秋水仙素对草莓离体叶片再生和多倍体诱导的影响

以草莓(Fragaria×ananassa Duch.)栽培品种'雪蜜'(2n=8X=56)的离体叶片为外植体,研究了不同浓度秋水仙素对愈伤组织诱导率、不定芽再生率以及多倍体植株诱导的影响,并采用流式细胞仪对多倍体植株的倍性进行鉴定.结果显示,用质量体积分数0.1%、0.3%、0.5%和0.7%的秋水仙素浸泡2、4和6 d,草莓离体叶片均能诱导出愈伤组织和不定芽,但随秋水仙素浓度的提高和处理时间的延长,愈伤组织诱导率和不定芽再生率均显著下降.用不同浓度秋水仙素处理均能产生多倍体植株,倍性为9X、10X、11X、12X、14X和16X;随秋水仙素浓度的提高,多倍体诱导率呈现先上升后下降的变化趋势.用质量体积分数0.3%秋水仙素浸泡处理4 d是最佳的草莓离体叶片诱导方法,不定芽再生率达到40.5%,多倍体诱导率为100.0%,并且诱导产生出16X的植株.     

大花萱草组培快繁体系的研究

以大花萱草的6个品种为试材,主要研究了其组织培养中的激素配比、外植体类型、基因型以及不定芽的生根条件。结果表明：适合于大花萱草紫蝶的最佳愈伤组织及不定芽诱导培养基为MS+1 mg·L^-1 6-BA+0.1 mg·L^-1 NAA,出愈率、分化率和平均出芽数分别为63.33%、91.11%和5.86;但是,上述指标在基因型间表现出差异;花茎是诱导不定芽的最佳外植体;1/2MS+0.2 mg·L^-1 NAA是紫蝶较适宜的生根培养基,生根率81.11%,平均生根数6.08;本实验建立了3个品种的再生体系,平均出芽数均在4个以上,其中“金娃娃”最高,是6.88。     

红掌遗传转化受体系统的建立Ⅰ离体高效培养体系的建立

以红掌叶片和叶柄为材料,研究了影响红掌愈伤组织的诱导和分化的因素.结果表明,基因型对愈伤组织诱导有显著的影响,Pink Champion愈伤组织诱导率最高,为90%.基本培养基对愈伤组织诱导影响显著.在改良MS培养基上,愈伤组织诱导率为85%,出愈伤多,质量高.消毒时间和接种方式对愈伤组织诱导率亦有显著影响.初展开叶片用氯化汞消毒8-10min,背面向下接入培养基,愈伤组织诱导率高.外源激素对愈伤组织的诱导和芽分化影响显著.单独使用BA不能诱导红掌叶片产生愈伤组织,高浓度BA、低浓度2,4-D时,愈伤组织诱导率高.在MS+BA0.5 mg·L-1+NAA 0.5 mg·L-1+CM 5%培养基上,芽分化率为97.5%,平均芽分化数为4.5个/块,芽粗壮.将分化出的芽转入1/2 MS+NAA 0.2 mg·L-1+AC 1 g·L-1培养基上诱导生根,生根率达100%.通过上述研究建立了红掌离体高效培养系统.     

不同基因型玉米愈伤组织诱导与植株再生研究

以5个玉米品系幼胚为外植体,研究了基因型、2,4-D浓度以及胚龄对愈伤组织诱导的影响;6-BA对愈伤组织分化的影响;以及IBA对再生芽生根的影响。结果表明：除。31外,其他基因型的外植体在相同条件下均可诱导出愈伤组织,但是不同基因型间存在显著差异;2,4-D浓度和胚龄显著影响愈伤组织的诱导,且2,4-D浓度为2．0mg／L,胚龄在11—13d之间时,玉米愈伤组织诱导率较高且质量较好。将愈伤组织转入分化培养基后,6-BA促进了愈伤组织的再分化;在生根培养基中,IBA促进了再生芽生根,经过炼苗后移栽获得再生植株。     

催吐萝芙木离体培养和植株再生体系的建立

为建立催吐萝芙木(Rauvolfia vomitoria Afzel.)的快繁再生体系,以茎段为外植体,比较了植物生长调节剂对其愈伤组织诱导、分化及生根的影响。结果表明,诱导愈伤组织的适宜培养基为MS+2,4-D 1.0 mg L~(–1)+TDZ 0.5 mg L~(–1)或MS+2,4-D2.0 mg L~(–1)+TDZ 0.5 mg L~(–1),出愈率达100%且生长状况良好;诱导丛生芽的最佳培养基为MS+6-BA 3.0 mg L~(–1)+NAA 0.1 mg L~(–1),出芽率为46.6%,平均出芽数为3.04。这为催吐萝芙木的快速繁殖和遗传转化研究奠定了基础。     

尾叶桉茎段再生系统的建立

本文以尾叶桉(Eucalypt urophylla)U6的无菌苗茎段为材料,选用MS培养基为基本培养基,研究不同IAA浓度对尾叶桉茎段愈伤组织诱导,不同IAA、6-BA浓度对尾叶桉茎段愈伤组织诱导芽、生根的影响。结果表明,当外植体的接种数一样,IAA的浓度为20.0mg/L时,愈伤组织诱导率达到最高,为95%,且此浓度下,出芽率、生根率最高,分别达43.3%和100%,而添加0.5mg/L6-BA则会抑制芽的再生、不能产生根。因此最适合诱导尾叶桉愈伤组织、再生芽和再生根的培养体系是MS+20.0mg/L IAA+30g/L蔗糖+8g/L琼脂。该再生系统的建立将为转基因技术改良桉树性状提供前提条件。     

韭菜胚根高效植株再生体系的建立

为了建立韭菜高频植株再生体系,以韭菜胚根的不同部位为外植体,研究了不同部位愈伤组织和芽的诱导。结果表明,胚根尖为诱导愈伤组织的最佳外植体,愈伤诱导率达到81.4%,出芽率达到73.3%;比较在不同激素配方上的愈伤组织诱导和芽的分化,在MS+1 mg/L BA+0.5 mg/L NAA+0.5 mg/L KT培养基中再生率最高,诱愈率达到80%以上,不定芽分化率达78.8%,繁殖系数为24.7;在MS+1 mg/L GA+0.5 mg/L KT不定芽伸长培养基中,30 d不定芽伸长为独立植株,比例为85.5%;独立的韭菜苗在1/2MS+0.3 mg/L IAA中生根率及根系状况最佳。通过该方法 130 d左右即可从胚根脱分化再生出植株。     

中药半夏单细胞悬浮培养、胚胎发生及植株再生

以悬浮细胞作为靶标材料,在COLC诱导获得多倍性细胞后,经诱导分化和再生形成纯合的多倍体植株,是一条可行且高效的多倍体诱导途径。但该途径的前提是成熟、高效的单细胞悬浮培养和植株再生体系建立。鉴于此,在获得优良胚性愈伤组织的基础上,对半夏细胞悬浮培养、细胞分化及再生植株所涉及的关键问题及其影响因素进行研究,结果获得了以近圆形细胞为主的高质量单细胞悬浮系,细胞生长曲线呈典型的"S"型,细胞密度可达2.0×106以上,活细胞率可达86.80%,近圆形细胞率可达82.64%;明确了其细胞胚胎发生的两种可能途径,并通过诱导细胞分化,成功获得了愈伤组织增殖,最终实现了高效的植株再生,愈伤分化率可达94.46%,丛生芽诱导率可达337.42%,幼芽成苗率可达90.75%,幼苗生根率可达95.40%,移栽成活率可达98.83%,从而建立了高效的半夏悬浮细胞培养和植株再生体系,为后续的基于单细胞水平的半夏多倍体诱导研究提供技术参考和科学依据。     

红掌花药培养

研究了发育时期、基因型、培养基、低温预处理等因素对红掌花药愈伤组织诱导的影响.结果表明,小孢子中晚期是红掌花药培养的适宜时期;基因型对花药膨大率有显著的影响;不同培养基上的Sweet Dream和Jungle Bush的花药膨大率差异显著;低温预处理明显提高Sweet Dream的花药膨大率.从Sweet Dream花药诱导出致密和疏松两种愈伤组织,两种愈伤组织芽分化率和生根率存在明显差异,致密愈伤组织的小苗生根率为95.00%,而疏松愈伤组织的小苗生根率为30.00%.Sweet Dream的花药再生植株与叶片再生植株在形态特征上有差异,染色体鉴定结果表明,花药再生植株均是二倍体.     

商陆离体再生体系的构建

目的:建立商陆离体再生体系。方法:选取商陆的幼茎、茎节、叶片、叶柄和顶芽为外植体,以MS作为基本培养基,通过添加不同浓度配比的植物生长调节剂分别进行愈伤组织、丛生芽和生根诱导,筛选商陆离体再生体系方案。结果:顶芽和幼茎为外植体诱导的愈伤组织出愈时间早,愈伤组织质量高,以培养基MS+6-BA0.5 mg/L+2.4-D 0.5 mg/L的诱导率最高,达到100%;其中,只有以顶芽产生的愈伤组织才能分化出丛生芽,芽分化培养基为MS+6-BA 2.0 mg/L+NAA 0.25 mg/L,诱导率为98%;诱导生根的适宜培养基为1/2 MS+NAA 0.3 mg/L,诱导率达100%。结论:建立和完善了商陆离体再生体系方案,为商陆遗传转化体系的构建奠定了基础。     

The effects of the physical characteristics of the culture medium on the development of red seaweeds in tissue culture

Explants of Gelidium versicolor, Grateloupia doryphora and Laurencia sp. were cultivated in Provasoli enriched seawater culture medium (PES) adjusted to several osmolalities (0.5, 0.7, 1.0 and 1.5 Os kg^–1) and solidities (agar concentration = 3, 8 and 15 g L^–1). Osmolality was adjusted by dilution of seawater with distilled water (50, 70 and 100% seawater) and by NaCl addition. Explants of Laurencia sp. and Grateloupia doryphora showed bud regeneration and callus formation. Explants of Gelidium versicolor only showed bud regeneration. Osmolalities of 0.5 and 1.05 Os kg^–1. inhibited or drastically reduced bud regeneration and callus formation. The highest callus formation and bud regeneration were observed at 0.7 to 1.0 Os kg^–1. An increase in the agar concentration of the culture medium was positively correlated with callus formation and negatively correlated with bud regeneration. An increase in the percentage of seawater increased the solidity of the culture medium and was positively correlated with callus formation. Glycerol was an effective carbon source for the vegetative propagation of axenic explants of Grateloupia doryphora, promoting growth and bud regeneration. An increase in glycerol concentration in the culture medium increased its osmolality, inhibiting the growth of the explants and their morphogenetic development.     

Efficient plant regeneration from leaves of rapeseed (Brassica napus L.): the influence of AgNO3 and genotype

Factors influencing reliable shoot regeneration from leaf explants of rapeseed (Brassica napus L.) were examined. Addition of AgNO₃ to callus induction medium was significantly effective for shoot regeneration in all three genotypes initially tested. When 48 genotypes subsequently were surveyed, a large variation of shoot regenerability was observed, ranging from 100 to 0% in frequency of bud formation and from 7.5 to 0 in the number of buds per explant. A significant correlation (r=0.84) was observed between the frequency of bud formation and the number of buds per explant. The shoot regenerability from leaf explants was not related to that from cotyledonary explants (r=0.28). Histological observations showed that an organized structure developed from calluses produced at vascular bundle tissues after 7 days of culture on callus induction medium, and they developed shoot apical meristems one week after transfer onto shoot induction medium. Regenerated plantlets were obtained 2 months after the initiation of culture and they normally flowered and set seeds. No alterations of morphology or DNA contents were observed in regenerated plants and their S1 progenies.     

Screening wheat genotypes for high callus induction and regeneration capability from anther and immature embryo cultures

Plant regeneration via tissue culture varies with the genotype and is an important factor in establishing cell selection and genetic transformation systems. To select genotypes – especially Japanese ones – with a high regeneration capability, we screened 107 wheat genotypes (78 domestic, 29 foreign) for callus induction and regeneration capability from anther and immature embryo cultures. For anther culture, 83 of 107 genotypes tested induced calli and 45 regenerated plants. Only 9 genotypes, however, produced green plants, 25 produced only albino plants, and 11 produced both green and albino plants. Glennson 81 was the highest in callus induction, followed by Orofen, Danchi–komugi and Chris. The genotypes with a relatively high regeneration capability were Framala 80 at 24% and Glennson 81 at 19%, these two genotypes produced only green plants. For immature embryo culture, 97 genotypes showed a 90% callus induction rate and 74 genotypes regenerated plants. Very few genotypes produced albino plants. The genotypes with a high regeneration capability were Genaro 81 at 90%, Chinese Spring at 80%, and Norin 75 at 75%. This revised version was published online in June 2006 with corrections to the Cover Date.     

Estimation of the callus formation and regeneration efficiency in spring varieties of barley zoned in Ukraine

Eight commercial varieties of spring barley (Getman, Tabora, Adagio, Galaktik, Europrestige, Korona, Nevada, and Stalker) were used for the in vitro culture establishment and the development of an efficient protocol for the induction of the callus formation and plant regeneration. Mature embroys of the above-mentioned genotypes were used as explants. The induction of callus formation, culture passage, and plant regeneration were carried out using a medium containing MS salts supplemented with casein hydrolysate (1 g/l), L-proline (690 mg/l), thiamine-HCI (1 mg/l), maltose (30 g/l), 2,4-D (2 mg/l), CuSO4 (12.5 mg/l), myoinositol (250 mg/1), and gelrite (3.5 g/l) at pH 5,6–5,8. The ability of callus formation was observed in all studied varieties, and the callus formation’s frequency varied from 65 ± 3.4% to 100%. The highest regeneration potential was revealed for the Korona (88 ± 2,8%), Europrestige (89 ± 6.5%), Tabora (93 ± 3.4%), Getman (99 ± 0.8%), and Nevada (100%) varieties. For all cultivars the generation of plants from a calli occurred via the organogenesis and somatic embryogenesis. The highest total regeneration potential was registered for the Getman variety (50 ± 5%), which was selected for the further development of effective genetic transformation protocols.     

影响小麦成熟胚培养及植株再生因素的研究

对3个不同栽培品种小麦的成熟胚进行离体培养,研究影响小麦愈伤组织诱导和植株再生的一些因素。结果表明,东农7742的苗分化率明显高于龙麦9814和龙麦26;高浓度的玉米素可明显提高芽的分化率;附加低浓度NAA的1/2 MS培养基可有效促进生根。可见,基因型对小麦愈伤组织的分化有很大的影响,附加一定的外源激素有利于提高植株的再生频率。     

Effect of gelling agents and antibiotics on adventitious bud regeneration from In vitro leaves of pear

Summary The effect of the type of gelling agent and of several antibiotics on the adventitious bud regeneration from in vitro leaves was tested on eight pear genotypes. The use of gellan gum (Phytagel™) in the medium instead of agar had a very strong positive effect on the rate of adventitious bud regeneration for all pear genotypes tested in this study. This gelling agent induced faster cell divisions than agar, thus more callus was produced on wound sites and subsequently more buds regenerated. Incubation on gellan gum medium during the first 20 d of bud induction was sufficient to induce a stimulatory effect on regeneration and limited the production of hyperhydric buds. In the prospect of Agrobacterium transformation, the effect of several antibiotics was tested. Cefotaxime (200 mg/l) plus ticarcillin/clavulanic acid (100 mg/l) could be used in the culture medium without affecting the frequency of bud regeneration. The inhibition of bud regeneration was obtained with different kanamycin concentrations according to the gelling agent in the medium. On gellan gum medium, a concentration of 100 mg/l of kanamycin was suitable. These conditions can be recommended for experiments on Agrobacterium-mediated transformation of pear, where bacterial inoculation and presence of antibiotics generally reduce and delay bud regeneration.     

Cytogenetic analysis of plants regenerated from colchicine-treated callus cultures of an interspecificHordeum hybrid

Summary Hybrids of Hordeum vulgare (HV) x H. jubatum (HJ) were synthesized for purposes of introgressive breeding, but were sterile and the recovery of pure diploid tillers by colchicine applications in vivo was difficult. Plant regeneration from colchicine-treated callus cultures of the hybrid (HV x HJ) was investigated as a means to produce high numbers of pure diploid, fertile intermediates. 10 of 50 plants regenerated in this manner exhibited variable chromosome numbers with means of approximately 37 (expected diploid number = 42). Cytological examinations of microsporogenesis in all such plants revealed a high incidence of bivalent formation at metaphase I (as compared to nearly complete asynapsis in the F₁), but spindle and chromosome abnormalities in later meiotic stages led to complete sterility. Approximately 40% of HJ plants regenerated from colchicine-treated calli appeared to be pure tetraploids of high fertility. These techniques are hence useful for high frequency production of diploid or polyploid plants.     

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.     

诱导突变高粱愈伤组织初探

本实验以突变体高粱的不同外植体 ,成熟种子、成熟胚、茎尖、和幼胚等为材料 ,诱导愈伤组织。经出愈率和生长状况观察 ,幼胚最好 ,成熟胚较好 ,成熟种子和茎尖略差。对于茎尖 ,2 ,4 D与KT搭配较好 ;成熟胚而言 ,不加细胞分裂素 ,加适量 2 ,4 D浓度效果较好 ,幼胚愈伤组织诱导和培养不需要细胞分裂素 ,加浓度为 2 .0mg/L2 ,4 D效果较好 ;若加KT ,同时要提高 2 ,4 D浓度 ;成熟种子培养 ,需要细胞分裂素