I have transformed nucleus of Nicotiana benthamiana with LBA4404 Agrobacterium strain harboring gene of interest that was Chloroplast Transit Peptide + aadA gene for spectinomycin resistance. I infected leaf discs with LBA4404 and grown them on Spectinomycin containing MS medium, spectinomycin seems to be working properly as the color of leaf discs has been bleached out and the callus formed is also swollen, it is almost 3 months and my callus didn't started shooting and rooting yet. I have used the appropriate Hormones for that purpose which are: BAP 500 uL/500 ml, NAA 50 uL/500 ml, Spectinomycin 750 uL/500 ml, Cefotaxime 500 uL/500 ml. Why am I not getting shoots and roots in my calli, is there any problem with the Hormonal or Spectinomycin concentrations that I am using? Kindly suggest me the best way to counter this problem. Reference papers would also be best in this regard.
Try with high BAP concentration for shoot induction (2 mg/l). Also pls refer http://www.opensciencepublications.com/wp-content/uploads/JPSR-2349-2805-3-150.pdf. May b this can be useful.
Do you have any control of explants? what is the response of control explants in the medium for shoot induction from the callus? Shoot induction from callus may require different concentrations of BAP or combination of BAP+ an Auxin (NAA/2,4-D). First standardize the method with control and then transform the explants with Agrobacterium. Also in the regeneration medium you have to remove/adjust the concentration of Cefotaxime.
Also please refer:
Article Transgenic Nicotiana benthamiana and grapevine plant transfo...
Sir, I have used the control wild type N. benthamiana leaf discs and they are showing color bleaching activity, curling and without any callus or swelling, whereas the transformed leaf discs have abundant mass of callus and are swollen but no shoot regeneration yet. I think I must try to grow them on Ceftaxime free media and use 1 mg/ml BAP and 500 ug/ml of Spectinomycin, any suggestions regarding these concentrations, Sir?
BAP (1 mg/l), IAA (0.1 mg/l) was suitable for callus induction in 20 days. This result was in accordance with previous studies. BAP (2 mg/l), IAA (1 mg/l), Kinetin (0.5 mg/l) was suitable for shoot regeneration in 25 days. This result differed from that of previous studies where BAP (1 mg/l) and NAA (0.1 mg/l) was used for both callus induction and shoot regeneration. Thus, this study forms the basis for invitro rising of Nicotiana benthamiana for transformation and genetic engineering studies ( Geethalakshmi et al. 2016). As far as Ceftaxime is concerned you can use very reduced concentration if you think there is still chances of contamination but none would be better. So please try out a regime of concentrations between 1mg/l to 4 mg/l of BAP + combination of IAA and Kinetin. You should get positive result. Refer to the paper for regeneration:
http://www.opensciencepublications.com/wp-content/uploads/JPSR-2349-2805-3-150.pdf. May b this can be useful.
