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agrobacterium–mediated Transformation of Kalanchoe laxiflora

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Horticultural Plant Journal 2019年第5期5卷 221-228页

作者： Xiling Wang Xinlu Chen Qunkang Cheng Kaikai Zhu Xiaohan Yang Zongming Cheng Department of Plant Sciences University of TennesseeKnoxvilleTN37996USA College of Biotechnology Southwest UniversityChongqing400715China Department of Entomology and Plant Pathology University of TennesseeKnoxvilleTN37996USA College of Horticulture Nanjing Agricultural UniversityNanjing210095China Biosciences Division Oak Ridge National LaboratoryOak RidgeTN37831-6422USA

Plants with crassulacean acid metabolism(CAM) generally utilize water 20%–80% more efficiently than non-CAM plants. The whole genomes of several CAM plants have been sequenced or are being sequenced. For effective genome characterization and genome editing of CAM plants,an efficient transformation system is essential. In this study, we developed an Agrobacterium-mediated transformation protocol for Kalanchoe laxiflora, an obligate CAM plant,by optimizing several factors affecting the transformation efficiency. agrobacterium strains AGL1, C58, EHA105,and GV3101 were all suitable for K. laxiflora transformation. Fifty-nine percent of the leaf explants yielded kanamycin-resistant and GUS-positive shoots. Polymerase chain reaction and quantitative real-time PCR(qRT-PCR) using gus A-, gus Plus-, npt II-and hpt-specific primers confirmed that the transgenes were integrated into K. laxiflora genome and expressed. This efficient transformation system will allow effective functional characterization of genes through over-or down-expression, knockout, or genome editing.

关键词： Kalanchoe laxiflora Agrobacterium-mediated transformation CAM plants

In planta Agrobacterium-mediated Transformation of Rice

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Rice science 2017年第3期24卷 181-186页

作者： Kumrop RATANASUT Weerawan ROD-IN Kawee SUJIPULI Department of Agricultural Science Faculty of Agriculture Natural Resources and Environment Naresuan University Phitsanulok 65000 Thailand

The floral-dip transformation, the simplest technique, is no requirement of tissue culture procedure, and can directly transfer the interest gene into plant reproductive cells. It has been successfully applied to various plant species. In this study, the optimal conditions of a floral-dip method for production of transgenic rice variety RD41 were explored. The simple and effective inoculation medium was composed of Murashige and Skoog(MS) medium, 5% sucrose, 44 nmol/L benzylaminopurine, and 0.075% surfactant Tween-20 with pH 5.7. The transformation efficiencies of agrobacterium tumefaciens strains AGL1 and EHA105 were compared with the agrobacterium density at OD_(600) = 0.8–1.0 and the co-cultivation at 25 ℃ for 48 h. A. tumefaciens strain EHA105 gave slightly higher transformation efficiency than AGL1, with statistically non-significant difference. The floral-drop transformation using the optimal floral-dip conditions showed higher transformation efficiency than the floral-dip method, but the dropped flowers turned brown and died within 2 d. Production of transgenic rice variety RD41 by the floral-dip method was achieved using A. tumefaciens strain EHA105 with the optimal conditions. Screening for the gus A gene by PCR using the gus A specific primers in the T_0 lines, there were 4 transgenic lines from 286 T_0 lines(1.4% transformation efficiency). However, histochemical glucuronidase(GUS) assay demonstrated that only three of four transgenic lines exhibited gus A expression. These results indicated that floral-dip transformation is a potential tool for production of the transgenic rice, which can be used for molecular breeding via genetic engineering in the future.

关键词： Agrobacterium-mediated transformation floral-dip floral-drop transformation efficiency in planta rice

Optimization of agrobacterium tumefaciens-mediated Transformation Systems in Tea Plant(Camellia sinensis)

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Horticultural Plant Journal 2017年第3期3卷 105-109页

作者： LV Qianru CHEN Changsong XU Yijuan HU Shunkai WANG Le SUN Kang CHEN Xuan LI Xinghui Tea Research Institute Nanjing Agricultural University Tea Research Institute Fujian Academy of Agricultural Sciences

In this study, an efficient plant regeneration protocol in vitro and transformation by Agrobacterium-mediated method of Camellia sinensis was achieved, which would lay the foundation for genetic improvement of tea plant by genetic engineering technology. The cotyledon callus of C.sinensis were used as the receptors for transformation by agrobacterium tumefaciens EHA105 containing PS1aG-3. Some factors which affected the result of Agrobacterium-mediated transformation of C. sinensis were studied on the basis of GUS transient expression system. The optimum system of Agrobacterium-mediated transformation was that the cotyledon callus were pre-cultured for 3 d, and then infected by EHA105 for 15 min followed by 3 d co-culture in the dark on the YEB medium containing 150 μmol·L^(-1) acetosyringone(AS). The transient expression rate of GUS gene was 62.6%. After being delayed selective culture for 3 d, infected callus were transferred into the differentiation medium and the root induction medium both of which were supplemented with 100 mg·L^(-1) spectinomycin, and then resistant seedlings of C. sinensis were obtained. The conversion rate was 3.6%.

关键词： Camellia sinensis cotyledon callus Agrobacterium-mediated GUS transient expression transformation ratio

Transformation of GbSGT1 gene into banana by an Agrobacterium-mediated approach

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Progress in Natural Science:Materials International 2007年第10期17卷 1241-1243页

作者： Li Shengjun1, Wen Ruiming2, Pei Xinwu1, Chen Shikai2, Li Weimin and Jia Shirong1(1. Biotechnology Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China 2. Xinyi Institute of Agricultural Science and Technology, Xinyi 523530, China) Biotechnology Institute Chinese Academy of Agricultural Sciences Beijing 100081 China Xinyi Institute of Agricultural Science and Technology Xinyi 523530 China

SGT1 is a homologue of the yeast ubiquitin ligase-associated protein. It controls some protein degradation and activates defense pathway in plants. Cotton GbSGT1 gene (Gossypium barbadense) has been isolated and characterized in previous work. In this study, the plant expression vector pBSGT1 with bar gene as a selection agent was constructed and transgenic banana was obtained via Agrobacterium-mediated transformation with the assistance of particle bombardment and screened with PCR and Basta spreading on banana plant leaves. Estimating of transgenic banana plants for resistance to Panama wilt is in progress.

关键词： transgenic banana Agrobacterium-mediated GbSGT1.

Genetic Transformation of GUS Gene into Musa acuminata cv.Mas(AA) by Agrobacterium-mediated Transformation(Abstract)

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Genetic Transformation of GUS Gene into Musa acuminata cv.Ma...

Agrobacterium-mediated Transformation of Embryogenic Calli of Anliucheng and Regeneration of Plants Containing the Chimaeric Ribonuclease Gene

第二届全国果树分子生物学学术研讨会

作者： HU Chun-hua,WEI Yue-rong,and YI Gan-jun (Fruit Research Institute,Guangdong Academy of Agricultural Science,Guangzhou,China 510640)

In order to establish a high efficient Agrobacterium-mediated genetic transformation system for banana.In this study,we explored the effects of different concentration of 2 antibiotics,and developed a new protocol of liquid medium selection system for Agrobacterium-mediated transformation.The results showed that the Hygromycin is more sensitivety than Kanamycin,the suitable concentration of Kanamycin on liquid medium cultivation of embryogenic cell suspensions(ECS) is 50mg/L;and the concentration of Hygromycin is 25mg/L.The Male-flower-derived ECS of Musa acuminata cv.Mas(AA),a major banana variety of the South East Asia region,were co-cultivated with agrobacterium strain EHA105 harboring a binary vector pCAMB1A1301 carrying nptII and gusA gene in M2 liquid medium supplemented with 100μM ACS.The infected ECS were co-cultivated first for 1 day at 60 rpm and tben for 3 days at 110 rpm on a rotary shaker.After 4days of co-cultivated in liquid medium,The infected ECS were then cultivated in M2 selective liquid medium containing suitable concentration of cefotaxime and hygromycin and subcultured every 10 days,Fine transgenic cell suspensions were obtained within 50 days of selection and confirmed by histochemical GUS assay.Regeneration of transformants after liquid-selection cultivation was carried out according to common method except for no antibiotic in medium.Compared to semi-solid co-cultivation and semi-solid selection transformation in other banana species,it is remarkable that liquid co-cultivation and selection was much more efficient for transformation of the ECS of banana.

关键词： banana Agrobacterium-mediated Transformation

来源： cnki会议评论

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Agricultural Sciences in China 2003年第2期2卷 127-131页

作者： LI Dong-dong, SHI Wei and DENG Xiu-xinNational Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University , Wuhan 430070 , P. R. China National Key Laboratory of Crop Genetic Improvement Huazhong Agricultural University Wuhan 430070 P. R. China

Anliucheng (Citrus sinensis Osbeck), a very seedy and widely spread acidless sweet orange cul-tivar in south of China, was transformed by the strain of agrobacterium Tumefaciens EHA105 carrying pTA29-barnase gene, which will induce pollen sterility in transgenic plants. The embryogenic calli of Anliucheng were co-cultivated with agrobacterium tumefaciens for 3 days, and then transferred to selective medium containing 50 mg I/1 basta (a kind of herbicide) for 5 weeks. The resistant calli were recovered and regenerated 118 embryoids. A total of 13 entire plants were obtained after micro-grafted on trifoliate orange. These regenerated plants were verified by PCR amplification and confirmed by PCR-Southern blotting analysis.

关键词： Citrus Calli pTA-barnase Genetic transformation Agrobacterium-mediated

Female reproductive system of Amaranthusas the target for agrobacterium-mediated transformation

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Advances in Bioscience and Biotechnology 2013年第2期4卷 188-192页

作者： Umaiyal Munusamy Siti Nor Akmar Abdullah Maheran Abd Aziz Huzwah Khazaai Department of Agriculture Technology Faculty of Agriculture Universiti Putra Malaysia Serdang Malaysia Department of Biomedical Science Faculty of Medicine and Health Sciences Universiti Putra Malaysia Serdang Malaysia

Agrobacterium-mediated transformation through floral dip and rapid selection process after transgenic event had become a preference as it will overcome the difficulties faced in tissue culturing procedures and lengthy time for screening transformed progenies. Therefore, in this study, three constructs, p5b5 (14,289 bp), p5d9 (15,330 bp) and p5f7 (15,380 bp) in pDRB6b vector which has hygromycin as a selectable marker gene were introduced individually into agrobacterium tumefaciens strain (AGL1). The cell suspension was applied to Amaranthus inflorescence by drop-by-drop technique and was left to produce seeds (T1). The T1 seeds were germinated and grown to produce seedlings under non-sterile condition. Hygromycin selection on seedling cotyledon leaves results in identification of 12 putative transformants, three from p5b5, four from p5d9 and five from p5f7. All positive putative transformants that were selected at the first stage through hygromycin spraying showed positive result in leaf disk hygromycin assay and in a construct specific polymerase chain reaction-based assay. A ~750 bp amplified hygromycin gene was further verified through sequencing. Our results suggest that Amaranthus inflorescences were able to be transformed and the transformed progenies could be verified through a combination of simple and rapid methods .

关键词： Agrobacterium-mediated Transformation Amaranthus Direct PCR Female Reproductive System Floral-Dip Hygromycin

Response of Explants of Calli Rice (Oryza sativaL.) Japonica cv. “Ilmi” to Gene Transformation Using agrobacterium tumefaciens-mediated

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American Journal of Plant Sciences 2013年第4期4卷 838-843页

作者： Fika Ayu Safitri Mohammad Ubaidillah Miswar Kyung-Min Kim Agrotechnology Study Program Faculty of Agriculture Jember University Jember Indonesia Division of Plant Biosciences School of Applied Biosciences College of Agriculture & Life Science Kyungpook National University Daegu South Korea

The AtBI-1 gene encoding the Arabidopsis thaliana Bax inhibitor was introduced into Japonica cultivars of rice (“Ilmi”) by Agrobacterium-mediated transformation, and a large number of transgenic plants were produced. The neomycin phosphotransferase II (NPTII) gene was used as a selectable marker. The activity of neomycin phosphotransferase could be successfully detected in transgenic rice calluses. Introduction of the AtBI-1 gene was also confirmed by PCR using AtBI-1 specific oligonucleotide primers in regenerated plants. Stable integration and expression of the AtbI-1 gene in plants were confirmed by GFP analysis.

关键词： AtBI-1 Agrobacterium-mediated GFP Transformation Rice

pYBA:A series versatile binary vectors for plant transformation

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pYBA:A series versatile binary vectors for plant transformat...