you will use BAP and NAA combination its enough  for shoot formation and NAA only enough for root formation 

Thank you Subramanian for your reply . In this case also concentration of NAA will be lower than BAP ? 

yes

Hi Mohd. I used 2.5mg/l of BAP and 1mg/l of NAA and took good result. 

Thank you Alibek for your reply. I will try to use NAA 

2mg Bap with out NAA also i got good shoots

I have used 2-8 mg/l BAP (different conc. of BAP) but i did't fount shoot regeneration. 

then i used BAP and kinetin (2-8mg/l) both in M.S media in this case also result was same no regeneration 

then i use BAP and 2,4-D in M.S again my experiment was fail.

MS medium with 2 mg/l Kinetin and 0.5 or 1.0 mg/l IAA (Add filter sterilised IAA after autoclaving); 3% sucrose, pH 5.8.  Can use NAA but 0.25 or 0.5 mg/l NAA (before autoclaving is OK) in place of IAAIncubate in 16 light/8 dark photoperiod.  Culture  leaf explants (or seedlings), you will get shoot directly with little callus in 3-4 weeks.  Subculture the shoots to MS or half-MS hormone free medium.  The shoots will elongate and root.

Best wishes.

Thank you  Martin for your answer i hope it will work

dear Mohd, you are using low auxin concentration than cytokinin its ok. but try to use another auxin like IAA/IBA than the 2, 4-D. it will give better results in shoot initiation. as 2, 4-D preferably enhances callus thts why you are not getting shoot proliferation.

Hi  Ali and thank you for your reply

pH of media always 5.6-5.8. and concentration of phytohormones are  as follow

experiment 1                         experiment 2                               experiment 3        

    BAP mg/l                    BAP+KINETIN mg/l                                  BAP+2,4D mg/l

              3                                   (2+2)                                                (3+0.75)mg/l

             6                                     (3+3)                                                (6+1.5)mg/l      

              8                                   (4+4)                                                (8+2)mg/l 

here we use 3 different concentration of phyto-hormones in 3 experiment. in experiment 1 we used only BAP in 2nd experiment we used kinetin with BAP and in 3rd experiment we used 2,4-D with BAP.

now we are using IAA with BAP and kinetin hoping to get some positive result.

if u now suggest me it will be valuable for me 

 Hi Mohd, did you solve your problem? Im following Clemente´s procedure altough some callus aren´t generating shoots. 

 Hi Stefano, my problem was not solved completely. we used different concentration and combination of cytokinin and auxin ratio very few callus show response. but the leaves of regenerated plants was very thick and dark green than the normal tobacco leaves. 

then we posted a Question related to this problem in research gate and we found, it is because of hyper-hydricity and try to solve this problem but not succeeded. if you understand my problem and if you have any protocol kindly share with me.