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a P.O. Box 110500, Univ. of Florida, Gainesville, FL 32611-0500
b North Florida Research & Education Center, Marianna, FL 32446-7906
c P.O. Box 748, Univ. of Georgia, Tifton, GA 31793
* Corresponding author (clover{at}ufl.edu).
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Abbreviations: AIT, artificially induced tetraploids • AT, apomictic tetraploids • EBN, endosperm balance number • RRPS, restricted recurrent phenotypic selection • STC, sexual tetraploid clones
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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All tetraploid cultivars released in the USA have been superior apomictic ecotypes selected from introduced germplasm (Gates et al., 2004). A segregating population can be generated for breeding purposes or for genetic analyses by making crosses between sexual induced tetraploid plants and apomictic tetraploid plants. Sexual tetraploids have been generated by treating both diploid seed and tissue cultured calli with colchicine (Forbes and Burton, 1961; Quarin et al., 2001). Quesenberry and Smith (2003) generated over 300 tetraploids by treating callus cultures from Tifton 9 with different chromosome duplication treatments.
These novel bahiagrass germplasm provide new opportunities for use in crop improvement of the species. Before the current germplasm can be used efficiently for breeding purposes, its reproductive behavior needs to be carefully studied and characterized. The first objective of this research was to determine the reproductive behavior of 20 new induced tetraploids, the cultivars Argentine and Wilmington, an experimental hybrid Tifton 7, and other promising bahiagrass accessions. A second objective was to determine and compare the fertility of current diploid and tetraploid cultivars, induced tetraploids, and other apomictic ecotypes under different methods of pollination.
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MATERIALS AND METHODS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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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 summer 2004 to the end of summer 2005. Two sexual tetraploid clones (STC), Q4188 and Q4205 (Quarin et al., 2003), the cultivars Argentine (PI 148996) and Wilmington (PI 434189), the experimental hybrid Tifton 7 (Burton, 1992), and accessions PI 315732, PI 315733 and PI 315734 (narrow leaf tetraploids with reduced flowering and darker green color phenotypically similar to Wilmington) were also divided into two clones and grown in a greenhouse from summer 2004 to the end of summer 2005. Q4188, Q4205, Argentine, and Tifton 7 were also grown in the field during summer 2005. Ramets of individual genotypes of diploids and tetraploids were transplanted to the field (Agronomy Forage Research Unit near Gainesville, FL) in early May, with 0.91-cm spacing within and between rows in two replications of a completely randomized design.
Ploidy Determination
Flow cytometry was used to corroborate the ploidy level of the 20 AIT and the tetraploid cultivar Argentine and to determine the ploidy level of PI 315732, PI 315733, and PI 315734. Young immature tillers were collected and approximately 0.5-cm length of immature leaves above the last developing node was immediately chopped in a petri dish containing 500 µL of CyStain UV solution A (Partec, Münster, Germany). The material was well mixed, collected, and filtered (50 µm filter). Using 1500 µL of CyStain UV solution B (Partec), the material was stained for 3 min. The sample was processed on a Ploidy Analyzer PA (Partec). A sample from known P. notatum diploid and tetraploid lines was analyzed after each 10 unknown samples as comparative standards. The analyzer was arbitrarily calibrated using the known diploid line of P. notatum at 100 units, so that a tetraploid was expected at 200 units.
Embryo Sac Observations
Inflorescences at anthesis (when the embryo sacs are usually fully developed) were fixed for 24 h in FAA (18 70% ethanol:1 37% formaldehyde:1 glacial acetic acid). The pistils were then dissected out of the florets and cleared using the procedure outlined by Young et al. (1979). The ovules were observed using a differential interference contrast microscope. A minimum of 20 ovules were observed from at least two different inflorescences. Single embryo sacs containing the egg apparatus, the binucleated central cell, and a mass of antipodals at the chalazal end were classified as sexual. In contrast, multiple or single embryo sacs with the egg apparatus, the central cell, and no antipodals, were classified as apomictic. Plants producing ovules with either sexual or apomictic embryo sacs were classified as facultative.
Pollination Techniques and Seed Set
Self-pollination was accomplished by enclosing two inflorescences of each clone in a glassine bag supported with a stake before anthesis. The bags were shaken each day shortly after anthesis to promote pollination. Mutual pollinations between pairs of different diploid genotypes were accomplished by enclosing an inflorescence from each of the two genotypes in a glassine bag. In addition, inflorescences from sexual and apomictic tetraploids were also enclosed together in glassine bags to determine the cross-fertility of the 4x sexual plants. Care was taken to select inflorescences at the same stage of maturity. Bags were shaken each day during anthesis. Two inflorescences from each diploid and tetraploid clone were bagged after anthesis, avoiding seed losses from open-pollinated inflorescences.
A month after bagging, the inflorescences were harvested and threshed, and florets containing caryopses were separated from empty florets using an air column. A sample of 25 seed from each genotype in all field treatments was dissected and observed for the presence of ergot sclerotia (Claviceps paspali Stevens and Hall) replacing the seed. After this procedure, the number of seed was adjusted for each genotype based on analysis of these samples. Ergot was only considered with field data, as none was observed in the greenhouse. Percent seed set was calculated by dividing the number of florets containing caryopses by the total number of pollinated florets and multiplying by 100.
Statistical Analysis
Fertility data were analyzed using PROC GLM of PC SAS (SAS Institute, 2004) as a completely randomized design with two replications of each genotype in each location (greenhouse and field). Statistical comparison of percent seed set was tested in the following order: (i) individual diploid population comparisons (Pensacola vs. Tifton 9) for self-, cross-, and open-pollination, (ii) genotype comparison within Pensacola and Tifton 9, and for each pollination method, (iii) pollination method (cross- or open- vs. self-fertility) comparison averaged over both diploid populations, (iv) individual genotype comparison among the 20 AIT for self- and open-pollination, (v) pollination method comparison (self- vs. cross- or open-pollination) averaged over the 20 AIT, (vi) ploidy comparison (all diploids vs. 20 AIT) for self-, cross-, and open pollination, (vii) genotype comparison of the two STC for self- and cross-pollination, (viii) pollination method comparison averaged over these two clones, (ix) tetraploid population (STC vs. AIT) comparison for self- and cross-pollination, (x) comparison of Argentine versus Tifton 7 for self-pollination, and (xi) comparison of pollination method (open- vs. self-pollination) averaged over Argentine and Tifton 7. The significance of years, locations (field and greenhouse), and the corresponding interactions were tested when appropriate. Duncan's Multiple Range Test was used for mean separations. Unless otherwise stated in the text, all differences refer to significance at P < 0.05.
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RESULTS AND DISCUSSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Although high reproductive variability has been reported for naturally occurring tetraploids (Martínez et al., 2001; Espinoza et al., 2006), highly sexual plants have not been found in natural populations. The low aposporic expression detected in two AIT 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), indicating that the genes involved in the generation of an apomictic embryo sac may be present at the diploid level. However, the detected apomictic expression was very low and should not be an impediment for using these induced tetraploid plants as the female parent for hybridization purposes. Thus, results indicate that these 20 sexual AIT plants should be considered as good female counterparts for breeding tetraploid bahiagrass.
Other Accessions
It was determined by cytometric analysis that PI 315732, PI 315733, and PI 315734 were tetraploids (2n = 4x = 40). In addition, most of the analyzed ovules from these three accessions contained apomictic embryo sacs (Table 1). However, a considerable number of ovules (20–51%) contained apomictic and sexual sacs within an individual ovule. Percentage of ovules containing only sexual embryo sacs varied from 7 to 14% (Table 1). Facultative apomictic plants are not uncommon in natural populations of bahiagrass. Martínez et al. (2001) recorded a large range of variation from obligate apomictic accessions (100% of the ovules containing apomictic sacs) to highly facultative (95% of the ovules containing sexual embryo sacs). Progeny from these three accessions appeared phenotypically uniform, indicating that the apomictic embryo sacs likely were predominantly the source of new embryos in seed from these genotypes (Quesenberry, personal observation). These three accessions were selected because of their cold hardiness, prostrate growth habit, reduced flowering, and darker green color. They also have narrow leaves similar to the cultivar Wilmington, an uncommon characteristic among tetraploid bahiagrass. With the above turf attributes and apomictic reproduction, these three tetraploid plant introductions could be evaluated as potential turf type cultivars or as male parents in crosses for producing potential turf cultivars.
Tetraploid Cultivars
Embryo sac observations indicated that Argentine and Wilmington were highly apomictic plants. Ninety-five percent of the analyzed ovules had multiple apomictic sacs for both cultivars; the other 3 (Argentine) to 5% (Wilmington) had multiple apomictic sacs in addition to one sexual sac (Table 1). Burton (1992) reported 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 detail. The experimental apomictic hybrid, known as Tifton 7, was generated throughout a long and intricate breeding approach (Burton, 1992). Although this apomictic hybrid has been shown to be significantly more productive than Argentine, it has remained an experimental hybrid due to concerns regarding seed production (Hanna, personal communication). It was also highly apomictic with 95% of its ovules containing only apomictic embryo sacs, 2% containing both apomictic and sexual sacs sharing the same ovule, and the last 3% containing aborted embryo sacs.
Fertility
Diploids
No significant differences were observed between Pensacola and Tifton 9 bahiagrass for self- or cross-pollinated seed set under either field or greenhouse conditions (Table 2). Years were different for self-fertility seed set, with the percent seed set in 2005 higher than that obtained in 2004. There were no interactions between population and year for self- or cross-fertility. The mean percent seed set for self-, cross-, and open-pollination was 12, 60, and 39%, respectively (Table 3). Although nine cycles of RRPS affected the expression of several characteristics such as day-length sensitivity and growth habit, in addition to herbage mass (Gates et al., 2004), it apparently did not affect fertility. These results represent strong evidence that fertility and herbage mass are independent traits and that it is possible to select for one without affecting the other.
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The average percent seed set for self-pollination was 12% (average of greenhouse and field data) for Pensacola bahiagrass and Tifton 9. However, significant differences in terms of self-fertility were observed among individual genotypes in each cultivar. There was no interaction between genotype and year for either cultivar. The percent self-seed set varied from 0 to 44% for Pensacola bahiagrass and from 0 to 35% for Tifton 9, indicating a marked variability among genotypes. More than 75% of the genotypes produced less than 15% seed when self-pollinated. However, several genotypes can be considered as moderately self-fertile. Self-sterility is a characteristic of a good breeding parent to avoid high levels of inbreeding in segregating populations.
The average percent seed set under cross-pollination was 60% for Pensacola and 59% for Tifton 9 (Table 2). Although significant differences were observed among genotypes in terms of self-fertility, no significant differences were observed among genotypes for cross-fertility for either population. This finding was corroborated by the results obtained with Pensacola under open-pollination in the field. However, some differences were observed among genotypes of Tifton 9 under open-pollination in the field.
Because no differences were detected between Pensacola and Tifton 9 in terms of general fertility, data from both populations were pooled and analyzed as a single population for other comparisons. Significant differences were observed for self- versus cross-fertility in the greenhouse, indicating that they are predominantly cross-pollinated with low but variable expression of self-fertility. There was no interaction between pollination method and year. This finding was corroborated by the differences observed between open- and self-fertility in the field (2005 data only) (Table 2). This finding is in agreement with previous reports regarding reproductive behavior of Pensacola (Burton, 1955). In the USA the present diploid cultivars and naturalized diploid ecotypes are characterized by large variability among individual plants for growth habit, photoperiod sensitivity, anther and stigma color, and persistence under grazing, indicating that this group of sexual plants mainly reproduces by cross-pollination. However, because the self-fertility of some genotypes was found to be reasonably high, a preliminary fertility test should be conducted before selected genotypes are used as progenitors for breeding purposes or for genetic analyses.
Tetraploids
Significant differences (P = 0.08) were detected among AIT genotypes for self-fertility in the greenhouse during the two years. There was no interaction between genotype and year. The average percent self seed set was 2% (greenhouse and field) (Table 3), varying from 0 to 12% in the greenhouse, and from 0 to 6% in the field. The variability among genotypes is not surprising based on the above findings of marked variation in self-fertility among diploid genotypes.
When the self-fertility experiment was conducted in the field during 2005, no significant differences were observed between locations (greenhouse vs. field), validating greenhouse conditions for these experiments (Table 3). In contrast, a hurricane destroyed the field experiment during 2004, demonstrating that field conditions are many times inappropriate for self-fertility analysis.
High variability was detected among the AIT genotypes for seed set under open-pollination conditions for both years. There was no interaction between genotype and year. The average percent seed set was 14%, varying from 8 to 35%, indicating a reduction in overall fertility for the AIT. However, some of them were moderately fertile and should be considered as potentially good sexual females for crosses using apomictic genotypes as pollen donor.
In agreement with the two diploid populations, this group of AIT produced significantly more seed under open-pollination or cross-pollination versus self-pollination (Table 3). There was no interaction between pollination method and year. The above finding is an indication that the mode of reproduction of diploid bahiagrass was not altered by chromosome duplication. However, the diploid population produced significantly more seed than the AIT under self-, cross-, and open-pollination conditions (Table 3). Irregular meiotic behavior was indicated as the reason for low fertility in first generation induced autotetraploids (Burnham, 1962). These twenty vigorous, sexual, cross-pollinated tetraploid plants represent a key to release of the genetic variability locked in highly heterozygous apomictic tetraploids for a breeding program that is being conducted at the University of Florida.
The sexual tetraploid clones Q4188 and Q4205 (STC) exhibited similar seed set under self-pollination. Similar to the AIT and diploid populations, the seed set for these two clones was not significantly different between the greenhouse and field (Table 3). Also, the two STC had similar seed set when cross-pollinated. The similarities between these two clones were expected because they had a common origin and were closely related (Quarin et al., 2003).
Self- versus cross-fertility of the two STC was not different (Table 3). The average percent seed set was 21% when self-pollinated and 27% when cross-pollinated. Contrasting with the AIT used in this study, a considerable amount of selfing can be expected when these two STC are used as female parents without emasculation for breeding purposes. The presence of inbreds among progeny can result in a bias in progeny evaluations, waste of money and time, and can decrease the probability of identifying superior hybrid combinations.
The two STC produced significantly more seed than the diploids and AITs when self-pollinated (Table 3). There was no interaction between population and year. This indicates that the breeding process for developing these clones indirectly increased their self-fertility or that an original parent was high in self-fertility. However, their overall fertility was not increased because they did not produce more seed than the other AIT when cross-pollinated (Table 3).
Homogeneous self-fertility was observed among the apomictic genotypes (Argentine and Tifton 7). The average percent seed set under self-pollination was 32%. They also produced a similar amount of seed in the field and greenhouse (Table 3).
No differences were detected for seed set under open-pollination between these apomictic genotypes. Burton (1992) reported that Tifton 7 produced more seed per hectare than Argentine; thus, a higher number of inflorescences per unit area would be the reason for that superiority. In our research Argentine and Tifton 7 produced similar amounts of seed under self-, cross-, and open-pollination (Table 3). The average percent seed set over these two genotypes was 30% under self-pollination, 49% under cross-pollination, and 36% under open-pollination. These results are in agreement with previous reports for other bahiagrass apomictic genotypes (Burton, 1948). In fact, this is a characteristic of most pseudogamous (pollination and fertilization of the polar nuclei are required for endosperm development) apomictic plants (Vogel and Burson, 2004). Apomictic tetraploids and sexual diploids produced similar amounts of seed when cross-pollinated (Table 3). This is an indication that the apomixis–self-compatibility system is as efficient as the sexuality–self-incompatibility system in terms of seed production. In contrast, apomictic tetraploids produced more seed than the induced tetraploids when open-pollinated (Table 3). Meiotic chromosome behavior for both natural apomictic and induced tetraploid plants was determined to be quite similar but irregular (Forbes and Burton, 1961). However, in apomictic plants meiosis is avoided in the female reproductive organs; thus, these irregularities do not affect seed set. Also, they produce seed well when self-pollinated, which indicates that potential variation in the ploidy level of the male gametes does not affect endosperm development and seed set. In agreement with this statement, Quarin (1999) reported that endosperm development appears to occur regardless of the ploidy level of the pollen donor in apomictic tetraploid bahiagrass.
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CONCLUSIONS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Received for publication August 25, 2006.
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REFERENCES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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This article has been cited by other articles:
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  C. A. Acuna, A. R. Blount, K. H. Quesenberry, K. E. Kenworthy, and W. W. Hanna Bahiagrass Tetraploid Germplasm: Reproductive and Agronomic Characterization of Segregating Progeny Crop Sci., March 17, 2009; 49(2): 581 - 588. [Abstract] [Full Text] [PDF]
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