In Vitro Selection Of Anti-Phe Cell Mutants In Tobacco Callus

The phenylpropane skeleton metabolism is one of the most important secondary metabolism pathways in tobacco, within which L-phenylalanine can be changed into a wide variety of natural products, and result in phenylalanine and other fragrants, which are important for tobacco aroma quality and disease resistance. Therefore, it is hopeful to improve the content of phenylalanine or other relative substance, and finally make progresses in improving the aroma quality and disease resistance in tobacco by improving the phenylpropane metabolism in it.Several methods such as Somaclonal variation, induced mutation and in vitro selection were combined implemented in this study. The effect of MS culture medium with different dose of L-Phe on the growing state, cell activity, PAL activity of tobacco callus; tobacco callus from different variety's sensitivity to filter substance are also investigated. The select system was established based on the result of our experiment. The anti-Phe cell line were screened out, and then induced differentiation. Finally, we obtain several regenerating plants, which can be used in tobacco breeding. The main results of our research are as follows:1.MS culture medium with different concentration of L-Phe inhibited the growth of tobacco callus. But this effect of low L-Phe concentration(1² mmol·L^-1) was not significant. When the dose was 3mmol·L^-1, 50% of callus' growth stopped, the structure of most tobacco callus became very compact. High L-Phe concentration(4 mmol·L^-1) deadly affected the growth of tobacco, made over 80% callus stop growing, and died after lOd.2. Different concentration of L-Phe in the MS culture medium has different effects on the cell activity of tobacco callus. 1-2 mmol·L^-1 L-Phe's has slightly inhibit effect, while the concentration is 4 mmol·L^-1, the inhibit effect are severely, the cell activity decline to a very low level. When the concentration was 3 mmol·L^-1, the cell activity declined about 50%. We suppose the dose of 3 mmol·L^-1 is suitable for the selection system.3.As the radiation dose increase, the death rate of tobacco callus also increase. Base on tobacco callus which cultured in similar condition, we suppose 40GY is suitable for mutation breeding.