Callus induction and RAPD analysis of Simarouba glauca DC

Callus induction for somatic embryogenesis from Simarouba glauca DC leaf explants of three genotypes (S. glauca 5, S. glauca 19 and S. glauca 21) was studied. Leaf explants (leaf segments from basal, middle and tip of the leaves) were cultured on two types of nutrient media; SGC1 and SGC2. Both media contained Murashige and Skoog (MS) medium with vitamins: 100 mg/L ascorbic acid, 0.5 mg/L 6-benzylaminopurine (BAP), 0.5 to 5.0 mg/L NAA (1-napthaleneacetic acid), and 3.0 g/L sucrose. The SGC2 media additionally contained 0.5 to 5.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D). SGC2 media generated better callusing response compared to SGC1, thus displaying the importance of using 2,4-D in combination with NAA for callus induction. MS medium containing 2.5 mg/L NAA (SGC1.5) was noted to be the most effective in the initiation of friable embryogenic callus. On the other hand, MS medium containing a combination of 2.0 mg/L NAA and 2.0 mg/L 2,4-D was effective in the early initiation of friable embryogenic callus. In addition, a higher frequency of callus formation was observed from basal leaf segment as compared to that from middle and apical leaf segments. A random amplified polymorphic DNA (RAPD) analysis was also performed to see the genetic differences between the three S. glauca genotypes used in this study. The performance of S. glauca 5 and S. glauca 19 for higher callus frequency over the S. glauca 21 could be attributed to the genotypic differences between these genotypes. Overall, our protocol using SGC 2.4 media yielded optimal results and is suitable for large scale micropropagation of S. glauca. Keywords: Simarouba glauca, somatic embryogenesis, callus, plant growth regulators, RAPD and biofuel.African Journal of Biotechnology, Vol 13(53) 4766-477

Determination of 3-Hydroxy-3-methylglutaryl CoA Reductase Activity in Plants

The enzyme 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase catalyzes the NADPH-mediated reductive deacylation of HMG-CoA to mevalonic acid, which is the first committed step of the mevalonate pathway for isoprenoid biosynthesis. In agreement with its key regulatory role in the pathway, plant HMG-CoA reductase is modulated by many diverse external stimuli and endogenous factors and can be detected to variable levels in every plant tissue. A fine determination of HMG-CoA reductase activity levels is required to understand its contribution to plant development and adaptation to changing environmental conditions. Here, we report a procedure to reliably determine HMG-CoA reductase activity in plants. The method includes the sample collection and homogenization strategies as well as the specific activity determination based on a classical radiochemical assay.This work was supported by grants of the Spanish Ministerio de Economía y Competitividad and the Spanish Ministerio de Ciencia e Innovación (BFU2011-24208 to N.C., BIO2009-06984 to A.F. and M.A., and BIO2009-09523 to A.B., including FEDER funds), the Spanish Consolider-Ingenio Program (CSD2007-00036 Centre for Research in Agrigenomics), and the Generalitat de Catalunya (2009SGR0026).Peer reviewe