油菜小孢子培养技术体系研究

小孢子培养在油菜的基础研究和应用研究中均具有十分重要的意义。自1982年Lichter首次在甘蓝型油菜中进行小孢子培养获得成功以来,国内外在油菜小孢子培养技术方面已取得大量研究成果,包括油菜小孢子胚状体发生的影响因素,小孢子植株的再生、成苗、大田移栽、染色体加倍等,近年来又对一些关键技术环节加以了改进,笔者在对这些研究成果进行总结的基础上针对中国国情建立了大田条件下油菜高效小孢子培养技术体系。用该体系对甘蓝型油菜和新疆野生油菜的体细胞杂种后代进行小孢子培养的出胚率达到300枚/皿以上,采用小孢子苗直接移栽大田技术,成活率达到89.0%。此外还成功构建了含127个DH系的黄籽油菜DH群体及含115个DH系的粒重分离群体。     

甘蓝型油菜小孢子培养技术及其单倍体应用研究进展

油菜小孢子培养已经成为油菜育种和生物技术的一项重要内容.由于影响小孢子成胚和成苗的因素较多,低诱导频率及成苗率严重限制了该技术在育种中的应用.为了全面了解小孢子胚状体发生能力及影响再生体系的各项因素,掌握该技术的研究动态和发展方向,总结了近20年来国内外影响小孢子培养的主要因素:供体植株基因型、供体植株生长条件及生理状态、小孢子发育时期、培养基组成、培养条件,及其在双单倍体育种、诱变育种、转基因育种等领域中的应用.     

甘蓝型油菜小孢子培养中基因型及处理条件对成苗的影响

为进一步提高甘蓝型油菜小孢子培养效率,研究了基因型对甘蓝型油菜小孢子胚产量和再生植株自然加倍率的影响,以及小孢子振荡培养过程中光照与后期低温处理对胚状体一次成苗率和再生植株自然加倍率的影响。结果表明,基因型对小孢子培养胚产量和再生植株自然加倍率影响较大,D 393的胚产量和自然加倍率很高,分别高达119.87胚·蕾~(-1)和79.87%,D 393的自然加倍率是D 524的2～3倍;光照对一次成苗率和再生植株自然加倍率没有影响,4℃低温处理15 d能提高小孢子一次成苗率0.8～3倍。本研究获得了一个胚产量和自然加倍率都很高的甘蓝型油菜DH系D 393;综合考虑一次成苗率和加倍效率,在甘蓝型油菜小孢子培养进程中振荡培养阶段可能不需要光照,而在转入B 5固体培养基前应该对胚状体进行适当低温和时长的处理,可提高小孢子培养效率。     

活性炭对甘蓝型油菜(Brassica napus L.)小孢子胚胎发生的影响

试验以大田环境下的多份甘蓝型油菜的品种或品系为材料,研究了活性炭对游离小孢子胚胎发生的影响.结果表明:活性炭对甘蓝型油菜游离小孢子培养胚状体发生有很好的促进作用,不仅提高了胚产量,而且有利于小孢子胚的正常发育,添加活性炭的固液双层培养基效果尤为显著.     

甘蓝型油菜小孢子培养影响因素的研究

本实验对四川成都生态区甘蓝型油菜进行游离小孢子培养,对小孢子培养的影响因素作了系统研究,结果表明：小孢子形成阶段的温度及昼夜温差、花蕾消毒液、小孢子培养浓度及株龄都是影响小孢子胚产量的因素,小孢子形成阶段的温度以10-15℃,昼夜温差5℃左右胚产量最高达到300枚/蕾,温度低于5℃或高于20℃胚产量都较低甚至不出胚;花蕾消毒液以0.1%的Hgcl2效果较好,培养浓度以3-4蕾/皿较好;2009年成都生态环境下,株龄从125天到150天,胚产量随着株龄增加而增加,到141天达到最高,后逐渐下降。     

活性炭对甘蓝型油菜（Brossica napus L．）小孢子胚胎发生的影响

试验以大田环境下的多份甘蓝型油菜的品种或品系为材料，研究了活性炭对游离小孢子胚胎发生的影响。结果表明：活性炭对甘蓝型油菜游离小孢子培养胚状体发生有很好的促进作用，不仅提高了胚产量，而且有利于小孢子胚的正常发育，添加活性炭的固液双层培养基效果尤为显著。     

单倍体育种技术在油菜育种材料创新上的应用研究Ⅰ甘蓝型油菜游离小孢子胚状体的诱导发生

利用游离小孢子培养技术,对9个甘蓝型杂交油菜(品种)组合的F1代进行离体小孢子培养.2月底,当供体植株的第一朵花开时,取长度在2.5～3.5mm的花蕾通过离心分离出小孢子,并在NLN13液体培养基中浅层培养诱导胚状体产生,培养30天左右,可见到多种类型的胚状体产生.其中,黔杂991、黔杂271和黔杂18分别诱导出了30个、12个和2个胚状体,其余组合未能产生胚状体.表明:不同基因型材料,不同培养密度和不同生理状态,其小孢子胚状体产生的频率差异较大.     

单倍体育种技术在油菜育种材料创新上的应用研究 I．甘蓝型油菜游离小孢子胚状体的诱导发生

利用游离小孢子培养技术，对9个甘蓝型杂交油菜（品种）组合的F1代进行离体小孢子培养。2月底，当供体植株的第一朵花开时，取长度在2．5－3．5mm的花蕾通过离心分离出小孢子，并在NLN13液体培养基中浅层培养诱导胚状体产生，培养30天左右，可见到多种类型的胚状体产生。其中，黔杂991、黔杂271和黔杂18分别诱导出了30个、12个和2个胚状体，其余组合未能产生胚状体。表明：不同基因型材料，不同培养密度和不同生理状态，其小孢子胚状体产生的频率差异较大。     

单倍体育种技术在油菜育种材料创新上的应用研究Ⅱ.胚状体培育及试管苗越夏

利用游离小孢子培养技术对9个甘蓝型杂交油菜品种(组合)的F1代进行离体小孢子培养.在NLN-13培养基中诱导胚状体发生,将胚状体放置于含有多菌灵和链霉素的培养基中培养,结果表明:①20mg/L浓度的多菌灵和链霉素可以提高胚状体的成苗率.②将胚状体接种于含有0.05mg/L 2,4-D和0.01mg/L6-BA的MS培养基中培养,愈伤组织能快速分化形成不定根和丛芽.     

花椰菜游离小孢子培养及植株再生研究

以186个花椰菜杂交品种及组合为试材,进行了游离小孢子培养研究。结果表明:基因型是胚状体发生的决定因素;24 h 32℃的高温前处理是胚状体发生的必须条件;单核靠边期到双核期的小孢子是进行胚状体诱导的最佳时期,胚状体发生频率最高;培养基中添加6-BA 0.1 mg/L,NAA 0.005 mg/L可以促进胚状体发育。将20 d的胚状体进行脱分化处理,可以大幅度提高胚状体的成活率,植株再生率为53%,再生植株中双单倍体占83.5%。     

甘蓝型黄籽油菜与黑籽油菜苗期生理特性的比较研究

油菜苗期的植株长势与抗性及后期产量密切相关。为探索甘蓝型黄籽油菜抗逆性较弱和产量较低的原因,以2对不同遗传来源的甘蓝型黄、黑籽油菜近等基因系为材料,研究了苗期植株的主要生理特性。结果表明,甘蓝型黄籽油菜苗期生长势弱,叶绿素含量和类胡萝卜素含量,光合速率和叶面积指数（LAI）均比相同遗传背景下的黑籽油菜低;与黑籽油菜相比,甘蓝型黄籽油菜无论是在不同的叶龄期,还是在植株的不同部位,均表现出“糖高氮低”的代谢特点,且越冬期叶片的硝态氮含量和硝酸还原酶活性也低;根颈粗和单株干物质重也明显小。甘蓝型黄籽油菜苗期生长势弱,冬前干物质积累偏少,进而影响其春后的生殖生长,这可能是其产量较低的重要原因之一。     

Functional genomics of microspore embryogenesis

Isolated plant microspores, when stressed and cultured in vitro, can be diverted from their normal gametophytic pathway towards sporophytic development, with the formation of haploid embryos and ultimately doubled-haploid plants. This process is called androgenesis or microspore embryogenesis, and is widely used in plant breeding programmes to generate homozygous lines for breeding purposes. Protocols for the induction of microspore embryogenesis and the subsequent regeneration of doubled haploid (DH) plants have been successfully developed for more than 200 species. These practical advances stand in stark contrast to our knowledge of the underlying molecular genetic mechanism controlling this process. The majority of information regarding the genetic and molecular control of the developmental switch from gametophytic to sporophytic development has been garnered from four intensely studied (crop) plants comprising two dicotyledonous species, rapeseed (Brassica napus) and tobacco (Nicotiana tabacum), and two monocotyledonous species, wheat (Triticum aestivum) and barley (Hordeum vulgare). In these species the efficiency of microspore embryogenesis is very high and reproducible, making them suitable models for molecular studies. In the past, molecular studies on microspore embryogenesis have focussed mainly on the identification of genes that are differentially expressed during this developmental transition and/or early in embryo development, and have identified a number of genes whose expression marks or predicts the developmental fate of stressed microspores. More recently, functional genomics approaches have been used to obtain a broad overview of the molecular processes that take place during the establishment of microspore embryogenesis. In this review we summarise accumulated molecular data obtained in rapeseed, tobacco, wheat and barley on embryogenic induction of microspores and define common aspects involved in the androgenic switch.     

番茄游离小孢子培养研究进展

雄核发育是小孢子或未成熟花粉细胞沿孢子体发育途径形成植株的过程，也是创制育种材料的有效途径之一。为建立重复性好、再生频率高的番茄小孢子诱导方法，文章通过回顾国内外研究进展，总结了番茄雄核番茄雄核发育各阶段（胚胎发生能力的获得、诱导小孢子分化形成胚状体以及胚状体再生形成单倍体植株）的主要影响因素，并简要归纳了现有的游离小孢子分离培养技术。分析表明，基因型是制约番茄小孢子诱导体系的关键因素，且需与发育时期、培养条件等多个影响因素同期发生，才能使雄核发育偏离配子体途径。最后，展望了番茄小孢子培养未来的研究方向与发展趋势，以期为番茄双单倍体技术的深入研究提供参考。     

萝卜花药培养技术研究

通过单倍体育种途径可快速获得纯合材料,增加有益性状的选择几率,加快育种进程。花药培养是获得单倍体的有效途径之一,已在十字花科芸薹属作物上取得巨大成功。但关于萝卜花药培养的报道极少。以15个不同类型的萝卜品种为试材,研究了花药离体培养中胚状体的诱导及再生。结果表明:基因型是限制花药培养成胚的关键因素,15个品种中,有4个品种获得了胚状体及再生苗。同时植株生长条件也对胚状体的形成产生影响。采用流式细胞仪(FCM)鉴定了再生株的倍性,再生株群体的倍性组成比较复杂,以二倍体为主。     

Efficient doubled haploid production in microspore culture of loose-curd cauliflower (Brassica oleracea var. botrytis)

We present an improved protocol for highly efficient production of doubled haploid loose-curd cauliflower plants (Brassica oleracea var. botrytis) via microspore culture. Our experiment explored factors such as donor plant treatment, flower bud pretreatment, embryo germination medium, and ploidy characterization of regenerated plants. Our technique efficiently produced embryos from both tight- and loose-curd donor plants, although the embryo yields were genotype dependent. We achieved a germination rate of around 30 % by employing a hormone combination of zeatin, indole-3-acetic acid, and 6-benzylaminopurine pretreatment culture. We also used 1–4 days of cold pretreatment of the flower buds, which were submerged into NLN-13 medium, to induce microspore embryogenesis. Analysis using an FCM Ploidy Analyzer showed that more than 50 % of regenerated plants were spontaneously doubled haploids, more than 25 % were tetraploids, and fewer than 7 % were haploid. Visual examination of plants in the field revealed that they had distinct phenotypic characteristics relating to their ploidy level. The efficient production of double haploids using our improved microspore culture technique is a promising approach that can be applied in loose-curd cauliflower breeding programmes and genetic research.     

Effect of donor plant age and inflorescence age on microspore culture of Brassica napus L.

Summary Effect of age of donor plants and age of inflorescence on embryogenesis in microspore culture of B. napus was examined. Microspores isolated from buds of older plants had a higher embryo yield than those of younger ones. The effect of the age of inflorescence showed a different pattern. In older plants, a higher embryogenesis response was observed in microspores isolated from buds of new inflorescences, while in young plants, microspores isolated from buds of old inflorescences showed high embryo yield. These different responses were considered to be attributable to a difference in the developmental stage of pollen at the time of microspore isolation. Our results indicated that microspores collected from older inflorescences and older plants have sufficient embryogenic potential when the optimum developmental stage of pollen was used. Frequency of embryo to plant conversion was influenced by the size of embryos subcultured, but not by donor plant age or the age of the inflorescence.     

Efficient production of doubled haploid Brassica napus plants by colchicine treatment of microspores

Summary The effect of colchicine on isolated microspore cultures of Brassica napus was evaluated in order to combine a positive effect of colchicine on the induction of embryogenesis with the possibility to induce chromosome doubling at an early developmental stage, thus avoiding the production of haploid or chimeric plants. Colchicine was added to the culture medium immediately after isolation of B. napus microspores. The cultures were incubated from 6 to 72 h with various concentrations of colchicine. Samples were taken from the regenerating embryoids after 6 weeks for ploidy determination by flow-cytometry.The highest diploidization rate was obtained after a 24 h treatment of microspores with 50 mg/l colchicine, leading to 80–90% diploid embroids. A concentration of 100 mg/l colchicine applied for the same duration resulted in a lower diploidization rate (76–80%). Treatment durations of 6 h were not long enough to induce a high rate of diploidization, whereas the application of 10 mg/l for 72 h was also very effective.A sample of the plants regenerated from the colchicine treated microspores was transferred to the greenhouse. The plants looked similar to normal diploid rapeseed plants and showed reasonable pod and seed set. Thus, an additional generation for seed increase in the greenhouse is rendered unnecessary. The advantage of applying a minimum volume of colchicine under controlled in vitro conditions means a considerable saving of time and labour in DH-breeding programs.     

Effect of activated charcoal on Brassica oleracea microspore culture embryogenesis

The effect of the addition of a 0.1 ml drop of activated charcoal (AC) on microspore culture embryogenesis was studied in nine morphotypes of Brassica oleracea. Embryo yields were significantly increased in all of the morphotypes by the addition of the 0.1 ml drop of AC to the microspore culture media. The magnitude of the response to the addition of AC varied with the different plants and morphotypes. The addition of AC never produced a detrimental effect. A qualitative improvement of the subsequent development of embryos to plants was also observed with the addition of AC. Data suggest that the addition of AC to microspore culture media promoted embryo production in different B. oleracea morphotypes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Efficient Yield of Embryoids by Culture of Isolated Microspores of Different Brassicaceae Species

Microspore culture of rapeseed (Brassica napus) is well established in practical plant breeding as a potent method to produce homozygous plants. Except for B. carinata the method has not yet been applied to other Brassicaceae species. The present study describes successful microspore culture of B. campestris, B. nigra, B. oleracea and Raphanus sativus, overcoming the break down of such microspore cultures possibly arising from the release and accumulation of toxic substances into the medium. The modified procedure is as follows: exchange of the incubation medium after one day of culture, addition of activated charcoal to the medium and aerating the three day old microspore culture by agitating the petri dishes on a specially constructed shaker.