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。     

籼型杂交稻亲本成熟胚愈伤组织再生体系的建立

本文给出了6个籼型杂交稻亲本成熟胚愈伤组织再生体系建立优化措施。采用6个有重要育种价值的杂交籼稻亲本成熟胚盾片诱导愈伤组织作为分化再生的外植体,通过调节2,4-D浓度、培养基成分、接种方式、激素组合和愈伤组织分化途径,建立适合籼稻遗传转化的再生体系。结果表明,MB培养基是较为合适的愈伤组织诱导培养基类型,6个品种在MB培养基上的愈伤组织诱导率均在60％-80％之间。半粒米的接种方式能够明显提高愈伤组织诱导率,提高幅度达到28．2％。通过调节2,4-D浓度和激素组合,愈伤组织诱导率最高可达到97．9％,两步分化法和适当干燥处理能够提高愈伤组织的分化效率,6品种愈伤组织分化率均在50％-90％之间。初步建立了6个杂交籼稻亲本品种成熟胚愈伤组织的再生体系,为以后遗传转化奠定基础。     

影响小麦成熟胚再生频率因素的研究(简报)

以小麦成熟胚为外植体,研究了基本培养基、预处理类型、接种方式、植物激素的浓度和不同组合以及分化培养基中是否添加抗生素对愈伤组织诱导和分化的影响．在此基础上建立了一套高效的小麦成熟胚植株再生系统。经过试验,我们选择在MS培养基上接种经无菌水预处理的纵切成熟胚作为起始的试验条件。在含2mg／L2,4-D的MS培养基上．初级愈伤组织的诱导频率可达80％以上．在继代培养基中添加0．5mg／L6-BA和0．2mg／LNAA可以显著提高胚性愈伤组织的产生。而在再生培养基中加入适当浓度的头孢霉素可以有效提高胚性愈伤组织再生出小植株的比例。利用该再生系统,我们从5个小麦优良主栽品种的成熟胚再生出了可育的植株,再生频率达15．3％-34．5％。     

不同杂种优势类群玉米幼胚愈伤组织的诱导及植株再生特性的研究

以Reid、唐四平头和其它种质等3个杂种优势类群共19份玉米自交系为试验材料,以玉米幼胚作为外植体,研究了基因型、培养基和激素对玉米幼胚愈伤组织的诱导及植株再生的影响,结果表明供试材料均能进行愈伤组织的诱导,但是仅有12个自交系能再生植株。N6和改良N6培养基有助于提高愈伤组织的质量及其生长速度,2,4-D在愈伤组织的诱导中起着关键性作用。在诱导培养基中添加0.2mg／L的6-BA或KT会使胚性愈伤组织的诱导频率下降以及降低愈伤组织的质量。在胚状体诱导培养基中添加1mg／L的KT能促进绿苗的分化,但是浓度过高会使丛生苗分化过多。此外,通过对不同杂种优势类群自交系玉米幼胚培养特性的分析,发现在唐四平头类群的4个自交系中,黄早四的绿苗分化率仅为0.5％,其它3个自交系不能再生植株。但是,从Reid和其它种质类群的供试自交系中筛选出了胚性愈伤组织的诱导频率和绿苗分化率均较高的、适合于遗传转化的受体材料,如3189／4380、4380／陕综5、8103、先早17、18-599红、18-599白、501、178和冀53。     

多年生黑麦草成熟胚再生体系的建立及基因枪转化

目的:建立以多年生黑麦草成熟胚为起始材料的再生体系,用于基因枪转化。方法:多年生黑麦草成熟种子在附加 5mg L 2,4 D的MS培养基上诱导愈伤组织,转至新继代培养基上产生胚性愈伤组织。分化培养基为无激素MS培养基。再生植株在培养基成分减半的无激素MS培养基生根,之后移栽至土壤。基于这一再生体系,用含有水稻几丁质酶基因RC2 4的质粒pARN6和含有草丁膦乙酰转移酶基因Bar的质粒pDB1,通过基因枪轰击胚性愈伤组织。用附加PPT的继代培养基进行转化植株的抗性筛选。结果:共获得 2 4 3株再生植株。通过PCR进行检测,获得1 8株整合有RC2 4基因的植株,1 5株整合有Bar基因的植株,同时转入 2个基因的植株 2株。     

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。     

几种影响籼稻成熟胚愈伤组织诱导及再生的因素

建立了适合5个籼稻品种成熟胚籼稻遗传转化的高效植株再生体系.N6基本培养基有利于籼稻愈伤组织的诱导和继代培养;N6大量元素和MS微量元素有利于愈伤组织的分化.降低分化培养基中蔗糖含量,加入适量山梨醇、Cu2 、Ag 和玉米素(2T)均可明显提高水稻愈伤组织的再生植株频率,5个品种分化频率均达到75%以上.     

新疆天山雪莲体胚诱导与分化研究

以新疆天山雪莲的叶片为外植体,分别用不同配方培养基诱导愈伤组织,后进行体胚诱导和分化培养形成再生雪莲植株.结果表明,诱导愈伤组织的最适培养基为MS 2,4-D 0.5 mg/L BA 1.5 mg/L,诱导率可达到100%;愈伤组织转移至MS 2,4-D 0.5 mg/L BA 1.5 mg/L培养基进行继代培养,增殖后的愈伤组织转移到MS 2,4-D 0.2 mg/L的液体培养基后成功诱导出雪莲体胚,出胚率达40%;将体胚接至MS ABA 0.5 mg/L培养基后,结果分化生长出大量的再生雪莲幼苗.     

甘蓝型油菜子叶和下胚轴再生植株无性系建立

以甘蓝型油菜(Brassica napus L.)豫油2号和6257的子叶和下胚轴为材料,在不同激素配比的MS培养基上诱导出了愈伤组织。将经过继代的部分愈伤转入分化培养基,分化结果表明:除基因型、外植体和分化培养基的激素配比对分化率有影响外,诱导愈伤培养基的激素配比对分化率也至关重要。豫油2号的子叶和下胚轴在最适诱导培养基(ZT 1+NAA0.5+2,4-D 0.2 mg/L)和最适分化培养基(ZT4+IAA 0.2 mg/L)组合中的愈伤分化率分别为12.5%和75%;6257的子叶和下胚轴在其最适诱导培养基(KT 2+NAA1+2,4-D 0.2 mg/L)和最适分化培养基(6-BA 4+IAA 0.02 mg/L)组合中的愈伤分化率分别为50%和37.5%。将其最适诱导培养基中的愈伤组织继代达8个月以上,建立了不同继代愈伤的再生植株无性系。     

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。     

The effect of auxin on plant regeneration of wheat,barley and triticale

One of the basic components of a medium influencing somatic embryogenesis of cereals from immature embryos is the type of auxin. According to some researchers, phytohormones can also play an important role during Agrobacterium-mediated transformation. In this first part of research, the influence of three types of auxins used alone or in combination of two on somatic embryogenesis and plant regeneration in three cereal species has been tested. Eight cultivars of barley, five cultivars of wheat and three cultivars of triticale have been used. Efficiency of plant development on two regeneration media, with and without growth regulators has been compared. Efficiency of regeneration characterized by frequency of explants that form embryogenic callus ranged from 25% for wheat cultivar Torka to 100% for two barley cultivars. Mean number of plantlets regenerating per explant differed significantly (from 2 to 58) depending on the type of auxin in inducing media, the type of regenerating media as well as cultivar. The biggest differences in regeneration efficiency were observed between barley cultivars, however regeneration of plants occurred in all combinations tested. The best regeneration coefficients for most barley cultivars were obtained after culture on dicamba or dicamba with 2,4-D. However, in the case of highly regenerating cv Scarlett, the most effective culture media contained picloram or 2,4-D alone. The highest values of regeneration coefficients for two triticale cultivars (Wanad and Kargo) were obtained on picloram (26.1 and 21.4, respectively) and for `Gabo' on picloram with dicamba (12.6). The range of mean number of regenerated plantlets was from 12 to 30. Dicamba alone or lower concentrations of picloram with 2,4-D were the best media influencing embryogenic callus formation in five wheat cultivars. However, the highest values of regeneration coefficients ranging from 10.6 to 26.8 were obtained at lower concentrations of picloram with 2,4-D or picloram with dicamba. R2 regeneration medium containing growth regulators was significantly better for plantlet development in several combinations (cultivar and induction medium) than the one without growth regulators. Generally, regeneration coefficients for all tested cultivars of three cereal species on the best media were high, ranging from 5.5 for barley cultivar Rodion to 51.6 for another barley cultivar Scarlett. Plantlets developed normally, flowering and setting seed.     

两个苏丹草品系成熟种子的组织培养和植株再生研究

该研究以苏丹草品系S722和Sa的成熟种子为外植体、MS培养基为基础培养基,2,4-D和NAA各3个浓度共6个处理对这两个苏丹草品系成熟种子进行愈伤诱导,探讨不同品系在不同植物生长物质浓度及植物生长物质组合中诱导愈伤组织和继代培养以及分化的能力。结果表明：苏丹草S722和Sa成熟种子的愈伤诱导率差异不显著,平均诱导率为17.19％。诱导培养基中2,4-D浓度为0.5或1 mg?L-1时,诱导效果最佳,而添加NAA不能提高愈伤诱导率。在继代培养中,设定2,4-D和6-BA各两个浓度共4个处理组合,处理1(2,4-D 1 mg?L-1＋6-BA 0 mg?L-1)的继代培养效果最佳。为了解不同植物生长物质对愈伤分化的影响,设定6-BA、NAA 各两个不同浓度、KT 3个不同浓度共5个处理组合对继代培养的愈伤进行分化培养。在5个处理中,处理1(6-BA 2 mg?L-1＋NAA 0 mg?L-1＋KT 0 mg?L-1)对 S722成熟种子诱导的愈伤分化率最高,达33.3％。在这两个苏丹草品系中,S722更容易分化培养。综上结果表明,2,4-D浓度为1 mg?L-1时诱导愈伤和继代培养效果较好,6-BA浓度为2 mg?L-1时分化效果较好。另外,针对不同苏丹草品系进行组织培养和植株再生时,适当调整植物生长物质浓度能提高植株再生的成功率。     

Improved Regeneration Efficiency from Mature Embryos of Barley Cultivars

A reliable protocol for plant regeneration from mature embryo derived calli of nine barley (Hordeum vulgare) cultivars has been developed. The auxins 2,4-dichlorophenoxyacetic acid, picloram and dicamba proved effective in inducing callus from mature embryos of most of the barley cultivars. The induced primary callus was loose, friable and translucent. It ultimately yielded creamy white and compact callus after 2 - 3 transfers on fresh medium of the same composition. Callus induction and regeneration capacity were highly cultivar dependent. Addition of a high concentration of picloram (4 mg dm^-3) promoted regeneration in 3 cultivars (Tallon, Grimmett and Sloop). In cv. Arapiles, abscisic acid and betaine were crucial in generating morphogenic callus from the mature embryos. Plants regenerated from these calli were hardy and developed roots readily when transferred to hormone free medium. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effect of abscisic acid and callus size on regeneration of american and international rice varieties

Embryogenesis and plant regeneration of Texas and international rice, Oryza saliva (L.), varieties (both indica and japonica types) were induced in culture on a regime consisting of the use of ABA or BAP in the subculture medium and small (10 mg) callus pieces on the regeneration medium. Ten 10 mg callus pieces on regeneration medium resulted in a 2- to 10-fold increase in plant regeneration over single 100 mg pieces. Plant regeneration of Texas rice cultivars (Lemont, Rico I, Rexmont and Skybonnet) and Taipei 309 was enhanced by the use of ABA in the subculture medium with a 2-fold and a 3- to 10-fold increase in plant regeneration with 2.6 mgL^–1 and 26 mgL^–1 ABA in the subculture media, respectively. Regeneration of plants from callus of IR36 and IR64 was not enhanced by ABA but by the use of BAP and Trp in the subculture medium or by 2,4-D alone. The subculture medium containing BAP and Trp produced a 5-fold increase in plant regeneration rate from IR64 callus and was equal to subculture medium containing only 2,4-D for IR36 callus. Both Lemont and IR36 were previously reported to be difficult to regenerate or non-regenerative, however, the use of ABA or BAP in the subculture medium, small callus pieces and visual selection of embryogenic callus allowed the regeneration of up to 20 and 22 plants from 100 mg of Lemont and IR36 callus, respectively.Abbreviations ABA abscisic acid - BAP benzylaminopurine - MS Murashige and Skoog - NAA napthaleneacetic acid - Trp tryptophan - 2,4-D 2,4-dichlorophenoxyacetic acid     

Clonal propagation byin vitro culture of Corchorus (jute)

Callus was produced on cotyledon, shoot tip, hypocotyl and root explants of twoCorchorus species on several media. Cytokinin was necessary for callus production on cotyledon explants. BothC.olitorius genotypes produced most callus on media with zeatin and either NAA or IAA, and theC.capsularis genotype produced most callus on media with IAA and either zeatin or BA. High frequencies of regenerated shoots were obtained from shoot tip explants of both species, from the apical meristem and from callus. Media with 2.0 mg 1⁻¹ BA were superior for both species, and media with zeatin were equally good forC.capsularis only. More regeneration was obtained for all genotypes after subculture of callus on media with 2.0 mg 1⁻¹ zeatin. Cotyledon callus produced less regeneration, also with differences between genotypes; explants of both genotypes ofC.olitorius produced regeneration on a medium with NAA and zeatin, and theC.capsularis genotype produced regeneration on a medium with IAA and BA. Limited regeneration from root explant callus was obtained forC.capsularis only on medium with BA and IAA. Regeneration was not obtained from hypocotyl callus. Further regeneration of shoots of both species was obtained from secondary callus after subculture, and from nodal segments of regenerated shoots and of seedling shoots cultured on basic MS medium without growth hormones. Roots were produced on about 80% of all shoots after transference to medium with 0.2 mg 1⁻¹ IBA, and rooted plantlets survived and flowered normally after transference to compost.     

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.     

Effect of genotype and medium composition on linseed (<Emphasis Type="Italic">Linum usitatissimum</Emphasis>) ovary culture

Breeding linseed (Linum usitatissimum L.) using haploid techniques allows breeders to develop new cultivars in a shorter time period. Many research groups successfully created new linseed genotypes through anther culture; however ovary culture has been the subject of only a few earlier studies. In the present study, the effect of genotype and growth regulators combination on callus induction and shoots regeneration in ovary culture of nine commercially important linseed cultivars was investigated. Ovaries were cultured on modified MS medium supplemented with three different combinations of plant growth regulators. Variable callogenic responses were expressed by all of the genotypes tested on different induction media. The results suggested that specific combination of growth regulators for callus induction must be designed for each genotype. Shoot regeneration from ovary derived callus is a critical phase of the whole gynogenetic process. Differences in adventitious shoot formation frequency among genotypes were demonstrated and four responsive genotypes have been selected. Ovary derived callus from cultivar ‘Mikael’ manifested the highest adventitious shoot formation frequency with a high number of shoots per explant. The optimum ratio of growth regulators for shoot regeneration was shown to depend on the genotype. Cultivars ‘Linola’, ‘Mikael’ and ‘Szaphir’ showed the highest shoot regeneration frequency when callus had originated on induction medium supplemented with 2 mg L⁻¹ BAP and 2 mg L⁻¹ NAA, while combination of 1 mg L⁻¹ BAP and 2 mg L⁻¹ IAA promoted shoot formation in ovary-derived callus of ‘Barbara’. The highest rate of shoots per explant has been obtained in second subculture.     

Evaluation of regeneration potential of mature embryo derived callus in Indian cultivars of barley (Hordeum vulgare L.)

A protocol for plant regeneration in Indian cultivars of barley (Hordeum vulgare L.) has been developed using mature embryo culture. The influence of various auxins 2,4-D (2,4-dichlorophenoxyacetic acid), Dicamba (3,6-dichloro-o-anisic acid) and Picloram (4-amino-3,5,6-trichloropicolinic acid) on the callus induction and subsequent plant regeneration revealed highest percent of callus induction form cultivar (cv) BL 2 on MSB₅ medium (MS salts + B₅ vitamins) supplemented with 6 mg l^?1 Picloram, but maximum number of shoot buds (6–13) were regenerated on MSB₅ medium containing 0.5 mg l^?1 Picloram. Regenerated shoots were rooted on half-strength MSB₅ medium. Plantlets were successfully transferred to soil and grown to maturity in greenhouse. The effect of copper sulphate revealed significant improvement in callus induction and plant regeneration when the concentration of CuSO₄ was increased to 3 μM (30 times higher than normal MS medium) for cv BL 2. Regeneration potential differed for different cultivars of barley used, with highest for cv BL 2 and lowest for cv BH 924. We conclude that the Indian barley genotypes exhibit plant regeneration from mature embryo cultures. The protocol has potential application in barley improvement through genetic engineering.     

Identification of a novel elite genotype for in vitro culture and genetic transformation of cotton

Hypocotyls of cotton (Gossypium hirsutum L.) cultivars cv. YZ-1, Coker 312 and Coker 201 were inoculated on Murashige and Skoog callus induction medium. YZ-1 exhibited a very high regeneration potential, with 81.9 % of the explants inoculated differentiated into embryogenic callus within 8–10 weeks. During the process of callus maintenance (subculture for 1 to 3 years), the total embryos number in Coker 312 and Coker 201 calli dropped sharply, and the percentage of embryo germination decreased. On the contrary, the callus of YZ-1 consistently maintains a high frequency of plant regeneration after long-time subculture. Transgenic kanamycin-resistant calli of Coker 201 partially lost the ability of somatic embryogenesis and plant regeneration. The stress produced by the transformation procedure slightly affected somatic embryogenesis and plant regeneration of YZ-1, which showed minimum loss of plant regeneration ability.