Genetic Transformation of Tomato Using Bt Cry2A Gene and Characterization in Indian Cultivar Arka Vikas

Authors
V. S. Hanur 1
B. Reddy 1
V. V. Arya 1
P. V. Rami Reddy 2
1 Division of Biotechnology, Indian Institute of Horticultural Research, Hessaraghatta Lake Post, Bangalore, 560089, India.
2 Division of Entomology, Indian Institute of Horticultural Research, Hessaraghatta Lake Post, Bangalore, 560089, India.
Abstract
Transgenic tomato plants of south Indian cultivar Arka Vikas were developed using Agrobacterium strain EHA 105, harbouring Bt Cry2A gene with a construct containing 35S CaMV promoter, OCS terminator and nptII selectable marker, through Agrobacterium-mediated transformation. This study was conducted to improve the regeneration and transformation protocol for south Indian cultivar Arka Vikas. Hypocotyl was used as explant source for transformation due to high regeneration efficiency, molecular analysis through PCR for putative transformants in T0 generation and qualitative ELISA method was performed for Bt protein expression followed by insect bioassays. Insect bioassay studies was conducted using neonate larva of helicoverpa armigera to screen the plants and the plants expressing good resistance with molecular and phenotypic characters were carried further for successive generations. The experimental results concluded that Bt gene was deployed in tomato cultivar successfully and had developed resistance to neonate larva of Helicoverpa armigera at laboratory conditions. These results signified that transgenic lines expressed substantial quantity of Bt Cry2A protein efficient in management of Helicoverpa armigera. Precise screening of transgenic T1 lines is highly important to obtain single copy number plants since the expression of Bt protein in successive generations promotes effective management of this pest in the future.

Keywords

1. Ahmed, M. and McCaffery, A. R. 1991. Elucidation of Detoxification Mechanisms Involved in Resistance to Insecticides against Third Instar Lrvae of a Field Strain of Helicoverpa armigera with Use of Synergists. Pestic. Biochem. Phys., 41: 41-52.
2. Atkinson, P. W. 2002. Genetic Engineering in Insects of Agricultural Importance. Insect Biochem. Molec., 32: 1237–1242.
3. Atwal, A. S. 1986. Agricultural Pests of India and South East Asia. Kalyani Publishers, New Delhi.
4. Carmen, S., Hernandez-Rodriguez, Adri Van Vliet, Bautsoens, N., Van Rie, J. and Ferre, J. 2008. Specific Binding of Bacillus thuringiensis Cry2A Insecticidal Proteins to a Common Site in the Midgut of Helicoverpa Species. Appl. Environ. Microb., 74(24): 7654- 7659.
5. Chandrashekara, K. N., Chandrashekara, C. and Boopal, K. 2012. Transgenics: A Genetic Engineering Approach to Develop Disease Resistant Plants. 18. In: “Eco-friendly Innovative Approaches in Plant Disease Management”. ISBN 817089375-5 International Book Distributors Publisher, PP. 303-324.
6. Saleem, F. and Shakoori, A. R. 2010. Characterization of Cry2A-type Gene(s) from Pakistani Isolates of Bacillus thuringiensis Toxic to Lepidopteran and Dipteran Insects. Pak. J. Zool., 42(3): 181-193.
7. Fraser, P. D., Roemer, S., Shipton, C. A., Mills, P. B., Kiano, J. W., Misawa, N., Drake, R. G., Schuch, W. and Bramley, P. M. 2002. Evaluation of Transgenic Tomato Plants Expressing an Additional Phytoene Synthase in a Fruit-specific Manner. P. Natl. Acad. Sci., USA 99: 1092–1097.
8. Ghosh, P. K. 2001. Genetically Engineered Crops in India with Special Reference to Bt Cotton. IPM Mitr., II: 8-21.
9. Giovannucci, E. 1999. Tomatoes, Tomato-based Products, Lycopene, and Cancer: Review of the Epidemiologic Literature. J. Natl. Cancer Inst., 91(4): 317-31.
10. Gurinder, J. R. and Chhabra, R. 2013. Transgenic Cotton: Methods and Protocols. Method Mol. Cell. Biol., 958. DOI: 10.1007/978-1-62703-212-4_2.
11. Kranthi, K. R. 2006. Bt-cotton: High Toxin Level in Fruiting Parts Is Most Critical for Bollworm Control. Curr. Sci. India, 90(3): 10.
12. Madhulatha, P., Pandey, R., Hazarika, P. and Rajam, M. V. 2007. High Transformation Frequency in Agrobacterium-mediated Genetic Transformation of Tomato by Using Polyamines and Maltose in Shoot Regeneration Medium. Physiol. Mol. Biol. Plant., 13: 191–198.
13. Mandaokar, A. D., R. K. Goyal, A., Shukla, S., Bisaria, R., Bhalla, V. S., Reddy, A., Chaurasia, R. P., Sharma, I., Alomar, P. A. Kumar, 2000. Transgenic Tomato Plants Resistant to Fruit Borer (Helicoverpa armigera Hubner). Crop Prot., 19: 307–312.
14. Manjunath. T. M. 2006. Bt-cotton: Protein Expression in Leaves is Most Critical. Curr. Sci. India, 90(3): 10.
15. McCormick, S. 1991. Tomato Transformation with Agrobacterium tumefactions. In: “Plant Tissue Culture Manual: Fundamentals and Its Applications”, (Ed.): Lindsey, K. Kluwer Academic Publishers, Dordrecht, B-6: 1-9.
16. Mueller, L. A., Tanskley, S. D., Giovannoni, J. J., van Eck, J., Stack, S., Choi, D., Kim, D. and Chen, M. 2005. The Tomato Sequencing Project: the First Corner Stone of the International Solanaceae Project (SOL). Comp. Function. Genomic., 6: 153–158.
17. Paramesh, H., Fakrudin, B. and Kuruvinashetti, M. S. 2010. Genetic Transformation of a Local Variety of Tomato Using Gus Gene: An Efficient Genetic Transformation Protocol for Tomato. J. Agri. Technol., 6(1): 87–97.
18. Raheja, A. K. 1996. IPM Research and Development in India: Progress and Priorities. In: “Recent Advances in Indian Entomology”, (Ed.): Lal, O. P. APC Publications Pvt. Ltd, New Delhi, PP. 115-126.
19. Rashid, H., Yokoi, S., Toriyama, K. and Hinata, K. 1996. Transgenic Plant Production Mediated by Agrobacterium in Indica Rice. Plant Cell Rep., 15: 727–730.
20. Romer, S., Fraser, P., Kiano, J., Shipton, C., Misawa, N. and Schuch, B. P. 2000. Elevation of the Provitamin a Content of Transgenic Tomato Plants. Nature Biotechnol., 18: 666–669.
21. Sambrook, J., Fritsch, E. F. and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 3.
22. Sesso, H. D., Buring, J. E., Norkus, E. P. and Gaziano, J. M. 2004. Plasma Lycopene, Other Carotenoids, Retinol and the Risk of Cardiovascular Disease in Women. Am. J. Clin. Nutr., 79(1): 47-53.
23. Shadang R., Dwivedi P., Hegde P., Ahmed N, 2007. Effects of Different Culture Media on Seed Germination and Subsequent in Vitro Development of Protocorms of Hygrochilus parishii (Veith & Rchb. f) Pfitz (Orchidaceae). Indian J. Biotechnol. 6:256–261.
24. Sidhu, J. S. and Chellan, S. 2010. Genetic Engineering of Vegetable Crops. In: “Handbook of Vegetables and Vegetable Processing”, (Eds.): Sinha, N. K., Hui, Y. H., Evranuz, E. O., Siddiq, M. and Ahmed, J. Wiley-Blackwell, Oxford, PP. 83–105.
25. Sneh, B., Schuster, S. and Broza, M. 1981. Insecticidal Activity of Bacillus thuringiensis Strains against the Egyptian Cotton Leafworm, Spodoptera littoralis Boisd. (Lepidoptera: Noctuidae), Entomophaga, 26: 179–190.
26. Sun, H. J., Uchii, S., Watanabe, S. and Ezura, H. 2006. A Highly Efficient Transformation Protocol for Micro-Tom: A Model Cultivar for Tomato Functional Genomics. Plant Cell Physiol., 47: 426-431.
27. Vageeshbabu, S., Hanur, K., Boopal, K. N., Srividya, S., Poovarasan, S. B., Saraswathi, M. S., Vijeth, V. A. and Rami Reddy, P. V. 2011. Development of Cry2A Bt Transgenic Tomato for Resistance to the Tomato Fruit Borer, Helicoverpa armigera. In Tumkur University and CSIR sponsored National Level Conference on “Dissecting the Complexities of Plant Biotechnology in the Post-Genomic Era”, 21st September, PP. 16-32.
28. Vageeshbabu, S., Hanur, H., Modak, P. M., Shamseer, K. B. and Srividya, K. N. 2009. Development of Bt Transgenic Eggplant and Tomato Resistant to Fruit Borers. In 7th Pacific Rim Conference on the “Biotechnology of Bacillus thuringiensis and Its Environmental Impact”, Held at the National Academy of Agricultural Sciences, 25-28 November, 2009, New Delhi, P2806, 65.
29. Vageeshbabu, S., Hanur, Srividya, K. N. K. Boopal, S. Poovarasan, M. S. Saraswathi, Vijeth V. Arya, P. V. Rami Reddy and V. Krishna 2012. Comparative Phenotyping Analysis of Cry1Aa3- and Cry2A-Bt Transgenic Tomato for Resistance Against Tomato Fruit Borer, Helicoverpa armigera. National Level Conference on “Biotechnology and Society–NCBS 2012, April 19-20, Kuvempu University.
30. Vageeshbabu, S., Hanur, K., Boopal, K. N., Srividya, S. Poovarasan, Shraddha Bhalewar, Saraswathi, M. S, Vijeth. V. Arya and P. V. Rami Reddy 2012. Resistance Phenotyping of Bt Transgenic Eggplant and Tomato. at International Conference on “Plant Biotechnology for Food Security”, February 21-24,
31. Vidya, C. S. S., Manoharan, M., Kumar, C. T. R., Savathri, H. S. and Sita, G. L. 2000. Agrobacterium Mediated Transformation of Tomato (Lycopersicum esculentum var. Pusa Ruby) with Coat Protein Gene of Physalis mottle Tymovirus. J. Plant Physiol, 156: 106-110.
32. Wing, R. A., Zhang, H. B. and Tanksley S. D. 1994. Map Based Cloning in Crop Plants: Tomato as a Model System. I. Genetic and Physical Mapping of Jointless. Mol. Genet Genomic., 242: 681-688.
33. Zalcucki, M. P., Clarke, A. R. and Malcolm, S. B. 2002. Ecology Behavior of First Instar Larval Lepidoptera. Annu. Rev. Entomol., 47: 361-393.
Journal of Agricultural Science and Technology
Volume 17, Issue 7
November and December 2015
Pages 1805-1814