Cytological Observations on Microsporogenesis and Pollen Development of Regenerated Stamen in Hyacinth

The present study consists of the cytological observations on the process of microsporogenesis and pollen development in the regenerated stamen of hyacinth; and a comparative study of the cytological changes in stamens of both regenerated and produced under natural condition. Results showed that the cytological changes of microsporogenesis and the pollen deveLopment in the regenerated stamen of the hyacinth were basically normal. But in the stage of the mature pollen there was an obvious cytological difference between both stamens in vitro and in nature. The mature pollen of the regenerated stamen consisted of three cells: one vegetative cell and two sperms, while mature pollen grain under natural condition was made up of two cells: one vegetative cell and one generative cell. This difference mainly resulted from different time and place of the generative cell division. The reason resulting in the differences and their influence on sperms were discussed.     

Maturation of stamens and ovaries on cultured ear inflorescences of maize (Zea mays L.)

Observations were made on the maturation of stamens and ovaries from cultured maize (Zea mays L.) ear inflorescences. Immature ears (5.1–10.0 mm long) of maize were cultured in kinetin medium to study microsporogenesis and pollen maturation in developing stamens. Male spikelets developed on ears cultured in kinetin medium. Meiosis-I began by 7 days of culture in the developing anthers and the mature tri-nucleate pollen grains were developed by 20 days of culture. Further, kinetin was required in the culture medium for at least initial 5 days to obtain the microspores in differentiated stamens.To observe the embryosac formation in developed ovaries, ears were cultured in control, kinetin (10.1–15.0 mm long ears) medium, and kinetin + gibberellic acid (5.1–10.0 mm long ears) medium. Formation of embryosacs was noticed in the developed ovaries which were sampled after 20 days of culture. This differential flower development using two growth regulators provides an opportunity to uncover the biochemistry and physiology of micro- and mega-gametophyte development in maize.     

曼地亚红豆杉‘Hicksii’花粉活力检测条件优化和适宜诸藏温度分析

采用单因素实验设计对适宜于曼地亚红豆杉‘Hicksii’(Taxus media‘Hicksii')花粉活力检测的TTC染色法和离体培养法的实验条件进行了选择,并采用优化条件研究了在25℃～-196℃条件下储藏13周花粉活力的变化.结果表明:采用TTC染色法测定的花粉活力均高于离体培养法.在TTC染色法的3个影响因素(染液pH、TTC浓度和染色温度)中,染液pH对检测结果有极显著影响,而染色温度和TTC浓度则无显著影响,但温度对染色速率有影响.在离体培养法的3个影响因素(培养基中蔗糖添加量、H3BO3和Ca(NO3)2浓度及培养温度)中,蔗糖添加量对检测结果有极显著影响,在含质量体积分数15％蔗糖的培养基上花粉活力最高,而在含质量体积分数20％和25％蔗糖的培养基上花粉均不能萌发;在含100和200 mg ·L-1H3BO3的培养基中添加200 mg·L-1Ca(NO3)2均能显著提高花粉活力;培养温度对花粉萌发速率有影响但对花粉活力没有明显影响.TTC染色法的最优检测条件为:用5.0g·L-1TTC染液(pH 7.0)在35℃下染色2.0h;离体培养法的最优检测条件为:用含质量体积分数15％蔗糖、100 mg·L-1H3BO3和200 mg·L-1Ca(NO3)2的培养基暗培养4d.在25℃、4℃、0℃、-20℃、-80℃和-196℃条件下储藏13周,‘Hicksii'花粉活力和保持时间有明显差异,其中,于-80℃和-196℃储藏3d 花粉就丧失活力;于25℃和-20℃储藏7周、4 ℃储藏10周,花粉仍有一定的活力;而在0℃条件下花粉活力最高,且储藏11周花粉仍有活力.推测曼地亚红豆杉‘Hicksii’花粉对低温的抗性较差,0℃为其适宜的储藏温度.     

Embryo rescue and plant regeneration in banana (<Emphasis Type="Italic">Musa</Emphasis> spp.)

An efficient regeneration protocol for zygotic embryos at varying maturity stages was developed for wild banana (Pisang Jajee (AA)). Embryo ontogeny was studied to determine the best maturity stage for embryo rescue, suitable media and culture conditions (light and dark) for germination and regeneration. The conversion of endosperm from transparent fluid into a semi-solid state was followed by visible embryo development, which commenced only after 70% embryo maturity. Zygotic embryos of Pisang Jajee at different maturity levels were excised and cultured on medium fortified with different concentrations of 6-benzyl adenine (BA) and indole acetic acid (IAA). Zygotic embryos produced callus or plantlets 25 days after initiation. The frequency of callus induction was greater in immature embryos irrespective of the media composition and decreased with increasing maturity. Fully matured embryos regenerated directly into plantlets without producing callus. Immature embryos required medium supplemented with plant growth regulators (PGRs) for successful regeneration. Although the culture conditions had no influence, dark conditions favoured callus induction and plant regeneration.     

Evaluation of a new culture method for mass-propagation of a photoperiod-temperature sensitive genic male sterile rice strain N19S

A newly established culture method was evaluated for application in mass-propagation of a photoperiod-temperature sensitive genic male sterile rice strain. It was found that the new culture method was very efficient for the regeneration of plants from callus of this strain, and a high concentration of 6-benzyladenine necessary for highly efficient plant regeneration culture did not seem to have negative effects on some agronomic traits investigated of the regenerated plants and their progenies. By the new culture method, a large number of plants could be regenerated in a relatively short time (approximately 8×10¹² plants within 300 days) from one naked seed-explant. Because of this, prominent drift of the PTGMS rice strain, which accumulates gradually during the course of propagation of the seeds through generations, in the response to photoperiod-temperature conditions could be avoided when this in vitro culture protocol is employed instead of seed propagation. Differences in rates of pollen fertility and seed setting were found between regenerated plants and seed-grown plants, and between their respective first and second progenies. These differences are also of important application value in two-line rice production: Higher pollen fertility of the regenerated plants can be applied with higher efficiency for the propagation of the strain seeds because they have much higher seed setting by self-fertilization; lower pollen fertility of the regenerated plant progenies will result in much lower seed setting by self-fertilization and is, therefore, conducive to the production of hybrid seeds of higher purity by natural crossing with the other line in the two-line system.     

黄金树大、小孢子发生及雌、雄配子体发育的细胞学观察

采用石蜡切片法对黄金树大、小孢子发生及雌、雄配子体发育的过程进行了详细的细胞学观察。结果表明：黄金树子房两室,中轴胎座,胚珠多数,倒生。单珠被,薄珠心,孢原细胞直接发育成大孢子母细胞。单孢原蓼型胚囊。黄金树雄蕊五枚,三枚退化,二枚可育。花药二室,腺质绒毡层,小孢子四分体排列方式为正四面体型,成熟花粉粒为四合花粉。     

Effects of High Temperature on the Cultures of Isolated Microspores in Brassica campestris ssp. pekinensis

The influence of high temperature (33℃) on embryogenesis in isolated microspore culture of Chinese cabbage (Brassica campestris spp. pekinensis ) was investigated by microscpopy of FDA and DAPI. The microspores cultured at constant temperature of 25 ℃ lost their viability quickly and only few viable microspores were found after 7 days of culture. The morphology of the cultured microspores became as turgescent as the "rugby" which was similar to the mature pollen of the Chinese cabbage. The first nuclear division of the microspores was asymmetric. The microspores lost their capacity of embryogenesis under such condition. In contrast, when the microspores were cultured at 33℃ for 24 h before they were transfered to the culture condition of 25 ℃, their developmental pattern was changed. Some of the microspores could remain viable even for 7 days in culture, they became rounded off. The symmetric nuclear division pattern was induced. The frequency of such division was about 40%. Of the several new cell division types observed, the symmetric type was more frequent (55%) than others. The microspores treated under 33℃ were able to form embryoids via embryogenesis. The critical period of high temperature treatment on microspore culture of Chinese cabbage was about the initial 12 h, if the cell division index of microspore was concerned, but the period was the initial 24 h if the frequency of embryogenesis was considered.     

丹参小孢子发生和雄配子体发育过程的解剖学研究

在显微水平上对丹参小孢子发生和雄配子体的发育过程及其与不同发育阶段花蕾的外部形态的相关性进行了研究。结果表明：丹参有2枚雄蕊,每个花药具2个花粉囊．小孢子母细胞减数分裂属同时型,小孢子在四分体中的排列属四面体型。成熟花粉粒属3细胞型并有6个萌发沟。花粉囊壁发育属双子叶型,由4层细胞构成,即表皮、药室内壁、中层和绒毡层。绒毡层细胞为腺质,二核。植株花蕾肉眼可见,大小在1～1.5mm时雄蕊孢原细胞开始分化;花蕾长至9～12mm。即从钟型花萼的钟口肉眼可见乳白色花瓣时．形成成熟的雄配子体,雄配子体具有3细胞的花粉粒。     

In vitro hermaphrodism induction in date palm female flower

This study explores and reports on the gain brought to the morphogenetic aptitude of female date palm inflorescences through in vitro hermaphrodism induction. It investigates the main factors involved in the process of sex modification through hormonal induction, such as the floral developmental stage and hormone combination and concentration. It demonstrates that the vestigial stamens (staminodes) of female date palm flowers display a new and high capacity to proliferate under particular in vitro conditions, without blocking carpel's development, leading to morphologically typical hermaphrodite flowers. This de novo activation of repressed stamens was found to occur rapidly. The isolated pollen mother cells appear in the obtained anther's locules and undergo an ordinary microsporogenesis process. The data show that hermaphrodism induction depended heavily on both the growth regulators applied and the flower's developmental stage. They also confirm the early theory that suggests that dioecious plants come from a hermaphrodite ancestor. Such hermaphrodism control can provide new prospects and opportunities for the investigation of the in vitro self-fertilization process. It can also be useful in improving the understanding of the genetic mechanism involved in sex organ development in date palm.     

温度和水分对不同基因型小麦未成熟胚体细胞胚胎发生以及分化能力的影响

对小麦未成熟胚盾片组织离体再生途径中,未成熟胚发育时期以及不同小麦品种的体细胞胚发生能力和体细胞胚的分化能力进行了研究,在所 试的14个小麦品种中,筛选出具有很强的体细胞胚发生能力和体细胞胚分化能力的4个品种,西农1376、盐2号、85＋1－3和宝丰7228。为进一步给小麦离体遗传操作打下基础,研究还对温度的影响进行了分析。通过低温手段解决了胚性愈伤组织随继代天数的延长体细胞胚分化能力快速降低的问题,同时研究还首次分析了干燥处理对小麦体细胞胚转换能力的影响,建立起一套高效的小麦离体培养再生体系,而且该体系从接种未成熟胚到再生植株移至土壤只需10－12周时间,避免了长期培养过程中存在的体细胞变异问题。     

楸树大小孢子发生与雌雄配子体发育的研究

运用石蜡切片法和整体透明法对楸树(Catalpa bungei C.A.Meyer)大、小孢子发生及雌、雄配子体发育过程进行了研究.结果表明:楸树可育雄蕊2枚,花药4室,药壁发育属双子叶型,腺质异型绒毡层.小孢子母细胞减数分裂为同时型,四分体后小孢子不分离形成正四面体型四合花粉,偶有左右对称型和十字交叉型.成熟花粉为二细胞型,无萌发孔.子房上位,2室,中轴胎座,胚珠多数,倒生,单珠被,薄珠心,具珠被绒毡层.单孢原直接发育为大孢子母细胞,四分体线形排列,合点端大孢子发育为功能大孢子,胚囊发育为蓼型.雄蕊发育早于雌蕊,花开后雌、雄蕊趋于同熟.研究认为:虽然楸树雌、雄蕊发育过程中均存在一定比例的败育,但其花而不实"并非雄性或雌性不育所致.推测与其授粉受精和胚后发育有关.     

Microsporogenesis in a normal line and in the ogu cytoplasmic male-sterile line of Brassica napus

Summary Under an intermediate temperature regime (23° C/18° C; day/night), microsporogenesis in stamens of the ogu cytoplasmic male-sterile (CMS) line of Brassica napus terminated by the tetrad stage, although in some cases degeneration of the sporogenous tissue occurred prior to meiosis. In most cases the tetrads were collapsed and bounded by a sparse exine, but contained many organelles. Also, the tapetum in CMS anthers was abnormal and often highly vacuolated by the tetrad stage. Under low temperature conditions (18° C/15° C; day/night), neither microsporogenous nor tapetal tissues were observed. In the normal stamens, no differences were observed under different temperature regimes. In conjunction with the adjoining paper, this study demonstrates that temperature conditions strongly affect the cytological processes associated with microsporogenesis in the CMS anthers.     

山玉兰花粉离体萌发和花粉管生长特性的研究

山玉兰(Magnolia delavayi)是木兰科木兰属的常绿乔木或大型灌木,是重要的园林造景、庭院绿化素材,也是重要的育种资源。山玉兰花粉的研究对其杂交育种的成败具有重要影响,但目前尚未见其花粉活力的相关报道。该研究以新鲜的山玉兰花粉为对象,采用悬滴培养法分析了温度、光照以及培养液的蔗糖和硼酸浓度对山玉兰花粉萌发的影响。结果表明:(1)山玉兰花粉萌发时,最适宜的温度为27℃。(2)光暗条件下,山玉兰花粉以浓度为5%的蔗糖培养效果最佳,其萌发率在16%以上;而硼酸浓度则以0.001%的培养效果最佳。(3)蔗糖与硼酸共同作用可有效促进花粉萌发和花粉管生长。其中,在光照条件下,以5.0%蔗糖+0.001%硼酸为最适宜的培养液,花粉萌发率达41.27%,花粉管长达281.49μm;而在黑暗条件下,则以5.0%蔗糖+0.01%硼酸为最适宜的培养液,花粉萌发率达45.71%,花粉管长达254.00μm。该研究结果为进一步开展人工辅助授粉、发掘山玉兰的种质资源工作奠定了基础。     

小麦幼胚培养再生单性雌花的研究

通过45个基因型的小麦(Triticum aestivum L.)幼胚培养,发现有11.1%的基因型从靠近再生芽基部的愈伤组织上分化形成花器官.再生花芽呈裸露的、多子房丛生的、具有茂盛羽毛状柱头而缺乏雄蕊、外稃、内稃和颖片的单性雌花. 组织切片观察发现,其雌蕊起源于再生芽附近的分生组织细胞,并通过次生雌蕊再生的方式形成丛生状,其羽毛状结构的发育先于子房中胚珠的分化. 除正常的单胚珠外,还发现双生胚珠分化.χ2独立性检验结果显示,花芽再生率存在强烈的基因型效应.小麦品种YA-1 表现突出(44.4%),其花芽再生潜力能在不同年份间较好地再现,说明YA-1的花芽再生能力具有相对稳定性.与脱分化培养基的效应相比,YA-1的花芽再生效率主要受继代培养基成分的影响. 其中,6-BA、NAA和加倍无机铁盐的配比较2,4-D和正常浓度无机铁盐的配比更有利于YA-1的幼胚培养再生花芽.同时,外植体实验表明,YA-1的幼穗和成熟胚培养无任何成花反应,而其幼胚外植体具有特异的花芽再生能力.据此认为,YA-1的幼胚培养有助于为小麦花发育机理研究建立理想的实验系统.     

A glycine-rich protein that facilitates exine formation during tomato pollen development

Formation of the unique and highly diverse outer cell wall, or exine, of pollen is essential for normal pollen function and survival. However, little is known about the many contributing proteins and processes involved in the formation of this wall. The tomato gene LeGRP92 encodes for a glycine-rich protein produced specifically in the tapetum. LeGRP92 is found as four major forms that accumulate differentially in protein extracts from stamens at different developmental stages. The three largest molecular weight forms accumulated during early microspore development, while the smallest molecular weight form of LeGRP92 was present in protein extracts from stamens from early microsporogenesis through anther dehiscence, and was the only form present in dehisced pollen. Light microscopy immunolocalization experiments detected LeGRP92 at only two stages, late tetrad and early free microspore. However, we observed accumulation of the LeGRP92 at the early tetrad stage of development by removing the callose wall from tetrads, which allowed LeGRP92 detection. Transmission electron microscopy confirmed the LeGRP92 accumulation from microspore mother cells, tetrads through anther dehiscence. It was observed in the callose surrounding the microspore mother cells and tetrads, the exine of microspores and mature pollen, and orbicules. Plants expressing antisense RNA had reduced levels of LeGRP92 mRNA and protein, which correlated to pollen with altered exine formation and reduced pollen viability and germination. These data suggest that the LeGRP92 has a role in facilitating sporopollenin deposition and uniform exine formation and pollen viability.     

UV-B 辐射对番茄花粉生活力的影响与内源激素和多胺的关系

以成熟期不同的两个番茄品种同辉(早熟型)和霞光(晚熟型)为试材, 模拟兰州地区12%和20%臭氧层减薄时增强的UVB辐射(分别为T1=2.54 KJ.m-2.d-1和T2=4.25 KJ.m-2.d-1), 研究了大田条件下增强UV-B辐射对其花粉生活力的影响以及雄蕊中4种内源激素(IAA、GAs、ZR和ABA)和多胺(Put、Spm和Spd)及脯氨酸的变化。结果表明: 辐射抑制了同辉的花粉萌发和花粉管伸长, 但只降低了霞光的花粉萌发率; 两种辐射明显降低了两品种番茄雄蕊中的GAs含量, 同时造成同辉雄蕊中Put和Spd含量明显增加, Spm和Put/Spd+Spm比值显著降低; 霞光中3种多胺含量都显著减少, 导致高辐射时Put/Spd+Spm比值上升; 同辉番茄雄蕊的脯氨酸含量不受影响, 但高辐射使霞光番茄雄蕊的脯氨酸含量降低。实验表明, 两品种番茄花粉生活力的变化与增强UV-B 辐射下雄蕊中GAs水平、Spm含量以及脯氨酸含量的减少有关。雄蕊中多胺和脯氨酸含量变化对UVB辐射的响应说明霞光品种对UV-B辐射更敏感。     

风信子花器官中HAP2基因的分离与表达研究(英文)

在离体条件下,以风信子花被片为外植体,通过控制激素的浓度可诱导花被片、雄蕊或胚珠的再生。近年来,在拟南芥和金鱼草等模式植物中已经分离出了许多控制花器官发育的同源异形基因,如AG,AP1,AP2,AP3等,其中AP2在控制花萼和花瓣形成过程中起重要作用,因此本文从风信子中分离AP2的同源基因,并对它在风信子再生系统中的表达进行了分析。根据AP2同源基因功能域的保守序列设计一对简并引物：5'-TGGGA（A/G）TC（G/T/C）CA（C/T）AT（C/T）TGGA-3'和5'-TCCCA(AGC)(CT)(GT)(AG)CC(AG) CA(CT)TT(AG)TG-3', 以再生的花被片为材料进行RT-PCR,扩增出大小约300bp的片段,序列分析表明该片段的氨基酸序列与AP2同源性高达89%。进而,利用5’和3’Race PCR,得到全长的cDNA。该基因命名为HAP2,GenBank登记号为AF134116,该基因全长1597bp,编码368个氨基酸(Fig.1）。与AP2相比,HAP2也含有10个氨基酸长的碱性功能域,其中KKSR为核定位信号。此外,HAP2也含有两个序列重复的68个氨基酸长的功能域（HAP2-R1,HAP2-R2）,HAP2-R1也含有能形成(-螺旋结构的核心区域,且与AP2-R1中的核心序列100%同源,而HAP2-R2中的核心区域与AP2-R2相比, 缺少9个氨基酸(Fig.2）。RT-PCR结合Southern 杂交结果表明(Fig.3）,HAP     

Links between early pollen development and aperture pattern in monocots

Summary. Although the pollen grains produced in monocots are predominantly monosulcate (or monoporate), other aperture types are also found within this taxonomic group, such as the trichotomosulcate, inaperturate, zonaperturate, di-, or triaperturate types. The aperture pattern is determined during the young-tetrad stage of pollen development and it is known that some features of microsporogenesis can constrain the aperture type. For example, trichotomosulcate pollen is always associated with simultaneous cytokinesis, a condition considered as derived in the monocots. Our observations of the microsporogenesis pathway in a range of monocot species show that this pathway is surprisingly variable. Our results, however preliminary, reveal that variation in microsporogenesis concerns not only cytokinesis but also callose deposition among the microspores and shape of the tetrads. The role played by these features in aperture pattern determination is discussed. Correspondence and reprints: Laboratoire Ecologie, Systématique et Evolution, Université Paris-Sud, 91405 Orsay Cedex, France.     

Pollen development of Cardiocrinum giganteum (Wall.) Makina in China

In this article, we studied the pollen morphology and wall development, microsporogenesis, male gametophyte development, and anther wall structure changes during pollen development of Cardiocrinum giganteum (Wall.) Makina from the genus Cardiocrinum (Endl.) Lindl. (Liliaceae) using paraffin sections, scanning and transmission electron microscopy, and fluorescence microscopy. The results showed that C. giganteum has oval-shaped pollen with a single sulcus and reticulate ornamentation. The exine is of the semi-tectum type and can be divided into the tectum layer, columellate layer and basal layer. Meiosis in the microsporocyte is accompanied by successive cytokinesis. The mature pollen is three-celled. The anther wall prior to maturity is built by one layer of epidermis, 1–2 layers of endothecium cells, 4–5 middle layers and 2 layers of tapetum, while upon maturity it is only built by one layer of epidermis, one layer of endothecium cells and one middle layer. The tapetal cells are secretory, with two or more nuclei. Ubisch bodies originate from rough endoplasmic reticulum except a few from mitochondria.     

三叶木通大小孢子发生和雌雄配子体发育研究

三叶木通（Akebia trifoliata）属于木通科（Lardizabalaceae）木通属（Akebia）,为雌雄异花,雌雄同株的木质藤本植物,具有药食两用的经济价值。本文运用石蜡切片技术观察了三叶木通的大小孢子发生和雌雄配子体发育过程中胚胎学特征,以期了解三叶木通有性生殖过程,分析该物种自然条件下结实率低的生殖原因,为其后续进行杂交育种和新品种培育提供理论基础。结果表明：三叶木通雄蕊6枚,每枚花药具4个小孢子囊,花药壁完全分化时由外到内依次为1层表皮、1层药室内壁、2～3层中层及1层绒毡层,且绒毡层为分泌型绒毡层。小孢子母细胞减数分裂的胞质分裂为同时型,小孢子四分体为四面体型排列,从四分体分离出来的小孢子经过进一步发育形成2-核成熟花粉细胞。小孢子囊中的小孢子四分体时期存在少量败育现象。雌蕊具1室子房,为侧膜胎座,胚珠横生,多枚,双珠被,厚珠心。大孢子母细胞减数分裂后形成的大孢子四分体呈线型排列,靠珠孔端的3个大孢子退化,合点端的1个大孢子发育成为功能大孢子,经3次连续有丝分裂并进一步发育为七细胞八核的蓼型胚囊。雌花中的雄蕊早期发育正常,但小孢子发育至单核靠边期不在进一步发育,...