The Effectiveness of Vernalization of Immature Embryos of Winter Wheat var. Grana as Related to Age and Exogenous Phytohormones

Starting from the 10th day after pollination, immature embryos of winter wheat var. Grana were isolated and then vernalized for 4 to 8 weeks on Murashige and Skoog nutrients containing BAP (0.5 or 2 mg/dm³), NAA (0.5 or 2 mg/dm³), or GA₃ (5 or 20 mg/dm³). Vernalized seedlings were cultivated in a greenhouse and the number of days to the heading of individual plants as well as the percentage of plants capable of generative development were estimated. The lower limit of size for 50 % survival of embryos strongly depended on the phytohormone used: from 0.9 mm in control, 1.1 mm in nutrient containing BAP, 1.2 mm for NAA, up to 1.7 mm in nutrient with GA₃. Exogenous GA₃ was lethal for embryos younger than 18 days but induced elongation of older embryos. Embryos isolated 2.5 to 4 weeks after pollination showed minimal requirements for the length of vernalization. BAP increased the percentage of heading plants originated from the youngest embryos. GA₃ improved partial vernalization, strongly increasing the percentage of heading plants, but did not change the time from the end of vernalization to heading. It has been postulated that GA₃ increases number of plants capable of overcoming the threshold of induction of generative development but does not accelerate the flowering process. Hormonized plants showed no deformation of generative development. As the effectiveness of vernalization increased, the heading of plants was faster but they were shorter, forming spikes with a smaller number of spikelets and producing fever lateral shoots. The very young embryos probably have in reserve sufficient amounts of auxins and gibberellins and therefore exogenous GA₃ decreases their viability or even exerts a deleterious effect. However, as the embryos' ageing, gibberellin starvation develops. This being especially pronounced during vernalization. The de novo synthesis or activation of gibberellins takes place during the second stage of vernalization. This is why exogenous hormone improves the effectiveness of partial vernalization, though it is not possible to substitute by gibberellins the vernalization requirements of immature embryos.     

Donor Plant Growth Conditions and Regeneration of Fertile Plants from Somatic Embryos Induced on Immature Zygotic Embryos of Sunflower (Helianthus annuus L.)

Immature zygotic embryos of the sunflower inbred line ‘Ha 300’, cultivated on a modified MS medium containing 6-benzyladenine and a high amount of sucrose, regenerated fertile plants via direct somatic embryogenesis. Plant regeneration from immature sunflower embryos is generally characterized by a relatively high experimental variability resulting from the interactions of multiple factors. We present here a study of some of the factors acting on the donor plants and their influence on the capacity to regenerate plants. Repeated experiments during a 2-year period with greenhouse-grown as well as field-grown plants led to the following conclusions: (i) The use of pesticides, unavoidable in the greenhouse, is compatible with routine regeneration of fertile plants, (ii) The plant growth retardants tested were useful for the production of healthy plants in the greenhouse and had no effect on the regeneration capacity.     

RNA Synthesis in Porcine Blastomere Nuclei Introduced into in Vitro Matured Ooplasm

The objective was to investigate the RNA synthesis in porcine blastomere nuclei upon transplantation into in vitro matured enucleated oocytes. Nuclei from 2- to 8-cell porcine embryos were introduced into the ooplasm of in vitro matured and enucleated porcine oocytes by electrofusion, and the resultant reconstructed embryos were cultured in vitro. Before fusion or at different intervals after this event embryos were incubated with [³H]-uridine, fixed, and histologically processed for autoradiography in order to detect RNA synthesis. About two thirds of the embryos were considered to depict normal development. All blastomeres displayed pronounced RNA synthesis before fusion, at 3 and 9 h after fusion the synthesis decreased or ceased, and at 24-49 h some embryos resumed synthesis at the 1- to 2-cell stage.     

牡丹‘凤丹’体细胞胚发生技术

为建立牡丹‘凤丹’体细胞胚发生培养体系,以牡丹‘凤丹’的合子胚、胚轴和子叶为外植体进行离体培养,研究了种胚发育时期不同质量浓度蔗糖处理及不同的培养基对牡丹体细胞胚发生的影响,结果表明,花后110 d是体细胞胚直接诱导合适的发育时期,诱导率为33.00%,3种外植体中,种胚的诱导率最高;利用质量浓度为90 g.L-1的蔗糖溶液处理种胚2 h,体细胞胚诱导效果最好,在MS+2,4-D 2.0 mg.L-1+6-BA 2.0 mg.L-1培养基上获得较高的体细胞胚,诱导率为38.33%。     

诱导不同基因型大豆未成熟子叶体细胞胚胎发生的研究

以未成熟子叶为外植体,研究25种基因型对大豆愈伤组织形成与体细胞胚胎发生的影响。结果表明,大豆不同基因型间愈伤组织诱导率变化较小,而基因型对体细胞胚胎发生的影响较大,依基因型不同体细胞胚诱导率变化在3.33%～83.33%。体细胞胚诱导率、平均胚数在基因型间的差异均达极显著水平,且两者间存在极显著相关关系。     

陕西关中中部灌区12个小麦品种幼胚脱分化特性研究

[目的]对陕西关中中部灌区推广的12个小麦品种幼胚脱分化特性进行研究,以筛选出优良转化受体的基因型。[方法]供试小麦品种为综合农艺性状优良的小偃22、西农9871、西农979、阎麦8911、西农889、西农2611、陕麦159、陕农138、西农2000、西农3517、小偃216和西农538。培养基A:MS基本培养基+2,4-D 2.0 mg/L+6-BA 0.5 mg/L+蔗糖30 g/L+琼脂4 g/L;培养基B:MS基本培养基+2,4-D 2.0 mg/L+6-BA 0.5 mg/L+AgNO32.5 mg/L+蔗糖30 g/L+琼脂4 g/L。培养温度25～26℃,暗培养,28～35 d继代培养1次。[结果]不同小麦品种之间愈伤组织的形成时间、愈伤组织诱导率、胚芽率和胚性愈伤组织诱导率明显不同,且在2种培养基间的波动趋势具有一致性;培养基中附加的AgNO3可以减轻胚发芽的发生概率而促进胚性愈伤组织产生;12个品种中,小偃22、陕农138和小偃216表现出较好的脱分化综合性状,可用于小麦的基因转化研究。[结论]该研究可为建立高效稳定的小麦植株再生体系奠定基础。     

不同菜用大豆品种的胚胎发生与再生

以8个菜用大豆品种的未成熟子叶为外植体,在MS(附加高浓度2,4-D)培养基诱导愈伤组织形成、体细胞胚胎发生与再生植株.结果表明:八个菜用大豆品种的未成熟子叶均可诱导产生愈伤组织与体细胞胚胎发生,出愈率为14.71%～100%.体细胞胚胎发生率变化在0～36.76%之间,绿子叶菜用大豆没有形成体细胞胚.体细胞胚可正常萌发产生小植株,移栽成活结实.菜用大豆的出愈率和体细胞胚胎发生率与基因型有关.     

水牛徒手克隆胚胎培养及冷冻条件的优化

本试验利用微滴、微穴和平板培养系统对徒手克隆(hand-made clone,HMC)重组胚进行体外培养;采用了40% EG(ethylene glycol,EG)、25% EG＋25% DMSO(dimethylsulphoxide,DMSO) 和20% EG＋20% DMSO＋0.5 mol/L蔗糖作为玻璃化冷冻液对HMC囊胚进行了超低温冷冻;并且比较了HMC与传统核移植的胚胎生产效率及囊胚冷冻存活率。结果表明,微穴系统的卵裂率要显著高于平板系统(P<0.05),极显著高于微滴系统(P<0.01);且微穴系统的囊胚率(40.0%)极显著高于平板(19.8%)和微滴系统(8.3%)(P<0.01)。采用20% EG＋20% DMSO＋0.5 mol/L蔗糖作为冷冻保护剂时HMC囊胚存活率极显著高于40% EG(P<0.01);HMC重组胚的融合率和囊胚率均高于传统核移植法(P<0.05;P<0.01),而HMC囊胚的冷冻存活率与传统核移植生产的囊胚没有显著差异。以上结果说明水牛HMC可以替代传统核移植法生产克隆胚胎,微穴体系最适合水牛HMC胚胎的体外培养,且采用20% EG＋20% DMSO＋0.5 mol/L蔗糖对HMC囊胚进行玻璃化冷冻可以取得良好的冷冻效果。     

不同外植体对多年生黑麦草愈伤组织诱导的影响

对多年生黑麦草的3个品种进行愈伤组织诱导,选取成熟种子、成熟胚、胚轴、胚根4种外植体。结果表明,胚轴、胚根诱导效果较差,成熟胚在愈伤组织的诱导率、鲜重及愈伤质量方面均优于成熟种子。2,4-D浓度在2~4mg/L,6-BA浓度为0.025mg/L时有利于成熟胚诱导产生愈伤组织;成熟种子为外植体时,2,4-D浓度在5~8mg/L,6-BA浓度在0.025mg/L或0.2mg/L有利于成熟种子诱导产生愈伤组织。2,4-D浓度为2mg/L,6-BA浓度为0.5mg/L时成熟胚和成熟种子诱导的愈伤组织鲜重均达到最大。特拉华在愈伤组织的诱导率、增殖速度及愈伤质量方面表现较优,托亚次之,尤文图斯较差。AC和LH有利于植株的再生。     

野牛草成熟胚植株再生及其影响因素研究

以野牛草Buchloe dactyloides成熟胚为外植体,对外植体灭菌方式及影响成熟胚再生的因素进行了研究.结果表明:70%酒精处理60 s,75% NaClO溶液(原溶液含有效氯为7%～10%)处理30 min,污染率最低为0.59%.6-BA 0.1 mg/L,2,4-D浓度增至3 mg/L时,愈伤组织诱导率达最高,为80.27%,但愈伤组织结构疏松、呈水渍状.附加5 mg/L硝酸银(AgNO3)或硫代硫酸银(STS)时,愈伤组织诱导率略有降低,愈伤组织结构紧密且表面有颗粒状突起,当AgNO3或 STS浓度为10 mg/L以上时,均不利于愈伤组织的诱导.愈伤组织继代培养过程中,3/4倍MS大量元素用量、聚乙烯吡咯烷酮(PVP) 200 mg/L、维生素C(Vc )200 mg/L及水解酪蛋白(CH) 1 000 mg/L可减轻愈伤组织褐化程度.不同诱导培养基所获得的愈伤组织与其植株再生能力有关,以不加任何植物生长调节剂的MS培养基(MS0)为分化培养基,仅在附加STS的诱导培养基中所得的愈伤组织能够形成再生植株,再生率为10%.