Physiology Study On Sex Differentiation Of Hemp (Cannabis Sativa L.)

Hemp(Cannabis sativa L.)is an annual herb, dioecious, with medicine, textile, and other aspects of economic value. Under natural conditions, the male plants mature earlier than the female plants, and they have different economic values. It was reported that external factors effected the sex differentiation of hemp. In view of this, we identified the sex of hemp at seeding stage, we also analyzed the impact of exogenous factors and physiological indices on the sex differentiation of hemp. Meanwhile, we studied the expression of GA20-oxidase gene(a key gene encoding key enzymes in GA3metabolic pathway), preliminarily discussed the physiological mechanisms and hormonal regulation of sex differentiation of hemp, lay the foundation for the physiological study of sex differentiation of hemp. The main results were as follows:1. Using the methods of TTC and NADH, we identified the sex of hemp at seedling(4blades stage), the results showed that:the two methods had significant difference (p<0.01), and the accuracy was91.48%and81.49%respectively when identified the individual plant of mixed population. We also used the specific marker linked with the male to identify the plants from six places(Yunnan, Tianshui, Longxi, Zhangye, Lingqiu, Changtu) and Yunmal, the results showed that only the plants from Yunmal and Lingqiu amplified the specific bands.2. We treated the seedlings of3-bladess with different concentrations of GA3,6-BA and IAA combined with different photoperiods of8h,12h and16h, the results showed that:GA3promoted the male differentiation of hemp, and the effects showed positive correlation with the concentration (50-200mg/L);6-BA promoted the female differentiation and the effects also showed positive correlation with the concentration (30-120mg/L). IAA could promote the male differentiation when treated with low concentration(30mg/L), but when treated with high concentration(60,120mg/L), IAA promoted the male differentiation. Short day treatment (8h) had no influence on sex differentiation, but long day treatment (16h) promoted the male differentiation. When treated with GA3,6-BA, IAA at3-bladess and5-bladess, the results showed that:6-BA (120mg/L) and IAA (30mg/L) promoted the female differentiation only at3-bladess, but GA3(200mg/L) promoted the male differentiation regardless of the stage, and the promotion effect was significant at3-blades.3. The differences of physiological and biochemical indexes in the processs of sex differentiation of hemp showed that:in leaves and flowers, the POD activity, soluble protein content, ZT content and IAA content were higher in male plants than those in female plants, but CAT activity, IAAO activity and reducing sugar content were lower than that of female plants; The Soluble sugars content in the leaves of female plants were higher than that of male plants, but in the flowers, male plants were higher; The GA3content in the leaves of male plants were higher than that of female plants, but in the flowers, female plants were higher. The activities of POD, CAT, IAAO and the content of ZT and soluble protein in leaves of male and female plants were higher than that of the3-blades stage, but the content of soluble sugars and GA3were lower; The IAA content in leaves was lower than that of3-blades stage in male plants, but higher in female plants-, The reducing sugar content was higher in male plant leaves than that of3-blades stage, but had no significant difference in female plants.4. After treated with hormones, we determined physiological and biochemical indicators of female and male plants, the results showed that:(1) GA3treatment. With the increase of GA3concentration, the activities of POD and IAAO presented the trend of decreasing in leaves of both male and female plants; The CAT activity in the leaves of female plants presented the trend of increasing at first, and then decreasing, but always increasing in male plants. The contents of soluble sugars and reducing sugar appeared dropping in the leaves of female plants, but increasing in male plants. The affect of GA3on the content of soluble protein in leaves of both male and female plants was not obvious.(2)6-BA treatment. With the increase of6-BA concentration, The POD activity in the leaves of female plants had no significant changes, but increased in male plants; The activity of CAT in the leaves of female plants presented the trend of increasing at first, and then decreasing, but always increasing in male plants. The IAAO activity in the leaves of female plants appeared increasing, but showed no significant changes in male plants; The contents of reducing sugar appeared increasing in the leaves of female plants, but presented increasing at first and then decreasing. The affect of6-BA on the content of soluble protein and soluble sugars in leaves of both male and female plants were similar with GA3 treatment.(3)IAA treatment. With the increase of IAA concentration, the POD activity appeared dropping in leaves of both male and female plants; The IAAO activity in the leaves of female plants appeared dropping, but showed no significant changes in male plants; The contents of reducing sugar in leaves of both male and female plants and the CAT activity in the leaves of female plants both increased at first and then tended to be stabilized, but always increased in male plants. The IAA had no significant effect on the contents of soluble protein and soluble sugars in leaves of both male and female plants.5. We isolated the fragment of the gene GA20ox (CSGA20ox). Gene expression analysis using semi-quantitative RT-PCR showed that the expression level of CSGA20ox had significant distinctives at different development stages, also different tissues. The expression level was highest at the leaf of3-blades stage, and the expression level of male leaf was higher than female plant at5-blades stage, and the expression level of female flower was higher than male flower at bud stage.