BAHIAGRASS GERMPLASM REPRODUCTIVE CHARACTERIZATION, AND BREEDING AT THE TETRAPLOID LEVEL By CARLOS A. ACUNA A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2006
Copyright 2006 by Carlos A. Acuna
To my wife, Lorena
iv ACKNOWLEDGMENTS I would like to start by thanking God for my existence and for the inspiration and support He has provided throughout my life. I th ank my wife, mother, and sister for their unconditional company, encouragement, and support. I would also like to thank my grandfather for sharing with me hi s love for agricultural systems. I want to thank my supervisory co mmittee chair (Dr. Ann Blount) for her confidence in giving me the opportunity to con tinue a graduate career in the USA. I thank Dr. Kenneth Quesenberry for offering his fr iendship, guidance, and knowledge that made my graduate school experience more interest ing. I am also grateful to Dr. Wayne Hanna for his professional example, and for his cri tical point of view. My thanks also go to Dr. Kevin Kenworthy, for agreeing to be on my supervisory committee. I also appreciate the technical support of Judy Dampier, Loan Ngo Hung, Laura Vidoz, and Brandy Williams. I sincerely thank Camilo Quarin for his cons tant advice and interest in my graduate career. I would also like to thank all my friends for their unconditional support.
v TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES............................................................................................................vii LIST OF FIGURES.............................................................................................................x ABSTRACT....................................................................................................................... xi CHAPTER 1 INTRODUCTION........................................................................................................1 2 LITERATURE REVIEW.............................................................................................4 Bahiagrass Races..........................................................................................................5 Geographical Distribution and Importance...........................................................6 Reproductive Biology............................................................................................7 Breeding Bahiagrass.....................................................................................................9 Breeding Diploids..................................................................................................9 Breeding Tetraploids...........................................................................................10 Objectives...................................................................................................................13 3 FERTILITY CHARACTERIZATION OF DIPLOID AND TETRAPLOID BAHIAGRASS...........................................................................................................14 Introduction.................................................................................................................14 Materials and Methods...............................................................................................15 Plant Material......................................................................................................15 Diploids........................................................................................................15 Tetraploids....................................................................................................16 Pollination Techniques........................................................................................16 Seed Set...............................................................................................................16 Statistical Analysis..............................................................................................17 Results and Discussion...............................................................................................18 Diploid Bahiagrass..............................................................................................18 Tetraploid Bahiagrass..........................................................................................20 Summary and Conclusions.........................................................................................23
vi 4 REPRODUCTIVE CHARACTERIZATION OF SEVERAL NATURAL AND INDUCED BAHIAGRASS ACCESSIONS..............................................................32 Introduction.................................................................................................................32 Materials and Methods...............................................................................................33 Plant Material......................................................................................................33 Ploidy Level Determination................................................................................34 Embryo Sac Observations...................................................................................35 Results and Discussion...............................................................................................35 Induced Tetraploids.............................................................................................35 Other Accessions.................................................................................................36 Tetraploid Cultivars.............................................................................................38 Summary and Conclusions.........................................................................................38 5 GENERATION AND EVALUATION OF TETRAPLOID HYBRIDS IN BAHIAGRASS...........................................................................................................44 Introduction.................................................................................................................44 Materials and Methods...............................................................................................45 Plant Material and Crosses..................................................................................45 Seed set, Germination, and Greenhouse and Field Operations...........................46 Field Evaluations.................................................................................................46 Laboratory Evaluations.......................................................................................47 Statistical Analysis..............................................................................................47 Selection Index....................................................................................................48 Results and Discussion...............................................................................................48 Progeny................................................................................................................48 Field Evaluations.................................................................................................49 Summary and Conclusions.........................................................................................54 6 CONCLUSIONS........................................................................................................66 APPENDIX SEED SET MEANS OF DIFFERENT BAHIAGRASS DIPLOIDS AND TETRAPLOIDS UNDER SELF-, CROSS-, AND OPEN-POLLINATION IN THE GREENHOUSE AND FIELD, ON 2004 AND 2005..................................70 LIST OF REFERENCES...................................................................................................81 BIOGRAPHICAL SKETCH.............................................................................................86
vii LIST OF TABLES Table page 3-1 Analysis of variance (ANOVA) tabl e showing the significance of diploid populations (Pensacola bahiagrass and Tifton 9) on seed set under selfpollination during 2004 and 2005, in the greenhouse..............................................25 3-2 ANOVA table showing the significance of the diploid population (Pensacola bahiagrass and Tifton 9) on seed se t under cross-pollination during 2004 and 2005, in the greenhouse............................................................................................25 3-3 ANOVA table showing the significan ce of diploid population (Pensacola bahiagrass and Tifton 9) on seed set under open-pollination during 2005, in the field.......................................................................................................................... .25 3-4 ANOVA table for the effect of locati on (field vs. greenhouse) on seed set for diploid bahiagrass (Pensacola bahiagra ss and Tifton 9) under self-pollination during 2005..............................................................................................................25 3-5 ANOVA table showing the significance of genotype on seed set for Pensacola bahiagrass under self-pollination during 2004 and 2005, in the greenhouse...........25 3-6 ANOVA table showing the significance of genotype on seed set for Tifton 9 under self-pollination during 2004 and 2005, in the greenhouse.............................26 3-7 Range in self-pollinated seed set on diploid bahiagrass genotypes.........................26 3-8 ANOVA table showing the significance of genotype on seed set for Pensacola bahiagrass under cross-pollination during 2004 and 2005, in the greenhouse.........26 3-9 ANOVA table showing the significance of genotype on seed set for Tifton 9 under cross-pollination during 2004 and 2005, in the greenhouse..........................26 3-10 ANOVA table showing the significance of genotype on seed set for Pensacola bahiagrass under open-pollination during 2005, in the field....................................26 3-11 ANOVA table showing the significance of genotype on seed set for Tifton 9 under open-pollination during 2005, in the field......................................................27
viii 3-12 ANOVA table showing the significance of the pollination method (selfand cross-pollination) on seed set fo r diploids during 2004 and 2005, in the greenhouse................................................................................................................27 3-13 ANOVA table showing the significance of the pollination method (selfand open-pollination) on seed set for diploi d bahiagrass during 2005, in the field........27 3-14 ANOVA table showing the significance of genotypes on seed set for the AIT under self-pollination during 2004 and 2005, in the greenhouse.............................27 3-15 Seed set under selfand open-pollination for 23 AI T genotypes in the field. Open-pollinations were accomplishe d during 2004 and 2005, while selfpollinations only during 2005..................................................................................28 3-16 ANOVA table showing the significance of the experimental location (field and greenhouse) on seed set for the AIT under self-pollination during 2005.................28 3-17 ANOVA table showing the significance of genotype for the AIT on seed set under open-pollination during 2004 and 2005, in the field......................................28 3-18 ANOVA table showing the significance of the pollination method (openand self-pollination) on seed set for the AIT during 2004 and 2005, in the field...........29 3-19 ANOVA table showing the significance of population (diploids, AIT, and STL) on seed set under self-pollination during 2004 and 2005, in the greenhouse..........29 3-20 ANOVA table showing the significance of genotype on seed set for Q4188 and Q4205 under self-pollination duri ng 2004 and 2005, in the greenhouse.................29 3-21 ANOVA table showing the significance of the experimental location (field and greenhouse) on seed set for Q4188 a nd Q4205 under self-pollination during 2005..........................................................................................................................2 9 3-22 ANOVA table showing the significance of genotype on seed set for Q4188 and Q4205 under cross-pollination du ring 2004, in the greenhouse..............................29 3-23 ANOVA table showing the significance of pollination method (selfand crosspollination) on seed set for Q4188 and Q4205 during 2004, in the greenhouse......30 3-24 ANOVA table showing the significance of population (STL and AIT) on seed set under cross-pollination dur ing 2004, in the greenhouse.....................................30 3-25 ANOVA table showing the significance of genotype on seed set for the apomictic tetraploids (Argentine, Tifton 7, PI 315733, and PI 315734), under self-pollination during 2004 and 2005, in the greenhouse.......................................30
ix 3-26 ANOVA table showing the significance of the experimental location (field and greenhouse) on seed set for the apomic tic tetraploids under self-pollination during 2005..............................................................................................................30 3-27 ANOVA table showing the significance of genotype on seed set for Argentine and Tifton 7 under open-pollination during 2005, in the field.................................30 3-28 ANOVA table showing the significan ce of the pollination method (Selfpollination and open-pollination) on seed set for Argentine and Tifton 7 during 2005, in the field.......................................................................................................30 3-29 Comparisons of seed set means from diploids (Pensacola bahiagrass and Tifton 9), AIT, STL, and apomictic tetraploids (Argentine and Tifton 7), under self-, cross-, and open-pollination in the field and greenhouse during 2004 and 2005.....31 4-1 Embryo sac types in 27 bahiagrass acce ssions. The first 20 accessions in the table are induced tetraploids, the last 7 are natural tetraploids................................40 5-1 Number of caryopses and plants gene rated from crosses made between sexual and apomictic tetraploids of bahiagrass...................................................................58 5-2 Plant diameter and plant height m eans from three measurements during the growing season.........................................................................................................58 5-3 Means separation for plant regrowth, frost resistance, and total number of inflorescences at the end of the growing season......................................................59 5-4 Parental lines evaluati on showing plant diameter a nd plant height means from three measurements during the growing season.......................................................59 5-5 Means separation for plant regrowth, frost resistance, and total number of inflorescences at the end of the growing season......................................................60 5-6 Means separation (G2) for herbage mass, frost resistance, and total number of inflorescences at the end of the growing season......................................................60 5-7 Contribution from each population to the top 20% of the genotypes......................61 5-8 Embryo sac types in 71 bahiagrass hybrid s (the first 63 accessions in the table are from G1, and the last 8 are from G2).................................................................62 A-1 Seed set means of different di ploid and tetraploid bahiagrass.................................70
x LIST OF FIGURES Figure page 4-1 Mature meiotic embryo sac in one ovule of the induced tetraploid plant 52 of Paspalum notatum showing the two polar nuclei with large nucleolus (arrow), and a mass of antipodals in the chalazal end (arrow head). Magnification: x312.541 4-2 Two mature aposporous embryo sacs in one ovule from PI 315732 showing their correspondent polar nuclei (a rrows). Magnification: x312.5...................................42 4-3 Histogram generated with a Partec Ploidy Analyzer PA for PI 315733 of Paspalum notatum. Because the analyzer was calibrated at 100 units for a known diploid bahiagrass (2n=2x=20), a clear p eak at 200 units indicated that PI 315733 was a tetraploid (2n=4x=40).......................................................................43 5-1 Tetraploid progeny of Paspalum notatum obtained by crossing sexual and apomictic genotypes. Different grow th habits can be recognized...........................64 5-2 Multiple aposporous embryo sacs in one ovule of an apomictic tetraploid hybrid of Paspalum notatum , 2-2-7x Tifton 7 #14. Ma gnification: x312.5........................65
xi Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science BAHIAGRASS GERMPLASM REPRODUCTIVE CHARACTERIZATION, AND BREEDING AT THE TETRAPLOID LEVEL By Carlos A. Acuna May 2006 Chair: Ann R. Blount Cochair: Kenneth H. Quesenberry Major Department: Agronomy Bahiagrass, Paspalum notatum FlÃ¼gge, an extremely persistent forage crop, is the base of the beef cattle production systems in Florida. Improved cultivars of this forage are needed to improve these production systems. Selection and efficient use of a breeding approach are defined by the reproductive m ode of a species. Our objectives were to characterize the reproduction mode of bahiag rass germplasm, and to initiate a breeding program, to generate more-productive genetic lines. The reproductive behavior of diploid and tetraploid bahiagrass was determined by a series of controlled pollination studies, in the greenhouse and field, in 2004 and 2005. Tw o sexual diploid populations (Pensacola bahiagrass and Tifton 9) were determined not to be different in terms of selfand cross-fertility, indicating that the general fertility of the crop was not affected by phenotypic mass selection. Sexual diploids, as a group, were determined to be primarily cross-pollinated, with low but variable levels of self-fertility.
xii A group of 20 artificially i nduced tetraploids was determined to be sexual and cross-pollinated. Self-f ertility was lower in this group th an in the diploids. In addition, two selected sexual tetraploids (Q4188 and Q4205) set similar amounts of seed when selfor cross-pollinated. In contrast, seven tetraploids were determined to be apomictic, with variable levels of expression. A segr egating population contai ning 822 plants was generated by crossing different tetraploid sexual and apomictic genotypes. Large variability was observed among progeny in terms of growth ha bit, reproductive expression, regrowth at end of the season, a nd frost resistance. A selection index was developed and the top 20% of progeny plan ts was identified based on the index. Apomictic male parents Argentine and Tift on 7, and sexual female parents 2-2-7, 106, 4-36-1, and Q4188 resulted in a higher pr oportion of superior progeny. A 3:1 ratio between sexual and apomictic (facultative + obligate apomictic) plan ts, and a 9:1 ratio between others (facultative apomictic + sexua l) and obligate apomictic plants were found in the progeny. Selected apomictic lines will undergo additional evaluations and superior sexual plants may be used as female parents in future crosses.
1 CHAPTER 1 INTRODUCTION Paspalum notatum FlÃ¼gge, bahiagrass, is a warm-season grass that has become widely distributed in warm and humid regi ons of the western hemisphere. It is the primary constituent of native grassland in southern Brazil, Paraguay, Uruguay and northeastern Argentina (Gates et al., 2004). Bahiagrass is grown throughout Florida and in the Coastal Plain and Gulf Coast regions of the southern United States (Chambliss and Adjei, 2006). It has been plan ted extensively as pasture and as ground cover on highway rights-of-way. As forage, bahiagrass is ch aracterized by its mark ed persistence under continuous stocking and intense defoliation (Bea ty et al., 1970). This characteristic is considered the main reason for the predominan ce of this species in native grasslands, as well as in beef cattle production systems of the southern USA. Bahiagrass is a perennial grass with st rong, shallow rhizomes formed by short, stout internodes usually covered with old, dr y leaf sheaths. Culms are simple, ascending, geniculate at the node between the firs t and the second elonga ted internodes, and otherwise erect (10 to 60 cm tall). Leaves are mostly crowde d at the base with overlapped keeled sheaths, glabrous, or with ciliate ma rgins mainly toward the summit. Blades are usually flat or somewhat folded toward the base, linear lanceolate 3 to 30 cm long, 3 to 12 mm wide, usually glabrous or ciliate toward the base, and rarely pubescent throughout. Inflorescences are su bconjugate with a short that are almost imperceptible common axis, having two racemes, rarely three, that are ascending or recurved-divergent in some races, with racemes 3 to 14 cm long. The rachis is glabrous, flexuous, and green
2 or purplish. Spikelets are solitary in two rows on one side of the rachis, obovate or ovate, shining, glabrous, 2.5 to 4 mm long, and 2 to 2.8 mm wide. Anthers and stigmata are usually purple, and the fruit are oval, about 1.8 mm long and 1.2 mm wide (Gates et al., 2004). Bahiagrass is highly diverse containing races with different ploidy levels. The most common races are diploids (2n=2x=20) and tetraploids (2n=4x=40) (Pozzobon and Valls, 1997). The most representative tetraploid form is known as common bahiagrass in that it is broad leaved, strong-root ed, and spreads slowly by st out rhizomes with short internodes. The dipl oid race belongs to Paspalum notatum var. saurae, known as Pensacola bahiagrass, and when compared to common bahiagrass, it is taller, spreads faster, has longer, narrower leaves, smaller spikelets, and can have more racemes per inflorescence (Burton et al., 1970). Persistence (even under conditions of low fertility, drought, flooding, and particularly, severe continuous grazing) makes bahiagrass a ve ry reliable feed source for low input beef cattle production. Most of the area planted with bahiagrass in the USA is used for pasture for extensive cow-calf production. It is also widely used as low-maintenance turf in the same regions wher e it is used as forage. Once established, it is persistent, requires low fertility and pest control inputs, and tolerates frequent close mowing. Extensive areas adjacent to highway s have been planted or sodded with bahiagrass in the southeastern USA. The dense sod it forms effectively limits soil erosion and provides a uniform turf. In addition, bahiagra ss is used in crop rotations especially in combination with crop legumes. Establis hment from seed makes bahiagrass more
3 attractive than many vegetatively propagated grasses for use in rotations (Gates et al., 2004). Bahiagrass shows a seasonal pattern of herbage accumulation, depending on latitude and altitude. Temperature, moisture, and daylength are the factors that determine its seasonal production cycles (Gates et al ., 2001; Mislevy et al., 2001). In the humid southeastern USA, for example, bahiagrass can be used for grazing or cutting from April to October. Even in lower latitudes such as peninsular Florida, US A, more than 85% of total annual production takes place during the six warmest months (April-September) of the year (Beaty et al., 19 80; Mislevy and Everett, 1981; Mislevy and Dunavin, 1993). Except for locations where winters are mild to warm, where moisture is available either from rain or irrigation, or where stockpiling is practiced, bahiagrass is not often used for winter grazing or haying (Gates et al., 2001) . "Winter" forage production is frequently insufficient, even for semi-intensive lives tock operations where stocking rates are medium to low. Currently, there is a need for new bahi agrass cultivars that can produce more foliage for a longer period of the year, and increase productivity of the cattle production systems. Thus, generation of new genetic materials should not only focus on increasing the productivity during the regular growing s eason, but also during the colder period of the year. In the selection process of more pr oductive materials the pe rsistence of the crop needs to remain; therefore, retaining the most important characteristic of bahiagrass as a forage.
4 CHAPTER 2 LITERATURE REVIEW The genus Paspalum L. belongs to the tribe Pani cea of the subfamily Panicoideae and consists of approximately 400 species (Chase, 1929). Several species are important forage and turf grasses, such as, Paspalum notatum FlÃ¼gge, P. dilatatum Poir., P. atratum Swallen, P. nicorae Parodi, P. vaginatum Swartz, P. plicatulum Michx., and P. guenoarum Arech. Many other species have the poten tial to become important forage or turf crops (Evers and Burson, 2004). P. notatum FlÃ¼gge (bahiagrass) is a perennial rhizomatous grass recognized as one of the majo r constituents of the native grasslands in the New World (Chase, 1929). In addition, it is one of the most important cultivated grasses in Florida and the southe rn part of the Gulf States of the USA. It has been planted extensively as pasture and utility turf on hi ghway right-of-ways (Gates et al., 2004). Races with different ploidy levels as well as linked reproductive behaviors make the crop highly diverse, and different approaches are needed for breeding purposes. This review is divided in two sections, section one is a characterization of the bahiagrass races, a description of the o ccurring natural races , their geographic distribution and importance, and finally thei r reproductive biology, a nd a description of the genetic control of apomix is. The second section relates to the different approaches used for breeding diploids and tetraploids of bahiagrass. Finally, after an explanation of what additional information is needed to in itiate a breeding program for the bahiagrass tetraploids, the objectives of this work are described.
5 Bahiagrass Races Most Paspalum species have a base chromosome number of x=10; however, base numbers of x=6 and x=9 have been proposed for the Paspalum almum Chase complex (Quarin, 1974), and P. contractum Pilger (Davidse and Pohl, 1974), respectively. Bahiagrass has a base chromosome numb er of x=10, and diploids (Burton, 1946), triploids (Tischler and Burson, 1995), tetr aploids (Burton, 1940) , and pentaploids (Tischler and Burson, 1995) are the known ploidy levels. The most important races are the tetraploids and the diploids. Chromosome numbers were recently determined for 150 naturally occurring bahiag rass accessions. Pozzobon and Walls (1997) examined 127 accessions and found that 116 (91%) were te traploids while the remaining 11 were diploids. Tischler and Burson (1995) st udied 23 accessions where 17 (74%) were tetraploids, four (17%) were diploids, one was triploid, and one was a pentaploid. Common bahiagrass is the typi cal tetraploid form of the species being broad leaved, strong-rooted, and spreads slowly by stout rh izomes with short in ternodes. The diploid form belongs to the botanical variety P. notatum var. saurae Parodi, and is usually referred to as Pensacola type bahiagrass. It is taller, spreads faster, has longer narrower leaves, smaller spikelets, and can have more racemes per inflorescence than common bahiagrass (Gates et al., 2004). Chromosome pairing behavior in induced autotetraploids, natural tetraploids, and induced x natural tetraploid hybrids indicates that the natu ral tetraploids originate by autopolyploidy (Forbes and Burton, 1961; Quar in et al., 1984). Mo reover, triploids hybrids from crosses between diploids and tetraploids plants have as much as 10 trivalents per pol len mother cell during meiosis I indicating complete homology between the genome of the diploid parent a nd both genomes from the tetraploid male
6 parent (Forbes and Burton, 1961). In addition, Stein et al . (2004) showed molecular evidence of tetrasomic inheritance for several lo ci of the tetraploid race. This supports the hypothesis that P. notatum is an agamic complex composed of several cytotypes of different ploidy levels, most of which are autopolyploids (Gates et al., 2004). Geographical Distribution and Importance The tetraploid races are distributed in open areas, savannas, and cultivated pastures from sea level to 2000 m from central Mexi co to Argentina and throughout the West Indies (Chase, 1929). Common bahiagrass is the primary constituent of many native pasturelands in southern Brazil, Paraguay, Uruguay, and northeastern Argentina (Gates et al., 2004). The original distribution of the var. saurae was confined to Corrientes, Entre Rios, and the eastern edge of Santa Fe provi nces in Argentina. The diploid populations are infrequent and usually restricted to we t sandy soils along rivers and flat sandy islands in the Parana River (Burton, 1967; Gates et al., 2004). Because the polyploid forms of the species showed an autoploid origin, and b ecause of the higher variability observed in tetraploid populations that were in contact with diploi d populations (Daurelio et al., 2004), this region is considered the ce nter of origin of this species. After its introduction into Florida, the var. saurae was brought into cultivation throughout the state, the Coastal Plain, and the Gulf Coast regions of the southern USA. It soon escaped from cultivation and rapidly b ecame naturalized throughout that region of the USA (Gates et al., 2004). It can now be found growing from Texas to North Carolina, extending into Arkansas and Tennessee (W atson and Burson, 1985). Several accessions of the tetraploid form were also introduced in the USA more than 50 years ago, and a few of them have persisted as cultivars. Na turalized populations of both Pensacola and tetraploid races occur in Australia (Gates et al. 2004). Bahiagrass is used as a pasture
7 grass in Japan (Sugimoto et al., 1985), Ta iwan (Jean and Juang, 1979) and Zimbabwe (Mills and Boultwood, 1978). Reproductive Biology Diploid and polyploid bahiag rass races show completely different and opposite methods of reproduction. Based on the high variability observed between many spaced clones in plant type, seed production, rate of spread, leaf length, leafiness, forage production, disease resistance, seed shattering, and anther color, Burton (1955) concluded that Pensacola bahiagrass reproduced se xually and was highly cross-pollinated. Moreover, he found that most Pensacola bahiag rass plants were self-sterile, fifty-seven clones that set 89.5% of seed when open-pol linated averaged only 6.0% seed set when selfed. Also, 694 of 705 mutual pollinations ma de between 47 Pensacola clones set seed well, indicating that most clones are cross-co mpatible. This characteristic is typical among diploids of the genus Paspalum . Quarin (1992) recorded that 75% of the sexual diploids studied in the genus showed allogamy due to self-incompatibility. Also, it is common in sexual ecotypes from species of closely related genera, such as Panicum virgatum L. (Talbert et al., 1983; Mar tinez-Reina and Vogel, 2002), and Thrasya petrosa Chase (AcuÃ±a et al., 2005). The basis of the ge netic control of self-incompatibility in the grasses was established in 1956 by Lundqvist and Hayman. They proposed that selfincompatibility in grasses is determined by the action of two independently segregating, polyallelic genes, S and Z. The pollen gr ain is specified gametophytically by the complementary interaction of S and Z. A polle n grain will be incompatible with a style that has the same alleles. Although, the S-Z syst em is considered to be characteristic of the Poaceae as a whole, only a limited number of species, mainly temperate, have been studied (Helpson-Harrison, 1982; MartinezReina and Vogel, 2002). It is also
8 characteristic of this system that it does not break down on the polyploidy level (Lundqvist, 1957). In 1970, Burton found that do ubling the chromosome number did not improve the self-fertility of a highly self-incompatible di ploid clone of bahiagrass. Studying different tetraplo id introductions, Burton (1948) found that several bahiagrass tetraploids set the same amount of seed under crossand self-pollination, and were pseudogamous. In addition, most of the seed obtained by pollinating a male-sterile white stigma plant with pollen from other tetr aploids gave rise to plants exactly like the female parent. The few obtained hybrids showed red stigma and 60 chromosomes, indicating that red was dominant over white, and that unreduced eggs originated from the hybrids. Burton suggested that one type of apomixis was the method of reproduction for the tetraploid races. Later cytoembryologica l studies revealed that the apomictic mechanism in bahiagrass was apospory (B urton and Forbes, 1 960). Pollination is essential for seed formation because the e ndosperm develops after the polar nuclei are fertilized. Endosperm development appears to occur regardless of th e ploidy level of the pollen donor. Moreover, pollen of cl osely related species, such as Paspalum cromyorrhizon Trin., also can produce fertile seed with hybrid endosperm, although the embryo results from the maternal genotype (Quarin, 1999). Triploid plants occur spor adically in nature (Gould, 1966; Quarin et al., 1989; Tischler and Burson, 1995). Because triploids reproduce by apomixis, the odd ploidy level is maintained through generations. When these triploids are pollinated by a diploid, some of the progeny are new apomictic tetrap loids that result by the fertilization of an unreduced egg cell (Burton and Hanna, 1986; Quar in et al. 1989; Daurelio et al., 2004).
9 The most recent and convincing study of the genetic system controlling apomixis vs. sexuality in bahiagrass indicates that se xuality is a recessive trait and that plants without aposporuos development are homozygous recessive. Although there was an excess of sexual (3 sexual: 1 apomictic) indi viduals in the progeny, the authors suggested that a single dominant gene controls apospory (Martinez et al., 2001). They hypothesized that a pleiotropic lethal eff ect with incomplete penetrance of the dominant allele was the reason for the distortion in the segregation pa tterns. More recently Stein et al. (2004) using molecular and cytogenetic technique s found preferential chromosome pairing (disomic inheritance) in the chromosome segment related to apopory. The presence of chromatin bridges with accompanying fragments indicated the existence of an inversion in that chromosome segment. This may be re sponsible for the distorted segregation ratio observed in the transference of apospory a nd the suppression of r ecombination observed around the apospory controlling locus. However, it is known that a plant can inherit all the genetic factors involved in the developmen t of a mature aposporous sac, but it can fail to produce seed (AcuÃ±a et al ., 2004). Thus, it is evident th at there are more genetic factors needed for the devel opment of mature and viable seed through an apomictic event. Also, controversial re sults indicate that a rise of ploidy level induces the expression of apomixis in Paspalum hexastachyum (Quarin and Hanna, 1980), and P. notatum (Quarin et al., 2001). These results indi cate that the gene s involved in the apomixis expression are present at th e diploid level (Quarin et al., 2001). Breeding Bahiagrass Breeding Diploids Sexual diploid germplasm has been improve d using two approaches. The discovery of self-incompatibility in most Pensacola plants suggested that commercial F1 hybrid
10 seed could be produced by harvesting year afte r year all seed produced in a field planted to alternate strips of two se lf-sterile cross-fertile clones (Burton, 1984). Two such clones, whose F1 hybrids yielded 17% more dry matter than a Pensacola check in clipping tests were sought and found by screening several dialleles. A pilot seed production field planted vegetatively produced seed to plant in pasture that gave 17% more live weight gain per acre than Pensacola bahiagrass when grazed for 4 years. Two of these hybrids, Tifhi 1 (Hein, 1958) and Tifhi 2, were released but the hand labor required to establish the seed fields and the cold injury to one pare nt clone in a severe wi nter kept them from becoming important on the farm (Burton, 1984). In 1961, G. W. Burton collected a mixture of Pensacola bahiagrass seed from 39 farms and from 75 clones of Tifhi 2 to initiate what is considered the longest effort to improve this forage crop (Burton, 1984; Gates et al., 2004). Using an innovative approach specially adjusted to the characterist ics of this species calle d restricted recurrent phenotypic selection (RRPS) he completed 23 cycles with the specific objective of increasing herbage mass. Tifton 9 was released as a cultivar from the ninth selection cycle (Burton, 1989). Because the selection was based on above ground herbage accumulation of individual plants growing in a noncompetitive environment, more advanced selection cycles (subsequent to cycle 9) were not persistent in a grazing environment (Gates et al., 2004). Breeding Tetraploids Because the naturally occu rring tetraploid bahiagrass types reproduce by apomixis, improvement using conventional breeding techniques has not been possible. All tetraploid cultivars released in USA were sel ected superior apomictic ecotypes (Gates et al., 2004). Beginning in 1936 and c ontinuing for more than 30 years, some 80 ecotypes of
11 bahiagrass from Brazil, Uruguay, Paraguay, a nd northern Argentina were collected and studied. Most of the 80 ecot ypes evaluated were typical, low-growing broadleafed common types that failed to survive a severe winter at Tifton, Georgia. All the common types were obligate apomictics and tetraploid s. Â‘ArgentineÂ’ and Â‘Paraguay 22Â’, PI 148996 and PI 158822 respectively, were the most wint erhardy and the most promising ecotypes. Compared with common bahiagrass in a 3year clipping test, Argentine produced twice as much dry matter and protein as common. Paraguay was more drought tolerant than Argentine and much more drought and frost resistant than common, Argentine has been used for lawn turf in Florida and both grasses are used for pastures (Burton, 1992). Ten sexual tetraploids were created by doubling the chromosomes of the sexual Pensacola bahiagrass using colchicine (F orbes and Burton, 1957). Two more genotypes, Q4188 (PI 619631), and Q4205 (PI 619632) were more recently selected in Argentina as sexual lines, introduced, and registered in US A (Quarin et al., 2003). Both originated from one of the 10 plants orig inally doubled in USA. This doubled tetraploid was crossed with an apomictic tetraploid generating a pl ant known as Q3664 which was determined to be facultative apomictic with high sexual expression. Q4188 was originated from a cross between Q3664 and an obligate apomictic genotype from Brazil. Q4205 was generated by self-pollination of Q3664. In 1956, crosses were made between three of the original sexual lines and two natural apomictic tetraploids. Also, the sexual F1s were used to generate apomictic F2 plants (Burton and Forbes, 1960). Thirty six apomictic F1 hybrids yielded 26% more than their 42 apomictic F2s. The top hybrid, after topping a 5year small plot test was named Tifton 54 (Burton, 1992). In 1983, G. W. Burt on generated a Â“sexual femaleÂ” (SF)
12 population by intermating 23 F1 hybrids between 4 sexual white stigma female plants and 6 red stigma apomictic plants. Another population Â“sexual female apomictic maleÂ” (SFAM) population intermated the SF23 populati on with an equal number of culms from six red stigma apomictics (T ifton 54, and Paraguay 22 were among them). The SF and SFAM populations were advanced through thre e RRPS generations. In 1987, the 6 plants of the 165 entries of each population were planted in 3-plant plots to permit the uniformity test for apomixis. Fresh weights of each plant were taken. Only 9 of the top yielding SF entries were apomictic, whereas 19 of the top SFAM entries were apomictic. In a replicated 3-year small plot test s eeded May 4, 1989, the best apomictic from the SFAM population, plant # 7, yiel ded significantly more dr y matter than Argentine bahiagrass. It also surpassed Argentine in er got resistant and percen t seed set (Burton, 1992). More recently, this plant # 7, err oneously called Tifton 7, produced superior yields than Argentine and Paraguay 22 in a 2-years experiment in Florida (Muchovej and Mullahey, 2000). To initiate a breeding program for the ba hiagrass tetraploids it is necessary to complete the reproductive characterizati on of the available lines. A complete characterization, including selfand cross-fe rtility and sexual expression, is needed for the artificially induced tetrap loids obtained by Quesenberry and Smith (2003). Selfand cross-fertility is needed to be determin ed for the sexual lines Q4188 and Q4205. Also, doubled tetraploids and diploids can be compar ed to determine the effect of chromosome duplication on the general fertility. In contrast , the degree of apomixis expression and the fertility of the natural tetraploids should also be determined.
13 The variability generated from crosses between selected sexual and apomictic tetraploids can be quantified based on the evaluation of agronomic characteristics. Several parental combinations should be sc reened for those matings that can generate bigger progeny with desirable characteristics. Sexual, facultative, and apomictic plants should be identified among the progeny usi ng a quick an inexpensive method. The selected sexual progeny can be used for furt her crosses with other superior apomictic F1s or with the original parents to generate new segregating populations. Selected apomictic plants in the F1 should be considered potential new cultivars and used for further evaluations. Objectives To determine and compare the fertility of the current dipl oid and tetraploid cultivars, artificially induced tetraplo ids, and other tetraploid ecotypes under different pollination methods. To determine the mode of reproduction of a group of natural and artificially induced bahiagrass tetraploids. To generate and evaluate a segregatin g population by hybridizing selected sexual and apomictic bahiagrass tetraploids.
14 CHAPTER 3 FERTILITY CHARACTERIZATION OF DIPLOID AND TETRAPLOID BAHIAGRASS Introduction Paspalum notatum FlÃ¼gge, bahiagrass, is currently the most important pasture and utility turf species of Florida. It is a perennial rhizomatous grass extremely tolerant to intense defoliation and mismanagement (Gates et al., 2004). It is also highly diverse containing races with different ploidy leve ls. Diploids and tetraploids have been introduced in southern USA and extensively cu ltivated for more than 60 years (Burton, 1967; Burton, 1992). Diploids reproduce se xually (Burton, 1955), while tetraploids reproduce asexually by apomixis (Burton, 1948). Most of the studied diploids set low amount of seed when self-pollinated, but set more seed when crosspollinated (Burton, 1955). Tetraploids previously studied did not di ffer in the amount of seed produced when selfand cross-pollinated (Burton, 1948). Because of their different reproductive characteristics, different breedi ng approaches have been used for diploids and tetraploids. Burton (1989) released a cultiv ar called Tifton 9, after nine cy cles of restricted recurrent phenotypic selection (RRPS) us ing diploid material, known as Pensacola bahiagrass. In addition to increased producti on this cultivar differs from the original Pensacola bahiagrass by its more upright growth ha bit and increased shorter day dry matter production (Gates et al., 2004). All tetraploid cultivars released in USA were superior apomictic ecotypes selected from introduced germplasm (Gates et al., 2004). A segregating population can be
15 generated for breeding purposes or for ge netic analyses by making crosses between induced sexual tetraploid plants and apomictic tetraploid plants. Sexual tetraploids have been generated by treating both diploid seed a nd tissue cultured calluses with colchicine (Forbes and Burton, 1961; Quarin et al., 2001) . In a more recent attempt to induce polyploidy, Quesenberry and Smith (2003) ge nerated over 300 tetraploids by treating callus cultures from Tifton 9 with differe nt chromosome duplication treatments. This new bahiagrass germplasm in The USA opens new avenues for use in crop improvement research. Before the current germ plasm can be efficiently used for breeding purposes, its reproductive behavior needs to be carefully studied and characterized. The objectives of this work were to determine th e fertility of current diploid and tetraploid cultivars, induced tetraploids, and other a pomictic ecotypes under different pollination methods, and to compare the fertility of diploi d, and induced tetraploid bahiagrasses. Materials and Methods Plant Material Diploids Two diploid bahiagrass populat ions, identified as Pensacola (WPG: Wide Gene Pool, obtained from Dr. G. Burton, Coastal Plain Experimental St ation, Tifton, GA) and Tifton 9, were used for this study. Twenty fi ve genotypes were randomly collected from each population, divided into two clones, transp lanted, and grown in pots in a greenhouse from the summer of 2004 to the end of the summ er of 2005. The five least self-fertile and the five most self-fertile genotypes (based on 2004 results) from each population were grown in the field during the summer of 2005.
16 Tetraploids Twenty of the most vigorous plants from a population of 300 artificially induced tetraploids (AIT) (Quesenberry and Smith, 2003) were divided into two clones and grown in the field and greenhouse from the summer of 2004 to the end of the summer of 2005. Two sexual tetraploid lines (STL) from Argentina, Q4188 and Q4205 (Quarin et al., 2003), and apomictic accessions (see chapte r 4), Argentine, Tifton 7, PI 315733, and PI 315734 were also divided into two clones and grown in a greenhouse from the summer of 2004 to the end of the summer of 2005. Q4188, Q4205, Argentine, and Tifton 7 were also grown in the field during the summer of 2005. Pollination Techniques Self-pollination was accomplished by isola ting two inflorescences from each clone in glassine bags supported with stakes just before anthesis. The bags were shaken each day during anthesis to favor pollination. Mutu al pollinations between pairs of different diploid genotypes were accomplished by en closing a panicle from each of the two genotypes in a glassine bag. In addition, in florescences from sexual and apomictic tetraploids were also enclosed in glassine bags in an attempt to determine the crossfertility of the sexual plants. Care was taken to choose panicles of equal maturity (ready to flower). Bags were shaken each day duri ng anthesis. Inflorescences from diploids, and tetraploids were individually bagged afte r anthesis avoiding seed losses from openpollinated inflorescences. The number of pollin ated spikelets is described for each case (Appendix). Seed Set After a month, the inflorescences were harvested, thresh ed, and spikelets containing caryopses were separated from em pty spikelets using an air column. Percent
17 seed set was calculated by dividing the num ber of spikelets cont aining caryopses by the total number of pollinated sp ikelets and multiplying by 100. Statistical Analysis Data were analyzed using PROC GLM of PC SAS (SAS Institute, 2003) as a completely randomized design with two repli cations of each genotype in each location. Statistical comparison of percent seed set was te sted in the following order: 1) individual diploid populations (Pensacola and Tifton 9) compared for self-, cross-, and openpollination, 2) among genotype comparison fo r Pensacola and Tifton 9, and for each pollination method, 3) pollination method (cross or openvs. self-fertility) compared over both diploid populations, 4) individual genotype am ong the 20 AIT compared for selfvs. open-pollination, 5) pollination met hod (selfvs. open-pol lination) for these 20 AIT, 6) population (all diploids vs. 20 AIT) under self-pollination, 7) genotype comparison the two STL under selfand cr oss-pollination, 8) pollination method for these two lines, 9) population (STL and AIT) under selfand cro ss-pollination, 10) genotype for the apomictic tetraploids under self-pollination, and 11) pollination method for Argentine and Tifton 7. The significance of years, locations (field and greenhouse), and the corresponding interactions were test ed when needed. DuncanÂ’s Multiple Range Test was used as a mean separation procedure when more than two means were compared, while least significant difference (LSD) was used when two means were compared. Unless otherwise stated in the text, all differences refer to significance at P < 0.05. The number of pollinated spikelets and th e average seed set for each genotype is described (Appendix), as well as the overall mean s, and coefficients of variation (CV) are described for each location (greenhouse and field) and for each year (2004 and 2005).
18 Results and Discussion Diploid Bahiagrass No significant differences were observe d between Pensacola bahiagrass and Tifton 9 in terms of seed set under greenhouse condi tions for selfand cro ss-pollination (Tables 3-1, 3-2 and 3-29). Also, these two populati ons did not differ for seed set when open-pollinated under field conditions (Tab le 3-3). Years were significant for self-fertility seed set, with 2005 percent seed set being higher than 2004 (Appendix). There were no interactions be tween population and year for se lfor cross-fertility. The mean percent seed set for self-, cross-, and open-pollination was 11, 60, and 39 respectively (Table 3-29). Although 9 cycles of RRPS affected the expression of several characteristics in addition to herbage mass, such as daylength sensitivity, and growth habit, it did not affect fertil ity. These results represent str ong evidence that fertility and herbage mass are independent traits, and that it is possible to sele ct for one of them without affecting the expression of the other. No significant differences were obser ved for diploid genotypes (Pensacola bahiagrass and Tifton 9) when comparing self-fertility between field and greenhouse results (Table 3-4). These results indicate th at greenhouse data accurately represented the self-fertility of this crop under field conditions. Wind, rai n, adverse climatic conditions in general make self-fertility determinations very difficult under field conditions. However, based on the results, field experiments for se lf-fertility determinations were deemed unnecessary when greenhouse data is available. It should be poi nted out that these results are for only one year as the bagged heads for self-fertility were destroyed by hurricane Frances in 2004.
19 The average percent seed set for self-pol lination was 11 for Pensacola bahiagrass and Tifton 9 (Table 3-29). However, significant differences in terms of self-fertility were observed among the genotypes of each diploi d population (Pensacola bahiagrass and Tifton 9) (Tables 3-5 and 3-6). There were no interaction between genotype and year for either cultivar. The percent seed set vari ed on average from 0 to 44 for Pensacola bahiagrass and from 0 to 35 for Tifton 9 (Appe ndix) indicating that there was a marked variability among the genotypes. More than 75 % of the genotypes set less than 15% of seed (Table 3-7). However, several genot ypes can be considered as moderately self-fertile. Self-steri lity should be considered as ch aracteristic of a good progenitor avoiding high levels of inbreed ing in segregating populations. The average percent seed set under cro ss-pollination was 60 for Pensacola and 59 for Tifton 9 (Table 3-29). The percent seed set varied from 8 to 99 for Pensacola, and from 6 to 95 for Tifton 9 (Appendix). Alt hough significant differences were observed among the genotypes in terms of self-fertility, no significant differences were observed in terms of cross-fertility for both populations (Pensacola and Tifton 9) (Tables 3-8 and 3-9). The results indicated that only the self-fertility varied among the genotypes of diploid populations. This hypothesis was co rroborated by the results obtained from Pensacola bahiagrass under open-pollination in the field (Table 3-10). However, some differences were observed among genotypes of Tifton 9 under open-pollination (Table 3-11). This differs from greenhouse results, and could be attribut ed to the high ergot ( Claviceps paspali Stevens & Hall) occurrence observed summer 2005. Because no differences were detected betw een Pensacola bahiagrass and Tifton 9 in terms of general fertility both populations can be analyzed as a singl e population for other
20 comparisons. Significant differences were observed for seed set in the greenhouse between selfand cross-fertility across both diploid bahiagrass cultivars, indicating that they are predominantly cross-poll inated with low but variable expression of self-fertility (Tables 3-12 and 3-29). This hypothesis was corroborated by the differences observed between openand self-fertili ty in the field (2005 data only) (Tables 3-13 and 3-29). Tetraploid Bahiagrass Significant differences ( P < 0.0824) were detected among induced tetraploid genotypes self-fertility (Tab le 3-14) in the greenhouse dur ing two years. The average percent seed set was 2 (gree nhouse and field), varying from 0 to 12 in the greenhouse, and from 0 to 6 in the field (Table 3-15). Th e results were expected because diploids in general showed marked vari ation in terms of self-f ertility among genotypes. The self-fertility experime nt was repeated in the fi eld during 2005. No significant differences for self-fertility were observed be tween both locations (g reenhouse and field), validating greenhouse conditions for these ex periments (Table 3-16). In contrast, a hurricane destroyed the field experiment dur ing 2004 showing that field conditions are many times inappropriate fo r self-fertility analysis. High variability was detected among the AIT genotypes for seed set under open-pollination conditions (Table 3-17). The average percent seed set was 14, varying from 8 to 35 (Table 3-15) i ndicating a reduction in overall fertility for AIT. However, some of them were moderately fertile a nd should be considered as potentially good sexual females for crosses using apomictic genotypes as pollen donor. In agreement with the two diploid po pulations this group of AIT produced significantly more seed under open-pollination versus self-pollination (Tables 3-18 and
21 3-29). This was an indication that the reproductive characte ristics of diploid bahiagrass were not altered by chromosome duplication. The diploid population set significan tly more seed than the AIT under self-pollination conditions (Table 3-19). Irre gular meiotic behavior was indicated as the reason for low fertility for the first genera tion of induced autotetraploids (Burnham, 1962). In contrast, Burton et al. (1970) showed that doubling chromosome number did not improve the self-fertility of seven selected highly incompatible Pensacola bahiagrass clones. The AIT clones used for this study may also have originated from highly self-incompatible genotypes (seed) that surv ived the chromosome doubling. That is a reasonable expectation since most of the dipl oid genotypes showed seed set of less than 15% (Table 3-7). The sexual tetraploid lines Q4188 and Q 4205 (STL) exhibited similar seed set under self-pollination (Table 3-20). Similar to the AIT and diploid populations, the seed set for these two lines was not significantly different between th e greenhouse and field (Table 3-21). Also, the two STL had similar s eed set when cross-pollinated (Table 3-22). The similarities between these two lines we re expected because they had a common origin and were closely rela ted (Quarin et al., 2003). The selfand cross-fertility of the two S TL were not significantly different (Tables 3-23 and 3-29). The average percent seed set was 21 under self-pollination, and 27 under cross-pollination. Contrasting with the AIT us ed in this study a considerable amount of potential self progeny can be expected when the two STL are used as female parents without emasculation for breeding purposes . Emasculation techniques need to be considered for the use of these lines in cro sses, otherwise a high pe rcent of their progeny
22 will result from self-pollination. The presen ce of inbreds among progeny represents a bias in the progeny evaluation, waste of money and time, and decreases the probability of identifying superior hybrid combinations. The two STL set significantly more seed than the diploids and AITs when self-pollinated (Tables 3-19 and 3-29). This indicates that the breeding process for developing these lines indirectly increased their self -fertility, or that an original line parent was self-fertile. However, the fertil ity of them was not increased in general because they did not set more seed than the other AIT when cross-pollinated (Table 3-24). Homogeneous self-fertility was observe d among the apomictic genotypes (Table 3-25). The average percent seed set unde r self-pollination wa s 24. There is little variability among these apomictic lines resulting in limited potential for increasing the fertility. They also set a similar amount of s eed in the field and greenhouse (Table 3-26). No differences were detected for s eed set under open-pollination between Argentine and Tifton 7 (Table 3-27 and Appe ndix). Burton (1992) reported that Tifton 7 produced more seed per hectare than Argentin e, so a higher amount of reproductive units per unit area should be the reason for that superiority. Argentine and Tifton 7 produced significantly more seed when open-pollinated than when self-pollinated under field conditions (Table 3-28) indi cating that mixed populations should produce higher seed yields. The average percent seed se t for these two genotypes was 30 under self-pollination and was 36 under open-pollina tion (Table 3-29). The results are in contrast with previous reports of no difference between self-f ertility and cross-fertility of
23 the apomictic lines (Burton, 1948) . Although the differences were statistically significant they do not represent a large practical difference. Summary and Conclusions Because Paspalum notatum FlÃ¼gge, bahiagrass, is of primary importance in cattle production systems as well as for the turf indus try in Florida there is a marked need for new cultivars. However, the available germplasm in USA needs to be characterized before plant breeders can use it efficiently. The fertility studies of di ploid and tetraploid bahiagrass germplasm reported here showed that: Pensacola bahiagrass and Tifton 9 are not different in terms of self-, and cross-fertility indicating that phenotypic selection for herb age mass did not affect the fertility of this species. Individual diploid genotypes differed in term s of self-fertility but not in terms of crossfertility. They produced significantly more seed when cross-pollinated than when self-pollinated indicating that bahiagrass di ploid germplasm can be considered as a cross-pollinated crop with low but va riable levels of self-fertility. The first generation of induced tetraploids showed low fertility. They also produced more seed under cross-pollination than under se lf-pollination. Low leve ls of self-fertility and moderate levels of cro ss-fertility of selected indi vidual genotypes make them good potential sexual parents for use in crosse s with apomictic lines as pollen donors. The two sexual tetraploid lines known as Q4188 and Q4205 did not differ from the other AITs in terms of cross-fertility. However, they exhibited similar seed set when selfor cross-pollinated indicating that emasculati on techniques will be needed for the use of these two lines as females to avoid high levels of selfing in their progeny.
24 The apomictic genotypes were homogeneous in terms of selfand cross-fertility. Although a small group was used, the low variabili ty in terms of seed set indicated that the number of reproductive units per area refl ects differences in seed yield among these lines. Argentine and Tifton 7 produced more seed under open-pollination than under self-pollination. Extraneously, the presence of hybrid endosperm should be considered as a positive factor for apomictic seed produc tion. However, a 6% difference will hardly justify the complications of a polycu lture for commercial seed production. Results show that greenhouse fertility da ta correlated well with field data. The complications of isolating the inflorescences in the field trying to avoid bias in the results related with a different environment were de termined to be unn ecessary when greenhouse data was available.
25 Table 3-1. Analysis of variance (ANOVA) table showing the si gnificance of diploid populations (Pensacola bahiagrass and Tifton 9) on seed set under selfpollination during 2004 and 2005, in the greenhouse Source DF Type I SS Mean Square F Value Pr > F Population 1 74.91 74.91 0.44 0.51 Year 1 1770.08 1770.08 10.29 0.0015 Pop*Year 1 569.70 569.70 3.31 0.07 Error 217 37336.28 172.06 Table 3-2. ANOVA table showi ng the significance of the di ploid population (Pensacola bahiagrass and Tifton 9) on seed se t under cross-pollination during 2004 and 2005, in the greenhouse Source DF Type I SS Mean Square F Value Pr > F Population 1 36.84 36.84 0.07 0.80 Year 1 613.86 613.86 1.08 0.3 Pop*Year 1 15.84 15.84 0.03 0.87 Error 105 59421.58 565.91 Table 3-3. ANOVA table showi ng the significance of diploid population (Pensacola bahiagrass and Tifton 9) on seed se t under open-pollination during 2005, in the field Source DF Type I SS Mean Square F Value Pr > F Population 1 225.45 225.45 0.44 0.51 Error 34 17266.00 507.82 Table 3-4. ANOVA table for the effect of lo cation (field vs. greenhouse) on seed set for diploid bahiagrass (Pensacola bahiagra ss and Tifton 9) under self-pollination during 2005 Source DF Type I SS Mean Square F Value Pr > F Location 1 283.01 283.01 1.16 0.28 Error 145 29757.88 205.23 Table 3-5. ANOVA table showi ng the significance of genotype on seed set for Pensacola bahiagrass under self-pollination during 2004 and 2005, in the greenhouse Source DF Type I SS Mean Square F Value Pr > F Genotype 24 11517.40 479.89 6.45 <0.0001 Year 1 1796.25 1796.25 24.15 <0.0001 Genotype*Year24 2062.62 85.94 1.16 0.3149 Error 65 4834.34 74.37
26 Table 3-6. ANOVA table showi ng the significance of genotype on seed set for Tifton 9 under self-pollination during 2004 and 2005, in the greenhouse Source DF Type I SS Mean Square F Value Pr > F Genotype 25 8225.70 329.03 2.51 0.0024 Year 1 131.76 131.76 1.00 0.32 Genotype*Year24 3432.40 141.02 1.09 0.3839 Error 54 7079.07 131.09 Table 3-7. Range in self-pollinated seed set on diploid bahiagrass genotypes Percent seed set 0.0-5.0 5.1-10.0 10.1-15.0 15.1-25.0 25.1-40.0 Number of Plants 20 11 8 8 4 Table 3-8. ANOVA table showi ng the significance of genotype on seed set for Pensacola bahiagrass under cross-pollination during 2004 and 2005, in the greenhouse Source DF Type I SS Mean Square F Value Pr > F Genotype 23 10943.73 475.81 0.66 0.84 Year 1 477.66 477.66 0.66 0.42 Genotype*Year17 4985.00 293.29 0.41 0.97 Error 26 18784.64 722.49 Table 3-9. ANOVA table showi ng the significance of genotype on seed set for Tifton 9 under cross-pollination during 2004 and 2005, in the greenhouse Source DF Type I SS Mean Square F Value Pr > F Genotype 21 12889.88 613.80 0.72 0.75 Year 1 345.87 345.87 0.40 0.54 Genotype*Year8 3062.14 382.77 0.45 0.87 Error 10 8561.38 8561.38 Table 3-10. ANOVA table showing the signi ficance of genotype on seed set for Pensacola bahiagrass under open-po llination during 2005, in the field Source DF Type I SS Mean Square F Value Pr > F Genotype 9 4321.42 480.16 1.14 0.4239 Error 9 3788.58 420.55
27 Table 3-11. ANOVA table showing the significance of genotype on seed set for Tifton 9 under open-pollination during 2005, in the field Source DF Type I SS Mean Square F Value Pr > F Genotype 8 7250.39 906.29 3.8 0.0382 Error 8 1905.61 238.20 Table 3-12. ANOVA table showi ng the significance of the pollination method (selfand cross-pollination) on seed set fo r diploids during 2004 and 2005, in the greenhouse Source DF Type I SS Mean Square F Value Pr > F Method 1 167862.12 167862.12 480.31 <0.0001 Year 1 3540.51 3540.51 10.13 0.0016 Method*Year 1 158.05 158.05 0.45 0.50 Error 326 113933.21 349.49 Table 3-13. ANOVA table showi ng the significance of the pollination method (selfand open-pollination) on seed set for diploi d bahiagrass during 2005, in the field Source DF Type I SS Mean Square F Value Pr > F Method 1 9553.78 9553.78 27.70 <0.0001 Error 74 25520.85 344.88 Table 3-14. ANOVA table showi ng the significance of genotypes on seed set for the AIT under self-pollination during 2004 and 2005, in the greenhouse Source DF Type I SS Mean Square F Value Pr > F Genotype 21 246.08 11.72 2.18 00824 Year 1 55.94 55.94 10.42 0.0072 Genotype*Year5 38.59 7.72 1.44 0.2799 Error 12 64.41 5.37
28 Table 3-15. Seed set under selfand open-pol lination for 23 AIT genotypes in the field. Open-pollinations were accomplishe d during 2004 and 2005, while selfpollinations only during 2005 Identification Self-polli nation Open-pollination No ------------% seed set-------------2 3.0 14.7 5 2.8 20.9 6 1.5 23.5 2-2-7 0.2 35.3 35 2.6 10.8 52 1.4 8.4 55 0.4 8.9 56 0.3 13.2 71 2.4 16.4 73 1.7 9.5 74 2.8 7.5 77 0.9 8.1 78 2.7 10.7 99 2.0 13.8 104 5.3 14.5 105 1.9 10.2 106 1.7 17.3 110 1.5 13.0 111 4.6 13.0 120 1.5 10.4 121 0.4 10.6 4-36-1 5.5 9.0 5-12-9 4.3 9.2 Average 2.2 13.4 Table 3-16. ANOVA table showing the significance of the expe rimental location (field and greenhouse) on seed set for the AIT under self-pollination during 2005 Source DF Type I SS Mean Square F Value Pr > F Location 1 5.28 5.28 1.11 0.2963 Error 72 343.67 4.77 Table 3-17. ANOVA table showi ng the significance of genotype for the AIT on seed set under open-pollination during 2004 and 2005, in the field Source DF Type I SS Mean Square F Value Pr > F Genotype 33 4414.73 133.78 2.66 0.0009 Year 1 96.22 96.22 1.91 0.1726 Genotype*Year22 589.56 26.80 0.53 0.9453 Error 50 2512.85 50.26
29 Table 3-18. ANOVA table showi ng the significance of the pollination method (openand self-pollination) on seed set for th e AIT during 2004 and 2005, in the field Source DF Type I SS Mean Square F Value Pr > F Method 1 6711.47 6711.47 165.51 <0.0001 Year 1 82.56 82.56 2.04 0.1550 Method*Year 1 15.49 15.49 0.38 0.5372 Error 326 8226.50 40.52 Table 3-19. ANOVA table showi ng the significance of population (diploids, AIT, and STL) on seed set under self-polli nation during 2004 and 2005, in the greenhouse Source DF Type I SS Mean Square F Value Pr > F Population 2 3904.19 1952.09 13.21 <0.0001 Year 1 1358.79 1358.79 9.20 0.0027 Pop*Year 2 459.56 229.78 1.56 0.2130 Error 263 38851.09 147.72 Table 3-20. ANOVA table showi ng the significance of genotype on seed set for Q4188 and Q4205 under self-pollination during 2004 and 2005, in the greenhouse Source DF Type I SS Mean Square F Value Pr > F Genotype 1 177.66 177.66 2.82 0.1685 Year 1 7.41 7.41 0.12 0.7489 Genotype*Year1 76.226 76.226 1.21 0.3331 Error 4 252.13 63.03 Table 3-21. ANOVA table showing the significance of the expe rimental location (field and greenhouse) on seed set for Q4188 and Q4205 under self-pollination during 2005 Source DF Type I SS Mean Square F Value Pr > F Location 1 43.25 43.25 0.65 0.4499 Error 6 397.38 66.23 Table 3-22. ANOVA table showi ng the significance of genotype on seed set for Q4188 and Q4205 under cross-pollination during 2004, in the greenhouse Source DF Type I SS Mean Square F Value Pr > F Genotype 1 5.52 5.52 1.04 0.4145 Error 2 10.59 5.29
30 Table 3-23. ANOVA table showi ng the significance of pollination method (selfand cross-pollination) on seed set fo r Q4188 and Q4205 during 2004, in the greenhouse Source DF Type I SS Mean Square F Value Pr > F Method 1 110.11 110.11 2.05 0.1742 Error 14 752.25 53.73 Table 3-24. ANOVA table showing the signi ficance of population (STL and AIT) on seed set under cross-pollination during 2004, in the greenhouse Source DF Type I SS Mean Square F Value Pr > F Population 1 61.43 61.43 1.12 0.3071 Error 14 765.29 54.66 Table 3-25. ANOVA table showi ng the significance of genotype on seed set for the apomictic tetraploids (Argentine, Tifton 7, PI 315733, and PI 315734), under self-pollination during 2004 and 2005, in the greenhouse Source DF Type I SS Mean Square F Value Pr > F Genotype 3 1632.59 544.20 1.46 0.3051 Year 1 140.37 140.37 0.38 0.5787 Genotype*Year3 286.59 95.53 0.26 0.8545 Error 7 2607.18 372.45 Table 3-26. ANOVA table showing the significance of the expe rimental location (field and greenhouse) on seed set for the apomictic tetraploids under selfpollination during 2005 Source DF Type I SS Mean Square F Value Pr > F Location 1 284.46 284.46 1.28 0.2828 Error 11 2453.79 223.07 Table 3-27. ANOVA table showing the signi ficance of genotype on seed set for Argentine and Tifton 7 under open-po llination during 2005, in the field Source DF Type I SS Mean Square F Value Pr > F Genotype 1 23.60 23.60 2.15 0.2163 Error 4 43.87 10.97 Table 3-28. ANOVA table showing the signi ficance of the pollination method (Selfpollination and open-pollination) on s eed set for Argentine and Tifton 7 during 2005, in the field Source DF Type I SS Mean Square F Value Pr > F Method 1 122.88 122.88 5.48 0.0413 Error 10 224.30 22.43
31 Table 3-29. Comparisons of seed set means from diploids (Pensacola bahiagrass and Tifton 9), AIT, STL, and apomictic tetraploids (Argentine and Tifton 7), under self-, cross-, and open-pollinat ion in the field and greenhouse during 2004 and 2005 Pollination method Self Cross Open Identification Greenhouse Field Greenhouse Field Tifton 9 11.4 Â† 17.7 60.3 Â‡ 41.8 Pensacola 10.6 15.6 59.1 36.8 Diploids 11 Â§# 16.8 Â¶ 60.1 39.1 AIT 2.2 2.2 Â†Â† 22.2 13.6 STL 22.5 Â‡Â‡ 20.7 26.7 Apomictic tetraploids 34 30 Â§Â§ 49 36.2 Â† LSD0.05 = 3.5 for mean comparison of population (Tifton 9 and Pensacola) seed set under self-pollination in the greenhouse on 2004 and 2005. Â‡ LSD0.05 = 9.3 for mean comparison of population (Tifton 9 and Pensacola) seed set under crosspollination in the greenhouse on 2004 and 2005. Â§ LSD0.05 = 4.3 for mean comparison of diploids (Tifton 9 and Pensacola) seed set under selfand crosspollination in the greenhouse on 2004 and 2005. Â¶ LSD0.05 = 8.5 for mean comparison of diploids (Tifton 9 and Pensacola) seed set under selfand openpollination in the field on 2005. # DuncanÂ’ test 0.05 = 7.7 for mean comparison of popu lation (diploids, AIT, and STL) seed set under selfpollination in the greenhouse on 2004 and 2005. Â†Â† LSD0.05 = 1.7 for mean comparison of AIT seed set under selfand open-pollination in the field on 2005. Â‡Â‡ LSD0.05 = 9.1 for mean comparison of STL seed set unde r selfand cross-pollination in the greenhouse on 2004. Â§Â§ LSD0.05 = 6.1 for mean comparison of apomictic tetrap loids (Argentine and Tifton 7) seed set under selfand open-pollination in the field on 2005.
32 CHAPTER 4 REPRODUCTIVE CHARACTERIZATION OF SEVERAL NATURAL AND INDUCED BAHIAGRASS ACCESSIONS Introduction Paspalum notatum FlÃ¼gge, bahiagrass, a vigorous a nd well adapted species, is the predominant pasture and utility turf in Florid a. It is characterized by having races with different ploidy levels. The tetraploid race ( 2n=4x=40) is the predominant form in open areas and savannas of the tropical and s ubtropical Americas, while the diploid race (2n=2x=20) grows naturally in a small area of northeastern Argentina (Gates et al., 2004). Both races have been introduced into the southern USA and are extensively cultivated as forage and turf for more than 60 years (Burton, 1967; Burton, 1992). Diploids reproduce sexually a nd are cross-pollinated (Bur ton, 1955), while tetraploids reproduce by aposporous apomixis and are pseudogamous (Burton, 1948). At anthesis, diploid spikelets show a single meiotic embr yo sac per ovule. This meiotic embryo sac is distinguished by having the egg apparatus at the micropylar end, a la rge central cell with two nuclei, and a mass of antipodal cells at the chalazal end. The ovules from apomictic plants bear either one or usually several a posporous embryo sacs, which are characterized by an egg cell, one or two synergids, a large central cell with two nuclei, and the lack of antipodals. In some ovules the meiotic sac develops together with one or more aposporous sacs. Facultative apomictic pl ants produce ovules containing aposporous sacs, and others containing a single meiotic embryo sac (Quarin et al., 1984; Martinez et al., 2001).
33 Sexual tetraploids have been generated by co lchicine treatment of diploid seed and callus (Forbes and Burton, 1961; Quarin et al., 2001). In a more recent attempt Quesenberry and Smith (2003) generated ove r 300 tetraploids by treating embryogenic callus from Tifton 9 with three different ch romosome doubling treatments (colchicine, trifluralin, and oryzalin). Quar in et al. (2001) detected th at two out of three induced tetraploids were facultative apomictic plants indicating that an unexpressed gene(s) for apomixis exists at the diploid level. All tetraploid cultivars released in the USA were superior apomictic ecotypes that were selected from introduced germplasm (G ates et al., 2004). A segregating tetraploid population can be generated for breeding purpos es or genetic analyses by making crosses between induced sexual plants used as female s and apomictic tetraploids used as males. However, the ploidy level, the reproductive characteristics, and the fertility of the selected parents need to be de termined before this approach can be efficiently used. The objectives of this work were to conf irm or determine the ploidy level and the mode of reproduction of 20 of the most vigor ous plants from a population of 300 induced tetraploids, and other promising bahiag rass accessions, and to characterize the reproductive behavior of the cultivars, Argen tine, Wilmington, and th e experimental line Tifton 7. Materials and Methods Plant Material Twenty of the most vigorous genotypes were selected from a population of 300 induced tetraploids. This population was generated by treating Tifton 9 embryogenic callus with different chromosome doubling ag ents, with the purpose of obtaining sexual tetraploids that could be used for breeding tetraploids of this species (Quesenberry and
34 Smith, 2003). Three more plant intro ductions, PI 315732, PI 315733, PI 315734, and one white stigmas (WS) genotype (collected in th e gardens of Corry Village, University of Florida, Gainesville, FL), were selected for the analysis. The cultivars Argentine (PI 148996) and Wilmington (PI 434189) and a breedi ng line Tifton 7 were also included in this study. Ploidy Level Determination Flow cytometry was used to corrobora te the ploidy level of the 20 induced tetraploids, and to determine the pl oidy level of PI 315732, PI 315733, and PI 315734. These three genotypes were selected because of their cold resistance, marked prostrate growth, and narrow leaves, an uncommon characteristic among the tetraploids of bahiagrass. Young leaves were collected a nd approximately 0.5 cm square leaf was immediately chopped in a petri dish contai ning 500Âµl of Partec CyStain UV solution A. The material was well mixed, collected, and filtered (50 Âµm filter). Using 1500 Âµl of Partec CyStain UV solution B the material wa s stained for 3 minutes. The sample was run on a Partec Ploidy Analyzer PA. A sample from known Paspalum notatum diploid and tetraploid lines was used as comparative standards for the analysis. The analyzer was arbitrarily calibrated using a diploid race of P. notatum at 100 units, so a peak for a tetraploid was expected at 200 units. In contrast, young, white inflorescences from WS were fixed in CarnoyÂ’s solution 1 (1 glacial acetic acid: 3 et hanol 95%). After 24 hours the ma terial was placed into a solution of ethanol 70% and stored in the re frigerator. The anthers were extracted from the spikelets and transferred to a slide. A drop of aceto-carmin (1g carmine powder, 45 ml glacial acetic acid, and 55 mL of dH2O) was deposited with the tissue, and the
35 material was broken trying to spread the polle n mother cells (PMCs) in the slide. The preparations were observed and analyzed usi ng a regular light tran smission microscope. Embryo Sac Observations Inflorescences at anthesis (when the embr yo sacs are usually fully developed) were fixed for 24 hours in FAA (18 Ethanol 70%: 1 Fo rmaldehide 37%: 1 glacial acetic acid). The pistils were then dissect ed out of the spikelets and cleared using the Young et al. (1979) technique. The ovules were observed using a differential interference contrast microscope. A minimum of 20 ovules from at least two different inflorescences were observed. Single embryo sacs containing the egg a pparatus, the central cell, and a mass of antipodals at the chalazal end were classified as meiotic. In contrast, multiple or single embryo sacs showing the egg apparatus, the cen tral cell, lack of an tipodals, and variable size and position, were classified as aposporous . Ovules containing bo th types of embryo sacs were classified as facultative aposporous. Results and Discussion Induced Tetraploids Flow cytometric analysis confirmed that the 20 genotypes from the artificially induced population were all tetraploid (2n=4x=40). Eight een of them produced only regular meiotic embryo sacs (Table 4-1; Figure 4-1). Although, tw o of them mostly showed meiotic embryo sacs, they also produ ced a few aposporous s acs (Table 4-1). To hybridize the natural occurring Paspalum notatum tetraploids as males, four main characteristics are needed for the female counterpart: 1) the same ploidy level, 2) high sexual expression, 3) high vigor, and 4) reasonable crossfertility. Successful development of the embryos and seed requires proper endosperm development. The endosperm (3n) is formed as a result of fertiliz ation of the polar nucle i or central cell (2n)
36 by a male nucleus (1n). The endosperm bala nce number (EBN) hypothesis states that normal endosperm development occurs when the ratio of maternal to paternal EBN contribution to their progeny is 2 to 1 (Johns ton et al., 1980). Any deviation from this ratio (2EBN maternal:1EBN paternal) will result in low or no seed set. This hypothesis explains the low seed set values obtained when crosses were made between genotypes with different ploidy levels in P. notatum (Hanna and Burton, 1986; Burton and Hanna, 1992), and the high seed set values for crosse s made at the same ploidy level (Burton, 1955; Burton and Forbes, 1960). Although large variability in terms of sexua l expression was recorded for naturally occurring tetraploids (MartÃnez et al., 2001), highly sexual plants ha ve not been found in natural populations. The low aposporic expression detected in two genotypes in this study was previously reported for other induced tetraploids in P. notatum (Quarin et al., 2001), and in P. hexastachyum (Quarin and Hanna, 1980), indicati ng that the genes involved in the generation of an aposporous embryo sac are present at the diploid level. However, the detected aposporic expression was very low and should not be an impediment for using these induced tetraploid plan ts as the female parent for hybridization purposes. In addition to being the most vigorous plants in a large population these 20 genotypes were reasonably cross-fertile (Chapter 3), and self-sterile. Thus, re sults indicated that these 20 sexual tetraploids should be considered as good female counterparts for breeding tetraploid bahiagrass. Other Accessions It was determined by cytometric an alysis that PI 315732, PI 315733, and PI 315734 were tetraploids (Figure 4-3). In addition, mo st of the analyzed ovules from these three accessions showed the presence of aposporous embryo sacs (Table 4-1; Figure 4-2).
37 However, a considerable number of ovules showed the presence of aposporous and meiotic sacs within an in dividual ovule. Percentage of ovules showing only meiotic embryo sacs varied from 7 to 14 (Table 4-1) . Facultative apomictic bahiagrass plants are not uncommon in natural populations of bahiag rass. MartÃnez et al . (2001) recorded a large range of variation from obligate apomictic accessions (100% of the ovules showing aposporous sacs) to highly f acultative (95% of the ovules sh owing meiotic embryo sacs). In other related research progeny from these three accessions appeared phenotypically highly uniform indicating that the aposporous embryo sacs likely were predominantly the source of new embryos in seed from these cl ones. However, it is evident that these genotypes have conserved the genetic factors necessary for normal meiosis. These three accessions were selected because of their cold hardiness, and marked prostrate growth habit. Also, they have narrow leaves sim ilar to the cultivar Wilmington, an uncommon characteristic among tetraploid bahiagrass. Each of these thre e tetraploid plant introductions could be considered as potential cultivars because they appear to be predominantly apomicticly propagated genotypes. However, an impediment could be the low seed set observed for this group (Chapter 3). It was also determined, by chromosome counting, that the local collection with white stigmas was a tetraploid (2n=4x=40). Mo st of the analyzed ovules (64%) showed multiple aposporous embryo sacs, 9% showed multiple aposporous sacs in addition to one meiotic sac, 9% had aborted sacs, and the other 18% of the ovules showed only one meiotic embryo sac (Table 4-1). No progeny te st was conducted for th is genotype, so it was not possible to know the functionality of the meiotic and aposporous embryo sacs. Because white stigma is a recessive trait (B urton, 1948) it will be easy to separate the
38 apomictic products (white stigma) from th e sexual products (purple stigma) when a regular accession with purple stig mas is used as a pollen donor. Tetraploid Cultivars Embryo sac observations indicated that the cultivars Argent ine and Wilmington were highly apomictic plants. Ninety five percent of the analyzed ovules had multiple aposporous sacs for both cultivars; the other 5% had multiple aposporous sacs in addition to one meiotic sac (Table 4-1). Burton (1992) published a general description of these two cultivars indicating that they were apomictic . However, this is the first description in which the mature embryo sacs were analyzed and quantified in deta il. The experimental line, known as Tifton 7, was generated througho ut a long and intricate breeding approach (Burton, 1992; Chapter 2). Although this line has been shown to be significantly more productive than Argentine, it has remained as an experimental line due to concerns regarding seed production. It was also highly apomictic with 95% of its ovules containing only aposporous embryo sacs, and 5% contai ning both aposporous and meiotic sacs sharing the same ovule. Although Burton (1992 ) stated that Tift on 7 was significantly more fertile than Argentine, our results s howed no significant differences in selfand cross-fertility between these two genotypes (Chapter 3). Summary and Conclusions Bahiagrass is a popular perennial pasture and utility turf in Florida and the southern Coastal Plain region of the USA. It grows naturally in South America where sexual diploids and apomictic tetraploid coexis t. Studies of ploidy level and method of reproduction in several natural and induced accessions of this species lead to the following conclusions:
39 Twenty artificially induced genotypes we re confirmed to be tetraploids and reproduce sexually. These vigorous and reasonably fertile plants can be used as females and represent the key to releas e the high genetic variability lo cked in highly heterozygous apomictic tetraploids. The accessions PI 315732, PI 315733, and PI 315734 were found to be tetraploids and facultative apomictic with high apos porous expression. These ecotypes with a marked prostrate growth, narrow leaves and good cold hardiness can be considered as good genetic sources for forage and turf breeding. A bahiagrass plant with white stigmas was found in an area not far from the former campus farm. Because white stigma is the only known recessive trait for this species, and can have a major role in genetic analyses, the plant was collected and studied. The plant was found to be tetraploid and facultative apomictic. The cultivars Argentine, Wilmington, and an experimental line, known as Tifton 7, were determined to be highly apomictic showing 100% of their ovules containing multiple aposporous embryo sacs. These lines are among the most productive bahiagrass tetraploids and will be used as pollen sources in a breeding program for increased forage yield.
40 Table 4-1. Embryo sac types in 27 bahiagrass accessions. The first 20 accessions in the table are induced tetraploids, th e last 7 are natural tetraploids Number of ovules with Accession Number of ovules Meiotic Aposporic Mei +Apo Aborted 2 23 22 0 0 1 5 20 20 0 0 0 7 40 34 1 0 5 35 27 27 0 0 0 52 55 55 0 0 0 55 28 23 3 1 1 56 20 20 0 0 0 71 31 30 0 0 1 73 20 19 0 0 1 74 26 25 0 0 1 77 40 40 0 0 0 78 23 22 0 0 1 99 30 30 0 0 0 104 24 24 0 0 0 105 24 24 0 0 0 106 25 25 0 0 0 110 26 26 0 0 0 111 22 22 0 0 0 3-13-9 24 24 0 0 0 3-16-4 30 30 0 0 0 PI 315732 35 5 22 7 1 PI 315733 33 3 12 17 1 PI 315734 41 3 30 8 0 WS 22 4 14 2 2 Argentine 62 0 59 2 1 Tifton 7 54 0 51 1 2 Wilmington 22 0 21 1 0
41 Figure 4-1. Mature meiotic embryo sac in one o vule of the induced tetraploid plant 52 of Paspalum notatum showing the two polar nuclei with large nucleolus (arrow), and a mass of antipodals in the chal azal end (arrow head). Magnification: x312.5
42 Figure 4-2. Two mature aposporous embryo sacs in one ovule from PI 315732 showing their correspondent polar nucle i (arrows). Magnification: x312.5
43 Figure 4-3. Histogram generated with a Pa rtec Ploidy Analyzer PA for PI 315733 of Paspalum notatum. Because the analy zer was calibrated at 100 units for a known diploid bahiagrass (2n=2x=20), a cl ear peak at 200 units indicated that PI 315733 was a tetraploid (2n=4x=40)
44 CHAPTER 5 GENERATION AND EVALUATION OF TETRAPLOID HYBRIDS IN BAHIAGRASS Introduction The tetraploid (2n=4x=40) race of bahiagrass, Paspalum notatum FlÃ¼gge, is the predominant grass in native savannas of S outh and Central America (Chase, 1929). Its area of distribution is an e normous region from central Argentina to central Mexico (Gates et al., 2004). The predominance of this grass in native grasslands is suspected to be related with its almost unique persisten ce under extensive grazi ng systems, and with its mode of reproduction. Tetraploid bahi agrass reproduces by aposporous apomixis (Bashaw et al., 1970), and is pseudogamous (Burton, 1948). As in the other apomictic plants, the meiotic process is suppressed only in the female reproductive organs, thus the plants produce normal meiotically reduced pollen. In the past, released tetraploid cultivars were just superior genotypes selected from germplasm introduction and evaluation, becau se hybridization of these apomictic genotypes was not possible (Gates et al., 2004). A major breakthrough occurred when sexual tetraploids were generated by doubli ng the chromosome number of the diploid sexual form of the same species ( Paspalum notatum var saure Parodi) (Burton and Forbes, 1960). These doubled au totetraploids were then successfully hybridized with tetraploid apomictic strains (B urton and Forbes, 1960). However, to date no cultivar has been released from hybrids generated using th is technique (Gates et al., 2004). Several sexual autotetraploid lines have been mo re recently reported (Quarin et al., 2001;
45 Quesenberry and Smith, 2003). Two of these se lected lines were released as sexual tetraploid bahiagrass germpl asm (Quarin et al., 2003). Bahiagrass has shown to be the best ad apted grass for extensive cattle production systems in Florida. To intensify these sy stems more productive cu ltivars are needed, especially cultivars that can increas e the length of the grazing season. The objectives of this work were to ge nerate segregating bahiagrass populations by hybridizing selected sexual and apomictic ba hiagrass tetraploids, to evaluate these populations in terms of growth habit, reproductive expression, regrowth at the end of the growing season, and frost resistance, and to select superior hybrids for further evaluations. Materials and Methods Plant Material and Crosses To generate a segregating population of bahi agrass at the tetraplo id level, crosses were made between induced sexual and a pomictic tetraploids. A group of 11 sexual tetraploids was used (Table 5-1). Nine of them were vigorous genotypes selected from a group of 300 induced tetraploids (Quesenberry and Smith, 2003), determ ined to be sexual (Chapter 4), and cross-pollinated (Chapter 3), and the other two were Q4188 and Q4205 (Quarin et al., 2003). Argentine, Tifton 7, PI 315732, PI 315732, and PI 315734, determined to be apomictic (Chapter 4), we re used as the pollen donors (Table 5-1). Crosses were made by isolating together one panicle from the sexual female and one or two panicles from the apomictic male inside of a glassine bag just before anthesis. Care was taken to choose panicles of equal maturity (ready to flower). All bags were shaken each day during anthesis.
46 Seed set, Germination, and Greenhouse and Field Operations Upon maturity each head from the sexual parents was threshed separately, the number of florets was counted, empty floret s were removed, and the number of florets containing caryopses was determined. The perc ent seed set was calculated to supply the data summarized in Table 5-1. The seed were scarified using concentrated sulfuric acid for 5 minutes, and were sown in rows in flats containing sterile germination medium. Individual seedlings were later transplanted to small pots in a greenhouse. Each genotype was divided into two clones and allowed to develop into vigorous plants before transplanting them in the field. A group of the progeny (G1) was transplanted on 1 June 2005, and another (G2) on 6 July. These two gr oups were evaluated separately. The two clones for each genotype were planted in separate blocks in the field. Individual populations as units were randomly positioned in each block to enable the statistical analysis for a randomized complete block desi gn. Individual plants were spaced 3 by 3 feet in the field. The plot was uniformly fertilized with 300 kg of 5-10-10 per hectare before transplanting. Field Evaluations The analyzed variables for G1 were pl ant diameter, plant height, number of inflorescences, regrowth, and frost resistance. A group of pa rents was evaluated together with the progeny from G1. In contrast, only number of inflorescences, regrowth, and frost resistance were the variables analyzed for G2 since these plants had a shorter growing season. Plant diameter was estimated using the average between the longest and the shortest diameters. Plant height was measur ed using a device consisting in a rectangular piece of clear plastic (20 cm x 30 cm) with an orifice in the center held horizontally from the corners with cords by the operator, and a vertical ruler. Star ting on 20 July, three
47 measurements for both variables were ta ken using 4 week intervals between each measurement. The inflorescences were count ed on 16 September. On 21 September all the plants were defoliated to approximatel y 6 cm above the soil level. Regrowth was visually estimated on 28 October using a 1 to 5 scale, 1 for plants showing the lowest amount of herbage and 5 for plants showing th e highest amount of herbage. Also, frost resistance was visually estimated on 28 Decem ber after two consecutive frost events on 23 and 24 December, with temperatures of -2 and -3 Â°C respectively, using a 1 to 5 scale, 1 for the least frost resistant, and 5 for the most frost resistant plant. Laboratory Evaluations In an attempt to generate information and to collect seed before the field evaluations were completed, a group of 72 plants was selected on 19 August. Inflorescences from this group were fixed at anthesis (when the embryo sacs are usually fully developed) in FAA (18 Ethanol 70%: 1 Formaldehide 37%: 1 glacial acetic acid). The pistils were dissected out of the spikel ets and cleared using th e Young et al. (1979) technique. The ovules were observed usi ng a differential interference contrast microscope. In addition, seed were collected from this group to have material for the next evaluation phase. Statistical Analysis Data were analyzed using PROC GLM of PC SAS (SAS Institute, 2003) as a randomized complete block design. The signifi cance of all the variables was analyzed among the different progeny as well as among individual genotypes. In the case that significant differences were det ected for one variable DuncanÂ’s Multiple Range Test was used as a mean separation procedure. Unless ot herwise stated in the text, all differences referred to are significant at P < 0.05.
48 Selection Index A selection index was generated to iden tify the superior 20 % of the genotypes. Maximum plant diameter (D3) was converted to a 1-5 scale by divi ding the range of the actual values in 5 intervals of equal size. Then, the average of maximum plant diameter, regrowth and frost resistance was calculated for each replication and for each genotype belonging to G1. Genotypes that produced in average less than 20 inflorescences were not included. One hundred and twenty one plan ts were identified, a nd the contribution of each population was calculated. For the genotype s in G2, the average of regrowth and frost resistance was calculated for each replic ation and for each genotype. Genotypes that produced on average less than 5 inflorescences were not included. Forty four genotypes were identified from G2, and also the cont ribution of each population was calculated. Results and Discussion Progeny Eight hundred and twenty three plants we re obtained from 16 crosses between sexual induced and apom ictic tetraploids of Paspalum notatum (Table 5-1) . One progeny containing 13 plants was obtained from two open-pollinated panicles of one sexual tetraploid surrounded in the field by Argentin e and Tifton 7 plants (71op) (Table 5-1). The average percent seed set was 25 varying from 10 to 35. The average percent germination was 34, ranging from 7 to 81. Seed originating from inflorescences of Q4205 showed marked lower germination. Because the group of 9 induced tetraplo ids came from a population that was determined to be sexual (Chapter 4), and cr oss-pollinated (Chapter 3) most of their progeny can be considered hybrids. In c ontrast, Q4205 and Q4188 showed no significant differences between their selfand cross-fe rtility (Chapter 3) indicating that a high
49 proportion of their progeny may be products of self-pollination. An estimation of the percentage of selfed plants among the Q 4205 progeny can be made based on the number of plants having white stigmas (white stigma is recessive to purple stigma in bahiagrass) like the female parent or purpl e stigma like the male parent in a group of 20 plants in G1. Sixty five percent (13 plants) of the plants showed the recessive white stigma trait indicating that they were pr oduced by self-pollination. Because inbreeding reduces vigor and yield of bahiagrass (Burton, 1955) as in many other species, and increases the evaluation costs, emasculation should be used when Q4205 and Q4188 are used as females for crosses. Field Evaluations Progeny were evaluated in two different gr oups (G1 and G2) that were transplanted to the field at different times. Plant diameter and height were measur ed at three different times during the growing season to represent the growth habit of the progeny included in G1. Progeny within G1 showed significant di fferences for both diameter and height ( P <0.001) throughout the growing season. Th e progeny were ranked based on their diameter 50 days after transpla nting (20 July) into the fiel d (Table 5-2). A range from 12.2 to 26.6 cm in diameter was observed for the first measurement. The progeny spread between 5 and 16 cm after a month and between 10 and 24 cm after two months. The rate of spread between the first and the second measurements was higher than between the second and the third measurements. This diffe rence could be relate d with the daylength of each period. In addition, with a few exceptions the progeny were ranked in a similar order in each of the three meas urements (Table 5-2) indica ting that the differences in terms of rate of spread were proportiona lly constant throughout the growing season.
50 Plant height for a given progeny did not vary significantly among the three measurements (Table 5-2), indi cating that the progeny did not increase in height from 20 July until the end of the growing season. Thes e results indicate that growth in height occurs primarily during early stages reachi ng maximum height soon after transplanting. Some progeny showed a marked verti cal or upright growth, such as Q4205xArgentine which averaged 41.5 cm of tall and only 22.7 cm in diameter on 20 September. In contrast, other progeny showed a marked horizontal or prostrate growth, such as 2-2-7xTifton 7 with an average height of 36.3 cm, and diameter of 45.3 cm on 20 September. Other progeny showed a more bala nced growth habit, such us 106xTifton 7 showing an average height of 41.8 cm and diameter of 36.6 cm on 20 September. The most successful forage grasses in Florida ha ve exhibit marked prostrate growth with distinct persistence under grazing, and have the capabil ity to spread and colonize new areas quickly. Thus, 4-36-1xArgentine , 2-2-7xTifton 7, 71op, 71xArgentine, and 106xTifton 7 may be considered the best comb inations having a more prostrate growth habit as a requisite for Florida livestock production systems. The progeny were determined to be significantly different ( P <0.0001) in terms of plant regrowth. The top five populations, 2-2-7xTifton 7, 4-36-1xArgentine, 106xTifton 7, 71xArgentine, and 71op were determined to be the most suitable combinations for extending the growing season (Table 5-3). Some progeny showed large variation in terms of plant regrowth, such as, 71xArgentine, varying from 1 to 4.75. Other progeny showed lower variation, such as 71op varying from 2.5 to 4. Interestingly the 5 progeny that spread faster between 1 June and 21 Septem ber showed better plan t regrowth between 21
51 September and 28 October. Results indicate th at these five populat ions have a better general vegetative vigor throughout the growing season. Large variability was also observed among the progeny in terms of frost resistance ( P <0.0001). The five progeny that showed be tter vegetative vigor were among the most frost resistant populations (Table 5-3). Some progeny exhibit greater variation, such as 71xArgentine varying from 1.5 to 4, while others such as 2-2-7xTifton 7 varied from 1.5 to 3.5. The progeny 106xTifton 7 was characterize d by its marked frost resistance. Other less vigorous progeny, such as Q4205xArgentine and 106xArgentine were among the most frost resistant plants. However, the higher proportion of bare soil around these progeny (more upright plants) coul d result in less frost damage. The reproductive expression of these pr ogeny was estimated based on the total number of inflorescences at the end of the s eason because seed production is a requisite for this species. A large variation am ong the progeny was detected (P<0.0001), 71x315733 showed an average of 2.1 inflorescen ces per plant to 58.6 inflorescences per plant for the 2-2-7xTifton 7 cross (Table 5-3). Because low reproductive expression would result in fewer seed, and high repr oduction expression could result in lower nutritive value both extremes may be consider ed negative. An evaluation of the duration of the reproductive period will be needed in subsequent seasons to complement these results. Plants that produced a sufficient number of inflorescences but in a short period of time would be beneficial for both harv esting procedures, and nutritive value. A group of parents was evaluated for the same variables together with their progeny. They were determined to be significan tly different in terms of plant diameter and height during the entire season (three m easurements). Some parental lines were
52 characterized by their marked prostrate growth such as 2-2-7, PI 315732, PI 315733, and PI 315734 (Table 5-4). Evidently the progeny fro m 2-2-7 has inherited its growth habit (Table 5-2). On the other extreme, Q4205, 106 and their progeny tend to grow more vertically being more upright plants (Table 5-2 and 5-4). The parental lines were also determined to be significantly di fferent in terms of regrowth, frost resistance, and number of in florescences. As expect ed, Tifton 7 appeared to be a superior parent because of its very good regrowth, excellent frost resistance, and sufficient inflorescence number (Table 5-5). Plants 55, 73, and 106 were characterized by the lack of inflorescences and their marked frost resistance. Q4188 was one of the best sexual tetraploids having very good regrow th, frost resistance, and good production of inflorescences. Apparently its frost resistan ce was inherited by its progeny (Table 5-3). The combination between 106 and Tifton 7 genera ted progeny that differentiated for frost resistance not only at the begi nning of the cool season (Table 5-3) but also in the middle of February (Data not presented). Large variation was also observed among another progeny group (G2) in terms of regrowth ( P <0.0001), frost resistance ( P <0.015), and total number of inflorescences ( P <0.0291). In general, Q4205xArgentine showed th e lowest performance in terms of the analyzed variables (Table 5-6). A very good combination was Q4205xTifton 7 showing the highest performance fo r all the variables. Plant breeding for crops that reproduce as exually has the advantage that every superior genotype is a potential new cultivar. Thus, comparisons among individual genotypes independent of thei r origin may be more important than comparisons among groups of plants with different origins. Highl y significant differences in terms of plant
53 diameter ( P <0.0001) and height ( P <0.0001) showed the large va riability detected among the genotypes of G1 as a whole in terms of growth habit during th e entire growing season (Figure 5-1). Also, significant variability ( P <0.0001) among the genotypes was observed for reproductive expression, from plants that di d not flower to plants that produced an average of 120 inflorescences. Significant di fferences were also de tected for regrowth after cutting at the end of the season ( P <0.0001), and for frost resistance ( P <0.016). Similar results were detected when compar isons were made among the genotypes of G2. The most important variables were combined in an attempt to identify the top 20 % of the 603 genotypes included in G1, and from the 219 genotypes included in G2. The progeny 71xArgentine contributed the highest number of plants 40% for the best 20% in G1, while Q4188xArgentine contributed 77.3% of th e top 20% of the plants in G2 (Table 5-7). Progeny from 2-2-7xTifton 7 and 106xT ifton 7 contributed 20 and 14% of the superior plants in G1 in spite of thei r reduced progeny numbers, 38 and 33 hybrids respectively. Also, Q4205xTifton 7 showed ve ry good performance, contributing 18% of the top 20% of th e plants in G2. The group of parents was also compared as one more individual genotype. Tifton 7 was the only apomictic tetraploid that appear ed among the top 20% of the plants in G1. The results were not surprising because this experimental line wa s generated trying to increase the extent of the growing season. The induced tetraploid, # 55, appeared also among the best 20%. Its marked frost resistan ce contributed to that position. However, this plant showed very low expression of the reproductive phase and some degree of apomixis which is a negative characteristic fo r a sexual tetraploid female. It is important
54 to recognize that parental lines can not be accurately compared based on their progeny large numbers of progeny from each line are evaluated. To gain time and realize genetic advance in the evaluation pro cess, a group of 71 plants was phenotypically selected on 19 A ugust. Both inflorescences at anthesis and seed were collected from all of them. The plan ts were classified as sexual, apomictic, and facultative apomictic based on embryo sac obser vations. Eight of them were classified as apomictic because most of their ovules cont ained aposporous embryo sacs (Figure 5-2; Table 5-8). Twelve plants were classifi ed as facultative apomictic because a low percentage of their ovules showed aposporous embryo sacs. The remaining 51 were classified as highly sexual because all of their ovules contained only a single meiotic embryo sac (Table 5-8). By grouping both apom ictic and facultative apomictic plants, it is possible to observe that 28% of the plants from this sample inherited the gene(s) for apospory. The ratio between sexual and apomictic plants was not significantly different from 3:1 which was in agreement with previous reports (Martinez et al., 2001). This is an indication that if progeny from self-pollinati on was present we selected this group against them. A more conclusive data will be genera ted after the embryo sac analysis of the top 20% of the plants during the following season. The three categories, the best sexual, apomictic, and facultative apomictic, will be used to advance the breeding process. In addi tion, seed from the apomictic plants can be used to establish small plots that can be mech anically clipped or graz ed to determine their behavior under defoliation. Summary and Conclusions Tetraploid bahiagrass, Paspalum notatum FlÃ¼gge, reproduces by apomixis, one type of asexual reproduction through seed. This reproduc tive characteristic has
55 previously limited crop improvement programs to germplasm introdu ction, evaluation, and release of superior lines. Through th is work we generated tetraploid hybrids by crossing several induced sexual tetraploid cl ones as females with apomictic lines as males. This breeding approach and evalua tion of the progeny lead to the following conclusions: Hybridization of sexual and apomictic tetraploids was an efficient process with an average seed set of 25% over all crosses. Determination of self-sterility should be a prerequisite when selecting females for use in crosses. Inbreeding depression was evident among progeny obtained by hybridizing Q4188 a nd Q4205. It is important to recognize that Q4205 has white stigmas, a recessive marker that was used to identify selfpollinations. However, given the cost of the entire process, of progeny generation, manipulation, and evaluation, emasculation should be used if female lines are known to have moderately high self-fertility. Large variability can be expected in the progeny generated by hybridizing apomictic tetraploids of bahiagrass as pollen parent. Significant variation in terms of growth habit, reproductive expression, s easonal growth, and frost resistance was observed among progeny. This is an indication th at breeding of this cr op is in its initial steps and an enormous potential is presen t in natural population s distributed throughout the New World. At least three combinations showed an excellent potential for increasing the productivity of this crop. One of them, 2-27xTifton 7 showed a vigorous and aggressive growth habit during the warmer part of th e season. In contrast, 106xTifton 7 showed a good vigor during the warmer part of the s eason, and excellent vigor at the end of
56 growing season. The progeny from 4-36-1xArg entine was at the top for almost all evaluated characteristics. The general contri bution from these three progeny to the best 20% of the hybrids was limited by their low number of progeny produced and evaluated. However, more crosses will be made using these combinations to increase their progeny. The parental lines also were different for all the analyzed variables. Tifton 7 was ranked as the best male based on its own perf ormance and also in the performance of its progeny. The induced tetraploids 55, 73, and 106 appeared among the most frost resistant plants. The main negative characteristics for th ese three plants were their upright growth habit, and low reproductive expression. Howe ver, the genotype known as 2-2-7 appeared as one of the best sexual lines showing pronounced prostrate growth, similar frost resistance and better reproductive expre ssion. In addition, Q4188 appeared among the best parents showing excellent vegetative a nd reproductive vigor and its particular erect and dense canopy. The progeny from both Q 4188 and Q4205 probably would be rated better if probable selfs were not part of the progeny mean. A ratio of 3:1 sexual: apomictic plants, wa s observed in a representative selected sample of progeny agreeing with previous re ports (Martinez et al ., 2001). However, a ratio of 9:1 sexual + facultative apomictic: ob ligate apomictic was detected. That is an indication that much larger progeny may be needed than with other clonally propagated sexual crops when the goal is to identify a s uperior agronomic genotype that is also an obligate apomictic. The detection of apomictic plants represen ts a handicap for this breeding approach that needs to be addressed. Screening the progeny using embryo sac observation is the quicker and cheaper known methodology, but it requires reproductive material. Thus,
57 field evaluations can not be done until the s econd season in the case that only apomictic plants will be included in the evaluation. Th e used of molecular markers linked to the trait would make possible earl y detection using ve getative material. However, the use of these techniques to process more than 800 sa mples would require considerable time and money. Apomictic, facultative apomictic, and sexua l plants are expected to be found among the top 20% of these progeny. Every superi or apomictic genotype is a potential new cultivar. Thus, seed will be harvested from these genotypes to establish small plots for further evaluations. The sexual and facultative pl ants can be used for future crosses with the same or additional apomictic males. A few of the best sexual plan ts could be released as sexual female germplasm, and would be available for other breeding projects.
58 Table 5-1. Number of caryopses and plants ge nerated from crosses made between sexual and apomictic tetraploids of bahiagrass Crosses Pollinated Spikelets Caryopses Seed Set Plants Germination x * No No % No % 55x315733 156 16 10.3 13 81.3 71xArgentine 716 216 30.2 140 64.8 71x315733 100 35 35.0 17 48.6 73x315734 119 39 32.8 22 56.4 106xTifton 7 245 40 16.3 33 82.5 106xArgentine 962 150 15.6 118 78.7 2-2-7xTifton 7 241 58 24.1 38 65.5 3-13-9xTifton 7 243 58 23.9 18 31.0 3-16-4xTifton 7 359 79 22.0 37 46.8 4-36-1xArgentine 86 11 12.8 7 63.6 4-38-6x315732 366 66 18.0 42 63.6 Q4188xArgentine 2260 613 27.1 237 38.7 Q4188x315733 96 23 24.0 11 47.8 Q4205xArgentine 2226 583 26.2 50 8.6 Q4205xTifton 7 1256 372 29.6 27 7.3 71op 248 81 32.7 13 16.0 Total 9679 2440 25.2 823 33.7 sexual 4x, apomictic 4x. Table 5-2. Plant diameter and plant height means from three measurements during the growing season Progeny D1 * D2 D3 H1 ** H2 H3 ----------------------------------cm--------------------------------4-36-1xArgentine 26.6 a 42.8 a 50.1 a 36.3 abc 39.6 ab 37.3 b 2-2-7xTifton 7 25.7 a 40.9 a 45.3 b 33.9 cde 35.3 cd 36.3 b 71op 19.9 b 33.6 b 38.7 c 37.2 ab 38.4 bc 38.0 b 71xArgentine 18.5 bc 33.1 b 38.7 c 34.6 bcd 38.0 bc 37.7 b 3-16-4xTifton 7 17.8 cde 25.7 cd 28.5 de 31.0 f 32.3 de 31.2 c Q4188xArgentine 17.8 bcd 28.7 c 28.6 d 32.1 def 38.7 ab 38.6 ab 106xTifton 7 17.0 cde 33.1 b 36.6 c 38.1 a 41.8 a 41.8 a 3-13-9xTifton7 15.7 def 26.3 cd 28.1 de 28.4 g 29.4 ef 28.1 cde 73x315734 14.5 efg 22.7 de 29.2 d 26.1 g 26.0 g 28.1 cde 106xArgentine 13.9 fg 24.4 de 27.0 def 31.5 ef 33.6 d 31.5 c 71x315733 13.3 fg 18.2 f 23.0 f 26.0 g 22.8 h 25.4 e 55x315733 13.0 fg 22.4 def 26.1 def 26.3 g 27.7 fg 29.4 cd 4-38-6x315732 12.5 g 20.7 ef 23.7 ef 26.1 g 26.3 fg 26.1 de Q4205xArgentine 12.2 g 20.7 ef 22.7 f 38.0 a 39.9 ab 41.5 a Means with the same letter are not significantly different. *D (plant diameter): D120 July, 50 days after tran splanting into the field, D217 August, and D322 September. **H (plant height): H121 July, H2-19 August, and H319 September.
59 Table 5-3. Means separation for plant regrow th, frost resistance, and total number of inflorescences at the end of the growing season Progeny Regrowth* Frost Resistance* No of Inflorescences** 2-2-7xTifton 7 3.4 a 2.8 bcd 58.6 a 4-36-1xArgentine 3.4 a 2.9 abc 40.4 b 106xTifton 7 3.4 a 3.1 a 30.8 bc 71xArgentine 3.1 a 2.9 abc 18.8 de 71op 3.06 ab 2.7 cd 35.8 bc 3-16-4xTifton 7 2.7 bc 2.7 bcd 2.5 f Q4188xArgentine 2.5 cd 3.0 ab 42.3 b 73x315734 2.2 de 2.5 de 3.8 f 55x315733 2.1 def 2.7 cd 4.1 f 3-13-9xTifton7 2.1 def 2.3 e 11.6 ef 106xArgentine 1.9 ef 3.1 a 6.7 f Q4205xArgentine 1.8 ef 3.1 a 22.3 d 4-38-6x315732 1.8 ef 2.8 bcd 2.6 f 71x315733 1.7 f 2.5 de 2.1 f *Plant regrowth and frost resistance are based on 1 to 5 visual rates, plants rated with 5 showed higher regrowth or higher frost resistance. Regrowth was estimated on 28 October, and frost resistance on 28 December. ** The number of inflorescences wa s determined on 16 December. Table 5-4. Parental lines evaluation showing pl ant diameter and plant height means from three measurements during the growing season Identification D1 D2 D3 H1 H2 H3 -------------------------------------cm------------------------------------PI315734 28.5 a 36.0 a 42.5 a 24.0 e 20.0 e 20.0 ef Q4188 26.1 a 35.0 a 33.0 abc 33.8 abcd 44.8 ab 44.3 a PI315733 26.0 ab 37.5 a 37.5 ab 27.0 cde 23.0 e 17.0 f PI315732 24.5 abc 28.0 abc 42.5 a 27.0 cde 28.0 de 24.0 def Argentine 20.4 abcd 31.7 ab 38 ab 33.6 abcd 35.1 cd 32.3 bcd Tifton 7 20.1 abcd 35.2 a 38.1 ab 43.1 a 45.4 a 45.5 a 2-2-7 18.5 abcd 32.1 ab 37.3 ab 26.2 de 24.5 e 27.5 cde 71 17.0 cd 30.3 ab 30.2 bc 35.0 ab 44.0 ab 37.7 ab Q4205 16.5 cd 23.3 bc 25.5 c 41.0 a 46.0 a 46.3 a 73 16.3 cd 23 bc 24.8 c 36.0 ab 35.0 cd 35.5 bc 55 15.0 d 29.3 abc 32.0 abc 40.5 a 45.0 ab 34.0 bc 3-16-4 13.7 d 28.8 abc 30.8 abc 30.8 bcde 33.0 cd 30.7 bcd 3-13-9 13.5 d 27.2 abc 31.3 abc 34.7 abc 36.3 bcd 33.7 bc 106 11.9 d 20.1 c 23.0 c 34 abc 38.0 abc 34.0 bc Means with the same letter are not significantly different. *D (plant diameter): D120 July, 50 days after tran splanting into the field, D217 August, and D322 September. **H (plant height): H121 July, H2-19 August, and H319 September.
60 Table 5-5. Means separation for plant regrow th, frost resistance, and total number of inflorescences at the end of the growing season Progeny Regrowth* Frost Resistance* No of Inflorescences** Tifton 7 3.6 a 3.2 bc 54.2 a Q4188 3.1 ab 3.5 bc 44.8 a 73 3.0 abc 3.0 bcd 0.0 c 55 3.0 abc 5.0 a 0.5 c PI315734 2.5 bcd 2.0 d 27.0 abc PI315732 2.5 bcd 2.0 d 15.0 bc Argentine 2.4 bcd 2.6 cd 33.1 ab Q4205 2.4 bcd 3.0 bcd 7.0 bc 71 2.3 bcd 4.0 b 6.3 bc 3-13-9 2.3 bcd 3.0 bcd 32.3 ab 2-2-7 2.2 bcd 3.0 bcd 43.3 a 3-16-4 2.2 bcd 3.7 b 13.7 bc PI315733 2.0 cd 2.0 d 16.0 bc 106 1.8 d 3.25 bc 0.0 c *Plant regrowth and frost resistance are based on 1 to 5 visual rates, plants rated with 5 showed higher regrowth or higher frost resistance. Regrowth was estimated on 28 October, and frost resistance on 28 December. ** The number of inflorescences wa s determined on 16 December. Table 5-6. Means separation (G2) for herbage mass, frost resi stance, and total number of inflorescences at the end of the growing season Progeny Herbage mass* Frost resistance* No of Inflorescences** Q4188xArgentine 2.3 a 3.4 ab 8.1 ab Q4205xTifton7 2.3 a 3.6 a 9.4 a Q4188x315733 1.9 a 3.3 ab 11.4 a Q4205xArgentine 1.3 b 3.1 b 4.5 b *Plant regrowth and frost resistance are based on 1 to 5 visual rates, plants rated with 5 showed higher regrowth or higher frost resistance. Regrowth was estimated on 28 October, and frost resistance on 28 December. ** The number of inflorescences wa s determined on 16 December.
61 Table 5-7. Contribution from each populati on to the top 20% of the genotypes Progeny G1 G2 ----------% --------2-2-7xTifton 7 20 4-36-1xArgentine 5 106xTifton 7 14 71xArgentine 40 71op 2.4 3-16-4xTifton 7 0 Q4188xArgentine 6 77 73x315734 0 55x315733 0 3-13-9xTifton7 1 106xArgentine 11 Q4205xArgentine 1 0 4-38-6x315732 0 71x315733 0 Q4205xTifton7 18 Q4188x315733 5
62 Table 5-8. Embryo sac types in 71 bahiagrass hy brids (the first 63 accessions in the table are from G1, and the last 8 are from G2) Number of ovules with Identification Plant No No of ovules Meiotic Aposporic Mei +Apo Aborted 106x315732 13 21 21 0 0 0 106xArgentine 9 22 22 0 0 0 106xArgentine 16 22 22 0 0 0 106xArgentine 21 20 17 0 0 3 106xArgentine 22 24 24 0 0 0 106xArgentine 32 21 21 0 0 0 106xArgentine 45 24 24 0 0 0 106xArgentine 47 28 28 0 0 0 106xArgentine 64 28 27 0 0 1 106xArgentine 93 20 4 4 11 1 106xTifton7 13 20 0 10 9 1 106xTifton7 14 20 19 0 1 0 106xTifton7 17 21 21 0 0 0 106xTifton7 20 20 20 0 0 0 106xTifton7 29 20 20 0 0 0 106xTifton7 33 23 23 0 0 0 2-2-7xTiftton7 3 20 0 20 0 0 2-2-7xTiftton7 5 26 26 0 0 0 2-2-7xTiftton7 6 22 21 0 1 0 2-2-7xTiftton7 9 26 26 0 0 0 2-2-7xTiftton7 10 20 20 0 0 0 2-2-7xTiftton7 13 23 23 0 0 0 2-2-7xTiftton7 14 25 0 20 5 0 2-2-7xTiftton7 16 20 13 0 7 0 2-2-7xTiftton7 17 22 22 0 0 0 2-2-7xTiftton7 21 22 0 18 4 0 2-2-7xTiftton7 24 20 20 0 0 0 2-2-7xTiftton7 33 20 19 0 0 1 2-2-7xTiftton7 37 20 20 0 0 0 3-13-9xTifton7 4 24 20 0 0 4 3-13-9xTifton7 9 23 23 0 0 0 4188xArgentine 13 21 12 0 0 9 4188xArgentine 16 26 9 0 0 17 4188xArgentine 25 20 6 0 0 14 4188xArgentine 45 20 6 0 0 14 4188xArgentine 67 26 9 0 0 17 4188xArgentine 76 23 5 0 0 18 4188xArgentine 97 21 7 0 2 12 4205xArgentine 4 20 20 0 0 0 4205xArgentine 13 22 22 0 0 0 4205xArgentine 17 29 25 2 0 2
63 Table 5-8. Continued Number of ovules with Identification Plant No No of ovules Meiotic Aposporic Mei +Apo Aborted 4-36-1xArgentine 1 26 21 1 1 3 4-36-1xArgentine 2 25 24 0 1 0 4-36-1xArgentine 7 40 40 0 0 0 4-38-6x315732 32 20 8 0 1 11 55x315734 12 20 18 0 0 2 71op 3 20 19 0 0 1 71op 11 20 20 0 0 0 71op 12 20 8 0 0 12 71xArgentine 28 21 21 0 0 0 71xArgentine 48 23 23 0 0 0 71xArgentine 61 25 17 5 2 1 71xArgentine 61 21 14 4 1 2 71xArgentine 62 26 26 0 0 0 71xArgentine 67 27 26 1 0 0 71xArgentine 83 20 20 0 0 0 71xArgentine 84 24 24 0 0 0 71xArgentine 93 31 28 1 1 1 71xArgentine 99 22 22 0 0 0 71xArgentine 131 22 21 0 0 1 71xArgentine 136 25 20 3 1 1 71xArgentine 137 24 20 3 0 1 73x315734 12 29 22 4 0 3 4205xTifton7 1 20 19 0 0 1 4205xTifton7 3 22 0 12 10 0 4205xTifton7 5 22 14 1 2 5 4205xTifton7 19 20 0 10 10 0 4188xArgentine 93 20 10 0 0 10 4188xArgentine 122 20 0 12 8 0 4188xArgentine 137 22 14 0 0 8 Q4188x315733 2 20 10 0 0 10
64 Figure 5-1. Tetraploid progeny of Paspalum notatum obtained by crossing sexual and apomictic genotypes. Different grow th habits can be recognized
65 Figure 5-2. Multiple aposporous embryo sacs in one ovule of an apomictic tetraploid hybrid of Paspalum notatum , 2-2-7x Tifton 7 #14. Magnification: x312.5
66 CHAPTER 6 CONCLUSIONS Bahiagrass breeding used for the generation of more productive cu ltivars is needed for the improvement of forage-livestock production systems in Florida and southern Coastal Plain region of the USA. Breeding systems that can be used effectively to improve a species are determined more by a speciesÂ’ mode of reproduction than by any other factor. Thus, studies were conducted to determine the reproductive behavior of the most important bahiagrass germplasm in Flor ida. In addition, a breeding program was initiated for the tetraploid race of this forage crop. In general, diploid bahiagrass can be considered as a cross-pollinated group. They produced significantly more seed under cros s-pollination than under self-pollination. Most genotypes are not different in terms of cross-fertility and are cross-compatible with each other. They do differ in the amount of seed produced under self-pollination with most plants setting low percent of seed wh en selfed, but some genotypes are moderately self-fertile setting more than 20% seed. Thus, sexual bahiagrass diploids are crosspollinated with low but variable levels of self-fertility. Although Pensacola bahiagrass and Tifton 9 are different in terms of productivity, growth habit, and seasonal growth, they are not different in terms of crossa nd self-fertility. This is an indication that phenotypic selection can be used for increasi ng the productivity of this crop without affecting its general fertility. A group of 20 artificially i nduced tetraploids obtained by Quesenberry and Smith (2003) reproduce sexually, and probably the en tire group (300 genot ypes) shares this
67 reproductive characteristic. The levels of apomix is expression, if pres ent, are expected to be very low and not significant for breeding purposes. They are cross-pollinated, and set more seed under cross-pollination than under self-pollination. This group of 300 autotetraploids represents a potential source of sexuality that may make possible the release of the natural variability in apomictic tetraploids. In fact, 9 of them were used successfully as females in crosses with apomic tic lines used as polle n donors. Variability was also observed among them in terms of growth habit, number of inflorescences produced, regrowth at the end of the growing season, and fros t resistance. Most of them have a defined upright growth habit, but a few of them have prostrate growth. It appears that these characters can be successfully transmitted to their progeny. For example, a genotype distinguishable for its marked prostrat e growth habit transfer red the trait to its progeny. The negative aspect of several of these lines is the very low production of inflorescences. Two sexual tetraploid lines known as Q 4188 and Q4205 (Quarin et al., 2003) are not different in their general fertility. They do not differ in terms of cross-fertility with the other group of induced tetraploids. Howeve r, they have the nega tive characteristic of producing significantly more seed under self-p ollination than the ot her group of induced tetraploids. In fact they produce similar amount of seed when selfand cross-pollinated. Q4188 is a vigorous plant having a char acteristic dense canopy uncommon among tetraploids of this species. High proportion of aborted em bryo sacs found in the progeny of this plant, indicates that the fertility of them should be low. The other sexual line, Q4205 has white stigmas, which is the only known recessive trait that can be used as a
68 marker for breeding purposes in this species. However, these two lines should not be used as female parents without emasculation, becau se florets are prone to self-pollination. The plant introductions 315732, 315733, a nd 315734 are tetraploids and are facultative apomictic with high apomixis ex pression. These three plants have a marked prostrate growth, characteristic narrow leav es, and an open canopy with very low leaf density. They are not frost resistant and they seem to become dormant rapidly at the end of the growing season. With some exceptio ns their progeny l acked vegetative and reproductive vigor. Thus, these thr ee plants are considered to be turf-types in a breeding program rather than high forage producers. Argentine and Tifton 7 are highly apom ictic plants showing aposporous embryo sacs in 100% of their ovules. There is low variability among the apomictic genotypes in terms of selfand crossfertility. Previous reports indicated that Tifton 7 produced more seed than Argentine (Burton, 1992). That difference is based on the number of inflorescences per unit area, but is not relate d with the reproductive efficiency of their reproductive units. Another important difference is that Tifton 7 has a longer growing season than Argentine because of its shor ter winter dormancy. Both lines can be considered as very good male parents because they transferred effici ently their desirable agronomic characteristics to their progeny. Pr ogeny from crosses between specific female parents with Argentine and Tifton 7 as male parents, showed vi gorous and aggressive vegetative growth, as well as tr ansgressive segregation for in florescence number and frost resistance. The generation of a segregating populat ion through hybridization between sexual induced and apomictic tetraploids can be consid ered an efficient approach. In general, the
69 different genotypes are cross-compatible, setting sufficient amounts of seed. Large variability between and within progeny can be generated using this approach offering infinite opportunities for recomb ination in breeding. Variability in terms of growth habit, reproductive expression, extent of the grow ing season, and frost resistance can be expected among progeny and among individual genotypes. Based on the performance of a particular progeny, combination of specific parents should hasten reaching specific breeding objectives. In this case, progeny that showed continue d growth and vigor, especially at the end of the typical growing s eason would be desirable as forage and is an objective of this breeding program. Apomixis diagnosis should be included in the general progeny evaluation. Because the segregation of this trait is not expected to be linked to any agronomic characteristic, we would anticipate that sexual plants w ill be found among the best plants. Sexual, facultative apomictic, and obligate apomictic pl ants are expected to be found within the top 20% of the plants. The analysis of a group of 71 phenotypically selected plants corroborated this assumption. Se xual and facultative apomictic plants were 9 times more common than the number of obligate apomictic plants. Selected sexual F1s can be crossed with selected apomictic F1s, other apomictic lines, or b ackcrossed with the best male parents to increase the number of plants w ith desirable characteristics. Seed from the apomictic plants will be used to establis h small plots to determine their productivity under mowing or timed defoliation.
70 APPENDIX SEED SET MEANS OF DIFFERENT BAHIAGRASS DIPLOIDS AND TETRAPLOIDS UNDER SELF-, CROSS, AND OPEN-POLLINATION IN THE GREENHOUSE AND FIELD, ON 2004 AND 2005 Table A-1. Seed set means of different diploid and tetraploid bahiagrass Identification * Plant No Pollination Loc Â¶ Year Pollinated spikelets Seed Set C9 1 Self GH 2004 886 1.4 C9 2 Self GH 2004 330 3.3 C9 3 Self GH 2004 981 0.8 C9 4 Self GH 2004 533 16.9 C9 5 Self GH 2004 571 11.6 C9 6 Self GH 2004 1639 0.8 C9 7 Self GH 2004 898 24.7 C9 8 Self GH 2004 181 1.1 C9 9 Self GH 2004 693 0.5 C9 10 Self GH 2004 150 12.5 C9 11 Self GH 2004 918 3.6 C9 12 Self GH 2004 725 6.2 C9 13 Self GH 2004 251 0.0 C9 14 Self GH 2004 710 31.4 C9 15 Self GH 2004 1175 8.6 C9 16 Self GH 2004 948 0.7 C9 17 Self GH 2004 654 0.0 C9 18 Self GH 2004 738 2.5 C9 19 Self GH 2004 1320 26.7 C9 20 Self GH 2004 512 7.2 C9 21 Self GH 2004 414 5.3 C9 22 Self GH 2004 1035 17.9 C9 23 Self GH 2004 871 32.8 C9 24 Self GH 2004 141.0 0.0 C9 25 Self GH 2004 727 20.7 C9 26 Self GH 2004 649 2.2 Overall mean 9.20 CV Â§ 1.14 C9 1 Self GH 2005 365 2.0 C9 2 Self GH 2005 808 19.3 C9 3 Self GH 2005 751 14.7 C9 4 Self GH 2005 150 52.8 C9 5 Self GH 2005 590 21.1
71 Table A-1. Continued Identification * Plant No Pollination Loc Â¶ Year Pollinated spikelets Seed Set C9 6 Self GH 2005 744 4.3 C9 7 Self GH 2005 698 25.9 C9 8 Self GH 2005 933 22.4 C9 9 Self GH 2005 899 0.2 C9 11 Self GH 2005 605 5.2 C9 12 Self GH 2005 325 18.6 C9 13 Self GH 2005 677 1.4 C9 14 Self GH 2005 1128 38.7 C9 15 Self GH 2005 1197 8.6 C9 16 Self GH 2005 688 4.8 C9 17 Self GH 2005 309 1.0 C9 18 Self GH 2005 801 0.5 C9 19 Self GH 2005 1250 10.7 C9 20 Self GH 2005 579 9.5 C9 21 Self GH 2005 754 14.9 C9 22 Self GH 2005 1158 5.9 C9 23 Self GH 2005 1222 5.5 C9 24 Self GH 2005 282 10.6 C9 25 Self GH 2005 1536 6.2 C9 26 Self GH 2005 1096 2.9 Overall mean 12.3 CV 1.03 C9 3 Self Field 2005 678 22.6 C9 6 Self Field 2005 761 2.2 C9 7 Self Field 2005 968 22.1 C9 13 Self Field 2005 892 13.0 C9 14 Self Field 2005 1042 47.0 C9 16 Self Field 2005 448 0.4 C9 17 Self Field 2005 388 20.9 C9 19 Self Field 2005 1206 33.0 C9 22 Self Field 2005 740 6.4 C9 23 Self Field 2005 1499 9.7 Overall mean 17.7 CV 0.82 C9-1xC9-13 1 Cross GH 2004 226 26.1 C9-5xC9-15 5 Cross GH 2004 361 80.6 C9-6xWPG-23 6 Cross GH 2004 445 38.2 C9-7xWPG-4 7 Cross GH 2004 169 94.7 C9-9xC9-20 9 Cross GH 2004 455 39.6 C9-13xC9-1 13 Cross GH 2004 297 30.3 C9-15xC9-5 15 Cross GH 2004 287 71.1 C9-15xC9-24 15 Cross GH 2004 147 90.5 C9-15xWPG-5 15 Cross GH 2004 125 6.4 C9-15xC9-6 15 Cross GH 2004 533 62.5 C9-20xC9-9 20 Cross GH 2004 167 86.8 C9-21xC9-26 21 Cross GH 2004 188 70.7
72 Table A-1. Continued Identification * Plant No Pollination Loc Â¶ Year Pollinated spikelets Seed Set C9-22xC9-12 22 Cross GH 2004 346 12.7 C9-23xC9-1 23 Cross GH 2004 224 68.3 C9-24xC9-15 24 Cross GH 2004 177 67.2 C9-26xC9-21 26 Cross GH 2004 243 43.6 Overall mean 55.6 CV 0.50 C9-3xWPG-6 3 Cross GH 2005 377 32.4 C9-3xWPG-14 3 Cross GH 2005 276 80.8 C9-4xC9-18 4 Cross GH 2005 142 76.8 C9-6xWPG-4 6 Cross GH 2005 278 90.6 C9-7xC9-8 7 Cross GH 2005 152 88.8 C9-8xC9-13 8 Cross GH 2005 475 45.3 C9-8xC9-7 8 Cross GH 2005 174 75.3 C9-9xWPG-2 9 Cross GH 2005 268 68.7 C9-9xC9-10 9 Cross GH 2005 218 20.2 C9-9xC9-13 9 Cross GH 2005 135 32.6 C9-10xC9-9 10 Cross GH 2005 230 46.5 C9-11xWPG-18 11 Cross GH 2005 158 39.9 C9-12xWPG-21 12 Cross GH 2005 332 89.8 C9-12xWPG-18 12 Cross GH 2005 150 95.3 C9-13xC9-9 13 Cross GH 2005 167 15.0 C9-13xC9-24 13 Cross GH 2005 263 58.2 C9-13xC9-8 13 Cross GH 2005 221 71.5 C9-15xWPG-15 15 Cross GH 2005 238 48.3 C9-16xC9-20 16 Cross GH 2005 170 73.5 C9-17xWPG-13 17 Cross GH 2005 149 34.2 C9-18xC9-4 18 Cross GH 2005 147 87.8 C9-20xC9-16 20 Cross GH 2005 180 70.6 C9-21xWPG-4 21 Cross GH 2005 132 59.1 C9-22xWPG-16 22 Cross GH 2005 204 67.6 C9-24xC9-13 24 Cross GH 2005 208 65.9 Overall mean 61.4 CV 0.38 C9 6 Open Field 2005 243 47.3 C9 7 Open Field 2005 1027 86.9 C9 13 Open Field 2005 710 36.5 C9 14 Open Field 2005 1198 44.6 C9 16 Open Field 2005 1503 30.4 C9 17 Open Field 2005 694 21.3 C9 19 Open Field 2005 1108 29.6 C9 22 Open Field 2005 1272 57.2 C9 23 Open Field 2005 704 16.4 Overall mean 41.1 CV 0.52 WPG 1 Self GH 2004 878 14.6 WPG 2 Self GH 2004 941 1.1
73 Table A-1. Continued Identification * Plant No Pollination Loc Â¶ Year Pollinated spikelets Seed Set WPG 3 Self GH 2004 943 11.3 WPG 4 Self GH 2004 734 3.8 WPG 5 Self GH 2004 484 1.3 WPG 6 Self GH 2004 599.0 2.2 WPG 7 Self GH 2004 593 1.4 WPG 8 Self GH 2004 1168 2.7 WPG 9 Self GH 2004 1038 12.9 WPG 10 Self GH 2004 835.0 0.6 WPG 11 Self GH 2004 525 3.5 WPG 12 Self GH 2004 368 8.9 WPG 13 Self GH 2004 927 27.4 WPG 14 Self GH 2004 1011 3.4 WPG 15 Self GH 2004 183.0 0.0 WPG 16 Self GH 2004 898 36.4 WPG 17 Self GH 2004 466.0 1.7 WPG 18 Self GH 2004 1251 2.1 WPG 19 Self GH 2004 795 4.0 WPG 20 Self GH 2004 346 0.0 WPG 21 Self GH 2004 889 0.6 WPG 22 Self GH 2004 514 0.2 WPG 23 Self GH 2004 607 5.7 WPG 24 Self GH 2004 1190 9.6 WPG 25 Self GH 2004 765 16.3 Overall mean 6.87 CV 1.31 WPG 1 Self GH 2005 886 21.0 WPG 2 Self GH 2005 1574 6.3 WPG 3 Self GH 2005 237 19.0 WPG 4 Self GH 2005 785 14.7 WPG 5 Self GH 2005 1165 12.8 WPG 6 Self GH 2005 1268 3.2 WPG 7 Self GH 2005 570 5.4 WPG 8 Self GH 2005 1342 10.1 WPG 9 Self GH 2005 752 17.4 WPG 10 Self GH 2005 432 2.1 WPG 11 Self GH 2005 1323 22.0 WPG 12 Self GH 2005 540 3.6 WPG 13 Self GH 2005 1697 25.0 WPG 14 Self GH 2005 986 20.4 WPG 15 Self GH 2005 732 28.1 WPG 16 Self GH 2005 701 52.4 WPG 17 Self GH 2005 1668 36.3 WPG 18 Self GH 2005 731 6.4 WPG 19 Self GH 2005 600 6.1 WPG 20 Self GH 2005 1016 0.4 WPG 21 Self GH 2005 668 8.0
74 Table A-1. Continued Identification * Plant No Pollination Loc Â¶ Year Pollinated spikelets Seed Set WPG 22 Self GH 2005 693 2.9 WPG 23 Self GH 2005 800 8.3 WPG 24 Self GH 2005 834 25.9 WPG 25 Self GH 2005 907 38.2 Overall mean 15.8 CV 0.83 WPG 1 Self Field 2005 397 25.1 WPG 2 Self Field 2005 1365 19.8 WPG 5 Self Field 2005 576 12.9 WPG 9 Self Field 2005 274 11.7 WPG 13 Self Field 2005 1155 29.6 WPG 16 Self Field 2005 973 35.9 WPG 20 Self Field 2005 796 1.2 WPG 21 Self Field 2005 699 7.9 WPG 22 Self Field 2005 653 2.7 WPG 25 Self Field 2005 688 9.6 Overall mean 15.6 CV 0.74 WPG-1xC9-4 1 Cross GH 2004 136 70.6 WPG-1xWPG-5 1 Cross GH 2004 177 90.7 WPG-1xWPG-25 1 Cross GH 2004 169 72.8 WPG-1xWPG-2 1 Cross GH 2004 82 9.8 WPG-2xWPG-1 2 Cross GH 2004 152 0.0 WPG-2xWPG-3 2 Cross GH 2004 470 92.0 WPG-3xWPG-2 3 Cross GH 2004 155 76.8 WPG-4xWPG-14 4 Cross GH 2004 191 49.2 WPG-4xC9-7 4 Cross GH 2004 169 89.9 WPG-5xC9-15 5 Cross GH 2004 114 93.0 WPG-5xWPG-1 5 Cross GH 2004 177 70.1 WPG-8xWPG-12 8 Cross GH 2004 314 28.3 WPG-8xWPG-17 8 Cross GH 2004 131 25.9 WPG-9xWPG-18 9 Cross GH 2004 217 57.6 WPG-9xWPG-8 9 Cross GH 2004 230 31.3 WPG-10xWPG-12 10 Cross GH 2004 203 87.7 WPG-11xWPG-15 11 Cross GH 2004 158 81.6 WPG-12xWPG-10 12 Cross GH 2004 112 99.1 WPG-13xWPG-25 13 Cross GH 2004 111 54.1 WPG-14xWPG-4 14 Cross GH 2004 170 7.6 WPG-14xWPG-18 14 Cross GH 2004 187 78.1 WPG-16xWPG-17 16 Cross GH 2004 195 38.4 WPG-17xWPG-25 17 Cross GH 2004 303 89.1 WPG-17xWPG-16 17 Cross GH 2004 285 60.9 WPG-17xWPG-8 17 Cross GH 2004 160 8.8 WPG-18xWPG-19 18 Cross GH 2004 225 41.3 WPG-18xWPG-9 18 Cross GH 2004 342 52.6 WPG-18xWPG-14 18 Cross GH 2004 210 34.3
75 Table A-1. Continued Identification * Plant No Pollination Loc Â¶ Year Pollinated spikelets Seed Set WPG-19xWPG-18 19 Cross GH 2004 144 77.1 WPG-20xWPG-22 20 Cross GH 2004 226 60.2 WPG-23xWPG-11 23 Cross GH 2004 160 63.1 WPG-23xWPG-14 23 Cross GH 2004 155 64.5 WPG-23xC9-6 23 Cross GH 2004 300 58.3 WPG-25xWPG-13 25 Cross GH 2004 149 39.6 WPG-25xWPG-17 25 Cross GH 2004 199 85.4 Overall mean 58.3 CV 0.47 WPG-1xWPG-17 1 Cross GH 2005 158 84.2 WPG-2xWPG-24 2 Cross GH 2005 198 76.3 WPG-2xC9-9 2 Cross GH 2005 431 67.5 WPG-3xWPG-25 3 Cross GH 2005 186 62.4 WPG-4xC9-6 4 Cross GH 2005 504 56.0 WPG-4xC9-21 4 Cross GH 2005 156 76.3 WPG-5xWPG-11 5 Cross GH 2005 180 74.4 WPG-5xWPG-19 5 Cross GH 2005 169 74.0 WPG-5xWPG-20 5 Cross GH 2005 172 82.0 WPG-6xWPG-7 6 Cross GH 2005 207 47.3 WPG-6xC9-3 6 Cross GH 2005 186 77.4 WPG-7xWPG-6 7 Cross GH 2005 72 75.0 WPG-8xWPG-9 8 Cross GH 2005 166 74.1 WPG-9xWPG-8 9 Cross GH 2005 192 59.4 WPG-10xWPG-11 10 Cross GH 2005 177 67.2 WPG-11xWPG-10 11 Cross GH 2005 139 67.6 WPG-11xWPG-15 11 Cross GH 2005 126 63.5 WPG-11xWPG-5 11 Cross GH 2005 482 60.6 WPG-13xWPG-23 13 Cross GH 2005 307 69.4 WPG-13xC9-17 13 Cross GH 2005 203 21.2 WPG-14xC9-3 14 Cross GH 2005 212 68.9 WPG-15xWPG-11 15 Cross GH 2005 155 38.7 WPG-15xC9-15 15 Cross GH 2005 150 65.3 WPG-16xC9-22 16 Cross GH 2005 82 48.8 WPG-17xWPG-1 17 Cross GH 2005 207 82.6 WPG-18xC9-12 18 Cross GH 2005 225 28.4 WPG-18xC9-11 18 Cross GH 2005 198 38.9 WPG-19xWPG-5 19 Cross GH 2005 305 58.0 WPG-20xWPG-5 20 Cross GH 2005 119 69.7 WPG-21xC9-12 21 Cross GH 2005 242 48.8 WPG-23xWPG-13 23 Cross GH 2005 184 51.1 WPG-24xWPG-2 24 Cross GH 2005 221 36.7 WPG-25xWPG-3 25 Cross GH 2005 136 90.4 Overall mean 62.5 CV 0.27 WPG 1 Open Field 2005 794 36.2 WPG 2 Open Field 2005 1744 37.4
76 Table A-1. Continued Identification * Plant No Pollination Loc Â¶ Year Pollinated spikelets Seed Set WPG 5 Open Field 2005 852 12.7 WPG 9 Open Field 2005 547 62.8 WPG 13 Open Field 2005 651 41.3 WPG 16 Open Field 2005 1608 30.1 WPG 20 Open Field 2005 1389 18.8 WPG 21 Open Field 2005 1074 29.5 WPG 22 Open Field 2005 827 60.2 WPG 25 Open Field 2005 573 50.4 Overall mean 38.0 CV 0.43 AIT 2 Self GH 2004 429 0.6 AIT 2-2-5 Self GH 2004 812 0.0 AIT 3-13-9 Self GH 2004 333 0.0 AIT 3-16-4 Self GH 2004 192 2.6 AIT 4-12-9 Self GH 2004 335 0.6 AIT 4-36-1 Self GH 2004 195 1.0 AIT 2-2-7 Self GH 2004 397 0.0 AIT 20 Self GH 2004 150 0.0 AIT 22 Self GH 2004 200 0.6 AIT 71 Self GH 2004 82 8.5 AIT 77 Self GH 2004 72 0.0 AIT 104 Self GH 2004 128 7.0 AIT 105 Self GH 2004 807 3.0 AIT 106 Self GH 2004 110 0.0 AIT 110 Self GH 2004 120 11.7 AIT 111 Self GH 2004 446 8.3 AIT 120 Self GH 2004 141 2.8 AIT 121 Self GH 2004 362 4.9 Overall mean 2.9 CV 1.28 AIT 2 Self GH 2005 167 0.0 AIT 2-2-7 Self GH 2005 187 0.0 AIT 35 Self GH 2005 213 0.0 AIT 52 Self GH 2005 432 2.4 AIT 55 Self GH 2005 275 5.5 AIT 56 Self GH 2005 715 2.3 AIT 104 Self GH 2005 208 0.5 AIT 105 Self GH 2005 712 1.4 AIT 111 Self GH 2005 418 0.9 AIT 121 Self GH 2005 86 0.0 Overall mean 1.3 CV 1.33 AIT 2 Self Field 2005 1372 3.0 AIT 5 Self Field 2005 616 2.8 AIT 6 Self Field 2005 484 1.5 AIT 2-2-7 Self Field 2005 563 0.2
77 Table A-1. Continued Identification * Plant No Pollination Loc Â¶ Year Pollinated spikelets Seed Set AIT 35 Self Field 2005 657 2.6 AIT 52 Self Field 2005 410 1.4 AIT 55 Self Field 2005 944 0.4 AIT 56 Self Field 2005 864 0.3 AIT 71 Self Field 2005 637 2.4 AIT 73 Self Field 2005 805 1.7 AIT 74 Self Field 2005 880 2.8 AIT 77 Self Field 2005 283 0.9 AIT 78 Self Field 2005 647 2.7 AIT 87 Self Field 2005 752 1.4 AIT 99 Self Field 2005 964 2.0 AIT 104 Self Field 2005 969 5.3 AIT 105 Self Field 2005 1184 1.9 AIT 106 Self Field 2005 929 1.7 AIT 110 Self Field 2005 926 1.5 AIT 111 Self Field 2005 920 4.6 AIT 120 Self Field 2005 669 1.5 AIT 121 Self Field 2005 598 0.4 AIT 4-36-1 Self Field 2005 328 5.5 AIT 5-2-9 Self Field 2005 254 4.3 Overall mean 2.2 CV 0.69 55x315733 55 Cross GH 2004 156 10.3 71xArg 71 Cross GH 2004 716 30.2 71x315733 71 Cross GH 2004 100 35.0 73x315734 73 Cross GH 2004 119 32.8 106xTif7 106 Cross GH 2004 245 16.3 106xArg 106 Cross GH 2004 962 15.6 2-2-7xTif7 2-2-7 Cross GH 2004 241 24.1 3-13-9xTif7 3-13-9 Cross GH 2004 243 23.9 3-16-4xTif7 3-16-4 Cross GH 2004 231 17.7 3-16-4xTif7 3-16-4 Cross GH 2004 128 29.7 4-36-1xArg 4-36-1 Cross GH 2004 86 12.8 4-38-6x315732 4-38-6 Cross GH 2004 366 18.0 Overall mean 22.2 CV 0.37 121x315732 121 Cross GH 2005 127 0.0 5x315733 5 Cross GH 2005 153 0.0 2-2-7x315734 2-2-7 Cross GH 2005 393 1.5 105x315733 105 Cross GH 2005 148 0.7 2xArg 2 Cross GH 2005 143 2.1 105x315733-19 105 Cross GH 2005 168 0.6 52x315733-16 52 Cross GH 2005 202 1.0 2x315733-6 2 Cross GH 2005 60 0.0 73x315734-2 73 Cross GH 2005 159 7.5 2-1-1x315732-1 2-1-1 Cross GH 2005 233 0.0
78 Table A-1. Continued Identification * Plant No Pollination Loc Â¶ Year Pollinated spikelets Seed Set 105x315732-1 105 Cross GH 2005 153 3.9 56xWilming 56 Cross GH 2005 217 1.8 99x315734-2 99 Cross GH 2005 140 0.0 2-2-7xArg 2-2-7 Cross GH 2005 96 16.7 2-2-7xTif7 2-2-7 Cross GH 2005 186 10.2 Overall mean 3.1 CV 1.57 AIT 2 Open Field 2004 482 14.8 AIT 6 Open Field 2004 165 19.9 AIT 2-2-7 Open Field 2004 657 40.2 AIT 20 Open Field 2004 775 2.9 AIT 22 Open Field 2004 136 17.9 AIT 35 Open Field 2004 156 10.3 AIT 52 Open Field 2004 1172 8.6 AIT 55 Open Field 2004 642 8.8 AIT 56 Open Field 2004 544 17.2 AIT 71 Open Field 2004 900 19.2 AIT 73 Open Field 2004 1000 10.6 AIT 74 Open Field 2004 612 8.4 AIT 78 Open Field 2004 326 13.4 AIT 87 Open Field 2004 590 11.2 AIT 99 Open Field 2004 469 14.6 AIT 101 Open Field 2004 435 13.7 AIT 105 Open Field 2004 654 7.9 AIT 106 Open Field 2004 621 14.7 AIT 110 Open Field 2004 475 13.3 AIT 111 Open Field 2004 570 14.4 AIT 120 Open Field 2004 394 11.4 AIT 121 Open Field 2004 524 10.4 AIT 5-42-9 Open Field 2004 600 21.1 AIT 4-49-9 Open Field 2004 246 11.2 AIT 3-16-4 Open Field 2004 698 10.7 AIT 3-13-9 Open Field 2004 234 10.7 AIT 4-36-1 Open Field 2004 280 9.2 AIT 5-12-9 Open Field 2004 325 12.8 AIT 2-13-3 Open Field 2004 453 8.1 AIT 5-11-6 Open Field 2004 381 3.7 Overall mean 13.0 CV 0.51 AIT 2 Open Field 2005 639 14.7 AIT 5 Open Field 2005 692 12.7 AIT 6 Open Field 2005 421 27.1 AIT 2-2-7 Open Field 2005 546 30.5 AIT 35 Open Field 2005 617 11.4 AIT 52 Open Field 2005 887 8.1 AIT 55 Open Field 2005 843 9.1
79 Table A-1. Continued Identification * Plant No Pollination Loc Â¶ Year Pollinated spikelets Seed Set AIT 56 Open Field 2005 759 9.2 AIT 71 Open Field 2005 756 13.7 AIT 73 Open Field 2005 764 8.4 AIT 74 Open Field 2005 836 6.6 AIT 77 Open Field 2005 683 8.1 AIT 78 Open Field 2005 846 8.0 AIT 87 Open Field 2005 557 3.9 AIT 99 Open Field 2005 744 12.9 AIT 104 Open Field 2005 1003 14.5 AIT 105 Open Field 2005 657 12.4 AIT 106 Open Field 2005 631 19.9 AIT 110 Open Field 2005 900 12.6 AIT 111 Open Field 2005 742 11.6 AIT 120 Open Field 2005 806 9.4 AIT 121 Open Field 2005 731 10.7 AIT 3-16-4 Open Field 2005 320 18.8 AIT 4-36-1 Open Field 2005 490 8.8 AIT 4-38-6 Open Field 2005 413 15.5 AIT 5-12-9 Open Field 2005 268 5.4 Overall mean 12.5 CV 0.49 4188x315733 4188 Cross GH 2004 96 24.0 4188xArg 4188 Cross GH 2004 2260 27.1 4205xArg 4205 Cross GH 2004 2226 26.2 4205xTif7 4205 Cross GH 2004 1256 29.6 Overall mean 26.7 CV 0.09 ArgxTif7 Argentine Cross GH 2004 256 56.1 Tif7xArg Tifton 7 Cross GH 2004 123 41.8 Overall mean 48.9 CV 0.21 4188 4188 Self GH 2004 698 31.3 4188 4188 Self GH 2005 266 23.2 4188 4188 Self Field 2005 840 21.1 4205 4205 Self GH 2004 988 15.7 4205 4205 Self GH 2005 270 20.0 4205 4205 Self Field 2005 347 12.7 Overall mean 20.7 CV 0.31 315733 315733 Self GH 2004 514 9.4 315733 315733 Self GH 2005 342 8.4 315734 315734 Self GH 2004 214 31.4 315734 315734 Self GH 2005 150 4.3 Overall mean 13.4 CV 0.91 Argentine Self GH 2004 398 32.0
80 Table A-1. Continued Identification * Plant No Pollination Loc Â¶ Year Pollinated spikelets Seed Set Argentine Self GH 2005 223 31.5 Argentine Self Field 2005 2423 28.8 Tifton7 Self GH 2004 1589 37.0 Tifton7 Self GH 2005 340 29.3 Tifton7 Self Field 2005 410 33.5 Overall mean 32.0 CV 0.09 Argentine Open Field 2005 1496 34.2 Tifton7 Open Field 2005 1605 38.1 Overall mean 36.2 CV 0.08 * Diploids: Tifton 9 (C9), Pensacola bahiagrass (WPG). Tetraploids: artificially induced tetraploids (AIT), Sexual tetr aploid lines (STL), PI 315732, PI 315733, and PI 315734, Tifton 7 (Tif7), Argentine (Arg). Â¶ Location: greenhouse (GH), and field. Â§ Coefficient of variation (CV).
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86 BIOGRAPHICAL SKETCH Carlos Alberto Acuna was born in Corpus Christy, northeastern Argentina, on the banks of the Parana River, on 2 May, 1977. Carlos attended Bachillerato Polivalente No 14 (high school), and graduated in 1994. In 1995, he moved to Corrientes, Argentina, and began his undergraduate studies at Facu ltad de Ciencias Ag rarias, Universidad Nacional del Nordeste. In 2001, he received hi s Agronomy Engineer degree. He worked for 4 years, including the last 2 years of his undergraduate career, with reproductive systems of warm-season grasses at IBONE, Co rrientes, Argentina. In May 2004 he began his graduate career at the Univ ersity of Florida under the di rection of Dr. Ann Blount and Dr. Kenneth Quesenberry. In May 2006 he was awarded a Master of Science degree in agronomy with an emphasis in Genetic a nd Plant Breeding. After graduation, Carlos plans to begin Ph.D. studies at the University of Florida, and con tinue learning on plant breeding. After completing graduate studies, he plans to go back to Argentina, and work with grassland improvement.