An Efficient Agrobacterium-mediated Genetic Transformation Using cry1F gene in Castor (Ricinus communis L.) for protection Against lepidopteran Insects

Authors

  • ROHAN V. KANSARA Department of Plant Molecular Biology and Biotechnology, Navsari Agricultural University, Navsari- 396450, Gujarat, India Author
  • SANJAY JHA Department of Plant Molecular Biology and Biotechnology, ASPEE Shakilam Agricultural Biotech Institute, Navsari Agricultural University, Athwa Farm, Surat-395007, Gujarat, India Author
  • VANRAJSINH H. SOLANKI Department of Plant Molecular Biology and Biotechnology, Navsari Agricultural University, Navsari- 396450, Gujarat, India Author
  • SUMANKUMAR JHA Department of Forest Biology and Tree Improvement, College of Forestry, Navsari Agricultural University, Navsari-396450, Gujarat, India Author
  • VISHAL S. SRIVASHTAV Department of Plant Molecular Biology and Biotechnology, Navsari Agricultural University, Navsari- 396450, Gujarat, India Author
  • VIVEK V. MEHTA Regional Horticulture Research Station, ASPEE college of Horticulture and Forestry, Navsari Agricultural University, Navsari-396450, Gujarat, India Author
  • HIREN K. PATEL School of Sciences, P.P. Savani University, Surat-394135, Gujarat, India Author
  • CHAITANYA S. MOGAL Department of Plant Molecular Biology and Biotechnology, Navsari Agricultural University, Navsari- 396450, Gujarat, India Author

DOI:

https://doi.org/10.25083/rbl/27.1/3282-3291

Keywords:

Ricinus communis, Agrobacterium-mediated transformation, Cry1F gene, Shoot apex, Spodoptera litura, qRT-PCR

Abstract

Castor (Ricinus communis L.) is an essential non-edible, pharmaceutical, and industrial oilseed as well as vulnerable to foliage feeders like Spodoptera litura  which resulted in a loss of production. This report focuses on the development of  an optimized protocol for the transformation of castor shoot apices by  Agrobacterium tumefaciens strain LBA4404 containing plasmid construct pBIN1F  harboring neomycin phosphotransferase (nptII), as selectable marker gene and  Bacillus thuringiensis var. aizawai (Bt) cry1F gene controlled by cauliflower mosaic  virus 35S promoter. Several parameters like O.D., concentration of kanamycin and  acetosyringone were optimized and produced a significant difference in the  transformation efficiency. Co-cultivation time and seedling age were factors, with overall transformation efficiency of 2.0% in 15 days-old seedlings and co-cultivated  for 3 days. The surviving and actively developing shoot apices were validated for  gene integration by molecular analysis after being preliminarily screened on  kanamycin. Furthermore, PCR, qRT-PCR and insect bioassay were used to confirm  the putative primary transformants. When bioassayed against newly hatched  Spodoptera litura hatchlings, these putative transgenics with cry1F gene caused  significant (≤93%) insect mortality. Cry1F gene expressing transgenic plants had  adequate defence against Spodoptera litura when exposed to castor. 

RBL271-13

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Published

2024-05-23