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Cell Research 2001年第2期11卷 149-155页

作者： HUANG JIAN QIU, ZHI MING WEI, HAI LONG AN, YU XIAN ZHU National Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China National La National Laboratory of Plant Molecular Genetics Institute of Plant Physiology and Ecology Shanghai Institute for Biological Sciences Chinese Academy of Sciences Shanghai China National Laboratory of Plant Genetic Engineering and Protein Engineering College of Life Science Peking University Beijing China

Immature embryos of rice varieties 'Xiushui11' and 'Chunjiang 11' precultured for 4d were infected and transformed by agrobacterium tumefaciens strain EHA101/pExT7 (containing the spider insecticidal gene). The resistant cant were transferred onto the differentiation medium and plants were regenerated. The transformation frequency reached 56%-72% measured as numbers of Geneticin (G418)-resistant calli produced and 36%-60% measured as numbers of transgenic plants regenerated, respectively. PCR and Southern blot analysis of transgenic plants confirmed that the T-DNA had been integrated into the rice genome. Insect bioassays using T1 transgenic plants indicated that the mortality of the leaffolder (Cnaphalocrasis medinalis) after 7d of leaf feeding reached 38%-61% and the corrected mortality of the striped stem borer (Chilo suppressalis) after 7d of leaf feeding reached 16%-75%. The insect bioassay results demonstrated that the transgenic plants expressing the spider insecticidal protein conferred enhanced resistance to these pests.

关键词： Rice agrobacterium tumefaciens spider insecticidal gene transgenic plant Leaffolder striped stem borer insect bioassay.

agrobacterium tumefaciens-mediated transformation of modern rose(Rosa hybrida)using leaf-derived embryogenic callus

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Horticultural Plant Journal 2021年第4期7卷 359-366页

作者： Guoqin Liu Yuan Yuan Hui Jiang Ying Bao Guogui Ning Liangjun Zhao Xiaofeng Zhou Hougao Zhou Junping Gao Nan Ma State Key Laboratory of Agrobiotechnology Beijing Key Laboratory of Development and Quality Control of Ornamental CropsDepartment of Ornamental HorticultureCollege of HorticultureChina Agricultural UniversityBeijing 100193China Faculty of Life Science Tangshan Normal UniversityTangshanHebei 063000China Key Laboratory of Horticultural Plant Biology College of Horticulture and Forestry SciencesHuazhong Agricultural UniversityWuhan 430070China College Horticulture&Landscape Architecture Zhongkai University of Agriculture and EngineeringGuangzhou 510225China

Rose(Rosa hybrida)is widely used for cut flowers and as garden plants.Stable and efficient transformation system is required for functional genomics of rose.Here,we established an efficient transformation method for rose using agrobacterium tumefaciens-mediated transformation of embryogenic callus.Expanding rose leaves were used as explants to induce somatic embryos,which were subjected to transformation with A.tumefaciens strain GV3101 using Green Fluorescence Protein(GFP)as a marker gene.It took about 8 months to generate transgenic shoots from embryogenic callus.PCR,RT-PCR,Southern and Western blotting,as well as stereoscopic fluorescence microscopy analysis demonstrated that GFP transgenes integrated stably into the rose genome.According to our data,a transformation efficiency of up to 6%can be achieved by following this optimized protocol.

关键词： Rose Rosa hybrida Leaf Somatic embryos agrobacterium tumefaciens Genetic transformation

Enemies atpeace:Recentprogressin agrobacterium-mediated cereal transformation

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The Crop Journal 2024年第2期12卷 321-329页

作者： Shaoshuai Liu Ke Wang Shuaifeng Geng Moammar Hossain Xingguo Ye Aili Li Long Mao Karl-Heinz Kogel State Key Laboratory of Crop Gene Resources and Breeding Institute of Crop SciencesChinese Academy of Agricultural SciencesBeijing 100081China Institute of Phytopathology Centre for BioSystemsLand Use and NutritionJustus Liebig UniversityHeinrich Buff-Ring 26-32D-35392GiessenGermany

agrobacterium tumefaciens mediated plant transformation is a versatile tool for plant genetic engineering following its discovery nearly half a century ago.Numerous modifications were made in its application to increase efficiency,especially in the recalcitrant major cereals plants.Recent breakthroughs in transformation efficiency continue its role as a mainstream technique in CRISPR/Cas-based genome editing and gene stacking.These modifications led to higher transformation frequency and lower but more stable transgene copies with the capability to revolutionize modern agriculture.In this review,we provide a brief overview of the history of agrobacterium-mediated plant transformation and focus on the most recent progress to improve the system in both the agrobacterium and the host recipient.A promising future for transformation in biotechnology and agriculture is predicted.

关键词： agrobacterium tumefaciens Cereal species Genome editing Genetic engineering Plant breeding

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Research Progress on agrobacterium tumefaciens-based Transgenic Technology in Brassica rapa

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Horticultural Plant Journal 2018年第3期4卷 126-132页

作者： LI Guoliang YUE Lixin LI Fei ZHANG Shifan ZHANG Hui QIAN Wei FANG Zhiyuan WU Jian WANG Xiaowu ZHANG Shujiang SUN Rifei Institute of Vegetables and Flowers Chinese Academy of Agricultural Sciences

Brassica rapa L.is cultivated globally and consumed in many areas worldwide.Using the transgenic agrobacterium-mediated transformation method,which is a reproducible and efficient technique,genes can be transferred into various B.rapa species.This review summarizes the processes involved in agrobacterium-mediated transformation of B.rapa,including surface seed sterilization,co-cultivation with A.tumefaciens,induction of callus/shoot/root formation,and confirmation of transgenic plants.In addition,factors such as the agrobacterium strain,plant genotype,explant age,transformation efficiency of the hybrid or inbred line,and the concentrations of N6-benzyl amino purine and naphthalene acetic acid,are discussed.And this review shows clearly how to do it,what to do,and what not to do in the transgenic agrobacterium-mediated in Brassica rapa.The information presented here lays the foundation for a simple and efficient method that resolves existing problems and improves overall transgenic B.rapa production,thereby benefiting both basic and applied research.

关键词： Brassica rapa agrobacterium tumefaciens transgenic transformation

Improvement of Plant Regeneration from Immature Embryos of Wheat Infected by agrobacterium tumefaciens

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Agricultural Sciences in China 2011年第3期10卷 317-326页

作者： TAO Li-li YIN Gui-xiang DU Li-pu SHI Zheng-yuan SHE Mao-yun XU Hui-jun YE Xing-guo National Key Facility of Crop Gene Resources and Genetic Improvement Key Laboratory of Crop Genetics and Breeding Ministry ofAgriculture~Institute of Crop Sciences Chinese Academy of Agricultural Sciences Beijing 100081 P.R.China

Wheat, one of the most important food crops, has been extensively studied with respects to plant regeneration and transformation employing the immature embryos as recipient tissues. However, the transformed tissues often become severely necrotic after co-cultivation with agrobacterium, which is one of the major obstacles in gene delivery. In this study, wheat varieties CB037, Kenong 199, Xinchun 9, Lunxuan 987, and Shi 4185 showed desirable culture potential or high infection ability in agrobacterium-mediated transformation. Similarly, optimal regeneration conditions were determined by testing their ability to inhibit the cell necrosis and cell death phenotype. Two auxins of 2,4-dichlorophenoxyacetic acid （2,4-D） and 3,6-dichloro-o-anisic acid （dicamba） were evaluated for highly significant effect on both callus and plantlet production, although they were genotype-dependent in wheat. Substitution of 2,4-D by dicamba enhanced the growth and regeneration ability of callus from the immature embryos of most genotypes tested. The callus growth state couldn’t be modified by adding antioxidants such as ascorbic acid, cysteine, and silver nitrate or organic additives such as thiamine HCl and asparagine to the media. On the contrary, the best tissue statement and plant regeneration was achieved by employing the media containing the simplest MS （Murashige and Skoog） components and dicamba without organic components and vitamins. Thereby, our results are thought to inhibit wheat cell necrosis effectively and suggested to be used for more wheat genotypes.

关键词： wheat immature embryos agrobacterium tumefaciens transformation genotypes plant regeneration

Reliable and Efficient agrobacterium tumefaciens-Mediated Genetic Transformation of Dianthus spiculifolius

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Horticultural Plant Journal 2020年第3期6卷 199-204页

作者： Jingang Wang Shiyue Liu Hongping Ma Ye Tao Shuang Feng Shufang Gong Jinzhu Zhang Aimin Zhou College of Horticulture and Landscape Architecture Northeast Agricultural UniversityHarbin 150030China Key Laboratory of Saline-Alkali Vegetation Ecology Restoration Northeast Forestry UniversityMinistry of EducationHarbin 150040China College of Life Sciences Northeast Forestry UniversityHarbin 150040China

Dianthus spiculifolius is a perennial herbaceous flower with strong environmental adaptability and is an important ornamental ground cover plant.In this study,seeds of D.spiculifolius were used as explants for callus induction,adventitious bud differentiation,and rooting by adding different concentrations of 2,4-dichlorophenoxyacetic acid(2,4-D),6-benzyl aminopurine(6-BA),and naphthaleneacetic acid(NAA)to Murashige and Skoog medium.The calli generated were co-cultured with agrobacterium tumefaciens EHA105 containing pBI121-GUS or pBI121-GFP plasmids for 30 min,and transgenic regenerated plants were obtained by kanamycin(30mg·L^−1)screening.RT-PCR confirmed the stable expression of the exogenous GUS and GFP genes in the D.spiculifolius.Theβ-glucuronidase(GUS)histochemical staining confirmed GUS gene expression in transgenic calli,adventitious buds,and regenerated plants of D.spiculifolius.The green fluorescent protein(GFP)visual analysis showed GFP gene expression in transgenic calli.Furthermore,subcellular localization analysis showed that the three organelle marker proteins were not only successfully expressed but also accurately localized to their corresponding organelles in D.spiculifolius callus cells.These results indicated a successful establishment of a reliable and efficient A.tumefaciens-mediated genetic transformation system,which will contribute to functional gene research and genetic improvement of D.spiculifolius.

关键词： agrobacterium tumefaciens Dianthus spiculifolius Callus Tissue culture Plant transformation

agrobacterium tumefaciens-mediated GUS gene transformation of Robinia pseudoacacia 'Idaho'

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Forestry Studies in China 2006年第4期8卷 63-68页

作者： Sun Hai-jun Li Min Chen Shou-yi He Si-jie Wang Hua-fang College of Biological Sciences and Biotechnology Beijing Forestry University Beijing 100083 P. R. China Institute of Genetics and Developmental Biology Chinese Academy of Sciences Beijing 100101 P. R. China

Based on the plant regeneration system, a GUS gene transformation system to Idaho locust （Robinia pseudoacacia ‘Idaho＇） mediated by agrobacterium tumefaciens was established. The successful transformation was confirmed by regenerating the shoots fi＇om the infected leaves in the presence of hygromysin; by histochemical X-gluc assays of 15-glucuronidase （GUS） and by PCR and PCR-Southern blotting analysis. The ratio of positive transgenic plants is 5.8% （5 out of 86 plants）. With this system, the target gene DREB was introduced into the leaves of Idaho locust. The transgenic plants regenerated, which was verified by PCR-Southern blot- ting. It is suggested that the transformation system could be a new, simple, reliable and practical route to gene transformation of R. pseudoacacia ＇Idaho＇ mediated with A. tumefaciens.

关键词： Robinia pseudoacacia ‘Idaho＇ agrobacterium tumefaciens G US gene transformation

Production of Transgenic Tall Fescue Plants with Enhanced Stress Tolerances by agrobacterium tumefaciens-Mediated Transformation

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Agricultural Sciences in China 2006年第5期5卷 330-338页

作者： WU Guan-ting CHEN Jin-qing HU Zhang-hua LANG Chun-xiu CHEN Xiao-yun WANG Fu-lin JIN Wei XIA Ying-wu Institute of Virology and Biotechnology Zhejiang Academy of Agricultural Sciences Hangzhou 310021 P.R. China Institute of Nuclear-Agricultural Sciences Zhejiang University Hangzhou 310029 P.R. China

In order to improve stress tolerances of turf-type tall fescue （Festuca arundinacea Schreb.）, agrobacterium tumefaciens strain EHA105 carrying plasmid pCMD containing stress tolerance-related CBF1 gene from Arabidopsis thaliana was used to transform mature seeds-derived embryogenic calli of four cultivars. A total of 112 transgenic plants were regenerated from 32 independent lines and verified by histochemical detection of GUS activity, PCR assay and Southern hybridization analysis. The transformation frequency ranged from 0.92 to 2.87% with apparent differences among the cultivars. Stress tolerances of transgenic plants were enhanced, which was shown by the facts that transgenic plants had distinct growth superiority and significantly higher survival rate than non-transformed ones under high salinity and high osmosis stresses, and that relative electronic conductivity of in vitro leaves treated with low and high temoeratures, dehvdration and high salinity stresses was 25-30% lower in transgenic plants than in control plants.In addition,it was observed that growth of transgenic plants was inhibited due to constitutive overexpression of CBF1 gene under normal environmental conditions.

关键词： tall fescue （Festuca arundinacea Schreb.） agrobacterium tumefaciens genetic transformation CBF1 gene stress tolerance

agrobacterium tumefaciens-mediated GUS gene transfer to Sophora japonica L.

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Forestry Studies in China 2006年第4期8卷 6-9页

作者： Zhang Xiao-ying Wang Hua-fang Yin Wei-lun Zhu Zhen College of Biological Sciences and Biotechnology Beijing Forestry University Beijing 100083 P. R. China Beijing Langshan Nursery Beijing 100042 P. R. China institute of Genetics Chinese Academy of Sciences Beijing 1 O0101 P. R. China

agrobacterium-mediated genetic transformation of Sophora japonica was standardized using the agrobacterium tumefa- ciens strain LBA4404 that harbored the binary vector pBI121 containing genes for fl-glucuronidase （GUS） and neomycin phos- photransterase （npt II）. S. japonica transformants were selected by the ability of the leaf explants to produce kanamycin-resistant calli that regenerated into kanamycin-resistant plantlets. Successful transformation was confirmed by histochemical assay for GUS activity, PCR analysis and Southern blot. The period of nearly two months was required for the regeneration of transgenic plantlets fi＇om the explants. The transformed plants resembled their parents in morphology.

关键词： β-glucuronidase （GUS） Sophorajaponica agrobacterium tumefaciens neomycin phosphotransterase （nptⅡ）,transformation

Adventitious shoot regeneration of Platanus acerifolia Willd. facilitated by Timentin,an antibiotic for suppression of agrobacterium tumefaciens in genetic transformation

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Forestry Studies in China 2007年第1期9卷 14-18页

作者： Li Zhi-neng Liu Guo-feng Fang Fang Bao Man-zhu Key Laboratory of Horticultural Plant Biology Ministry of Education College of Horticulture and Forestry Sciences Huazhong Agricultural University Wuhan 430070 P. R. China

The effects of Timentin and cefotaxime （Cef） on shoot regeneration of the London plane tree （Platanus acerifolia Willd.） and their use for the suppression ofagrobacterium tumefaciens in agrobacterium-mediated genetic transformation were compared. Shoot regeneration was significantly reduced on the media with Cef at concentrations from 100 to 500 mg·L^-1. Timentin showed negative effect on plant regeneration at concentrations of 100 and 500 mg·L^-1; however, 300 mg·L^-1 Timentin was shown to facilitate shoot regeneration significantly and the regeneration frequency increased from 64% （control） to 88%. Effective suppression of A. tumefaciens could be obtained with 500 mg·L^-1 Cef, but plant regeneration was completely inhibited at this level. The A. tumefaciens on infected P. acerifolia leaf tissues was visually undetectable after three subcultures on a medium with 300 mg·L^-1 Timentin. Considering the effect of Cef and Timentin on plant regeneration and suppression of Agrobacteria, Timentin at 300 mg·L^-1 is the preferred application in .4. tumefaciens-mediated transformation ofP acerifolia.

关键词： Platanus acerifolia agrobacterium tumefaciens leaf explant plant regeneration genetic transformation antibiotic,Timentin