Optimization of Regeneration and Transformation of Fagonia indica

Abstract

Fagonia indica (syn. F. cretica), commonly known as Dhamasa, is an important medicinal plant. It contains many important compounds especially active against breast and liver cancer. In vitro propagation of this plant to increase the mass is an important step to obtain large amount of these compounds. Furthermore, genetic engineering of this plant is very valuable in improving the genetic diversity as well as to increase secondary metabolites. The aim of this study is to optimize different tissue culture conditions as well as to establish a significant protocol for Agrobacterium tumifaciens mediated transformation of F. indica. Treatment of seeds and explants with 70% ethanol, 0.1% mercuric chloride, 5% sodium hypochlorite and terbinafine (1mg/ml) resulted in significant control of bacterial and fungal contamination. The seed germination efficiency is maximum on MS medium i.e. 45% as compared to ½ MS (30%), RMOP (10%) and B5 (6.11%). Out of three explants (shoot apical meristems, nodes and cotyledons) chose for in vitro propagation, shoot apical meristems has maximum regenerative potential. The optimized callus induction medium which gave 100% efficiency is MS medium supplemented with 1:1 of NAA:BAP (0.5 mg/l). The best results for shoot induction medium include MS supplemented with BAP (0.1mg/l) only. During optimization experiments, somatic embryos and protocorm like bodies are formed in number of combinations of calli and shoot induction media. The roots were induced on media containing BAP, NAA and IAA in different combinations. A. tumifaciens strain C58C1 harbouring a binary vector p35GUSint containing neomycin phosphotransferase (nptII) as selectable marker and β-glucuronidase (GUS) as a reporter gene, was used for plant transformation. The optimal concentration of Cefotaxime and Kanamycin for elimination of A. tumifaciens and transgenic explants was found to be 300 mg/l and 50mg/l respectively. Transient GUS expression of A. tumifaciens-mediated transformation showed that bacterial density of OD600 value of 0.5, co-infection time of 7 minutes, cocultivation time of 1 day and acetosyringone concentration of 200 µM for explants proved to be best variables for successful transformation, as maximum transformation efficiency was obtained with these optimized conditions. Out of five explants i.e. stipules, leaves, shoot apical meristems, nodes and callus, the maximum transformation efficiency was observed in callus. The tissue culture and transformation conditions optimized in this study can be helpful for future research of F. indica genetic engineering.