An efficient in vitro plant regeneration system was established for elite, recalcitrant Malaysian indica rice, Oryza sativa L. CV. MR 219 using mature seeds as explant on Murashige and Skoog and Chu N6 media containing 2,4-dichlorophenoxy acetic acid and kinetin either alone or in different combinations. L-proline, casein hydrolysate and L-glutamine were added to callus induction media for enhancement of embryogenic callus induction. The highest frequency of friable callus induction (84%) was observed in N6 medium containing 2.5 mg l(-1) 2,4-dichlorophenoxy acetic acid, 0.2 mg l(-1) kinetin, 2.5 mg l(-1) L-proline, 300 mg l(-1) casein hydrolysate, 20 mg l(-1) L-glutamine and 30 g l(-1) sucrose under culture in continuous lighting conditions. The maximum regeneration frequency (71%) was observed, when 30-day-old N6 friable calli were cultured on MS medium supplemented with 3 mg l(-1) 6-benzyl aminopurine, 1 mg l(-1) naphthalene acetic acid, 2.5 mg l(-1) L-proline, 300 mg l(-1) casein hydrolysate and 3% maltose. Developed shoots were rooted in half strength MS medium supplemented with 2% sucrose and were successfully transplanted to soil with 95% survival. This protocol may be used for other recalcitrant indica rice genotypes and to transfer desirable genes in to Malaysian indica rice cultivar MR219 for crop improvement.
Purification of lipase produced by L. mesenteroides subsp. mesenteroides ATCC 8293 was conducted for the first time using a novel aqueous two-phase system (ATPS) composed of Triton X-100 and maltitol. The partitioning of lipase was optimized according to several parameters including pH, temperature, and crude load. Results showed that lipase preferentially migrated to the Triton X-100 rich phase and optimum lipase partitioning was achieved in ATPS at TLL of 46.4% and crude load of 20% at 30 °C and pH 8, resulting in high lipase purification factor of 17.28 and yield of 94.7%. The purified lipase showed a prominent band on SDS-PAGE with an estimated molecular weight of 50 kDa. The lipase was stable at the temperature range of 30⁻60 °C and pH range of 6⁻11, however, it revealed its optimum activity at the temperature of 37 °C and pH 8. Moreover, lipase exhibited enhanced activity in the presence of non-ionic surfactants with increased activity up to 40%. Furthermore, results exhibited that metals ions such as Na⁺, Mg2+, K⁺ and Ca2+ stimulated lipase activity. This study demonstrated that this novel system could be potentially used as an alternative to traditional ATPS for the purification and recovery of enzymes since the purified lipase still possesses good process characteristics after undergoing the purification process.