3.2. Effect of cytokinins (TDZ &BAP) and Auxin (IBA) on Adventitious shoot proliferation from Callus
Analysis of variance showed that both of percentage of shoot regeneration and mean number of adventitious shoots per callus were significantly (P<0.01) affected by the type and concentration of PGRs, accessions and interaction effect of the two factors.
The highest percentage of shoot regeneration from callus (66.67%) was observed for Shewa Robit accession in MS medium supplemented with combination of 0.5mg/L TDZ and 0.1 mg/L IBA followed by 64% and 61.33% for Metema and Adami Tulu accessions, respectively, on the same PGRs concentrations as for Shewa Robit. Meanwhile, the lowest percentage (39.2-40.8%) of shoot regenerations were recorded for all accession when the callus was cultured on media supplemented with 0.25mg/L TDZ (Table 4a). However, MS supplemented with BAP alone and combination with IBA did not induce shoot regeneration from callus culture. Calli that were cultured on this medium (BAP supplemented) was proliferated large quantities of healthy, green callus, but failed to differentiate shoots on all accessions type (Fig. 3b). Even on subculturing to respective media it continued to form massive callus rather than formation of organogenesis. And also, on the control medium the shoot regeneration was not noticed. However, the addition of TDZ to the medium improved the shoot induction potential of the calli as many shoot primordia were observed after 45 days of culture (Table 4a; Fig. 3c). Similar results were verified by Khurana-Kaul et al. (2010) who also showed that the combination of TDZ and IBA was more effective than the combination of BAP and IBA in Jatropha shoot regeneration using leaf segments as explants. Aishwariya et al . (2015) also reported that Thidiazuron (TDZ) is among the most active cytokinin like substances and it induces greater in vitro shoot proliferation than many other cytokinins in many plant species. Variation in the activity of different cytokinins can be explained by their differential uptake rate in different genomes, translocation rates to meristematic regions and metabolic processes in which the cytokinin may be degraded or conjugated with sugars or amino acids to form biologically inert compounds also reported (Kaminek, 1992; Kumar et al ., 2011).
The highest adventitious shoot number (11shoot) was recorded for Shewa Robit callus cultured on MS media supplemented with 0.5mg/L TDZ and 0.1mg/L IBA followed by (10.4) and (9 shoot) shootlet per callus for Adami Tulu and Metema accessions, respectively. Whereas, the lowest shoot number (3.8-4) was recorded for all accessions, when the callus subcultured on MS media supplemented with 0.25mg/L TDZ (Table 4b). In this study, the number of shoots per callus was increased as the concentration of TDZ was increased from 0.25 to 1mg/L. These results suggest that TDZ plays a very important role in the formation of adventitious shoot buds of Jatropha, and these effects may be involved in stimulating de novo synthesis of auxins by increasing the levels of IAA and its precursor, tryptophan, as well as increase in contents of endogenous cytokinin (Murthy et al ., 1995; Murthy and Saxena, 1998). Besides, the ability of TDZ to induce high shoot regeneration efficiency in plant tissue has been reported for a number of species (Feyissa et al ., 2005; Landi and Mezzetti, 2006).