Journal of Biotechnology & Biomaterials
Open Access

Abstract

Genetic transformation studies in apple rootstock MM111 were carried out to standardize a protocol for Agrobacterium mediated chitinase gene transfer in apple. Antibiotics sensitivity, shoot regeneration experiments and time for Agrobacterium infection were carried out to study their effect on relative growth of explants and to obtain high frequency of callus induction/shoot regeneration. Leaf explants resulted in high frequency regeneration on MS medium supplemented with 0.8 mg/l TDZ and 0.5 or 1.0 mg/l NAA while only callus formation was achieved with other combinations. It was observed that callus induction decreased on increasing the concentration of hygromycin from 1-5 mg/l whereas all the explants died at 6 mg/l. 5 mg/l hygromycin was considered for selection of putative shoots. Agrobacterium tumefaciens LBA4404 strain harboring a chitinase (chi-11), hygromycin phosphotransferase (hpt) and phosphinothricin acetyltransferase (bar) genes obtained from Dr S Muthukrishnan, KSU, USA was used for transformation experiments. Minimum callus induction (5.11%) and maximum shoot regeneration (2.40%) was achieved with 25 minutes of infection. 500 mg/l cefotaxime was found the best to control Agrobacterium growth after four blots and its concentration can be reduced to 200-300 mg/l for further callus/shoot regeneration. Two and three days preculturing combined with 96 hours co-cultivation proved effective for getting putative shoots on selective medium containing 5 mg/l hygromycin and 500 mg/l cefotaxime.

Biography

Arjun Chauhan has completed his Masters in Biotechnology and he is currently pursuing his PhD in Molecular Biology and Biotechnology from Dr. Yashwant Singh Parmar University of Horticulture and Forestry, Himachal Pradesh, India.

Email: arjunsrf@rediffmail.com