Study On Regeneration Of Tobacco Plantlet And Their Nodulation

Study on Regeneration of Tobacco Plantlet and their Nodulafion Abstract After the completely spreading of the forth true leaf of the sterile seedling of tobacco, took off the forth young leaf and cut it into an 5mm2 piece on the condition of no bacteria. Then these pieces were inoculated on MS medium which contained NAA(0.lmgIl), 6-BA(l.Omg/l), sucrose(3%) and agar(0.85%). The culture temperature was 28 0C, the intensity of illumination were 30001x and 12h photoperiod. In the forth day of culture, the leaf pieces generated calluses. In the tenth day, budlets appeared on the faces of the calluses. In the twelfth day, a lot of clustered buds which could be see by eyes. After 1-2 weeks, took off the regeneration seedlings which should be 1cm long at least, then they were inserted into root-forming medium (MS, 0.lmgll IAA, 3% sucrose and 0.85% agar). 3 days later, successions they generated roots. The 2 weeks old regeneration plantlets were transplanted to basins which were full of soil. They were cultured on the condition that the temperature was about 250C, the intensity of illumination was 50001x, and the illumination period was 1 Oh per day. 3 month later, we could see that the survival rate of the transplanted plantlet was 100%. Moreover, the growing situation was pretty good.The observation of histology and cytology showed that the regeneration of tobacco plantlets were mainly achieved by the way of calluses. Using different consistency 2,4-D to induce R. leguminosarum to intrude the regenerated tobacco plantlet could make plenty of para-nodules form at the root of the plant. Its quantity and rate had relation to 2,4-D and the consistency of rhizobium. Its quantity and rate would increase complying with the consistency in a certain extent of consistency. If the consistency exceeded the certain extent, the quantity and the rate would not increase but decrease. In our condition, using 2,4-D of 0.3mg/I and rhizobiuin of 5 x 108 rhizobia per litre to generation nodules could get a good effect. We observed a lot of rhizobia in the para-nodule under light microscopic. They mainly existed in the intercellular layer and intracellular spaces. There only were a small quantity of rhizobia ir the host cell, but not found in the root tip cell.