Floral morphology affects seed productivity through pollination efficiency in radish (<Emphasis Type="Italic">Raphanus sativus</Emphasis> L.)

To examine the effect of stigma position and size on seed productivity through pollination efficiency in radish, the numbers of self and cross pollen grains on the stigmas and the seed productivity under insect-pollination were compared among four bred lines. Lines with a small stigma or a high stigma relative to the anthers on long stamens tended to receive fewer self and total (self + cross) pollen grains but showed a higher cross-pollination percentage (ratio of cross pollen grains to total pollen grains on stigmas) than those with a large stigma or a lower stigma. Additionally, a higher cross-pollination percentage was associated with a higher outcrossing percentage. This result suggests that variations in the two stigma traits affect the outcrossing percentage via the cross-pollination percentage. Therefore, it should be possible to prevent loss of F₁ purity by selecting new parental lines with floral morphology that favors cross-pollination percentage.     

Effect of floral morphology on pollination in Brassica rapa L

Summary Flower structure, especially the anther–stigma separation (ASS), is well known to affect pollination efficiency, and thus to potentially increase or decrease seed production in crops. Therefore, investigating the relationship between flower characteristics and pollination ability is crucial to a full understanding of mechanisms to improve F₁ seed production in Brassica rapa. We used image analysis to measure three flower characteristics: short stamen height (SSH); long stamen height (LSH); and pistil height (PH) in seven cultivars. We calculated the ratio of PH to LSH as an index of anther–stigma separation (ASS). We investigated the number of pollen grains (NPG) deposited on the stigma and the seed-set percentage (SSP) under open-pollination and self-pollination conditions (with and without insects, respectively). Nested ANOVA indicated significant differences between the seven cultivars in the floral characteristics except for PH. Moreover, much larger variation was observed in NPG and SSP than in floral characteristics. Although stepwise multiple regression analysis indicated that plants with relatively high PHs produced more seed under self-pollination, the number of seeds that resulted from self-pollination did not affect seed production because of incompatibility. Therefore, the effect of the spatial position of pistils on the F₁ seed production was low. Possibly other factors such as the total pollen production and visiting times of pollinators were important factors in the low yields observed in some cultivars     

Floral biology in the Zanzibar type clove (Syzygium aromaticum) in Indonesia

Summary Investigations of the floral biology of Zanzibar type cloves were carried out on five mature trees. The majority of flowers opened in the afternoon. Anther dehiscence occurred shortly after anthesis and stamens were shed almost 2 days later. Nectar appeared around the base of the stigma as anthers dehisced and remained in evidence for approximately 60 hours. Three days after anthesis a constriction around the tip of the stigma became apparent. The stigma tip subsequently withered, turned brown and was shed after a further four days. Maximum pollen viability and stigma receptivity were attained simultaneously, 48 hours after anthesis. Indications concerning the breeding system in clove are discussed and a procedure for artificial pollination is suggested     

Study of morpho-physiological, phenological and reproductive behaviour in protogynous lines of Indian mustard [Brassica juncea (L.)]

Understanding the relationship between floral and reproductive traits are critical to understand the evolution of plant species in brassica and need for selecting lines as parental lines in hybrid seed prodcution. The protogyny nature of Brassica species are characterized based on the variations in floral morphology, protogyny interval, stigma receptivity, pollen biology, compatibility and seeds set. The variation in stigma morphology features (stigma exsertion prior to anthesis) and protogyny interval (ranged from 8 to 10 days) formed phenotypically distinct. Studies on stigma receptivity and pollen viability revealed significant variability and found to be at its peak up to 3 days after anthesis and reduced drastically thereafter. Compatibility crosses and post pollination events such as pollen deposition on the surface of the stigma and culminating with the entry of the pollen tube into the embryo sac, was rapid and completed within 2 days after pollination, whereas in in-compatible cross not even single pollen was germinated on stigmatic surface and consequently no pollen tube growth even after sixth day of pollination. In compatible crosses average seed set ranged from 3 to 8 seeds per pod from cross pollination and no seed set in self. The investigation revealed that variation exists for protogyny interval, stigma receptivity and compatibility in the protogynous lines of Indian mustard and this can be exploited for the production of hybrids without emasculation.     

狭瓣辣木的开花动态及花粉活力和柱头可授性

为了观测狭瓣辣木和多油辣木改良种PKM-1开花散粉时期花部形态、花粉萌发率和柱头可授性的变化规律,本研究采用琼脂培养基萌发法测定花粉萌发率,用联苯胺-过氧化氢法测定柱头可授性,结合田间花部性状随着时间变化的实际观测进行分析。结果表明,狭瓣辣木花朵开花之初,花药即完全打开并开始大量散粉,柱头直立,高于雄蕊;花朵开放后,柱头直立不变,高度逐渐低于雄蕊。PKM-1花朵开花之初,花药微微闭合,2 h后完全打开并开始大量散粉,柱头微微弯曲,高度低于雄蕊;花朵开放后,柱头逐渐弯曲,高度逐渐高于雄蕊。狭瓣辣木花粉萌发率在开花第2天达到最大值77%,呈现先上升后下降的趋势。PKM-1花粉萌发率在开花第1天达到最大值69%,之后迅速降低;狭瓣辣木和PKM-1柱头可授性均呈上升趋势,二者在开花第3天柱头可授性最强。在开花第1天,狭瓣辣木柱头具有微弱活性,而PKM-1柱头没有活性。在自然授粉条件下,狭瓣辣木花期柱头形态不利于其杂交授粉;在开花第2天采集花粉,在开花第3天柱头可授性最强时人工授粉,可以提高狭瓣辣木结实率和杂交成功率。本研究对于制定辣木杂交育种措施、提高辣木人工授粉效率和产量有重要意义。     

Pollen grain germination and pollen tube growth following in vivo and in vitro self and interspecific pollinations in annual Cicer species

Summary Germination of pollen grains and growth of pollen tubes were studied to determine the cause of barreness in crosses among annual Cicer species. In vivo and in vitro time-course studies and fluorescent microscopy revealed no pollination incompatibility among the selfs, crosses and reciprocals of C. arietinum L., C. reticulatum Lad. and C. cuneatum Rich. In general, Cicer pollen grains germinated and grew on styles of Cicer species. Pollen tube growth was characterized by irregularly spaced and intermittent callose deposits. Failure of seed formation in interspecific pollinations may be attributed to the slowness of pollen tube growth or collapse of fertilized ovules. In addition to these causes, shortness of stamens and sparsity of pollen grains were responsible for flower drop in natural selfs. Although the number of pollen tubes entering the micropyle in interspecific pollinations was low, it may be possible to grow the fertilized ovules on an artificial medium to obtain F₁ plants.     

Induction of an alloplasmic male-sterile Brassica oleracea by substituting cytoplasm from ‘Early Scarlet Globe’ radish (Raphanus sativus)

Summary Alloplasmic male-sterile Brassica oleracea L. was synthesized in a backcrossing program through amphidiploid Raphanobrassica by using Early Scarlet Globe radish (Raphanus sativus L.) as the donor of cytoplasm and B. oleracea broccoli and cabbage as recurrent pollen parents. Persistence of radish chromosomes and high female sterility were encountered in the first four backcrosses. Following use of colchiploid 4x broccoli as pollen parent, a BC₅ plant was obtained that had 2n=3x+1=28 chromosomes, improved seed set, and no radish traits. The BC₆ with recurrent 2x broccoli contained male-sterile plants with 2n=18 or 19 chromosomes, increased seed set, and broccoli morphology. Subsequent generations segregated for male-sterile and restored male-fertile plants, some with variable development of stamens and pollen. Leaf color of the alloplasmic plants, especially seedlings, was lighter green than normal.     

Variation of prezygotic barriers in the interspecific hybridization between oilseed rape and wild radish

Escape of transgenes from genetically modified oilseed rape, Brassica napus, into wild radish, Raphanus raphanistrum, depends on sexual compatibility. The variation in prezygotic barriers of two different cultivars for interspecific hybridization with a population of wild radish was investigated by hand crossing and fluorescence microscopy of pistils. Significant differences were observed between oil seed rape cultivars in their ability to accept wild radish pollen germinating onto their stigma and the rate of fertilization of ovules. Some differences among the pollen donor plants were also detected. These results suggest that the rate of interspecific hybridization in the field would depend upon the oilseed rape cultivar and the genotype composition of the local wild populations. The implication of S-related genes, as revealed through identification by pistil tissue prints of class I and II S-types of SLG (S-Locus Glycoprotein) and SLR1 (S-Locus Related),and immuno-IEF, was not significant. This revised version was published online in August 2006 with corrections to the Cover Date.     

Floral Traits and Seed Production of Sorghum A-, B- and R-lines under Chilling Field Temperatures

In A-lines of sorghum ( Sorghum bicolor L. Moench), seed production under field conditions with manual pollination is generally lower than that in self-pollinated B-lines. This may be associated with floral differences. Six pairs of A/B-lines and four R-lines were evaluated during 2005 and 2006 at Montecillo, State of México (2240 m altitude). Rachis length, number of primary branches and fertile flowers per panicle, 100-seed weight, seed number, seed yield and seed set per panicle were evaluated. In the A-lines, the pistil characteristics were also measured and in the male-fertile lines, the size of anthers and the amount and size of viable pollen were recorded. Compared with the A- and B-lines, the R-lines exhibited significantly higher (P ≤ 0.05) numbers of flowers, seed set and seed yield per panicle and they also produced more pollen grains of larger size and with greater viability during their longer flowering period (FP). Between A- and B-lines, there were differences (P ≤ 0.05) in most of the yield traits, which also interacted with year. The proportion of viable pollen in B- and R-lines (75 %) was not considered to be a factor that might account for their low seed production. Chilling temperatures (3.5–8.4 °C) during the FP could have affected stigma receptivity in the three different line types and thus may have reduced seed set in the male-fertile lines.     

Control pollination and pollen management in Sesbania sesban (L.) Merr.

Sesbania sesban was amiable to controlled cross pollination when the emasculation and pollination operations were performed in the morning. A medium of 10% sucrose solution was found to be optimal for in vitro germination of the pollen grains. The pollen is quite tolerant of orthodox storage conditions enabling controlled crosses to be performed among accessions that flower in differing seasons of the year.     

Effect of selection for pollen grain size on various traits in common bean

Summary Selection among microgametophytes usually exploits variation in pollen grain germination. Studies of variation in pollen grain size in common bean (Phaseolus vulgaris L.) suggested that selection for size might lead to changes in sporophytic traits. To determine whether microgametophytic selection based on size would affect pollen grain size in subsequent generations or sporophytic traits that were correlated with pollen grain size, pollen grains from three crosses were separated into two size categories by sieving and then used to pollinate cv. Diacol Calima. Selection resulted in changes in pollen grain diameter for pollen from F₁, F₂ and F₃ plants for all crosses. In vitro germination indicated no differences between vigor of large and small grains, but extraction and sieving reduced germinability. F₁ seed from two of the crosses with size-selected pollen varied in weight according to pollen grain size, but in subsequent generations, the effect disappeared. Both size categories of selected pollen resulted in F₂ progeny with reduced numbers of seeds per pod as compared to controls, suggesting that the size selection process may have resulted in indirect selection for traits reducing seed set. The overall results suggested that genes determining pollen grain size in bean have little or no effect on sporophytic traits such as seed size and seed yield.     

Overcoming pre-fertilization barriers in the wide cross between Chrysanthemumgrandiflorum (Ramat.) Kitamura and C. nankingense (Nakai) Tzvel. by using special pollination techniques

In our earlier study, pre-fertilization barrier was one of the main factors causing the failure of the wide hybridization between Chrysanthemum grandiflorum ‘Yuhuaxingchen’ and C. nankingense, and seriously restricted the utilization of the excellent chrysanthemum germplasm, C. nankingense. In order to overcome the pre-fertilization barrier and obtain some hybrids, we carried out wide hybridization between them again in this study, and tentatively adopted three pollination techniques including mentor pollen, delayed pollination, and gibberellic acid treatment. The average number of pollen grains germinating on stigma at 24 h after pollination and the percentage of embryo were investigated with a temporary mount method and the technique of fluorescence microscopy. It was found that, compared with the control, delayed pollination, gibberellic acid treatment, and mentor pollen significantly increased the average number of pollen grains germinating on each stigma at 24 h after pollination by approximately 152, 235, and 182%, respectively. In addition, all the three pollination techniques significantly increased the percentage of normal embryos at different periods after artificial pollination in comparison to the control. Furthermore, seed sets of the crosses using the three special pollination techniques were 1.1, 1.4, and 2.0%, respectively, whereas we did not obtained any seeds in the control. Taken together, these results suggest that delayed pollination, gibberellic acid treatment, and mentor pollen are three useful pollination techniques for overcoming pre-fertilization barriers and increasing seed set in the wide hybridization between C. grandiflorum ‘Yuhuaxingchen’ and C. nankingense, and they may be widely applied for increasing breeding efficiency in other chrysanthemum wide hybridization facing similar pre-fertilization barriers.     

Morpho-physiological and phenological attributes of reproductive biology of tea (<Emphasis Type="Italic">Camellia sinensis</Emphasis> (L.) O. Kuntze) in Sri Lanka

A comprehensive knowledge on reproductive traits is a prerequisite in utilizing the existing tea germplasm effectively for crop improvement to develop superior planting material for grower acceptance and market profitability. The Sri Lankan tea germplasm was characterized based on reproductive traits viz. floral morphology, pollen biology, stigma receptivity and phenology of flowering and fruit set. The variability in floral morphology, especially the style morphological features, formed phenotypic clines rather than distinct groups. Studies on pollen biology and stigma receptivity revealed significant variability and asynchrony that could result unequal reproductive success among the genotypes. Four distinct patterns were predictable among the genotypes based on flower and fruit abundance and the time of flowering and fruit set. Three well marked flowering periods occurred in February to April, July and in November. Nonetheless, major flowering period coincided February and March in all the genotypes allowing free crossing between the different genotypes. Profuse mature fruit crop was obtained in February to May. Approximately 26% success was achieved in tea controlled hybridization programmes. Fruits carry two seeds on average and became mature in 8–9 months after pollination. Zygotic development in tea takes more than 1 month after pollination and early embryonic development continued for 4 months after pollination. The indexing of the morpho-physiological diversity and the phenological calendars of flowering and fruit set made available in the study are of significant importance in effective utilization of the tea germplasm for crop improvement.     

Pollen grain size in four ploidy levels of genusAvena

Summary Pollen grain size in many genera positively correlated with chromosome number. In this study, oat (Avena) pollen grain size was examined in diploids (2×=14, one species, for accessions), tetraploids (4×=28, five species, 20 accessions), hexaploids (6×=42, one species, eight cultivars), and in 10 octoploid (8×=56) accessions. Mature anthers ready to dehisce pollen were sampled from one to six plants per accession, and pollen grains were squeezed out of the anther with tweezers. Oat pollen grains are slightly elliptical, and the length of the major axis was found to be highly correlated with the minor axis width (r=0.94^**). Pollen grain length, 39.3 m for diploids, 41.3 m for tetraploids, 47.0 m for hexaploids, and 48.8 m for octoploids, was positively correlated (r=0.86^**) with ploidy level. No genomic or species effects appeared to influence this trait. Only tetraploidAvena vaviloviana accession PI 412767 produced two distinct class sizes of pollen grains, 99% normal (43.0 m) and 1% large (52.7 m).     

Production of 2n pollen of Asiatic hybrid lilies by nitrous oxide treatment

Tetraploid varieties of lilies have superior agronomic traits such as large flowers and resistance to physiological disorders. In the present study, we attempted to induce 2n pollen of Asiatic hybrid lilies by arresting the meiotic process with nitrous oxide (N₂O) gas. To determine which meiotic stage is optimal for induction of 2n pollen, plants with attached buds at different meiotic stages were treated with N₂O for 24 h in a pressure-tolerant cylinder. A few 2n pollen grains were induced using plants with anthers in prophase I, whereas mixed pollen grains of differing size were produced using plants undergoing meiotic metaphase predominantly in anthers. Although normal lily pollen grains are elliptical, nitrous oxide exposure induced giant pollen grains that appeared spherical. Flow cytometry analysis showed that the giant pollen grains were diploid. When mixed pollen that included normal and giant pollen was crossed to tetraploid cultivars, the resulting seedlings were tetraploid and aneuploid, indicating that the giant pollen grains were diploids that could generate tetraploid seedlings through fusion to diploid eggs supplied from a tetraploid female parent. Thus, treatment with N₂O is useful for the production of 2n lily pollen and may provide a new approach for tetraploid lily breeding.     

The breeding system of rubber (Hevea brasiliensis): an evaluation of controlled hand pollination methods

Summary Controlled hand pollinated pistils of rubber were observed using fluorescence microscopy to assess the efficiency of the universally-employed method for the production of progeny for plant breeding. The controlled hand pollination method conducted in the morning resulted in the deposition of a mean of 15.6 pollen grains on the stigma, with no stray pollination observed. Over 36% of the pistils had the potential to set fruit.Pollinations conducted in the afternoon at the normal time of anthesis, had double the fruit set potential of morning pollinations as measured by penetration of ovules by pollen tubes. Pollinator efficiency also varied, with excessive damage to stigmas resulting in reduced pollen germination and tube growth. There were differences between clones in both female and male fertility, in the proportion of pistils with more than three carpels and in the production of small abnormal stigmas. There was no difference in pollen tube growth following self- or cross-pollination, indicating that the self-sterility mechanism of rubber operates in the ovary. Pollen could be stored for 5 days at 5°C and 75% RH with a 22% loss of fertility.     

Regeneration and somaclonal variation in apomictic Paspalum dilatatum Poir

Summary In an attempt to incorporate variation into a uniform obligate apomict, plants of apomictic common dallisgrass, Paspalum dilatatum Poir., were regenerated from callus derived from immature inflorescences. Plants developed through both organogenesis and embryogenesis. A total of 682 regenerants were produced and more than 400 were transplanted into a field nursery and screened for somaclonal variation. Eventually 20 regenerants were selected, increased, and planted into a replicated nursery along with normal common dallisgrass. The characteristics examined were maturity date, plant height, number of racemes per inflorescence, number of spikelets per raceme, pubescence, stigma and anther color, ergot resistance, seed germination, seed set, pollen stainability, method of reproduction, and chromosome number. There were differences among the regenerants and between them and common dallisgrass for all traits except chromosome number, stigma and anther color, and ergot resistance. One of the more important regenerants had significantly higher seed set than common dallisgrass. All regenerants reproduced by aposporous apomixis but some exhibited a high degree of abortion while others had more aposporous embryo sacs per ovule than common dallisgrass. These findings demonstrate that common dallisgrass can be regenerated through tissue culture and that somaclonal variation is expressed in some of the regenerants, even though some of the altered traits are deleterious.     

Evaluation of soybean germplasm conserved in NIAS genebank and development of mini core collections

Genetic variation and population structure among 1603 soybean accessions, consisted of 832 Japanese landraces, 109 old and 57 recent Japanese varieties, 341 landrace from 16 Asian countries and 264 wild soybean accessions, were characterized using 191 SNP markers. Although gene diversity of Japanese soybean germplasm was slight lower than that of exotic soybean germplasm, population differentiation and clustering analyses indicated clear genetic differentiation among Japanese cultivated soybeans, exotic cultivated soybeans and wild soybeans. Nine hundred ninety eight Japanese accessions were separated to a certain extent into groups corresponding to their agro-morphologic characteristics such as photosensitivity and seed characteristics rather than their geographical origin. Based on the assessment of the SNP markers and several agro-morphologic traits, accessions that retain gene diversity of the whole collection were selected to develop several soybean sets of different sizes using an heuristic approach; a minimum of 12 accessions can represent the observed gene diversity; a mini-core collection of 96 accession can represent a major proportion of both geographic origin and agro-morphologic trait variation. These selected sets of germplasm will provide an effective platform for enhancing soybean diversity studies and assist in finding novel traits for crop improvement.     

Pollen tube growth and fertilization after different in vitro pollination techniques of Aechmea fasciata

The effect of cut style and placental pollination on fertilization efficiency was studied for compatible Aechmea fasciata plants. These alternative in vitro pollination techniques resulted in lower penetration rates of the ovules by pollen tubes in comparison to pollination on the stigma. An explanation was found in the intervention of the normal pollination process,through which less pollen-pistil interactions were built up. After cut style pollination the percentage of ovule penetration by pollen tubes increased when a longer style part was left at the ovary. Probably fewer factors that control pollen tube growth are present in the lower style part. Pollen germinated on the ovules after placental pollination but only rarely penetration of the micropyle by a pollen tube occurred. Activation of the ovary, induced by in vivo prepollination for 6 hours, and pollination two days after anthesis did not increase the fertilization percentage. Grafting a style with active growing pollen tubes to ovules on the placenta (placental grafted style pollination) resulted in a higher fertilization percentage. Pollen tube growth through the style was essential for pollen tube guidance to the ovules and penetration of the micropyle. This revised version was published online in August 2006 with corrections to the Cover Date.     

Pollen production in soybeans with respect to genotype,environment, and stamen position

Summary Our objective was to determine the average numbers of pollen grains from fertile plants (Ms ₁) and the average numbers of coenocytic microspores from genetic male sterile plants (ms ₁ ms ₁) in soybeans, Glycine max L. Merr. Comparisons were made between the average numbers of pollen grains and the average numbers of coenocytic microspores with respect to environment where plants were grown and to stamen position in the flower. Five male sterile lines were used. They included the North Carolina ms ₁ mutant, the cultivar Hark with the ms ₁ gene, and mutants identified as the Urbana, Tonica, and Ames male steriles. Three environments used were the Agronomy Farm, University of Georgia, Athens, Georgia; the Agronomy and Agricultural Engineering Research Center, Iowa State University, Ames, Iowa; and the Agronomy Greenhouse, Iowa State University, Ames, Iowa.Pollen production from fertile plants varied from 374 to 760 pollen grains per anther among genetic lines and environments. This variation may be an important consideration in selecting a male parent to use as a pollinator for hybrid seed production.Among fertile plants, the average numbers of pollen grains per anther of the separate stamen and and of the lower whorl of stamens were significantly different only in greenhouse-grown plants. Among male sterile plants, the average numbers of coenocytic microspores per anther of the separate stamen and of the lower whorl of stamens were significantly different in three genotype x environment combinations. These three exceptions did not conform to any genetic or environmental pattern. Deviations from the expected ratio of 4 pollen grains from fertile plants: 1 coenocytic microspore from sterile plants were attributed to initial differences in the average number of microspore mother cells between the two genotypes.Joint contribution: Agricultural Research Service, USDA, North Central Region, and Journal Paper No. J-8910 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa 50011; Project 2107.