大白菜生物技术研究进展

综述了国内外在大白菜(Brassica rapa ssp. pekinensis)单倍体培养、基因工程和分子标记方面的最新研究成果。重点讨论了大白菜小孢子培养的影响因素、取材要点、培养条件、植株再生和倍性鉴定以及大白菜的转基因载体选择、受体系统建立、遗传转化与转基因植株鉴定等。对小孢子培养及转基因研究的原理、方法和关键技术进行了总结。展望了大白菜生物技术研究的应用前景。     

大白菜生物技术研究进展

综述了国内外在大白菜(Brassica rapa ssp.pekinensis)单倍体培养、基因工程和分子标记方面的最新研究成果.重点讨论了大白菜小孢子培养的影响因素、取材要点、培养条件、植株再生和倍性鉴定以及大白菜的转基因载体选择、受体系统建立、遗传转化与转基因植株鉴定等.对小孢子培养及转基因研究的原理、方法和关键技术进行了总结.展望了大白菜生物技术研究的应用前景.     

麦类作物小孢子培养与遗传转化研究进展

小孢子作为单个单倍体细胞,通过诱导培养能再生成纯合的二倍体植株,不仅为分子生物学、遗传学、形态发生学研究提供稳定的材料,也对小麦等作物的转基因研究提供新方向和新思路。本文主要介绍麦类作物小孢子培养的基本步骤及关键因素,简要介绍了以小孢子为材料进行小麦基因遗传转化的方法。     

菊科植物单倍体研究进展

单倍体培养是快速获得菊科纯合系的重要途径。目前已进行单倍体研究的菊科植物共有13个种,其中9个已成功获得单倍体植株。菊科中诱导单倍体的途径有花药培养、小孢子培养、离体雌核培养、远源杂交和辐射花粉诱导单倍体。本文详细论述了不同外植体发育时期、预处理、培养基、培养条件等因素对单倍体植株诱导再生的影响。对菊科植物单倍体诱导的几种途径进行对比总结,指出研究中存在的问题并提出思路和建议。     

水稻OsMS2基因在花药发育中的功能分析

拟南芥MS2(MALE STERILITY 2)是一个调控花药花粉发育的关键基因。水稻OsMS2(Os03g07140)基因与拟南芥MS2的序列具有高度同源性。利用RNA干扰技术研究OsMS2基因在水稻花药发育过程中的功能。与野生型水稻相比, 转基因 植株营养生长阶段正常, 但雄性育性降低。转基因植株雄性育性降低与RNA干扰引起的OsMS2基因表达水平降低有关。进一步对转基因植株花药进行细胞学观察, 结果表明OsMS2基因表达水平的降低导致绒毡层细胞退化延迟, 小孢子壁的形成出现异常。扫描电镜观察结果显示, 小孢子壁光滑, 不能形成正常的外壁。以上结果表明OsMS2基因在水稻花药发育过程中起重要作用。     

水稻DsMS2基因在花药发育中的功能分析

拟南芥MS2（MALE STERILITY2）是一个调控花药花粉发育的关键基因。水稻OsMS2（Os03g07140）基因与拟南芥MS2的序列具有高度同源性。利用RNA干扰技术研究OsMS2基因在水稻花药发育过程中的功能。与野生型水稻相比,转基因植株营养生长阶段正常,但雄性育性降低。转基因植株雄性育性降低与RNA干扰引起的OsMS2基因表达水平降低有关。进一步对转基因植株花药进行细胞学观察,结果表明OsMS2基因表达水平的降低导致绒毡层细胞退化延迟,小孢子壁的形成出现异常。扫描电镜观察结果显示,小孢子壁光滑,不能形成正常的外壁。以上结果表明OsMS2基因在水稻花药发育过程中起重要作用。     

水稻OsMS2基因在花药发育中的功能分析

拟南芥MS2（MALE STERILITY2）是一个调控花药花粉发育的关键基因。水稻OsMS2（Os03g07140）基因与拟南芥MS2的序列具有高度同源性。利用RNA干扰技术研究OsMS2基因在水稻花药发育过程中的功能。与野生型水稻相比,转基因植株营养生长阶段正常,但雄性育性降低。转基因植株雄性育性降低与RNA干扰引起的OsMS2基因表达水平降低有关。进一步对转基因植株花药进行细胞学观察,结果表明OsMS2基因表达水平的降低导致绒毡层细胞退化延迟,小孢子壁的形成出现异常。扫描电镜观察结果显示,小孢子壁光滑,不能形成正常的外壁。以上结果表明OsMS2基因在水稻花药发育过程中起重要作用。     

植物花粉培养研究进展

花粉培养又称为游离小孢子培养,指将发育到一定阶段的花粉从花药中游离出来成为分散或游离状态,通过培养使花粉粒脱分化,进而发育成完整植株的过程。花粉培养的主要目的是获得单倍体植株,进而得到双单倍体(double haploid,DH)植株,最终获得纯合系物种。本文对花粉培养形成植株的物种信息进行了收集整理,概述了国内外花粉培养的一些最新研究进展,包括影响花粉培养形成胚的因素以及提高花粉胚产量的措施,并对花粉培养的前景进行了展望。     

植物花粉培养研究进展

花粉培养又称为游离小孢子培养,指将发育到一定阶段的花粉从花药中游离出来成为分散或游离状态,通过培养使花粉粒脱分化,进而发育成完整植株的过程。花粉培养的主要目的是获得单倍体植株,进而得到双单倍体(double haploid,DH)植株,最终获得纯合系物种。本文对花粉培养形成植株的物种信息进行了收集整理,概述了国内外花粉培养的一些最新研究进展,包括影响花粉培养形成胚的因素以及提高花粉胚产量的措施,并对花粉培养的前景进行了展望。     

甜(辣)椒单倍体培养研究进展

介绍了甜(辣)椒花药培养和游离小孢子培养的研究概况,花药培养应用相对成熟,游离小孢子培养尚未获得突破性进展。对影响花药培养的各关键因素(包括材料基因型、供体植株生长状态、小孢子发育时期、培养基、培养方法、变温处理、培养条件等)进行了综述,并讨论了甜(辣)椒单倍体培养存在的问题和进一步研究方向。     

Twinned microspore-derived embryos of canola (<Emphasis Type="Italic">Brassica napus</Emphasis> L.) are genetically identical

Microspore culture is used extensively in several crop species to generate diverse populations of homozygous, doubled haploid lines for breeding and genetic analyses. In our canola (Brassica napus L.) doubled haploid breeding programme we regularly observe conjoined microspore-derived embryos, most commonly twins, joined either at the base of the hypocotyl or along the length of the hypocotyl axis. The aim of this study was to determine if twinned embryos were genetically identical or non-identical in order to gauge their value for breeding and linkage analysis. Microsatellite marker fingerprinting of 12 pairs of twinned embryos produced by microspore culture of heterozygous F₁ lines revealed that pairs of twins were genetically identical. Based on this finding, we recommend breeders and geneticists using microspore culture technology to retain only one embryo from each pair of twinned embryos.     

Inducing homozygosity in transgenic barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) by microspore culture

Homozygosity was induced in transgenic barley by microspore culture. Spikes of transgenic barley plants carrying microspores in the late uni-nucleate stage were cold pretreated. Teflon rod maceration and a density of 100 000 viable micropores per plate were used. The developed calli were regenerated and plantlets were treated with colchicine. The microspore culture of 16 mother plants (three transgenic lines) resulted in 927 green regenerants. Of these plants, 476 were transferred to soil, 380 were transgenic, 358 reached maturity and 350 were fertile with a normal seed-set carrying a yield of 6.9 kg. A production efficiency of 0.8 fertile transgenic doubled haploid barley plants per spike used for microspore isolation was recorded. The produced transgenic seeds were used in malting experiments.     

Indica rice anther culture: can the impasse be surpassed?

During the past two decades numerous papers have been published on anther culture of rice. These studies clearly indicate that while anther culture is a technique that can be adopted for breeding japonica rice, it being a useful adjunct in indica rice breeding is still some way away. The main reasons why anther culture cannot be utilized for indica rice breeding is analyzed, and aspects that may be manipulated to achieve progress are presented in this review. The two stages of rice anther culture, callus induction and green plant regeneration, are genetically determined traits that show quantitative inheritance. Indica rice is known to have a recalcitrant genetic background that supports these traits poorly. While improvement of the genetic background through recombination or gene transfer remains possible, manipulation of culture media, particularly the nitrogen and carbon sources, has brought about substantial improvements in indica rice anther culture. Adjustments to pre- and post-culture conditions, that include application of various stresses on anthers before and after culture, also have had beneficial effects. The importance of reducing the tissue culture phase to achieve direct embryogenesis is discussed with special reference to improving green plant regeneration potential. The necessity to understand the processes involved in microspore embryogenesis is highlighted in order to support empirical knowledge and achieve a breakthrough in technology. In this regard, rice genome sequence information may be leveraged to elucidate functions of genes involved in microspore embryogenesis.     

Combination of reversible male sterility and doubled haploid production by targeted inactivation of cytoplasmic glutamine synthetase in developing anthers and pollen

Reversible male sterility and doubled haploid plant production are two valuable technologies in F₁-hybrid breeding. F₁-hybrids combine uniformity with high yield and improved agronomic traits, and provide self-acting intellectual property protection. We have developed an F₁-hybrid seed technology based on the metabolic engineering of glutamine in developing tobacco anthers and pollen. Cytosolic glutamine synthetase (GS1) was inactivated in tobacco by introducing mutated tobacco GS genes fused to the tapetum-specific TA29 and microspore-specific NTM19 promoters. Pollen in primary transformants aborted close to the first pollen mitosis, resulting in male sterility. A non-segregating population of homozygous doubled haploid male-sterile plants was generated through microspore embryogenesis. Fertility restoration was achieved by spraying plants with glutamine, or by pollination with pollen matured in vitro in glutamine-containing medium. The combination of reversible male sterility with doubled haploid production results in an innovative environmentally friendly breeding technology. Tapetum-mediated sporophytic male sterility is of use in foliage crops, whereas microspore-specific gametophytic male sterility can be applied to any field crop. Both types of sterility preclude the release of transgenic pollen into the environment.     

转修饰cry1Ac基因籼稻明恢81经花药培养获得抗虫DH系

对基因枪法获得的明恢81转修饰的cry1Ac基因当代植株进行花药培养,共接种花药2600枚,获得83份花培植株,其中双倍体植株43份,单倍体植株40份,PCR结果表明含有cry1Ac基因的植株55份,花培植株群体中转基因与非转基因植株的比值为2：1（55／28）,进一步结合Southern blot和ELISA分析,于花培植株当代筛选到转基因纯合株系36份,外源蛋白表达量上,花药来源于同一克隆的DH系的不同植株之间基本一致,最高的Cry1Ac含量达0．25％,田间抗虫性试验表明,经花药培养纯合获得的部分转基因纯合系植株对二化螟（chilo suppressalis)表现出高抗,而且主要农艺性状保持不变,以上结果表明水稻花药培养可以加速转基因的纯合与良种利用。     

Isolated wheat microspore culture

The use of doubled haploid plants in a wheat breeding program requires an efficient haploid production system. While the techniques for producing doubled haploids from anther culture are well established, those for isolated microspores are complicated and inefficient. Four methods of isolating microspores from anthers (blending, stirring, macerating, and floating) were compared. Isolated microspores were washed and cultured in liquid medium. The effects of pre-isolation mannitol conditioning, cell density, culture dilution, and sucrose centrifugation on microspore viability were evaluated. Isolation by blending gave the highest initial microspore viability (75%). Mannitol conditioning and purification by sucrose centrifugation had a detrimental effect on initial viability. An initial microspore density of 2 × 10⁵ microspores per ml was necessary for continued microspore viability. One hundred and nine haploid or spontancously doubled haploid plants were regenerated from microspores isolated without mannitol conditioning using the blending method. Based on this research, blender isolation with an initial density of 2 × 10⁵ microspores per ml is recommended for isolated microspore culture.Abbreviations LSmean least square mean - MES 2-N-morpholinoethane sulfonic acid - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphtaleneacetic acid