Protocol optimization and evaluation of rice varieties response to <i>in vitro</i> regeneration

Abstract

Understanding the control of related molecular mechanisms is impossible without efficient and reproducible regeneration and stable genetic transformation. The key factors for enhancing successful regeneration are genotypes, tissue source of explants, combination and concentration of growth regulators, and culture conditions. In the present study different methods of regeneration were tested in a set of 12 rice accession representing indica, japonica, aromatic and wild groups. The highest frequency of shoot regeneration was achieved using mesocotyls derived from in vitro grown seeds cultured on Murashige and Skoogs, basal medium without growth regulators, when cultured on medium supplemented with Benzyl Adenine (BA) 0.5 mg/l under subdued light at 25°C ± 2°C. Under these conditions mesocotyls (Plurality of meristems) produced 3 to 4 tiller shoots in primary culture. One seed/One single mesocotyls segment produced over 5 to 9 shoots, arising primarily through direct organogenesis after 3 weeks of culture. Through callusing phase different rice cultivars produced different percentage of callusing however Basmati 370 gave high percentage in response to callussing, organogenesis and root formation. In between these two methods, the direct shoot regeneration gave 80% - 100% result besides the wild species in respect with their genotype whereas the indirect organogenesis gave only 10% - 30% of plantlets. So the direct multiplication from mesocotyls is an efficient method for plantlet regen eration of rice cultivars through in vitro culture. Tissue culture derived plants when evaluated with rice markers no variability is found. For regeneration genotypes and its trigger ing factors like medium composition, culture conditions and type of explants are playing a combined role.