Agrobacterium tumefaciens-mediated transformation of Dendrobium lasianthera J.J.Sm: An important medicinal orchid

IF 3.5 Q3 Biochemistry, Genetics and Molecular Biology

Journal of Genetic Engineering and Biotechnology Pub Date : 2018-12-01 DOI:10.1016/j.jgeb.2018.02.002

Edy Setiti Wida Utami , Sucipto Hariyanto , Yosephine Sri Wulan Manuhara

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引用次数: 8

Abstract

A protocol for genetic transformation mediated by Agrobacterium tumefaciens and production of transgenic Dendrobium lasianthera has been developed for the first time. The 8-week-old protocorm explants were used as target of transformation with Agrobacterium tumefaciens strain LBA4404 carrying plasmid pG35SKNAT1. Several parameters such as infection period, Agrobacterium density, concentration of acetosyringone, and co-cultivation period were evaluated for the transformation efficiency. The data were analyzed using one-way analysis of variance (ANOVA) and Duncan's Multiple Range Test (DMRT) with p < 0.05. Subsequently, KNAT1 gene expression was confirmed by polymerase chain reaction (PCR) analysis. The highest efficiency of transformation (70%) obtained from protocorm explants infected with Agrobacterium culture was at the OD₆₀₀ concentration of 0.6 for 30 min, and co-cultivated with acetosyringone 100 µM for 5 days. The results of confirmation by PCR analysis show that the KNAT1 gene has been integrated and expressed in the genome of Dendrobium lasianthera transgenic.

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农杆菌介导的lasianthera石斛的转化:一种重要的药用兰花

首次建立了农杆菌介导的转基因石石斛的遗传转化和转基因石石斛的生产方案。以8周龄的原球茎外植体为目标，转染质粒pG35SKNAT1的农杆菌LBA4404。对感染时间、农杆菌浓度、乙酰丁香酮浓度、共培养时间等参数进行了转化效率评价。数据分析采用单因素方差分析(ANOVA)和Duncan多元极差检验(DMRT)， p为 < 0.05。随后，通过聚合酶链反应(PCR)分析证实了KNAT1基因的表达。当OD600浓度为0.6时，与乙酰丁香酮100 µM共培养5 天，农杆菌培养的原球茎外植体转化效率最高(70%)。PCR分析结果证实，KNAT1基因已在转基因石斛基因组中整合表达。

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来源期刊

Journal of Genetic Engineering and Biotechnology Biochemistry, Genetics and Molecular Biology-Biotechnology

CiteScore

5.70

自引率

5.70%

发文量

159

审稿时长

16 weeks

期刊介绍： Journal of genetic engineering and biotechnology is devoted to rapid publication of full-length research papers that leads to significant contribution in advancing knowledge in genetic engineering and biotechnology and provide novel perspectives in this research area. JGEB includes all major themes related to genetic engineering and recombinant DNA. The area of interest of JGEB includes but not restricted to: •Plant genetics •Animal genetics •Bacterial enzymes •Agricultural Biotechnology, •Biochemistry, •Biophysics, •Bioinformatics, •Environmental Biotechnology, •Industrial Biotechnology, •Microbial biotechnology, •Medical Biotechnology, •Bioenergy, Biosafety, •Biosecurity, •Bioethics, •GMOS, •Genomic, •Proteomic JGEB accepts