterripening is associated with non-enzymatic biochemical reactions among carbohydr ates (sugars) and proteins, called Maillard and Amadori react ions (Wettlaufer and Leopold, 1991; Berma-Lugo and Leopold 1992; Murthy et al. 2000, 2003). Afterripening in Asteraceae occurs at seed moisture contents of 5 to 12% (Schutz et al. 2002). Prediction of afterripening duration is confounded by initial dormancy level since seed lots of one species stored under the same conditions lose dormancy at different rates, and require distinct dormancy breaking treatments (Steadman et al., 2003; Benech-Arnold et al., 1999; Taylor et al. 2000; Ramagosa et al. 2001; Chono et al. 2006). Temperature controls germination through its in herent regulation of me tabolic rates. The degree of afterripening determines the temperatur e range at which germination occurs (Leon and Knapp, 2004). Non-dormant seeds generally germ inate in a wide temperature range (Bewley
87 and Black, 1994; Baskin a nd Baskin, 1998; Pritchard et al. 1999), but germination is inhibited above or below certain limits. Germinati on failure at supraoptimal temperatures (thermoinhibition) is associated with de novo abscisic acid (ABA) biosynthesis in lettuce (Gonai et al 2004) and Arabidopsis (Toh et al 2008) seeds. Abscisic acid is known to impose and maintain dormancy (Finch-Savage and Leubne r-Metzger, 2006). Fluridone, an ABA biosynthesis inhibitor, generally increases the high limit temperature for germination (Yoshioka et al., 1998). In many seeds, light regulates germ ination (Loercher, 1974; Shinomura et al., 1994; Poppe and Schafer, 1997; Milberg et al., 2000; Yamaguchi et al. 2004; Leon and Owen, 2003). Lettuce seeds respond to light at seed mo isture content from 4-32% (Vertucci et al., 1987). Gibberellins have been used to substitute for light in dark-imbibed, light-requiring seeds (Braun and Khan, 1975; Hilhorst and Karsen, 1988; Yoshioka et al., 1998; Yamaguchi and Kamiya, 2002; Yamaguchi et al 2004; Mollard et al. 2007). Cold stratifica tion is associated with enhanced de novo GA biosynthesis and sensitivity, and substitutes for light requirement in positively photoblastic lettuce genotypes (Lewak and Khan, 1977; van der Woude and Toole, 1980). In many species nitrate breaks dormanc y (ISTA, 1985; Hilhorst and Karsen, 1988; AOSA, 1998; Alboresi et al., 2005); Giba et al. (2003) reported that nitr ate action is enhanced by light. Nitrates break down and donate nitric oxide (NO), a signaling molecule that plays an important role in seed dormancy loss (Bethke et al., 2006a; 2006b). Dormancy alleviation through afterripening c ould be due to an acquired capacity to degrade ABA during imbibition (Romagosa et al. 2001; Debeaujon and Koornneef, 2000; Olszewski et al., 2002; Yamaguchi and Ka miya, 2002; Yamaguchi et al 2004; Kucera et al., 2005). ABA biosynthesis in imbibe d seeds is associated with nonafterripened seeds (Schopfer
88 and Plachy, 1984, 1985; Leon-Kloosterziel et al., 1996; Leung and Giraudat, 1998; Toorop et al. 2000; Schmitz et al. 2002; Leubner-Metzger 2003; Benech-Arnold et al., 1999, 2006; Kucera et al. 2005; Finch-Savage and LeubnerMetzger, 2006). Abscisic ac id specifically inhibits endosperm rupture in most seeds as observed in lettuce (Argyris et al. 2008), Lepidium sativum and Arabidopsis thaliana (Muller et al., 2006). Embryo envelopes are effective in imposing dormancy (Jones, 1974; Watkins and Cantliffe 1983; Groot and Karsen, 1992; Welbaum et al. 1998; Sung et al., 1998; Finch-Savage a nd Leubner-Metzger, 2006). Although dormancy in native Coreopsis seeds can be partly modulated by dry afterripening (Norcini and Aldrich, 2007a), other abiotic f actors are involved as well (Kabat et al. 2007). To elucidate the roles of these factor s, effects of temperat ure, light regimen, GA, nitrate, cold stratification and embryo e nvelopes on dormancy were examined during afterripening under dry conditions. 5.2 Materials and Methods 5.2.1 Plant Material Previously described seed lots of C. floridana and C. lanceolata (Chapter 3, section 220.127.116.11) were used. Wh en seeds were stored at 10oC and 50% relative humidity seed moisture content ranged from 7 to 9% in both species. Experiments with C. lanceolata seeds began on 20 June 2007, and with C. floridana, on 1 December 2007. Germination te sts were carried out biweekly in each species for 8 weeks in C. floridana and 12 weeks in C. lanceolata Cold stratification and potassium-nitrate dormancy-breaking experiments were conducted after 60 days of dry storage of each species. 5.2.2 Germination Tests A thermo-gradient table, Type db 5000 (Van dok and de Boer Machinefabriek BV, Enkhuizen, The Netherlands) provided tem peratures of 10, 15, 20, 25 and 30oC in continuous
89 light or dark. An Isotemp in cubator model 304R (Fis her Scientific, Fair Lawn, NJ) set at 20oC in continuous light or dark wa s used for other assays. Four replications of 25 seeds each were placed on two blue blotter papers (Anchor Paper Company, St. Paul, MN) moistened with de-ionized water in 5 cm glass Petri dishes. Dark conditions were achieved by wrappi ng Petri dishes with aluminum foil. Number of germinated seeds (radicle protrusion of 2 mm or greater) were recorded at 7 and 14 days. Intermediate darkgermination counts were made under a dim-gr een light (25W, A19, Specialty 90912; General Electric Company, Cleveland, OH). 5.2.3 Gibberellic Acid and Potassium Nitrate Except for moistening blotter papers with 100 M GA4+7 (Plant Protection Limited, Yalding, United Kingdom) or KNO3 (Fisher Scientific Company, Fair Lawn, NJ) at 1, 10, 50 and 100 mM, germination tests were conducted at 20oC as described previously. At intermediate counts, the blotter papers were re-moistened with GA or the appropria te nitrate solution. 5.2.4 Cold Stratification Seeds were sown in Petri dishes as in dark germination tests described previously, and placed in dark at 5oC in a Precision incubator (model 816; Precision Scientific Group, Chicago, IL). Four replicates of 25 seed s each were then transferred to 20oC in light or dark for germination tests. Germination tests were conducted every 7 days up to 42 days for C. floridana and up to 28 days for C. lanceolata Germination evaluations we re performed as described previously. 5.2.5 Removal of Seed Coverings Pericarps, testa and endosperms of C. floridana and C. lanceolata seeds imbibed for 24 hours were excised from embryos using a dissecting microscope (Model, ASZ37L3, Bausch and
90 Lomb, Rochester, NY) and tweezers. Fifteen em bryos per replicate and three replicates per treatment were used in germination tests. Intact seeds were used as controls. 5.2.6 Data Analysis Data from e ach species was analyzed separate ly. Germination percentages were adjusted for viability based on pre-germination TZ tests. This correction was done for C. lanceolata only because C. floridana had 100% viable seeds. If necessary, an arcsine-square root transformation was done. Corrected data were analyzed usi ng PROC GLM (SAS Version 9.1; SAS Institute, Cary, NC). Treatment means were separated usi ng the least significant difference (LSD) test at = 0.05. 5.3 Results 5.3.1 Temperature and Light At harvest, C. floridana seed germination in light was 100% at 10 to 25oC and declined to 83% at 30oC. In dark it was consis tently about 20% at 10 to 20oC, dropped to about 10% at 25oC before it further declined to about 5% at 30oC (Figure 5-1). In C. lanceolata seeds incubated at 10 to 25oC, germination progressively increased with increasing dry storage duration at each sampling tim e (Figure 5-2). Until seeds were totally nondormant, that is fully afterripened, germination in light was greater than in dark. Germination was greatest at 15 and 20oC and peak germination (99%) in intact seeds was attained after 150 days of afterripening (Figures 5-3 and 5-4). At 30oC, germination was low in light and dark at all sampling times regardless of the degree of afterripening (Fi gures 5-3 and 5-4). 5.3.2 Gibberellic Acid, Potassium Nitr ate and Cold Stratification In intact dark-imbibed C. floridana seeds, GA totally overcame dormancy. In C. lanceolata, GA only marginally alleviated dormancy in light or dark (Figure 5-2). Potassium nitrate did not alleviate dormancy in dark-imbibed, 60-day afterripened C. floridana seeds, but
91 10 mM potassium nitrate promoted germin ation from 43 to 61% in light-imbibed C. lanceolata seeds (Figure 5-5); but 50 and 100 mM KNO3 inhibited germination. Forty-two days of cold stratification in 60-day afterripened C. floridana seeds promoted germination from 20 to 60% in dark (Figure 5-6), but in C. lanceolata cold stratification reduced germination in dark-imbibed, 60-day afterripened seeds (Figure 5-7). 5.3.3 Germination After Removal of Embryo Coverings In C. floridana seeds imbibed and incubated in the dark, intact seeds had 20% germination, seeds without pericarp 50% germination, and naked embryos 100% germination (Figure 5-8); however in light, a ll three seed treatments had 100% germination. Germination in dark-imbibed C. floridana seeds did not improve as the afterr ipening period increased. After 30 days afterripening, C. lanceolata intact seeds had 26% germinat ion in dark and 65% in light, seeds without pericarp germinated 37% in dark and 67% in light, and excised embryos had 90% germination in dark and 97% in light. Seeds of C. lanceolata imbibed intact lost dormancy after 150 days of afterripening, when pericarp plus testa were removed seeds lost dormancy in 90 days, whereas naked embryos were virtually non-dormant at harvest (Figure 5-9). 5.4 Discussion When im bibed in light or dark, C. lanceolata seeds required 150 days dry afterripening for complete dormancy loss. Coreopsis lanceolata seeds afterripened in 180 days (Norcini and Aldrich, 2007a), and a similar period was reported for C. basalis (Carpenter and Ostmark, 1992). Differences in afterripening duration within a species could be caused by environmental conditions during seed maturation, storage conditi ons and genetics (El-Keblawy and Al-Ansari, 2000; Leubner-Metzger, 2002; Steadman et al., 2003; Cristaudo et al. 2007; Tarasoff et al. 2007). Termination of dormancy was coincident w ith a loss of response to GA and light (Figure 5-2). Light effects on dormancy relief were dependent on afterripening period in Polygonum
92 aviculare seeds (Batlla and Benech-Arnold, 2005). Af terripening conditions used in this study were similar to those used by Shutz et al. (2002) in four Asteraceae species (Millotia myosotidifolia, Podotheca gnaphalioides, P. chrysantha and Ursinia anthemoides ), and by Norcini and Aldrich (2007a) in C. basalis, C. floridana, C. lanceolata and C. leavenworthii seeds. Afterripening alleviates dormancy in other Asteraceae such as Artermisia tridentate (Meyer et al., 1990) and Tagetes minuta (Karlson et al. 2008). Potassium nitrate (10 mM) diminished dorma ncy in partially afte rripened (60 days afterripening) C. lanceolata seeds imbibed in light (Figure 5-5) Similarly, nitrates alleviated dormancy in Arabidopsis but the response to nitrate and light was dependent on afterripening duration (Giba et al. 2003; Alboresi et al. 2005; Ali-Rachedi et al. 2004; Finch-Savage et al. 2007). Although nitrate often alleviates dorma ncy (ISTA, 1985; AOSA, 1998), it only partially did so in non-afterripened C. lanceolata seeds. In the natural environment, nitrate sensing by seeds could be an adaptation for detecting wa ter availability and/or soil fertility (Giba et al. 2003). Since C. lanceolata is a colonizer, soil water and nutrient availability may indicate gaps in vegetation to insure seedling survival and rapid plant establishment in a competition-free microsite. Cold stratification reduced germ ination in partially afterrip ened (60 days afterripening) C. lanceolata seeds (Figure 5-7). In pr evious experiments (Chapter 4), similar behavior was observed in seeds of four C. lanceolata accessions, C. basalis and C. pubescens Cold stratification at 5oC induced secondary dormancy in northern USA accessions of C. lanceolata seeds (Banovetz and Scheiner, 1994). Inhibiti on of germination under cold conditions is a probable mechanism to avoid germination during cold winters which are like ly not favourable to plant establishment.
93 Removal of the pericarp, testa and endosperm in C. floridana seeds alleviated dormancy in dark. Intact seeds exhibite d no dormancy in light (100% ge rmination). In light-requiring lettuce genotypes, endosperm imposed dormancy in dark (Jones, 1974; Psaras et al., 1981; Nijsse et al. 1998), and endosperm enhanced dormancy in many Asteraceae (Leubner-Metzger, 2003; Finch-Savage and Leubner-Met zger, 2006). The most freque nt ways by which primary dormancy is imposed are by the seed coat (physical) and embryo ( physiological) dormancy (Baskin et al. 2003; Leubner-Metzger, 2003; 2005; Ali-Rachedi et al. 2004; Muller et al., 2006; Donohue et al., 2008). In C. floridana and C. lanceolata seeds imbibed at supraoptimal temperatures the endosperm imposed dormancy by acting as a physical barrier to radicle protrusion (Chapter 3). In many Asteraceae species, dormancy is alleviated by light (Karlson et al. 2008). Although initial germin ation in dark-imbibed excised embryos was high, slight afterripening effects were obser ved (Figure 5-8), and this was consistent with reported seed behavior in C. floridana (Norcini and Aldrich, 2007a). Exogenous GA relieved dormancy of dark-imbibed C. floridana seeds. Likewise GA relieved dormancy in light-requiring lett uce seeds (Braun and Khan, 1975; Psaras et al. 1981; Vertucci et al., 1987; Nijsse et al. 1998; Yoshioka et al., 1998). In light-requiring species, light is postulated to overcome dormancy by stimulating de novo GA biosynthesis (Shinomura et al. 1994; Poppe and Schafer, 1997; Milberg et al., 2000; Yamaguchi et al. 2004; Leon and Owen, 2003) and enhancing embryo sensitivity to GA (Hilhorst and Karsen, 1988; Yamaguchi and Kamiya, 2002; Roth-Bejerano et al. 1999; Leubner-Metzger, 2003). Cold stratification of C. floridana seeds partially relieved dor mancy when germinated in dark (Figure 5-6). Cold stratificatio n also alleviated dormancy in other Asteraceae species, Guizotia scabra, Parthenium hysterophorus and Verbesina encelioides (Karlson et al., 2008), in
94 Arabidopsis thaliana (Ali-Rachedi et al. 2004; Finch-Savage et al. 2007), as well as other genera (Noronha et al., 1997; Leon and Owen, 2003; Leon et al., 2006; 2007). Lightrequirement for germination in some lettuce genotypes could be substituted for by cold stratification (Lewak and Khan, 1977; van der Woude and Toole, 1980). Coreopsis floridana flowers and produces seeds in fall, and ecologica lly, cold stratification is likely to promote germination when soil temperatures rise in spring as occurs with temperate species (Bewley and Black, 1994). In C. floridana seeds, the extent of dormancy a lleviation is dependent on cold stratification duration. Potassium nitrate did not relieve dormancy of dark-imbibed C. floridana seeds. Nitrate is used to break dormancy in seeds of several species (ISTA, 1985; Hilhorst and Karsen, 1988; AOSA, 1998; Giba et al., 2003; Alboresi et al. 2005). Nitric oxide alleviated dormancy in lightrequiring dark-imbibed lettuce seeds (Beligni an d Lamattina, 2000). Nitrates break dormancy in Arabidopsis thaliana (Alboresi et al. 2005; Bethke et al ., 2006a, 2007) seeds probably by donating the NO molecule to the ni tric oxide pathway (Javanovic et al ., 2005; Bethke et al ., 2006a, 2006b, 2007). The disparity in pot assium nitrate effects between C. floridana and C. lanceolata may be attributed to natural habit differences. Coreopsis floridana seeds lack responsiveness to nitrate probably because water and nutrients are not likely to be limiting in their wetland habitat. Being an upland colonizer species, C. lanceolata seeds might be sensitive to nitrate as a means to detect gaps in vegeta tion where water and nutrients would not be limiting for seedling establishment. Optimal germination temperatures were 15 and 20oC in fresh and afterripened C. floridana and C. lanceolata seeds (ISTA, 1985; Carpenter and Ostmark, 1992; Banovetz and Scheiner, 1994; AOSA 1998) Germination of C. floridana seeds at elevated temperatures
95 (30oC) was promoted by light (Figure 5-1), but in C. lanceolata (at 25oC), afterripening duration dictated germination response (Figures 5-2, 5-3 a nd 5-4). Light-requiring seeds tolerate higher imbibition temperatures (Taylorson and Hendricks 1972; Shinomura et al., 1994; Poppe and Schafer, 1997; Steadman, 2004). In earlier studies with Coreopsis seeds (Chapter 3), results suggested that pericarp, testa a nd endosperm inhibited germination at supraoptimal temperatures. Reduced germination at supraoptimal temperatures was associated with endosperm resistance in seeds of lettuce (Cantliffe et al. 1984; Abeles, 1986; Sung et al 1998; Nascimento et al 2001; Leubner-Metzger, 2003; Gonai et al. 2004) and Arabidopsis thaliana (Yoshioka et al., 1998; Ramagosa et al., 2001; Tamura et al. 2006; Toh et al. 2008). Although the two species had a common optima, germination at elevated temperat ures seems to be species dependent and could be an ecological adaptation because C. floridana is a wetland and C. lanceolata an upland species. Disparities in seed dormancy behavior between C. floridana and C. lanceolata might be related to differences in natural ecological habitats. In C. floridana, like in most wetland species, soil disturbance and exposure of seeds to adequate light is necessary for germination (Kettenring et al ., 2006). Coreopsis lanceolata is an upland species that flowers in spring but seeds germinate in late summer and fall when soil temp eratures are low, thus seeds may afterripen in the warm summer soil temperatures. Germinati on at optimal and elevated temperatures in C. floridana seeds is regulated by light, but in C. lanceolata, this is modulated by afterripening duration. Afterripening requirement of C. lanceolata seeds has been reported under different storage conditions (Norcini and Al drich, 2007a), but this study dem onstrated that these seeds can also be afterripened under low temperatures (10oC) at a seed moisture co ntent of 8.1%. Removal of pericarp, testa and endospe rm relieves dormancy in C. floridana and C. lanceolata seeds,
96 indicating that there is potent ial to manipulate embryo covers to overcome dormancy. Seed producers should have prior knowledge of afterripening requirements for Coreopsis seeds to enable them to devise marketing plans a nd deliver non-dormant seeds to end users. 5.5 Summary Effects of temperature a nd light, embryo envelopes, potassium nitrate (KNO3), gibberellic acid (GA4+7) and cold stratification were examined to determine influence on dormancy alleviation during afterripening (dry st orage). Seeds were produced in a greenhouse, harvested and bulked over 4 weeks, dried to 8.7% ( C. floridana ) and 8.1% ( C. lanceolata ) moisture content and stored in moistu re-proof plastic bags in dark at 10oC. Freshly harvested C. floridana seeds germinated at 100% in light but only 20% germinated in dark. Gibberellic acid overcame dormancy in dark-imbibed seeds. Cold stratification for 42 days at 5oC partially overcame (60% germination) dormancy in dark-inc ubated seeds. Potassium nitrate did not affect germination in C. floridana seeds. Removal of pericarp in C. floridana led to 50% germination while naked embryos germinated 100% in the dark. In intact seeds of C. lanceolata afterripening (dry storage) for 150 days reli eved dormancy. Removal of endosperm overcame dormancy in fresh C. lanceolata seeds. Potassium nitrate (1 0 mM) partially overcame dormancy in 60-day afterripened C. lanceolata seeds, germination increasing to 61% from 43%. In C. lanceolata afterripened seeds germinated normally and light or GA did not substitute for afterripening, whereas cold stratification enhanc ed dormancy. Light is required to break dormancy in C. floridana while in C. lanceolata, dry afterripening is paramount before germination can be optimized.
97 A LSD=8 Temperature ( o C) 01 01 52 02 53 0 Germination % 0 20 40 60 80 100 B LSD=5 Temperature ( o C) 01 01 52 02 53 0 Germination % 0 20 40 60 80 100 Figure 5-1. Effects of five incubation temper atures at two dry afterripening periods on germination in C. floridana seeds in light (broken lines) and dark (solid lines). A) At zero days afterripening. B) At 75 days afterripening. LSDs are as displayed in each graph.
98 Afterripening period (days) 0406080100120140160 Germination % 0 20 40 60 80 100 Ctrl+D Ctrl+L GA+D GA+L Figure 5-2. Progressi ve dormancy loss with increasi ng dry afterripening duration in C. lanceolata seeds. Seeds were imbibed in light or dark in water (control) or on GA substrate in light or dark. Germ ination tests were conducted at 20oC. L, light; D, dark; Ctrl, control; GA, gibberellic acid. LSD=12.
99 A LSD=10 Afterripening period (days) 0406080100120140160 Germination % 0 20 40 60 80 100 B LSD=13 Afterripening period (days) 0406080100120140160 Germination % 0 20 40 60 80 100 Figure 5-3. Effect of dry afterripening duration and imbibition temperature on germination in C. lanceolata seeds in light (broken lines) or dark (solid lines). A) 10oC. B) 15oC. C) 20oC. D) 25oC. E) 30oC. LSDs are displayed in each graph.
100 C LSD=11 Afterripening period (days) 0406080100120140160 Germination % 0 20 40 60 80 100 D LSD=12 Afterripening period (days) 0406080100120140160 Germination % 0 20 40 60 80 100 Figure 5-3. Continued
101 E LSD=5 Afterripening period (days) 0406080100120140160 Germination % 0 20 40 60 80 100 Figure 5-3. Continued
102 A LSD=11 Temperature ( o C) 01 01 52 02 53 0 Germination % 0 20 40 60 80 100 B LSD=8 Temperature (oC) 01 01 52 02 53 0 Germination % 0 20 40 60 80 100 Figure 5-4. Influence of various incubation temperatures on dor mancy at two dry afterripening periods in C. lanceolata seeds imbibed in light (broken line s) or dark (solid lines). A) After 30 days, and B) 150 days afterripening. LSDs are shown in each graph.
103 KNO 3 Concentration (mM) 020406080100 Germination % 0 20 40 60 80 100 Figure 5-5. The effect of incr easing concentrations of KNO3 on dormancy relief in 60-day dry afterripened C. lanceolata seeds in light (broken line) and dark (solid line). Germination tests were conducted at 20oC. Non-germinated seeds were dormant. LSD = 10. Chilling period (days) 01 02 03 04 0 Germination % 0 20 40 60 80 100 No-CS+D CS+D Figure 5-6. Germination respons e of 60-day dry afterripened C. floridana seeds after various cold stratification (5oC) durations in dark. Cold stratified and non-cold stratified seeds had 100% germination in light. CS cold stratification; No-CS, no cold stratification; D, dark. LSD = 13.
104 Chilling duration (days) 051015202530 Germination % 0 20 40 60 80 100 Control+D Control+L Chilling+D Figure 5-7. Cold stratification (5oC) effects on 60-day dry afterripened C. lanceolata seeds. Germination of non-chilled seeds in light (C ontrol+L) or dark (C ontrol+D); or chilled seeds in dark (Chilling+D). In chilled seeds incubated in light, total germination after 14 days was similar to light control but germination was slower. LSD = 11.
105 Afterripening period (days) 02 04 06 08 0 Germination % 0 20 40 60 80 100 E+D S-P+D S+D Figure 5-8. Influence of increasing dry afterripening duration on germination (at 20oC) of intact C. floridana seeds in dark; or when pericarp was removed in dark; or when naked embryos were imbibed in dark. In light, al l treatments germinated to 100%. D, dark; S, intact seeds; P, perica rp, E, naked embryo. LSD = 14.
106 Afterripening period (days) 040 60 80 100 120 Germination % 0 20 40 60 80 100 E+D E+L S-P+D S-P+L S+D S+L Figure 5-9. Influence of embryo envelopes on C. lanceolata seed germination at increasing dry afterripening periods of inta ct seeds imbibed in light or dark; or when pericarp was removed in light or dark; and in naked embr yos in light or dark. Germination tests were conducted at 20oC. L, light; D, dark; S, intact seeds; P, pericarp, E, naked embryo. LSD = 15.
107 CHAPTER 6 SEED PRIMING ALLEVIATES DORMANCY IN Coreopsis floridana AND PERM ITS GERMINATION AT HIGH TEMPERATURES IN C. floridana AND C. lanceolata 6.1 Introduction Coreopsis floridana and C. lanceolata are important native w ildflowers used in Florida for ecological restoration, reclamation, and beautification of roadsides an d public parks. Some seeds are expensive if used in large quantities for roadside restoration ($110 and $220 per kilogram; Norcini and Aldrich, 2008), increasin g seeding rates to achieve acceptable field establishment will increase costs for public use areas. Seeds of these native species remain dormant under many environmental conditions (N orcini and Aldrich, 2007; 2008) and germinate poorly at supraoptimal temperatures (thermoinhi bition) found in the southeastern USA resulting in reduced stand establishment. In C. floridana, light overcomes dormancy and in C. lanceolata dry afterripening for 5 to 6 months is often re quired (Norcini and Aldr ich, 2007a; Chapter 5). Optimal germination temperatures are 15 to 20oC for both species (ISTA, 1985; Banovetz and Scheiner, 1994; AOSA, 1998). Daily soil te mperatures at 10 cm depth exceed 30oC during the summer in Florida (FAWN, 2008), which means that soil temperatures in the seed zone are likely much higher. Seed priming is a technology that improve s germination uniformity, seedling vigor and stand establishment under a variety of field e nvironments (Cantliffe, 1981). Priming regulates seed hydration to permit pre-germinative biological activity, but stops prior to radicle emergence. Seeds are primed in various liquids (osmoprim ing or osmoconditioning) or in hydrated inert solid-matrix materials (solid matrix priming [S MP] or matriconditioning) (Cantliffe, 2003). Use of priming technology to mitigate dormancy and/or promote germination at elevated temperatures facilitates effici ent sowing programs in restoration projects (Walmsley and Davy, 1997). While priming could increase costs pe r seed, stand establishment under diverse
108 environments is maximized and thus total seed costs many times are reduced (Cantliffe, 1981; Cantliffe et al ., 1984; Cantliffe and Abebe, 1993; Cantliffe, 2003). From anatomical studies, C. floridana and C. lanceolata seeds consist of an outer dry pericarp, testa and a single ce ll-layer of endosperm surrounding an embryo (Chapter 3). Lettuce seeds have similar anatomy (Borthwick and Robbins, 1928; Jones, 1974; Psaras et al 1981; Nijsse et al ., 1998). In previous studies (C hapter 3), thermoinhibition in C. floridana and C. lanceolata seeds was alleviated by removal of endospe rm. Additionally, rem oval of these same tissues relieved dormancy in dark-imbibed C. floridana seeds. Lettuce (same family as Coreopsis ) seeds exhibited thermoinhibition (Cantliffe et al ., 1984; Abeles, 1986; Sung et al 1998; Nascimento et al 2000; 2001, 2005; Leubner-Metzger, 2003; Gonai et al. 2004), and endosperm removal relieved this problem (Guedes et al ., 1981; Psaras et al., 1981; Dutta et al., 1997; Sung et al., 1998; Nonogaki and Morohashi, 1999; Leubner-Metzger, 2003; Nascimento et al 2005). Endosperm removal alleviates dormanc y in dark-imbibed light-requiring lettuce seeds (Jones, 1974; Psaras et al., 1981; Nijsse et al ., 1998). Priming overcame thermoinhibition in seeds of lettuce (Guedes and Cantliffe, 1980; Cantliffe et al ., 1984; Cantliffe, 1991; Sung et al ., 1998; Nascimento and Cantliffe, 1998; Nascimento et al 2000, 2001; Nascimento, 2003), and Tagetes minuta (Taylor, 2007). Priming induces endos perm weakening in lettuce seeds (Sung et al 1998; Nascimento et al 2000; 2001, 2005), before irreversib le cell elongation and division occur (Cantliffe et al ., 1984). Since removal of tissues enveloping the em bryo is not economically practical, seed priming techniques that weaken embryo envelopes and/or promote embryo growth might be utilized to overcome dormancy and hasten germination of Coreopsis seeds. This study explored
109 the possible beneficial effects of osmopriming and SMP technique s in alleviating dormancy of C. floridana and for circumventing thermoinhibition of both C. floridana and C. lanceolata seeds. 6.2 Materials and Methods 6.2.1 Plant Material The same seed lots of C. floridana and C. lanceolata as described in Chapter 5, section 5.2.1 were used. Seed priming experiments for both species were conducted in May and June 2008. 6.2.2 Osmotic Priming Protocol Seed samp les (0.5 g) were primed in 30 ml of priming solution in 50 ml test tubes in a 30% (wt:vol) aerated solution of polyethylene glycol 8000 (PEG) (F isher Scientific, Fair Lawn, NJ) (-1.2 Mpa) for 3, 4, 5, 6 or 7 days at 15C w ith constant cool white fluorescent light (10 molm-2s-1). Seeds were primed in PEG only, PEG plus gibberellic acid (GA4+7) (Plant Protection Limited, Yalding, United Kingdom), or PEG plus 6-benzyladenine (BA) (Sigma Chemical Company, St. Louis, MO). Gibberellic acid and BA were added at 100 mg/liter PEG priming solution. A Tetratec aquarium pump (T etra Sales, Blacksburg, VA) provided aeration. Air from the aquarium pump was hydrated by bu bbling through de-ionized water to minimize evaporation from the priming solution. Primed seeds were washed thoroughly in de-ionized water, damp dried on paper towel, and placed on raised wire-gauze in a container for further drying at 15C and 50% RH for 3 days. 6.2.3 Solid Matrix Priming (SMP) Protocol Emathlite clay (0.5 g, Cat Litter, Pu blix Supe rmarkets Inc. Lakeland, FL) was mixed with 0.25 g seeds and various volumes (0.2, 0.5, 1.5 or 2.5 ml) of distilled water, 100 mg BA/liter water, or 100 mg GA/liter water in clear plastic bottles. Seeds were primed at 15oC for 4 days under white light (10 molm-2s-1) on a vertically rotating (1 cy cle/5 minutes) wheel (Lab-line
110 Instruments, Inc., Melrose Park, IL). Bottles were held in horizontal slots on the rotating wheel to ensure adequate light exposure. Primed seed s were washed and dried as described previously. 6.2.4 Germination Tests Constant temperatures of 20 or 30oC were provided in an Isotemp incubator (model 304R, Fisher Scientific, Fair Lawn, NJ) in con tinuous dark. Darkness wa s achieved by wrapping Petri dishes with aluminum foil and maintaining da rkness in the incubator. Four replications of 25 seeds were placed on two blue blotter papers (Anchor Paper Company, St. Paul, MN) moistened with de-ionized water in 5 cm glass Pe tri dishes. Germination was recorded daily for 14 days. Intermediate dark-germination counts were made under a green light (25W, A19, Specialty 90912, General Electric Company, Cleveland, OH) in a dark room. Germination was defined as 2 mm protrusion of the radicle. 6.2.5 Statistical Analysis Germination data were analyzed separately for each species. Data were adjusted for viability as determined by a TZ test (ISTA 2003). If necessary data were arcsine-square root transformed and analyzed by general linear mo del methods (SAS Version 9.1; SAS Institute, Cary, NC). Treatment means were separate d by the least significant difference (LSD; =0.05) test. Mean germination time (MGT, average time to total germination) was computed using Seed Calculator Version 3.0 (Plant Research International B.V., Wageni ngen, The Netherlands). 6.3 Results and Discussion 6.3.1 Coreopsis floridana Seed priming for 6 to 7 days in PEG im proved germination (23 to 63% after 7 days) of C. floridana in dark at 20oC (Figure 6-1). However, germination at 30oC in dark was not improved by PEG priming (Figure 6-1). Non-primed seeds when imbibed in light at 20oC had 100% germination, and at 30oC germination was 67%. In dark, non-primed seeds had 23%
111 germination at 20oC and 12% at 30oC (Figure 6-1). In celery ( Apium graveolens ), the cultivar Earlybelle failed to germinate at supraoptimal temperatures after priming in PEG alone (Tanne and Cantliffe, 1989). Adding GA to the PEG (100 mg GA/L PEG) pr iming solution promoted germination at 20oC from 23 to 67% after 3 days of priming, and from 12 to 31% at 30oC after 4 days priming (Figure 6-2). Increasing prim ing duration past 3 days did not improve germination at 20oC, but at 30oC germination was promoted. Prim ing celery seeds in PEG plus GA4+7 improved germination in dark and at supraoptimal temper atures (Tanne and Cantliffe, 1989), and in darkimbibed lettuce seeds, GA stimulated hypocotyl elongation and cotyledon expansion (Ikuma and Thimann, 1963), thereby overcomi ng the restricting endosperm. When C. floridana seeds were primed in PEG plus BA (100 mg BA/L PEG), germination was 100% after 3 days priming at both 20 and 30oC in dark (Figure 6-3). Priming duration past 3 days was not necessary. Priming seeds in vari ous concentrations of BA at 25, 50, 75 and 100 mg BA/L PEG for 4 days led to 100% germination when seeds were imbibed and germinated at 20 or 30oC in dark. Mean germination time (MGT) was reduced from 4.6 days at 20oC to 3.7 days at 30oC for non primed seeds, to less than 2 days at both temperatures when seeds were primed in PEG+BA (Figure 6-4). Increasing BA c oncentration from 25 to 100 mg/liter PEG had no influence on further reduction of MGT. Less than 5% of C. floridana seeds germinated with cotyledons protruding first (abnormal). This ha s been common in other species. Benzyladenine promoted dark-germination of other Asteraceae seeds such as Bidens pilosa (Valio et al ., 1972). When BA was added to PEG priming solution, celery seeds germinated in dark and at supraoptimal temperatures (Tanne and Cantliffe, 1989), while in lettuce se eds, a similar priming treatment promoted germination at supraoptimal temperatures (Cantliffe, 1991). In the case of
112 lettuce, at high BA concentrations cotyledons expanded and ruptured endosperm first followed by radicle protrusion. Due to its ch emical composition and structure (N6substituted aminopurine), synthetic BA used in these experiments mimics cytokinins (aminopurine derivatives); cytokinins regul ate cell cycle (Taiz and Zeiger 2002), and promote cell division and expansion (Pozsar and Matolcsy, 1968; Sussex et al., 1975; Khan, 1971; Taiz and Zeiger, 2002). It is noteworthy that BA has been reported to occur natu rally in some plant species as an aromatic cytokinin (Sakakibara, 2006; Strnad, 1997). Kinetin stimulates cotyledon expansion in imbibed lettuce seeds (Ikuma and Thimann, 1963). Solid matrix priming with BA and osmopr iming in PEG+BA both improved germination of C. floridana seeds in dark at both 20 and 30oC (Figures 6-3 and 6-5). When using GA, however, C. floridana seeds had 85% germination at 20oC in dark when solid matrix primed, and 65% germination when osmoprimed (Figure 6-2 and 6-5). Germin ation was 71% at 30oC after SMP plus GA and 31% after osmopriming plus GA. Increasing the volume of BA or GA priming solution in SMP reduced MGT when seeds were primed for 4 days (Figure 6-6). At both 20 and 30oC, MGT was similar for 0.5 to 2.5 ml BA priming solution, but when using GA, 0.5 ml had the shortest MGT at 20oC while at 30oC it was 1.5 ml. Priming celery seeds using SMP overcame thermoinhibition (Parera et al., 1993). 6.3.2 Coreopsis lanceolata In C. lanceo lata seeds, priming in 100 mg BA/L PE G for 5, 6 or 7 days, produced the same final germination (~86%) at 30oC, but the MGT was shortest at the longest priming duration (Figure 6-7). When imbibed at 20oC, non-primed C. lanceolata seeds germinated 98% but at 30oC only 2% germinated. Optimizing seed priming factors leads to rapid uniform germination and seedling emergence in C. lanceolata When other seed priming conditions were
113 held constant, priming effect was determined by priming duration in diffe rent lettuce cultivars (Cantliffe, 1981). Seed priming technologies have potential to enhance native Coreopsis seed germination under diverse environmental conditions, and this study is the first to re port success in priming C. floridana and C. lanceolata seeds. To overcome dormancy, and promote maximum germination and seedling uniformity in C. floridana, it is recommended that seed producers and sellers adopt seed priming technology for the benefit of end users. Primed seeds of C. floridana and C. lanceolata will also produce maximal germination at high soil temperatures. Using SMP with emathlite clay and BA at a ratio of 1:1:0.5 (c lay [weight]:BA [volume] :seed [weight]) produced better results, and is recommended. This SMP method will be sustainable because the clay is easy to separate from seeds and is locally available, and disposal may not be an environmental hazard. Use of primed Coreopsis seeds maximizes genetic diversity by permitting most seeds to germinate, and would facilitate successful sowing programs even during the hot Florida summers. The goal of this research is to iden tify priming methods that result in acceptable field establishment, thus promoting widespread use of these native wildflowers in revegetation of roadsides and ecological restoration sites. 6.4 Summary Germination of the wildflowers C. floridana and C. lanceolata is erratic due to seed dormancy. Coreopsis floridana seeds do not germinate in dark, and both species have reduced germination above 25oC. Osmopriming and SMP techniques were used to circumvent these problems. Seeds of both species were produced in a passively ventilat ed greenhouse, harvested and cleaned before storage in moistu re-proof bags in darkness at 10oC for 6 months ( C. floridana ) or 12 months ( C. lanceolata ). Osmopriming was accomplished using PEG-8000 at a water potential -1.2 Mpa. Emathlite clay was used for SMP. Gibberellic acid (GA4+7) or BA
114 were added at 100 mg/liter PEG to some osmoprim ing treatments, while 100 mg GA or BA per liter water were added to emathlite in SMP to evaluate possible enhancement of the priming effect. Coreopsis floridana seeds primed in PEG improved germination from 23 to 63% at 20oC but at 30oC germination only increased from 12 to 33%. When BA was added to PEG, 100% germination was promoted at both 20 and 30oC, but addition of GA to PEG priming solution promoted germination from 23 to 67% at 20oC and from 12 to 31% at 30oC in C. floridana seeds imbibed in dark. SMP of C. floridana seeds with BA led to similar positive effects on germination as osmopriming, while SMP seeds with GA had 85% germination, compared to osmoprimed plus GA which led to 63% germinat ion. Using SMP and BA at a ratio of 1:1:0.5 (clay [weight]:BA [volume]:seed [weight ]) produced the best results. In C. lanceolata osmopriming with BA increased germina tion percentage from 2 to 86% at 30oC. Priming reduced the MGT in both species. In C. floridana and C. lanceolata, seed priming can maximize germination and thus improve stand es tablishment under diverse environments
115 Priming duration (days) 0246 Germination % 0 20 40 60 80 100 20 30 Figure 6-1. Germination of C. floridana seeds in dark after priming in PEG for 3, 4, 5, 6 or 7 days in light. Germination tests were conducted at 20oC (LSD = 20) or 30oC (LSD = 9) for 14 days. Priming duration (days) 012345 Germination % 0 20 40 60 80 100 20 30 Figure 6-2. Germination of C. floridana seeds in dark at 20oC (LSD = 20) or 30oC (LSD = 9) after priming in 100 mg GA4+7/L PEG for 3, 4 or 5 days in light. Non-primed seeds are denoted by zero days priming durati on. Non-primed seeds imbibed on 100 mg GA4+7/L water substrate led to 97% germination at 20oC and 68% at 30oC. Germination test duration was 14 days.
116 Priming duration (days) 012345 Germination % 0 20 40 60 80 100 20 30 Figure 6-3. Germination of C. floridana seeds in dark at 20 or 30oC after priming in 100 mg BA/L PEG for 3, 4 or 5 days in light. Non-primed seeds are denoted by zero days priming duration. LSD = 9. Non-primed seeds imbibed on 100 mg BA/L water substrate had 54% germination at 20oC and 34% at 30oC. Test duration was 14 days. BA concentration (mg/liter PEG) 020406080100 Mean germination time (days) 0 1 2 3 4 5 20 30 Figure 6-4. Mean germination time (MGT) in C. floridana seeds in dark after osmopriming in PEG plus various BA concentrations for 4 days in light. LSD = 1.0. BA treatments germinated 100%, and non-primed seeds 23% at 20oC and 12% at 30oC. Germination tests were conducted for 14 days.
117 A Volume (ml)/0.5 g clay 0.00.51.01.52.02.53.0 Germination % 0 20 40 60 80 100 C GA BA B Volume (ml)/0.5 g clay 0.00.51.01.52.02.53.0 Germination % 0 20 40 60 80 100 C GA BA Figure 6-5. Germination of C. floridana seeds in dark after 4 days of SMP in various volumes of solutions in light. A) Germination at 20oC, LSD = 19. Non-primed seeds had 23% germination at 20oC. B) Germination at 30oC, LSD = 22. Non-primed seeds had 12% germination at 30oC. SMP was conducted with emathlite clay plus 100 mg BA/L water (BA) or 100 mg GA/ L water (GA), or water cont rol (C). Regardless of volume, BA or GA concentrations were maintained the same. Germination test duration was 14 days.
118 A Volume (ml)/0.5 g clay 0.00.51.01.52.02.53.0 Mean germination time (days) 0 2 3 4 5 6 7 C GA BA B Volume (ml)/0.5 g clay 0.00.51.01.52.02.53.0 Mean germination time (days) 0 2 3 4 5 6 7 C GA BA Figure 6-6. Mean germination time (MGT) of C. floridana seeds in dark after SMP in clay (0.5 g) with various volumes of benzyladenine (BA, 100 mg/L water) or gibberellic acid (GA, 100 mg/L water) or water control (C) in light. Regardless of volume, BA or GA concentrations were maintained the same. A) Germination at 20oC, LSD = 1.1. Nonprimed seeds had a MGT of 7.2 days at 20oC. B) Germination at 30oC, LSD = 2.5. Non-primed seeds had a MGT of 6.3 days at 30oC. Germination test duration was 14 days.
119 Priming duration (days) 02468 Mean germination time (days) 0 4 6 8 10 12 14 Figure 6-7. Mean germination time (MGT) of C. lanceolata seeds in dark at 30oC after osmopriming in PEG plus BA (100 mg/L PEG) for various durations in light. LSD = 1.1. All BA priming treatments had 86% final germination and non-primed seeds had 2%. Germination tests were conducted for 14 days.
120 CHAPTER 7 CONCLUSION In Florida, there is growing dema nd for prem ium value, pre-variety germplasm of locally or regionally specific ecotype seeds of native Coreopsis ( Asteraceae ) for use in ecological restoration, reclamation and along roadsides. Dema nd of this seed has led to a concomitant rise in seed production; however, unlike seeds of domesticated crops, moderate to substantial dormancy is common among seeds of pre-variety ge rmplasm of native flowers. Seed dormancy continues to present a major challe nge to seed producers, testing agencies and end users, because it affects seed quality assessment and therefor e value to producers and the amount purchased by end users (Norcini and Aldrich, 2008). Coreopsis species, commonly known as tickseed, ha ve a wide distribution range from North to South America (Smith, 1975; Tadesse et al. 1995). There are 28 Coreopsis species native to the U.S. (USDA-NRCS, 2007), and ecotype seeds of at least ei ght species are being commercially produced (Norcini a nd Aldrich, 2007a). Dormancy in cultivated populations is variable within and among, freshly harvested or stored pre-variety seed lots of native Coreopsis (Kabat et al ., 2007; Norcini and Aldrich, 2007a; Norcini et al ., 2004, 2006). To gain insight about seed dormancy in pre-variety germplasm of native Coreopsis species, anatomical and physiological studies were conducted. Coreopsis seeds (achenes) were comprised of a dicotyledonous embryo enclosed by an outer pericarp, testa and endosperm (Chapter 3) The ploidy ratio of endosperm to embryo was 3:2 as determined by DNA fluorescence techniques While this is consistent with other angiosperms, this has not been reported elsewhere for Coreopsis In C. floridana and C. lanceolata the lateral endosperm was a single cell-laye r, but micropylar endosperm was 2-3 celllayers thick in C. lanceolata This is the first such report detailing the anatomy of Coreopsis
121 seeds, and paves way for future research in seed physiology of this species. Germination results showed that pericarp, testa and endosperm modulated dormancy of C. floridana and C. lanceolata and especially so at elevated temperatures In dark-imbibed C. floridana seeds, the same tissues imposed dormancy (Chapters 3 and 5). The different germination behavior between the two species might be linked to their natural ecological habitat. Coreopsis floridana and C. lanceolata germination requirements were consistent with those of wetland and upland species, respectively. Dormancy is important for the survival of some plant species because it enables them to evade harsh and unsuitable environmental conditions which do not support seedli ng growth and establishment. Intact seeds of C. floridana tolerated germination at higher temperatures than C. lanceolata seeds, a possible adaptation for wetland species. These results on anatomy and germination are important for devising pre-germination seed treatments th at weaken embryo envelopes for overcoming dormancy. In C. lanceolata seeds, EBM activity was associated with germination, and activity was detected at 90 hours imbibition while germinati on occurred at 96 hours (Chapter 3). Conditions not favorable to germination such as abscisic acid (ABA), tetcyclacis (gibberellic acid [GA] biosynthesis inhibitor) and hi gh imbibition temperature (30oC) inhibited this enzymes activity. Coreopsis floridana seeds did not exhibit EBM activity at any time throughout germination, implying that another system might be involved in endosperm digestion during germination in this species, however, this was not explored further. This is th e first report of EBM activity in C. lanceolata seeds, and this mechanism may be im portant to circumventing dormancy in nonafterripened C. lanceolata seeds.
122 Optimum germination temperatures for Coreopsis seeds were 15 and 20oC in light and germination was low above 25oC (Chapters 3, 4 and 5). In ro adside sowings, germination will be reduced and erratic when soil temperatures exceed 25oC and sowing should be confined to periods when soil temperatures are below this threshold. Gibberellic acid and cold stratification (5oC) promoted germination in dark-imbibed C. floridana and C. leavenworthii seeds but maximum germination was not achieved. However, in C. basalis, C. lanceolata and C. pubescens GA had no effect while cold stratificati on reduced germination (Chapters 4 and 5), indicating that seeds of these spec ies should not be sown into cold soils during Florida winters to avoid inducing dormancy, and precautions should be taken since C. lanceolata is normally sown in fall. Coreopsis floridana flowers and produces seed in fall and seeds might be adapted to germinate in spring after cold stratification during the winter. Pota ssium nitrate (10 mM) promoted germination of non-afterripened C. lanceolata seeds in light, but did not totally overcome dormancy. All this information is importa nt to nursery managers and other end users, because it enables them to know what germ ination conditions are required to optimize germination. Coreopsis lanceolata seeds required 150 days of dry afterripening at 10oC to overcome dormancy, but fresh (non-afterripened), naked C. lanceolata embryos germinated 100% (Chapter 5). Seed producers desiring seed lo ts with minimal dormancy should allow C. lanceolata seeds to dry afterripen for at least this period before they sell th e seeds. They should also rely on viability tests such as the tetrazolium (TZ) or ex cised embryo test to ascert ain the suitability of a seed lot for afterripening, otherw ise non-viable seeds may be mistak enly left to afterripen with no consequent improvement in germination. Af terripened seeds should be stored under low humidity (<50%) and low temperatures (~10oC) to promote longevity. Seed producers should
123 therefore have prior knowledge of afterripening requirements so th at they can devise marketing plans. While C. floridana seeds had 100% germination in lig ht when freshly harvested, these seeds had 23% germination in dark; however, naked embryos germinated 100% in dark. For species like C. floridana which do not require afterripening (C hapter 5), low temperature storage soon after conditioning is advisable to maintain viability. Pre-ge rmination seed treatments that help weaken endosperm have potential to alleviate dormancy in dark-imbibed C. floridana and non-afterripened C. lanceolata seeds. In lettuce, another Asteraceae endosperm removal alleviated dormancy (Leon-Kloosterziel et al., 1996; Debeaujon and Koorneef, 2000; LeubnerMezger, 2003). Afterripening requirem ent has been reported before in C. lanceolata although under different storage conditions (Norcini and Aldrich, 2007a), and this study demonstrated that seeds of this species can dry afte rripen under low temperatures (~10oC) at a seed moisture content of 8.1%. Norcini a nd Aldrich (2007a) reported an af terripening period of 6 months whereas in this study C. lanceolata seeds afterripened in 5 months, a minor discrepancy which could be due to the influence of different st orage conditions and initial dormancy level. Naked embryos of C. floridana seeds germinated 100% in dark, and germination of naked embryos was also 100% at 30oC in both C. floridana and C lanceolata Whole nonprimed C. floridana seeds had 23% germination at 20oC and 12% at 30oC when imbibed in dark, while whole non-primed C. lanceolata seeds had 2% germination at 30oC. However, removal of these tissues in large sowing programs is not practical. This study suggested that technology such as seed priming might be used to maximize and promote germination of Coreopsis seeds under diverse environments (Chapter 6). Coreopsis floridana seeds germinated 100% in dark after priming, and in both C. floridana (100%) and C. lanceolata (86%) germination was maximized at high temperature (30oC). While priming would increase costs slightly, the benefits
124 far outweigh any extra costs, because germination and plant establishment is maximized (Cantliffe, 2003). Results here suggest that us ing solid matrix priming (SMP) with emathlite clay and 100 mg benzyladenine (B A) per liter water solution gives the best results at a ratio of 1:1:0.5 (clay [weight]:BA [volume]:s eed [weight]). This study is the first to report success in priming C. floridana and C. lanceolata seeds, a technology that could be adopted by seed producers or sellers to benefit end users. Based on this study, recommendations to prom ote germination and establishment of native Coreopsis species include: 1) sowing Coreopsis seeds when soil temperatures are below 25oC in order to avoid thermoinhibition, 2) C. floridana seeds require light for germination, and should be sown on the soil surface to maximize light perception, 3) testing for viability and dormancy at harvest in C. lanceolata seeds and allowing for afterripening under appropriate conditions such as 10oC at a seed moisture content of 8.1% if necessary, and 4) use of seed priming techniques to overcome dark-dormancy in C. floridana and maximize germination in high soil temperatures for both C. floridana and C. lanceolata Additionally, future research is recommended on: 1) investigating seed matu ration conditions that favor production of high quality Coreopsis seeds with low dormancy, 2) studying pr e-sowing seed treatment methods that weaken the endosperm to overcome dormancy in freshly harvested C. lanceolata seeds, and 3) further exploration of seed priming technology to promote germination under diverse environments.
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150 BIOGRAPHICAL SKETCH Dzingai Rukuni was born in Bulawayo, Zimbab we. He graduated with a Bachelor of Science honors degree, in agricultu re, from the University of Zimbabwe in 1989. He worked for the Forestry Commission of Zi mbabwes Research and Deve lopment Division as a seed physiologist from January 1991 to July 1995. In Ju ly 1995 he left for New Zealand and started a Master of Applied Science honors degree program in seed and crop science at Massey University in Palmerston North. He specialized in Seed Technology and completed in July 1997, and left for Zimbabwe to rejoin the Fo restry Commission in the same capacity until September 2001. In October 2001 he joined the Tobacco Research Board as a senior seed physiologist until January 2006, when he left to begin his doctoral studies in seed physiology at the University of Florida in Gainesville. His research focused on seed dormancy in Florida native wildflower Coreopsis species.