Diversity of abnormal embryo sacs in indica/japonica hybrids in rice demonstrated by confocal microscopy of ovaries

Embryo sac abortion occurs frequently in the spikelet of indica/japonica hybrids in rice. To provide a comprehensive understanding of embryo sac sterility in typical indica/japonica hybrids, a simple eosin B staining procedure for use with confocal microscopy was developed to observe the mature embryo sacs of 35 indica/japonica F₁s making up an incomplete diallel cross design (5 × 7). A diversity of abnormal embryo sacs existed in the hybrids. The main types were classified into five categories: (i) embryo sac degeneration (ESD), (ii) embryo sac without egg apparatus, (iii) embryo sac without female germ unit, (iv) embryo sac with abnormal polar nuclei and (v) abnormal small embryo sac (ASES). The frequencies of abnormal embryo sacs in the 35 hybrids ranged from 26.0% to 76.3%, with an average of 48.6%. The highest frequency of abnormality was 31.3% ESD. The frequencies of various abnormalities varied with parental genotype and environment.     

Seasonal changes in the incidence of apomixis of diploid,triploid, and tetraploid plants of Paspalum cromyorrhizon

Summary Changes in incidence of apomixis were determined at different intervals of the flowering period in a highly sexual diploid cytotype of Paspalum cromyorrhizon Trin., a natural tetraploid cytotype of the same species, and in the triploid and tetraploid intraspecific hybrids that were produced by pollinating the 2x cytotype with pollen of the tetraploid. Reproductive behaviour was determined from observations of embryo sacs in mature ovules. Gametophytic apomixis in this species in characterized by aposporous embryo sacs which differ from normal sacs by their number, orientation inside the ovule, their dimensions and shape, and the absence of antipodal cells.The potential for apomictic reproduction increased in relation to the ploidy level, suggesting a gene dosage effect in the incidence of apomixis. In the three ploidy levels, the highest incidence of apomixis was observed when the plants reached the maximum number of flowering heads. These findings suggest that the same environmental conditions that favour flowering should also be responsible for increasing the incidence of apomixis. An additional experiment under controlled conditions indicated that the light regime should be one of the factors that govern the amount of apomictic reproduction. Thus, polyploidy and apomixis should be of special significance in the dispersion and evolution of this grass species. The 4x facultative apomictic cytotype is far more frequent in nature than the highly sexual 2x. Moreover, most of the seeds produced in a flowering season should arise asexually. So, apomixis rapidly increases the number of plants bearing the successful maternal genotype, and sexual reproduction becomes important in adverse environmental conditions.     

Evidence of sexuality in induced tetraploids of Brachiaria brizantha (Poaceae)

Difficulties in obtaining new breeding lines of Brachiaria (Trin.) Griseb., an important forage grass in Brazil, are mostly related to differences in ploidy among the accessions, and to apomixis, an asexual mode of reproduction. Usually, sexual accessions are diploid while apomicts are polyploid. Induced tetraploids of Brachiaria brizantha (A. Rich.) Stapf have been successfully obtained and this paper presents the results of a study of their reproductive modes and fertility. Despite frequent meiotic aberrations during microspore development, the induced tetraploids produced viable pollen and produced progeny after controlled self-pollination. Similarly to the original diploid sexual progenitor, embryo sacs of the Polygonum type with confirmed meiotic origin were present in the induced tetraploids suggesting chromosome doubling did not alter the reproductive mode. The embryo sac of the Polygonum type was also observed in progenies obtained after self and open pollination. Nevertheless, embryo sacs of the Polygonum and the Panicum types within the same ovule were observed in some progenies obtained after open pollination, probably having resulted from hybridization with tetraploid, apomictic plants. Indeed, the compatibility of the progeny with tetraploid, apomictic B. brizantha was confirmed by the formation of mature caryopses after controlled pollination. Evidence is presented that the induced tetraploids and their progeny are sexual plants and that they are compatible with natural tetraploid B. brizantha. The induced tetraploids will be useful for analyses of apomictic inheritance as well as in the development of sexual tetraploid lines in Brachiaria breeding programs.     

Developmental abnormalities in the ovule and embryo sac of ulluco (Ullucus tuberosus) and their effect on seed set

Summary Ulluco (Ullucus tuberosus) is a vegetatively propagated crop plant that flowers abundantly but sets only few seeds. We examined if the low seed/ovule ratio can be explained by morphological abnormalities in ovules, embryo sacs or embryos. In 35 clones growing in the actual area of cultivation in Ecuador and in 5 clones grown in Finland flowers were analysed by clearing technique. About half of all analysed clones contained ovules and embryo sacs whose development was arrested, in two clones there were embryo sacs whose cell patterns were abnormal and in four clones there were double embryo sacs. There were both normal and abnormal ovules and embryo sacs in the same plant. Genetic load is suggested to be one of the causes for the low sexual fertility found in ulluco. It may be incapable of maintaining homeostasis of development of the ovule and embryo sac in a varying environment. The occurrence of developmental abnormalities should be studied in each clone intended for inclusion in traditional breeding programmes.     

Interspecific hybridisation betweenLimonium perigrinum Bergius andLimonium purpuratum L.

Summary Interspecific hybrids betweenLimonium perigrinum andL. purpuratum were obtained usingL. perigrinum as the female parent. No hybrids were produced by the reciprocal cross. Twelve- to 15-day-old embryos were rescued and cultured within their embryo sacs on modified B5 or KM medium. After two to three days the embryos were excised from their embryo sacs and re-plated on to fresh medium. When the embryo-derived plantlets had attained a length of 1 cm they were transferred to a modified MS medium containing BA and NAA for shoot proliferation. Plantlets were transferred to modified MS medium supplemented with IBA for 24 hours for root initiation then to a modified growth-regulator-free MS medium for root growth. After a further 28 days the plantlets were transferred to soil-less medium for acclimatisation. The hybrid characteristics of one of the 15 embryo-derived plants were determined by flow cytometry and by examination of morphological features. The mean DNA contents of 2C nuclei fromL. perigrinum, the hybrid andL. purpuratum were 13.98 pg, 16.81 pg and 19.37 pg, respectively. Mitotic and meiotic chromosome counts fromL. perigrinum andL. purpuratum showed that both parents and their hybrids had identical chromosome numbers (2n=24), and that the species were closely related. Morphological analyses of leaves and flowers showed that the hybrids displayed a number of features intermediate between both parents.Abbreviations B5 Gamborg et al. (1968) B5 medium - BA benzyl adenine - IBA indole-3-butyric acid - KM Kao and Michayluk (1975) medium - MS Murashige and Skoog (1962) medium - NAA napthalene-1-acetic acid - PVP polyvinylpyrrolidone     

High embryo sac fertility and diversity of abnormal embryo sacs detected in autotetraploid indica/japonica hybrids in rice by whole‐mount eosin B‐staining confocal laser scanning microscopy

An eosin B staining procedure for use with confocal microscopy (WE‐CLSM) was used to examine the mature embryo sacs in 24 typical autotetraploid (2n = 4x = 48) indica/japonica hybrids and to compare with their original diploid (2n = 2x = 24) indica/japonica hybrids in rice. Four of the 24 hybrids showed >80% embryo sac fertility, and 13 hybrids over 70%. The average embryo sac fertility of the 24 autotetraploid indica/japonica hybrids was 68.08%, which was 17.33% higher than that in diploid hybrids. When compared with the diploid hybrids, 79.17% of the autotetraploid hybrids had higher embryo sac fertility. A diversity of abnormal embryo sacs occurred in autotetraploid indica/japonica hybrids. Embryo sac abnormalities were similar for both autotetraploid and diploid hybrid, but their frequencies were different. Some novel abnormal types were found in autotetraploid hybrids, such as enlarged egg apparatus. The embryo sac fertilities and frequencies of various abnormalities in autotetraploid hybrids varied with parental genotype and environment. The average seed set of the autotetraploid hybrids were higher than their diploid hybrids in different growing seasons.     

Transfer of powdery mildew resistance from Brassica carinata to Brassica oleracea through embryo rescue

Interspecific hybrid plants and backcross 1 (BC₁) progeny were produced through sexual crosses and embryo rescue between Brassica carinata accession PI 360883 and B. oleracea cvs Titleist’and‘Cecile’to transfer resistance to powdery mildew to B. oleracea. Four interspecific hybrids were obtained through application of embryo rescue from crosses with B. carinata as the maternal parent, and their interspecific nature confirmed through plant morphology and random amplified polymorphic DNA (RAPD) analysis. Twenty‐one BC₁ plants were obtained through sexual crosses and embryo rescue although embryo rescue was not necessary to produce first backcross generation plants between interspecific hybrids and B. oleracea. All interspecific hybrids and eight of the BC₁ plants were resistant to powdery mildew.     

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.     

Reproductive pathways of seed development in apomictic guinea grass (<Emphasis Type="Italic">Panicum maximum</Emphasis> Jacq.) reveal uncoupling of apomixis components

One hundred and sixty accessions representing global germplasm of guinea grass (Panicum maximum Jacq.), an important apomictic (aposporous) fodder crop, were subjected to study on reproductive diversity in apomictic seed development utilizing ovule clearing and flow cytometric seed screen (FCSS). Single seed FCSS of selected 14 tetraploid and five hexaploid lines demonstrated uncoupling between the three apomixis components, viz. apomeiosis, parthenogenesis and functional endosperm development, in natural as well as experimental populations, though it differed across ploidy levels and genotypes. Reconstruction of reproductive pathways yielded a total of eight different pathways of seed development, arising by uncoupling/recombination between apomixis components. Amongst these, two pathways involving modifications in embryo-sac (ES) (presence of two polar nuclei in aposporous ES that fuse prior to fertilization) and fertilization process (fusion of only one polar nucleus in a sexual ES) have been reported for the first time. Some of the combinations, such as M_I (haploids arising from parthenogenetic development of reduced egg cell), were found viable only in hexaploid background. Germplasm lines with higher expression of individual components were also identified. These components (including autonomous endosperm development) were also experimentally partitioned in hexaploid progenies (derived from a tetraploid parent viz. accession IG 04-164) that showed segregation in their reproductive capacities, and are reported for the first time. Occurrence of several apomixis recombinants (phenotypic) in guinea grass lines suggested their hybrid origin, favors a multigene model for apomixis, with their penetrance affected by modifiers and epigenetic mechanisms, in contrast to earlier reports of single locus control. Implications of partitioning components on apomixis research are discussed.     

Abnormalities of endosperm development inLilium hybrids

Summary Of 191 interspecific crosses attempted between 27 different species representing different subgenera ofLilium only 14 gave rise to seeds and only 6 of these were viable. Sterility was associated with various types of abnormality in endosperm development. Many of these irregularities involved chromosome breakage and reunion but others entailed abnormal DNA replication and chromosome coiling. In one cross the individual endosperm nuclei contained four stranded structures, the behaviour of which at division was similar to that of bivalents at meiosis. The embryo-sac ofLilium is of theFritillaria type, containing both haploid and triploid polar nuclei. As a consequence the balance between the number of chromosome sets in the embryo, endosperm and maternal tissue is 2:5:2 and not 2:3:2 as is more commonly found in diploid species. It is suggested that sterility results from genetic imbalance of the endosperm itself rather than interaction of the endosperm with either embryo or maternal tissue.     

Influence of photoperiod on the frequency of sexual embryo saes in facultative apomictic buffelgrass

Summary The frequency at which sexual embryo sacs occur in some facultatively apomictic species is influenced by photoperiod. This research was conducted to study changes in the frequency of sexual embryo sacs (SES) in field-grown accessions of buffelgrass [Pennisetum ciliare (L.) Link] and to determine the influence of photoperiod on the expression of sexuality under controlled environmental conditions. In a 3-yr field study, mean frequency of SES collected from two facultative buffelgrass accessions (PI 409266 and 409277) ranged from 2.5 to 8.6 percent. Within each year, the greatest number of SES were observed during spring and summer, while a reduction in SES occurred during the late summer and early fall. Under controlled environmental conditions equal numbers of SES were observed from plants grown at 8, 12, and 16 h. For the two facultative accessions of buffelgrass examined in this study, it was concluded that no relationship existed between photoperiod and frequency of SES under either field or controlled environmental conditions.     

Sporophyte induction studies in ferns in vitro

Sporophytes induction from spore-derived gametophytes and the morphogenic potential of homogenised sporophytes were performed in several ferns: Adiantum capillus-veneris, Asplenium adiantum-nigrum, Ceterach officinarum, Davallia canariensis, Dicksonia antarctica, Dryopteris dilatata, Dryopteris filix-mas, and Polypodium cambricum. The influence of growth rate, the formation of sexual organs, and the homogenisation of the gametophyte on sporophyte formation were studied. Gametophytic growth and sporophyte development were not antagonistic events and no correlation was found between the proportion of sexual phenotypes and rate of sporophyte formation. A pre-treatment of the rhizome with BAP exhibited swelling and upon transferral to Murashige and Skoog (MS) basal medium developed numerous fronds. Homogenised sporophytes of A. capillus-veneris, D. canariensis, and P. cambricum cultured on MS basal medium regenerated sporophytes while on MS with 4.4 μM BAP favoured formation of aposporous gametophytes and/or dedifferentiated cellular aggregates. In A. adiantum-nigrum, BAP induced either aposporous gametophytes or sporophytes.     

Introgression of Cajanus platycarpus genome into cultivated pigeonpea, C. cajan

Summary Cajanus platycarpus, an incompatible wild species from the tertiary gene pool of pigeonpea (C. cajan (L.) Millspaugh), has many desirable characteristics for the improvement of cultivated varieties. To necessitate such transfers, embryo rescue techniques were used to obtain F₁ hybrids. The F₁ hybrids were treated with colchicine to obtain tetraploid hybrids, that were selfed to obtain F₂, F₃ and F₄ progenies. All of the hybrids and subsequent progenies had an intermediate morphology between the two parents. Backcrossing of the tetraploid hybrids with cultivated pigeonpea was not possible given embryo abortion, with smaller aborted embryos than those obtained in the F₀ parental cross.As a route of introgression, diploid F₁ hybrids were backcrossed with cultivated pigeonpea and BC₁ progeny obtained by in vitro culture of aborting embryos. BC₂ plants were obtained by normal, mature seed germination. Although embryo rescue techniques had to be used to obtain F₁ and BC₁ plants, it was possible to produce BC₂ and subsequent generations through direct mature seed. Every backcross to cultivated pigeonpea increased pollen fertility and the formation of mature seeds.Special project assistant till December, 2003.     

Cytology and reproductive behavior of diploid, tetraploid and hexaploid germplasm accessions of a wild forage grass: Paspalum compressifolium

Summary Fourteen germplasm accessions of Paspalum compressifolium native from southern Brazil were cytologically and embryologically analysed. The study revealed that one accession was diploid (2n=20), twelve were tetraploid (2n=40) and one was hexaploid (2n=60). This is the first report of diploid and hexaploid cytotypes for this species. Studies on microsporogenesis, megasporogenesis, and embryo sac development indicated that the diploid cytotype had regular meiotic behavior and reproduces sexually. Tetraploid cytotype usually had an important proportion of chromosomes that associated as quadrivalents during meiosis and reproduced by mean of aposporous apomixis. The hexaploid cytotype showed irregular meiotic behavior with about one third of the chromosomes associated as multivalents and reproduced by aposporous apomixis. Thus, P. compressifolium could be an agamic complex. Breeding in this complex is possible due to the presence of diploid sexuals which can be treated by colchicine and pollinated by apomicts.     

In vivo embryoids from anthers of Narcissus biflorus Curt

Summary Mature anthers of some plants of Narcissus biflorus Curi., cultivated under subtropical conditions at Jammu, were found to contain monads, microspore mother cell embryo sacs, and multinucleate embryoids. In addition, multicellular embryoids resembling those obtained in vitro through anther culture were also observed. All these structures arise from the microspore mother cells which do not follow regular course of microsporogenesis.     

An embryological study of a Pisum sativum x Vicia faba cross

Summary Flowers of Pisum sativum plants grown in the field and greenhouse were self-pollinated and pollinated with Vicia faba. Germination of pollen and pollen tube growth were slower in the Pisum x Vicia cross. About 2% fertile ovules in the field and 8% in the greenhouse were obtained from the Pisum x Vicia cross, compared to 26% and 48% for Pisum x Pisum. Development of the Pisum x Vicia embryo was normal in form, though much slower than the Pisum x Pisum embryo, and about the sixth day it began to collapse. The greatest difference between the two types of crosses was observed in the endosperm. While the Pisum x Pisum endosperm nuclei divided normally and endosperm developed throughout the ovule cavity and around the embryo, the Pisum x Vicia endosperm nuclei formed only scattered masses of densely stained nuclei which were not always in close proximity to the hybrid embryo. It was concluded that faillure of some ovules to develop following Pisum sativum x Vicia faba cross pollination was the result of slow germination of pollen and slow growth of the Vicia pollen tubes, with subsequent lack of fertilization. Collapse of fertilized ovules was associated with abnormal and limited development of the hybrid endosperm, possibly leading to lack of nourishment and eventual collapse of the hybrid embryo.     

关于糜胚胎发育的研究

糜为禾本科黍族作物.子房内有1倒生胚珠,双珠被,厚珠心.胚囊类型为蓼型,8核7细胞胚囊.成熟胚囊受精前1助细胞已解体.反足细胞4～5个,极核未融合.双受精过程中,卵受精后有三核仁现象,二极核与精核先后受精后,胚乳核先于合子分裂,游离核阶段约1.5～4天、第5天开始积累淀粉.合子休眠约20小时,第1次分裂为横裂,约12天胚分化完成,约22～28天籽粒成熟.反足细胞于花后6天胚分化开始,胚乳淀粉大量积累时消失.籽粒发育过程中的落粒现象应深入探讨.     

The crossing of common wheat (Triticum aestivum L.) with cultivated rye (Secale cereale L.). II. Fertilization and early post-fertilization developments

Summary Fertilization and early seed development was studied in the variety Chinese Spring of common wheat (Triticum aestivum L.) after pollination with rye (Secale cereale L.) and selfing, and in the common wheat variety Hope after selfing. In all three combinations the first pollen tube reached the micropyle in about 40 min after pollination. When pollinated with rye the migration of the sperm nuclei to the egg cell and the polar nuclei was delayed by about an hour. In the subsequent development until 72 hours after pollination the average cellular and nuclear doubling times of embryo and endosperm were about 16 and 8 hours for the interspecific combination. 18 and 9 hours for Chinese Spring selfed and 20 and 12 hours for Hope selfed.     

细胞质雄性不育陆地棉的细胞质效应

哈克尼西棉细胞质与陆地棉核互作不但引起花粉母细胞的败育，而且也影响到胚囊育性，具体表现与保持系比较，不育系的胚珠显著变小，不孕籽率显著变高和异常胚囊的增多，不育系不杂交，Ｆ１的花粉育性接种正常，但自交结铃率仍较低，表明哈克尼西棉不育细胞地对Ｆ１的育怀有一定程度的负效应。然而，这种负效应可以通过优良不育系和强恢复系的选与得以克服或减轻到不显著水平，在强优势组合（Ｆ１）中不育细胞质的负效应，不足以Ｆ１     

Triple fusion of the primary endosperm nucleus as a cause of interspecific cross-incompatibility in Avena

Summary Using five diploid, two tetraploid and three hexaploid Avena species (x=7) 78 reciprocal crosses were made. Of these 74 were successfull, though the frequency of seed set varied greatly. The development of hybrid seeds differed widely between cross combinations, especially in reciprocal crosses. Hybrid seeds were grouped into four types, (1) normal kernels, well developed and germinative (designated as D+); (2) shrivelled-empty kernels, which do not germinate (E–); (3) small viable kernels (Rd+) and (4) small inviable kernels (Rd–). Some rare intermediate types were found in certain crosses.The results are well interpreted in terms of a hypothesis of polar-nuclei activation in which the strength of the activating action of the male nucleus, and the reaction of the female nucleus at double fertilization are expressed as activating value(AV) and response value(RV), respectively. The degree of seed failure is closely related to the activation index(AI) of the polar nuclei, AV/2RV (or × 100). In a selfed plant the activation index is always 1/2=0.5 (or50%), and this usually results in the formation of normal seeds. If AI deviates from 50% the endosperm often stops developing or degenerates.Based on the degree of abnormality of hybrid seeds, activating values of the ten species were arbitrarily assigned from 0.4 in A. ventricosa (2x) to 3.0 in A.sterilis (6x). For comparison A.strigosa (2x) was used as a standard and set at 1.0. Among the 74 succesful interspecific crosses the AI of the polar nuclei varied widely, from 7 to 375% Generally speaking, AI values of less than 20, 20–30, 30–80, and more than 80% show the R-, Rd+, D+ and E-kernel types, respectively. In other words, if the AV of the pollen parent is < 40 or >160% that of the maternal parent hybrid seeds fail to develop. Thus in double fertilization of Avena or probably angiosperms, the triple fusion of two polar nuclei with a secondary male nucleus is apparently a sexual isolation mechanism, being a barrier to interspecific hybridization.Professor Emeritus of Kyoto University, Faculty of Agriculture, Kyoto.