Amal Thomas Cheeran, Karthika Palanisamy, Dhandapani Gurusamy, Selvakumar Subramaniam, Vasanth Krishnan
Molecular Biology Laboratory, Department of Botany, School of Life Sciences, Bharathiar University, Coimbatore, Tamil Nadu, 641046, India.
PG Research Department of Botany, Kongunadu Arts and Science College, Bharathiar University, Coimbatore, Tamil Nadu, 641029, India.
J Genet Eng Biotechnol. 2020 Mar 24;18(1):9. doi: 10.1186/s43141-020-00023-z.
Recalcitrant nature is a major constraint for the in vitro regeneration and genetic transformation of leguminous species members. Therefore, an improved genetic transformation in horse gram has been developed via in planta method, in which Agrobacterium strain harboring binary vector pCAMBIA2301 was used for the transformation. Several factors affecting in planta transformations were put forth viz. Agrobacterium cell density, co-cultivation, and sonication combined with vacuum infiltration duration which were optimized.
Germinated seeds were sonicated and vacuum infiltrated with different densities of Agrobacterium culture and co-cultivated in half-strength MS medium with 100 μM of acetosyringone for 48 h. Seedlings were washed with cefotaxime and sowed in vermiculite soil for maturation. T plants were subjected to histochemical and molecular analysis to ensure transformation efficiency. Among various combinations analyzed, maximum transformation efficiency (20.8%) was attained with seeds of 5 min sonication combined with vacuum infiltration with 0.6 optical density of Agrobacterium culture.
It concludes that a different Agrobacterium cell density with sonication combined with vacuum infiltration has improved transgenic efficiency in horse gram plants. This simple and efficient method is feasible for the stable expression of foreign genes that could be beneficial for future food security.
顽拗性是豆科植物成员体外再生和遗传转化的主要限制因素。因此，通过体内方法开发了一种改良的鹰嘴豆遗传转化方法，其中携带二元载体pCAMBIA2301的农杆菌菌株用于转化。提出了几个影响体内转化的因素，即农杆菌细胞密度、共培养以及与真空渗透持续时间相结合的超声处理，并对其进行了优化。
将发芽的种子进行超声处理，并用不同密度的农杆菌培养物进行真空渗透，然后在含有100μM乙酰丁香酮的半强度MS培养基中共培养48小时。用头孢噻肟洗涤幼苗，并播种在蛭石土壤中使其成熟。对转基因植株进行组织化学和分子分析以确保转化效率。在分析的各种组合中，超声处理5分钟并结合0.6光密度的农杆菌培养物进行真空渗透的种子获得了最高的转化效率（20.8%）。
得出结论，不同的农杆菌细胞密度结合超声处理和真空渗透提高了鹰嘴豆植株的转基因效率。这种简单有效的方法对于外源基因的稳定表达是可行的，这可能对未来的粮食安全有益。
