Optimization of factors for efficient recovery of transgenic peanut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.) |
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Authors: | Siddharth Tiwari Rakesh Tuli |
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Institution: | (1) Plant Molecular Biology and Genetic Engineering Division, National Botanical Research Institute, Rana Pratap Marg, Lucknow, 226001, India;(2) Present address: National Agri-Food Biotechnology Institute (NABI), Department of Biotechnology, Govt. of India, C-127, Industrial Area, Phase VIII, SAS Nagar, Mohali, 160071, Punjab, India;(3) National Agri-Food Biotechnology Institute (NABI), Department of Biotechnology Govt. of India, C-127, Industrial Area, Phase VIII, SAS Nagar, Mohali, 160071, Punjab, India |
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Abstract: | De-embryonated cotyledon explants of peanut were co-cultivated under different conditions with Agrobacterium tumefaciens harbouring pIG121hm plasmid carrying intron-containing β-glucuronidase as a reporter while hygromycin phosphotransferase and neomycin phosphotransferase as selectable marker genes.
Co-cultivation duration and temperature, various antioxidants and their concentrations, bacterial strains and explant characteristics
(incised and non-incised) were examined either alone or in combinations for optimization of transient expression of the reporter
gene. Up to 81% transformation was recorded when non-incised explants were co-cultivated with strain EHA101 for 5 days at
21°C on shoot induction medium containing 100 mg/L l-cysteine. Addition of the optimized concentration of augmentin (200 mg/L) along with cefotaxime (200 mg/L) to the shoot induction
medium not only effectively eliminated bacterial growth, but also facilitated high frequency of shoot induction. The 40 mg/L
hygromycin concentration prevented complete shoot regeneration of non-transgenic explants thus considered for the regeneration
of transgenics. Resistant shoots were successfully transferred to soil either by grafting or in vitro rooting. Survival rate
of the grafted shoots was nearly 100% in glass-house conditions. The optimized protocol took around 3 months to generate healthy
plants. Polymerase chain reaction, Southern blot hybridization, histochemical tests, segregation and hygromycin-leaf assays
of selected transgenic plants showed integration of the transgene into peanut genome. No chimeras were noticed during the
study. |
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