1. KAUTIR (Kerala Agricultural University Theses Information and Retrieval)
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Item Particle bombardment mediated genetic transformation of Cassava (Manihot esculenta Crantz)(Department of Molecular Biology and Biotechnology, College of Agriculture, Vellayani, 2025-01-06) Akilan, L K; Anuradha, T.Cassava, a starchy root crop, serves multiple purposes including use as food, feed, and in various industrial applications. Enhancing its genetic traits is essential to improve both biotic and abiotic stress tolerance and boosting overall productivity. The genetic transformation of cassava is genotype-specific, necessitating cultivar specific protocols for effective transformation. The study entitled “Particle bombardment mediated genetic transformation of cassava (Manihot esculenta Crantz) was conducted in the Department of Molecular Biology and Biotechnology, College of Agriculture, Vellayani, Thiruvananthapuram from 2023-2024. The objective was optimisation of conditions for particle bombardment mediated genetic transformation of embryogenic callus of cassava (Manihot esculenta Crantz) variety H-226, which has not yet been developed for Indian cassava varieties. The embryogenic calli development potential of cultivar H-226 was assessed using an established protocol achieving an induction rate of 42% . These calli were used as target tissue for biolistic transformation experiments using the binary vector, pCAMBIA 1305.1 which contains the GUS reporter gene and Hygromycin selectable marker gene hptII. A sensitivity test on the embryogenic callus revealed that 50 mg L-1 of hygromycin was optimal for effective screening of both non transformed and transformed calli . The key physical parameters influencing transformation efficiency including target distance and the plasmid DNA-to-microcarrier ratio, were evaluated and optimized. The results demonstrated that a bombardment distance of 6 cm and a plasmid DNA-to-microcarrier ratio of 1:2, combined with a helium pressure of 650 psi, provided optimal conditions for biolistic transformation. To confirm genetic transformation, a GUS histochemical assay was conducted 48 hours post bombardment, identifying an average of 13 GUS-positive spots per callus, confirming successful gene transfer. Further verification through PCR using gene specific primers for the GUS and hptII genes confirmed the stable integration of the transgenes in the transformed calli. 51 This study partially optimized a protocol for stable transformation of cassava embryogenic calli using particle bombardment. Molecular analysis of the regenerated transgenic plants is required to fully validate the transformation efficiency and to evaluate its potential for genome editing applications in cassava