Cytological screening of rice TGMS lines

An investigation was undertaken to develop new stable thermo‐sensitive genic male sterility (TGMS) rice lines in intermated progenies (IMPs) of TGMS lines by using an anther clearing technique. The results indicate that both pre‐ and post‐meiotic genetic systems operate during anther development for the expression of sterility in TGMS lines. In all the TGMS lines, sterile anthers were small with empty pollen grains of irregular shape, except for TS 16, which showed pollen‐free anthers. This indicates that the sensitive stage of TS 16 is around stage IV (stamen and pistil primordia) of panicle development. Distinct differences were observed between sterile and fertile phases with respect to anther size, shape and colour of the pollen grains in TS 18 and TS 29. The pollen grains at the sterile phase were small and irregular in shape while in the fertile phase they were plump and larger with a yellow colour, establishing that the occurrence of sterility in TS 18 and TS 29 is post‐meiotic. Three distinct classes of pollen fertility percentage viz. <20%, 50‐70% and >90% were observed in IMPs. Anther clearing in IMPs showed distinct developmental patterns of pollen production with respect to distinct classes of pollen fertility. Less than 20% pollen fertility was observed in hybrids such as TS 15 × TS 16, TS 15 × Co 47, TS 18 × TS 16 and TS 18 × Co 47 which hold promise for developing new TGMS lines with a good plant type and acceptable quality.     

Effects of short-term storage on germinability of pistachio pollen

The effects of short‐term storage under room conditions (constant 25°C and 35% relative humidity), refrigeration (4°C) and freezing (‐20°C) on the germinability of pollen of pistachio, Pistacia vera L., were studied and the effects of desiccation and pollen age on its germinability were reassessed. It was found that: (1) weight loss as a result of desiccation was positively correlated with germinability; (2) pollen grains stored in room conditions only germinated after prehydration, which restored germinability even after 7 days of exposure; (3) refrigerated (4°C) pollen grains retained their germinability for at least a week; (4) frozen pollen grains irreversibly lost most of their germinability; (5) 2‐day‐old pollen grains, within the anthers, retained their germinability under room conditions. These findings will contribute to the improvement of pistachio pollen preservation and help to find a simple and inexpensive method for its short‐term storage, while retaining its germinability.     

Effect of high temperature on the production of 2n pollen grains in diploid roses and obtaining tetraploids via unilateral polyploidization

To integrate the gene pool of a wild species (primarily diploid) into a cultivated pool (primarily tetraploid), a crossing between a dihaploid cultivated rose and a hybrid of Rosa wichurana allowed to obtain interspecific diploid hybrids that produced 2n pollen grains. A return to a tetraploid level sought by breeders can then be considered using sexual polyploidization, obtained by crossing a tetraploid cultivated rose with these hybrids. Application of a high‐temperature regime led to a small but significant increase in the percentage of 2n pollen grains in these hybrids of up to 4.6%. This result was obtained by applying high temperatures close to 32°C during the day to plants cultivated in a glasshouse during recurrent cycles of bloom. Crosses were made between an unreleased tetraploid hybrid tea rose, as a female, and the diploid hybrid that produces the most 2n pollen grains. Tetraploid (42.1%) and triploid (57.9%) offspring were obtained. The use of these 2n pollen grains of the first division restitution type should facilitate the introgression of complex traits of interest.     

Cytological mechanism of 2n pollen formation and unilateral sexual polyploidization in Lolium

Summary Plants of Lolium perenne, L. multiflorum and L. hybridum (331, 51 and 18, respectively) were screened for 2n pollen production. The screening was based on the size differences that are expected to be found between n and 2n pollen of a plant. It was found that 28 plants of perennial ryegrass-belonging to 13 cultivars-produced produced pollen grains of heterogenous size (big and small). The estimated frequency of big pollen grain production was higher than 10% in ten out of 28 plants and in three of them it reached a value of 100%. Eight plants obtained from two 4x × 2x crosses, in which the male parent had been previously identified as producing pollen grains of heterogenous size, was tetraploid. The cytological mechanism of big pollen grain formation observed in three plants consists in the lack of spindle formation and, consequently, of chromosome migration in anaphase II. From these breeding and cytological results, it was concluded that the big pollen grains observed were viable 2n pollen grains. The nuclei of the 2n pollen grains produced by this mechanism are genetically equivalent to those formed by the restitution of the second meiotic division. The value of these gametophytes in a breeding program of Lolium is discussed.     

Factors affecting seed set in the crosses between <Emphasis Type="Italic">Dendranthema grandiflorum</Emphasis> (Ramat.) Kitamura and its wild species

Reproductive barriers often exist in the crosses between Dendranthema grandiflorum (Ramat.) Kitamura and its wild species and seriously result in low seed set, consequently reducing breeding efficiency. For the purpose of revealing the factors leading to low seed set, we investigated pollen viability, germination behavior of pollen grains on stigmas and embryo development in the crosses between D. grandiflorum and three wild species, D. nankingense (Nakai) Tzvel., D. indicum (L.) Des Moul. and D. zawadskii (Herb.) Tzvel. using technique of paraffin section, and light, fluorescence and scanning electron microscopy. The results indicated pollen viability of three wild species ranged from 20 to 25%. In the cross between D. grandiflorum and D. nankingense, very few pollen grains germinated on stigmas after pollination and most of them germinated abnormally. In addition, normal embryos were observed in 12% ovaries at 8 days after pollination and thereafter all the embryos aborted. In other two crosses, many pollen grains germinated on stigmas and pollen tubes penetrated stigmas normally after pollination. Moreover, normal embryos were observed in over 50% ovaries from 8 to 15 days after pollination in the cross between D. grandiflorum and D. indicum, and seed set was 59%. In the cross between D. grandiflorum and D. zawadskii, normal embryos were observed in 52% ovaries at 8 days after pollination. After that, however, most embryos degenerated and seed set was only 9%. These data suggest that pollen viability has no significant effects on seed set of the three crosses. Very few germinated pollen grains on stigmas and abnormal growth of most pollen tubes before fertilization, and embryo abortion are the main factors causing failure of the cross between D. grandiflorum and D. nankingense, whereas only embryo abortion is a main factor resulting in low seed set in the cross between D. grandiflorum and D. zawadskii and no barriers occur in the cross between D. grandiflorum and D. indicum.     

Reproductive development response to high daytime temperature in field pea

Flowering plants are highly sensitive to heat stress during reproductive phase, which covers development from floral initiation to seed maturity. The objectives of this study were to diagnose high temperature effects on pollen production and morphology, production of reactive oxygen species (ROS) in pollen grains and ovules in pea cultivar “CDC Golden.” This study also investigated timing and duration of heat exposure at specific developmental stages of floral buds, open flowers and early set pods on flower and pod abortion, seed development and seed yield in “CDC Golden” and a second cultivar “CDC Sage.” The experiments were conducted in growth chambers with two temperature regimes (24/18°C and 35/18°C day/night temperature for 4–14 days) during reproductive development. Heat stress reduced the number of pollen grains per anther, induced smaller pollen grains and increased ROS production in pollen grains, but it did not affect ROS accumulation in ovules and ovule number per ovary. Heat exposure when young floral buds were visible at the first reproductive node was more detrimental to flower retention, seed set, pod development and seed yield compared to heat exposure started later when flowers at the second reproductive node were fully open.     

Male fertility and cytology of triploid hybrids between tetraploidSolanum commersonii (2n=4x=48, 2EBN) and Phureja-Tuberosum haploid hybrids (2n=2x=24, 2EBN)

Summary Solanum commersonii Dun. is a diploid (2n=2x=24, 1EBN) wild species of potential value for potato breeding. It is a reproductively isolated species and cannot be crossed with Tuberosum haploids (2n=2x=24, 2EBN) or other diploid 2EBNSolanum species. In order to overcome the EBN barriers, triploid hybrids were produced between Phureja-Tuberosum haploid hybrids, which form 2n pollen grains by parallel spindles, and tetraploidS. commersonii. Microsporogenesis analysis of the triploids indicated a trend towards low values of chromosome distribution at Anaphase I; lagging chromosomes were often observed as well. Despite these abnormalities, the percentage of stainable pollen was very high, ranging from 5.0% to 74.3%. A high variation in pollen grain diameter was also evident. Parallel and tripolar orientation of spindles at Metaphase II of microsporogenesis was a common feature of all the triploids analyzed, but dyads and triads were observed at a very low frequency. Therefore, also the frequency of 2n pollen was very low; the different size of stainable pollen appears to represent the ploidy levels which are possible according to the distribution of chromosomes in Anaphase I. The results obtained also suggest thatS. commersonii could have minor genes acting at the end of meiosis in such a way that, despite the presence of parallel/tripolar spindles, dyads/triads are not formed.Contribution no. 124 from the Research Center for Vegetable Breeding.     

美国梾木(Cornus spp.)花粉活力及萌发特性

为了了解美国梾木花粉活力及萌发特性,采用扫描电镜技术对13个美国梾木无性系花粉形态进行观测与方差分析,并采用荧光显微技术和TTC染色法对无性系2号花粉萌发特性及花粉活力进行观察。结果显示:(1)不同无性系间花粉大小差异不显著,但外壁纹饰差异较大,极面沟距的变异系数最高且遗传最为稳定。(2)花序开花率为75%时花粉活力最高,整花4℃条件下短期保存花粉活力较高。(3)授粉后,多数花粉可在柱头上正常萌发,72 h后花粉管即可进入子房。研究认为,花粉外壁及极面沟距可作为鉴定美国梾木无性系的形态指标,美国梾木花粉活力及萌发特性是抗逆及丰产杂交组合的重要保证。     

Soybean Pollen Anatomy,Viability and Pod Set under High Temperature Stress

High temperature (HT) stress is one of the major environmental factors influencing yield of soybean (Glycine max L. Merr.) in the semi‐arid regions. Experiments were conducted in controlled environments to study the effects of HT stress on anatomical changes of pollen and their relationship to pollen function in soybean genotype K 03‐2897. Objectives of this study were to (a) quantify the effect of HT stress during flowering on pollen function and pod set and (b) observe the anatomical changes in pollen grains of soybean plants grown under HT stress. Plants were exposed to HT (38/28 °C) or optimum temperature (OT, 28/18 °C) for 14 days at flowering stage. HT stress significantly decreased in vitro pollen germination by 22.7 % compared to OT. Pollen from HT stress was deformed; it had a thicker exine wall and a disintegrated tapetum layer. HT stress decreased pod set percentage (35.2 %) compared to OT. This study showed that decreases in pollen in vitro germination by HT stress were caused by anatomical changes in pollen, leading to decreased pod set percentage under HT stress.     

The effect of pollen load and pollen grain competition on fertilization success and progeny performance inFagopyrum esculentum

Summary Flowers of cultivated buckwheat (Fagopyrum esculentum Moench) often receive natural pollen loads of fewer than 10 pollen grains. The cultivated varieties also have high genetic variability. These observations raise the question of whether seed production in buckwheat is often limited by pollen delivery, and whether small increases in pollen load could result in gametophytic selection through pollen grain competition. In greenhouse-grown buckwheat plants, embryo sac penetration by pollen tubes was universal with 10 or more pollen grains. However, seed production increased with pollen load up to 30 grains per flower. Larger pollen loads, which intensify selection among gametophytes, resulted in more vigorous progeny. Seedlings produced from high pollen load (15–20 pollen grains) were larger (40% by weight) than those from low pollen load (5 pollen grains). These results are evidence that pollen grain competition can occur in buckwheat with benefits for progeny performance.     

Anther and pollen grain culture of rice (Oryza sativa L.)

Summary Factors favouring callus proliferation and subsequent regeneration of plants from pollen grains of rice anthers (Oryza sativa L., cvx. Bahia, Girona, Balilla × Sollana and Sequial) were determined. Cultivar differences in response were found, such as a high rate of haploid plant regeneration.In addition, isolated pollen grain culture was used to induce tissue proliferation outside the anther walls. The frequency of callus formation from isolated pollen grains was very low. It was necessary to preculture the anthers before pollen grain separation, in order to accomplish a successful development later. Root differentiation was observed in some of the obtained callus.     

Induction of 2n pollen in tulips by arresting the meiotic process with nitrous oxide gas

Triploid tulips have agronomically desirable traits such as vigorous growth and large flower size, but only a portion of all cultivated tulips is triploid. To apply 2n pollen to polyploid breeding of tulips, the polyploidizing agent, nitrous oxide gas (N₂O), was applied to bulbs. In tulips, meiosis in anthers occurs inside the bulbs from mid- to late-October. When meiosis in anthers (excised from bulbs) reached metaphase I, we treated other bulbs of the same clones with N₂O for 24–48 h. Most of the treated plants produced pollen grains with a wide-ranging or bimodal size distribution, indicating a mixture of n, 2n and aneuploid pollen grains. The use of pollen containing a relatively high proportion of giant pollen grains tended to yield larger numbers of triploids in the progeny. The number of giant pollen grains could be increased when N₂O-treated pollen grains were suspended in 10% sucrose and then sieved through a nylon mesh. Very few polyploids were observed in some cross combinations, even those involving pollen with a relatively high proportion of giant grains. Even so, this low polyploid yield most likely is due to a triploid block, because the capsules obtained in the crosses of the diploid×N₂O-treated plants contained some abnormal seeds, which were mostly triploid. Embryo culture was useful in rescuing abnormal embryos. The present study reveals that 2n pollen can be produced at high frequency using N₂O during tulip breeding.     

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.     

Pollen tube growth in crosses between Porteresia coarctata and Oryza sativa

Summary Comparative study with fluorescence microscopy revealed that the pollen tubes of the self-pollinated rice variety BR-9 reached the ovary within 75 min after pollination. In P. coarctata it took 150 min. In the cross between P. coarctata and BR-9, pollen tubes reached the ovary in 165 min but seed setting was not observed. In the cross between BR-9 and P. coarctata, small and deformed pollen tubes were formed and they failed to grow through the stylodium.     

A statistical mixture model for estimating the proportion of unreduced pollen grains in perennial ryegrass (Lolium perenne L.) via the size of pollen grains

Summary The size of pollen grains is commonly used to indicate the ploidy level of pollen grains. In this paper observations of the diameter of pollen grains are evaluated from one diploid accession of perennial ryegrass (Lolium perenne L.), which was expected to produce diploid (unreduced) pollen grains in addition to haploid pollen grains. The considerable overlap of the diameter distributions of haploid and diploid pollen grains severely hampers the accurate estimation of the proportion of diploid pollen grains. To overcome this problem we develop in this paper a statistical normal mixture model and we describe a method to test for the production of diploid pollen grains from a diploid parent, and to estimate the proportion of diploid pollen grains.     

Pattern of inheritance of a self-fertility gene in an autotetraploid perennial ryegrass (Lolium perenne) population

A mutation causing self-fertility (SF) in perennial ryegrass was studied at the tetraploid level. The aim of this work was to determine a) whether SF remains functional in a tetraploid population and b) whether the SF mutation expresses dominance in heterozygous pollen grains. A tetraploidized plant carrying SF alleles was self-pollinated to create a segregating F₂ population. In the F₂ individuals, pollen compatibility ranged between 38% and 84% showing that SF remained functional. The SF locus genotype was the main determinant of pollen compatibility explaining 78% of the variation. The observed segregation was significantly different from the expected under both SF being dominant or recessive models (P_(χ2)≤0.001) and tended to be intermediate between them, indicating partial dominance or additive gene action. The frequency of the different genotypes suggested that pollen grains homozygous for the mutation have a competitive advantage over heterozygous pollen and that pollen compatibility is affected by the interaction with additional loci. The implications of our results for breeding polyploid grasses are discussed.     

Production of an interspecific hybrid between Brassica fruticulosa and B. rapa

An interspecific hybrid between a wild species, Brassica fruticulosa (2n = 16, FF) and a crop Brassica species, B. rapa (2n = 20, AA) was synthesized using sequential ovary‐ovule culture on MS medium supplemented with casein hydrolysate. Morphological, molecular and cytogenetic analysis confirmed the true hybrid nature of the offspring. The F1 plants (2n = 18) were intermediate in morphology, highly pollen‐sterile as well as self‐sterile. A maximum of three bivalents per PMC was recorded, but 14 I + 2 II was the most common meiotic configuration. Normal pollen fertility and regular bivalent (18 II) formation was observed in the amphiploid sectors of the hybrid plants. The F₁ hybrid harboured significantly lower aphid populations than the crop Brassica parent.     

Male gamete development and early tapetal degeneration in cytoplasmic male-sterile pepper investigated by meiotic, anatomical and ultrastructural analyses

Cytoplasmic male‐sterile (CMS) pepper (Capsicum annuum L.) is a useful germplasm for heterosis breeding, however, the process of male gamete development and the sterility mechanism is unclear. In the present study, the developmental sequence in CMS pepper and the causative factors responsible for pollen abortion were investigated through combined meiotic analysis, paraffin section observations, and transmission electron microscopy. Results from meiotic analysis showed that meiosis in a male‐sterile inbred line, CMS 21A, was normal, and uni‐nucleate pollen could be released from the tetrads of both CMS 21A and the fertile maintainer line 21B. However, only the uni‐nucleate pollen from CMS 21B could develop into functional gametes, while the uni‐nucleate pollen from the 21A went through a process of rupture, and the cellular components were released into the anther sac. Observations of paraffin sections viewed with the light microscope showed that the tapetum of CMS 21A at the uni‐nucleate stage swelled abnormally and was pressed against pollen grains of the locule. Further observations with transmission electron microscopy revealed that the mitochondria in CMS 21A tapetum were highly vacuolated, and there was no accumulation of sporopollenin on the surface of CMS 21A pollen, suggesting a close relationship between early degeneration of tapetum and CMS.     

Chromosome pairing in haploid plants of radish derived from alien monosomic addition lines

Six haploid plants of radish were obtained via alien monosomic addition lines (2n = 19). One plant was derived from anther culture of an Raphanus sativus‐Brassica oleracea addition line, one plant from the selected smaller seed of an R. sativus‐Sinapis arvensis addition line and four plants from an R. sativus‐B. rapa addition line. During metaphase I of pollen mother cells, two plants exhibited the chromosome pairing of (0‐3)II+ (3‐9)I, three showed (0‐1)III + (0‐3)II + (3‐9)I and the remaining plant (0‐2)II + (5‐9)I. Trivalents seemed to be formed by the pairing between two larger chromosomes and the smaller one with somewhat loose pairing. All haploid plants were inferior to the radish cv. ‘Shogoin’ (2n = 18) with respect to vegetative growth. Their flowers were smaller, with sterile pollen grains, but a few normal flowers with fertile pollen sporadically developed and then produced a few seeds. It is suggested that the radish genome (R, n = 9) might comprise three pairs of homoeologous chromosomes, with the remaining three chromosomes carrying the homologous region(s) that results in a trivalent formation.     

亚麻受精过程及其经历的时间

应用石蜡制片技术, 观察亚麻的受精过程及其各阶段经历的时间。结果表明, 亚麻开花时落到柱头上的花粉随即萌发, 花粉管在花柱引导组织的细胞间隙中生长, 进入子房后经子房内表面, 沿胎座经珠柄进入珠孔, 进入1个助细胞, 在卵细胞与中央细胞之间释放2个精子;精子脱掉细胞质鞘后,形成精核与细胞质体;2个精核同时分别进入卵细胞和中央细胞, 并与卵核及次生核融合;精核与次生核融合速度稍快;观察到合子中雌、雄性核仁融合的过程。受精各阶段经历的时间为,花粉落到柱头上随即萌发;开花后4.5 h左右, 花粉管长入胚珠的珠孔;4.5~5.5 h,花粉管进入1个助细胞并释放精子;5.5~6.5 h为精卵融合和精核与次生核的融合期;7.5~8.5 h初生胚乳核分裂;12.5 h之后,合子分裂;6.5~12.5 h为合子静止期。