芥蓝游离小孢子培养及植株再生研究

对12个基因型的芥蓝进行了游离小孢子培养及植株再生研究.结果表明:供体基因型对诱导胚胎发生至关重要;最佳热激温度为32℃,24h;单核靠边期到双核期的小孢子最适合进行胚胎诱导;NLN培养基中的最佳蔗糖浓度为130 g·L-1;添加活性炭可以提高成胚速度和质量;15～25 ℃的供体植株生长的环境温度决定着游离小孢子培养的成败,将子叶型胚胎转移到MS固体培养基上可直接发育成植株.     

Structural observations during androgenic microspore culture of the 4c1 genotype of Zea mays L.

Summary The capacity of the maize genotype 4c1 to regenerate microcalli and embryos from cultured microspores has been examined by comparing various cold pretreatments and culture media, using microspores and pollen at different stages of development. Viability of cultured cells was tested with FDA and their development was traced with light and fluorescence microscopy using DAPI as a nuclear dye.It was found that a pre-incubation of dissected flowers floating in a liquid nutrient medium at 8°C during 10–14 days was most successful for the induction of cell division. Among the developmental stages tested only the microspores appeared to regenerate. Subculture at 25°C in the same liquid medium, supplemented with 0.1 mg/l TIBA, gave highest rates of microspore division, i.e. up to 70% at 4 to 6 days of culture.All pathways described earlier for maize androgenic embryogenesis were observed within the 4c1 genotype. Symmetric divisions occurred in cultured microspores but most frequently asymmetric divisions lead to the formation of microcalli within 12 days of culture. In at least 60% of all dividing microspores cells were derived from the generative nucleus. Microcalli further developed either into loose or compact calli. Compact calli formed embryo-like structures.Abbreviations DAPI 4,6-diamidino-2-phenylindole - Dicamba 3,6-dichloro-2-methoxy benzoic acid - 2,4D 2,4 dichlorophenoxyacetic acid - FDA fluorescein diacetate - PAA phenylacetic acid - TIBA 2,3,5-triiodobenzoic acid - YP medium Yu-Pei basal salt medium     

从油菜(Brassica napus L. cv. Topas)裂外壁小孢子培育出胚状体与小植株

油菜(Brassica napus L. Cv. Topas)小孢子经高温(32℃)预培养导致外壁开裂, 形成裂外壁小孢子. 微室饲养培养技术结合定位追踪观察证实裂外壁小孢子具有胚胎发生能力. 33.3%的裂外壁小孢子能启动细胞分裂, 其中13.3%的裂外壁小孢子能持续分裂, 并遵循胚胎发生途径. 与完整小孢子不同, 第一次分裂既有均等分裂, 亦有不均等分     

茄子游离小孢子培养中小孢子发育的细胞学观察

本文以茄子（Solanum melongena L.）杂交F1代的植株为试验材料，通过游离小孢子的培养，对小孢子脱分化、再分化形成再生植株的发育途径，进行了较为详细的形态细胞学观察。其结果如下：（1）小孢子进行均等分裂和营养细胞分裂两种方式都能形成胚状体或愈伤组织。生殖细胞仅分裂1-2次。（2）多细胞小孢子从花粉沟或萌发孔突出来脱掉花粉壁。（3）花药组织在胚状体形成过程中起着重要作用。（4）从获得的再生植株中，随机取37株进行根尖细胞染色体观察发现，22株为单倍体，14株为二倍体，1株为四倍体。     

Effect of Cultivar, Incubation Temperature, and Stage of Microspore Development on Anther Culture in Wheat and Triticale

Effects of incubation temperature and developmental stage of microspores on polyhaploid production in three wheat cultivars‘Pavon 76′, ‘Kitt’, and ‘Chris’ and one triticale cultivar, ‘T81′, were studied using a one-step medium. Calli failing to differentiate on the one-step medium were placed on a medium containing 1 mg/l indole-3-acctic acid (IAA) and 2 mg/1 6-furfurylaminopunne (KIN). Anthers containing either early- or late-uninucleate microspores were incubated in dark at 26, 28 or 32°C lor 3 days prior to transfer to 26°C. Averaged over temperatures and microspore stages, frequency of calli and green plantlets were 8.9 % and 3.4 %, respectively, for wheat cultivar‘Pavon 76′, 8.4 % and 1.6 % for cultivar ‘Kitt’, 4.5 % and 0.25 % for cultivar ‘Chris’, and 2.9 % and 0.12 % for the triticale cultivar‘T81′. However, cultivar-by-developmental-stage interaction was significant for frequency of callus induction. Temperature had no significant effects on callus induction and plantlet regeneration. Anthers containing early-unmucleate microspores produced no polyhaploids.     

Improved Culture System for Microspores of Barley to Become a Target for DNA Uptake

For the winter barley cultivar ‘Igri’, a microspore isolation and regeneration procedure is described which allows the production of such vigorous micro-spore fractions that this single-cell system can be used as a target for DNA uptake. Up to 60 % of the vigorous microspores isolated from the anther, and cultured in liquid modified MS medium, formed embryoids and/or calli. Such preparations were used for trials in DNA uptake with the plasmid pBI 221. Transformation trials were performed with polyethylene glycol as the inducing agent. With this treatment, a relative increase of fluorescence could be shown under UV light indicative of transient expression of the uidA gene.     

Transformation studies in Hordeum vulgare using a highly regenerable microspore system

Summary A highly regenerable, isolated microspore system for barley, Hordeum vulgare L. cv. Igri, has been developed which is amenable to transformation studies using particle bombardment. The system allows DNA to be delivered to microspores at the single cell stage and both transient and stable transformation events have been demonstrated. The potential advantages of using isolated microspores as the target tissue in routine transformation systems are discussed.     

甘蓝型油菜基因型对离体小孢子染色体加倍的反应

采用秋水仙碱处理甘蓝型油菜不同基因型的离体小孢子,研究了小孢子再生植株的染色体加倍特性 及其与基因型的关系。将发育到单核晚期一双核早期的小孢子分别接种于含10、20、50、100 mg／L秋水仙碱的 NLN液体培养基中处理24 h或48 h进行染色体加倍,然后将处理过的小孢子转接于不含秋水仙碱的相同培 养基中诱导胚状体。21种基因型小孢子再生植株中双单倍体比率为23．94％～99．42％。方差分析的结果表 明,基因型相似的品系和杂种之间染色体加倍率差异不显著,基因型不同的一些品系和杂种间差异显著。由 此说明,基因型对秋水仙碱处理离体小孢子诱导染色体加倍的反应不同。     

INFLUENCE OF VARIOUS PHYSICAL PARAMETERS ON ANTHER CULTURE OF BARLEY

This study describes the effect of some parameters on anther culture of barley to optimize the plant regeneration condition of this species. The embryo formation and plant regeneration from anthers of three barley cultivars (‘Igri’, ‘Saida’ and ‘Libya’) were investigated. The effects of length of flag leaves, stage of microspore development, and pre-treatments (mannitol or cold pre-treatment) of anthers were investigated. Results showed significant responses. Anthers at the mid uninucleate to mid-late uninucleate stage gave the best anther culture response showing 80, 60, and 30% anther development in ‘Igri’, ‘Saida’ and ‘Libya’ barley cultivars, respectively. The use of mannitol (0.3 M) or 20 days cold pre-treatments showed the best results for embryo and green-plant production.     

影响甘蓝小孢子DH植株生长的几个因素

旨在筛选甘蓝小孢子DH植株健壮生长的最适培养基和光照强度。为缩短小孢子DH植株培养时间并为获得健壮植株创造条件,从而加快甘蓝DH系育种进程。以甘蓝‘秦甘50’为材料进行游离小孢子培养至诱导分化出不定芽,转接到NAA质量浓度不同的2种培养基上(1/2MS+0、0.1、0.2、0.3mg/L NAA和MS+0.1mg/L NAA),不同光照强度[40、60、80、100μmol/(m2·s)]及添加不同质量浓度NH4NO3(0、0.4、0.8、1.2g/L)培养,研究DH植株的生长势,获得甘蓝小孢子DH植株最适培养基和培养条件。结果表明,在NAA质量浓度不同的培养基中,1/2MS+0.1mg/L NAA培养基中小孢子DH植株生长速度最显著,生根快、植株长势强;在80μmol/(m2·s)光照强度下培养小孢子DH植株净生长高度显著,10.3d再生出根系,根质量较好,叶片大而厚;MS培养基中添加质量浓度为0.4g/L的NH4NO3,小孢子DH植株生长健壮,35d内植株净生长高度最大为56.25mm。以上结果说明,甘蓝小孢子DH植株快速健壮生长的适宜培养基为1/2MS+0.1mg/L NAA+8g/L琼脂+30g/L蔗糖;在80μmol/(m2·s)光照强度下或MS培养基中添加质量浓度为0.4g/L的NH4NO3均能够促进甘蓝小孢子DH植株根茎叶生长旺盛。