Submit Manuscript | http://medcraveonline.com Abbreviations: KAN, Kanamycin; AS, Acetosyringone; CN, Cotyledonary node; LL, Leafet; DC, De emryonated cotyledon; nptII, Neomycin phosphotransferase; NAA, α-Naphthalene acetic acid; BAP, 6-Benzylaminopurin Introduction Groundnut/Peanut (Arachis hypogaea L.) has great potential in third world countries to reduce hunger and malnutrition. 1 It is widely grown in the semi-arid tropics and India ranks second by 12.5% in world production. 2 The production of groundnut is constrained by different biotic and abiotic factors by causing decrease in yield. 3 To develop stress tolerance through conventional breeding takes longer time and passes through various incompatibility barriers. To overcome these, the biotechnological tools play a great role for developing stress tolerance through introduction of alien genes into elite germplasm of crop plants through genetic transformation technology. 4-6 The various factors that affect signifcant differences in T-DNA delivery and transformation included explant type, duration of pre-culture of ex plant, incubation period in bacterial suspension, the concentration of A. tumefaciens during infection, the use and concentration of inducers such as Acetosyringone during infection, antibiotics used for selection and the extent of the co-culture period were optimized to establish a transformation protocol of common bean (Phaseolus vulgaris). 7 and in chickpea. 8 Legumes in general are recalcitrant to tissue culture and Agrobacterium mediated transformation is also highly genotype specifc. 9 Several reports have been published on effcient regeneration from diverse explants of peanut, 10,11 but less success has been recorded with genetic transformation of A.hypogaea. This is due to lack of effcient protocols to induce adventitious shoot buds after the transformation. 12-14 Recently we have developed the reproducible regeneration protocol in groundnut cv ICG 13942. 15- 17 The aim of this work was to develop a reproducible and effcient method of A. tumefaciens mediated genetic transformation and to evaluate the factors that affect the genetic transformation effciency by using marker genes (nptII; uidA) and later the same was used for Agrobacterium mediated transformation with the Tc Chitinase-I gene to develop the resistance to Tikka disease in cv ICG 13942 for the frst time. Therefore, in the present report we describe the successful transformation and regeneration of putatively transformed plants in groundnut cv ICG 13942. Material and methods Plant material, culture initiation and maintenance The mature seeds of groundnut cv ICG 13942, obtained from the germplasm bank of ICRISAT, Patancheru, Hyderabad, Telangana, India were used. Dried seeds were washed under running tap water for 10-15 min followed by treating with Tween-20 (5%-v/v) for 5 min. Later these were rinsed twice in sterile distilled water. Afterwards the seeds were surface sterilized with 0.1% (w/v) HgCl 2 for 8 min followed by rinsing in sterilized distilled water for 3-4 times under aseptic conditions and soaked for 24 h in sterile distilled water. The experiments were conducted on modifed MS 18 medium (MMS medium) containing MS basal salts and B 5 vitamins, 19 100 mg/L myo- inositol and 30 g/L sucrose. The pH of the medium was adjusted to Adv Plants Agric Res. 2018;8(3):275‒282. 275 © 2018 Marka et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and build upon your work non-commercially. Optimization of factors affecting Agrobacterium- mediated genetic transformation in groundnut (Arachis hypogaea L.) Volume 8 Issue 3 - 2018 Rajinikanth Marka, Rama Swamy Nanna Department of Biotechnology, Kakatiya University, Plant Biotechnology Research Laboratory, India Correspondence: Rama Swamy Nanna, Plant Biotechnology Research Laboratory, Department of Biotechnology, Kakatiya University, Warangal-506009, Telangana, India, Email swamynr.dr@gmail.com Received: October 24, 2017 | Published: June 04 2018 Abstract The effciency of Agrobacterium-mediated genetic transformation of groundnut is dependent on various factors such as type and age of explants, antibiotics used for selection, Acetosyringone (AS), duration of co cultivation and infection period. Hence, we have attempted to standardize these parameters to improve the Agrobacterium mediated genetic transformation effciency in groundnut cv ICG 13942. To evaluate the optimization of factors affecting Agrobacterium mediated genetic transformation in which we used three different explants (Deembryonated cotyledon-DC, Leafet-LL, Cotyledonary node-CN) and studied, the effect of inoculum concentration (0.5, 1.0 and 1.5 OD 600 of A. tumefaciens), pre culture period (1-11 days), co cultivation period (1-7 days), Acetosyringone concentration (25-200mg/L) and antibiotic (Kan) concentration (25-150mg/L) on the effciency of genetic transformation. The transformation was verifed by GUS staining and by means of PCR amplifcation of the uidA and nptII genes. The optimized factors for A. tumefaciens LBA 4404 harboring pBAL2 binary vector mediated transformation in groundnut cv ICG 13942 are: Pre culture period-3 days, Concentration of A. tumefaciens-1.0 at OD 600 , Co-cultivation period-4 days, Kan concentration-100mg/L for DC &CN, 75mg/L for LL explants, Concentration of AS-100mg/L during infection and co cultivation and Cefotaxime concentration-250mg/L. Thus, the optimized protocol showed the enhanced transient transformation frequencies such as 18.13% for DC, 15.33% for LL and 12.32% for CN explants. Keywords: agrobacterium tumefaciens, arachis hypogaea, GUS expression, npt-II, polymerase chain reaction Advances in Plants & Agriculture Research Research Article Open Access