Hada Alkesh, Krishnan Veda, Punjabi Mansi, Basak Nabaneeta, Pandey Vanita, Jeevaraj Theboral, Marathe Ashish, Gupta Amit K, Jolly Monica, Kumar Arun, Dahuja Anil, Manickavasagam Markandan, Ganapathi Andy, Sachdev Archana
Division of Biochemistry, Indian Agricultural Research Institute, New Delhi 110 012, India.
Department of Biotechnology and Genetic Engineering, Bharathidasan University, Tiruchirappalli 620 024, India.
Plant Biotechnol (Tokyo). 2016;33(5):341-350. doi: 10.5511/plantbiotechnology.15.0901a. Epub 2016 Dec 17.
Soybean like many other crops, in this genomic era, has well-established genomic database which provides a wide range of opportunities for improvement through genetic manipulation. But the growing demand for soybean transgenics with increased production and improved quality has been handicapped due to inefficient transformation strategies and hence an efficient, stable and reliable transformation system is of prime requisite. In the present study, -mediated transformation was standardized by refining the glufosinate selection system in terms of dosage (0-6 mg l) and degree of exposure. The cotyledonary node explants (with and without wounding) initially cultured on a non-selective shoot induction medium for 10 days before transferring them to the selective SIM with an optimized concentration of 5.0 mg l ammonium glufosinate, showed least selection escape frequency. Wounded cotyledonary node explants infected with harboring pBIN-bar construct, showed an improved regeneration efficiency of 55.10% and transformation efficiency of 12.6% using Southern blotting in T plants. Southern analysis of T plants confirmed the integration of gene into the genomic DNA and the positive T plants segregated in 3 : 1 ratio. This is the first report, to our knowledge, of a high transformation efficiency using -mediated cot node-glufosinate system in an Indian soybean genotype.
与许多其他作物一样,在这个基因组时代,大豆拥有完善的基因组数据库,这为通过基因操作进行改良提供了广泛的机会。但是,由于转化策略效率低下,对产量增加和品质改良的大豆转基因产品的需求不断增长受到了阻碍,因此,高效、稳定和可靠的转化系统是首要需求。在本研究中,通过在剂量(0 - 6 mg l)和暴露程度方面优化草铵膦选择系统,对农杆菌介导的转化进行了标准化。子叶节外植体(有伤口和无伤口)最初在非选择性芽诱导培养基上培养10天,然后转移到含有5.0 mg l草铵膦铵盐优化浓度的选择性芽诱导培养基上,其选择逃逸频率最低。用携带pBIN-bar构建体的农杆菌感染有伤口的子叶节外植体,在T代植株中使用Southern杂交分析显示再生效率提高到55.10%,转化效率提高到12.6%。对T代植株的Southern分析证实了基因整合到基因组DNA中,并且阳性T代植株以3∶1的比例分离。据我们所知,这是关于在印度大豆基因型中使用农杆菌介导的子叶节 - 草铵膦系统获得高转化效率的首次报道。