PHD-Department of Biochemistry and Biotechnology

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Browsing PHD-Department of Biochemistry and Biotechnology by Author "Gacheri, Limbua Purity"

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    Regeneration and Transformation of Selected Kenyan Groundnut (Arachis Hypogaea L.) Genotypes with Isopentenyl Transferase (IPT) Gene Towards Drought Tolerance
    (Kenyatta University, 2018) Gacheri, Limbua Purity
    Groundnut (Arachis hypogaea L.) is an important crop in terms of income and nutrition. The seeds are nutritional source of vitamin E, niacin, falacin, calcium, phosphorus, magnesium, zinc, iron, ribloflavin, thiamine and potassium. It helps in reducing the risk in developing type II diabetes and cardiovascular disease. Despite its importance, groundnut yield is limited by environmental factors such as drought. The prediction of climate change indicate that drought will occur more often due to factors like global warming and this will reduce groundnut production thereby affecting its prices and the prices of its derived products such as groundnut butter. Strategies towards improvement of groundnuts towards drought tolerance have involved both genetic engineering (GE) and non-genetic engineering approaches such as marker assisted selection. Breeding is however laborious, lengthy and carries along undesired alleles. The objective of this work was to regenerate and transform selected Kenyan groundnut (Arachis hypogaea L.) genotypes with isopentenyl transferase (IPT) gene towards drought tolerance.The cotyledonary nodes of six Kenyan adapted Groundnuts genotypes (ICGV 12991, CG 7, Red Valencia, ICGV 90704, Chalimbana and JL 24) were transformed using Agrobacterium tumefaciens strain EHA 101 carrying PNOV-IPT binary vector containing an IPT gene which was driven by SARK promoter and terminated by TNOS terminator. The vector also contained phospomannose Isomerase enzyme (PMI) gene for selection of transformed tissues. Putative transformants were tested for presence of the transgene by PCR designed to amplify IPT gene sequence. Gene expression was confirmed by RT–PCR. Transformation frequency was calculated as a percentage of the number of putative transformants divided by the total number of infected cotyledonary nodes. This ranged from 9.87% for ICGV 90704 to 19.77% for JL 24. Transformation efficiency was calculated as a percentage of the number of PCR positive plants divided by the total number of cotyledonary nodes infected. This ranged from 0% for ICGV 12991 and Chalimbana to 1.74% for JL 24. As a prerequisite for transformation, in vitro regeration protocol for ICGV 12991, CG 7, Red Valencia, ICGV 90704, Chalimbana and JL 24 groundnut genotypes was optimized using cotyledonary nodes explants. Vapor sterilization showed four hours as the optimal for obtaining clean viable explants. BAP 5 Mg/L combined with 1 Mg/L TDZ was the best concentration for shoot induction and BAP at 5 Mg/L was the best for elongation of shoots.The data suggest the possibility of transforming groundnuts with ipt gene and regenerating normal transgenic plants. The recommendations are that drought stress assays should be done for RT – PCR positive plants so as to determine the efficacy of IPT gene to confer drought stress tolerance under drought conditions. Other groundnut genotypes should also be transformed using ipt for drought tolerance.