秋水仙碱对大麦离体培养小孢子存活与成苗的影响

以4份大田生长的优良大麦品种／品系为材料,采用超速旋切法分离小孢子进行培养,提取液和预处理液中添加适当浓度的秋水仙碱可明显提高大麦小孢子的存活率,胚状体的成苗潜力。     

秋水仙碱对大麦离体培养小孢子存活与成苗的影响

以4份大田生长的优良大麦品种/品系为材料,采用超速旋切法分离小孢子进行培养.提取液和预处理液中添加适当浓度的秋水仙碱可明显提高大麦小孢子的存活率、胚状体的成苗潜力.     

小麦的小孢子培养

小孢子培养是在花药培养的基础上发展起来的一种高效再生体系, 其在植物育种中的作用一直受到关注。但是, 由于培养技术的限制, 小麦(Triticum aestivum)小孢子培养还未能推广应用。近年来小麦小孢子培养发展很快, 展现出广阔的应用前景。     

小麦的小孢子培养

小孢子培养是在花药培养的基础上发展起来的一种高效再生体系,其在植物育种中的作用一直受到关注.但是,由于培养技术的限制,小麦(Triticum aestivum)小孢子培养还未能推广应用.近年来小麦小孢子培养发展很快,展现出广阔的应用前景.     

大麦不同基因型游离小孢子的直接培养再生及培养体系的优化

以微搅拌法建立了小孢子直接游离的预处理和培养程序。在大田生长的４个对培养反应不同的大麦基因型上,以新鲜幼穗游离小孢子进行直接培养,均成功地诱导了胚状体并获得再生绿色植株。小孢子的发育进程说明,直接游离的小泡子在预处理过程中的发育要慢于在花药中预处理的小孢子,而且其培养效率也较低。直接游离小孢子的培养密度以０．８～１．０×１０５／ｍｌ较理想,至少应不低于６×１０４／ｍｌ．８％－１０％的糖浓度可明显提高小孢子分裂频率和胚状体诱导频率。实验结果也表明两种培养基ＦＨＧ和ＭＮ６无明显差异,均适宜于直接游离的小孢子培养,并对游离小孢子直接培养在理论和应用上的意义进行了讨论     

源于小孢子培养的大麦耐盐变异体获取

以大麦品种‘花30’作为供试材料,比较了甲基磺酸乙酯（EMS）和平阳霉素处理小孢子60Co γ-射线辐照处理离体穗和干种子,对300mg·L-1NaCl胁迫培养下游离小孢子的愈伤组织产量和愈伤组织在0.3％NaCl胁迫筛选下的绿苗产量的影响。结果表明,EMS处理离体小孢子和60Co γ-射线辐照千种子的愈伤组织产量和绿苗产量明显优于平阳霉素处理小孢子和60Co γ-射线辐照离体穗。以16份源于种子辐照处理的再生植株自交一代种子为供试材料,比较了在0．3％NaCl胁迫下种子的发芽率和幼苗的成活率以及植株的分蘖数、株高和单株产量。结果表明,‘花30’发芽率为0,供试的16份耐盐变异体中,有14份材料在NaCl胁迫下的发芽率优于‘花30’,鉴定出4份耐盐性明显优于‘花30’的变异体材料。选择耐盐变异体作为供试材料,测定了变异体中Na＋／H＋逆向转运蛋白基因NHXl、NHX2和NHX3和编码甜菜碱醛脱氢酶（BADH）的两个同工酶基因曰肋,和BBD2的表达模式和表达量,结果表明变异体耐盐性的提高与这些基因的表达量存在联系。     

大麦直接游离小孢子培养中的脱分化启动和胚胎发生

采用DNA特异荧光染料,在共聚焦激光扫描显微镜下,对直接游离的大麦小孢子,在预处理过程中的脱分化启动,以及培养过程中的胚胎发生进行了较详尽的细胞学特征的观察,并在主要发育途径上与低温预处理后的小孢子进行了比较.研究结果表明:(1)小孢子的脱分化启动在预处理的12h内就已开始.完成脱分化启动的小孢子在细胞学上的主要特征为:细胞体积明显增大;核与核仁体积也显著增加,核仁极其明显,且高度浓缩,核/质比例高.(2)不同的预处理方法,都是促使离体小孢子完成脱分化启动,从而激发胚胎发生过程.(3)预处理方法可通过改变离体小孢子第1次有丝分裂的方式,而形成不同的主要发育途径.经低温预处理以形成A途径为主,直接游离以形成B途径为主.     

甘蓝和青花菜杂种小孢子培养

对甘蓝(Brassicaoleraceavar.capitata)×青花菜(Brassicaoleraceavar.italica)的20个杂种及相应的父母本进行游离小孢子培养,并对影响甘青杂种小孢子胚胎发生的主要因子进行探讨,适于小孢子培养的培养基为1/2NLN,附加0.5mgL-1NAA、0.05mgL-1BA、5mgL-1AgNO3、0.2mgL-12,4-D和0.1mgL-1活性炭。结果有14个杂种能产生胚状体,诱导率70%;不同杂种间小孢子胚胎发生频率存在很大差异,最高的是绿洲808×夏宝,平均每蕾16.2个胚。诱导杂种胚状体发生的最佳时期是小孢子单核靠边期至双核期,34℃热激2d有利于小孢子细胞对称分裂。在含糖170gL-1的液体培养基中培养3d,添加低糖(含糖110gL-1)的培养液,可显著提高出胚率。     

基因型与培养条件对羽衣甘蓝小孢子胚胎发生的影响

本文详细地研究了小孢子发育时期、基因型与培养条件对羽衣甘蓝小孢子胚胎发生的影响,建立了一个稳定、高频地获得小孢子胚胎的有效体系。结果表明,不同基因型材料相同大小的花蕾其小孢子发育时期存在很大差异,需针对不同基因型材料选取适合大小的花蕾。供试的37个基因型中,有20个获得了胚状体,占供试材料的54．1％,其中基因型‘桃舞’获得了最高的出胚率,为123．6个·皿-1。自交系的出胚率比商业品种和F,代杂种的出胚率要低得多,且自交代数越高,小孢子的胚胎发生能力就越弱。在热激培养48hN加液培养对小孢子的发育能起到积极作用,向培养基中添加激素和活性炭对小孢子的胚胎发生无促进作用。     

大麦铁蛋白基因(HvFer1)cDNA的克隆和表达

根据玉米铁蛋白ZmFer1的氨基酸序列,采用同源序列法进行序列拼接和引物设计,RT-PCR扩增获得了1个源自二叶期大麦叶片mRNA的大麦铁蛋白基因cDNA片段,HvFer1(GenBank登录号为EF440353)。HvFer1全长1023 bp,5′非翻译区56 bp,3′非翻译区202 bp,开放阅读框编码254个氨基酸。序列分析表明,此蛋白与已知植物铁蛋白高度同源,相似性介于56.7%～83.7%之间,N端具27个氨基酸的信号肽,C端(84～247)具有1个类似铁蛋白的功能域。RT-PCR表达谱分析显示,HvFer1在大麦的茎、叶、幼穗和幼根均能表达,茎和幼穗中表达量略高些。此外,HvFer1受PEG和盐胁迫的强烈诱导表达,中度铁胁迫也可不同程度地增加HvFer1的表达量。     

Comparison of Anther and Microspore Culture in the Embryogenesis and Regeneration of Rye (Secale cereale)

Anther culture in solid and liquid medium and isolated microspore culture were compared in rye genotypes with potential agronomic characteristics. Some important factors influencing androgenic capacity were optimised. Three weeks cold pre-treatment of spikes and two days mannitol pre-treatment of anthers maximized callus and green plant yield in both culture methods. Intensity order of the culture methods in callus and green plant production was: isolated microspore culture, anther culture in liquid medium and anther culture in solid medium. Genotype ability of embryogenesis followed the same pattern in both cultivation methods. Kinetin (BA) with genotype dependent concentrations created the most effective regeneration conditions.     

The Effect of cAMP on Phosphohydrolase Activity of the Barley Root Cells

We studied the effects of cAMP on phosphohydrolase activity in the microsomal fraction and its 0.5 M KCl extract. In both preparations, cAMP inhibited the activity of acid phosphatases. In the microsomal fraction, a 50% inhibition of acid cation-dependent (Mg²⁺, Cu²⁺) phosphatase was induced at pH 6.0 by 10 M cAMP; whereas, for cation-independent phosphatase, this effect was exerted only by 50 M cAMP. In the KCl extract, the sensitivity of both cation-dependent and cation-independent acid phosphatases to cAMP was higher than in the microsomal fraction. Cyclic AMP did not reduce the activity of nucleosidases. Selectivity in the cAMP action on acid phosphatases is assumed to be one of the mechanisms controlling the biochemical activity outside the barley root cells.     

The Effect of High Temperatures on the Photosynthetic Activity of Intact Barley Leaves at Low and High Irradiance

Light curves of CO₂ fixation by barley seedling leaves preliminarily heated at 30–43°C for 5 min were measured. The slope of the linear part of the light curve decreased after leaf heating at temperatures above 35°C; whereas, at a high light level, the photosynthesis rate decreased only at temperatures of 40°C and higher. The linear relationships between the photosynthetic CO₂-fixation rate and a photon flux density up to 1400 mol/(m² s) were found in leaves preheated at 42°C; this indicates the strong nonphotochemical dissipation of absorbed light quanta. The lowering of the oxygen concentration from 21 to 1% led to a CO₂ fixation maximum quantum yield and a photosynthesis-rate increase at the highest light intensity in leaves preheated at temperatures above 40°C as compared to the control leaves. Nevertheless, the linear relationship between the photosynthetic CO₂ fixation and the light intensity was found in leaves heated at 42°C at O₂ concentrations of both 21 and 1%. The latter fact suggests that the proton gradient of the thylakoid membrane, which causes an increase in the nonphotochemical dissipation of the quanta absorbed, could also be formed due to the cyclic electron transport over photosystem I.     

Effects of Paclobutrazol on Response of Two Barley Cultivars to Salt Stress

The seeds of two barley (Hordeum vulgare L.) cultivars (a drought resistant cv. Tokak-137/57 and a drought sensitive cv. Erginel-90) were imbibed either in distilled water (control) or in a solution containing 40 mg dm⁻³ paclobutrazol (PBZ) and air dried. Seeds were germinated and grown in a glasshouse for 21 d and seedlings were subjected to salt stress by treating them with 100 and 200 mM NaCl for 12 d. The height of shoots was significantly decreased and root length was increased in PBZ-treated plants prior and after NaCl stress for 12 d leading to an increase in root to shoot ratio. Leaf chlorophyll and carotenoid contents in PBZ treated plants were increased in controls and especially in plants subjected to salt stress. PBZ induced increase in superoxide dismutase (SOD) activities was higher in cv. Tokak-157/37, than in cv. Erginel-90. However, an increase in SOD activity was not accompanied by an increase in peroxidase activity. This revised version was published online in July 2006 with corrections to the Cover Date.     

Cryopreservation of Embryogenic Suspension Cultures of Barley (Hordeum vulgare L.)*

Anther-derived suspension cultures of Hordeun vulgare L. were cryopreserved by slow cooling and storage in liquid nitrogen for up to 55 days. Cells were pre-cultivated in L3 suspension medium supplemented with sorbitol. For freeze preservation the cells were treated with different combinations of cryoprotectant agents such as DMSO, proline, glycerol and sucrose. After rapid thawing high viabilities of up to 77% could be achieved. Cell growth commenced 2- 3 weeks later. The frequency of plantlet regeneration was 1%.     

Barley anther culture: The effect of position on pollen development in vivo and in vitro

Locule structure and organization were studied in vivo and in vitro to determine whether the disposition of pollen within barley anthers affected the response of pollen in culture. Following release from the meiotic tetrads, juvenile barley microspores become peripherally organized around the locule, with the single pollen pore oriented towards the tapetum. Scanning electron micrographs of transverse sections from freeze fractured anthers showed that some microspores failed to locate the tapetal surface and occupied a position in the centre of the locule where they continued to develop as small, abnormal pollen grains (dimorphic pollen). Previous evidence has suggested that in some species dimorphic pollen could be the source of embryonic pollen in vitro. Cultured anthers frequently dehisced to reveal a mass of dividing pollen grains, however those anthers that remained intact retained the original locule structure and could be freeze fractured permitting examination of the developing pollen in situ. This showed that pollen embryogenesis was not restricted to dimorphic pollen, and that any grain could become Embryogenic irrespective of position.     

Effect of Increasing Concentrations of Heavy Metals on the Growth of Barley and Wheat Seedlings

The tolerance to increasing doses of lead and cadmium salts on the growth and survival of barley (Hordeum vulgare L.) and wheat (Triticum aestivum L.) seedlings were studied. Seedlings grown under controlled conditions were treated with dilute (0.001–0.005 mM) solutions of either lead nitrate or cadmium bromide for 1, 4, or 7 days. Subsequently, they were incubated for 7 days in solutions of the same compounds, but at sublethal or lethal concentrations (0.05–10 mM). Plant pretreatment with low concentrations of heavy metals induced an increase in their tolerance to the metals, because pretreated plants could tolerate heavy metals at high concentrations. It is concluded that plant tolerance to increasing concentrations of heavy metals is related to the activation of protective and adaptive processes in their tissues.