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CROP BREEDING, GENETICS & CYTOLOGY

Stigma Receptivity and Seed Set in Protogynous Buffelgrass

^a Dep. of Soil & Crop Sciences, Texas A&M Univ., College Station, TX USA
^b USDA-ARS, 430 Heep Center, Texas A&M Univ., College Station, TX 77843-2474 USA

b-burson{at}tamu.edu

	ABSTRACT

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussion REFERENCES

 
Buffelgrass, Pennisetum ciliare (L.) Link syn = Cenchrus ciliaris L., is an important warm-season perennial forage grass that is widely grown throughout the arid tropics. It has perfect florets, and emasculation is thought to be required to produce controlled hybrids. This is a tedious, difficult undertaking because of the small floret size. The flowering behavior of buffelgrass is such that the stigmas are exserted from the floret prior to anthesis, which is referred to here as the protogynous interval. This investigation was conducted to determine the duration of the protogynous interval in 447 buffelgrass accessions and to ascertain stigma receptivity during the protogynous intervals. Protogynous intervals in a field nursery near College Station, TX, for all accessions ranged from 1 to 4 d. Six accessions with protogynous intervals ranging from 1 to 3 d were used to investigate stigma receptivity under both self- and cross-pollinated conditions in a greenhouse. Pollen germination and tube growth were observed with fluorescent microscopy at different time increments following pollination. Across all accessions, pollen germinated within 15 min of contacting the stigma, and pollen tubes grew to the micropyle within 2 to 6 h, depending on the accession and pollen source. Mean seed set ranged from 11 to 76% and from 22 to 80% among accessions following self- and cross-pollination, respectively. This investigation revealed that variation exists for protogynous interval within buffelgrass, and the stigmas are receptive when exserted from the floret and remain receptive throughout duration of the protogynous interval regardless of whether it occurs 3, 2, or 1 d prior to anthesis. These findings demonstrate that protogyny can be used to produced controlled hybrids in sexual buffelgrass without emasculation.

	INTRODUCTION

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussion REFERENCES

 
BUFFELGRASS is a warm-season, perennial bunch grass that is an important forage in many of the drier regions of the world, including parts of the southwestern USA. The grass reproduces primarily by apomixis, with the mechanism being apospory followed by pseudogamy (Fisher et al., 1954; Snyder et al., 1955). Most buffelgrass accessions are obligate apomicts (Fisher et al., 1954; Snyder et al., 1955); however, facultative apomicts that reproduce by sexual and apomictic means also have been reported (Bray, 1978; Sherwood et al., 1980). Even though apomixis is prevalent within the species, Bashaw (1962) reported a unique sexual plant that has been used in genetic studies. Pseudogamous apomicts, such as buffelgrass, require fertilization of the polar nuclei for endosperm development. Therefore, viable pollen is a necessity for seed set in apomictic buffelgrass. Pollen viability also is important for hybridization in apomicts because when a facultative apomict is used as the female parent, new genotypes are produced when the reduced egg cells in meiotically derived embryo sacs are fertilized. Another fertilization event in apomict plants occurs when the unreduced egg in an apomictic sac is fertilized by a reduced sperm nucleus. This phenomenon is known as the fertilization of an unreduced egg (2n + n) or B_III hybridization (Bashaw and Hignight, 1990). Fertilization of an unreduced egg provides a means for producing new genotypes by the incorporation of whole alien genomes while retaining the entire somatic chromosome complement of the apomictic female parent. Bashaw and Hignight (1990) demonstrated that 2n + n fertilization can be used to develop unique apomictic buffelgrass germplasm.

A range of chromosome numbers has been reported for buffelgrass with the most common number being 2n = 4x = 36 (Fisher et al., 1954). The species apparently is a segmental allotetraploid because its chromosomes typically pair as one or two quadrivalents and 16 or 14 bivalents during diakinesis of meiosis I. However, it is not uncommon to find plants with 2n = 5x = 45 and 6x = 54 chromsomes as well as aneuploids of these three ploidy levels (Fisher et al., 1954; Bashaw and Hignight, 1990).

Like most warm-season perennial grasses, buffelgrass is predominately cross-pollinated. Some accessions exhibit a protogynous flowering behavior where the stigmas are extruded from the florets prior to anther exsertion (Fisher et al., 1954; Snyder et al., 1955). The period between stigma exsertion and initiation of anthesis is referred to here as the protogynous interval. Little is known about the duration of this protogynous interval or the receptivity of the exserted stigmas prior to anthesis in buffelgrass. There are conflicting reports in the literature regarding stigma receptivity in buffelgrass. Snyder et al. (1955) indicated that the stigmas were exserted and apparently were receptive as early as 36 to 48 h prior to anthesis; however, Bashaw and Funk (1987) reported that the stigmas began to emerge 2 d before anthesis but were not receptive until the day of anthesis.

In the past, successful hybridization in buffelgrass has been accomplished by hand emasculation or with a gametocide prior to pollination. Because of the small size of the florets, hand emasculation is a tedious, difficult process which often results in damaging the pistil in an emasculated floret. An experimental gametocide that prevents anther extrusion from the floret has been used to produce controlled crosses in buffelgrass (Bashaw and Hignight, 1990). Unfortunately, this gametocide is no longer available and hand emasculation is the only option for producing controlled hybrids. Since protogyny might be a means of producing hybrids in buffelgrass without emasculation, this investigation was undertaken to (i) quantify the variation of protogynous intervals, (ii) determine the receptivity of stigmas during the different protogynous intervals, and (iii) determine seed set under both self- and cross-pollinated conditions during the different intervals for both sexual and apomictic buffelgrass accessions.

	Materials and methods

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussion REFERENCES

 
Plant Materials
Seed of 447 buffelgrass accessions in the National Plant Germplasm System (NPGS) were germinated and 10 seedlings of each accession were transplanted into individual pots in a greenhouse. After the seedlings had produced from 3 to 5 tillers, five plants of each accession were transplanted into a field nursery on the Texas A&M University Research Farm near College Station, TX, in April 1996 and were used to determine the variation for protogynous interval in this population. The interval between stigma exsertion and anthesis was determined by monitoring the florets in the upper one-third of a minimum of five inflorescences within each accession. Stigma exsertion date was recorded on a tag that was attached to the culm supporting the inflorescence, and the florets on each tagged inflorescence were examined daily to determine when anthesis had occurred.

Six accessions (PI 295657, PI 315679, PI 409367, PI 409407, PI 409704, and S 12103) were further evaluated for stigma receptivity and seed set because of their different protogynous intervals. PI 409407 and PI 409367 had 1-d protogynous intervals (short), PI 409704 and S 12103 had 2-d protogynous intervals (intermediate), while PI 315679 and PI 295657 had 3-d protogynous intervals (long). Five accessions (PI 295657, PI 315679, PI 409367, PI 409407, and PI 409704) were obligate apomicts or highly apomictic, whereas S 12103 was sexual. All had 2n = 4x = 36 chromosomes except for PI 409704, which is a pentaploid with 45 chromosomes. Clones of each accession were planted into pots and grown in a greenhouse (35°C/25°C day/night) under a 12-h photoperiod with 1000-W high intensity discharge lamps. The protogynous interval of these six accessions was checked and verified under greenhouse conditions.

Pollination Techniques
The plant materials used to determine stigma receptivity and pollen tube growth and seed set were grown in a greenhouse under the above mentioned conditions. Individual inflorescences of each accession were placed into glassine bags prior to stigma exsertion. At approximately 0800 h of the following morning, the bag was removed from each inflorescence and the florets of each involucre were examined to determine if stigma exsertion had occurred. Once stigma exsertion had initiated, the date was recorded and each stigma was examined with a 16x hand lens to determine if pollen was present. All involucres with contaminated stigmas were removed from the inflorescences as were those with florets in which stigma exsertion had not occurred. To determine when the stigmas were receptive, stigmas of florets on different individual inflorescences were hand-pollinated on consecutive days following stigma exsertion. Some inflorescences were pollinated the morning of stigma exsertion and the remaining inflorescences were enclosed in glassine bags. The following morning the bags were removed from some of the remaining inflorescences and the stigmas of florets on these inflorescences were pollinated. This was continued each morning until the day of anthesis at which time the remaining inflorescences with exserted unpollinated stigmas were pollinated prior to anther dehiscence. This protocol was followed for each accession and depending on the protogynous interval of the accession, pollinations were made on (i) 3, 2, 1, and 0 d for those with the long interval, (ii) 2, 1, and 0 d for those with the medium interval, and (iii) 1 and 0 d for those with the short interval. After each inflorescence was pollinated, it was enclosed again in a glassine bag. This procedure was followed for those inflorescences used to determine stigma receptivity and pollen tube growth under both self- and cross-pollinated conditions, as well as those used to measure seed set for self- and cross-pollinated conditions.

Stigma Receptivity and Pollen Tube Growth
To determine stigma receptivity and pollen viability, 10 involucres were removed from selected inflorescences of each accession at different time intervals following pollination, fixed in FAA for 30 min., and stored in 70% (v/v) ethanol. Involucres were collected 15 min after pollination and thereafter at hourly intervals up to 6 h. Pistils were dissected from the primary florets and prepared for examination with fluorescent microscopy by a modification of Kho and Baër's (1968) technique. Pistils were placed in 1 M NaOH for 15 min, transferred into a 0.1% (w/v) aniline blue solution for at least 30 min, and examined with a Zeiss¹ Standard 14 microscope equipped with a F1 Epi-fluorescence condenser illuminated with an Osram HBO 50W high pressure Hg lamp (Carl Zeiss, Inc., Thornwood, NY). Pollen germination was determined by counting the number of germinated and non-germinated pollen grains on each stigma. Pollen tube growth was recorded as the maximum distance the tubes had grown into each pistil.

Stigma receptivity was determined for both self- and cross-pollinated conditions. For self-pollinations, the stigmas were dusted with pollen collected from plants of the same accession that were growing in a different greenhouse. Birdwoodgrass, P. ciliare (L.) Link var. setigerum (Vahl.) Leek, (PI 193444) pollen was used for the cross-pollination studies.

Seed Set
Seed set also was determined for all accessions under both self- and cross-pollinated conditions. The same pollination protocol described above was used for seed set determinations. Once the respective inflorescences were cross- or self-pollinated, they were immediately bagged and harvested 28 d later. A minimum of 200 primary florets was removed from the involucres of each inflorescence, and these were counted and threshed to dislodge the caryopses. Percentage seed set was calculated by dividing the number of caryopses by the number of primary florets harvested and multiplying by 100.

All data were tested for normal distribution and then analyzed by SigmaStat version 2 (SPSS, 1997). Data were analyzed by paired t-tests or ANOVA as appropriate. Significant differences were determined at the 0.05 probability level.

	Results and discussion

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussion REFERENCES

 
Protogynous Intervals
The protogynous intervals were checked in the field nursery during June and July, 1996. Plant growth and flowering during this period appeared normal as both temperature and rainfall were similar to the 30 yr means. Of 447 accessions examined, 68 (15%) exserted their stigmas 1 d prior to anthesis, 343 accessions (77%) exserted their stigmas 2 d before anthesis, and the remaining 36 (8%) exserted their stigmas 3 d prior to anthesis. These findings demonstrate variability for length of the protogynous interval within buffelgrass. This range could be an important criterion in selecting accessions to use in a hybridization program, especially if early stigma exsertion can be used as a mechanism to circumvent emasculation in making controlled crosses.

Six of the accessions were grown in a greenhouse under a 12-h photoperiod the following winter and the protogynous interval of each accession was the same as when the plant was grown in the field the previous summer. This suggests that protogynous interval is a consistent trait that is not influenced by different environmental conditions.

Self-Pollination
Pollen Germination and Tube Growth
Pollen tubes were observed within 15 min of contacting the stigmas in all six accessions, regardless of the protogynous interval or the day within the protogynous interval when pollinations were made. Mean pollen germination under self-pollination ranged from 51 to 84% across all six accessions (Table 1) . There were significant (P < 0.05) differences in pollen germination between the three protogynous interval groups. The two accessions (PI 409367 and PI 409407) selected for the short-interval group produced pollen of lower viability. The reason for their lower germination is unknown. Accession PI 409704 of the intermediate group also had a relatively low mean germination of 59% (Table 1). The most likely reason for the lower germination is that this accession is a meiotically irregular pentaploid with 45 chromosomes; the other accessions are stable tetraploids with 36 chromosomes. During meiosis, the 45 chromosomes associate essentially as 18 bivalents and 9 univalents. The univalents tend to lag behind the other chromosomes during anaphases I and II, and form micronuclei at telophases I and II which often are not incorporated into the nuclei. Consequently, the resultant pollen grains lack chromosomes and viability is reduced. Pollen germination for the remaining accessions (PI 285657, PI 315679, and S 12103) was 80% or higher (Table 1). Differences in pollen germination between these three accessions and the two selected for the short-interval group appear to be genotype specific. Thus, germination does not appear to be associated with the protogynous interval or influenced by the length of time the stigmas have been exserted from florets prior to pollination.

Pollen tube growth through the pistil under self-pollinated conditions appeared to be slower in buffelgrass than other warm-season grasses. In dallisgrass, Paspalum dilatatum Poir., and P. juergensii Hackel, the tubes grew to the micropyle within 45 min after pollination (Burson, 1987). The same was true for kleingrass, Panicum coloratum L.; however, in P. deustum Thunb. and P. antidotale Retz., from 1.75 to 2 h were required for the tubes to reach the micropyle (Burson and Young, 1983). An important finding from this study is that the pollen tubes were observed at the micropyle, whether pollination occurred 3, 2, 1, or 0 d prior to anthesis. These results support the conclusions of Snyder et al. (1955), who reported that buffelgrass stigmas were receptive up to 48 h prior to anthesis.

Seed Set
Seed set under self-pollination ranged from 11.5 to 75.7% (Table 2) . Previous studies have reported self-pollinated seed set in buffelgrass to range from 2 to 84% ( Read and Bashaw, 1969) and 27 to 91% (Hignight et al., 1991). Mean seed set for the short, intermediate, and long protogynous interval groups was 24, 40, and 60%, respectively (Table 2). Significant (P < 0.05) differences were observed between the three interval groups, as well as between the accessions within the short and long interval groups. Those accessions with lower pollen germination generally had lower seed set. Seed set was not influenced by what day during a protogynous interval pollination occurred. Seed set data demonstrate that the pollen tubes are proceeding through the micropyle into the female gametophyte and fertilization is occurring. This is most evident in the sexual accession S 12103 which requires double fertilization for seed development. It is also true for the five apomictic accessions because they are pseudogamous and are believed to require fertilization of the polar nuclei for endosperm development.

For all accessions, regardless of which day within the three protogynous intervals that the stigmas were pollinated, birdwoodgrass tubes grew to the micropyle within 2 to 5 h following pollination and in most cases within 2 to 4 h (Table 3). Approximately the same amount of time was required for the tubes to grow to the micropyle in interspecific crosses among three Paspalum species; however, in two of the crosses, tubes reached the micropyle within only 30 to 45 min after pollination (Burson, 1987). Findings from our study demonstrate that stigmas of these six buffelgrass accessions are receptive to birdwoodgrass pollen when they are exserted from the floret and remain receptive until anthesis regardless of the protogynous interval.

Seed Set
When the six accessions were crossed with birdwoodgrass, seed set ranged from 22.0 to 80.4% (Table 4) . Significant (P < 0.05) differences were observed among the protogynous interval groups with a mean seed set of 33, 50, and 69% for the short, intermediate, and long interval groups, respectively. Overall, cross-pollination resulted in 21% higher seed set than self-pollination, with the only exception being the sexual accession, S 12103. These findings are similar to those reported by Hignight et al. (1991) who reported that when buffel-grass was open-pollinated, 23% more seed were produced than when self-pollinated. This is consistent with the fact that buffelgrass evolved as an out-crossing species. Similar increases have been reported by pollinating Bothriochloa accessions with pollen from Dicanthium species (Dewald and Harlan, 1961).

This investigation revealed that buffelgrass germplasm varied in protogynous interval from 1 to 4 d in length. We also determined that buffelgrass stigmas are receptive to both buffelgrass and birdwoodgrass pollen when they are exserted from the floret, and they remain receptive until anthesis. These findings demonstrate that the protogynous nature of buffelgrass can be used to produce hybrids in sexual genotypes without having to make tedious, time-consuming hand emasculations. Seed set in apomictic genotypes that produce small quantities of seed might be increased if pollinated with other sources.

	NOTES

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussion REFERENCES

 
Joint contribution from the Crop Germplasm Research Unit, USDA-ARS and Dep. of Soil & Crop Sciences, Texas A&M Univ. Part of a thesis submitted by G.S. Shafer in partial fulfillment of the requirements for a M.S. degree.

¹ Mention of a trade mark or 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.

Received for publication November 30, 1998.

	REFERENCES

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussion REFERENCES

 

• Bashaw E.C. Apomixis and sexuality in buffelgrass. Crop Sci. 1962;2:412-415.[Free Full Text]
• Bashaw E.C., Funk R. Apomictic grasses. In: Fehr W.R., ed. Principles of cultivar development. Vol. 2. New York: Macmillan Publ. Co, 1987:40-82.
• Bashaw E.C., Hignight K.W. Gene transfer in apomictic buffelgrass through the fertilization of an unreduced egg. Crop Sci. 1990;30:571-575.[Abstract/Free Full Text]
• Bray R.A. Evidence for facultative apomixis in Cenchrus ciliaris. Euphytica 1978;27:801-804.
• Burson B.L. Pollen germination, pollen tube growth and fertilization following self and interspecific pollination of Paspalum species. Euphytica 1987;36:641-650.
• Burson B.L., Young B.A. Pollen-pistil incompatibilities and interspecific-incompatibility among Panicum antidotale, P. coloratum, and P. deustum. Euphytica 1983;32:397-405.
• Dewald C.L., Harlan J.R. Stigma removal studies on certain accessions of Bothriochloa intermedia and Dichanthium annulatum. Crop Sci. 1961;1:15-17.
• Fisher W.D., Bashaw E.C., Holt E.C. Evidence for apomixis in Pennisetum ciliare and Cenchrus setigerus. Agron. J. 1954;46:401-404.[Free Full Text]
• Hignight K.W., Bashaw E.C., Hussey M.A. Cytological and morphological diversity of native apomictic buffelgrass, Pennisetum ciliare (L.) Link. Bot. Gaz. (Chicago) 1991;152:214-218.
• Kho Y.O., Baër J. Observing pollen tubes by means of fluorescence. Euphytica 1968;7:298-302.
• Read J.C., Bashaw E.C. Cytotaxonomic relationships and the role of apomixis in speciation in buffelgrass and birdwoodgrass. Crop Sci. 1969;9:805-806.[Abstract/Free Full Text]
• Sherwood R.T., Young B.A., Bashaw E.C. Facultative apomixis in buffelgrass. Crop Sci. 1980;20:375-379.[Abstract/Free Full Text]
• SPSS. SigmaStat statistical software user's manual, Version 2.0 for Windows. Chicago, IL: SPSS Inc, 1997.
• Snyder L.A., Hernandez A.R., Warmke H.E. The mechanism of apomixis in Pennisetum ciliare. Bot. Gaz. (Chicago) 1955;116:209-221.

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