Effect of pollination time on the frequency of 2n + n fertilization in apomictic buffelgrass. (Crop Breeding, Genetics & Cytology).
Apomixis is an asexual form of reproduction in which seeds are produced without fertilization of the egg cell. In the aposporous form of apomixis, the female gametophyte (embryo sac) develops from an unreduced somatic nucellar cell in the ovule. Consequently, all cells, including the egg cell, in the mature female gametophyte of an apomict have an unreduced chromosome number (2n). In most apomicts, the unreduced egg cell initiates division near or at the time of anthesis and develops autonomously into an embryo without being fertilized by a sperm nucleus from the pollen; this is referred to as parthenogenesis.
Unreduced gametes occur in both sexual and apomictic plants. In sexual plants, most unreduced gametes result from the failure of chromosome reduction at the first meiotic division or the failure of cytokinesis at the second meiotic division during megasporogenesis and/ or microsporogenesis. Regardless of the mechanism, the resultant unreduced "gametophyte" has the same chromosome number as the sporophyte. In apomicts, the development of an unreduced egg in the female gametophyte is normal; therefore, 2n + n fertilization should occur at a higher frequency in apomicts than in sexual plants.
In apomictic species where sexual genotypes are not available to use as the female parent, fertilization of an unreduced egg is a method of creating new genotypes. Intraspecific Kentucky bluegrass, Poa pratensis L., hybrids were produced by crossing facultative apomicts with highly apomictic ecotypes, and occasionally, an unreduced egg was fertilized by a reduced male gamete resulting in an apomictic 2n + n hybrid (Pepin and Funk, 1971). Some of these 2n + n hybrids were superior to the n + n hybrids as well as the existing apomictic ecotypes and were released as improved cultivars (Bashaw and Funk, 1987). Burson and Hussey (1996) reported that most 2n + n [F.sub.1] hybrids between Pennisetum flaccidum Griseb. and P. mezianum Leeke were more winter hardy and produced more forage than the n + n [F.sub.1] hybrids. These findings demonstrate that the fertilization of an unreduced egg has potential for producing superior apomictic germplasm. However, the unpredictability and low frequency of this event limits its usefulness for improving apomictic species.
The control of 2n + n fertilization is poorly understood. Pepin and Funk (1971) recovered high numbers of 2n + n intraspecific Kentucky bluegrass hybrids when pollinations were made before anthesis. They hypothesized that early pollination may have contributed to the fertilization of unreduced eggs in aposporous embryo sacs of the facultative apomictic female parent. Martinez et al. (1994) investigated the effect of pollination time on the recovery of 2n + n hybrids by pollinating stigmas of apomictic tetraploid bahiagrass, Paspalum notatum Flugge, from 1 to 5 d before anthesis. All 2n + n hybrids recovered were from florets pollinated 2 and 3 d before anthesis. They hypothesized that early pollination prevented parthenogenetic development of the unreduced egg cell and this permitted the egg to be fertilized. These findings suggest a relationship between time of pollination and the recovery of 2n + n hybrids, but neither study established definitively a relationship between these two events.
Buffelgrass is an important forage grass in the semi-arid, subtropical areas of the world. It is an excellent species to investigate this purported relationship. The grass reproduces primarily by apomixis with the mechanism being apospory followed by pseudogamy (Fisher et al., 1954; Snyder et al., 1955). Most accessions are either obligate apomicts (Fisher et al., 1954; Snyder et al., 1955) or facultative apomicts (Bray, 1978; Sherwood et al., 1980); however, a few sexual plants exist (Bashaw, 1962). When apomictic buffelgrass is pollinated in controlled hybridization studies, 2n + n hybrids are sometimes recovered (Bashaw and Hignight, 1990; Bashaw et al., 1992; Hussey et al., 1993). Buffelgrass has a protogynous flowering behavior where its stigmas are exserted from the florets 1 to 3 d before anthesis depending on the genotype. The stigmas are receptive when exserted from the floret and remain receptive throughout the duration of the protogynous interval or until they are pollinated (Shafer et al., 2000). This study was undertaken to determine if the timing of pollination (days before anthesis) influenced the frequency at which 2n + n progeny are recovered in apomictic and sexual buffelgrass.
MATERIALS AND METHODS
Six buffelgrass accessions with protogynous intervals ranging from 1 to 3 d were used for this study (Table 1). Ten clones of each accession were grown in 35-cm pots in a greenhouse (35[degrees]C/25[degrees]C day/night) under a 12-h photoperiod with 1000-W high energy discharge lamps. Shortly after emergence from the leaf sheaths, individual inflorescences were enclosed in glassine bags. Every morning thereafter, the bag was removed from each inflorescence to determine if stigmas were beginning to be exserted. Once stigma exsertion occurred, the date was recorded and each stigma was examined with a 16x hand lens to determine if pollen was present. All involucres with contaminated florets as well as those with florets in which stigma exsertion had not initiated were removed from each inflorescence. Approximately half of these inflorescences were self-pollinated with pollen from plants of the same accession growing in another greenhouse. The remaining inflorescences were cross-pollinated with birdwoodgrass pollen. Birdwoodgrass accession PI 193444 was used as the pollinator because it produces large quantities of viable pollen (Read and Bashaw, 1969). After pollination, each inflorescence was enclosed in a glassine bag. All unpollinated inflorescences also were enclosed in individual glassine bags. The following morning the glassine bags were removed from the unpollinated inflorescences, reexamined for the presence of pollen, and self- and cross-pollinated as described above. This protocol was repeated daily until anther exsertion. On that day, the stigmas were self- or cross-pollinated before anther exsertion and dehiscence. The number of consecutive days that pollinations were made depended on the length of the protogynous interval of the individual accession (Table 1). The time from stigma exsertion until the anthers are exserted is referred to as the protogynous interval. Involucres on individual inflorescences of PI 409397 and PI 409407 were pollinated on two consecutive days (1 and 0 d before anthesis), PI 409704 and S 2103 for three consecutive days (2, 1, and 0 d), and PI 295657 and PI 315679 for four consecutive days (3, 2, 1, and 0 d). The day of anthesis is designated as 0 d.
After approximately 6 wk, the bagged inflorescences were removed from each plant, and the involucres were removed from each inflorescence, counted, hand threshed, and cleaned. The total number of caryopses recovered from each protogynous interval of each accession was counted. A single caryopsis was planted into a commercial soil mix in an individual 5- by 5-cm cell in a 30- by 30-cm plastic potting flat. Each flat consisted of 36 individual 5- by 5-cm cells. After all the caryopses were planted, the flats were placed in a warm greenhouse and kept moist to promote germination. Following germination, the seedlings were allowed to grow to a height of approximately 15 cm before they were clipped to promote tillering.
Two or three young leaves about 3 to 4 cm long were removed from each seedling and taken into the laboratory to determine the DNA content of each seedling. Common buffelgrass, 2n = 4x = 36, was used as an internal standard. A piece of leaf about 1 [cm.sup.2] was cut from a leaf blade of each seedling and placed into a 15-cm petri dish along with a similar sample from common buffelgrass. A drop of a commercial buffer solution (Partec GmbH, Munster, Germany) (1) was added to the dish, and both leaf samples (standard and unknown) were finely chopped with a double-edged razor blade until the tissue was thoroughly macerated. Approximately 0.5 mL of the buffer solution was added to the chopped tissue in the petri dish. This solution was resuspended several times with a pipette and filtered through a 30- [micro]m filter to eliminate the debris. After 5 min, 2.5 mL of a commercial Partec DAPI (4'-6'-diamidino-2phenylindole) solution were added to the filtered buffer solution containing the nuclei and the solution was resuspended. After about 5 min, the sample was analyzed with a Partec CA II flow cytometer. A minimum of 600 cells was analyzed for each sample. Common buffelgrass was used as the internal standard for every seedling analyzed because its 2C DNA content (3.08 pg) and chromosome number (2n = 4x = 36) were known. It was possible to determine the ploidy level of the plant being analyzed by comparing the [G.sub.1] peak of the unknown seedling with the [G.sub.1] peak of the internal standard. Because PIs 409367, 409407, 295657, 315679, and S 2103 have 36 chromosomes, all offspring that resulted from regular apomictic reproduction (2n + 0) or sexual reproduction (n + n) would have 36 chromosomes. The DNA content of these tetraploid offspring was similar to that of the internal standard (common buffelgrass) and their [G.sub.1] peaks were similar. With additional genomes, the 2n + n progeny would have a higher chromosome number (2n = 6x = 54) and their [G.sub.1] peaks would differ from the standard (2n = 4x = 36).
Because PI 409704 is a pentaploid (2n = 5x = 45), common buffelgrass was used as an internal standard for all PI 409704 seedlings. Progeny resulting from apomictic reproduction (2n + 0) had a [G.sub.1] peak associated with 45 chromosome plants that was higher than the [G.sub.1] peak of the internal standard. Progeny with [G.sub.1] peaks higher than the 45 chromosome plants were suspected 2n + n products. These plants were reanalyzed with Sorghum bicolor (L.) Moench as the internal standard which has a 2C DNA content of 1.74 pg.
The ploidy level of some of the predicted 2n + n plants was confirmed by counting the number of chromosomes in their root tip cells. Root tips were collected from plants growing in pots in a greenhouse between 0900 and 1000 h and prepared as previously described (Burson, 1991).
All data were analyzed with the PROC GLM and PROC REG procedures of the SAS Statistical Software Package (SAS, 1999). Regression analysis was used to determine the relationship between day of pollination and number of 2n + n hybrids recovered.
Germination of the self-pollinated seed produced from each protogynous interval over all accessions ranged from 34.8 to 85.3% with a mean of 68.2%. There were no trends or patterns associated between percent seed germination and the day when the pollinations were made.
Nuclear DNA content of leaf cells was determined for 1962 self-pollinated plants of which 1766 had a mean 2C DNA content of 3.09 pg, nearly identical to that of the internal standard, common buffelgrass (3.08 pg). These plants were considered to be tetraploids with 36 chromosomes. One hundred sixty-eight plants were progeny from PI 409704 and their mean 2C DNA content was 4.15 pg which indicated they were pentaploids with 45 chromosomes. Of the remaining 28 plants, 25 had a 2C DNA content of 4.76 pg suggesting they were hexaploids with 54 chromosomes. Three plants from PI 409704 had a 2C DNA content of 5.52 pg, and they were thought to be aneuploids with approximately 67 chromosomes. These 28 plants are the products of 2n + n fertilization from self-pollination (Table 2).
Of the six buffelgrass accessions, 2n + n plants were recovered from four of the five apomictic accessions and none were recovered from the sexual accession S 2103 (Table 2). This was expected for the sexual parent because unreduced gametes usually are produced at a much lower frequency in sexual plants than in apomicts.
Of the four apomictic accessions that produced 2n + n progeny, there was no significant difference in the overall frequency of 2n + n hybrids recovered. Across protogynous intervals, the frequency of 2n + n hybrids produced was 1.0, 1.8, 0.8, and 2.9% for Pis 409367, 409704, 295657, and 315679, respectively. When averaged across all genotypes, a complex polynomial relationship between the day of pollination and frequency of 2n + n hybrids recovered was observed (Fig. 1). However, for the five apomictic accessions, the frequency of 2n + n hybrids recovered from the 0, 1, 2, and 3 d protogynous interval groups was 1.7, 0.7, 3.1, and 1.3%, respectively. The highest frequency of 2n + n fertilization was in those florets pollinated 2 d before anthesis (Fig. 1).
[FIGURE 1 OMITTED]
Root tip counts on five plants with a mean 2C DNA content of 5.59 pg confirmed that they were hexaploid (2n + n) plants with 54 chromosomes.
Percent germination of seed from the buffelgrass x birdwoodgrass crosses ranged from 23 to 91% with a mean of 60.9%. No relationship between germination and protogynous interval was observed.
The 2C DNA content was determined for 3072 cross-pollinated seedlings of which 2992 had a DNA content similar to their maternal parent (n + n and 2n + 0), while the remaining 80 plants had a higher DNA content (Table 3). Of the 3072 plants, 2721 had a 36-chromosome buffelgrass plant as their maternal parent. Of these 2721 plants, 2649 had a mean 2C DNA content of 3.11 pg compared with 3.08 pg for the common buffelgrass standard. The remaining 72 offspring had a mean DNA content of 4.77 pg suggesting these plants are hexaploids with 54 chromosomes (36 + 18 = 54) and are the products of 2n + n fertilization. The 45-chromosome accession, PI 409704, was the maternal parent of the remaining 279 cross-pollinated seedlings, and 271 of these had a mean DNA content of 4.12 pg, which is similar to that of the maternal parent (4.15 pg). The remaining eight plants had a mean DNA content of 5.41 pg indicating that they were septaploids with 63 chromosomes (45 + 18 = 63) resulting from fertilization of unreduced gametes (Table 3).
When buffelgrass was crossed with birdwoodgrass, a significant ([r.sup.2] = 0.97) linear relationship was observed between the day of pollination and the frequency of 2n + n hybrids recovered (Fig. 1). The overall frequency of 2n + n hybrids recovered from all six buffelgrass accessions over all protogynous intervals was 2.6% (Table 3). Contrary to the self-pollinated study, 2n + n offspring were recovered from the sexual plant (S 2103) when it was crossed with birdwoodgrass.
There was a difference among the five apomictic accessions for the number of 2n + n hybrids recovered (Table 3). If the protogynous intervals are ignored and only the total number of 2n + n hybrids from each cross are considered, the mean frequency of 2n + n fertilization was 0.5, 0.5, 2.9, 3.4, and 3.5% for Pis 409367 x 193444, 409407 x 193444, 409704 x 193444, 295657 x 193444, and 315679 x 193444 hybrids, respectively. These data demonstrate that a higher frequency of 2n + n hybrids was produced by the latter three crosses. Because accessions Pis 409704, 295657, and 315679 have longer protogynous intervals, data indicate the length of the protogynous interval influences the frequency of 2n + n fertilization when cross-pollinated. When the number of 2n + n hybrids recovered from the different protogynous intervals are examined across hybrids, this pattern is even more evident. Across all five apomictic accessions, the frequency of 2n + n fertilization at 0, 1, 2, and 3 d protogynous intervals was 1.0, 2.0, 4.3, and 4.0, respectively. This demonstrates that as the length of the protogynous interval increases up to 2 or 3 d, the number of 2n + n hybrids produced also increases ([r.sup.2] = 0.86).
The highest frequency of 2n + n fertilization (8.2%) occurred when the stigmas were pollinated 2 d before anthesis in the PI 409704 x PI 193444 cross (Table 3). In comparing the number of 2n + n hybrids recovered to the different protogynous intervals when the pollinations were made for all crosses, the frequency of 2n + n fertilization was higher when pollinations were made 2 or 3 d before anthesis (Table 3; Fig. 1). The only exception was the PI 409367 x PI 193444 cross.
The overall frequency at which 2n + n hybrids were recovered under both self- and cross-pollinated conditions was higher than expected. When only the apomictic accessions are considered, the frequency of 2n + n fertilization when self- and cross-pollinated was 1.6 and 2.9%, respectively, and when the sexual maternal parent (S 2103) is included, the frequencies dropped to 1.4 and 2.6%, respectively. The major question is whether or not early pollination increases the frequency of 2n + n fertilization. Under self-pollinated conditions, regression analysis revealed a complex polynomial relationship between time of pollination and number of 2n + n hybrids recovered (Fig. 1). For the two accessions that were pollinated over four time intervals, there was a trend to recover more 2n + n hybrids when pollinated 2 d before anthesis. However, when cross-pollinated, the number of 2n + n hybrids recovered increased linearly as the day of pollination increased to 3 d before anthesis (Fig. 1). These data demonstrate that when buffelgrass is crossed with birdwoodgrass more 2n + n hybrids are recovered from pollinations that are made 2 or 3 d before anthesis. The frequency of 2n + n fertilization was 5 and 8% for some crosses (Table 3).
The recovery of three 2n + n hybrids from the S 2103 x PI 193444 cross was unexpected (Table 3). An argument could be made that the unreduced eggs that were fertilized were not the products of sexual reproduction and that S 2103 is a facultative apomict. If that were true, then apomictic sacs could be produced and unreduced eggs in these sacs could have been fertilized to produce the 2n + n individuals. This is unlikely because more than 1000 mature ovules from this accession have been examined cytologically and no evidence of apomictic development has been observed. Another possibility is that the three hybrids did not result from the fertilization of an unreduced egg (2n + n) but are the products of the fertilization of a reduced egg by an unreduced sperm (n + 2n). However, unreduced male gametes have not been reported in PI 193444. Further, if this accession produced unreduced male gametes, some of the hybrid offspring from the five apomictic accessions should have been octoploids (2n = 8x = 72; 2n + 2n) rather than hexaploids (2n = 6x = 54; 2n + n), but all the offspring were either tetraploids (2n + 0) or hexaploids (2n + n) (Tables 2 and 3). Therefore, the three hexaploid S 2103 x PI 193444 hybrids probably originated from the fertilization of an unreduced egg, suggesting that unreduced female gametes occasionally develop in sexual genotypes of this species.
Another concern is the 2n + n plants recovered from both self- and cross-pollinations made on the day of anthesis (Tables 2 and 3). If early pollination is the primary criterion for the fertilization of an unreduced egg, how did these 2n + n plants originate since they were pollinated on 0 d? This behavior has been reported in other grass species (Harlan and de Wet, 1975; Espinoza and Quarin, 2000). However, depending on the accession, the frequency of this event in this study ranged from 0.9 to 3.8% (Tables 2 and 3) which is higher than what normally occurs in other species. An explanation for these higher frequencies is that the controlled pollinations (both self and cross) were made approximately 4 h before anthesis. This 4-h period may be enough time to allow the unreduced egg cell to be fertilized before the onset of parthenogenesis because the time required from pollination until the pollen tube enters the embryo sac is between 2 and 3 h for most buffelgrass accessions (Shafer et al., 2000).
Findings from this study demonstrate unequivocally that early pollination of apomictic buffelgrass by bird-woodgrass increases the frequency of the fertilization of an unreduced egg. Besides supporting Martinez's et al. (1994) findings in apomictic bahiagrass, this study provides credibility to their theory because a greater number of plants were examined in this study. Martinez et al. (1994) examined only 59 progeny, whereas we examined 5034 offspring. The frequency of 2n + n fertilization was higher in bahiagrass, but the large number of buffelgrass plants analyzed demonstrates this phenomenon is not an aberration.
Upon establishing a relationship between early pollination and an increased frequency of 2n + n fertilization in apomicts, close examination of the products from crosses where other protogynous, apomictic species were used as the female parent tend to support this hypothesis. For example, 2n + n hybrids were recovered from interspecific crosses with protogynous, apomictic accessions of Pennisetum orientale L. and P. flaccidum as the female parents (Bashaw et al., 1992; Hussey et al., 1993; Wipff, 1995). Kindiger and Dewald (1994) reported the frequency of 2n + n fertilization in some eastern gamagrass, Tripsacum dactyloides (L.) L., crosses as high as 27%. Eastern gamagrass is a monecious species with the female and male florets on the same inflorescence, and the stigmas of the female flowers normally are exserted several days before anther exsertion in the male flowers. This protogynous flowering behavior allows the female florets to be pollinated early and may account for the high number of 2n + n plants. As mentioned earlier, a higher frequency of 2n + n hybrids was recovered when apomictic Kentucky bluegrass plants were pollinated early (Pepin and Funk, 1971). However, early pollination does not guarantee 2n + n fertilization in an apomict. Espinoza and Quarin (2000) hand emasculated and pollinated common dallisgrass, Paspalum dilatatum Poir., florets 2 to 3 d before anthesis but no 2n + n hybrids were produced. The only 2n + n hybrids recovered were from crosses made on the day of anthesis. Apparently the success of 2n + n fertilization from early pollination is species dependent. There also is a difference among genotypes within a species and whether or not they are self- or cross-pollinated (Tables 2 and 3).
A plausible explanation for this phenomenon has not been presented. The only hypothesis that attempts to explain why early pollination increases the chance of the fertilization of an unreduced egg was proposed by Martinez et al. (1994). They hypothesized that early pollination prevented the parthenogenetic development of the unreduced egg cell and this allowed the unreduced egg cell to be fertilized. Using electron microscopy, Vielle et al. (1995) studied the ultrastructural changes that occur in the egg apparatus of sexual and apomictic embryo sacs of buffelgrass before and shortly after pollination. Their findings provide insight into why the frequency of the fertilization of unreduced eggs in apomictic embryo sacs increase when pollinated early. In aposporous sacs, a cell wall completely covered the plasma membrane of the unreduced egg cell several hours before a pollen tube entered the female gametophyte. Whereas, in sexual embryo sacs, a cell wall only covered the micropylar end of the reduced egg cell and the plasma membrane of the cell was exposed on the chalazal end. Even 4 h after pollination, the cell wall had not covered the chalazal end of the reduced egg cell. These findings provide a plausible explanation for this phenomenon in that a cell wall that forms around the unreduced aposporous egg cell provides a physical barrier that prevents the pollen tube from penetrating the cell. Since this occurs before anthesis, it prevents the unreduced egg cell from being fertilized. However, if the stigmas are pollinated before the cell wall has developed and before the initiation of parthenogenesis, the unreduced egg cell can be fertilized. This may explain how the frequency of 2n + n fertilization is increased when the stigmas are pollinated 2 or 3 d before anthesis.
Findings from this study demonstrate that when apomictic buffelgrass is pollinated with birdwoodgrass pollen, early pollination increases the frequency of 2n + n fertilization. There are differences between genotypes and an effect of the male parent on this event. However, in protogynous, apomictic buffelgrass, when pollinations are made 2 or 3 d before anthesis, the frequency of 2n + n fertilization is high enough that this approach could be used as a tool to create new apomictic genotypes. This reproductive phenomenon may have application as a breeding tool to improve other apomictic species where sexual types are not available to use as the maternal parent in a cross.
Table 1. Buffelgrass and birdwoodgrass germplasm used. Method of Protogynous Country of Accession 2n reproduction interval origin Buffelgrass PI 409367 36 Apomictic 0-1 d South Africa PI 409407 36 Apomictic 0-1 d South Africa PI 409704 45 Apomictic 0-2 d South Africa S 2103 36 Sexual 0-2 d USA ([dagger]) PI 295657 36 Apomictic 0-3 d Zimbabwe PI 315679 36 Apomictic 0-3 d USA ([double dagger]) Birdwoodgrass PI 193444 36 Apomictic -- Australia ([section]) ([dagger]) This line was selected from a sexual plant discovered in a field of common buffelgrass. ([double dagger]) This accession was obtained by USDA-ARS National Plant Introduction System from USDA-NRCS (formerly USDA-SCS) in the 1960s, and the original germplasm was probably introduced from Africa. ([section]) This germplasm was originally introduced into western Australia from Afghanistan (Dr. Don Loch, personal communication). Table 2. Frequency of 2n + n fertilization in six buffelgrass accessions when self-pollinated 0 to 3 d prior to anthesis. Day pollinated Seedlings Accession prior to anthesis Caryopses analyzed no. PI 409367 1 126 98 0 143 97 PI 409407 1 27 14 0 51 21 PI 409704 2 76 51 1 94 68 0 108 52 S 2103 2 152 111 1 137 66 0 138 48 PI 295657 3 507 344 2 212 165 1 202 169 0 131 80 PI 315679 3 156 127 2 212 134 1 116 69 0 299 250 Overall 2887 1962 2n 2n + n 2n + n Accession plants plants plants no. % PI 409367 97 1 1.0 96 1 1.0 PI 409407 14 0 0 21 0 0 PI 409704 50 1 2.0 68 0 0 50 2 3.8 S 2103 111 0 0 66 0 0 48 0 0 PI 295657 342 2 0.6 162 3 1.8 168 1 0.6 80 0 0 PI 315679 123 4 3.1 127 7 5.2 68 1 1.4 245 5 2.0 Overall 1934 28 1.4 Table 3. Frequency of 2n + n fertilization in six buffelgrass accessions when pollinated by birdwoodgrass 0 to 3 d prior to anthesis. Day pollinated prior to Seedlings Accession anthesis Caryopses analyzed no. PI 409367 x PI 193444 1 177 119 0 414 97 PI 409407 x PI 193444 1 258 189 0 50 27 PI 409704 x PI 193444 2 173 73 1 109 43 0 258 163 S 2103 x PI 193444 2 271 207 1 136 88 0 162 115 PI 295657 x PI 193444 3 319 240 2 365 245 1 205 136 0 132 62 PI 315679 x PI 193444 3 342 233 2 954 528 1 556 352 0 174 155 Overall 5055 3072 2n 2n + n 2n + n Accession plants plants plants no. % PI 409367 x PI 193444 119 0 0 96 1 1.0 PI 409407 x PI 193444 188 1 0.5 27 0 0 PI 409704 x PI 193444 67 6 8.2 43 0 0 161 2 1.2 S 2103 x PI 193444 207 0 0 86 2 2.3 114 1 0.9 PI 295657 x PI 193444 228 12 5.0 237 8 3.3 133 3 2.2 62 0 0 PI 315679 x PI 193444 226 7 3.0 506 22 4.2 339 13 3.7 153 2 1.3 Overall 2992 80 2.6
The authors thank Drs. H. James Price and J. Spencer Johnston for verifying the DNA content of the common buffelgrass accession that was used as an internal standard.
(1) Mention of a trade mark of a proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.
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Byron L. Burson, * Mark A. Hussey, Jo M. Actkinson, and Gail S. Shafer
B.L. Burson and J.M. Actkinson, USDA-ARS, Crop Germplasm Research Unit, 430 Heep Center, Texas A&M Univ., College Station, TX 77843-2474; M.A. Hussey, Dep. of Soil & Crop Sciences, Texas A&M Univ., College Station, TX 77843-2474; G.S. Shafer, Yoder Bros., Inc., Chualar, CA 93925. Joint contribution of the USDA-ARS, Crop Germplasm Research Unit, Southern Plains Agric. Res. Center and the Dep. of Soil & Crop Sciences, Texas A&M Univ. Received 21 Feb. 2001. * Corresponding author (firstname.lastname@example.org).
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|Author:||Burson, Byron L.; Hussey, Mark A.; Actkinson, Jo M.; Shafer, Gail S.|
|Article Type:||Statistical Data Included|
|Date:||Jul 1, 2002|
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