低温和PEG预处理对小麦愈伤组织形成及IAA氧化的影响

研究了低温和ＰＥＧ预处理对小麦成熟胚愈伤组织形成及ＩＡＡ氧化的影响。结果表明,低温和ＰＥＧ预处理促进两种小麦成熟胚愈伤组织形成,但对其ＩＡＡ氧化的影响不同;其中低温引起‘郑引１号’ＩＡＡ氧化酶活性降低,但对‘保７０５９’的影响不大;而ＰＥＧ使‘保７０５９’的ＩＡＡ氧化酶活性增加,但对‘郑引１号’无明显影响;两种处理均使两种小麦ＩＡＡ过氧化物酶活性降低。     

低温与植物生长调节剂预处理对小麦成熟胚培养特性的影响

为提高小麦成熟胚愈伤组织的分化率,以3个黄淮麦区冬小麦品种(系)‘小偃22’、‘西农1013’和‘千斤早’为材料,研究了4℃低温和3种植物生长调节剂(TDZ、2,4-D、多效唑)预处理小麦种子对小麦成熟胚愈伤组织分化特性的影响。结果表明:4℃低温和植物生长调节剂预处理对小麦成熟胚出愈率无显著影响,但能够显著促进愈伤组织的分化,并表现出处理及基因型间差异。‘小偃22’、‘西农1013’、‘千斤早’感应低温预处理的最好时间节点分别为6h、12h、12h,其成熟胚愈伤组织的分化率比对照分别显著提高16.2%、14.2%、12.8%;3个材料分别在10mg/L、5 mg/L、20 mg/L TDZ预处理下成熟胚愈伤组织分化率最高,比对照分别显著提高39.1%、29.7%、16.7%;3个材料的最佳2,4-D预处理浓度均为10mg/L,其愈伤组织分化率分别比对照显著提高17.3%、11.2%、25.2%;3个材料的成熟胚愈伤组织分化率分别在10mg/L、20mg/L、20mg/L多效唑预处理时最高,分别比对照显著提高6.2%、11.6%、7.2%。研究表明,低温和植物生长调节剂预处理均可有效提高小麦成熟胚愈伤组织的分化率,但促进植物生长的调节剂(TDZ和2,4-D)对小麦成熟胚愈伤组织再分化的效应远大于抑制植物生长的调节剂(多效唑)。     

小麦遗传转化受体系统建立的研究

选用‘小偃22’和‘宁春16’小麦品种的成熟胚和幼胚进行培养,研究不同种类的胚和培养因子对愈伤组织诱导和分化的影响。结果表明,幼胚和成熟胚的愈伤组织诱导率无明显差异,但较高浓度的2,4-D有利于成熟胚的诱导,而幼胚培养时2,4-D浓度的影响效果因品种而异;两种外植体分化率的高低与KT/IAA的配比均有密切关系,但高浓度的激素水平不利于成熟胚的分化;诱导培养基中低浓度的2,4-D有利于所诱导的愈伤组织的分化。同时,在诱导培养基中添加低浓度的KT能显著提高两品种成熟胚愈伤组织的分化率;各种培养基处理与品种间都存在显著的互作效应,‘小偃22’成熟胚培养的最佳培养基组合为MSD 3.0 mg/L 2,4-D和MSD 0.5 mg/LIAA 1.0 mg/L KT,幼胚培养为MSD 4.0 mg/L 2,4-D和MSD 0.5 mg/L IAA 1.0 mg/L KT;‘宁春16’成熟胚培养为MSD 4.0 mg/L 2,4-D和MSD 1.0 mg/L IAA 1.0 mg/L KT,幼胚培养时为MSD 1.0 mg/L 2,4-D和MSD 2.0 mg/L IAA 2.0 mg/L KT。     

PEG胁迫及复水对不同抗旱性小麦幼苗脯氨酸代谢关键酶活性的影响

以两种不同抗旱性小麦品种幼苗为试验材料,采用PEG模拟干旱胁迫处理,探究干旱胁迫及复水对小麦幼苗叶片与根系脯氨酸累积及关键酶活性的影响。结果显示:(1)PEG胁迫下抗旱品种‘普冰143’根长和根干重下降不大,而水敏感品种‘郑引1号’根长和根干重下降显著;且于胁迫处理36h时‘普冰143’根系脯氨酸含量增加(75.0%)显著大于‘郑引1号’(37.7%),复水24h后均恢复至对照水平。(2)PEG胁迫下‘普冰143’叶片中谷氨酸合成途径关键酶P5CS和鸟氨酸合成途径关键酶δ-OAT活性均显著增加,且‘普冰143’叶片脯氨酸两条合成途径关键酶活性均得以加强;PEG胁迫处理36h时,‘郑引1号’叶片中P5CS活性增加显著,δ-OAT活性变化较小,且‘郑引1号’叶片脯氨酸合成可能以谷氨酸途径为主;但在PEG胁迫下两个不同抗旱性品种的根中P5CS、δ-OAT活性均变化较小。(3)PEG胁迫处理36h时‘普冰143’叶片脯氨酸降解酶PDH活性显著下降,而‘郑引1号’叶片PDH活性显著增加,复水后抗旱品种叶片该酶活性显著增加,水敏感品种恢复至对照水平;但PEG胁迫处理下两个不同抗旱性品种的根中PDH活性均显著下降。研究表明,PEG胁迫下小麦叶片是合成脯氨酸的主要部位,抗旱品种‘普冰143’根系脯氨酸持续积累与叶片中高的脯氨酸合成关键酶活性及脯氨酸转运有关。     

小麦成熟胚愈伤组织诱导及分化研究

以2个小麦品种成熟胚为外植体进行离体培养,研究了不同预处理、不同2,4-D浓度及与KT组合、不同蔗糖浓度等因素对愈伤组织诱导及分化的影响。结果表明:4℃低温预处理可提高愈伤组织的出愈率及再生苗率,2个材料的出愈率及再生苗率均达到90%和30%以上;在不同预处理条件下,2,4-D浓度对出愈率及再生苗率的影响与基因型有关,2,4-D浓度为1~2 mg/L更有利于愈伤组织诱导及分化;附加KT能缓解高浓度2,4-D对再生苗率的抑制作用,而对于在1、2 mg/L 2,4-D的培养基中附加KT则不表现这种作用;蔗糖浓度则在30 g/L条件下更有利于愈伤组织诱导。因此通过4℃低温预处理,在MS基本培养基中附加1~2mg/L 2,4-D及30 g/L蔗糖亦可促进小麦成熟胚愈伤组织的诱导和分化。     

2,4-D和激动素对烟草愈伤组织IAA氧化酶和细胞分裂素氧化酶活性的影响

2,4-D和激动素(KT)均显著降低烟草愈伤组织中IAA氧化酶和细胞分裂素氧化酶的活性,KT的影响更显著.在MS中的愈伤组织IAA氧化酶活性最高,MS 2,4-D中的次之,MS KT和MS 2,4-D KT中的最低.愈伤组织在MS中继代6 d时,细胞分裂素氧化酶活性出现明显的高峰,在其它3种培养基中则没有.     

蔗糖与激素对小麦幼穗体细胞无性系形成及生长特性的影响研究

通过在诱导培养基中添加不同浓度的蔗糖和激素,研究了蔗糖与激素对小麦幼穗体细胞无性系形成及生长特性的影响。结果表明;较高浓度的2,4-D有利于小麦幼穗愈伤组织的形成与生长,但低浓度的2,4-D更有利于胚性愈伤组织的形成,低浓度的KT能显著促进小麦幼穗愈伤组织的生长和胚性愈伤组织的形成,而高浓度的KT不利于小麦幼穗体细胞无性系的形成和生长;蔗糖有利于小麦幼穗胚性愈伤组织的形成,在2.5%-5.5%浓度范围内,随蔗糖浓度的提高,胚性愈伤组织的形成率也随之提高。     

水分胁迫下小麦类脱水素基因表达的半定量RT-PCR分析

以‘郑引1号’小麦为材料,经水分胁迫后进行RT-PCR扩增,结果在胁迫条件下获得500bp的脱水素基因(wzy1-1)。利用半定量RT-PCR方法,分析‘陕合6号’和‘郑引1号’小麦在正常供水条件下及PEG6000胁迫后18、24和42h,以及复水6和12h时wzy1-1在叶片中的表达。结果表明:2个小麦品种中wzy1-1在水分胁迫后18h均有表达,至胁迫24、48h时表达量较18h均有所增多;复水6h后该基因表达迅速降低,至复水后12h表达消失。且该基因在‘陕合6号’(抗旱性较强)叶片中表达量较‘郑引1号’(抗旱性较弱)高,表明该基因的表达与小麦的抗旱性密切相关。     

渗透胁迫条件下小麦成熟胚愈伤组织的诱导及植株再生的研究

用不同浓度的PEG6000及NaCl对5个小麦品种的成熟胚组织培养物进行处理,研究在渗透胁迫条件下基因型和激素对成熟胚愈伤组织的诱导及分化的影响。结果表明,小麦整株水平与细胞水平的抗性存在一定相关,不同基因型对干旱与盐胁迫的敏感程度不同,成熟胚愈伤组织的诱导率和植株再生率表现出明显的差异。初步得到了晋麦47、长武134、红芒麦的耐旱愈伤组织以及晋麦47、长武134的耐盐愈伤组织,并获得了晋麦47和长武134具有一定抗性的再生芽。     

吲跥乙酸及椰子乳汁对向日葵离体胚生长的影响

五、摘要根据向日葵胚(约5毫米长)的离体培养试验,我们得出下列结论: 1.IAA(0.05—20ppm)对于生长有促进作用,表现在鮮重的显著增加。IAA有引起胚愈伤组织形成的作用,1ppm IAA约有1/4的胚长愈伤组织,5ppm的约2/3,10ppm以上的100％胚长愈伤组织。IAA在低浓度时(0.05—0.1ppm)对根的形成与生长有促进作用,在高浓度时(10—20ppm)对根的形成与生长有明显抑制作用。 2.椰子乳汁(经高压消毒)对苗的生长有显著促进作用,对于根的生长有抑制作用,这种现象在培养前两周差别特别明显,在后期差别逐渐缩小。高浓度的椰子乳汁(20％以上)也可以引起愈伤组织的形成,但不如IAA有效。 3.当IAA和椰子乳汁混合处理时,对于生长的影响及愈伤组织的形成表现得更为明显。而椰子汁与IAA的作用有某些类似之处,但不能表明椰子乳汁中起作用的物质只是一种IAA。     

Analysis of biochemical and physiological changes in wheat tissue culture using different germplasms and explant types

To select adequate wheat germplasms for genetic transformation, tissue culture efficiency of 21 different wheat lines (Einkorn, Emmer, Durum wheat, etc.) were compared, along with two different explants, namely, immature embryo and mature embryo. The results showed that the average differentiation rate and regeneration rate of immature embryo calli (46.5 and 20.82 %) were better than those for mature embryo calli (14.03 and 4.37 %). The best genotypes for immature embryo callus culture were ‘Ningchun 16’ and ‘Ei 15’, ‘Xiaoyan 22’, followed by ‘Durum 332’ and ‘Tr 256’. The best genotypes for mature embryo callus culture were ‘Ying 4286’, ‘Yunyin 01’, and ‘Xiaoyan 22’. To analyze how physiological and biochemical settings influence the totipotency of calli, different physiological and biochemical indices were analyzed. Differences between immature embryo callus and mature embryo callus were significant, as well as differences of most indices among different wheat types. The interaction effects between explant types and genotypes were also significant. Correlation analysis results showed that the total phenol and soluble sugar contents were significantly correlated with callus differentiation and regeneration rates.     

High efficiency plant regeneration by somatic embryogenesis from callus of mature embryo explants of bread wheat (Triticum aestivum) and grain sorghum (Sorghum bicolor)

Summary Tissue culture methods were developed for reproducible induction and maintenance of embryogenic (E) callus established from developmentally mature embryo explants of bread wheat (Triticum aestivum) and grain sorghum (Sorghum bicolor). Embryogenic callus was obtained by culturing seeds and mature embryos of wheat on Linsmaier and Skoog’s (LS) medium containing 5 or 2 mg/liter 2,4-dichlorophenoxyacetic acid (2,4-D), respectively, and for sorghum mature embryos on LS medium containing 2 mg/1 2,4-D plus 0.5 mg/liter kinetin. Plant regeneration from E callus was achieved for several months and quantified on a fresh-weight basis of E callus. Phenotypically normal plants were regenerated from E callus cultured on LS medium supplemented with 0.1 mg/liter IAA plus 0.5 mg/liter benzyladenine (BA) for wheat and 1.0 mg/liter IAA plus 0.5 mg/1BA for sorghum. Wheat research was funded by the United States Agency for International Development, Washington, DC, cooperative agreement DNA-4137-A-00-4-53-00. Sorghum research was supported by the Gas Research Institute, Chicago, IL, contract 5084-260-0973. Expert technical asistance was provided by Nitschka S. ter Kuile, Barbara J. Ashton, Laurie Osborne, Erin Scott, and Kathleen M. Petersen.     

Callus formation and plant regeneration from immature and mature embryos of rye (Secale cereale L.)

Summary An efficient protocol was developed to regenerate entire plants from immature embryos of elite genotypes of rye as a prerequisite to plant transformation. Three winter genotypes and one spring genotype were tested using both immature and mature embryos as explants. Four types of callus initiation media and five kinds of regeneration media were tested in all possible combinations. Immature embryos gave much higher levels of plant regeneration than mature embryos, but mature embryos could be induced to regenerate plants for all genotypes and media tested, although at low levels. A minimum stage of embryo development must be reached before embryos can be cultured successfully. Genotypic effects were less pronounced than those reported for inbred cereal species such as wheat and barley, but there was an effect of genotype on percentage of callus formation. There was a significant interaction between genotype and initiation media. Composition of the initiation media affected both the percentage of callus formation from embryos and subsequent frequencies of plant regeneration. Composition of the regeneration media had no effect on level of plant regeneration. Immature embryos of all genotypes tested could be induced to produce 90–100% callus on appropriate initiation media and all regenerated shoots from approximately one-half to three-quarters of the calluses produced.     

Cytokinin control in subcellular localization of indoleacetic acid oxidase and peroxidase

IAA oxidase and peroxidase were found in all subcellular fractions of tobacco callus cells. The bound and cytoplasmic fractions differed greatly in IAA oxidase/peroxidase ratio and in isoperoxidase composition. The IAA oxidase/peroxidase ratio was particularly high in the plasma membrane-rich fraction. Kinetin had profound effects on IAA oxidase and peroxidase. The appearance of fast migrating isoperoxidases in response to 0·2 μM kinetin was found only in cytoplasmic, plasma membrane and ribosome-rich fractions; a high concentration of kinetin inhibited their formation. High kinetin concentrations also lowered the specific activity of IAA oxidase and peroxidase in all subcellular fractions, but the effect was much greater on peroxidase than on IAA oxidase, thus resulting in a drastic increase in IAA oxidase/peroxidase ratio. Evidently the activities of IAA oxidase and peroxidase were not equivalent and should be considered separately.     

The effect of auxins, time exposure to auxin and genotypes on somatic embryogenesis from mature embryos of wheat

The effects of different factors on the embryogenesis and plant regeneration from mature embryos of Russian spring and winter genotypes were studied. Embryogenic callus induction was achieved on MS medium supplemented with different concentrations of 2,4-D (2,4-dichlorophenoxyacetic acid), 2,4,5-T (2,4,5-trichlorophenoxyacetic acid) or Dicamba (3,6-dichloro-o-anisic acid). Although all auxins were able to induce callus from explants with high frequency (98–100%), Dicamba was more effective for the induction of embryogenic callus (21.8–38.3%). Maximum embryogenic callus formation and high number of regenerated plants were observed at 12 mg l⁻¹ of Dicamba. The time exposure to Dicamba (7, 14, 21 and 28 days) had a significant effect on efficiency of somatic embryogenesis. When contact of explants with callus induction medium was increased from 7 to 21 days the rate of somatic embryogenesis and number of regenerated plants per embryogenic callus gradually increased from 13.0 to 38.4% and 3.6 to 8.0%, respectively. Supplement of additional auxins (indoleacetic acid (IAA), indolebutyric acid (IBA), and naphthaleneacetic acid (NAA)) to callus induction medium with Dicamba had a positive effect on the rate of embryogenic callus formation, while the average number of regenerated shoots was not affected. The best rate of somatic embryogenesis was observed at the addition of 0.5 mg l⁻¹ IAA with Dicamba (61.0%). The optimum combination of Dicamba and IAA increased the efficiency of somatic embryogenesis and plant regeneration from seven spring and winter wheat genotypes, thought overall morphogenic capacity was still genotype dependent.     

水分胁迫对小麦根质膜H~＋－ATPase活性与H~＋分泌的影响

在渗透势为－０．５和－１．０ＭＰａＰＥＧ处理下,不抗旱的郑引一号小麦根ＰＭＨ＋－ＡＴＰａｓｅ活性分别下降４５％和６５％,抗旱品种陕合六号则增加了１１％和１２％。小麦根组织Ｈ＋分泌与ＰＭＨ＋－ＡＴＰａｓｅ活性的变化趋势基本一致,即随着胁强增加,郑引一号Ｈ＋分泌下降,陕合六号Ｈ＋分泌是先升高后略降。Ｎａ３ＶＯ４和ＤＣＣＤ对Ｈ＋分泌有不同程度的抑制,品种之间没有明显的差异。外源电子受体Ｆｅ（ＣＮ）６３－的加入促进了小麦根Ｈ＋分泌,陕合六号增加２５％－３９％,郑引一号增加２１％－４５％。与此同时,Ｎａ３ＶＯ４没有表现出对Ｈ＋分泌的抑制作用。     

水分胁迫对棉花叶片中IAA从含量、IAA氧化酶和过氧化物酶活性的影响

整株干旱降低盐棉46号叶片中的IAA总量,叶龄愈小下降愈多。幼叶中IAA总量的下降主要是结合态IAA减少的结果。气干和-1.7MPa PEG溶液渗透胁迫处理也降低离体成熟叶片的IAA总量,其变化与叶片含水量呈直线相关(r=0.905)。整株干旱处理提高各叶片的过氧化物酶活性,叶龄愈小提高愈多,但IAA氧化酶活性无显著变化。离体和整株干旱时IAA总量的下降,可能是过氧化物酶活性增加所致。