水杉愈伤组织诱导及植株再生

通过愈伤组织诱导器官发生途径,建立了水杉（Metasequoia glyptostroboides）的植株再生体系,探讨了不同外植体（种胚、幼叶切块、茎段、根段）和植物生长调节剂对不定芽直接再生和愈伤组织诱导器官发生的影响。结果表明：以种胚、无菌苗叶片、茎段和根作为外植体,在MS补加2,4-D、NAA和6-BA不同组合的培养基上都能诱导得到愈伤组织,其中种胚诱导愈伤组织效果最好,诱导率可达100%,茎诱导效果次之,诱导率为97.1%。诱导愈伤组织效果较好的培养基有：MS＋1.0mg·L-12,4-D＋0.5mg·L-16-BA、MS＋0.1mg·L-16-BA＋1.0mg·L-1NAA、MS＋0.5mg·L-16-BA＋1.0mg·L-1NAA、MS＋1.0mg·L-16-BA＋1.0mg·L-1NAA、MS＋0.5mg·L-16-BA＋2.0mg·L-1NAA、MS＋1.0mg·L-16-BA＋2.0mg·L-1NAA和MS＋0.5mg·L-12,4-D＋0.5mg·L-1NAA。以愈伤组织在MS培养基上植株再生效果最好,再生率为62.5%。     

诱导茶树成熟胚培育成幼苗的研究

以茶树品种——农抗早的成熟胚为外植体离体培养,初次研究了2,4-D、6-BA等不同植物激素对茶树成熟胚诱导成幼苗的影响。结果表明,在合适的激素组合条件下,愈伤组织诱导为97%,在附加2.5mg/L 6-BA 0.5mg/L NAA的MS培养基上,愈伤组织的芽分化率为14.2%,附加1.5mg/L NAA的1/2 MS培养基,幼苗的根分化率为5%。     

Development of Agrobacterium-mediated transformation technology for mature seed-derived callus tissues of indica rice cultivar IR64

Indica rice cultivar IR64 is most recalcitrant to regenerate, which affects the transformation efficiency especially when mature seed-derived callus tissues are used as explants. Therefore, a simple, rapid and improved genetic transformation protocol has been developed for the indica rice cultivar IR64 using Agrobacterium-mediated genetic transformation. With different hormonal combination tested, the maximum callus induction was observed on MS medium supplemented with 2.5 mg/l 2,4-D and 0.15 mg/l BAP from the scutellum explants. Three weeks old scutellum derived callus explants were immersed in Agrobacterium suspension (strain LBA4404, OD600=1.0) and co-cultured at 26±2°C in dark for 2 d. The maximum transformation efficiency (12%) was achieved with infection of callus explants for 20 min along with use of 150 μm acetosyringone. The maximum plant regeneration was observed on MS medium supplemented with 3 mg/l BAP, 1 mg/l Kinetin and 0.5 mg/l NAA. The maximum root induction was observed on MS medium along with 10 g/l glucose and 20 g/l sucrose. The integration of the transgene in T1 transgenic plants was confirmed by polymerase chain reaction and Southern blot analyses. The copy number of transgenes has been found to vary from 1 to 2 in transgenic plants. By using this improved method we have successfully raised transgenic rice plants within 3 mo from seed inoculation to plant regeneration.     

何首乌愈伤组织的诱导

研究了外植体、光暗条件、植物激素等因子对何首乌愈伤组织诱导的影响 ,以及 6 -BA和何首乌组织提取液对愈伤组织增殖的影响。结果表明 :茎段的愈伤组织诱导优于带侧芽茎段和叶片 ;光照有利于愈伤组织的诱导 ;4因子 4水平的正交实验表明 ,对何首乌茎段愈伤组织诱导的影响 2 ,4 -D >6 -BA >IBA >IAA ,最佳激素搭配是 :MS +2 ,4 -D 2mg L +6 -BA 1mg L +IBA1mg L或MS +2 ,4 -D 2mg L +6 -BA 2mg L +IAA 1mg L ;MS培养基中附加 6 -BA或组织提取液均促进愈伤组织的增殖。     

药用植物金荞麦愈伤组织诱导与植株再生

目前转基因技术已成为植物定向遗传改良的重要手段,而建立稳定高频的离体再生系统是实现遗传转化的基础和前提.本试验以25 ～30 d苗龄的金养麦(Fagopyrum dibotrys)无菌苗叶片、茎节间、叶柄为外植体进行愈伤组织诱导与植株再生研究.结果表明:叶片在MS +2,4-D 4.0 mg/L +6-BA 1.0 mg/L培养基上愈伤组织诱导率达到89％.茎节间在MS +2,4-D 2.0 mg/L +6-BA 2.0 mg/L培养基上愈伤组织诱导率为87％.叶柄在MS +2,4-D 4.0 mg/L +6-BA 2.0 mg/L+ IBA 0.2 mg/L培养基上的最高诱导率仅为54％.愈伤组织分化不定芽的适宜培养基为MS +6- BA2.0 mg/L +TDZ0.2 mg/L +NAA0.2 mg/L;金荞麦不定芽在1/2 MS +NAA 0.5 mg/L的培养基上生根效果最好.组培再生植株经炼苗后移栽到田间成活率达80％以上,且生长表现正常.高频完整再生体系的建立,为金荞麦进一步遗传操作和扩大药材资源奠定了基础.     

百合体细胞胚胎发生和植株再生

以切花百合(Lilium)品种‘黄天霸’(‘Manissa’)花器官为外植体诱导体细胞胚胎发生与植株再生。结果表明,不同花器官、不同激素配比对愈伤组织形成均具有显著影响。花丝为最佳外植体,激素对愈伤组织诱导的影响效应为NAA>6-BA>2,4-D,最适培养基为MS+1.0 mg.L-1NAA+0.2 mg.L-16-BA;激素诱导体细胞胚胎发生的影响效应为2,4-D>KT>6-BA,最佳培养基配方为MS+1.0 mg.L-12,4-D+0.2 mg.L-1KT+1.0 mg.L-16-BA;MS培养基添加IBA可促进体细胞胚萌发成苗,体细胞胚芽成苗的最佳培养基为MS+0.2 mg.L-16-BA+1.0 mg.L-1IBA。     

小叶龙竹（Dendrocalamus barbatus）愈伤组织诱导及植株再生

以小叶龙竹种子为外植体,通过研究MS培养基中不同植物生长调节剂浓度组合对外植体愈伤组织诱导和不定芽分化的影响以及不同配比对生根的作用,建立了稳定的繁殖再生体系。试验结果表明愈伤组织诱导的最适培养基为MS＋2,4-D5．0mg·L-1;不定芽分化的最适培养基为MS＋2,4-D0．5mg·L-1＋6．BA1．0mg·L-1＋KT0．25mg·L-1;小苗的最适生根培养基为1／2MS＋6-BA0．5mg·L-1＋NAA1．0mg·L-1＋IBA0．4mg·L-1,建立的繁殖再生体系将为进一步利用分子生物学技术对竹类植物进行遗传改良奠定基础。     

Plant regeneration and stable transformation in the floricultural plant Cleome spinosa, a C3 plant closely related to the C4 plant C. gynandra

Cleome spinosa is widely used as a garden ornamental in many countries. Here we determined the optimal conditions for plant regeneration from different tissue explants grown in vitro. Induction medium containing MS salts, MS vitamins, 3% sucrose, 1 mg l?1 BA, 200 mg l?1 timentin, and 0.8% agar was sufficient for shoot regeneration of all the tissue explants examined, including leaf, hypocotyl, and cotyledon. Subsequently, an Agrobacterium tumefaciens-mediated method was developed to transform the vector pCHS, which carries the transgenes Petunia chalcone synthase (chs) and selection marker neomycin phosphotransferase II (nptII), into C. spinosa. From a total of 368 cotyledon explants, 13 putative transgenic lines were regenerated from selection medium supplemented with 50 mg l?1 kanamycin and 200 mg l?1 timentin, and transferred to the greenhouse. Genomic PCR and Southern blot analyses revealed that the nptII transgene was present in all 13 transgenic plants. Similarly, when the Petunia chs transgene was used as a probe in Southern blot analysis, single or multiple hybridization bands were detected in 12 out of the 13 transgenic plants. In addition, T? progeny assay from selected transformants showed that the nptII transgene can be transmitted in a Mendelian manner from transgenic parents into their progeny. This is the first report of stable transformation of the C? dicotyledon C. spinosa, which will facilitate functional comparison of cell-type specific genes with counterpart C? dicotyledon C. gynandra using transgenic approaches.     

郁金樱愈伤组织诱导及植株再生

为建立郁金樱(Cerasus serrulata var. lannesiana cv. ‘Grandiflora’)再生体系,以多年生母株小叶、一年生嫁接苗小叶、腋芽诱导小叶和增殖一代小叶为外植体,探讨不同外植体和植物激素组合对郁金樱愈伤组织诱导、不定芽分化、增殖和生根的影响。结果表明,4种外植体均可诱导出愈伤组织,...     

Expression of anthocyanins and proanthocyanidins after transformation of alfalfa with maize Lc

Three anthocyanin regulatory genes of maize (Zea mays; Lc, B-Peru, and C1) were introduced into alfalfa (Medicago sativa) in a strategy designed to stimulate the flavonoid pathway and alter the composition of flavonoids produced in forage. Lc constructs included a full-length gene and a gene with a shortened 5'-untranslated region. Lc RNA was strongly expressed in Lc transgenic alfalfa foliage, but accumulation of red-purple anthocyanin was observed only under conditions of high light intensity or low temperature. These stress conditions induced chalcone synthase and flavanone 3-hydroxylase expression in Lc transgenic alfalfa foliage compared with non-transformed plants. Genotypes containing the Lc transgene construct with a full-length 5'-untranslated region responded more quickly to stress conditions and with a more extreme phenotype. High-performance liquid chromatography analysis of field-grown tissue indicated that flavone content was reduced in forage of the Lc transgenic plants. Leucocyanidin reductase, the enzyme that controls entry of metabolites into the proanthocyanidin pathway, was activated both in foliage and in developing seeds of the Lc transgenic alfalfa genotypes. Proanthocyanidin polymer was accumulated in the forage, but (+)-catechin monomers were not detected. B-Peru transgenic and C1 transgenic populations displayed no visible phenotypic changes, although these transgenes were expressed at detectable levels. These results support the emerging picture of Lc transgene-specific patterns of expression in different recipient species. These results demonstrate that proanthocyanidin biosynthesis can be stimulated in alfalfa forage using an myc-like transgene, and they pave the way for the development of high quality, bloat-safe cultivars with ruminal protein bypass.     

荞麦组织培养及高频植株再生体系的建立

通过对荞麦(Fagopyrum esculentum Moench)不同外植体、不同激素配比的比较研究,建立了荞麦离体培养高效植株再生体系。荞麦子叶切段在含2．0 mg/L 2,4-D和1．0 mg/L 6-BA的MS培养基上愈伤组织诱导率为89．6％,而下胚轴切段在含2．0 mg/L 2,4-D和1．0-2．0 mg/L 6-BA MS培养基上愈伤组织诱导率高达100％。在2．0 mg／L 6-BA、0．1 mg,L IAA和1 mg／L KT的MS培养基上通过愈伤组织间接分化或外植体直接分化形成不定芽。来自子叶和下胚轴的愈伤组织的分化率分别为42．5％和73．6％,下胚轴的分化率明显高于子叶。将生长状态良好的不定芽转至含1．0 mg/L IBA和0．5mg/L NAA的1/2 MS培养基上生根,生根率达到100％。再生植株移栽到盆土中,成活率达91．6％,并且生长状态和特征均表现正常。     

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。     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of niger [<Emphasis Type="Italic">Guizotia abyssinica</Emphasis> (L. f.) Cass.] using seedling explants

A protocol was developed for Agrobacterium-mediated genetic transformation of niger [ Guizotia abyssinica (L.f.) Cass.] using hypocotyl and cotyledon explants. Hypocotyls and cotyledons obtained from 7-day-old seedlings were co-cultivated with Agrobacterium tumefaciens strain EHA101/pIG121Hm that harbored genes for beta-glucuronidase (GUS), kanamycin, and hygromycin resistance. Following co-cultivation, the hypocotyl and cotyledon explants were cultivated on MS medium containing 1 mg/l 6-benzylaminopurine (BA) for 3 days in darkness. Subsequently, hypocotyl and cotyledon explants were transferred to selective MS medium containing 1 mg/l BA, 10 mg/l hygromycin, 10 mg/l kanamycin, and 500 mg/l cefotaxime. After 6 weeks, hypocotyls and cotyledons produced multiple adventitious shoot buds, and these explants were subcultured to MS medium containing 1 mg/l BA, 30 mg/l hygromycin, and 30 mg/l kanamycin. After a further 3 weeks, the explants (along with developing shoot buds) were subcultured to MS medium containing 1 mg/l BA, 50 mg/l kanamycin, and 50 mg/l hygromycin for further selection. Transgenic plants were obtained after rooting on half-strength MS medium supplemented with 0.1 mg/l alpha-naphthaleneacetic acid, 50 mg/l kanamycin, and 50 mg/l hygromycin and were confirmed by GUS histochemical assay and polymerase chain reaction analysis. Genomic Southern blot hybridization confirmed the incorporation of the neomycin phosphotransferase II gene into the host genome.     

火龙果愈伤组织诱导与植株再生

为了建立火龙果愈伤组织诱导与植株再生体系,以火龙果茎段、幼苗和子叶为外植体进行离体培养试验。结果表明：茎段诱导愈伤组织的最优培养基为1／2MS＋2,4-D2．0mg·L^-1＋6-BAO．5mg·L^-1,诱导子叶愈伤组织的最适培养基是1／2MS＋2,4-D2．0mg·L^-1＋6-BA1．0mg·L^-1,诱导愈伤组织分化的最优培养基为1／2MS＋6-BA4．0mg·L^-1＋NAA0．5mg·L^-1,最佳生根培养基为1／2MS＋6．BA1mg·L^-1＋NAA0-3mg·L^-1。     

中华结缕草(Zoysia sinica Hance)组织培养和再生植株研究

以中华结缕草(Zoysiasinica Hance)成熟种子为外植体在附加2.5mg/L2,4-D、0.25mg/L6-BA和1～2mg/LVB₁的改良MS培养基(MS_m)上愈伤组织的诱导率最高为43.0%。愈伤组织的最佳继代培养基为MSm附加0.1mg/L6-BA和2.0mg/L2,4-D。在无生长调节物质的MS培养基(MS₀)上,外观呈白色到淡黄色、含有密实颗粒的愈伤组织再生率为30%～60%。     

中华结缕草(Zoysia sinica Hance)组织培养和再生植株研究

以中华结缕草(Zoysia sinica Hance)成熟种子为外植体在附加2．5mg／L2,4-D、0．25mg／L 6-BA和1～2mg／L VB1的改良MS培养基(MSm)上愈伤组织的诱导率最高为43．0％。愈伤组织的最佳继代培养基为MSm附加0．1mg／L 6-BA和2．0mg／L 2,4-D。在无生长调节物质的MS培养基(MS0)上,外观呈白色到淡黄色、含有密实颗粒的愈伤组织再生率为30％～60％。     

In vitro plant regeneration in the genus Cucumis

Plants were regenerated from cotyledonary and root explants of cucumber (Cucumis sativus L.) cultivars and breeding lines of diverse sex type, growth habit, and processing quality and from cotyledonary explants of muskmelon (C. melo L.). Somatic embryogenesis was induced on a medium consisting of Murashige and Skoog (MS) salts supplemented with 1.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 1.0 mg/l α-naphthaleneacetic acid and 0.5 mg/l 6-benzylaminopurine. Embryos matured on the same medium without 2,4-D, and developed into normal plants on a hormone-free MS medium. Cucumber plants were also regenerated from cotyledonary protoplasts using a modified tomato protocol.     

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     

轮叶党参的组织培养及植株再生研究

以轮叶党参为材料,研究了不同外植体、激素组合、培养基及光照条件对愈伤组织诱导及植株再生的影响.结果表明,叶片是轮叶党参组织培养较为合适的外植体.外植体在添加0.5 mg·L~(-1) 2,4-D+1.0 mg·L~(-1) 6-BA+0.5 mg·L~(-1) KT的MS培养基上愈伤组织诱导率最高可达100%;愈伤组织转移到附加0.75 mg·L~(-1) 6-BA+0.5 mg·L~(-1) NAA的MS培养基进行继代培养,增殖后的愈伤组织转移到附加0.5 mg·L~(-1) 6-BA+0.2 mg·L~(-1) NAA的1/2MS分化培养基进行分化,其分化率达89.4%;将分化出的芽转接到附加0.2 mg·L~(-1) IAA+0.2 mg·L~(-1) NAA的1/2MS培养基,生根率达92.5%.暗培养诱导出愈伤组织后转到16 h·d~(-1)光照条件下,愈伤组织增殖倍数和分化率显著提高,再生苗健壮,长势强.     

风信子花被外植体年龄对花器官分化的影响

离体培养风信子(Hyacinthus orientalis L．)不同年龄的花被外植体诱导花器官直接再生的实验表明;1．在MS附加6-BA 2 mg／L,2,4-D 0.1 mg／L的培养基上,年龄V的外植体大量衰老,基本丧失器官再生能力,处于年龄段Ⅱ～Ⅳ的外植体可发生玻璃化反应。2．玻璃化反应的外植体转移至MS附加6-BA 0.2 mg／L、NAA 0.005 mg／L的培养基上继续培养30 d后可再生正常的花被片,表明降低培养基中外源激素浓度能够阻止玻璃化反应继续发生。3．在MS附加6-BA 2 mg／L．2,4-D 0.1 mg／L的培养基上,外植体形态学下部可再生雌蕊状早期结构。平均每块外植体分化雌蕊状早期结构数以年龄Ⅲ的外植体最多。