| Pogostemon cablin(Blanco) Benth is a plant from exogenous genus. Its aboveground part could be commonly used as a Traditional Chinese Medicine. This study includes four parts: The first part is to optimize the cells suspension culture system of P. cablin based on the techniques from our laboratory. The second part is to explore the effects of Salicylic Acid and Methyl Jasmonate on growth and the content of patchouli alcohol of suspension cells from P. cablin. The third part is to research the main influence factors on process of protoplast isolation and purification, taking the protoplasts yield and viability as evaluation index, and an efficient system is established finally. The fourth part is to culture the protoplast of P. cablin, by using shallow liquid culture methods, different mediums, different conditions etc, and the first division of protoplasts is observed successfully. The main results are as follows:(1) Optimization of the suspension cells culture system of P. cablinThe optimization of suspension cells culture system of P. cablin shows: The first induction time of P. cablin loose callus is 21 days. The method “To divide loose callus into 2-4 blocks in the case of keeping the integrity and growth polarity properly, then2-3 little loose callus are inoculated to a new medium ” is the most beneficial way to subculture the loose callus of P. cablin. The 8 g/100 mL inoculation size is beneficially to subculture the suspension cells which can get a large number of dispersed homogeneously suspension cells.(2) Effects of elicitors on growth and patchouli alcohol content of suspensioncells from P. cablin.Adding 1 mg/L Salicylic Acid and 5 mg/L Methyl Jasmonate in different stages of P. cablin suspension cells growth curve, and the cell fresh weight, dry weight and patchouli alcohol content are determined to study the impacts of Salicylic Acid and Methyl Jasmonate after the culture.Adding Salicylic Acid in different stages of P. cablin suspension cells growth curve, there are no significant differences between the growth situations of treated suspension cells and the control groups. However, when the adding time are 8 days and 15 days, the differences of cells fresh weight and dry weight are extremely significant. Taking 8 days as the adding time, Salicylic Acid can promote the cell growth. The cells fresh weight reaches 558.8474 g/L and cells dry weigh is 12.0912g/L, which are the highest. All Salicylic Acid treatment groups can improve the contents of patchouli alcohol, and there are significant differences between them, but it is significantly different compared with the the control groups. When add Salicylic Acid in the early stage of the logarithmic phase, 9 days, the contents of patchouli alcohol is 0.0502 mg/g, higher than control groups that is 0.0061 mg/g, and 8.292 times of the control groups.Adding Methyl Jasmonate in different stages of P. cablin suspension cells growth curve, there are no significant differences between the growth situations of treated suspension cells and the control groups. However, the differences of cells fresh weight between 9 days and 15 days are extremely significant. Taking 9 days as the adding time, Methyl Jasmonate does not have inhibition on the cell growth. The fresh weight reaches 523.9138 g/L and cells dry weigh is 10.8918 g/L, which are the highest. All Methyl Jasmonate treatment groups can promote the contents of patchouli alcohol,and there are significant differences between the treating groups and control groups.When adding Methyl Jasmonate in the early stage of the logarithmic phase, 9 days,the contents of patchouli alcohol is 0.1684 mg/g, which is the highest, and 27.6065 times of the control groups.(3) Isolation and purification of protoplasts from suspension cells of P. cablin.Taking suspension cells as material, protoplast yield and vitality as examiningindex, the effects of enzyme combination, enzyme treatment time, mannitol concentration, growth time after subculture of suspension cells, different aperture mesh and centrifugal conditions on protoplast isolation and purification are studied.The best way to isolate and purify cells suspension protoplasts of P. cablin. is:Using 1.5% cellulase, 0.8% pectinase and 0.5% hemicellulase as the best enzyme combination, and the best mannitol concentration is 0.4 mol/L, then prepare the enzyme solution and mix with the suspension cells grew 9-14 days. After putting it on the shaker for treating 12 hours with 26℃and 50 r/min, a large amount of protoplasts are isolated. Filtering the mixture by 40, 100, 200 meshes in turn to collect the crude extract of protoplasts. Then, in centrifugation conditions of 600 r/min for 5 min to purify further, high quality of suspension cells protoplasts are obtained finally. The yield reached 13.05×105/g, and the vitality is 80.98% 。(4) Culture of the protoplasts from suspension cells of P. cablin.The study explored the culture methods of protoplasts, including shollow liquid culture, micro drop culture, solid-liquid double layer culture and nurse culture. As for shollow liquid culture, different culture mediums and different light conditions are compared to show the influenceTaking MS medium, 0.1 mg/L 2,4-D and 3 mg/L 6-BA as the protoplasts medium for shollow liquid culture. After 12 days, protoplasts which cultured under the dark condition appear the first cell division. With the weak light conditions,protoplasts could apprar the first cell division after 15-18 days. However, protoplasts which culture under the light of 12 h/d need for 25 days to get the first cell division.The results show the time of protoplasts first division is different, and the dark culture conditions may be beneficial to the growth of protoplasts. In micro drop culture, the cytoplasm of protoplasts becomes strong. But the protoplasts go brown later, so no cell division are observed. Two mediums are used in the solid-liquid double culture,and the shape of protoplasts are changing, but no observation of cell division. Using nurse culture to cultivate the protoplasts 30 days, there are no small callus regeneration formed. |
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