Transgeni根癌农杆菌介导的小麦转基因植株再生(英文)

根癌农杆菌菌株Ａｇｌ Ⅰ的Ｔｉ 质粒ｐＵＮＮ－２ 带有Ｕｂｉ１ 启动子驱动的ｎｐｔ Ⅱ基因。７ 种基因型小麦幼胚或胚性愈伤组织用于农杆菌介导的转化实验。经过不同浓度巴龙霉素的筛选,３ 种基因型小麦产生抗性愈伤组织并再生植株。再生植株经ＰＣＲ 和Ｓｏｕｔｈｅｒｎ 杂交鉴定为转基因植株,转化频率（ 再生转基因植株的小麦愈伤组织数／ 用于转化实验的愈伤组织数） 为３．７％ ～５ ．９％ 。小麦基因型及转化材料的起始生理状态是影响ＴＤＮＡ转移的重要因素。     

根癌农杆菌介导的小麦转基因植株再生

根癌农杆菌菌株Ａｇｌ Ⅰ的Ｔｉ质粒ｐ＾ＵＮＮ－２带有Ｕｂｉ１启动子驱动的ｎｐｔⅡ基因。７种基因型小麦幼胚或胚性愈伤组织用于农杆菌介导的转化实验。经过不同家度巴龙霉素的筛选,３种基因型小麦产生抗性愈伤组织并再生植株。再生植株经ＰＣＲ和Ｓｏｕｔｈｅｒｎ杂交鉴定为转基因植株,转化频率为３．７％－５．９％。小麦基因型及转化材料的起始生理状态的影响Ｔ－ＤＮＡ转移的重要因素。     

基因枪法向小麦导入几丁质酶基因的研究

利用基因枪法,以菜豆几丁质酶基因转化小麦幼胚愈伤组织。在轰击压力1300psi,轰击距离6cm、100μg金粉/枪和轰击距离9cm、150μg金粉/枪的2种处理条件下,获得4株春小麦东农7742转化植株,转化频率分别为0.36％和0.56％。经PCR和PCR-Southern杂交分析,证实菜豆几丁质酶基因已整合到T0和T1小麦基因组中。采用氨基葡萄糖法测定几丁质酶活力,结果表明,转基因小麦的几丁质酶活力明显高于对照株;转基因植株对白粉病症状减缓,并获得一株赤霉菌接种未扩展的转基因T1植株。     

用基因枪法将抗除草剂基因导入小麦栽培品种的研究

利用基因枪法将抗除草剂bar基因导入西南地区的 3个小麦栽培品种 ,共获得 7个转基因植株 ,转化频率在 0 .45 %～ 1 .2 %之间 ,转化周期缩短至 3个月左右。对抗性植株进行PCR和PCR_Southern杂交检测 ,初步确定bar基因已导入小麦基因组。做转基因植株叶片对除草剂PPT的抗性试验 ,有 4株呈抗性 ,3株呈部分抗性 ,表明bar基因已在小麦植株中得到表达。     

农杆菌介导亚洲百合转化体系的建立及转GsZFP1基因研究

以亚洲百合‘耀眼’(Cedeazzle)无菌苗小鳞片为遗传转化受体,并利用农杆菌介导法将锌指转录因子基因GsZFP1转化‘耀眼’无菌苗小鳞片,初步建立亚洲百合‘耀眼’的遗传转化体系,通过筛选获得了50株疑似转化植株,通过用PCR方法对获得的50株疑似抗性植株进行检测,结果显示20株呈阳性,PCR阳性转化率为40%;将得到的阳性植株进行RT-PCR检测,获得12株RT-PCR阳性植株。结果表明,锌指转录因子基因GsZFP1在亚洲百合‘耀眼’中得到表达。     

GFP基因在新疆小拟南芥和拟南芥中的表达分析

以质粒pMCB30为模板,扩增GFP基因,连接到载体pCMBIA2300-35S-OCS上,构建过量表达载体p35S:GFP,将其转入农杆菌GV3101.通过农杆菌介导法将p35S:GFP载体分别转入新疆特色植物小拟南芥和拟南芥中.T0代经含有卡那霉素的1/2MS培养基筛选,获得了T1代转基因小拟南芥2株,T1代转基因拟南芥9株.通过激光共聚焦显微镜观察,在转基因小拟南芥和拟南芥的根尖细胞中均可检测到GFP绿色荧光蛋白;对转基因植株进行PCR扩增,均可检测到GFP基因,表明GFP基因已成功转入小拟南芥和拟南芥中.该研究建立了小拟南芥的遗传转化体系,为进一步利用GFP基因和进一步研究小拟南芥的功能基因奠定基础.     

马铃薯遗传转化体系的优化及玉米淀粉分支酶基因SBEⅡb的导入

以马铃薯脱毒试管苗茎段为转化受体材料,建立并优化了农杆菌介导的马铃薯遗传转化体系。通过农杆菌介导法将玉米淀粉分支酶基因(Starch branching enzyme b,SBEⅡb)的过表达载体转化马铃薯,接种762个茎段,共获得35株抗性植株。经PCR检测获得了4株转基因阳性植株;对转基因植株进一步进行GUS活性组织化学染色,发现转基因植株的茎段与试管薯均被染上蓝色,表明外源SBEⅡb基因已整合到马铃薯基因组,且正常表达。     

Optimization of genetic transformation system of potato and introduction of maize starch branching enzyme gene SBEⅡb into potatoFAN

以马铃薯脱毒试管苗茎段为转化受体材料,建立并优化了农杆菌介导的马铃薯遗传转化体系.通过农杆菌介导法将玉米淀粉分支酶基因(Starch branching enzyme b,SBEⅡb)的过表达载体转化马铃薯,接种762个茎段,共获得35株抗性植株.经PCR检测获得了4株转基因阳性植株;对转基因植株进一步进行GUS活性组织化学染色,发现转基因植株的茎段与试管薯均被染上蓝色,表明外源SBEⅡb基因已整合到马铃薯基因组,且正常表达.     

农杆菌介导抗储粮害虫转基因小麦(Triticum aestivum L.)的获得及分析

以本实验室选育的小麦优良品系的胚性愈伤组织为材料,采用农杆菌介导将抗虫基因豇豆胰蛋白酶抑制剂基因CpTI转入小麦培养细胞,经筛选获得抗卡那霉素的愈伤组织并再生植株。经PCR和实时PCR检测、PCR-Southern和Southernblot验证,确定了3株独立再生植株为含有CpTI的转基因植株。农杆菌菌浓度、侵染时间及转化处理方式对小麦转化率均有明显影响。3株转基因植株正常可育并结籽,形成转基因株系。外源基因在转基因植株T1代中的分离呈多样性,部分株系(转基因株系T-Ⅰ、T-Ⅲ)表现出孟德尔遗传规律。抗虫试验表明,3株转基因植株T2代籽粒对储粮害虫麦蛾具有一定的抗性,转基因株系T-Ⅰ、T-Ⅱ、T-Ⅲ及非转基因植株的T2代籽粒虫蛀率分别为19·8%、21·9%、32·9%和58·3%。转基因植株T1代群体农艺性状调查显示,3个株系具有良好的农艺性状,为小麦的遗传改良提供了新的种质抗虫材料。     

用基因枪法将抗除草剂基因导入小麦栽培品种的研究

利用基因枪法将抗除草剂bar基因导入西南地区的3个小麦栽培品种,共获得7个转基因植株,转化频率在0.45%～1.2%之间,转化周期缩短至3个月左右。对抗性植株进行PCR和PCR_Southern 杂交检测,初步确定bar基因已导入小麦基因组。做转基因植株叶片对除草剂PPT的抗性试验,有4株呈抗性,3株呈部分抗性,表明bar基因已在小麦植株中得到表达。     

农杆菌介导法向玉米茎尖导入抗草甘膦EPSPS基因的研究

以玉米自交系郑58的茎尖为受体,通过农杆菌介导法将抗草甘膦EPSPS基因转入玉米中,研究以茎尖为受体的农杆菌转化体系的可行性。98株转化苗,经过300 ppm的除草剂(农达)筛选,共获得13株转基因植株,经PCR检测,其中7株表现阳性,转化率达7.14%。初步证明外源基因已经整合到玉米基因组中,以玉米茎尖作为受体的转化系统用于基因转化是可行、高效的。     

Transgenic wheat progeny resistant to powdery mildew generated by Agrobacterium inoculum to the basal portion of wheat seedling

To improve the transformation efficiency of wheat (Triticum aestivum L.) mediated by Agrobacterium tumefaciens, we explored the possibility of employing the basal portion of wheat seedling (shoot apical meristem) as the explants. Three genotypes of wheat were transformed by A. tumefaciens carrying β-1, 3-glucanase gene. After vernalization, the seeds to be transformed were germinated. When these seedlings grew up to 2∼5 cm, their coleoptile and half of the cotyledon were cut out, and the basal portions were infected by A. tumefaciens. A total 27 T₀ transgenic plants were obtained, and the average transformation efficiency was as high as 9.82%. Evident segregation occurred in some of the T₁ plants, as was indicated by PCR and Southern blotting analysis. Investigation of the T₂ plants revealed that some transformed plants had higher resistance to powdery mildew than the controls. Northern blotting revealed that β-1, 3-glucanase gene was normally expressed in the T₂ plants, which showed an increased resistance to powdery mildew. The results above indicate that the exogenous gene has been successfully integrated into the genome of wheat, transmitted and expressed in the transgenic progeny. From all the results above, it can be concluded that Agrobacterium inoculum to the basal portion of wheat seedling is a highly efficient and dependable transformation method. It can be developed into a practicable method for transfer of target gene into wheat.Tong-Jin Zhao and Shuang-Yi Zhao contributed equally to this paper.     

Transgenic maize plants of tropical and subtropical genotypes obtained from calluses containing organogenic and embryogenic-like structures derived from shoot tips

A highly efficient system for the production of transgenic maize plants starting from tropical and subtropical genotypes was developed. The method is based on particle bombardment of organogenic calli derived from shoot tips. Six tropical maize genotypes were successfully transformed and regenerated using this protocol. Genetic transformation was confirmed by Southern blot analysis of T0 plants and segregation analysis of the resistance marker in the T1 progeny. Plant transfer into the greenhouse was 100% successful, and no problems of fertility were observed with the transgenic plants produced with this transformation protocol.     

Shoot apical meristem: In vitro regeneration and morphogenesis in wheat (Triticum aestivum L.)

Summary We report a less genotype-dependent in vitro regeneration system capable of producing multiple shoot clumps and whole plants in four different wheat genotypes. Shool apical meristems from 7-d-old-seedlings produced axillary and adventitious shoots and somatic embryos on media containing N⁶-benzyladenine (BA) and 2,4-dichlorophenoxyacetic acid (2,4-D). All four genotypes responded positively to shoot multiplication depending upon media composition. Scanning electron microscopies of cultures showed a proliferating budding state that gave rise to adventitious shoots and somatic embryos on further multiplication. The percentage of relative shoot apical meristem multiplication was 80–90%, and the average number of shoot meristems per multiplied shoot was 40–50 in all genotypes. Among different concentrations of phytohormones, 2 and 4 mgl⁻¹ BA (8.8 and 17.7 μM) in combination with 0.5 mg l⁻¹ 2,4-D (2.26 μM) gave the best results. Actively multiplying shoot clumps were recovered with high frequency among 3-mo.-old cultures. These shoot clumps regenerated normally and produced fertile plants containing viable seeds. This in vitro system might prove useful for the production of transgenic plants of wheat in a relatively genotype-independent manner.     

Efficient genotype-independent cotton genetic transformation and genome editing

Cotton (Gossypium spp.) is one of the most important fiber crops worldwide. In the last two decades, transgenesis and genome editing have played important roles in cotton improvement. However, genotype dependence is one of the key bottlenecks in generating transgenic and gene-edited cotton plants through either particle bombardment or Agrobacterium-mediated transformation. Here, we developed a shoot apical meristem (SAM) cell-mediated transformation system (SAMT) that allowed the transformation of recalcitrant cotton genotypes including widely grown upland cotton (Gossypium hirsutum), Sea island cotton (Gossypium barbadense), and Asiatic cotton (Gossypium arboreum). Through SAMT, we successfully introduced two foreign genes, GFP and RUBY, into SAM cells of some recalcitrant cotton genotypes. Within 2–3 months, transgenic adventitious shoots generated from the axillary meristem zone could be recovered and grown into whole cotton plants. The GFP fluorescent signal and betalain accumulation could be observed in various tissues in GFP- and RUBY-positive plants, as well as in their progenies, indicating that the transgenes were stably integrated into the genome and transmitted to the next generation. Furthermore, using SAMT, we successfully generated edited cotton plants with inheritable targeted mutagenesis in the GhPGF and GhRCD1 genes through CRISPR/Cas9-mediated genome editing. In summary, the established SAMT transformation system here in this study bypasses the embryogenesis process during tissue culture in a conventional transformation procedure and significantly accelerates the generation of transgenic and gene-edited plants for genetic improvement of recalcitrant cotton varieties.     

农杆菌介导的半夏凝集素基因(Pinellia Ternate Agglutinin Gene,pta)对小麦的遗传转化及鉴定

以甘肃主要推广春小麦品种陇春22幼胚为转基因受体材料,建立了农杆菌介导的小麦遗传转化体系。以预培养4天的幼胚愈伤组织为受体,C58c1农杆菌菌株为供体,将含有半夏凝集素基因的重组质粒pBIpta转入了小麦,经G418 25 mg/L抗性筛选、PCR检测和荧光定量PCR检测共获得转基因植株3株,外源基因的插入拷贝数分别为2、1、3。同时对转基因小麦的T₁代植株进行了PCR检测和抗虫性分析,表明半夏凝集素基因在转基因植株的后代中得到了遗传并有一定的抗蚜虫作用。     

Transgenic plants from shoot apical meristems of <Emphasis Type="Italic">Vitis vinifera</Emphasis> L. “Thompson Seedless” via <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation

Shoot apical meristem explants of Vitis vinifera “Thompson Seedless” were used for Agrobacterium-mediated genetic transformation. It was determined that the meristems had to be subjected to a dark growth phase then wounded to obtain transgenic plants. Morphological and histological studies illustrated the role of wounding to expose apical meristem cells for transformation. A bifunctional egfp/nptII fusion gene was used to select kanamycin resistant plants that expressed green fluorescent protein (GFP). Kanamycin at a concentration of 16 mg L⁻¹ in selection medium resulted in recovery of non-chimeric transgenic plants that uniformly expressed GFP, whereas 8 mg L⁻¹ kanamycin allowed non-transgenic and/or chimeric plants to develop. Polymerase chain reaction (PCR) and Southern blot analyses confirmed the presence of transgenes and their stable integration into the genome of regenerated plants. Up to 1% of shoot tips produced stable transgenic cultures within 6 weeks of treatment, resulting in a total of 18 independent lines.     

Formation and maintenance of the shoot apical meristem

Development in higher plants is characterized by the reiterative formation of lateral organs from the flanks of shoot apical meristems. Because organs are produced continuously throughout the life cycle, the shoot apical meristem must maintain a pluripotent stem cell population. These two tasks are accomplished within separate functional domains of the apical meristem. These functional domains develop gradually during embryogenesis. Subsequently, communication among cells within the shoot apical meristem and between the shoot apical meristem and the incipient lateral organs is needed to maintain the functional domains within the shoot apical meristem.     

Agrobacterium-mediated transformation of Lupinus mutabilis L. using shoot apical explants

A procedure for regenerating plants of Lupinus mutabilis from shoot apices, from which the leaf primordia and initial cell layer(s) of the apical meristem were removed, has been used to generate transgenic plants following Agrobacterium tumefaciens-mediated gene delivery. Transformation competent cells, from which buds developed, were located at the periphery of the apical meristem. Kanamycin resistant plants were obtained which expressed β-glucuronidase activity. Integration of the neomycin phosphotransferase II and β-glucuronidase genes into the genomes of transgenic plants was confirmed by non-radioactive DNA-DNA hybridisation. This is the first report of the generation of transgenic plants in L. mutabilis.     

Pathogen resistant Transgenic Plant from Dwarfing Rootstock of Cherry by Introducing Antibacterial Polypeptide Genes

A shoot regeneration system of high frequency from shoot of Chinese cherry ( Prunus pseudocerasus Lindl. ) dwarfing rootstock was established and 19 transformed plants were obtained from the dwarfing rootstock by introducing gus gene and antibacterial polypeptide genes, using Agrobacteriuan tumefaciens (Smith et Townsend) Corm as mediator. The results of Southern blot analysis proved that antibacterial polypeptide gene was integrated into the cherry genome. The results of tumor test, X-gluc reaction and bacterial-resistant test of leaf extract against Agrobacterium tumefaciens C58 indicated that antibacterial polypeptide gene may be expressed efficiently in the transformed plant. The tube shoot was characteristic in its resistance to root nodule disease. This study on the characteristics of germ line transformation of shoot meristem suggested that it was a highly efficient transformation system.