3.1. Effects of BAP and 2,4-D on Callus Induction
The analysis of variance (ANOVA) showed that there is highly significant (P<0.01) difference due to the main effect of PGRs combinations and concentrations on mean number of days taken to form calli formation and callus fresh weight. However, there were no significant (P>0.05) effects recorded due to difference in accession and the interaction effect of PGRs and accessions on days to callus formation and callus fresh weight. Besides to this, significant differences for percentage of callus induction were observed among the PGRs combinations and concentrations as well as interaction of PGRs with accessions. However, the percentage of callus induction was not significantly (P>0.05) different among the accessions.
The minimum number of days (13.6 days) to callus induction was recorded for explants cultured on MS media supplemented with combination of 1mg/L BAP and 1mg/L 2,4-D followed by (14 days) on MS media supplemented with 1.5mg/L BAP and 1mg/L 2,4-D. Whereas, the longest time (18.87 days) taken to form callus was observed when callus induction medium was supplemented with combination of 2mg/L BAP and 0.5mg/L of 2,4-D (Table 2). From these results, increasing BAP levels in media within constant rate of 2,4-D led to an increase in the mean days taken to form calli. The media which supplemented with BAP alone did not induce a callus. This indicated that presence of auxins in the media play a crucial role in the callus formation of Jatropha explants in vitro (Rajore and Batra, 2007; Kumar et al ., 2008; Kumar et al. , 2015). The result of this study also revealed that the number of days to callus emergence decreased with increased in concentration of 2,4-D (0.5-1mg/L) in the treatments. Kumar et al. (2015) also reported that leaf explants Jatropha started callusing within two weeks on the media containing 2, 4-D (10μM). However, further increasing 2,4-D levels in media led to an increase in the mean days taken to form calli. Radhakrishnan et al . (2001) reported that the cells used up 2,4-D as required and any excess began to actively show the herbicidal effects that therefore slowed down the callus induction process.
The best callus formation (100%) was observed when MS medium was supplemented with combination of BAP (1.0 mg/L) and 2, 4-D (1.0 mg/L) for all accessions. Whereas, the lowest percentage (47.8-48.6%) of callus formation was recorded for all the three accessions explants cultured on media supplemented with 2.0mg/L BAP and 1.5mg/L 2,4-D (Table 3). This indicated that callus induction frequency decreases with further increasing the concentration of both 2,4-D and BAP. Several authors reported that appropriate concentrations and combinations of cytokinins and auxins are important to produce Jatropha callus (Costaet al., 2015; Kumar et al ., 2015). On the other hand, the MS media containing only 2,4-D, even at a low concentration resulted in a better callus formation (Fig. 2c). Similar observation was also made by Thao et al . (2003) and Soomro and Memon (2007) concluding that 2,4-D was pre-requisite for callus formation in many of plant species. Meanwhile, no callus formation was observed and the explants only showed leaf expansion on medium containing BAP alone (Fig.2b). These results are supported by Kaewpoo and Techato, (2009) who used different concentration of BAP (1mg/L, 2mg/L and 3mg/L) to induce callus from embryo cultures of Jatropha. The authors finding report showed that BAP alone has induced shoot rather than callus. It has long been suggested that strong auxins such as 2,4-D are mainly efficient in promoting cell clumping and further developing of the callus.
In case of Callus fresh weight the highest calli weight (2.23g) was recorded on MS media supplemented with combination of 1.0mg/L of BAP and 1.0mg/L 2,4-D followed by (2.06g) on media with 1.5mg/L of BAP and 1.0mg/L 2,4-D (Table 2). Whereas, the lowest overall mean calli weight (0.98g) were observed for explant induced on MS media containing high levels of BAP (2.0mg/L) and 2,4-D (1.5mg/L) were used. This results confirmed that despite 2,4-D being an effective auxin in producing callus in Jatropha, it was active for callus induction when used in small amounts (Soomro and Memon , 2007). Radhakrishnan et al . (2001) also reported that high 2,4-D concentration has been shown to have herbicidal effects on plants causing cell growth inhibition and at highest cell death.