Transformation of Tomato with Cry2ax1 by Biolistic Gun Method for Fruit Borer Resistance

Tomato (Solanum esculentum L.) is an important vegetable crop cultivated throughout India. The crop is extensively damaged by alepidopteran insect, Helicoverpa armigera Hubner (fruit borer). To mitigate this problem, an attempt was made to generate transgenic tomato plants resistant to fruit borer. The regeneration protocol being pre-requisite for any transformation studies, 1.0 mg L⁻¹ BAP (Benzyl amino purine) in combination with 0.2 mg L⁻¹ IAA (Indole-3-acetic acid) was standardized as shoot regeneration media for tomato cv. Pusa Ruby using leaf explant. The highest callus initiation (100%) and an average number of shoots (3.9) were obtained in the above media. Rooting was observed at 2.0 mg L⁻¹ of IBA(Indole-3-butyric acid) with a highest average number of roots (1.06). The transformation system has been established by Biolistic Gun method using plant binary vector pCAMBIA2300 mobilized into the Agrobacterium strain LBA4404 carrying cry2AX1 under the control of CaMV 35S promoter. The callus of tomato was bombarded twice from three different distances between tissue and macro carrier assembly. The highest transformation efficiency (75%) was obtained at distance 9 cm and transformants were screened using 50 mg L⁻¹ of kanamycin. Out of 72 explants bombarded, only six explants developed completely but only two putative transformed plants were successfully hardened in the greenhouse. The integration and expression of the cry2AX1 gene in transformed plants were confirmed by PCR and Quantitative ELISA. The cry2AX1 protein accumulated in leaf quantified by ELISA has recorded 38.9 and 79.9 ng ml⁻¹ respectively compared to 129 ng ml⁻¹ of protein sample in positive control.

Regeneration protocol was standardized for tomato.

Transformation studies performed using Biolistic gun.

Integration of cry2AX1 gene was confirmed using PCRand quantitative ELISA.