根癌农杆菌介导转录因子CBF1基因对草莓的转化

以草莓(Fragaria ananassa Duch)品种“达斯莱克特“(Darselect)为试材,用根癌农杆菌介导的方法,将转录因子CBF1基因导入草莓叶盘细胞,经多次筛选获得了转基因植株.转化植株经PCR检测,证实了CBF1基因已经整合到草莓的基因组中.以电解质渗透法检测了植株的抗寒性,结果显示转基因草莓的抗寒能力较未转化植株有明显提高,且不同转基因株系之间提高程度有着差异.     

Agrobacterium rhizogenes-mediated genetic transformation and regeneration of a conifer:Larix decidua

Summary Gene transfer and plant regeneration systems have been developed for European larch (Larix decidua Mill.) in our laboratory. Aseptically germinated young seedlings were hypocotyl wound-inoculated withAgrobacterium rhizogenes strains 11325 containing a wild-type Ri (root-inducing) plasmid. Swollen stems appeared at infected wounds followed by either abundant hairy roots or adventitious shoot buds that developed within 3 to 4 wk after inoculation. No symptoms were seen on wounded but uninoculated seedlings. We demonstrated agrobacteria attached to larch cells by examination of scanning electron micrographs. Subsequently, calli derived from symptomatic tissues exhibited phytohormone autotrophic growth. Adventitious buds were elongated and rooted in vitro before being transferred to the greenhouse where the transformed whole plants grew normally. Transformants tested positive for opine production and transformation was further confirmed by Southern blot analysis with larch genomic DNAs isolated from both proliferated calli and needle tissue of transgenic plants.     

去除转基因植物标记基因的研究进展

得到转基因植物以后 ,标记基因就失去了筛选的作用。但它的存在引起公众对转基因植物的安全性以及环境效应的担心 ,所以在目的基因转入后 ,要去除标记基因。该文主要就利用共转化、转座子、同源重组、位点特异重组酶等去除标记基因的方法进行了总结 ,并对各种方法的优缺点进行了比较 ,对该技术未来的发展趋势也进行了展望。     

Agrobacterium-mediated transformation and regeneration of cotton plants

Cotton (Gossypium hirsutum L.) was transformed by the EHA101 strain of Agrobacterium tumefaciens harboring a binary vector pGA482GG plasmid carrying the marker genes for neomycin phosphotransferase II (nptII) determining resistance to kanamycin and β-glucuronidase (GUS). The cotyledons, hypocotyls, shoot meristem tissue, and its segments taken from in vitro growing seedlings were used as explants. Explants were cultured in a Murashige and Skoog (MS) medium containing various hormone combinations to induce shoot regeneration. The highest frequency of shoot formation was obtained from the shoot meristem. After selection in the MS medium containing kanamycin (50 mg/l), these tissues were tested by histochemical GUS assay. Shoots regenerated from excised shoot meristems or their halves were cultured for 4–6 weeks to obtain rooted plants, which then produced fully-developed plants and seeds in pots. Genomic integration of the kanamycin-resistance gene was detected by the PCR analysis. Seed germination percentage was 95% after the F1 seeds of transgenic cotton plants were cultured on half-strength MS medium supplemented with 50 mg/l kanamycin. Thus, a protocol for effective Agrobacterium-mediated genetic transformation of cotton was optimized. Published in Russian in Fiziologiya Rastenii, 2006, Vol. 53, No. 3, pp. 462–467. The text was submitted by the authors in English.     

Plant regeneration from leaf explants of Aloe barbadensis Mill. and genetic fidelity assessment through DNA markers

An efficient plant regeneration protocol was developed from leaf explants of Aloe barbadensis Mill on Murashige and Skoog’s (MS) medium supplemented with 2.0 mg/l 6-benzyladenine (BA) or Kinetin (Kn), 0.25–0.5 mg/l NAA (1-napthalene acetic acid) and 3 % (w/v) sucrose within 4 weeks of culture. The maximum number of shoot buds were obtained on MS medium supplemented with 2.0 mg/l BA, 0.5 mg/l NAA, 40 mg/l Ads (adenine sulphate) within 4–6 weeks of subculture. Inclusion of 0.25–0.50 mg/l gibberellic acid into the medium, the shoot buds became elongated. Repeated subculture on regeneration medium induces higher rate of shoot regeneration. The root induction from excised microshoots was achieved on half-strength MS medium supplemented with 0.25–1.0 mg/l NAA or indole-3-butyric acid (IBA) and 2 % (w/v) sucrose. Maximum percentage of rooting was achieved on medium having 0.5 mg/l NAA with 3 % (w/v) sucrose. About 80 % of in vitro raised plantlets were hardened in the greenhouse and successfully established in the soil. Both Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeat (ISSR) markers were used to detect the variability among the regenerated plants developed in vitro. The results showed that there was no polymorphism among the regenerated plantlets. This study will help for propagation of quality planting material of Aloe barbadensis for commercialization.     

Plant regeneration from protoplasts of Triticum aestivum L. cv. Nakasoushu

A fast-growing, small, granular, embryogenic callus was selected from primary calli induced from the Japanese wheat cultivar Nakasoushu and the Australian wheat cultivar Bodallin. Regenerable and fine suspension cultures were induced three to six months after liquid culture was initiated and were characterized by dense cytoplasm and active division. These suspension cultures routinely provided high yields of protoplasts with about 90% viability when incubated in a modified KMP (Kao and Michayluk, 1975) medium containing 1 mg l^-1 2,4-D (2,4-dichlorophenoxyacetic acid), and 1 mg l^-1 zeatin. Nakasoushu and Bodallin protoplasts divided at frequencies of 8.6% and 11.1%, respectively, in agarose-solidified media. When Nakasoushu protoplasts were cultured with effective nurse cells of sorghum and wheat, protoplast division increased to 16.9% and 12.6%, respectively. Plating efficiencies varied from 0.03% to 2.5%. After subculture, protocolonies yielded embryogenic calli and somatic embryos, from which green plants were eventually regenerated. Whole plants obtained from Nakasoushu protoplasts were fertile, demonstrating the first report of Japanese cultivars in wheat protoplast cultures. This revised version was published online in June 2006 with corrections to the Cover Date.     

Agrobacterium-mediated transformation and regeneration of guayule

In vitro grown shoot tissue of facultative apomictic lines of guayule (Parthenium argentatum Gray), a rubber producing desert shrub, were transformed by Agrobacterium-mediated DNA transfer and regenerated into complete plants. Guayule shoots of lines 11591, UC101 and UC104 were inoculated with A. tumefaciens strains LBA4404 or PC2760 harboring the binary vector pCGN1557. Axillary shoots were regenerated from transformed cells and rooted in vitro in the presence of kanamycin. Genetic transformation in all cases was verified by Southern blot analysis. Transgenic plants were grown to maturity in the greenhouse and, as predicted for apomictic species, all seed produced possessed kanamycin resistance. Because apomicts have limitations for gene transfer by normal sexual crosses, this method offers a new means of transferring genes into this species.Abbreviations BA benzyladenine - EDTA ethylene diamine tetraacetate - kan_R kanamycin resistance - MS salts salts of Murashige and Skoog medium (1962) - NAA naphthalene acetic acid - NPT-II neomycin phosphotransferase - SDS sodium dodecyl sulfate     

The isopentenyl transferase gene is effective as a selectable marker gene for plant transformation in tobacco (Nicotiana tabacum cv. Petite Havana SRI)

 A selection method for transformed cells which does not inhibit regeneration is important for the establishment and optimization of a transformation protocol. We have assessed the 35S-ipt gene from Agrobacterium tumefaciens as a selectable marker gene. The identification of ipt-expressing cells from nontransformed cells enabled morphological selection without the use of kanamycin and also allowed for the elimination of a high proportion of nonexpressing cells. Ipt selection of tobacco leaf discs (Nicotiana tabacum cv. Petite Havana SRI) resulted in a 2.7-fold higher transformation frequency compared to kanamycin selection. Overexpression of the ipt gene favored plant regeneration from transformed cells, and the transformation frequency of the ipt plus kanamycin selection resulted in a 1.6-fold higher transformation frequency than kanamycin selection alone. These results indicate that this procedure might provide a strategy whereby transgenic plants can be efficiently obtained and some of the problems related to the use of antibiotics diminished. Received: 1 November 1999 / Revision received: 26 June 2000 / Accepted: 18 July 2000     

Heterologous expression of the AtDREB1A gene in chrysanthemum increases drought and salt stress tolerance

DNA cassette containing an AtDREB1A cDNA and a nos terminator,driven by a cauli- flower mosaic 35S promoter,or a stress-inducible rd29A promoter,was transformed into the ground cover chrysanthemum(Dendranthema grandiflorum)'Fall Color'genome.Compared with wild type plants,severe growth retardation was observed in 35S:DREB1A plants,but not in rd29A:DREB1A plants.RT-PCR analysis revealed that,under stress conditions,the DREB1A gene was over-expressed constitutively in 35S:DREB1A plants,but was over-expressed inductively in rd29A:DREB1A plants.The transgenic plants exhibited tolerance to drought and salt stress,and the tolerance was significantly stronger in rd29A:DREB1A plants than in 35S:DREB1A plants.Proline content and SOD activity were increased inductively in rd29A:DREB1A plants than in 35S:DREB1A plants under stress conditions.These results indicate that heterologous AtDREB1A can confer drought and salt tolerance in transgenic chrysanthemum,and improvement of the stress tolerance may be related to enhancement of proline content and SOD activity.     

Heterologous expression of the AtDREB1A gene in chrysanthemum increases drought and salt stress tolerance

DNA cassette containing an AtDREB1A cDNA and a nos terminator, driven by a cauliflower mosaic 35S promoter, or a stress-inducible rd29A promoter, was transformed into the ground cover chrysanthemum (Dendranthema grandiflorum) ‘Fall Color’ genome. Compared with wild type plants, severe growth retardation was observed in 35S:DREB1A plants, but not in rd29A:DREB1A plants. RT-PCR analysis revealed that, under stress conditions, the DREB1A gene was over-expressed constitutively in 35S:DREB1A plants, but was over-expressed inductively in rd29A:DREB1A plants. The transgenic plants exhibited tolerance to drought and salt stress, and the tolerance was significantly stronger in rd29A:DREB1A plants than in 35S:DREB1A plants. Proline content and SOD activity were increased inductively in rd29A:DREB1A plants than in 35S:DREB1A plants under stress conditions. These results indicate that heterologous AtDREB1A can confer drought and salt tolerance in transgenic chrysanthemum, and improvement of the stress tolerance may be related to enhancement of proline content and SOD activity.     

角质细胞生长因子2基因(KGF2)的克隆及其对油菜的遗传转化

近年来,植物表达人源蛋白的研究逐渐增多。本研究以人角质细胞生长因子(KGF2)基因cDNA为基础,根据植物偏好密码子进行改造,并利用PCR方法合成KGF2基因全长cDNA。在此基础上构建了KGF2的植物油体系统表达载体p1390-YO-KGF2,并采用农杆菌介导法对甘蓝型油菜无菌苗的子叶进行转化。通过PCR、Southern杂交和Western blotting检测,证明外源基因KGF2已经转入油菜中并得到成功表达。     

Establishment of far‐red high irradiance responses in wheat through transgenic expression of an oat phytochrome A gene

A transgenic wheat line over‐expressing an oat phytochrome A gene under the control of the constitutive maize ubiquitin promoter was generated using a biolistic particle delivery system from immature wheat embryos. The resulting line showed increased levels of total phytochrome A protein in both dark‐grown and light‐grown plants. When grown under continuous far‐red light, seedlings of this line showed additional inhibition of the coleoptile extension in comparison with wild‐type seedlings. Unlike the response of wild‐type seedlings to continuous far‐red, this additional inhibition was dependent on fluence rate and was not observed under half‐hourly pulses of far‐red delivering the same total fluence as the continuous irradiation treatment. These observations suggest that increase in phytochrome A levels in wheat leads to the establishment of a far‐red high irradiation reaction in this monocotyledonous plant. Exposure to continuous red light caused a similar inhibition of coleoptile extension in both the wild types and the transgenic seedlings. When wild‐type seedlings were grown under continuous far‐red, their coleoptiles remained completely colourless and first leaves remained tightly rolled. In contrast, transgenic seedlings grown in the same conditions produced significant levels of anthocyanins in their coleoptiles and their first leaves became unrolled. Taken together, our data suggest that the increased levels of phytochrome A in wheat can change the type of response of some developmental processes to light signals, leading to the generation of a high irradiance reaction which is otherwise absent in the wild types under the conditions used.     

Regeneration of diploid and tetraploid plants from callus-derived protoplasts of Agapanthus praecox ssp. orientalis (Leighton) Leighton

protoplasts was developed for the Liliaceous ornamental plant, Agapanthus praecox ssp. orientalis (Leighton) Leighton `Royal Purple Select' (2n=2x=32).Viable protoplasts were routinely isolated from leaf-derived embryogenic calluses with yields of 0.8 to 1.5x10 protoplasts per g FW of calluses. Protoplasts started to divide 5 to 7 days after isolation, and protoplast-derived colonies consisting of 50 to 100 cells were obtained after 1 month. A plating efficiency of 0.8% was obtained after 2 months of culture using a gellan gum-solidified medium containing 1 mg 1^-1 each of PIC and BA under continuous illumination. Protoplastderived calluses produced somatic embryos at a frequency of 46.7 % on PGR-free medium, whereas 68.3 % of the calluses regenerated adventitious shoots on a medium containing 1 mg 1^-1 BA. Somatic embryos and adventitious shoots developed into plantlets, which were successfully transplanted to pots. Flow cytometric analysis and chromosome observation revealed that both diploid and tetraploid plants were regenerated from protoplasts.     

Protoplast transformation of recalcitrant alkaliphilic Bacillus sp. with methylated plasmid DNA and a developed hard agar regeneration medium

Among the diverse alkaliphilic Bacillus strains, only a little have been reported to be genetically transformed. In this study, an efficient protoplast transformation procedure was developed for recalcitrant alkaliphilic Bacillus sp. N16-5. The procedure involved polyethylene glycol-induced DNA uptake by the protoplasts and subsequent protoplast regeneration with a developed hard agar regeneration medium. An in vivo methylation strategy was introduced to methylate the exogenous plasmid DNA for improving the transformation efficiency. The transformation efficiency reached to 1.1×10(5) transformants per μg plasmid DNA with methylated plasmid pHCMC04 and the developed hard agar regeneration medium. This procedure might also be applicable to the genetic transformation of other Bacillus strains.     

双孢蘑菇萎锈灵抗性基因定点突变的载体构建与遗传转化

为建立更为安全、有效的双孢蘑菇遗传转化体系,构建了双孢蘑菇琥珀酸脱氢酶的铁硫蛋白亚基Agsdi1突变(His突变为Leu)表达载体pAgsdi1,并通过农杆菌介导方法转化双孢蘑菇W192,经萎锈灵筛选以及PCR扩增和MnlⅠ酶切验证后获得了转化菌株.验证结果表明,点突变的铁硫蛋白亚基Agsdi1可以作为双孢蘑菇有效的抗...     

Plant regeneration and genetic transformation of Lotus angustissimus

Culture conditions have been established for callus induction and growth from different explants in L. angustissimus L. Calli were obtained from hypocotyls, leaves, stems, cotyledons and roots cultured on media containing 2,4-dichlorophenoxyacetic acid or -naphthaleneacetic acid with kinetin, N⁶ – ² or benzyladenine in different combinations and concentrations. Only those calli induced in presence of -naphthaleneacetic acid with benzyladenine or kinetin produced shoots. Calli induced from hypocotyl explants were the most efficient in regeneration of shoots. Transformation with an Agrobacterium rhizogenes binary vector carrying the plasmid pBI 121.1 is reported. The percentage of cotransformation was estimated by testing GUS activity in hairy roots. The integration of Ri T-DNA and the NPTII gene in transformed plants was confirmed by molecular analyses and in vitro culture of transgenic tissues in the presence of kanamycin.Abbreviations BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - 1AA indole-3-acetic acid - NAA -naphthaleneacetic acid - 2iP N⁶ – ² - PA proanthocyanidins - NOS nopaline synthase - NI TII neomycin phosphotransferase - GUS -glucuronidase - CaMV cauliflower mosaic virus     

Heterologous expression of the AtDREB1A gene in chrysanthemum increases drought and salt stress tolerance

DNA cassette containing an AtDREB1A cDNA and a nos terminator, driven by a cauliflower mosaic 35S promoter, or a stress-inducible rd29A promoter, was transformed into the ground cover chrysanthemum (Dendranthema grandiflorum) ‘Fall Color’ genome. Compared with wild type plants, severe growth retardation was observed in 35S:DREB1A plants, but not in rd29A:DREB1A plants. RT-PCR analysis revealed that, under stress conditions, the DREB1A gene was over-expressed constitutively in 35S:DREB1A plants, but was over-expressed inductively in rd29A:DREB1A plants. The transgenic plants exhibited tolerance to drought and salt stress, and the tolerance was significantly stronger in rd29A:DREB1A plants than in 35S:DREB1A plants. Proline content and SOD activity were increased inductively in rd29A:DREB1A plants than in 35S:DREB1A plants under stress conditions. These results indicate that heterologous AtDREB1A can confer drought and salt tolerance in transgenic chrysanthemum, and improvement of the stress tolerance may be related to enhancement of proline content and SOD activity.     

Small-scale field tests with transgenic potato,cv. Bintje,to test resistance to primary and secondary infections with potato virus y

The coat protein (CP) gene of the potato virus Y (PVY) strain N605 has been cloned into a plant binary expression vector and introduced into the potato variety Bintje. The transformed lines, Bt6, that contained two copies of the CP gene showed complete resistance to the homologous strain PVY-N605 and a good resistance to the related strain PVY-O803 in the greenhouse. The good resistance of Bt6 to primary and secondary infections by PVY was confirmed in two successive field tests where the virus was transmitted by its natural aphid vector.     

Effect of aminoglycoside antibiotics on in-vitro morphogenesis from cultured cells of chrysanthemum and tobacco

Successful genetic transformation of plants requires non-chimeric selection of transformed tissues and their subsequent regeneration. With rare exceptions, most transformation protocols still rely heavily on antibiotics for selecting transgenic cells that contain an antibiotic-degrading selectable marker gene. Here, the morphogenic capacity of in-vitro expiants of chrysanthemum and tobacco stems and leaves (control and transgenic) changed with the addition of aminoglycoside antibiotics (AAs). In a test of 6 AAs, phytotoxicity occurred at concentrations of 10 to 25 and 50 to 100 ng ml.⁻¹ in chrysanthemum and tobacco expiants, respectively. Light conditions as well as expiant source and size also had significant effects. The use of transverse thin cell layers (tTCLs), in conjunction with high initial AA selection levels, supported the greatest regeneration of transgenic material (adventitious shoots or callus) and the lowest number of escapes. Flow-cytometric analyses revealed no endoduplication in chrysanthemum, even at high AA levels. However, this phenomenon was observed in tobacco calli (8C or more), even at low AA concentrations (i.e., 5 to 10 μg mL^-1).     

在培养基中加入抗生素防止万年青茎段培养污染的研究

以玛丽安万年青茎段为试验材料,将浓度为50mg·L1的青霉素、氯霉素、利福平、庆大霉素、先锋霉素6号、链霉素和卡拉霉素7种抗生素分别经过过滤灭菌后加入到MS基本培养基,从中筛选出防止污染效果较好的两种抗生素,分别按50、100、150mg·L1的浓度加入到MS培养基中培养,以筛选出最适合浓度;然后再筛选出利福平和氯霉素的最佳浓度组合。结果表明:7种抗生素中以利福平和氯霉素防止污染效果较好,未污染率分别为68.42%和58.33%,成活率分别为65.79%和52.78%,利福平和氯霉素的浓度均为150mg·L1时防止污染的效果最佳;25mg·L1利福平+50mg·L1氯霉素和50mg·L1利福平+50mg·L1氯霉素组合,未污染率分别达到78.46%、85.18%,极显著高于50mg·L1利福平单独处理的效果。