低温预处理对甘蓝型油菜小孢子胚胎发生的影响

研究了大田环境下栽培的甘蓝型油菜小孢子胚状体诱导过程中低温预处理的作用,结果表明:两个供试材料经过低温预处理,小孢子胚胎发生能力明显提高,同时,不同基因型要求的预处理时间不同。G369的胚产量在低温3d时达到最高,是对照的27.2倍,而Topas的胚产量在低温2d时达到最高,是对照的15.4倍。试验还统计了子叶型胚状体发生频率,发现与对照相比,处理后子叶型胚状体的频率呈下降趋势,表明低温预处理使小孢子胚发育同步性降低。     

中国白菜小孢子离体培养和双单倍体植株再生

摘 要: 采用略加修改的NLN培养基（无机大量元素1/2）和甘蓝型油菜小孢子的胚状体诱导方法,培养了5个中国菜用小白菜品种和1个中国大白菜品种的小孢子,进行了双单倍体的诱导。结果表明,不同品种的小孢子产胚量差异很大,在接种的6个基因型中,有5个诱导出胚,诱导成功率83.33％ 。其中的中萁青抗热605产胚量最高,是8.676个胚／10蕾。这些胚在添加了NAA和PP333（多效唑）的B5培养基发育成健壮的小植株。关于6个基因型的小孢子胚胎发生能力,还有待进一步研究。     

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。     

浅析几个因子对甘蓝型油菜游离小孢子胚胎发生的影响

为了快速纯舍杂交后代材料,提高重组基因型的选择几率,对40个杂交F1代组合进行小孢子分离培养。其中,有9个材料产生小孢子胚（占22.5％）,仅4个材料（10.0％）获得了再生植株并种植大田,表明基因型是影响小孢子胚胎发生的一个主要因素。4个杂交组合材料的胚胎发生率为0.08～0.86胚／蕾,平均为0.25胚／蕾。细胞学检测表明,本试验杂交组合材料的最适取样花蕾长度为3.5mm。大田取样采取切取花序,然后在4℃冰箱中插水加散射光保存4～5d不影响胚胎发生。热激培养2d后小孢子的膨大率是衡量小孢子胚胎能否发生的一个有效指标。     

结球甘蓝小孢子培养技术体系的优化研究

以20份类型丰富的F1及亲本甘蓝为试材,探讨了不同甘蓝类型、F1及其亲本材料、供体植株的生长温度、供体植株的花期、高温热激时间、NLN培养基pH值等因素对甘蓝小孢子胚胎产量及质量的影响,为建立高效、完善的结球甘蓝小孢子培养技术体系提供依据。结果表明:不同叶球类型与小孢子的产胚率无明显相关性,但不同栽培季节及熟性的甘蓝品种间却存在显著差异,其中春秋类型的早、中熟甘蓝要明显好于晚熟的越冬甘蓝;用产胚能力强与产胚能力弱的材料分别做亲本杂交,所得杂交种的出胚率介于两亲本之间,表明小孢子产胚能力是一种受基因调控的遗传性状;供体植株的生长温度对于结球甘蓝胚状体的产生并无显著影响;从胚胎发生频率及胚胎质量来说,初花后10 d内(开花初期至盛花中期)是取样的最佳花期阶段;对于大多数基因型而言,32.5℃热激预培养48 h较24 h更有利于胚胎发生,热激32.5℃48 h并结合NLN-pH6.2为最佳处理组合,比目前在甘蓝类蔬菜中常用的32.5℃24 h+NLN-pH5.8的培养体系在基因型反应范围及胚胎发生频率方面都有显著提高。     

大白菜内因对小孢子胚胎发生能力的影响

以15个大白菜杂交种为试材,采用游离小孢子培养方法,研究基因型、花蕾长度对小孢子胚发生的影响。结果表明:不同试材的小孢子胚发生频率有明显差异,有11个品种诱导出胚状体,最高出胚率为每蕾54.17胚;诱导胚状体发生的最佳花蕾长度不同品种略有不同,多数品种在2.5～3.5mm时胚发生频率较高,其次是蕾长2.0～2.5mm时有较高发生频率。     

甘蓝型油菜离体小孢子胚胎发生能力的遗传分析

对一套甘蓝型油菜6×6半双列杂交组合和1个F2群体进行了离体小孢子培养。 不同基因型(组合)间产胚率差异极显著, 小孢子产胚率的广义遗传力为0.85。 F1小孢子产胚率具有明显的杂种优势, 平均优势率为10.52%。 小孢子产胚率的一般配合力和特殊配合力方差均达到极显著。 根据一般配合力的大小, 可将供试亲本分为高和低配合力     

基因型和供体植株生长环境对大白菜游离小孢子胚胎发生的影响

１７个用于大白菜常规育种的基因型在ＮＬＮ－１３液体培养基中进行游离小孢子培养，结果有１６个基因型经由小孢子胚胎发生途径获得小孢子胚。这些基因型包括从早熟晚熟的各种类型。各基因之间在小孢子胚胎发生频率方面存在很大差异，产量最高的两个基因型Ｔ１１和ＣＣ１１，平均每百花蕾分别达３５９２８和３４１３２个胚。供体植株生长环境对小孢子胚胎发生具有明显影响；在１０／２０℃（液／昼）的部分控温温室生长的供体植株，     

通过花药漂浮培养提高花椰菜小孢子胚胎发生率

通过花药漂浮培养,成功地诱导两个秋花椰菜基因型的小孢子胚胎发生.在液体培养基与固、液体双层培养基上,小孢子胚胎产量相仿,但后者褐色胚产生较多.在液体培养基中加入62.5mg/L的硝酸银对诱导小孢子胚胎发生效果较好.试验结果表明,采用花药漂浮培养方法可以明显提高花椰菜小孢子胚产量.     

辣椒花药培养研究进展

归纳了辣椒花药培养过程中影响雄性胚胎发生的诸多因素,包括供试材料、小孢子发育时期、基本培养基、植物生长调节剂、培养基添加物质和温度胁迫处理,总结了小孢子胚胎发生的细胞学观察、再生株的倍性水平和单倍体基因组加倍研究,指出尽管人类已从细胞和分子方面对辣椒花药培养中诱导小孢子胚胎发生和形成胚状体有了进一步的认识,但尚未完全了解小孢子是如何被激发进入孢子体发育途径。当小孢子胚胎发生之谜完全揭开后,辣椒花药培养技术将会应用于更加广泛的领域。     

Effect of thidiazuron on microspore embryogenesis and plantlet regeneration in Chinese flowering cabbage (Brassica rapa. var. parachinenis)

Traditional breeding methods require more than 6 years to obtain homozygous inbred lines, while isolated microspore culture (IMC) is an effective way to cultivate double haploid homozygous lines in only 2 years. However, low embryogenesis induction frequency in Chinese flowering cabbage remains a key obstacle to the practical application of this technique. Thidiazuron was added at different concentrations to NLN‐13 medium to estimate its effects on microspore embryogenesis and plantlet regeneration. Results showed that three genotypes responded positively. Optimum thidiazuron concentrations produced embryo yields of up to 14.67 embryos per bud and increased microspore embryogenesis frequency with up to 100% survival. Plantlet regeneration rates were up to 81.67%, and the treatment groups showed lower callus formation. We obtained up to 552 diploid plants from the tested genotypes, and the percentage of doubled haploid at different TDZ concentrations showed slight differences, and doubled haploid rates in the three genotypes were above 70%. They showed a high uniformity and can be directly used for hybrid breeding. This method accelerates microspore application in Chinese flowering cabbage hybrid 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。     

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。     

Regeneration of fertile doubled haploid plants from colchicine-supplemented media in wheat anther culture

The effect of colchicine added to induction medium for the production of fertile doubled haploid plants after in‐vitro anther culture was studied in wheat, Triticum aestivum L. For this, one winter and two spring wheat varieties were used. Anther cultures of the three genotypes were treated with 0.03% colchicine for 3 days at the beginning of microspore induction. Colchicine had no significant effect on anther response and embryoid production of the genotypes examined. However, in the winter wheat genotype ‘Mv Szigma’, colchicine caused a significant reduction in microspore‐derived structures. A significant decrease was also observed in plant regeneration ability of two genotypes (‘Vergina’ and ‘Acheloos’) after colchicine treatment. In addition, a significant reduction of the albinos produced was observed in all genotypes after olchicine treatment. In contrast, the regenerants obtained from the colchicine‐supplemented induction media produced significantly higher percentages of fertile plants in all genotypes. However, the level of fertility, was significantly different among the fertile plants obtained. This, together with the observation that in the case of the winter wheat variety the colchicine treatment resulted in 100% completely fertile plants with a high seed‐setting ability indicate that there is space for further improvement of the method when it is applied to spring cultivars. Finally, the increased number of seeds per 100 plated anthers obtained from all three genotypes after colchicine treatment, clearly demonstrates that the addition of colchicine to induction medium was superior to the conventional anther culture method and it could therefore be introduced into wheat breeding programmes.     

甘蓝未受精子房离体培养体系的建立

利用供试的甘蓝CMS材料对影响甘蓝未受精子房离体培养诱导的主要因素（包括外植体的基因型、外植体发育时期、外源激素类型及浓度、高温预处理温度及时间、灭菌所用次氯酸钠浓度及时间等）进行研究,旨在对各种处理条件下诱导率情况进行分析。通过单因素、多因素等试验方法筛选出能够诱导成苗的未受精子房离体培养体系,明确甘蓝未受精子房离体培养发育途径。结果表明：基因型RM的诱导效果最好;当外植体处于花初期时最易诱导成功;诱导未受精子房产生愈伤组织的适宜培养基MS+0.5 mg/L 6-BA+ 2.0 mg/L 2,4-D+ 0.1 mg/L NAA,愈伤组织及雌核发育胚总诱导率可达28.57%;诱导愈伤组织分化不定芽的适宜培养基为MS+ 0.1 mg/L NAA+ 0.2 mg/L 6-BA+ 0.05mg/L TDZ,不定芽分化频率可达57.12%;将不定芽转移至1/2MS+ 0.1 mg/L NAA培养基中可诱导生根,发育成完整植株;33℃高温诱导1 天的平均芽点数及芽点诱导率均最高,分别为2.85 和100%;次氯酸钠浓度为2%、处理时间为15 min时,平均芽点数和芽点诱导率均最高,分别为2.93和97%。     

大麦成熟胚胚性愈伤组织的高频诱导和植株再生

大麦成熟胚的初代愈伤组织是水质、柔软的非胚性愈伤组织，在继代培养中能以一定频率出现胚性愈伤组织。本文研究了基因型、有机添加物和胚切割等因素对大麦胚性愈伤组织诱导频率的影响。结果表明：供试的１０个大麦品种在胚性愈伤组织诱导率和植株再生率上有显著差异；培养基中添加水解酷蛋白、Ｌ－脯氨酸以及提高维生素Ｂ１和肌醇的浓度可以促进胚性愈伤组织的形成；胚芽段比全胚或胚根段更易形成胚性愈伤组织。胚性愈伤组织转入无     

An improved in vitro technique for isolated microspore culture of barley

A detailed procedure for isolated microspore culture of barley is presented along with examples of response across genotypes. Over 30 genotypes, including winter and spring growth habit and 2-row and 6-row genotypes, have shown an essentially genotype independent response, averaging about 10,000 embryos per 5 cm petri culture plate. The regeneration frequency, checked on samples of 500 embryos per plate ranged from 36 to 97% with most genotypes being in the range of 70 to 90%. About 70 to 80% of the plants regenerated have been completely fertile doubled haploids, thus eliminating the need to double the chromosome number of plants. Many little details are critical to success of the microspore procedure and while it saves much time compared to anther culture, greater attention to details and cleanliness is essential. This revised version was published online in August 2006 with corrections to the Cover Date.     

Microspore culture of white cabbage, Brassica oleracea var. capitata L.: Genetic improvement of non-responsive cultivars and effect of genome doubling agents

For the efficient application of haploid induction procedures in cabbage breeding, a sufficient number of regenerants should be achieved in a broad spectrum of genotypes. However, the majority of genotypes are somewhat recalcitrant. The efficiency of microspore culture was tested by crossing a responsive (28.7 embryos per Petri dish) and a non- responsive (0.1 embryo) cabbage cultivar. The embryo yield of one progeny was intermediate (18.9) while two were superior to the best parent cultivar (52.9 and 64.0 embryos). Thus, genes for haploid embryogenesis, present in responsive lines, can be effectively transmitted to responsive × non-responsive hybrids. Abscisic acid-induced desiccation of embryos was used for the efficient regeneration of plants. High germination percentages (54.7-70.6%) followed by normal plantlet development were achieved. Spontaneous genome doubling measured at the plantlet stage differed markedly in untreated genotypes. The percentage of diploids ranged from 21 to 67%. The effects of two antimitotic drugs applied to freshly isolated microspores were determined in two experiments. In the first experiment, trifluralin (0.5 and 1.0 mg:l) had no effect on embryo induction while oryzalin partly (0.125-0.25mg/l) or completely (0.5.mg/l) inhibited the formation of embryos. In the second experiment, higher concentrations of trifluralin increased the proportion of diploidized plants. Application of anti-mitotic drugs to microspores did generally not improve the overall production of haploid plants, which was higher in an untreated control.