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。     

提高油菜游离小孢子胚诱导频率的研究

以11个甘蓝型油菜基因型为材料,采用NLN培养基进行游离小孢子培养,对如何提高可培养的基因型范围和产胚率进行了研究。结果表明,11个油菜基因型中有10个基因型可以诱导出胚,培养成功率达90.9%,表明采用NLN培养基进行游离小孢子培养油菜基因型范围比较宽,但各基因型间小孢子胚产量差别很大,每花蕾产胚量为0.08~3.53个,TR4和TR9两个基因型每花蕾产胚可达3.23,3.53个。以TR4和TR9两个基因型为试材,进一步改进培养基和培养方法,采用NLN培养基中添加激素和活性炭方法,可大大提高产胚率,产胚量分别达到7.11和10.05个/蕾;接种后,小孢子经33℃高温预处理可显著影响产胚量。子叶形小孢子胚在光下适当培养后转入B5 BA 0.2 mg/L NAA 0.02 mg/L继代培养基上,大多数胚能长成绿芽,B5 6-BA 0.2 mg/L 3%蔗糖 1%琼脂培养基有利于小孢子胚长成植株。     

几种抗生素对离体培养中植物形态发生的影响

抗生素羧苄青霉素(Carbenicillin),头孢霉素Ⅱ(Keflodin)和Claforan影响甘蓝型油菜(Brassica napus L.)游离小孢子培养胚胎发生,其浓度越高,影响越大.羧苄青霉素促进辣椒(Capsicum annuum L.)子叶外植体愈伤组织发生,同时,还影响油菜小孢子胚再生植株及辣椒子叶再生株的生根,产生粗短根甚或发育为瘤状物.     

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。     

甘蓝型油菜矮秆突变体DS-1和DS-2

甘蓝型油菜矮秆突变体ＤＳ－１和ＤＳ－２甘蓝型油菜矮秆突变体ＤＳ－１和ＤＳ－２产生于离体诱变的甘蓝型油菜小孢子胚状体的胚性培养物。１９９３年３月，采用ＮＬＮ液体培养基（Ｌｉｃｈｔｅｒ，１９８２）对生长于大田的甘蓝型油菜品系９２－Ｂ１０植株的单核期小孢子...     

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。     

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.     

利用小孢子培养选育榨菜胞质甘蓝型油菜CMS恢复系研究

以榨菜胞质甘蓝型油菜恢复系材料“06 B033”为母本,优质油菜新品种(系)为父本配制7个杂交组合,并对其F1代进行小孢子培养.结果发现,不同父本的杂交组合在小孢子培养成胚率上表现极显著差异;对获得的DH群体进行对榨菜胞质甘蓝型油菜CMS的恢保性鉴定.结果表明,有5个DH株系对榨菜胞质甘蓝型油菜CMS表现良好的恢复性.     

甘蓝型油菜小孢了培养胚发生能力的遗传分析

用4×4完全双列杂交研究了甘蓝型油菜小孢子培养胚状体发生能力的遗传规律。结果表明多数F1的胚产量与具高胚发生能力的亲本相近，部分F     

活性炭对小白菜游离小孢子培养的影响

以24个小白菜品种为材料,通过在NLN培养基中添加不同浓度活性炭,研究其对游离小孢子培养的影响.结果表明:在NLN-13培养基中添加活性炭(0.5 g/L),有利于小孢子胚的诱导和形成,供试材料(N4,N8)添加活性炭处理胚诱导率较未添加活性炭的处理分别提高19,66.5倍.采用添加活性炭(0.5 g/L)的NLN培养基对24份小白菜品种进行游离小孢子培养,有20份材料诱导出胚,培养成功率为83.3%.表明添加活性炭的培养基对小白菜游离小孢子培养有较好的效果.不同基因型间胚诱导率差别较大,每花蕾诱导胚数为0.4～86.7个.依据胚诱导率的高低可将其分为极易诱导、易诱导、难诱导和不能诱导出胚4类.B5 6-BA 0.2 mg/L NAA 0.02 mg/L 活性炭0.5 g/L 3%蔗糖 1%琼脂培养基有利于幼胚长成植株.     

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.     

Optimization of culture conditions for improved plant regeneration efficiency from wheat microspore culture

The production of haploid plants through microspore culture is a very important tool for plant breeding. However, progress in microspore culture for many species has been hampered by a number of factors that have resulted in low recovery of regenerated green plants. In this study, a series of experiments were conducted to increase the regeneration of haploid green plants from isolated wheat microspores. The use of different basal media and variations in media components resulted in the increased recovery (approximately double) of regenerated haploid wheat plants. Our findings demonstrate that CHB medium, in combination with 2,4-d, was a better medium for embryoids induction and plant regeneration than medium MC17 with either 2,4-d or PAA growth hormones. Wheat microspores cultured without ovary co-cultivation did not respond. Furthermore, high efficiency of microspore derived embryoids (up to 296 MDEs per 100 anthers) and green plant regeneration (up to 71 green plants per 100 anthers) were achieved by the use of gelrite instead of agarose as a gelling agent, and by the addition of media additives such as spent medium or MET.     

Selection in vitro for erucic-acid content in segregating populations of microspore-derived embryoids of Brassica napus

Microspore culture of Brassica napus under optimized conditions leads to the regeneration of microspore-derived embryoids that, at the late cotyledonary stage, contain large amounts of storage lipids, equal or similar in composition to those found in seeds of the homozygous donor plants. At that stage, the microspore-derived embryoids are large enough to allow the dissection of one cotyledon under aseptic conditions and the determination of its fatty-acid composition. The remaining part of the embryoid can be cultured further and regenerated to give a plant. This offers the possibility of early selection for fatty-acid composition in segregating populations of microspore-derived embryoids. In order to verify this hypothesis, embryoids were generated from microspores of F| plants derived from a cross between doubled haploid lines of the low-erucicacid cv. ‘Duplo’ and the high-erucic-acid cv. ‘Janetzki’. The contents of eicosenoic acid (C20: 1) and erucic acid (C22: 1) in the cotyledons and in the seeds derived from plants regenerated from the remaining parts of the embryoids were highly correlated (rs = 0.85**, P = 0.01). This indicates that, in breeding programmes for high erucic acid, the majority of the microspore—derived embryoids can be discarded at an early stage in vitro. Only microspore-derived embryoids with a high content of C20: 1+C22:1 in the cotyledons need to be transferred to the greenhouse. This report also deals with the addition of abscisic acid (ABA) to the embryoid culture medium to increase the correlation, and discusses the possible application of this system for the selection of high-oleic or low-linolenic types in corresponding microspore-derived embryoid populations.     

Glucosinolates Determined by HPLC in the Seeds of Microspore-Derived Homozygous Lines of Rapeseed (Brassica napus L.)*

Microspore culture was employed to measure the relative efficiencies of anther culture and isolated microspore culture for the regeneration of embryoids and plants of Brassica napus. The yield of embryoids and plants was at least 10-fold greater from isolated microspores than from anther cultures. Approximately 1400 microspore-derived homozygous line's, the parental varieties and the corresponding F₂ plants were grown in a field trial. Important agricultural characteristics, such as morphological homogeneity, growth rate, onset of flowering and seed setting were evaluated subjectively and seed yield and glucosinolate content of individual plants were determined. The relative concentrations of up to S different glucosinolates in these seeds were measured via an automated high performance liquid chromatography (HPLC) system. The alkenyl and indole glucosinolates, the two most important categories of glucosinolates, were found in varying proportions and were independently determined in these line's. Our results do not support the previously suggested connection between low concentrations of glucosinolates and weak growth and/or poor seed yield. Additionally, no evidence was found that the lines derived from isolated microspore culture were subjected to unexpected selection pressures that might adversely affect the diversity of the lines obtained. These results demonstrate that microspore culture is a powerful tool not only for genetic analysis bur also for practical plant breeding.     

诸葛菜小孢子培养及其单倍体减数分裂染色体配对观察

诸葛菜是一种极有价值的观赏、蔬菜、饲料和油料作物种质资源。为建立诸葛菜小孢子胚状体诱导再生植株技术,并为诸葛菜染色体组的起源与进化研究提供相关数据资料,本研究通过对诸葛菜游离小孢子的培养,研究了热激培养时间和活性炭浓度对胚状体产量的影响,并采用常规压片法对诸葛菜单倍体减数分裂染色体配对行为进行了观察。结果表明,添加活性炭和热激培养对胚状体诱导是必需的。在直径6 cm培养皿中培养4 mL密度为1花蕾花粉mL^-1的小孢子NLN悬液时,每皿添加1 mg活性炭和32℃热激3 d的培养条件下子叶形胚状体和总胚状体产量最高,分别为每花蕾0.92±0.18个和1.32±0.25个。子叶形胚状体在1/2 MS培养基上萌发率为27.73%。花粉植株中自然加倍率为25%,加倍植株染色体数为24,单倍体植株染色体数为12。诸葛菜单倍体减数分裂染色体的平均配对构型为n=12=6.352Ⅰ+2.008Ⅱ+0.384Ⅲ+0.12Ⅳ,具有二价体及三价体和四价体的细胞比例高达96%,少量细胞的12条染色体联会形成3个四价体,说明诸葛菜很可能是起源于染色体基数x=3的同源八倍体。本试验结果对于诸葛菜新材料新品种选育和基础研究具有重要参考价值。     

番茄雄性不育突变体小孢子发育的细胞学研究

利用光镜和透射电镜对番茄雄性不育突变体９５３０５的小孢子发育进行了细胞学观察。结果表明,小孢子的败育发生在小孢子母细胞时期、四分体时期以及单小孢子时期。绒毡层细胞行为异常,表现为小孢子母细胞时期提早被破坏解体,或绒毡层细胞多层径向分化,异常肥厚,挤压单核期的小孢子。此外败育方式还有单核中期小孢子无法进行外壁沉积,或小孢子的细胞质被降解成空壳,以及花药畸形,花药上长出许多附属物等异常现象。     

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.     

嫩茎花椰菜花药和花粉培养中的胚胎发生

Futura、GV和GC三个品种嫩茎花椰菜单核阶段的花药和花粉,接种在(mg/L):①2,4-D0.1+NAA0.1+10％蔗糖;②2,4-D0.5+NAA1+BAP0.5+Kt0.5+GA_30.5+10％蔗糖的改良B_5固体和液体培养基上,给予不同温度和持续时间的热处理,最后培养在25℃光照下。接种后三周,从花药中产生出胚状体。转移到含2％蔗糖的改良B_5基本培养基上长成植株。观察了花药中花粉粒发育成胚状体的过程,分析了胚状体植物的染色体倍性。液体培养中的分离花粉粒,发育成一些膨大的胚性细胞和多细胞原胚。     

甜椒小孢子发育的细胞学观察

为研究甜椒小孢子发育时期的染色鉴定方法以及探讨小孢子发育时期与花器形态的相关性,采用不同染色方法进行甜椒小孢子的细胞学观察,并对小孢子发育期的细胞学特征以及小孢子不同发育时期与花器外观形态的关系进行研究。结果表明,甜椒小孢子经酒精盐酸处理后,用醋酸洋红染色液的染色效果最佳;甜椒小孢子的发育经历四分体期、单核早期、单核中期、单核后期、双核期直至花粉粒成熟期,各时期的特征明显;甜椒小孢子各发育时期与花蕾外部形态具有密切的相关性。