Development of transgenic Bt crops with stable and high level of Bt protein expression over generations under different environmental conditions is critical for successful deployment at field level. In the present study, progenies of transgenic cotton Coker310 event, CH12 expressing novel cry2AX1 gene were evaluated in T3 generation for stable integration, expression and resistance against cotton bollworm, Helicoverpa armigera. The cry2AX1 gene showed stable inheritance and integration in the T3 progeny plants as revealed by PCR and Southern blot hybridization. The expression of Cry2AX1 protein on 90 days after sowing (DAS) was in the range of 1.055 to 1.5 µg/g of fresh leaf tissue except one plant which showed 0.806 µg/g of fresh leaf tissue and after 30 days (i.e., on 120 DAS) three plants recorded in between 0.69 to 0.82 µg/g and other plants are in range of 0.918 to 1.058 µg/g of fresh leaf tissue. Detached leaf bit bioassay in T3 progeny on 110 DAS recorded mortality of 73.33 to 93.33 per cent against H. armigera and severe growth retardation in surviving larvae. These results indicate that the expression of chimeric cry2AX1 is stable and exhibits insecticidal activity against H. armigera in T3 progeny of CH12 event of transgenic cotton.

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Study of Expression of Indigenous Bt cry2AX1 Gene in T3 Progeny of Cotton and its Efficacy Against Helicoverpa armigera (Hubner)

Factors that influence transformation efficiency such as age of explants, plant growth regulator concentration, antibiotic concentration, Agrobacterium density, and infection time were optimized with a view to establish a high-throughput transformation protocol for tomato (Solanum lycopersicum L.) cv. PKM-1. Seedling explants were transformed using a binary vector carrying nptII gene, conferring kanamycin resistance by Agrobacterium mediated transformation. Transformation efficiency of 27 % and 17 % respectively was observed when cotyledon and hypocotyl explants were collected from 7 and 9 day old seedlings, co-cultivated with an Agrobacterium suspension of O.D600 of 0.4 and 0.2 for 30 and 10 minutes infection time respectively. Better shoot development was observed when shoots induced on cotyledonary and hypocotyl explants were cultured on modified MS medium containing 0.5 and 1 mg/L zeatin respectively. Supplementation of medium with 200 mg/L timentin effectively suppressed Agrobacterium overgrowth. Furthermore, the time required from inoculation of explants with Agrobacterium to transfer of transgenic tomato plants to soil was only 70 days as compared to 3 to 4 months in standard tomato transformation protocols.

https://www.chemijournal.com/archives/2017/vol5issue6/PartO/5-6-33-245.pdf

A rapid Agrobacterium-mediated transformation protocol for tomato (Solanum lycopersicum L.) cv. PKM-1

Genetic Manipulation of Tomato (Solanum lycopersicum) cv. PKM-1 using cry2AX1 Gene for Insect Resistance

https://doi.org/10.1016/j.sjbs.2022.103292

Highly efficient Agrobacterium-mediated transformation and plant regeneration system for genome engineering in tomato

Tomato crop is infested by large number of insect pests, causing significant yield loss. Fruit borer (Helicoverpa armigera) feeds on fruits which leads to quality detoriation and economic loss. Genetic engineering is recognised as a viable tool to engineer resistance in crops against insect pests. Transgenic tomato plants over-expressing cry2AX1 gene under the control of CaMV35S promoter were developed. The T0 and T1 plants were characterized by PCR for the presence and segregation of transgenes. Segregation of cry2AX1 gene in T1 progeny plants followed the Mendelian pattern of monogenic inheritance of 3:1 ratio. The expression of Cry2AX1 at 55 days after sowing ranged from 0.034 to 0.219 μg/g of fresh leaf tissue and 0.030 to 0.249 μg/g of fresh leaf tissue at 80 days after sowing. Insect bioassay of detached leaf bits performed on the ELISA positive transgenic tomato plants against Helicoverpa armigera and Spodoptera litura resulted in mortality of neonates upto 100 per cent in H. armigera and 53 per cent in S. litura.

E. I. Bamishaiye

Papers

A rapid Agrobacterium-mediated transformation protocol for tomato (Solanum lycopersicum L.) cv. PKM-1

Genetic Manipulation of Tomato (Solanum lycopersicum) cv. PKM-1 using cry2AX1 Gene for Insect Resistance

Efficacy of cry2AX1 Gene in Transgenic Tomato Plants against Helicoverpa armigera and Spodoptera litura