高粱SSA-1无融合生殖胚胎学研究

经常规石蜡切片法,在光学水平观察了高粱（Ｓｏｒｇｈｕｍ ｂｉｃｏｌｏｒ （Ｌ．） Ｍｏｅｎｃｈ） ＳＳＡ－１ 无融合生殖的胚胎发生。高粱ＳＳＡ－１ 的无融合生殖为无孢子生殖和二倍体孢子生殖两种类型。两种生殖类型的单核胚囊经３ 次有丝分裂形成７ 细胞（８ 核）的成熟胚囊,由卵细胞、２ 个助细胞、２ 个极核和３ 个反足细胞组成。反足细胞迅速分裂增殖,形成由２０—３０ 个细胞组成的细胞团。此外,还具有一定频率的无孢子生殖多孢原和多胚囊现象。在未授粉情况下,卵细胞自发分裂形成典型的禾本科类型单子叶胚。经切片统计表明,ＳＳＡ－１ 的无融合生殖频率为４２％ ,证明该系为一兼性无融合生殖系。文中还讨论了ＳＳＡ－１ 无融合生殖过程的特点。     

Embryological Study on Apomixis in a Sorghum Line SSA-1

The Developmental process of apomictic embryo sac and embryo in a sorghum (Sorghum bicolor (L.) Moench. ) line SSA-1 was observed under light microscope, using the method of conventional paraffin sectioning. The result showed that the apomictic development conforms apospory and diplospory. The uninucleate embryo sac underwent mitotic divisions for three times to form a seven-celled or eight-nuclei mature embryo sac including an egg, two synergids, two polar nuclei and three antipodals. The antipodals divided and multiplicated to form an antipodal mass. Moreover, aposporous multiarchesporial cells and multiple embryo sacs were infrequently observed. Without pollination, the egg divided autonomously to form a typical graminaceous mature embryo. The authors counted the apomictic sections in the whole sections and the result showed that the frequency of apomixis was 42%, indicating the facultative apomictic property in the line SSA-1. The characteristics of apomictic process in the line SSA-1 is also discussed.     

水蔗草兼性无融合生殖胚胎学研究

对水蔗草 (ApludamuticaL .)的生殖方式进行研究 ,结果表明水蔗草进行兼性无融合生殖。胚囊发育分为两种类型 ,即有性生殖的蓼型和无孢子生殖的大黍型。无融合生殖胚囊频率为 6 0 .74%。在大孢子母细胞发育至四分体后 ,珠孔端的 3个大孢子解体。合点端的大孢子未解体时 ,邻近大孢子的 1个珠心细胞开始特化 ,形成无融合生殖的原始细胞 ,由该原始细胞发育形成有 1个卵细胞、1个助细胞和 2个极核的四核胚囊。     

水蔗草兼性无融合生殖胚胎学研究

对水蔗草(Apluda mutica L.)的生殖方式进行研究,结果表明水蔗草进行兼性无融合生殖.胚囊发育分为两种类型,即有性生殖的蓼型和无孢子生殖的大黍型.无融合生殖胚囊频率为60.74%.在大孢子母细胞发育至四分体后,珠孔端的3个大孢子解体.合点端的大孢子未解体时,邻近大孢子的1个珠心细胞开始特化,形成无融合生殖的原始细胞,由该原始细胞发育形成有1个卵细胞、1个助细胞和2个极核的四核胚囊.     

Sexual and apomictic development in Hieracium

 Most members of the genus Hieracium are apomictic and set seed without fertilization, but sexual forms also exist. A cytological study was conducted on an apomictic accession of H. aurantiacum (A3.4) and also H. piloselloides (D3) to precisely define the cellular basis for apomixis. The apomictic events were compared with the sexual events in a self-incompatible isolate of H. pilosella (P4). All plants were maintained as vegetatively propagated lines each derived from a single plant. Sexual P4 exhibited characteristic events of polygonum-type embryo sac formation, showed no latent apomitic tendencies, and depended upon fertilization to set seed. In contrast, D3 and A3.4 were autonomous aposporous apomicts, forming both embryo and endosperm spontaneously inside an unreduced embryo sac. The two apomicts exhibited distinct mechanisms, but variation was also observed within each apomictic line. Seeds from apomicts often contained more than one embryo. A degree of developmental instability was also observed amongst germinated seedlings and included variation in meristem and cotyledon number, altered phyllotaxis, callus formation, and seedling fusion. In most cases abnormal seedlings developed into normal plants. Such phenomena were not observed following germination of hybrid seeds derived from crosses between sexual P4 and the apomictic plants. The three plants can now be used in inheritance studies and also to investigate the molecular mechanisms controlling apomixis. Received: 11 February 1998 / Revision accepted: 23 July 1998     

赤苎无融合生殖细胞胚胎学研究

对赤苎(Boehmeria silvestrii (Pamp.)W.T.Wang)细胞胚胎学研究表明,其生殖模式属无融合生殖的二倍体孢子生殖(diplospory),但其未减数胚囊的发育途径不同于已报道的类型。大孢子母细胞的减数分裂I在到达终变期时停滞,染色体呈单价体状态并维持较长的时间。在尚未到达以核膜、核仁消失,纺锤体出现为特征的中期I前,大孢子母细胞由终变期直接“跳”入间期,从而始终保持了二倍体水平。减数分裂Ⅱ正常进行并产生二倍体二分孢子。珠孔端孢子退化,合点端孢子经3次分裂形成包括1个卵细胞、2个助细胞、2个极核和3个反足细胞的八核胚囊。胚和胚乳分别起源于卵和次生核未受精的自发分裂。胚乳属核型,其发育早于胚。     

Reproductive development in apomictic populations of <Emphasis Type="Italic">Arabis holboellii</Emphasis> (Brassicaceae)

Megasporogenesis, megagametogenesis and seed formation were analyzed cytologically in populations of Arabis holboellii originating from North America (Colorado) and Greenland. The Colorado population contained only triploid plants, while the Greenland population consisted of diploid and triploid plants. The penetrance of the apomictic trait was assessed at the level of embryo sac development. All populations showed facultative apomeiotic embryo sac development; however the penetrance of this trait differed between the populations. Apomeiotic and meiotic embryo sac development were characterized by diplosporous dyad formation (Taraxacum-type) and meiotic tetrad formation (Polygonum-type), respectively. Flow cytometric analyses of single mature seeds from all three populations suggest that only unreduced gametes participate in viable seed development. Pseudogamy was the predominant mode of endosperm formation; however, autonomous endosperm development was also observed. The fertilization of unreduced egg cells with unreduced pollen was observed at a low frequency in the Greenland populations. The mechanisms of apomictic reproduction in A. holboellii are discussed.     

Pseudogamous Apomixis in Maize and Sorghum in Diploid-Tetraploid Crosses

Apomictic seed development is a complex process including formation of unreduced embryo sac, parthenogenetic embryo development from the egg cell, and endosperm formation either autonomously, or due to fertilization of polar nuclei by the sperm (under pseudogamous form of apomixis). In the latter case, an obstacle to the normal endosperm development is disturbance of maternal (m) -to-paternal (p) genomic ratio 2m: 1p that occurs in the cases of pollination of unreduced embryo sac with haploid sperms. Usage of tetraploid pollinators can overcome this problem because in such crosses maternal-to-paternal genomic ratio is 4m: 2p that provides formation of kernels with plump endosperm. Using tetraploid lines as pollen parents we observed formation of plump kernels on the ears and panicles of diploid maize and sorghum accessions. These kernels had hybrid endosperm and diploid maternaltype embryo or hybrid embryo with different ploidy level (2n, 3n, 4n). The frequencies of plump kernels on the ear ranged from 0.2-0.3% to 5.7-6.2% counting from the number of ovaries. Maternal-type plants were found in two maize lines, their frequency varying from 10.7 to 37.5% of the progeny plants. In CMS-lines of sorghum pollinated with tetraploid sorghum accessions, the frequency of plump kernels ranged from 0.6 to 14.0% counting from the number of ovaries; the frequency of maternal-type plants varied from 33.0 up to 96.1%. The hybrid nature of endosperm of the kernels that gave rise to maternal-type plants has been proved by marker gene expression and by SDS-electrophoresis of endosperm proteins. These data testify to variable modes of seed formation under diploid × tetraploid crosses in maize and sorghum both by amphi- and by apomixis. Therefore, usage of tetraploid pollinators might be a promising approach for isolation of apomixis in maize and sorghum accessions.     

Endosperm formation in aposporous Crataegus (Rosaceae, Spiraeoideae, tribe Pyreae): parallels to Ranunculaceae and Poaceae

Apomixis in Crataegus is primarily aposporous and requires pollination. The embryo sac is of the Polygonum type. A combination of meiotically unreduced embryo sacs with apparently reduced pollen would violate the usual requirement for a 2 : 1 ratio of maternal to paternal contributions to the endosperm. We therefore investigated the origin of endosperm in seeds of sexual diploids and apomictic polyploids of the sister genera Crataegus and Mespilus. Flow-cytometric DNA measurements from embryo and endosperm in mature seeds were converted to ploidy levels using leaf-tissue information. The diploids had triploid endosperm. In c. 60% of seed from polyploids, one sperm apparently contributes to the endosperm, while 25% or more may involve two sperm. Additional results suggest that trinucleate central cells also occur. Fertilization of meiotically unreduced eggs is indicated. The ratio of maternal to paternal contributions to the endosperm in these apomictic Crataegus is not constrained to 2 : 1. They thus resemble some Sorbus (Pyreae) and very distantly related Ranunculus (Ranunculaceae). It is suggested that Paspalum (Poaceae) may have similarly flexible endosperm ploidy levels.     

几种具无融合生殖特性的植物多胚和多苗现象的观察

报道6种具无融合生殖特性的植物种子的胚数和萌发实生苗数的观察结果。金桔(Fortunellamargarita (Lour.)Swingle)、蜜桔(Citrus unshiu Marcoritch)和花椒(Zanthoxylum bungeanum Maxim.)具珠心胚,含多胚种子频率分别为97.50％、100％和45.00％;多胚种子的胚数范围分别为2～49,3～54和2～6。草地早熟禾(Poa pratensis L.)、滨草(Elymus rectisetus)和湖北海棠(Malus hupehensis(pampon.)Rehd.)具非减数配子体无融合生殖特性,含多胚种子频率依次为34.25％、8.11％和37.50％;前两种的多胚种子中胚数范围为2～3,后者为2～15。蜜桔、草地早熟禾和湖北海棠种子萌发多苗的频率分别为22.00％、6.14％和2.22％。描述了多胚种子中胚的形态、位置和分布。对6种植物含单胚种子的胚的来源进行了分析。初步结论:1.具无融合生殖现象的植物种子含多胚和萌发多苗的特性可作为寻找具无融合生殖特性的植物的形态学指标;2.对其筛选的材料进行大孢子发生、胚囊形成和早期胚胎发育的研究,以期阐明多胚来源和生殖类型。     

Further Embryological Studies on the in Vitro Apogamy in Oryza saliva L.

In the cultured young ovaries of rice, the processes of megagametophyte develop- ment could be switched to the formation of various abnormally organized embryo sacs and then to the initiation of synergid apogamy. The main pathway leading to apogamy was found to go via a linearly oriented 4-nucleate embryo sac to the formation of a li- nearly oriented egg apparatus, from which it was usually the chalazal synergid giving rise to an apogamous proembryo, and the micropylar synergid degenerated. The proembryo thus produced was located at the base of a vacuolated egg cell (Plate Ⅰ, 1–7). The second pathway went through a nonlinearly oriented 4-nucleate embryo sac to the formation of an egg apparatus in which the two synergids were located at one side of the egg and oriented longitudinally. In this case it was often the chalazal synergid that could be triggered to apogamy, resulting in a hook-shaped proembryo embracing the egg cel1 from one side (Plate Ⅰ, 8–11). When ovaries with nearly matured embryo sac were cultured, in a few cases where apogamy was induced, the proembryos observed were all situated at one side of the egg and were hook-shaped (Plate Ⅰ, 12). All these pathways are summarized in a diagram (Fig. 23). Some interesting changes were observed in the synergid and the egg cell of the cultured ovaries by PAS reaction and mercuric bromphenol blue staining. The egg cells, in contrast to in vivo condition, often contained abundant starch grains,. The synergids and synergid proembryos were rich of cytoplasmic protein (Plate Ⅱ, 13, 14). We supposed that the egg may supply some nutrients as well as stimulants to the developing synergid in the course of apogamy. The distribution of starch and protein in apogamous embryoids during subsequent development was also described in this paper (Plate, 15–22).     

Embryological Observation on the Apomictic Rice-ApⅢ (Shuang 13)

Since 1988s, the phenomenon of multiple seedlings and multiple embryoes of apomictic rice, Ap Ⅲ (Shuang 13) has been interestingly concerned. Embryological investigation of Ap Ⅲ has been carried out with the whole dissection and conventional sectioning technique. The main conclusions are as follows: 1.8. 9 % of 5000 matured caryopsises, contained one embryo in a caryopsis, from which single seedling arose, while 8.9 % twin embryoes and 1.2 % triplet embryoes of which developed two seedlings and triple seedlings respectively; 2. In over 700 young caryopsises, 90.0%～95.0% contained only one embryo, 5.0%～7.0% twin embryoes, except a few of the ovules with absorptive embryo sac or embryo and endosperm. Triplet embryoes caryopsis could not be traced further due to their low frequency; 3. Authors' materials showed that all the single, twin and triplet embryoes were situated at the micropylar end in one embryo sac of young ovule and no adventitious embryo was seen outside the embryo sac of ovules. From the aforementioned results, it can be infered that albeit single embryo is derived from the egg cell yet it could be possible twin or triplet embryocs may be developed from the egg cell and one or two of the synergids either or not through fertilization.     

龙须草无融合生殖的胚胎学证据

采用石蜡切片技术对龙须草(Eulaliopsis binata(Rotz)C.E.Hubb)进行了系统的胚胎学研究,证明龙须草为禾本科植物中一种新的无融合生殖材料.龙须草无融合生殖方式为无孢子生殖,在胚珠发育早期,多个珠心细胞特化为无孢子生殖原始细胞,由原始细胞发育为单核胚囊,经两次有丝分裂形成4核胚囊,进一步分化形成两种类型的成熟胚囊:(1)具1个卵细胞,1个助细胞和2个极核,占观察总数的67.6%;(2)具1个卵细胞,2个助细胞和1个极核,占观察总数的32.4%.胚囊发育属大黍型.多个无孢子生殖原始细胞可以同时发育,最后形成2个或多个胚囊,其比例为17.7%.胚珠内没有有性胚囊的发育.胚的发生有两种类型:(1)早发生胚(74%),开花前1～2 d,极核未分裂前卵细胞分裂形成胚;(2)迟发生胚(26%),开花后2～3 d,极核分裂形成多个胚乳游离核后,卵细胞启动分裂形成胚.存在多胚现象,多胚来自不同胚囊内卵细胞的孤雌生殖,多胚发生率为13%.胚乳由极核不经受精自发分裂产生.     

龙须草无融合生殖的胚胎学证据

采用石蜡切片技术对龙须草(Eulaliopsisbinata(Rotz)C.E.Hubb)进行了系统的胚胎学研究,证明龙须草为禾本科植物中一种新的无融合生殖材料。龙须草无融合生殖方式为无孢子生殖,在胚珠发育早期,多个珠心细胞特化为无孢子生殖原始细胞,由原始细胞发育为单核胚囊,经两次有丝分裂形成4核胚囊,进一步分化形成两种类型的成熟胚囊:(1)具1个卵细胞,1个助细胞和2个极核,占观察总数的67.6%;(2)具1个卵细胞,2个助细胞和1个极核,占观察总数的32.4%。胚囊发育属大黍型。多个无孢子生殖原始细胞可以同时发育,最后形成2个或多个胚囊,其比例为17.7%。胚珠内没有有性胚囊的发育。胚的发生有两种类型:(1)早发生胚(74%),开花前1~2d,极核未分裂前卵细胞分裂形成胚;(2)迟发生胚(26%),开花后2~3d,极核分裂形成多个胚乳游离核后,卵细胞启动分裂形成胚。存在多胚现象,多胚来自不同胚囊内卵细胞的孤雌生殖,多胚发生率为13%。胚乳由极核不经受精自发分裂产生。     

Reconstruction of reproductive diversity in Hypericum perforatum L. opens novel strategies to manage apomixis

The mode of reproduction was characterized for 113 accessions of the tetraploid facultative apomictic species Hypericum perforatum using bulked or single mature seeds in the flow cytometric seed screen (FCSS). This screen discriminates several processes of sexual or asexual reproduction based on DNA contents of embryo and endosperm nuclei. Seed formation in H. perforatum proved to be highly polymorphic. Eleven different routes of reproduction were determined. For the first time, individual seeds were identified that originated from two embryo sacs: the endosperm from an aposporous and the embryo from the legitimate meiotic embryo sac. Moreover, diploid plants were discovered, which apparently reproduce by a hitherto unknown route of seed formation, that is chromosome doubling within aposporous initial cells followed by double fertilization. Although most plants were tetraploid and facultative sexual/apomictic, diploid obligate sexuals and tetraploid obligate apomicts could be selected. Additionally, genotypes were detected which at a high frequency produced embryos either from reduced parthenogenetic or unreduced fertilized egg cells. The endosperm developed most frequently after fertilization of the central cell in aposporous embryo sacs (pseudogamy) but in few cases also autonomously. The genetic control of apomixis appears to be complex in H. perforatum. Basic material was developed for breeding H. perforatum, and strategies are suggested for elucidation of inheritance as well as evolution of apomixis and for molecular approaches of apomixis engineering.     

Embryological Studies on Apomixis in Pennisetum squamulatum

Studies on the formation and development of the embryo sac of the apomictic material of Pennisetum squamulatum Fresen indicated that normal archesporial cell did form with consequent development of a megaspore mother cell and later meiotic division to give rise to a triad. But invariably the megaspore mother cell and the triad underwent degeneration after formation. During the period of formation or degeneration of the megaspore or the triad a number of nucellar cells around the degenerated sexual cell became much enlarged. Frequently, one of the enlarging nucellar cells near the micropylar end became vacuolated and then developed into an aposporous uninucleate embryo sac, which underwent two further mitotic divisions to form an aposporous four-nucleate embryo sac, where the four nuclei remained in the micropylar end. Thus in the mature aposporous embryo sac there were one egg cell, one synergid and one central cell (containing two polar nuclei). Antipodal cells were completely lacking. The pattern of development of the aposporous embryo sac resembles the panicum type. There were two types of embryo formed during apomictic development namely ( 1 ) The pre-genesis embryo--embryo formed without fertilization, 1 to 2 days before anthesis, and (2) The late-genesis embryo--derived from the unfertilized egg cells, 3 to 4 days after anthesis. In the late-genesis embryo type, the egg cell divided after the secondary nucleus has undergone division to form the endosperm nuclei. All egg cells developed vacuoles before they differentiated into embryos. The development of the aposporous embryo followed the sequence of the formation of globular, pearshaped embryo and full stages of differentiation. The unfertilized secondary nucleus divides to form free endosperm nuclei after being stimulated by pollination. The development of the endosperm belongs to the nuclear-type.