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agrobacterium-mediated transformation of herbicide resistance in creeping bentgrass and colonial bentgrass

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Journal of Zhejiang University Science 2003年第3期4卷 346-351页

作者：柴明良汪炳良 KIMJae-yeoul LEEJong-min KIMDoo-hwan DepartmentofHorticulture ZhejiangUniversityHangzhou310029China DeoartmentofHorticulturalScience KonkukUniversitySeoul143-701Korea DepartmentofBiologyScience TheUniversityofAucklandNewZealand

Embryogenic calli were induced from the seeds of creeping bentgrass ( Agrostis palustris Huds.) cv. Regent and colonial bentgrass ( Agrostis Tenuis Sibth. Fl. Oxen.) cv. Tiger. The embryogenic calli were precultured on fresh medium for 4-7 days and then co cultivated with agrobacterium tumefaciens , LBA4404, which contains plasmid vector pSBGM harboring bar coding region, synthetic green fluorescent protein (sGFP) coding region and matrix attachment region (MAR). After 3 days of co cultivation, the calli were washed thoroughly and transferred to MS medium containing 2 mg/L of 2, 4 D, 12-15 mg/L phosphinothricin (PPT) and 250 mg/L of cefotaxime. After 2-3 months of selection, the actively growing calli of 'Regent' and 'Tiger' were transferred to MS medium with 12-15 mg/L PPT and 250 mg/L cefotaxime for regeneration. The putative transformants were maintained on MS medium with 3 mg/L PPT for long period but control died within 1 month. After establishing in greenhouse, the transformants also showed strong resistance to 0.4% of herbicide Basta but control plants died within 2 weeks. Under confocal microscope, both young leaves and roots showed significant GFP expression. PCR analysis revealed the presence of a DNA fragment of GFP gene at the expected size (380 bp) in the transformants and its absence in a randomly selected control plant.

关键词： Agrostis palustris Agrostis tenuis agrobacterium Herbicide resistance sGFP(synthetic green fluorescent protein)

agrobacterium tumefaciens CAN TRANSFORM Triticum aestivum AND Hordeum vulgare OF GRAMINEAE

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Science China Chemistry 1990年第1期33卷 27-34页

作者：邓万银林晓影邵启全 Institute of Genetics Academia Sinica Beijing 100012 PRC

So far there is no suitable vector system for the genetic engineering of monocotyledonous plants, especially cereal crops. It is first reported in this paper that agrobacterium tumefaciens strains T37, A208 and B6 can transform some varieties of Triticum aestivum and Hordeum vulgare to form swellings and tumors. Itis shown in the experiments that phenolic compound acetosyringone may promote the transformation process. In addition, inoculating agrobacteria on appropriate plant tissues is also proved to be the key step in achieving successful transformation. The host range of A. tumefaciens and the possibility of utilizing Ti plasmid as the vector for the gene transfer of cereal crops are discussed.

关键词： agrobacterium tumefaciens Triticum aestivum Hordeum vulgarz acetosyringone transformation of cereal crops.

agrobacterium-mediated transformation: state of the art and future prospect

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Chinese Science Bulletin 2000年第17期45卷 1537-1546页

作者： Wei Li Guangqin Guo Guochang Zheng Lanzhou Univ Cell Biol Lab Lanzhou 730000 Peoples R China

Great progress has been made in recent years in studies on the mechanism of agrobacterium-mediated transformation and its application. Many details of the key molecular events within the bacterial cells involved in T-DNA transfer have been elucidated, and it is notable that some plant factors which were elusive before are purified and characterized. Vast kinds of species, which were either recalcitrant to or not included in the host range of agrobacterium, can' now be transformed by this bacterium, and they include the very important cereal species, gymnosperms, yeast and many filamentous fungi. The simple in vivo transformation of tissue in intact plants and the 'agrolistic' methods to transform recalcitrant plants are the two novel technical achievements. Combined with other powerful techniques such as bacterial artificial chromosome, very large DNA fragment can be transformed into the plant genome by agrobacterium. Further studies will elucidate more plant-encoded factors involved in T-DNA

关键词： agrobacterium -mediated transformation fungi monocots large DNA fragment.

Soybean hairy roots produced in vitro by agrobacterium rhizogenes-mediated transformation

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The Crop Journal 2018年第2期6卷 162-171页

作者： Li Chen Yupeng Cai Xiujie Liu Chen Guo Shi Sun Cunxiang Wu Bingjun Jiang Tianfu Han Wensheng Hou National Center for Transgenic Research in Plants Institute of Crop Science Chinese Academy of Agricultural Sciences Ministry of Agriculture Key Laboratory of Soybean Biology (Beijing) Institute of Crop Science Chinese Academy of Agricultural Sciences

Soybean is one of the world's most important oil and protein crops. Efficient transformation is a key factor for the improvement of soybean by genetic modification. We describe an optimized protocol for the agrobacterium rhizogenes-mediated transformation of soybean and the induction of hairy root development in vitro. Cotyledons with 0.5-cm hypocotyls were cut from 5-day-old seedlings and used as explants. After infection and co-cultivation,hairy roots were produced in induction culture medium after 10–12 days. Using this method, 90%–99% of the infected explants of five different cultivars produced hairy roots within one month. Observations using reporter constructs showed that 30%–60% of the hairy roots induced were transformed. Based on high transformation efficiency and short transformation period, this method represents an efficient and rapid platform for study of soybean gene function.

关键词： agrobacterium rhizogenes Hairy root Protocol Soybean Transformation

Evaluation of parameters affecting agrobacterium-mediated transient expression in citrus

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Journal of Integrative Agriculture 2017年第3期16卷 572-579页

作者： LI Fang DAI Su-ming DENG Zi-niu LI Da-zhi LONG Gui-you LI Na LI Yi Alexandra Gentile National Center for Citrus Improvement Changsha/Horticulture and Landscape College Hunan Agricultural University Changsha 410128 P.R.China Department of Plant Science University of Connecticut Connecticut 06269 USA Department of Agriculture and Food Science University of Catania Catania 95123 Italy

agrobacterium-mediated transient expression assays are a convenient alternative to stable expression because they are simple,easy to perform,and achieve gene expression *** study investigated the factors affecting transient gene expression efficiency in citrus by observing the cryo-sectioning of leaf samples under a laser confocal *** factors included the composition of the infiltration buffer,the agrobacterium cell density,the leaf development stage,the incubation temperature,and plant *** highest transient expression level of yellow fluorescent protein（YFP）was detected in Mexican lime（Citrus aurantifolia）on the third day after the intermediate-aged leaves were infiltrated with the improved infiltration buffer 1（15 mmol L^-1 2-（N-morpholino）ethanesulfonic acid,10 mmol L^-1 MgCl2,and 200μmol L^-1acetosyringone）,which had an optical density of 0.8 and was incubated at 22°***,this transient expression assay was applied to other citrus *** note,trifoliate orange（Poncirus trifoliata）and kumquat（Fortunella obovate）had higher expression efficiency than other six genotypes of the Citrus *** study provides research basis for the selection of optimization strategies in transient gene expression and improves the method for available genome investigation in citrus.

关键词： citrus agrobacterium transient expression efficiency yellow fluorescent protein (YFP)

STUDIES ON INTRODUCTION OF FOREIGN GENES INTO CULTURED CELLS OF Oryza sativa INDICA USINC agrobacterium Ti PLASMID SYSTEM

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Science China Chemistry 1991年第1期34卷 54-63页

作者：李宝健欧阳学智许耀 Biotechnology Research Centre Zhongshan University Guangzhou 510275 PRC

Either bacterial attachment or cellulose fibrillar elaboration was hardly observedduring the cocultivation of the cultured suspension cells of Oryza sativa Indica with thestrain C58C1 Rif^r of agrobacterium tumefaciens that was not specially pretreated. Onthe other hand, quite a lot of agrobacterium cells were found to adhere to the surface ofcultured rice cells and a number of cellulose fibrils were produced around the specifiedbacteria when phenolics-pretreated bacteria were cocultivated with rice suspension cellsin common culture media, especially in complex culture solutions. The complex culturesolution was the bacterium-free filtrate of hormone-containing MS medium which hadbeen utilized to incubate carrot cells and the newly wounded hypocotyl segments fromtomato and agrobacterium cells. Detecting experiments demonstrated that both NPT Ⅱ andNOS genes, located on the T-DNA segment of chimaeric plasmid pGV3850 :: 1103neo, weretransferred and expressed in the cultured cells of O. sativa Indica in the optimum trans-formation condition of the present experiment. Integration of the foreign genes into riceDNA was confirmed by Southern blot hybridization.

关键词： rice cell transformation Vir gene activation phenolic compounds interaction of agrobacterium cells with host cells scanning electron microscope

TRANSFORMATION OF Lotus corniculatus *** BY agrobacterium tumefaciens

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Science China Chemistry 1991年第8期34卷 932-937页

作者：虞剑平邵启全 Institute of Genetics Academia Sinica

A simple and effective system for the transformation and regeneration of a leguminous plant has been developed. The cotyledon explants of Lotus corniculatus (var. Leo) were infected by agrobacterium tumefaciens that contained a non-oncogenic Ti plasmid vector. The vector carried a chimaeric npt-Ⅱ gene and a nopaline synthase gene (nos). On the selective medium that contained kanamycin, 40% of the explants formed buds within 3 weeks. Developed shoots were cut off and transferred to rooting medium. Normal-looking plants were recovered and grew well after being transplanted into soil, bloomed and set seeds. Foreign genes were integrated onto the L. corniculatus genome, expressed, and inherited through sexual reproduction, which was proven by nopaline detection, NPT-Ⅱ enzyme activity detection and DNA hybridization test of the transformed plants and the nopaline detection of the progeny.

关键词： plant transformation Legumineae Lotus corniculatus agrobacterium tumefaciens L.

Optimisation of Adventitious Shoot Regeneration and agrobacterium-mediated Transformation in Canna × generalis(Canna Lily)

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Horticultural Plant Journal 2019年第1期5卷 39-46页

作者： Rani Singh Arvind Kumar Dubey Indraneel Sanyal Plant Transgenic Laboratory Division of Molecular Biology and Biotechnology CSIR-National Botanical Research Institute

Canna being ornamental plants has a significant role in agriculture, medical, economy and food industry. Canna has a limited vase life due to the rapid loss of moisture from its perianth. For improving its market value, cuticularisation of the perianth can be achieved by the expression of a heterologous cutin producing gene, using the tissue culture and transformation protocol developed in this study. Efficient, rapid and direct adventitious shoot regeneration was successfully established in Canna × generalis using recalcitrant rhizome explants. The explants were cultured on MS medium supplemented with 6-benzylaminopurine(6-BA), thidiazuron(TDZ), and kinetin. Among the four genotypes taken for tissue culture, the ‘Trinacria variegata' was the best responding cultivar. And 2 mg · L^(-1)6-BA or 1.5 mg · L^(-1) TDZ along with 0.1 mg · L^(-1) IAA was optimum for their regeneration. The highest regeneration was achieved in ‘Trinacria variegata'(36%) on 6-BA, 33% on TDZ while kinetin failed to evoke any regenerative responses. Regeneration was enhanced by supplementation of 100 mg · L^(-1) ascorbic acid(AsA), while, 100 mg · L^(-1) of l-cysteine or 100 mg · L^(-1) dithiothreitol(DTT), inhibited regeneration. Shoots were observed to develop 3–5 fibrous roots on MS medium supplemented with 0.5 mg · L^(-1) indole-3-butyric acid(IBA). The plantlets were transplanted into pots and acclimatised in glasshouse with 100%survival. For transformation of Canna, rhizome explants were co-cultivated for 60 min in agrobacterium suspension. The explants were washed with 500 mg · L^(-1) cefotaxime solution, subjected to 100 mg · L^(-1) kanamycin selection followed by excision of the shoots and culturing them on IBA-supplemented media for root development. Transgene integration in the putative transformants was confirmed by PCR assay and copy number by Southern blot hybridisation analysis.

关键词： Canna regeneration rhizome UidA agrobacterium transformation ascorbic acid 6-BA TDZ

Structural Basis of Interaction of Bipartite Nuclear Localization Signal from agrobacterium VirD2 with Rice Importin-o

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Molecular Plant 2014年第6期7卷 1061-1064页

作者： Chiung-Wen Chang, Simon J. Williams, Rafael Miguez Cou?ago Bostjan Kobe School of Chemistry and Molecular Biosciences Institute for Molecular Bioscience and Australian Infectious Diseases ResearchCentre University of Queensland Brisbane Qld 4072 Australia

Dear Editor, In the eukaryotic cell, the nuclear envelope separates genomic DNA from the rest of the cell, but ensures a molecu- lar communication between the nucleus and the cytoplasm through the nuclear pore complexes （NPCs）. Nuclear trans- port of macromolecules is an energy-dependent process, exemplified by the classical nuclear import pathway. In the cytoplasm, proteins containing classical nuclear localization signals （cNLSs） are recognized by a hetero-dimeric transport receptor （importin-α （Impα）：importin-β （Impl3） complex）. Impa is employed as an adaptor protein that specifically rec-ognizes the classical nuclear localization signal （cNLS） from the cargo protein （Marfori et al., 2011）. Translocation of the trimeric cargo：transport receptor import complex through the NPCs is mediated by transient interactions between Impβ and nucleoporins in the NPC. The directionality of the trans- port is imparted by the asymmetric distribution, between the cytoplasm and the nucleus, of the distinct nucleotide-bound （GTP/GDP） states of the small GTPase Ran （Tran et al., 2007）.

关键词： agrobacterium RESEARCH PLANT proteins PLANT proteins -- Structure PLANT cell nuclei CRYSTAL structure CARRIER proteins -- Molecular structure CARRIER proteins RICE -- Research