骨干玉米自交系丹598遗传再生体系的建立

目的:以玉米骨干自交系丹598的幼胚为外植体,诱导愈伤组织建立遗传再生体系。方法:探讨胚龄、培养基种类、2,4-D浓度对愈伤组织诱导的影响。结果:在授粉后16～18 d,2,4-D浓度为2.0 mg/L时诱导最佳;设置N6、NB、改良NB、MS、MB等5种培养基,筛选出改良NB培养基为最佳诱导培养基;分化培养基中添加1 mg/L激动素、0.5 mg/L 6-卞基嘌呤和0.5 mg/L萘乙酸能促进绿苗分化和根系生长。结论:建立了玉米自交系丹598的优良再生体系,为以后的基因转化工作打下了良好基础。     

白刺花胚性愈伤组织诱导及体细胞胚发生和萌发

探讨不同因素对白刺花下胚轴、子叶2种外植体胚性愈伤组织诱导及体细胞胚发生和萌发的影响。以B5和MS为基本培养基,研究2,4-D、6-BA和TDZ对白刺花下胚轴和子叶胚性愈伤组织的诱导;在MS培养基上添加不同浓度2,4-D,研究胚性愈伤组织增殖情况;采用ABA,探究对体细胞胚发生的影响。结果表明:下胚轴比子叶更易诱导胚性愈伤组织,筛选出2种外植最佳的胚性愈伤组织诱导培养基均为MS+2.0 mg/L 2,4-D+0.5 mg/L TDZ+0.5 mg/L 6-BA,胚性愈伤组织诱导率分别为77.3%和41.0%。15.0 mg/L ABA、0.2 mg/L 2,4-D和2.0 mg/L 6-BA有利于体细胞胚发生,1/3MS+0.2 mg/L NAA+0.1 mg/L 6-BA+2.0 g/L活性炭+25 g/L蔗糖+7 g/L琼脂的培养基可使体细胞胚萌发率达80%以上,再生植株移栽成活率高达90%。白刺花外植体种类及培养基类型均会影响胚性愈伤组织的诱导,其中下胚轴诱导效果优于子叶;MS培养基较适合启动细胞脱分化形成愈伤组织,2,4-D对胚性愈伤组织的增殖保持有调控作用,ABA有利于体细胞胚的发生。     

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。     

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。     

几种匍匐翦股颖成熟胚愈伤组织诱导及植株再生

利用成熟种子作为外植体,分析了2,4-D对匍匐翦股颖胚性愈伤组织诱导与植株再生体系的影响,并对体细胞胚的发生过程进行了观察.实验结果表明,在2.0 mg/L 2,4-D 0.1 mg/L 6-BA时胚性愈伤组织的诱导频率最高.随着2,4-D浓度的增加,愈伤组织的诱导和分化能力明显下降.在再生过程中采用1.0 mg/L的6-BA达到了比较好的效果,愈伤组织的再生频率大部分在90%以上.同时发现适当提高肌醇浓度可以使苗长得较为粗壮.在实验中发现匍匐翦股颖的体细胞胚发生过程为球形胚、心形胚、鱼雷胚和子叶胚.     

农杆菌介导的高效玉米遗传转化体系的建立

为了建立玉米高频再生及高效遗传转化体系, 对影响玉米胚性愈伤组织诱导的11个因素及影响胚性愈伤分化的9个因素用正交实验方法进行研究。结果显示, 基因型对胚性愈伤诱导有极显著影响。6-BA、培养基、AgNO3、2,4-D、ABA对胚性愈伤诱导的影响达到显著水平。多重比较分析显示ABA 2 mg/L每间隔1代添加对胚性愈伤诱导率有显著影响。在影响分化的因素中, 基因型和6-BA浓度表现出极强的主效应, NAA、培养基、KT、2,4-D对分化产生显著影响。Southern blotting 分析表明, 25 mg/L潮霉素选择压下抗性愈伤率作为转化体系优化指标是可靠的。在影响转化效率的因素中, acetosyringone (AS)使用浓度因基因型不同而表现出敏感度差异, 共培养温度24~25℃、农杆菌浓度和浸泡时间0.7 OD×15 min, 以及pH值5.5~6.2是最高转化率的优选组合。在整合后的玉米遗传转化体系中, 黄早4和综31自交系以抗性愈伤率为指标的GUS基因稳定转化率分别达到48.6%和46.2%。     

猕猴桃高频直接再生体系的建立

为了建立猕猴桃高频再生体系,以MS为基本培养基,猕猴桃(Actinidia deliciosaQinmei)茎及叶片为外植体,研究了2,4-D、6-BA和NAA在美味猕猴桃愈伤组织形成及分化过程中的作用。方差分析结果表明,6-BA能够显著促进愈伤组织形成,6-BA和NAA可以显著促进愈伤组织形成和分化,而2,4-D抑制愈伤组织形成。附加2.0 mg/L 6-BA、1.0 mg/L NAA和600 mg/L水解酪蛋白的MS培养基是茎段培养的最佳培养基,在该培养基上,以再生的无菌苗为起始材料,一个月时叶圆盘的直接再生频率达到100%,平均每个叶圆盘产生9.33个芽,其中23.21%芽高度超过0.5 cm。     

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。     

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％。     

党参的离体培养及植株再生的研究

在附加激素的MS培养基上,培养党参下胚轴和无菌芽切段,诱导产生愈伤组织并且再生植株。经过两年多(15个世代)的继代培养,建立了党参体细胞无性系。实验结果表明:(1)培养基MS 0.4mg/L 2,4-D 0.8mg/L Kt 2.0mg/L IAA对愈伤组织诱导及继代培养,MS 0.2mg/L 6-BA诱导外植体产生丛芽和愈伤组织再分化,MS 0.5mg/L NAA 0.2mg/L 6-BA及MS 0.2mg/L NAA诱导生根效果最好。(2)愈伤组织再分化经过胚状体途径。     

High-frequency plant regeneration through callus initiation from mature embryos of maize (<Emphasis Type="Italic">Zea Mays</Emphasis> L.)

An efficient maize regeneration system was developed using mature embryos. Embryos were removed from surface-sterilized mature seeds and sliced into halves. They were used as explants to initiate callus on induction medium supplemented with 4.0 mg l^–1 2,4-dichlorophenoxyacetic acid (2,4-D). The induction frequency of primary calli was over 90% for all inbred lines tested. The primary calli were then transferred onto subculture medium supplemented with 2.0 mg l^–1 2,4-D. Following two biweekly subcultures, embryogenic calli were formed. Inclusion of a low concentration (0.2 mg l^–1) of 6-benzylaminopurine (BA) in the subculture medium significantly promoted the formation of embryogenic callus. The addition of silver nitrate (10 mg l^–1) also supported an increased frequency of embryogenesis. The embryogenic callus readily formed plantlets on regeneration medium supplemented with 0.5 mg l^–1 BA. The regenerated plantlets were transferred to half-strength Murashige and Skoog medium supplemented with 0.6 mg l^–1 indole-3-butyric acid to develop healthy roots. The regenerated plantlets were successful on transfer to soil and set seed. Using this system, plantlets were regenerated from seven elite maize inbred lines. The frequency of forming green shoots ranged from 19.8% to 32.4%. This efficient regeneration system provides a solid basis for genetic transformation of maize.Abbreviations BA 6-Benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - IBA Indole-3-butyric acid - KT KinetinCommunicated by M.C. Jordan     

Callus Formation and Plant Regeneration from Cultured Embryos of Diploid and Tetraploid Winter Ryes (Secale cereale L)

The ability of immature, mature and endosperm-supported mature embryos of diploid and tetraploid winter ryes (Secale cereale L) was tested to compare the callus induction and plant regeneration. Immature embryos were obtained from field grown rye. Immature embryos were aseptically excised and placed, with the scutellum upwards, on the callus culture medium consisted of Murashige and Skoog (MS) mineral salts supplemented with 2 mg l^?1 2,4-dichlorophenoxyacetic acid (2,4-D). Mature embryos were aseptically excised the imbibed seeds and placed, scutellum up, on MS medium supplement with 2 mg l^?1 2,4-D. Endosperm-supported mature embryos were moved slightly (not set free) in the imbibed mature seeds. The seeds with moved embryos were placed furrow downwards in dishes containing 8 mg l^?1 2,4-D for callus induction. The developed calli and regenerated plant were maintained on hormone free MS medium. Comparison of the responses of the three explants used indicated that endosperm-supported mature embryo was the most useful explant for plant regeneration in both diploid and tetraplold ryes. This is the first report of winter ryes plants having been regenerated from endosperm-supported mature embryos.     

Effect of 2,4-Dichlorophenoxyacetic Acid and NaCl on the Establishment of Callus and Plant Regeneration in Durum and Bread Wheat

Optimal callus induction and plant regeneration were obtained in bread and durum wheat by manipulating the NaCl concentration in the induction medium. Immature embryos from a high regeneration line of spring wheat (Triticum aestivum L.), 'MPB-Bobwhite 26', and an elite durum wheat (Triticum turgidum var. durum L.), 'Mexicali', were cultured in E3 induction medium consisting of Murashige and Skoog (MS) medium, 2.5 mg l^–1 2,4-dichlorophenoxyacetic acid (2,4-D), 2% sucrose and 0.9% Bacto agar. The treated embryos were transferred to E3 liquid medium supplemented with various levels of 2,4-D and NaCl. Incubation on medium containing 2.5 mg l^–1 2,4-D for 45 days produced callus and plant regeneration in 'MPB-Bobwhite 26', but lower callus yield and plant regeneration in 'Mexicali', indicating that 2,4-D alone was not sufficient for callus induction and plant regeneration in this durum variety. Callus yield and regeneration frequencies were higher in 'Mexicali' embryos that were incubated in media containing 2 mg l^–1 2,4-D and 2 mg l^–1 NaCl. The presence of NaCl in the medium beyond the initiation phase was detrimental to plant regeneration. The use of NaCl in the callus formation could form the basis for improved transformation of durum wheat varieties.     

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。     

三叶半夏悬浮培养下的体细胞胚胎发生及植株再生

用三叶半夏幼嫩叶片诱导产生的胚性愈伤组织建立了胚性细胞悬浮系,研究了悬浮培养下体细胞胚胎的发生及植株的再生。结果表明,胚性悬浮细胞在附加1．0mg／L2,4-D、0．2 mg／LBA和300mg／L LH的MS液体培养基中产生大量的球形胚,转入液体分化培养基(MS 0．1 mg／LNAA 0．2 mg／L BA 300 mg／L LH)中进一步发育成心形胚、鱼雷形胚和子叶形胚。收集成熟胚转移到MS固体分化培养基上培养获得了再生植株。另外还观察到某些成熟胚上产生了许多次生胚。     

A comparison of callus induction and plant regeneration from different embryo explants of triticale (x Triticosecale wittmack)

Immature, mature and endosperm-supported mature embryos of six triticale cultivars (BDMT-98-8S, Melez-2001, Mikham-2002, Presto, Tacettin Bey and Tatlicak-97) were cultured in vitro to compare the levels of callus induction and plant regeneration. Immature embryos, 15-18 days after anthesis, were aseptically excised and placed with the scutellum upwards on a callus culture medium consisting of Murashige and Skoog (MS) mineral salts supplemented with 2 mg l(-1) 2,4-dichlorophenoxyacetic acid (2,4-D). Mature embryos were aseptically excised from the imbibed seeds and placed scutellum up on MS medium supplement with 2 mg l(-1) 2,4-D. Endosperm-supported mature embryos were moved slightly in the imbibed mature seeds. The seeds with moved embryos were placed furrow downwards in dishes containing 8 mg l(-1) 2,4-D for callus induction. The developed calli and regenerated plants were maintained on hormone-free MS medium. Variability among the genotypes was observed for all the types of embryo culture. Immature embryos from "Presto" and endosperm-supported mature embryos from "Mikham 2002" had excellent regeneration capacities (92.0% and 97.3%, respectively) and the highest number of plants regenerated growing in soil (9 and 13, respectively). A comparison of the responses of the three explants used indicated that the endosperm-supported mature embryo was the most useful explant for plant regeneration in triticale.     

Efficient callus induction and plant regeneration from mature embryo culture of winter wheat (Triticum aestivum L.) genotypes

Immature and mature embryos of 12 common winter wheat (Triticum aestivum) genotypes were cultured in vitro to develop an efficient method of callus formation and plant regeneration from mature embryo culture, and to compare the responses of both embryo cultures. Fifteen days after anthesis, immature embryos were aseptically dissected from seeds and placed with the scutellum upwards on a solid agar medium containing the inorganic components of Murashige and Skoog (MS) and 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). Mature embryos were moved slightly in the imbibed seeds. The seeds with moved embryos were placed furrow downwards in dishes containing 8 mg/l 2,4-D for callus induction. The developed calli and regenerated plants were maintained on 2,4-D-free MS medium. Plants regenerated from both embryo cultures were vernalized and grown to maturity in soil. Regenerated plantlets all maintained the hexaploid chromosome number. A strong genotypic effect on the culture responses was found for both explant cultures. Callus induction rate, regeneration capacity of callus and number of plants regenerated were independent of each other. Mature embryos had a high frequency of callus induction and regeneration capacity, and therefore, being available throughout the year, can be used as an effective explant source in wheat tissue culture. Received: 4 February 1997 / Revision received: 1 April 1997 / Accepted: 5 May 1997     

Embryogenesis and plant regeneration from tissue culture of Canada wildrye,Elymus canadensis L.

Somatic embryogenesis and plant regeneration of Canada wildrye (Elymus canadensis L.) from tissue culture was investigated by culturing immature embryos and inflorescences on MS medium containing 2 mg/l 2,4-D. The optimum size of explants for maximum embryogenic callus formation was 1.0 to 1.5 mm for embryos and 4 to 6 cm for inflorescences. Plant regeneration from the subcultured embryogenic callus was attempted monthly using hormone-free MS medium or MS medium with 0.5 mg/1 2,4-D and 0.3 mg/l GA3. Three hundred and fifty seven plantlets were regenerated from the callus cultures of both explant sources during a six month period. Ten chlorophyll deficient plants accounting for 2.8% of the total regenerants were observed. One plant with white striped leaves survived and was found to be an octoploid.Abbreviations GA3 gibberellic acid - MS Murashige and Skoog (1962) - 2,4-D 2,4-dichlorophenoxyacetic acid