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).