Using Isolated Embryo Sacs and Early Proembryos for Localization of Calmodulin mRNA Before and After Fertilization in Nicotiana

An in situ hybridization technique for localization of calmodulin(CaM) mRNA in isolated entire embryo sacs and proembryos in Nicotiana tabacum L.cv.W38 has been developed. This technique can be applied to small amounts of materials in which a whole view of CaM mRNA distribution can be obtained. The authors revealed that CaM mRNA expression changes dramatically before and after fertilization. Especially interesting is that a prominent CaM mRNA band appears between the egg apparatus and polar nuclei temporarily during the period of pollination and fertilization. The band disappears just prior to fertilization and expands to a fan_shaped region that occupies the micropylar portion of the embryo sac. After fertilization, CaM mRNA accumulates in the elongated zygotes with higher concentration in their chalazal portion than in the micropylar portion. Such an asymmetrical pattern continues to manifest in the early proembryos. It is supposed that CaM mRNA may be involved in the early events and signaling steps associated with double fertilization and zygote polarization in higher plants.

CHANGES OF MICROTUBULAR ORGANIZATION DURING THE EMBRYO SAC DEVELOPMENT IN RICE

Changes in the patterns of microtubular distributions and organization in various stages of development of the embryo sac in rice ( Oryza saliva L.) were different. In the megasporocyte, most microtubules were radially distributed but some were longitudinally oriented. Similar distributional patterns were seen in the dyad cells and the functional megaspore. Microtubules in the uni-nucleate embryo sac were mostly randomly distributed, although some radiated type was also present. The pattern of distribution of the microtubules in the two and four-nucleate embryo sac was quite similar and the microtubules were mainly of the perinuclear type. Microtubules in the cells of the eight-nucleate embryo sac were more complex. In the egg cell, the microtubules were mostly randomly distributed whereas in the synergids they were predominantly in longitudinal alignment. Those in the central cell were transversely aligned. The antipodal mass had very few microtubules and few longitudinally aligned ones were present in the cytoplasm.

Further Studies on Microtubule Organizational Changes During Megagametogenesis in Rice Embryo Sac

Changes in the pattern of microtubule distribution and organization during megagametogenesis in the embryo sac of rice (Oryza sativa L. cv. IR36) were re-examined using a modified polyethylene glycol sectioning technique before immuno-fluorescence staining of microtubules. In the sectioned materials the pattern of distribution and structural organization of the microtubule cytoskeleton were quite well preserved. Fine details of the patterns of structural changes and re-organization of the microtubule cytoskeleton in the major stages of development during embryo sac megagametogenesis (viz. functional megaspore, uni-nucleate, 2-nucleate, 4-nucleate, 8-nucleate and mature stage) could be clearly observed and easily followed. Some new organizational patterns of microtubules associated with the probable movement and positioning of the polar nuclei were observed.

A New Type of Embryo Sac in Cardiopteris and Its Systematic Implication

报道心翼果属 (CardiopterisWall.exRoyle)一种新的胚囊类型 :胚珠无珠被、直生、薄珠心。成熟胚囊中 ,卵器位于合点端 ,反足细胞位于珠孔端。这样的胚囊在所有进行过胚胎学研究的被子植物中系首次发现。合子和胚胎也位于合点端。心翼果属的这一新特征无疑是特异性的 。

Changes of Calmodulin Distribution in the Embryo Sac of Oryza sativa Before and After Fertilization: an Immunogold Electron Microscope Study

Changes of calmodulin (CaM) distribution in the embryo sac of rice (Oryza sativa subsp. Japonica) at various stages before and after fertilization have been investigated by using immunogold electron microscopy. Before pollination, both cytoplasm and vacuoles of the egg cell, synergids and central cell were labeled by gold particles. A small amount of gold particles were localized in the nucleus, endoplasmic reticulum, mitochondria and dictyosomes. From pollination to fertilization, CaM amount increased in these cells, especially rich in the starch of amyloplasts. Increase of gold particles in the central cell began about 2 h earlier than that in the egg cell. There was no distinct difference of CaM amount between the degenerated and the persistent synergids. It is interesting to observe an obvious change of CaM distribution form during pollination and fertilization from scattered single particles to clustered particles, and back again to single particles after the fertilization finished. CaM was also localized extracellularly in the embryo sac wall as well as in the wall and intercellular space of nucellus cells. The extracellular CaM also changes in its amount and form after pollination. These results suggest that CaM, either intra- or extra-cellular, may play important roles in fertilization and zygote formation.

Abnormal Structure of Embryo Sac in Autotetraploid Rice

The structures of mature embryo sacs in 13 genetic stock lines of autotetraploid rice （Oryza sativa L.）, including indica, japonica and javanica, were studied by using the whole-mount stain-clearing laser scanning confocal microscopy （WCLSM）. Among the 13 autotetraploid rice, the majority of ovaries possess normal polygonum-type embryo sacs, while a few ovaries were characterized by abnormal embryo sacs. The abnormalities of embryo sacs could be classified into six categories, i. e. no female germ unit, abnormal polar nuclei, embryo sac degeneration, no egg apparatus, small embryo sac and ＇double set＇ of embryo sacs. The frequency of abnormal embryo sac in japonica （26.6%） was higher than that in indica （19.34%）. In addition, the major abnormalities in each autotetraploid line varied, suggesting that the abnormalities may be related to the genotypes of the varieties.

Identification of an S5^n allele in Oryza rufipogon Griff. and its effect on embryo sac fertility

Oryza rufipogon exhibits abundant genetic diversity and contains many elite genes. In this work, a total of 441 accessions of O. rufipogon were genetically examined using the functional marker S5n, revealing that 18 accessions carried the S5n allele with a heterozygous genotype (S5nS5i/j). Of these accessions, 14 were from Guangdong Province, 2 from Guangxi Zhuang Autonomous Region, 1 from Hainan Province and 1 from Jiangxi Province. Further sequence analysis spanning both sides of the deletion in S5n allele of O. rufipogon confirmed that this allele is identical with that of 02428, a wide-compatibility variety of domesticated rice. The segregation ratio of the 3 genotypes (S5iS5i/S5jS5j:S5nS5i/j:S5nS5n) among 15 self-bred progenies of O. rufipogon significantly deviated from the Mendelian expectation (1:2:1) in that the proportion of plants exhibiting S5n heterozygous and homozygous genotypes was much lower in the progeny. Sequence analysis of full-length S5n alleles indicated that a few base pairs were different between O. rufipogon and O. sativa, and it was presumed that S5n may be an ancient allele. The fertility of the embryo sac in plants with an S5n heterozygous genotype was low, suggesting the existence of other genes controlling embryo sac fertility in the wild rice.

Inheritance and mapping of embryo sac abortion in hybrid between Indica and Japonica rice (Oryza sativa L.)

A doubled haploid (DH) population derived from anther culture of F\-1’s between an Indica var. Zhai\|Ye\|Qing 8 and a Japonica var. Jing\|Xi17 as well as two backcross populations derived from this DH population were used to investigate inheritance of the embryo sac abortion at early megagametogenesis occurring in Indica/Japonica rice crosses. Two major loci, dominant and complementary \%esa\|1\% and \%esa\|2\%, located on chromosomes 6 and 12 respectively, were detected. Genetic analysis indicated that embryo sac fertility is mainly regulated by the gametophytic genotype at these two loci.

Embryogenesis of Polyembryonic Rice ApⅢ: Structural and Histochemical Studies of Egg Apparatus Around Fertilization

The structural and histochemical changes of the egg apparatus in the polyembryonic rice ( Oryza sativa L.), ApⅢ with the highest frequence of additional embryos among the polyembryonic rice investigated, before and after fertilization were studied and compared with those of normal and other polyembryonic rices in a similar developmental period. A total of 2 932 ovules were observed and each of them contained only a single embryo sac with a set of egg apparatus. Among 1 655 embryo sacs, there were 1 643 embryo sacs (99.27%) with one normal egg apparatus in each embryo sac, and only 12 embryo sacs (0.73%) from the remainder with 4_celled egg apparatus, i.e. two eggs and two synergids. Neither the numerous poly_egg apparatus and egg_like cells, nor the double set of embryo sacs each containing one egg apparatus and other abnormal egg apparatus in single ovary, which were reported by earlier investigators to have high frequency of embryo production in SB_1 and ApⅣ, were observed. The egg cell was located at the subterminal site of the micropylar end of embryo sac. The cytoplasm of egg cell was rich in protein materials and polysaccharide grains, which did not disappear until the division of zygote. The prominent nucleus was closely surrounded by protein and polysaccharide grains, which did not disappear until the division of zygote. No cytological difference was found between egg cells from the normal and abnormal egg apparatus. The two synergids were fully developed and situated at the upper most part of the micropylar end of the mature embryo sac. In most embryo sacs, the synergids were flask_shaped with longer necks, and a widened cap_shaped top, in close contact with the micropyle. The synergids had a well developed filiform apparatus. The characteristic appearance of the filiform apparatus as well as the cap_neck region of synergids before and after pollen tube penetration were easily distinguishable from the egg cell. The structure, the stainability with Coomassie Brilliant Blue and PAS reaction, the process of accumulation, distribution and disapperance of the cytoplasmic protein materials and polysaccharide grains of the two synergids, the persistent and rarely the receptive synergids before and after pollen tube penetration, were closely similar to those of egg cell of the same developmental stage. In comparison with normal and other polyembryonic rice reported, the size of nucleus and nucleolus and their stainability also strongly resembled those of egg cell. Based on the results observed, the main conclusions are summarized as follows: (1) the additional embryos very frequently developed in the young and mature seed of polyembryonic rice ApⅢ were produced by one or two synergids of normal egg apparatus, rarely by 4_celled egg apparatus; (2) during fertilization, the synergids, in addition to the natural specific function of introducing pollen tube and transferring sperms to egg cell and central cell, could be closely associated with the potentiality to breed one or two additional embryos; and (3) as compared with that of normal or other polyembryonic rice it is firstly disclosed that in a few embryo sacs of ApⅢ, the cytoplasmic and nuclear structure, the active anabolism and catabolism of protein and polysaccharide materials and the delayed disorganization at the mid_basal region of the receptive and persistent synergid still remained unchanged before the division of zygote. Such salient features could be the predisposition for the origin of additional embryos in ApⅢ.

砂引草(紫草科)大小孢子发生和雌雄配子体发育及其分类学意义

砂引草属(Messerschmidia L.)在紫草科(Boraginaceae Juss.)分类系统里位置不稳定,为更好地理解砂引草属的分类学位置,利用常规石蜡制片技术结合光学显微镜观察了砂引草(Messerschmidia sibirica L.)的大小孢子发生及雌雄配子体发育特征。结果如下:(1)花药4室,成熟花药壁由表皮、药室内壁、1层中层和绒毡层共4层细胞构成,双子叶型花药壁发育类型,分泌型绒毡层,成熟绒毡层细胞含2核,表皮宿存,药室内壁不规则2层,具纤维性加厚;(2)小孢子母细胞减数分裂过程中胞质分裂为同时型,小孢子四分体四面体型排列,成熟花粉粒为2-细胞型;(3)胚珠倒生,单珠被,珠孔狭长,具珠被绒毡层,假厚珠心,部分珠心组织宿存至成熟胚囊时期;(4)胚囊发育类型为蓼型,成熟胚囊梭形,极核在受精前融合,反足细胞退化早。砂引草胚胎学特征与天芥菜属(Heliotropium)其他种类胚胎学特征十分相似,稍有不同,鉴于胚胎学特征在属内较为稳定,支持分子系统中将砂引草属置于天芥菜属的分类学处理。

不同禽胚和接种途径分离GoAstV JL01株的效果研究

鹅星状病毒(Goose astrovirus,GoAstV)可导致1~15日龄雏鹅痛风甚至死亡。为探究有效分离GoAstV方法,将RT-PCR鉴定为GoAstV阳性的雏鹅肝脏、肾脏研磨液通过尿囊腔、绒毛尿囊膜及卵黄囊途径分别接种至SPF鸡胚、SPF鸭胚及非SPF鹅胚,通过多次传代观察胚体剖检变化、死亡情况、核酸、病毒载量检测等,综合判断GoAstV JL01株在禽胚上的最佳分离方式。结果表明,病料经尿囊腔途径接种非SPF鹅胚及SPF鸭胚,绒毛尿囊膜途径接种非SPF鹅胚、SPF鸡胚及SPF鸭胚,卵黄囊途径接种SPF鸭胚均无法使胚体发生显著的剖检变化及规律性死亡,RT-PCR及RT-qPCR检测表明,病毒无法在相应胚体、尿囊液及胚体其他组织中有效增殖,卵黄囊途径接种SPF鸡胚所传的10代胚体均发生发育迟缓、出血等特征性病变,除F_(1)代其余9代均死亡,卵黄囊和胚体中病毒载量为10^(4.5)~10^(6.0)copies·μL^(-1)。因此,卵黄囊途径接种8日龄SPF鸡胚为GoAstV JL01株最适分离方式。研究为GoAstV分离提供可借鉴路径。

Lipid transport to avian oocytes and to the developing embryo

Studies of receptor-mediated lipoprotein metabolic pathways in avian species have revealed that physiological intricacies of specific cell types are highly analogous to those in mammals. A prime example for the power of com- parative studies across different animal kingdoms, elucidated in the chicken, is that the expression of different lipo- protein receptors in somatic cells and oocytes are the key to oocyte growth. In avian species, yolk precursor transport from the hen＇s liver to rapidly growing oocytes and the subsequent transfer of yolk nutrients via the yolk sac to the developing embryo are highly efficient processes. Oocytes grow from a diameter of 5 mm to 2.5-3 cm in only 7 days, and the yolk sac transfers nutrients from the yolk stored in the mature oocyte to the embryo within just 2 weeks. The underlying key transport mechanism is receptor-mediated endocytosis of macromolecules, i.e., of hepatically synthesized yolk precursors for oocyte growth, and of mature yolk components for embryo nutrition, respectively. Recently, the receptors involved, as well as the role of lipoprotein synthesis in the yolk sac have been identified. As outlined here, lipoprotein degradation/resynthesis cycles and the expression of lipoprotein receptors are not only coordinated with the establishment of the tbllicular architecture embedding the oocyte, but also with the generation of the yolk sac vasculature essential for nutrient transfer to the embryo.

腾冲红花油茶花芽分化及雌配子体发育的形态学观察

[目的]观察腾冲红花油茶花芽分化与雌配子体发育进程,旨在掌握腾冲红花油茶花芽分化整体进程,明确各分化时期的内部结构特征,完善腾冲红花油茶的生殖生物学基础数据,为腾冲红花油茶的栽培管理提供参考依据。[方法]以腾冲红花油茶为研究材料,采用石蜡切片的方法对其花芽分化及雌配子体的发育过程进行解剖观察。[结果]1)腾冲红花油茶的花芽分化经历了分化始期、萼片形成期、花瓣形成期、雌雄蕊形成期、子房花药形成期、雌雄蕊成熟期等6个时期,历时长达8个月,分化期间历经较长时间的低温环境。2)腾冲红花油茶子房有3~4个心室,每个心室多枚胚珠,每枚胚珠发育状况不一致,胚珠为倒生型胚珠,具双层珠被,胚珠发育属于薄珠心型。9月初,珠心中央的薄壁细胞迅速分裂形成孢原细胞,孢原细胞继续发育形成大孢子母细胞,在大孢子母细胞发育过程中珠心组织开始被溶解吸收。3)腾冲红花油茶雌配子体发育由大孢子母细胞进行两次减数分裂形成4个功能大孢子,进而发育成为四核胚囊,四核胚囊通过一次有丝分裂发育形成七细胞八核胚囊。胚囊发育类型为五福花型,胚囊为四孢子胚囊。其胚囊发育类型与普通油茶、攸县油茶的葱型胚囊有明显差异。[结论]腾冲红花油茶花芽分化历经6个时期;子房有3~4个心室;倒生胚珠,薄珠心型;胚囊发育类型为五福花型,胚囊为四孢子胚囊。

温室栽培黄金梨种子败育初探

【目的】通过比较温室栽培和露地栽培黄金梨从萌芽期至开花期的胚珠、胚囊发育情况,初步探讨温室栽培黄金梨种子败育的原因。【方法】调查记录了温室、露地栽培黄金梨萌芽期至开花期的气温变化规律,并以2种栽培条件下的黄金梨为试材,对该时期的花芽进行采样,利用石蜡切片法对比观察黄金梨的胚珠、胚囊发育过程及其形态特点,并用测微尺测量了开花期胚珠、胚囊的大小;于花后20 d统计坐果率,并对成熟期的果实进行种子败育情况调查。【结果】露地栽培黄金梨种子败育率低,坐果率高,温室黄金梨种子败育率高,坐果率低。温室栽培条件下,不适宜的高温处理缩短了黄金梨从现蕾到开花所需时间;同时造成开花期胚囊发育滞后,胚囊发育不完全(未成熟),50%的胚囊仍处于单核、二核阶段,11.1%的胚囊处于四核阶段,且胚囊的长度和宽度均小于露地黄金梨。【结论】温室黄金梨种子败育率高、坐果率低的重要原因是开花前的高温处理导致胚囊发育滞后、发育不完全。

‘无子瓯柑’E3泛素连接酶基因CsRNF217对转基因烟草育性的影响

【目的】为了验证‘无子瓯柑’Citrus suavissima‘Seedless’E3泛素连接酶基因CsRNF217对雄蕊育性的影响,采用异源转化获得过表达CsRNF217的转基因烟草Nicotiana tabacum,分析其对烟草育性的影响。【方法】采用亚历山大染色法、花粉离体培养法、杂交授粉后的结实率分析野生型烟草及转基因烟草自交1代(T1)阳性株系的花粉活力及胚囊育性,利用半定量RT-PCR分析基因CsRNF217在转基因烟草T1阳性株系花药中的表达强弱。【结果】‘无子瓯柑’CsRNF217在转基因烟草自交T1株系中高效表达,转基因株系的花粉染色活力和离体萌发率、自交结实率、以及与野生型的正反交结实率均显著低于野生型(P<0.05)。【结论】过表达CsRNF217的烟草植株花粉育性显著下降,同时伴随胚囊育性下降的现象,推测CsRNF217基因对‘无子瓯柑’雌雄育性存在负向调控的作用。

引导花粉管生长与防止多管入囊的分子基础

被子植物中,成熟花粉落在柱头上萌发出花粉管。花粉管在珠柄信号和珠孔信号的引导下,准确生长进入胚珠内的胚囊中,然后破裂释放两个精细胞完成双受精。被子植物建立了一套精细的调控机制,保证有且仅有一根花粉管进入胚囊完成双受精,从而保证遗传信息的稳定传递。本文对近年来国内外在花粉管引导与多管入囊阻断的机理研究进行了综述,并对花粉管导向的后续机制研究及利用其克服远缘杂交进行了展望。

Production of Partial New-typed Brassica Napus by Introgression of Genomic Components from B. rapa and B. carinata

A breeding strategy for widening the germplasm of Brassica napus was proposed by introgression of the A^r subgenome of B. rapa （A^rA^r） and C^c of B. carinata （B^cB^cC^cC^c） into natural B. napus （AnAncncn）. The progenies with 38 chromosomes that were derived from the self-pollinated seeds of pentaploid hybrids （A^rA^nB^cC^cC^n） were used for further research. Some of the partial new-typed B. napus showed normal meiotic behavior, high portion of germinated pollen and normal embryological development. This indicates that the selected new-typed B. napus had a balanced genetic base. Molecular analysis showed that about 50% of the genome in the new-typed B. napus was replaced by A^r and C^c subgenome from B. rapa and B. carinata. Considering the genetic diversity among different lines of new-typed B. napus, it was deduced that the introgression of the genomic components from B, rapa and B. carinata could widen the genetic diversity of rapeseed.

水稻和小麦胚乳发育的比较

以水稻盐粳 2 35和小麦扬麦 5号为研究材料 ,用整体解剖和树脂包埋切片等方法比较研究了这两种作物胚乳细胞分裂、分化和充实的特点 .两者发育的主要差异有 :(1)水稻胚乳囊呈香蕉形 ;小麦胚乳囊呈南瓜子形 .小麦的游离核与胚乳细胞要比水稻大 .(2 )水稻游离核的分裂以无丝分裂为主 ,而小麦游离核的分裂以有丝分裂为主 .水稻游离核及细胞的分裂速度较小麦快 .(3)水稻胚乳细胞中淀粉体约在花后第 4天出现 ,蛋白质体约在花后第 5天出现 ;小麦胚乳细胞中淀粉体约在花后第 7天出现 ,而蛋白质体约在花后第 9天出现 .(4 )水稻胚乳淀粉体中含有多个淀粉粒 ,而小麦胚乳淀粉体中仅含有一个淀粉粒 .(5 )水稻胚乳含两种蛋白质体类型 ,即PB1和PB2 ,而小麦胚乳只含一种蛋白质体 .(6 )水稻胚乳的背部有多层糊粉层细胞 ,其细胞壁上没有内突 ;而小麦腹部 (沟 )维管束的糊粉层细胞壁上有内突 。

利用激光扫描共聚焦显微镜观察同源四倍体水稻胚囊的形成与发育

激光扫描共聚焦显微镜是研究植物胚囊的形成与发育过程以及胚囊变异的理想工具。同源四倍体水稻是二倍体水稻染色体加倍形成的。本文利用激光扫描共聚焦显微镜直接观察经透明处理的水稻子房,研究加倍的基因剂量对于四倍体水稻的胚囊形成与发育的影响。结果表明,广陆矮4号四倍体大部分的胚囊形成与发育的过程基本是正常的,一部分胚囊出现异常现象,在成熟胚囊时期异常频率为10 61%;L202四倍体的异常频率较高,成熟胚囊时期异常频率达到45 65%。

甜樱桃胚珠、花粉发育与温度变化的关系

为探明甜樱桃性细胞发育规律及其所需的温度条件,利用石腊切片方法观察了露地栽培条件下,甜樱桃胚珠、胚囊和花粉发生发育过程的形态变化特征及其所需最低和最高温度。结果表明,甜樱桃的胚珠、胚囊和花粉发育,是在春季伴随着温度逐渐升高完成的;花前21￣35d,随着气温的升高,小孢子母细胞由相互连接在一起逐渐分离呈游离状态,在开花前21d的萌芽期前后完成2次分裂,由一个细胞分裂成四个细胞(四分体)形成花粉粒,此过程是在较低的温度(-0.1￣12.8℃)条件下完成;萌芽以后,胚珠的发育速度加快,开花前14d左右完成胚珠发育过程,胚囊的形成是在开花前7d内完成;甜樱桃的胚珠由单层珠被、珠心及胚囊构成。试验结果可为甜樱桃设施栽培的温度管理提供参考。