Genetic Transformation of Cry 1AC Gene to Counter Fruit and Shoot Borer of Brinjal (Solanum Melongena L.)

Brinjal shoot and fruit borer (Leucinodes orbonalis Guen) causes 50–70% loss to marketable yield of brinjal (Solanum melongena L.) and farmers spray heavy doses of pesticides for controlling the cryptic pest. This is a matter of concern for food safety, environmental degradation, pest resistance and economics of the crop. Use of biotechnological tools is an efficient and precise way to develop intrinsic plant resistance in genotypes of interest and transformation of Bt-gene is a tested option to control Lepidopteron pests. The present work was carried out to develop transgenic brinjal, wherein regeneration protocol was standardized and Cry1Ac gene was transferred in elite genotype of brinjal. Developing regeneration baseline, hypocotyl, cotyledon and leaf explants of in vitro raised seedlings of BL-5 were cultured on MS medium fortified with BAP and kin (2.0 mgl⁻¹ BAP, 2.0 mgl⁻¹ BAP + 1.0 mgl⁻¹ kin, 2.5 mgl⁻¹ BAP, 2.5 mgl⁻¹ BAP + 1.0 mgl⁻¹, 3.0 mgl⁻¹ BAP, 3.0 mgl⁻¹ BAP + 1.0 mgl⁻¹, 2.0 mgl⁻¹ BAP + 1.0 mgl⁻¹) for shoot regeneration and incubated at 16 hrs light/8 hrs dark cycles at 25±2°C. The regenerated buds were then elongated on half strength MS medium supplemented with 0.3 mgl⁻¹ BAP. The elongated plantlets were cut aseptically and transferred to MS basal medium for root induction. Cotyledon found best for plant regeneration (66.47%) and number of buds explant -1 (21.76) on MS medium was fortified with 2.5 mgl⁻¹ BAP and 1.0 mgl⁻¹ Kin. hypocotyl was found to have poor response for regeneration response in all the media types. Regenerated plants with well-developed root system established in the field after hardening in a glass house for flowering and fruiting. The cotyledon being best explant for regeneration was subjected to hygromycin sensitivity assay at 5, 10 and 15ppm concentration. With 10 ppm Hygromycin selection pressure, cotyledon induced 6.90% buds, which were turned pale afterwards and did not elongate. However, 5 ppm Hygromycin selection pressure regenerated 54.62% cotyledon explants with few buds as compared to 87.14 % regeneration efficiency in control explants (without selection pressure). For genetic transformation, cotyledon explants were co-bombarded with Cry1Ac gene construct (Altosaar) driven by Ubi promoter and NOS terminator and GUS gene construct driven by CaMV35S promoter and NOS terminator. Cry1Ac and GUS were maintained as plasmids in E.coli strain JM109. Cotyledons were arranged in the centre of the target plate containing osmotic medium (MS + 0.4M mannitol + 30gl-1 + 8gl-1 agar) for four hours and the plasmid DNA of Cry1Ac and GUS constructs were isolated separately, mixed into 9:2 proportions and coated on to the tungsten particles. The DNA was co-bombarded on to the target plate at 9 cm distance with 900psi pressure. The bombarded cotyledons were subjected to three selection cycles of 15, 10 and 5ppm hygromycin concentration. The overall selection efficiency after three selection cycles of 15 days each was 17.8%, where the plants were initially selected with 15 ppm Hygromycin in regeneration medium fortified with 2.5 mgl⁻¹ BAP and 1.0 mgl⁻¹ Kin and then transferred to elongation medium (MS + 0.3 mgl⁻¹ BAP) with 10ppm and 5ppm in succeeding cycles. GUS expression in cotyledon explants (76.92%) and elongating shoots (66.66%) and PCR analysis confirmed the presence of Cry 1Ac in two out of four plants in BL-5 genotype of brinjal.