Development of Inplanta Transformation system for Tomato (Solanum lycopersicum L.)

Abstract

Efficient and reproducible transformation system in tomato is required to circumvent tedious and prolonged tissue culture steps and to bioengineer the tomato rapidly. The majority of published protocols for tomato transformation require excellent regeneration capacity which is absent in our most elite cultivars. Furthermore, these protocols require expert personnel and costly equipment that may not be accessible to researchers serving in developing countries. Tissue culture based transformation system is also time consuming and somatic variation may arise. The induction, establishment and maintenance of callus in sterile condition are very difficult in bioengineering of tomato. Hence, present study is an endeavour to develop tissue culture independent transformation protocol for tomato. Solanum lycopersicum beta-lycopene cyclase mRNA (β-Lcy) gene was isolated from tomato and two different constructs were developed under different promoters and plant selection markers. The construct having β-Lcy gene under Ubiquitin promoter with bar gene as plant selectable marker was used in present study. The ovaries of closed and opened flowers were targeted for genetic transformation by Agrobacterium. Moreover, the apical meristems of germinated seeds were also targeted for genetic manipulation with the help of vacuum infiltration. Ovary injection method was found very disruptive and survival percentage of flowers was quite low. Survival percentage of opened flowers was 5% and closed flowers was 2.96% after injection of Agrobacterium suspension to their respective ovaries while the survival percentage of vacuum infiltrated germinated seeds was quite high from 44 to 52%. Different in planta transformation methods showed significant effect on genetic transformation efficiency. Injection of Agrobacterium suspension to ovaries of closed flowers was found most efficient with transformation efficiency of 1% followed by opened flowers (0.63%) and vacuum infiltrated germinated seeds method (0.56%) respectively. Statistical analysis revealed that there is no significant difference among varieties with respect to transformation efficiency with all three methods of In planta genetic transformation.