Micromanipulation and in vitro fertilization with single pollen grains of maize

Summary The manipulation of single pollen grains of maize was studied. The effects of delivering substances both locally to the grain wall, tube or tip by a microcapillary and directly into the pollen grain by microinjection, and single grain pollination were investigated. Germination was induced by adding small amounts of water locally to the grains with either a microcapillary or with a waterdelivering emulsion without any other ingredients in the medium. The grains were overlayered by mineral or silicone oil so that tube growth proceeded without the grains bursting. There was no apparent penetration of high-molecular-weight substances (FITC-dextran, ethidium bromide labelled DNA) into the living grain either before or after pollination. Neither could the penetration of these substances be detected in both dry, viable and hydrated grains, tubes and tube tips, with or without treatment with Triton X-100 and dimethyl sulfoxide. By microinjection, however, the delivery of high-molecular-weight substances into grains was possible. Such injected grains successfully pollinated stigmas of cultured ear segments. Pollination with pore-injected grains was most efficient (mean 26%). No difference in fertilization rates between mass pollination (mean 41%) and single grain pollination (mean 39%) could be found. A mean fertilization rate of 29% could be obtained after microinjection. Seedlings developed 3 weeks after being pollinated by means of the in vitro pollination and fertilization method.     

Performance of microsatellites for parentage assignment following mass controlled pollination in a clonal seed orchard of loblolly pine (Pinus taeda L.)

Mass controlled pollination (MCP), involving large-scale application of pollen on physically isolated female reproductive organs, has been a lower cost alternative to controlled pollination for the commercial production of genetically improved seeds. Nevertheless, rare are the studies that examined the efficacy of operational MCP and no such assessment has been done in loblolly pine to date. The success of MCP was assessed by a microsatellite-based investigation of the realized versus expected parentage of a set of 300 Pinus taeda offspring in 19 families generated in two subsequent rounds of MCP in 2005 and 2006 in a clonal seed orchard in Brazil. Multi-locus combined probability of parentage exclusion both theoretical and realized from actual testing was >99 % for single parent and parent pair testing when using nine or ten markers. Parentage assignments carried out under a maximum likelihood framework revealed a significantly higher success rate of MCP in 2006 (84 %) following technical improvements adopted to minimize pollen contamination and maximize male reproductive success, although significant variability in the correct maternity and full parentage was seen among individual families. The observed patterns of unexpected parentage indicated that this variability likely resulted from mislabeling of clonal ramets of the parents used in the crosses which impacted both maternity and paternity. Preventing pollen contamination will not be sufficient for successful MCP if inaccuracies exist in the identity of the clonal plants that ultimately provide pollen and female strobili, showing that DNA marker auditing and correction of identity of all ramets in a clonal seed orchard should be a standard practice in the operational implementation of MCP.     

Mechanism of peroxidase isoenzyme induction in pollinated Nicotiana alata styles

Summary A comparative study on the induction of peroxidase isoenzymes, specifically number 10 (P-10) in Nicotiana alata styles revealed significant differences between the various plants of an inbred progeny. In some plants the ageing-induced increase in P-10 activity was very low, whereas in some others, it was relatively high. Pollination accelerated this increase, independent of the pollen genotype. Fertilization was followed by a considerable increase in the activity of several peroxidase isoenzymes, including P-10 in all the plants.Two plants that differed greatly with regard to P-10 induction were used in additional experiments in order to ascertain the mechanism involved in the induction of P-10. The increase in P-10 activity due to pollination or fertilization can partly be explained on the basis of auxin and auxin-induced ethylene activity. The differences in P-10 induction between various plants of the inbred progeny were probably due to differences in their sensitivity to ethylene.     

中棉×戴维逊氏棉胚胎发育的研究

以棉花栽培种中棉作母本,野生种戴维逊氏棉作父本进行杂交试验,并用中棉自交作对照,比较研究了杂交情况下花粉粒的萌发、花粉管的生长、受精作用及胚和胚乳的发育过程,得到以下结果:(1)中棉×戴维逊氏棉花粉粒的萌发及花粉管在异己花柱中的生长基本正常,有花粉管胚珠的频率约20％,为中棉自交的1/4左右;(2)在杂交情况下,有花粉管进入的胚珠基本上能实现受精;(3)杂种胚乳在授粉后7天发育异常,11天开始解体,16天才有部分胚珠的胚乳开始形成细胞壁;(4)杂种胚不分化或畸形分化,在授粉后11—22天坏死。     

Comparative analysis of callus formation and regeneration on cultured immature maize embryos of the inbred lines A188 and A632

Induction, maintenance, differentiation and embryogenic capacity of callus obtained from immature embryos by culture on induction medium, proliferation medium, maturation medium and regeneration medium, respectively, were compared for two inbred lines of maize, i.e. A188 and A632. The callus of inbred line A188 was embryogenic and maintained embryogenic capacity for at least 1 year. Immature embryos of inbred line A632 formed callus that was not embryogenic. It only produced roots. When sucrose was replaced by sorbitol to induce or improve embryogenesis, again only A188 formed embryogenic callus. Subculture of this callus, however, allowed 4 week intervals in stead of 2 week intervals without loss of embryogenic capacity. When A188 was pollinated with A632 pollen, embryogenic callus was obtained from cultured immature "F₁" embryos, showing that embryogenic capacity was inherited, maternally. The callus did not differ from the embryogenic callus generated on selfed A188 embryos. When A632 was pollinated with A188 pollen, embryogenic callus was obtained too, showing that embryogenic capacity was also inherited paternally, though the embryogenic capacity diminished quickly, and the stability of the callus was lower than in the reciprocal cross. This revised version was published online in June 2006 with corrections to the Cover Date.     

The mating system of an hydrophilous angiosperm Posidonia australis (Posidoniaceae)

The hydrophilous seagrass Posidonia australis has a wide range of multilocus outcrossing rates (t), which vary from 0 to 0.89, with "apparent' outcrossing rates varying from 0 to 0.42 among the seven populations sampled. This pattern of outcrossing rate indicates that water pollination (hydrophily) is less uniform than wind pollination and more similar to animal pollination in its variability. Variation in levels of outcrossing between populations may be due to differences in water movement; for example, open bays have greater pollen dispersal and higher outcrossing rates. Considerable pollen movement within meadows was inferred from a high frequency of nonmaternal alleles in the pollen pool. The distribution of genetic diversity among populations (GST = 0.229) suggests moderate gene flow on the local scale. These results demonstrate that successful submarine cross-pollination occurs in the hydrophile P. australis, which has a diverse mating system with populations that range from predominantly inbred to predominantly outcrossed.     

Floral biology and pollen viability of parental lines of onion hybrids

Parental lines (inbred and hybrid male-sterile lines and inbred pollinators) of prospective F, hybrids from the NVRS spring-sown bulb onion breeding programme were examined to study their floral biology and aspects of pollination under glasshouse conditions. Inbreeding depression occurred in both pollinator and male-sterile inbreds in terms of reduced flower numbers per umbel and a greater proportion of defective pistils. Male-sterile and male-fertile lines differed in their stigma and style characteristics and pattern of development. At flower opening, the styles of male-sterile lines were longer and the stigmatic knob became receptive relatively sooner. The inbred male-sterile line II/3 ms appeared to have a reduced receptive area on its stigmatic knob and remained receptive for a relatively shorter period. Individual lines differed for time of flowering, nectar content and its sugar concentration, and in their effectiveness as male and female parents as judged from pollen tube growth down the style in specific pairings. Assessment of pollen viability using the fluorescein diacetate test did not correlate with assessments based on pollen grain germination and subsequent growth down the style, perhaps as a result of differential receptivity in the inbred lines tested. These results are discussed in relation to future hybrid seed production.     

荞麦亲和与不亲和授粉后柱头和花粉管中ATP酶的超微细胞化学定位

利用磷酸铅淀淀技术对荞麦（Fagopyrum esculentum Monch.)pin型植株分别进行亲和授粉和不亲和授粉后的柱头、花粉粒、花粉管进行了ATPase的超微细胞化学定位。结果表明（1）亲和授粉和不亲和授粉后0.5h,柱头细胞的ATPase活性反应水平较低或基于无酶活性;柱头表面、柱头上附着的花粉粒内ATPase活性在不亲和授粉时较低,亲和授粉时较高,花粉粒内ATPase主要定位于线粒体和精子细胞;（2）授粉后1.5h,不亲和授粉的柱头细胞及花粉管的ATPase活性均较低,花粉管停止生长,细胞质开始解体;而亲和授粉的柱头细胞及花粉管的ATPase活性均较高,ATPase主要定位于柱头细胞的质膜、胞基质以及花粉管的壁、质体的膜、高尔基体、线粒体上。根据不同时期不同部位ATPase活性的差异,我们认为荞麦发生自交不亲和时,花粉管在花柱中停止生长不仅是因此花粉管得不到花柱中的营养物质而引起的,可能也与花粉管自身物质代谢发生障碍有关。     

Patterns and consequences of self-pollen deposition on stigmas in heterostylous Persicaria japonica (Polygonaceae)

In order to test the "anti-interference" hypothesis for adaptive significance of reciprocal herkogamy, patterns of illegitimate pollination and effects of self-pollen on the legitimate pollination and fertility were investigated in a naturally pollinated experimental population of distylous Persicaria japonica. Pollen deposition was compared among the emasculation treatments, i.e., emasculation of a single flower from individual inflorescences, emasculation of all the flowers of individual inflorescences, and no emasculation control. In both morphs, considerable illegitimate pollination was found to occur in all the treatments, and there was no significant difference in incompatible pollen load among the treatments. Therefore, it is suggested that herkogamy of P. japonica can sufficiently reduce both intraflower and intra-inflorescence self-pollinations, but not interinflorescence geitonogamous and-or interclonal illegitimate pollination. Measurements of pollen load after the repetitive pollen addition showed that space on the stigma surface may not limit the legitimate pollination under natural pollination conditions. Seed sets after legitimate pollination following prior self-pollination did not differ from the controls without self-pollination. Therefore, there is little possibility that incompatible pollen load interferes with either pollination or fertilization by compatible pollen, suggesting that "anti-interference" is unimportant for adaptive significance of reciprocal herkogamy at least in P. japonica.     

Pollen aperture heteromorphism. Variation in pollen-type proportions along altitudinal transects in Viola calcarata.

Some species produce pollen grains with different aperture numbers within a single individual (pollen aperture heteromorphism). In the pansy Viola diversifolia, aperture number is positively correlated with pollen germination speed, and negatively correlated with viability. In V. calcarata, young five-aperturate pollen grains germinate faster than four-aperturate ones. Heteromorphism could thus be favoured when pollination is unpredictable, as plants produce both very competitive and long-lived pollen grains. Depending on the efficiency of the pollinators, different proportions of pollen types will be optimal. In insect-pollinated species, such as V. calcarata, pollination efficiency generally decreases as elevation increases. We therefore expect a decrease in mean aperture number as altitude increases. This was found in four transects (out of six). Pollinator activity therefore has a potential impact on pollen morphology.     

Early ovule development following self- and cross-pollinations in Eucalyptus globulus Labill. ssp. globulus

The study was conducted to identify the self-incompatibility mechanism in Eucalyptus globulus ssp. globulus. Controlled self- and cross-pollinations were conducted on individual flowers from three mature trees that had self-incompatibility levels of 76, 99.6 and 100%. Flowers were harvested at 4, 6 and 8 weeks after pollination. Embryology was investigated by bright field microscopy on material harvested at 4 and 6 weeks after pollination. Fertilization had taken place at 4 weeks after pollination with zygotes and free nuclear endosperm visible. There was a greater proportion of healthy, fertilized ovules in the cross- compared with the self-pollination treatment, and approx. half the ovules examined from both pollen treatments were not fertilized or were degenerating. By 6 weeks after pollination a few zygotes were starting to divide. The number of healthy, fertilized ovules was still greater in the cross-pollination treatment, but the number of healthy fertilized ovules was lower in both treatments compared with 4 weeks after pollination, and many ovules were degenerating. Fertilized ovules were significantly larger than non-fertilized or degenerating ovules and this difference was detectable by eye at 6 and 8 weeks after pollination. The mechanism of self-incompatibility appears to have both late pre- and post-zygotic components.     

On the Embryonic Development and Ovule Culture of Interspecific Hybrids Between Populus simonii Carr and P.pyramidalis Borkh

The embryonic development following P. simonii Cart. × P. pyramidalis Borkh. is described in the present paper. The majority of pollen grains of P. pyramidalis Borkh. may germinate on the stigma of P. simonii Cam and the pollen tubes grow normally through the style and enter the embryo sac from the micropyle. Fertilization occurs as usual 4–7 days after pollination. A lot of proembryos and heart-shaped embryos are abortive; however, the others may develop normally and grow into mature embryos. Some of the endosperms appear normal and others may degenerate at free nuclear stage or cease to develop further at cellular stage. The ovules containg immature hybrid embryos of 19 days, 22 days, 26 days and 29 days after pollination at various developmental stages (heart-shaped stage, torpedo-stage and cotyledonary elongation stage) are excised and inoculated on nutrient agar for culture. The results show that: ( 1 ) Mll0 medium ( 1/2 MS+IAA 0.01 mg/L+BA 0.1 mg/L+sucrose 2% ) is the best of all the media used; (2) immature hybrid embryos of various developmental stages contained in ovules cultured in vitro may grow into normal plantlets.     

Evidence for inbreeding depression and post-pollination selection against inbreeding in the dioecious plant Silene latifolia

In many species, inbred individuals have reduced fitness. In plants with limited pollen and seed dispersal, post-pollination selection may reduce biparental inbreeding, but knowledge on the prevalence and importance of pollen competition or post-pollination selection after non-self pollination is scarce. We tested whether post-pollination selection favours less related pollen donors and reduces inbreeding in the dioecious plant Silene latifolia. We crossed 20 plants with pollen from a sibling and an unrelated male, and with a mix of both. We found significant inbreeding depression on vegetative growth, age at first flowering and total fitness (22% in males and 14% in females). In mixed pollinations, the unrelated male sired on average 57% of the offspring. The greater the paternity share of the unrelated sire, the larger the difference in relatedness of the two males to the female. The effect of genetic similarity on paternity is consistent with predictions for post-pollination selection, although paternity, at least in some crosses, may be affected by additional factors. Our data show that in plant systems with inbreeding depression, such as S. latifolia, pollen or embryo selection after multiple-donor pollination may indeed reduce inbreeding.     

Self‐pollination and parthenocarpic ability in developing ovaries of self‐incompatible Clementine mandarins (Citrus clementina)

This study aimed to determine if self‐pollination is needed to trigger facultative parthenocarpy in self‐incompatible Clementine mandarins (Citrus clementina Hort. ex Tan.). ‘Marisol’ and ‘Clemenules’ mandarins were selected, and self‐pollinated and un‐pollinated flowers from both cultivars were used for comparison. These mandarins are always seedless after self‐pollination and show high and low ability to develop substantial parthenocarpic fruits, respectively. The time‐course for pollen grain germination, tube growth and ovule abortion was analyzed as well as that for carbohydrates, active gibberellins (GA₁ and GA₄), auxin (IAA) and abscisic acid (ABA) content in the ovary. ‘Clemenules’ showed higher pollen grain germination, but pollen tube development was arrested in the upper style 9 days after pollination in both cultivars. Self‐pollination did not stimulate parthenocarpy, whereas both un‐pollinated and self‐pollinated ovaries set fruit regardless of the cultivar. On the other hand, ‘Marisol’ un‐pollinated flowers showed greater parthenocarpic ovary growth than ‘Clemenules’ un‐pollinated flowers, i.e. higher ovule abortion rate (+21%), higher fruit set (+44%) and higher fruit weight (+50%). Further, the greater parthenocarpic ability of ‘Marisol’ paralleled higher levels of GA₁ in the ovary (+34% at anthesis). ‘Marisol’ ovary also showed higher hexoses and starch mobilization, but lower ABA levels (?64% at anthesis). Self‐pollination did not modify carbohydrates or GA content in the ovary compared to un‐pollination. Results indicate that parthenocarpy in the Clementine mandarin is pollination‐independent with its ability to set depending on the ovary hormone levels. These findings suggest that parthenocarpy in fertile self‐incompatible mandarins is constitutively regulated.     

荞麦亲和与不亲和授粉后柱头和花粉管中ATP酶的超微细胞化学定位

利用磷酸铅沉淀技术对荞麦(Fagopyrum esculentum Month.)pin型植株分别进行亲和授粉和不亲和授粉后的柱头、花粉粒、花粉管进行了ATPase的超微细胞化学定位。结果表明(1)亲和授粉和不亲和授粉后0.5h,柱头细胞的ATPase活性反应水平较低或基本无酶活性;柱头表面、柱头上附着的花粉粒内ATPase活性在不亲和授粉时较低,亲和授粉时较高,花粉粒内ATPase主要定位于线粒体和精子细胞;(2)授粉后1.5h,不亲和授粉的柱头细胞及花粉管的ATPase活性均较低,花粉管停止生长,细胞质开始解体;而亲和授粉的柱头细胞及花粉管的ATPase活性均较高,ATPase主要定位于柱头细胞的质膜、胞基质以及花粉管的壁、质体的膜、高尔基体、线粒体上。根据不同时期不同部位ATPase活性的差异,我们认为荞麦发生自交不亲和时,花粉管在花柱中停止生长不仅是因为花粉管得不到花柱中的营养物质而引起的,可能也与花粉管自身物质代谢发生障碍有关。     

Disruption of endosperm development: an inbreeding effect in almond (Prunus dulcis)

A homozygous self-compatible almond, originated from self-fertilization of a self-compatible genotype and producing a reasonable yield following open pollination, exhibited a very high fruit drop rate when self-pollinated. To investigate whether fruit dropping in this individual is related to an abnormal development of the embryo sac following self-fertilization, histological sections of ovaries from self and cross-pollinated flowers were observed by light microscopy. Additionally, the presence of pollen tubes in the ovary and fruit set were determined for both types of pollination. Despite pollen tubes reached the ovary after both pollinations, differences in embryo sac and endosperm development after fertilization were found. Thus, while for cross-fertilized ovules a pro-embryo and an endosperm with abundant nuclei were generally observed, most self-fertilized ovules remained in a previous developmental stage in which the embryo sac was not elongated and endosperm nuclei were absent. Although 30 days after pollination fruit set was similar for both pollination types, at 60 days it was significantly reduced for self-pollination. These results provide evidence that the high fruit drop in this genotype is the consequence of a disrupted development of the endosperm, what could be an expression of its high level of inbreeding.     

Perpetuation of the self-incompatible rare species Swainsona laxa R. BR by pollination in vitro and in situ

Seed set in the rare plant Swainsona laxa grown in the glasshouse was prevented by self-incompatibility and the lack of cross pollination due to loss of pollen viability before the flowers opened. Fertilization of decapitated, excised pistils in vitro overcame the incompatibility and resulted in viable seed production. Presence of the calyx was not necessary. Manual cross pollination in situ using pollen collected from flower buds before opening also produced seed.     

The determination of multiple microsatellite genotypes and DNA sequences from a single pollen grain

Pollen plays important roles in the reproduction and gene flow of flowering plants, and its haploid DNA sequence provides useful information for studies of plant evolution and genealogy. We describe a new method for multiple microsatellite genotyping and DNA sequencing from a single pollen grain. The haploid DNA was extracted from a single pollen grain by using a simple DNA extraction method, and multiple microsatellite genotypes and DNA sequences of multiple chloroplast loci were determined. Using nine pairs of microsatellite primers, more than 90% of genotypes were successfully determined, and 71% and 100% of DNA sequences were determined at two chloroplast DNA loci, the trnL intron region and the trnL/trnF intergenic spacer region, respectively. This simple method of genetic analysis for a single pollen grain will facilitate detailed study of pollination, evolution and genealogy.     

Floral Structure, Stigma Receptivity and Pollen Viability in Relation to Protandry and Self-incompatibility in Silky Oak (Grevillea robusta A. Cunn.)

The reproductive biology of Grevillea robusta growing underexotic conditions in Kenya and Australia is reported. The speciesshowed both protandry and a self-incompatibility mechanism.The stigma was wet and papillate with a distinct groove in themiddle. The anthers dehisced prior to anthesis, when the perianthopened. Stigmatic receptivity began 1 d after anthesis, withthe greatest pollen germination rates and longest pollen tubesobtained 2 d after anthesis. Nectar secretion commenced withpollen dehiscence and was abundant at anthesis. Most stigmaticgrooves opened widely 1–2 d after anthesis and stigmasshowed taller papillae and abundant secretion. Controlled pollinationsgave a greater fruit set from cross-pollination (5.9% in Apriland 17.5% in July) than open-pollination (0.1% in April and3.3% in July). No fruit set from self-pollination was obtainedin April, and very few fruit set for geitonogamous (two outof 1622; 0.1%) or for autogamous (one out of 2707 flowers; 0.04%)pollination treatments in July. Following self-pollination,growth of pollen tubes was poorer than in other treatments,and was generally arrested in the upper style. Cross-pollinatedflowers produced normal and straight pollen tubes, while self-pollentubes had growth abnormalities. Most of the open-pollinatedflowers were found without pollen or with only self-pollen ontheir stigmas indicating that the amount of cross-pollen reachingthe stigma under open-pollination may be a factor limiting seedproduction. Flowers shed soon after the fertilization phasewere those with ungerminated pollen or no pollen. Although avery low rate of selfing may occur, G. robusta presents a self-incompatibilitysystem and allogamy is its primary breeding behaviour.Copyright2000 Annals of Botany Company Grevillea robusta, silky oak, Proteaceae, protandry, controlled pollinations, receptivity, pollen-tube growth, self-incompatibility, pollination