EMBRYO SAC DEVELOPMENT IN SOYBEAN: THE CENTRAL CELL AND ASPECTS OF FERTILIZATION

Development of the soybean central cell and events associated with fertilization are described. Central cell development involves the simultaneous formation of numerous multigrain amyloplasts, development of wall ingrowths, reduction of the cell's major vacuole, and nuclear migration. As these processes reach completion, a phase of lytic activity begins. This activity ultimately results in both the breakdown of most multigrain amyloplasts in the central cell and the reestablishment of the cell's large vacuole. While these processes occur, the egg and synergid cells continue development. Examination of the egg apparatus after plasmogamy but prior to karyogamy reveals several changes. Whereas the synergid containing the pollen tube is degenerate and the distribution of organelles in the persistent synergid has changed, the egg cell with a free sperm nucleus exhibits higher levels of metabolite storage and changes in its wall structure. The walls of the newly formed sporophyte are similar to those of egg apparatus cells (Folsom and Cass, Canadian Journal of Botany 68: 2135–2147 [1990]) in that they seem to lack a middle lamella. This layer becomes apparent only in the two-celled proembryo. While these changes occur in the egg, zygote, and proembryo, synergids undergo different degradative pathways.     

MEGASPOROGENESIS AND MEGAGAMETOGENESIS IN SOYBEAN,GLYCINE MAX

Megasporogenesis and megagametogenesis were examined in Glycine max with light, fluorescence, and electron microscopy. Megasporogenesis results in a linear tetrad of four megaspores. Megagametophyte development is of the Polygonum type, with the functional chalazal megaspore undergoing three successive mitotic divisions to produce an eight-nucleate, seven-celled mature megagametophyte. The central cell becomes packed with starch. At fertilization, the antipodals are degenerate, the polar nuclei have fused, starch is diminished, and the egg occupies most of the micropylar portion of the megagametophyte. Several pollen tubes were occasionally observed at each micropyle, yet only one was involved in fertilization. Pollen tube entry occurs through a slightly reduced, viable synergid cell. Endosperm development precedes embryo growth. These results describing normal development allow important comparison with genetic mutants of soybean that affect female fertility.     

影响大豆体细胞胚诱导因素的研究

体细胞胚的诱导是大豆体外再生的关键。基因型,诱导光周期,外植体的英位,蔗糖浓度等因素,可导致诱导频率及正常胚比例不同,影响植株再生。本研究选用黑龙江省主栽大豆基因型的未成熟子叶,在含高浓度生长素的MSB培养基上诱导体细胞胚产生。合丰25和东农7819为优选基因型,生育前期下部英位大小为2－4mm未成熟子叶体细胞胚发生效果最好;四种光周期下体细胞胚诱导频率相近,但连续弱光了正常胚比例高;NAA诱导优于2－4,D;10mg/1NAA与1．5％蔗糖配比组合最佳。     

MALE STERILITY IN SOYBEAN (GLYCINE MAX). I. PHENOTYPIC EXPRESSION OF THE ms2 MUTANT

The effects of a nuclear male-sterile mutant (ms2) of soybean, Glycine max (L.) Merr., on anther development were analyzed by means of light- and electron-microscopy. The structure of microspore mother cells (MMCs) in male-sterile plants was identical to that of male-fertile plants. Meiosis was completed, and tetrads of microspores formed. Microspores degenerated after the deposition of primexine and probacullae. The sheath of callose surrounding microspores did not dissolve. No structural abnormalities of the microspores were detected before the onset of degeneration. The tapetal and anther wall layers were characterized by aberrant development. Tapetal abnormalities included premature vacuolation, a persistent inner tangential cell wall, failure to differentiate normal concentrations of endoplasmic reticulum and dictyosomes, disruption of plastids, and premature degeneration. Malfunction of the tapetal layer preceded, and may have induced, microspore degeneration. Gross anther morphology was not influenced until advanced stages of development.     

水稻胚囊发育过程中微管的变化

对水稻(Oryza sativa L.)胚囊发育过程中微管变化的研究表明,微管在胚囊发育的不同阶段变化多样。在大孢子母细胞阶段微管分布主要呈辐射状,部分纵向排列。二分体和功能大孢子具类似的微管分布,而在单核胚囊微管主要是随机分布,部分呈辐射状。两核和四核胚囊的微管组成和分布非常相似,主要分布于细胞核周围。而八核胚囊的微管分布较为复杂,胚囊中的细胞做管分布各异,在卵细胞中呈随机分布,在助细胞中大多数呈纵向分布,而在中央细胞中呈横向分布,微管在反足细胞中非常分散,细胞质中有少量纵向排列的微管。     

利用聚鸟氨酸提高大豆原生质体外源基因的转化效率(简报)

有不少利用PEG法把外源基因导入到大豆原生质体,获得了转基因植株的报道,目前PEG的转化效率有所提高,但还是不能满足转化的需要,如何提高原生质体的转化效率是基因转化工作中的关键问题。本实验为了解决这个难题,以大豆幼子叶原生质体为材料,利用聚鸟氨酸(PLO:Poly-L-Ornithine,MW:114900,SIGMA)对Bt基因的转化进行了探讨。     

DNA,RNA, AND PROTEIN COMPARISONS BETWEEN NODULATED AND NON-NODULATED MALE-STERILE AND MALE-FERTILE GENOTYPES OF SOYBEAN (GLYCINE MAX L.)

Four soybean (Glycine max L. Merr.) lines isogenic except for loci controlling male sterility (ms₁) and nodulation (rj₁) were developed to study the effects of reproductive development and nitrogen source on the nucleic acid and protein levels within the leaves. Changes in DNA, RNA, protein, and cellular viability were measured from flowering (77 days after emergence) until maturity (147 days after emergence) in leaves of nodulated and non-nodulated male-sterile and fertile soybean genotypes. Leaf nuclei from the sterile genotypes yielded DNA amounts that were significantly higher than those from the fertile lines. The average DNA values for the nodulated sterile and nodulated fertile lines at 147 days after emergence were 7.01 and 2.45 picograms, respectively. The average 2C DNA amount as determined from dividing root-tip nuclei was 2.83 picograms, which indicated occurrence of endopolyploid mechanisms in the sterile lines and age-related loss of DNA in fertile lines. Similar to DNA findings, the RNA and protein values in the sterile lines were significantly higher than those values observed in the fertile lines, suggesting an increased capacity to synthesize protein. The soybean leaf nuclear DNA declined, especially in the fertile lines in terms of the percent endopolyploid nuclei as well as the average DNA content during maturation. The DNA decline in leaves of fertile genotypes suggests that the leaves may be exporting nucleosides and phosphates to the seeds during embryo formation. In the sterile lines, due to the reduced pod-set, these ready reserves of nucleosides and phosphates tended to accumulate in the chromatin of the leaf nucleus as manifested by the DNA specific Feulgen stain. By the end of the study (147 days after emergence), the nodulated fertile genotypes had experienced a dramatic loss in DNA, RNA, and protein. The nodulated sterile genotypes, however, indicated 65% more DNA, 59% more RNA, and 53% more protein as compared to the nodulated fertile genotypes at 147 days after emergence. The sterile lines also indicated the slowest increase in the death of cells, while the fertile lines indicated the fastest increase in nonviable cells, as shown by trypan blue staining. The fertile lines displayed normal monocarpic senescence throughout the study. The reproductive structures of fertile plants utilized the molecules in seed production, whereas in the sterile lines, these accumulated in leaf cells.     

水稻胚囊壁的形成与发育观察

通过透射电镜对水稻(Oryza sativa L.)功能大孢子形成开始至胚囊成熟期间胚囊壁的形成与发育进行观察,结果表明:水稻胚囊壁是在原有功能大孢子壁的基础上,通过与其周围退化珠心细胞留下的壁相叠合,使壁加厚。功能大孢子近合点端壁存在胞间连丝,其中个别胞间连丝可保留到八核胚囊。胚囊壁上内突最早于四核胚囊近珠孔端发生。八核胚囊形成后,内突的发育在胚囊不同的细胞中表现不同,其中以中央细胞最具特点,表现为先在中央细胞与珠心相接的近珠孔端和近合点端两个区域的胚囊壁上形成,以后近珠孔端胚囊壁上的内突大量增加,而近合点端的却增加不明显,中部胚囊壁上的内突出现的时间相对较晚。到胚囊成熟时,近珠孔端胚囊壁上内突的分布密度最大,中部次之,近合点端的最小,三个区域上内突的形态各异。反足细胞与珠心相接的胚囊壁上内突的形成时间较早,但以后的发育却相对缓慢,数量增加不明显。2个助细胞交界处胚囊壁上的丝状器在胚囊未明显膨大时已形成。卵细胞除在与助细胞交界处的壁外,其它部位不形成明显的内突结构。     

STIGMA,STYLE, AND OBTURATOR OF SOYBEAN,GLYCINE MAX (L.) MERR. (LEGUMINOSAE) AND THEIR FUNCTION IN THE REPRODUCTIVE PROCESS

Soybeans have a wet stigma overtopped by a pellicle that originates from the cuticle. There are numerous exudate-filled, axially oriented channels between cells of the transmitting tissue in the stigma and style. Pollen tubes grow in these channels and receive nutrition and mechanical guidance. Transmitting-tissue cells of the obturator are secretory also, but the obturator in soybean does not appear to control direction of pollen tube growth mechanically. The significant function of transmitting tissue in soybeans is to provide nutrition and to control direction of pollen tube growth.     

FLORAL DEVELOPMENT OF A FLOWER-STRUCTURE MUTANT IN SOYBEANS,GLYCINE MAX (L.) MERR. (LEGUMINOSAE)

A flower-structure mutant with cleistogamous flowers (but often with an exposed style and stigma) and very low seed set was found in soybeans (Glycine max (L.) Merr.). The mutant, assigned Genetic Type Collection Number T269, is controlled genetically by duplicate recessive genes, fs₁ and fs₂. A study of flower development in T269 plants was undertaken to determine the cause of the low seed set. Both normal and mutant flower buds were observed with a light microscope by using paraffin serial sections and with a scanning electron microscope. Measurements of various floral structures were taken to verify differences observed between mutant and normal flowers. Young mutant flower buds had longer carpels and larger receptacles than did normal flower buds. These two factors caused the sepals to be positioned abnormally, which, in turn, prevented normal development of the petals. The abnormal petal development prevented staminal tube elongation, and a spatial separation between the anthers and stigma existed at anthesis, preventing self-pollination. Observations of the gynoecium of mutant flowers revealed that megasporogenesis and megagametogenesis were normal but that other features of ovule ontogeny were abnormal. In all ovules examined, the outer integuments failed to form micropyles. In addition, many ovules were positioned abnormally. The degree of aberration varied even within a carpel, but we estimated that at least 75% of the ovules were too aberrant to be functional. Therefore, the low seed set on T269 plants was due both to a lack of self-pollination and to partial female sterility. It is the only naturally occurring structural sterile reported in soybeans to date.     

水稻中央细胞发育期间超微结构变化的观察

本文通过透射电镜对水稻受精前胚囊中央细胞发育过程中超微结构的变化进行观察。结果表明,八核胚囊形成后很快就进行细胞化形成7个细胞,其中刚形成的中央细胞由1个大液泡、2个极核（珠孔端和合点端各1个）和一些含有丰富细胞器的胞质组成。中央细胞以后的发育主要是极核的发育和极核周围胞质的变化。极核发育经历以下过程：a．2个核都膨大呈“椭圆”形。核周围胞质呈不对称分布。b．2个核分别向胚囊中央移动并相互靠近。之后2个极核调整排列方式,由纵排（即与胚囊纵轴平行）变成横排。此时期有细胞质“桥”联结珠孔端卵器、2个极核和合点端反足细胞器。c．横排的极核移向卵器,并排列于卵细胞之上。此时胚囊未明显膨大,但极核相靠近的两边核膜有许多处已形成“融合桥”,核周围的胞质也起较大的变化,如质体内淀粉消失和光面内质网增加等。极核进一步发育直至胚囊成熟期间,极核排列方式及其周围胞质组成未观察到明显的变化,但胚囊体积明显增大。     

大叶杨配囊及胚珠的形成和发育

本文应用细胞化学方法研究了大叶杨胚珠、胚囊的形成和发育过程中核酸、蛋白质及不溶性多糖的分布和消长。大孢子母细胞、大孢子四分体及功能大孢子中含较少不溶性多糖,但却含丰富的RNA和蛋白质。功能大孢子经分裂发育成八核的蓼型胚囊。四核胚囊开始积累细胞质多糖,成熟胚囊中除反足细胞外充满淀粉粒。反足细胞形成后不久即退化。助细胞具多糖性质的丝状器,受精前两个助细胞退化。卵细胞核对Feulgen反应呈负反应。二极核受精前由胚囊中部移向卵器,与卵器接触后融合形成次生核。发育早期的胚珠为厚珠心,双珠被。晚期,内珠被退化,故成熟胚珠为单珠被。四核胚囊时期,珠孔端珠心组织退化,胚囊伸向珠孔形成胚囊喙。合点端珠心组织含丰富的蛋白质和核酸,这一性质与绒毡层性质相似,可能涉及胚囊的营养运输。胚囊的营养来源于子房和胎座细胞内贮存的淀粉粒。     

大豆下胚轴可溶性蛋白中钙激活的蛋白激酶

大豆（Ｇｌｙｃｉｎｅ ｍ ａｘ Ｌ．） 下胚轴可溶性蛋白提取液进行自磷酸化,以ＳＤＳ－ＰＡＧＥ电泳分析其标记产物时发现,当有较高浓度的Ｃａ２＋ 存在于反应液中时,有一条１８ ｋＤ蛋白带被高强度标记,同时也可观察到另一条标记强度不高的６７ ｋＤ蛋白带． 当反应时间延长到１５ 或３０ｍ ｉｎ 时,它们的标记强度都逐渐减弱,最终从放射自显影底片上消失;在反应液中加入钙螯合剂ＥＧＴＡ 时,则只有６７ ｋＤ 被高强度标记;在磷酸化反应过程中加入非标记ＡＴＰ,蛋白中的３２Ｐ逐渐被非标记磷取代,表明反应体系处于磷酸化－脱磷酸化的平衡过程中,并有结果显示这一过程是钙依赖性的． 组蛋白Ｈ１ 可以使反应进程加快,表明提取液中的蛋白激酶可以利用它作为底物． 综合结果表明,１８ ｋＤ和６７ ｋＤ蛋白可能是具有自磷酸化能力且对Ｃａ２＋ 敏感的蛋白激酶,它们对Ｃａ２＋ 的不同反应,使得钙信号的传递更具可控性     

A CYTOCHEMICAL STUDY OF EMBRYO SAC DEVELOPMENT IN STELLARIA MEDIA

Megasporogenesis and embryo sac development in Stellaria media were investigated using cytochemical methods for the demonstration of nucleic acids, proteins, and polysaccharides. RNA concentrations were high in the archesporial cells, low in the megaspore mother cell, and increased again to high concentrations with the formation of the megaspore and 2-, 4-, and early 8-nucleate embryo sac. RNA levels were also high in the egg and primary endosperm nucleus but low in the synergid and antipodal cells. Nucleolar size and vacuolation were indicative of RNA synthetic activity. Protein concentrations were parallel in concentration and distribution to those observed for RNA. Polysaccharides were conspicuously absent from all stages except the synergids and nucellar cells. Feulgen-stained DNA was demonstrable in the antipodal cells, megaspore mother cell, and megaspore cell, but was not visible in the 2-, 4-, or early 8-nucleate embryo sac. Feulgen staining was also absent from the egg and primary endosperm nucleus but was visible in the synergids and antipodals. Histones were difficult to visualize anywhere except in the egg cytoplasm and the nuclei of the antipodals.     

影响农杆菌介导的大豆子叶节遗传转化的因素

利用携带pCAMBIA1301质粒（含hpt和gus基因）的超毒根癌农杆菌菌株EHA105对大豆子叶节外植体进行遗传转化,研究了影响农杆菌介导的大豆子叶节遗传转化的因素。研究结果表明．农杆菌侵染液和共培养培养基中添加200μmok/L乙酰丁香酮和50mg／L抗坏血酸可以有效促进农杆菌对大豆子叶节的转化。农杆菌与子叶节共培养后羧苄青霉素（250mr／L）和头孢霉素（100mg／L）结合使用能有效抑制农杆菌过度繁殖并提高转化芽诱导频率;在转化细胞的分化和转化芽伸长过程中,改进的筛选策略可以明显改善对转化芽的筛选效果,从而提高转化频率。应用优化后的转化体系．获得了3个国内大豆主栽品种的转基因植株,PCR阳性植株频率为3．8％～7．6％。转化植株叶片总DNA的PCR和Southern blot实验表明,T-DNA上的外源基因已经整合到大豆基因组中。     

GYNOECIUM AND DEVELOPMENT OF EMBRYO SAC,ENDOSPERM, AND SEED IN PHOLISTOMA (HYDROPHYLLACEAE) RELATIVE TO TAXONOMY

The objective of this research was to provide data for a taxonomic evaluation of Pholistoma and for a later survey of evolutionary morphology within the tribe Hydrophylleae. The unilocular ovary bears specialized unicellular prickles. Ovary and capsule wall consist of five or seven cell layers, of which three develop thickened walls for protection and dehiscence. Two parietal placentae enlarge to fill the entire locule. The four to eight ovules per ovary, some of which degenerate, are irregularly ventral and half-epitropous to half-hypotropous. Each ovule is tenuinucellate, unitegmic, and anatropous with a feeble vascular strand. A large amount of ovular parenchyma is produced from two meristems, one of which is the integumentary tapetum. Cells of the ovular epidermis grow into giant cells, each with a tubular portion penetrating deep into the seed interior, thereby producing a most unusual seed coat. The embryo sac is of the Polygonum type, the embryo of the Onagrad type. The endosperm is cellular with a micropylar and a chalazal endosperm haustorium. Both haustoria produce lateral branches. The mature endosperm is porously pitted, due to the giant cells of the testa. Great similarity between the three Pholistoma species, and in particular the presence in all species of two lateral haustorium branches, of gigantic, funnel-shaped epidermal cells, and of a porously pitted endosperm, strongly supports the notion that these three species constitute a natural entity.