不同培养基对水稻胚性愈伤组织诱导及分化的影响

以水稻成熟胚为材料诱导愈伤组织,统计在不同基本培养基上的愈伤诱导率以及绿苗分化率,分析不同基本培养基及外源激素的含量和比例对愈伤组织生长及分化的影响。结果表明,试验材料对基本培养基具有选择性,MS培养基对籼稻种胚愈伤的诱导培养效果较好,NB培养基则更适合粳稻种胚愈伤的诱导培养;诱导继代培养基中加入多种氨基酸组合可有效提高出愈率和分化率,特别是粳稻的愈伤组织的诱导和分化需要多种氨基酸的共同作用;不同基因型水稻材料对激素和氨基酸组合的需求不同。     

基因型对春麦花粉植株诱导率影响的研究

花药培养中,不同基因型供体对于花粉植株的影响很大.几年来的研究结果表明,用花培品系做亲本配置杂交组合;不同世代的基因型,尤其是F_1代花药供体植株;亲本基因型在提高花粉愈伤组织出愈率、花粉绿苗分化及花粉绿苗率方面均有明显的效果.     

Cu2+对小麦花药脱分化和再分化特性的影响

以3个普通小麦杂交组合的F1群体为材料,研究了小麦花药离体培养中Cu2 对花药组织脱分化和再分化特性的影响.结果表明,诱导培养基中的Cu2 对小麦花药愈伤组织的诱导及再分化均有影响,均表现为低浓度下的促进作用和高浓度下的抑制作用,最适Cu2 浓度为0.15μmol/L.在该浓度下,3个供试材料的出愈率分别比对照提高了239.14%、214.72%和80.00%,反应率分别比对照提高了156.70%、151.86%和65.96%,绿苗分化率分别比对照提高了200.05%、241.87%和333.49%.再分化培养基中的Cu2 对小麦花药愈伤组织的再分化有明显的负向效应,3个供试材料中,均以不附加Cu2 的对照的绿苗分化率最高,分别为70.00%、66.67%和39.29%,附加不同浓度的Cu2 后,绿苗分化率显著下降.     

水稻抗s－氨乙基半胱氨酸细胞突变体的筛选

应用水稻花药培养技术,通过对抗s－氨乙基半胱氨酸(AEC)突变体的选择,研究了细胞水平筛选高含量赖氨酸水稻种质的效果。结果表明,AEC可抑制水稻花药(粉)愈伤组织的形成与生长,用O．5mmol,L AEC进行诱导或继代筛选,可获得抗性愈伤组织并再生绿苗,但仅有少数个体自然加倍正常结实,并保持对AEC的抗性。经多代选择与鉴定,获得了丰产优质突变体A一87306,其糙米游离赖氨酸含量0．43％,较对照增加34．38％。初步认为以AEC为外源胁迫因子,离体筛选水稻高赖氨酸含量的细胞突变体是有效可行的。     

紫外光照射小麦花药的研究

紫外光照射小麦花药进行组织培养,通过2年的试验结果初步表明:紫外光对诱导愈伤组织和分化绿苗有一定的作用,从多数试验材料的愈伤组织诱导率与绿苗分化率,照射1秒的比照射2秒、6秒、8秒和未照射的对照高。     

籼稻花药培养的研究——提高愈伤组织诱导率与分化率及根芽分化

3年来通过对籼稻(Oryza sativa subsp.hsien)花药培养的研究,愈伤组织的诱导率从0.2%提高到平均为2.85%,最高达18.75%;绿苗分化率从5%以下提高到平均为13.1%最高达到45.5%。在6个籼稻品种、38个籼×籼杂种中成功地诱导出了绿苗。本文着重研究了提高诱导率的有关因素。以单核晚期花粉的花药愈伤组织诱导频率最高。籼稻花粉去分化过程相对地要求较低浓度的培养基,籼稻花药对高浓度的改良 RM-1964培养基完全没反应。用杂种的诱导率较亲本材料为高。以易于诱导愈伤组织或易于分化绿苗的品种作亲本的杂种后代,诱导率与绿苗分化率亦较高。籼稻形成愈伤组织的高峰是在接种后的50—60天之间。愈伤组织分化的高潮是在转移后10—30天之间。籼稻愈伤组织的分化以先分化根的类型最多,在先分化根的愈伤组织中有36.74%能再分化出芽来。     

从“湖北光敏感核不育水稻”的未受精子房和花药培养出单倍体植株

采用10种诱导培养基,培养湖北光敏感核不育水稻农垦58品种的未受精子房和花药。共培养未受精子房2790个,获得胚囊愈伤组织17块,最高诱导频率达3.33%,其中2块分化出绿苗。培养花药16740个,获得花药愈伤组织15块,最高诱导频率为0.92%,其中3块分化出苗,2丛白苗,1株绿苗。胚囊植株和花粉植株经根尖染色体检查为单倍体,2n=x=12。实验证明,液体培养、2,4-D0.2-0.5 mg/1、低温预处理对诱导胚囊愈伤组织及花粉愈伤组织的形成具良好效果。     

甘蓝胞质雄性不育系及其保持系花药和叶片生化特性分析

对甘蓝胞质雄性不育系CMS158及其保持系花药激素、游离氨基酸和叶片叶绿素含量进行了比较研究。结果表明,不育系花药KT和IAA含量极显著低于保持系,GA含量不育系和保持系没有显著差异。游离氨基酸总量不育系花药极显著高于保持系,脯氨酸和胱氨酸含量不育系花药极显著低于保持系,赖氨酸含量两系无明显差异,苏氨酸在两系中均未检出,其余氨基酸含量均是不育系花药高于保持系;不育系花药中丝氨酸含量最高,保持系花药中脯氨酸含量最高,两系中天冬氨酸和脯氨酸含量的差异最大。叶片叶绿素含量不育系和保持系无显著差异。     

水稻花药液体培养下影响愈伤组织诱导和分化的一些因素的研究

水稻花药液体漂浮培养能大幅度提高愈伤组织诱导频率,但这些愈伤组织的分化能力很低。为了提高液体培养下愈伤组织的分化频率,试验了5种诱导培养基和4种分化培养基。结果表明,诱导培养基对愈伤组织分化能力的高低起主要作用,其中以过滤灭菌的马铃薯提取液培养基的效果最好,绿苗分化率可高达50％;分化培养基对愈伤组织分化频率的影响较小,且不甚规律。浮在液面上的愈伤组织比沉在培养液底部的愈伤组织有较高的分化能力。愈伤组织转移时间的早晚对分化频率也有很大影响。     

高粱不同类型胚性和非胚性愈伤组织的生理生化差异

植物体细胞胚胎发生过程中伴随着复杂的生理生化变化,为进一步揭示胚性愈伤组织的再生潜力,该研究以高粱Sb19未成熟胚诱导产生的两种胚性愈伤组织和一种非胚性愈伤组织为材料,通过测定各愈伤组织中可溶性蛋白、游离脯氨酸和可溶性糖的含量,采用方差分析法对高粱体细胞胚胎发生过程中不同类型愈伤组织的生理生化指标进行了差异比较研究。结果表明:(1)高粱两种胚性愈伤组织中可溶性蛋白、游离脯氨酸和可溶性糖的含量均显著高于非胚性愈伤组织,表明胚性愈伤组织中的代谢活性高于非胚性愈伤组织,能够为体细胞胚胎发生提供更多的物质能量基础。(2)两种类型胚性愈伤组织之间生理生化差异显著,其中,Ⅱ型胚性愈伤组织中可溶性蛋白和游离脯氨酸含量均显著高于Ⅰ型胚性愈伤组织,相反,Ⅱ型胚性愈伤组织中可溶性糖含量显著低于Ⅰ型胚性愈伤组织,这种生理生化差异在一定程度上影响了后期的分化。该研究结果为愈伤组织的胚胎发生能力与生化代谢的关系提供理论依据。     

Identification of Flavones in Thirteen Liliaceae Species

Changes in the levels of free and protein amino acids in the callus of Hiproly barley were studied during differentiation. Adventitious roots were formed in the callus after 90 days of cultivation on modified White’s medium containing 1 μm indoleacetic acid (IAA) and 200 μm adenine sulfate, and callus placed on Murashige–Skoog’s medium without plant hormones formed adventitious roots after 30 days of cultivation. During differentiation, protein amino acids in the callus decreased, then increased, without an appreciable compositional change in the protein amino acids. The amount of free amino acids in the callus increased with root formation. The major free amino acids during differentiation were glutamine, asparagine, alanine, and proline. Glutamine increased until roots were found in the callus after cultivation. Asparagine gradually increased during differentiation.     

Root exudates of plants

Summary Comparisons have been made of the amount and composition of seed and seedling exudates in barley, wheat, cucumber, and bean. Except in the case of wheat a greater proportion of the total nitrogen content in the seed exudates was formed by protein and peptide nitrogen than by nitrogen of free amino acids. In contrast, the greater part of the total nitrogen in the exudates of seedlings was formed by free amino acid nitrogen, except in the case of barley. Peptides represented 8 to 26 per cent of the protein and peptide fraction in seed exudates, the highest amount being found in bean. On an equal weight basis, the spectrum of amino acids released from seeds and seedlings differed little between barley, wheat and cucumber; greater differences were observed in bean exudates. Seedlings exuded reducing substances to a considerably greater extent than seeds. The spectrum of reducing sugars in seed and root exudates differed greatly, especially in the case of keto sugars. Differences in the organic acid spectra were small, except for bean plants, the seedling exudate of which contained more organic acids with a richer spectrum than seed exudate. Both seeds and seedlings of cucumber exuded a small quantity and a poor spectrum of organic acids.     

Anther Factor(s) in Barley Anther Culture

The effects of anther tissues were studied systematically on microspores forming multicellular units and furthermore on pollen callus formation in the anther culture of Hordeum vulgare (cv. Sabarlis). Anther productivity was found to be greatly enhanced by use of medium previously conditioned by anthers. In 15 experiments observed, anthers produced 26 times on average more calli in the conditioned medium than in control, in a few cases, even more than 80 times more calli formed. According to this, the authors supposed that cultured anthers released some components, anther factor (s) (AF), which is important to androgenesis in the culture. To achieve high yields of callus, culture was restricted to anthers which had been subpected to cold pretreatment. The temperature stress could not be replaced by the AF. However, for conditioning medium, anthers at binuclear stage were found to be more effective than the test anthers either with or without the pretreatment. Anthers from other 8 barley varieties were also effective for conditioning, as the difference of anther productivity still existed in the culture with conditioned medium between various genotypes tested. Anther response and callus yield were increased in both the culture of anthers at mid and late-uninuclear stage by use of conditioned medium. AF interacted synergistically with m-inositol. Cytological observation showed that AF increased apparently the formation of MPGs, while m-inositol mainly stimulated callus formation from MPGs. To some extent, the effect of exogenous hormone(s) could be replaced by AF. The anther response and pollen callus yield could be much enhanced by increasing anther inoculation density, which also raised the AF level in the culture. Thus, by use of the temperature stress prior to anther culture and culture of test anthers in conditioned medium with m-inositol, or at higher inoculation density, a very high production of pollen callus could be obtained in barley anther culture. For meeting the more specialized requirements of less responsive species or genopypes, the principles given here may be provide some basic information.     

The effect of nitrogen and sulphur on changes in nitrogen fractions of barley plants at various early stages of growth and on yield and amino acid composition of grain

Summary In a pot experiment the effects were estimated of varying nitrogen and sulphur applications on the total and nitrate nitrogen, free amino acid and amide and total amino acid concentrations of barley plants at five early stages of growth.Variations with time in contents of total and nitrate nitrogen differed at low and high nitrogen levels. Concentrations of free amino acids and amides were influenced by light intensity preceding sampling, age of plant and by levels of nitrogen and sulphur. At the fifth harvest (55 days old plants), high nitrogen addition without sulphur caused a fivefold increase in asparagine content and lesser increases in glycine and serine. Alterations in total amino acid contents with time were largely dependent on variations in total nitrogen. Sulphur deficiency, as judged by contents of S-amino acids and amides, became more pronounced with time. A positive interaction was found between effects of nitrogen and sulphur on the yield and cystine and methionine content of grain. Absence of sulphur reduced the cystine content of grain much more than that of methionine, particularly at the high nitrogen level.     

Engineering of the aspartate family biosynthetic pathway in barley (Hordeum vulgare L.) by transformation with heterologous genes encoding feed-back-insensitive aspartate kinase and dihydrodipicolinate synthase

In prokaryotes and plants the synthesis of the essential amino acids lysine and threonine is predominantly regulated by feed-back inhibition of aspartate kinase (AK) and dihydrodipicolinate synthase (DHPS). In order to modify the flux through the aspartate family pathway in barley and enhance the accumulation of the corresponding amino acids, we have generated transgenic barley plants that constitutively express mutant Escherichia coli genes encoding lysine feed-back insensitive forms of AK and DHPS. As a result, leaves of primary transformants (T₀) exhibited a 14-fold increase of free lysine and an 8-fold increase in free methionine. In mature seeds of the DHPS transgenics, there was a 2-fold increase in free lysine, arginine and asparagine and a 50% reduction in free proline, while no changes were observed in the seeds of the two AK transgenic lines analysed. When compared to that of control seeds, no differences were observed in the composition of total amino acids. The introduced genes were inherited in the T₁ generation where enzymic activities revealed a 2.3-fold increase of AK activity and a 4.0–9.5-fold increase for DHPS. T₁ seeds of DHPS transformants showed the same changes in free amino acids as observed in T₀ seeds. It is concluded that the aspartate family pathway may be genetically engineered by the introduction of genes coding for feed-back-insensitive enzymes, preferentially giving elevated levels of lysine and methionine.     

Seasonal changes in the free amino acids of oat and barley phloem sap in relation to plant growth stage and growth of Rhopalosiphum padi

The concentrations and composition of free amino acids in phloem sap from two cultivars of oats and barley, both susceptible to the aphid Rhopalosiphum padi, were determined by means of high performance liquid chromatography. Sap was collected from excised aphid stylets at three developmental stages (seedlings, tillering plants and plants undergoing stem elongation) from plants given or not given fertiliser and grown outdoors. In connection, the growth of individual R. padi nymphs was estimated at the same phenological stages on plants grown in the greenhouse. The content of free amino acids was consistently higher in seedlings than in plants at the early tillering stage. Only in seedlings did the addition of fertiliser increase amino acid levels. Barley phloem sap contained more free amino acids than that of oats when fertiliser was added and at later developmental stages. Phloem sap of oats and barley showed similar patterns in their composition of free amino acids at the seedling stage, but as the plants grew older the patterns became increasingly different. Plants given fertiliser had higher amounts of dicarboxylic amino acids (glutamic and aspartic acid) than unfertilised plants. The concentrations of γ-amino butyric acid, glycine, histidine, and methionine were very low in all treatments. The relative growth rates of R. padi nymphs were low when amino acid content was low and vice versa. The results are discussed in relation to host plant suitability and plant resistance mechanisms.     

Response of flax genotypes to doubled haploid production

Forty-four flax genotypes with a diverse genetic background were evaluated for anther culture response using a standard anther culture protocol in order to determine the feasibility to initiate a routine haploid production system in applied breeding programs. A strong genotype effect on callus induction and shoot regeneration in anther culture was found in this study. A number of genotypes, including two low cadmium content lines 96-11785 and 96-11826, a high oil content line 96-22109 and a high linolenic acid content line M 4919 were identified as highly responsive. The impact of the findings in this study on flax breeding was discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

大麦HvLEC1基因的克隆及其表达特征分析

植物LEC蛋白是NF-Y转录因子的一类B亚基,在植物胚状体形成过程中起重要作用。为了研究大麦小孢子体外培养形成胚状体的机理,本研究利用RACE技术在大麦中克隆了一个新的LEC基因,该基因cDNA全长为1004 bp,开放阅读框全长为597 bp,编码198个氨基酸,其蛋白1~59位氨基酸含有LEC结构域,命名为HvLEC1。HvLEC1在大麦的根、茎、叶和小孢子培养过程中均能表达,其中小孢子培养7 d时表达量最高,且HvLEC1在大麦品系BI04中的表达量比基19高,BI04愈伤产量也比基19高,表明HvLEC1表达量和愈伤产量有相关性,受盐胁迫后HvLEC1在大麦的根中快速上调表达,提示HvLEC1可能不仅参与小孢子胚状体发生,而且参与盐胁迫响应。     

Influence of some exogenous amino acids on the production of maize embryogenic callus and on endogenous amino acid content

The effects of four exogenous amino acids (proline, glycine, asparagine and serine) on the production of maize embryogenic callus and on its endogenous amino acid content have been investigated. For this purpose, an established embryogenic line of Type 1 callus from the inbred W64Ao2 has been used. From the results it may be concluded that a concentration of proline exceeding 6 mM is negative for the production of embryogenic callus. When proline is eliminated from the medium, other amino acids tested in certain concentrations yield a percentage of embryogenic callus production that exceeds or equals that of proline. The endogenous free proline content in embryogenic callus is significantly higher than that in non-embryogenic callus regardless of proline presence in the medium. The only exception are the glycine-containing media, in which endogenous free alanine of embryogenic callus increases at the expense of endogenous free proline. This study suggest a positive role of endogenous free proline or alanine accumulation in the embryogenic callus production which might be related to an adaptation to the metabolic changes produced by in vitro culture and embryogenesis induction. Furthermore, these results indicate that treatments with amino acids that are different from proline can be used to improve the efficiency of embryogenic callus production from well established maize callus cultures.Abbreviations Ala alanine - Asn asparagine - 2,4-d 2,4-dichlorophenoxyacetic acid - EC embryogenic callus - nEC non-embryogenic callus - Gaba gamma-aminobutyric acid - Glu glutamic acid - Gly glycine - Pro proline - Ser serine     

Ethylene production and embyogenesis from anther cultures of barley (Hordeum vulgare)

Summary Ethylene production was measured in cultured barley (Hordeum vulgare L.) anthers. The pattern of ethylene production and the content of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) were different among cultivars. Ethylene production appeared to be related to embryogenesis (callus and embryo production). In cultivars in which anthers had low amounts of ACC and produced ethylene slowly, the addition of ethylene promotors (Ethrel or ACC) increased embryogenesis. However, in the cultivar Klages, in which anthers had high amounts of ACC and produced ethylene rapidly, the addition of an ethylene production inhibitor (putrescine) increased embryogenesis. Thus, an optimum level of ethylene production appears to be important for embryogenesis. The differences in anther response and callus production among cultivars may be due to both the capacity to produce ethylene and the sensitivity to high ethylene levels.