What combination of plant growth regulators did you use for callus induction and which media and PGRs did you subculture the callus to? Also after what during in indcution medium was the callus subcultured? Knowledge of these factors may point out the problem
Dear Sir
1. For callus induction - 2,4-D (0.1mg/l) + kinetin (0.5mg/l) (friable callus) one subculture after two weeks on same medium. Then transfer to callus multiplication medium.
2. callus multiplication - 2,4-D (0.01mg/l)+2ip (0.5mg/l) two subculture after two weeks on same medium (friable callus)
3. callus multiplication - IBA(0.1mg/l) + TDZ(0.5mg/l) two subculture after two weeks on same medium (compact nodular)
I suggest you explore with a variety of PGR treatments for the regeneration, including the following:
Transfer from the TDZ + IBA medium to:
(a) a PGR-free medium, subculture twice on this medium, at two weeks interval. Idea is to allow endogenous levels of the PGRs to reduce to levels that could allow morphogenesis.
(b) a range of media with progressively higher cytokinin to auxin ratio to see if shoot organogenesis will occur from the nodular callus; You have used 5:1 ratio of TDZ to IBA. You could use media with the range from 6:1 to 10; i.e. from 0.6TDZ:0.1IBA, to 1mg/l TDZ :0.1mg/l) IBA.
(c) a medium with very low TDZ e.g. 0.005 or 0.05 mg/l but no IBA.
And probably it may be a good idea to have both types of callus, friable and compact, on these media. Some friable callus masses do regenerate efficiently.