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Abstract ［Objectives］ This study was conducted to establish a tissue culture regeneration system in Bama hemp (Cannabis sativa L.).
［Methods］ Using hemp seeds as explants, a regeneration system was established through explant sterilization, callus induction, callus differentiation, and rooting culture.
［Results］ The results showed that the best sterilization effect was achieved when sterilizing with 75% ethanol for 30 s, followed by 0.1% HgCl2 solution for 9 min, with a contamination rate as low as 11.4%. In presence of 3 mg/L 2,4D and 0.1 mg/L6BA, the callus induction effect from hemp seeds was better. The formula for better differentiation of callus was MS+2.0 mg/L 6BA+0.2 mg/L NAA. IBA had a promoting effect on the rooting of hemp aseptic plantlets. The highest rooting rate reached 80% when MS+0.3 mg/L IBA were used.
［Conclusions］ This study established a hemp seed regeneration system to provide technical support for the conservation and breeding of hemp germplasm resources.
Key words Hemp seeds; Tissue culture; Regeneration system
Received: July 17, 2019Accepted: October 11, 2019
Supported by Guangxi Natural Science Foundation (2017JJB130027); The Basic Ability Enhancement Program for Young and Middleaged Teachers of Guangxi (2017KY0724); Masters Degree Authorization Unit Construction Authorization Point (GXW［2018］7).
Yanfeng NONG (1988-), female, P. R. China, lecturer, master, devoted to research about plant tissue culture and plant resources development research.
*Corresponding author. Email: nongyan889@126.com.
Hemp (Cannabis sativa L.) is an annual herb in Cannabis of Cannabaceae, which is dioecious. Its fruit is called hemp seed. Hemp is one of the two special crops used as both medicine and food in Guangxi. Hemp seed is a national geographical indication agricultural product, mainly distributed in the Bama Yao Autonomous County of Hechi City, Guangxi and surrounding ethnic minority settlements［1］. Hemp seed is rich in oil, and has an oil content of 30%, higher than the fatty oil content of soybean, so hemp oil is the main source of edible oil in the region［2-3］. In addition, local people have also produced edible products such as hemp sprouts, hemp soup and hemp cake, which have good economic value［4-5］. Hemp oil contains 70%-80% of unsaturated fatty acids, which has antioxidation, antitumor and immuneenhancing effects. It can significantly reduce the content of highdensity protein cholesterol, thereby reducing the morbidity of diseases such as heart disease, stroke and hypertension. Linolenic acid contained in hemp oil also has a good preventive effect on cardiovascular diseases such as hypertension and atherosclerosis［6-7］. The traditional propagation method of hemp is seed propagation, but due to its dioecism, low pollination efficiency and other factors, the reproduction rate is not high, and the risk of artificial cultivation is higher［8-11］. In order to protect the germplasm resources of hemp while meeting the food consumption needs of the market, artificial propagation should be intensified. The use of tissue culture technology can speed up the propagation of hemp and make up for the lack of traditional propagation. In this study, the tissue culture technique of Bama hemp seeds was used as the material to determine the best sterilization time, select the best induction medium, differentiation medium and rooting medium, and establish an efficient and rapid propagation technology system, so as to fill the technical blank of the hemp tissue culture. This study will provide technical support for the preservation and breeding of hemp variety germplasm resources.
Materials and Methods
Experimental materials
Hemp seeds were collected from Bama Yao Autonomous County, Hechi City, Guangxi.
Experimental methods
Sterilization of experimental materials
The hemp seeds were soaked in tap water for 2 to 3 h. After laying wet gauze in culture dishes, the soaked hemp seeds were added and then covered with a layer of wet gauze for germination. The next day, the seeds germinated. The germinated hemp seeds were stripped off and added into a small beaker, soaked in a 1% potassium permanganate solution for 15 min, and rinsed with running water for 10 min. The pretreated seeds were treated with 75% ethanol (15 s, 30 s) and 0.1% HgCl2 (7, 8, 9, 10 min). Each treatment was inoculated with 35 seeds and was done in triplicate. After 7 d, the contamination rate was calculated according to according to following equation: Contamination rate=Total number of contaminated seeds/Number of inoculated seeds*100%.
Callus induction
MS was used as the basic induction medium, and the mixed media with different concentrations of 2,4D (0.5-4.0 mg/L) was set to directly induce calli from hemp seeds when fixing 6BA at 0.1 mg/L. The growth of induced calli on these media was observed and compared. Each treatment was inoculated with 35 seeds and was done in triplicate. The callus induction rate was calculated according to following equation: Callus induction rate=Number of growing calli/Total number of inoculated seeds*100%.
Differentiation culture With MS as the basic medium, the calli were differentiated in different concentrations of 6BA (0.5-3.0 mg/L) when fixing NAA at 0.2 mg/L, and the callus differentiation of hemp seeds on these media was compared. Each treatment was inoculated with 35 calli and was done in triplicate. The callus differentiation rate and average number of buds were calculated according to following equations: Callus differentiation rate=Number of differentiated callus blocks/Total number of inoculated calli*100%, and Average number of buds=Total number of adventitious buds differentiated /Total number of inoculated calli.
Rooting culture
MS was used as the basic medium, and different concentrations of IBA (0.1-0.9 mg/L) were set to induce rooting of healthy aseptic plantlets. Each treatment was inoculated with 35 calli and was done in triplicate. The rooting rate was calculated according to following equation: Rooting rate=Number of roots/Total number of aseptic buds*100%.
Culture condition
The culture conditions were 12-14 h/d for light and 25-27 ℃ for temperature.
Results and Analysis
Effect of different sterilization treatments on explants
As shown in Table 1, the treatment with 75% ethanol was carried out for 15 and 30 s, respectively, and the treatment with 0.1% HgCl2 was carried out for 7, 8, 9 and 10 min, respectively. It can be seen from the table that in the case of the same ethanol concentration (75%) and different sterilization time, the contamination rate decreased over the time of sterilization with 0.1% HgCl2. The survival rate of explants was observed to be 100% in the experiment, and the differences between various treatments were extremely significant. The comprehensive results showed that the sterilization effect was best when the seeds were sterilized with 75% ethanol for 30 s and then sterilized with 0.1% HgCl2 for 9 min, and the contamination rate was as low as 11.4%.
Callus induction
As shown in Table 2, explants inoculated with five different 2,4D concentration gradients all showed callus growth at a fixed 6BA concentration of 0.1 mg/L. The callus induction rate obtained with 0.5 mg/L 2,4D concentration was 42.9%, which was the lowest, while the callus induction rate obtained with 3 mg/L 2,4D concentration was 80.0%, which was the highest. The calli showed a size constantly increasing, and were fluffy and pale yellow. It can be seen from Table 2 that with the increase of 2,4D concentration, the callus rate of hemp seeds increased with the increase of the 2,4D concentration at first and then showed a downward trend after the 2,4D concentration increased to a certain extent. The differences between various treatments reached the extremely significant level. The study showed that the induction effect of callus from hemp seeds was the best at the 2,4D concentration of 3 mg/L and the 6BA concentration of 0.1 mg/L. Differentiation culture
As shown in Table 3, the inoculated calli were able to induce adventitious bud growth in the four treatments, and most of them were 3 to 4 cm long after 30 d of culture and had distinct stems and leaves. In Table 3, the 6BA concentration of 0.5 mg/L produced a differentiation rate of 22.9%, which was the lowest, and the average number of buds was only 0.4; the 6BA concentration of 2 mg/L gave a differentiation rate of 82.9%, which was the highest, and the average number of buds was 2.0. The differences in the differentiation rate reached the extremely significant level. The treatment of 0.5 mg/L 6BA plus 0.2 mg/L NAA was significantly different in the average number of buds from other three treatments, while there were no significant differences between any two of other three treatments. It can be concluded from the table that with the increase of the 6BA concentration, the callus differentiation rate and the average number of buds increased, and then showed a decreasing trend after the 6BA concentration increased to a certain extent. Considering the comprehensive differentiation rate and the average number of buds, the effect of 2.0 mg/L 6BA combined with 0.2 mg/L NAA was better on callus differentiation.
Rooting culture
Adventitious buds obtained from callus differentiation grow into robust aseptic plantlets after strong seedling culture, and the aseptic plantlets can grow into complete plants after rooting induction. IBA is a common plant growth regulator that induces rooting. In this study, IBA had a promoting effect on the rooting of hemp aseptic plantlets, and the treatment effect was obvious. The rooting rate was the highest at 80%, and the lowest at 40%, and the difference between 0.6 mg/L IBA and 0.9 mg/L IBA treatment was not significant. In comparison, MS + 0.3 mg/L IBA was the best. It was found that the roots of the aseptic plants were whiter and thicker, and the length was about 2 cm.
Discussion and Conclusions
In this study, the hemp seeds sprouted from the shell were used as the explants for sterilization. The test results showed that sterilizing with 75% ethanol for 30 s and then with 0.1% HgCl2 for 9 min was the best. In the sterilization test, dried hemp seeds were directly sterilized as explants, but the sterilization effect was not satisfactory. The initial contamination rate after sterilization was extremely low, but the contamination rate gradually increased at the beginning of seed germination, which might be caused by more endophytic bacteria in the seeds. Therefore, in this study, the seeds that had just sprouted from the shell were used as explants to reduce the source of endophytic bacteria. In this study, calli were induced with different concentrations of 2,4D and 6BA. The results showed that the medium containing 2,4D 3.0 mg/L+6BA 0.1 mg/L had the best effect on callus induction of hemp seeds, and the induced calli were larger and denser. When inducing calli with 2,4D alone, the induction rate was low, and the calli were white and loose. 6BA combined with NAA promoted the differentiation of callus, achieving a high differentiation rate, but the average number of buds differentiated was smaller. In the next stage, different cytokinins and auxins can be used to induce differentiation. In rooting culture, lower concentrations of auxins could induce rooting and achieved a rooting rate higher than 40%; and the roots were whiter, and there were 2 to 5 new roots in each aseptic plantlets. Prospects
Bama hemp seed is one of the national geographical indication agricultural products. Hemp is one of the crops used both as medicine and food, and has important economic value and medicinal value in the ethnic food culture resources in northwest Guangxi, but the seedling resources are lacking. The use of tissue culture biotechnology can solve current problems. This study established a regeneration system through hemp seeds, which provides a technical basis for seed production, and provides technical support for the development and utilization of hemp germplasm resources. On the basis of this study, cultivation and breeding can be further explored in the next step.
References
［1］ QIN HY. Analysis on the tourism development of ethnic food culture resource in northwest of Guangxi［J］. Journal of Huaihua University, 2016, 35(2): 16-18. (in Chinese)
［2］ GAO Z, ZHANG ZJ, LI HZ, et al. Research progress of the comprehensive processing and product development of hemp resources［J］. Journal of The Chinese Cereals and Oils Association, 2019, 34(3): 141-146. (in Chinese)
［3］ CONG RH, XU CF, ZHENG MM, et al. Liposoluble nutrients in hemp seed oils from different production area［J］. Chinese Journal of Oil Crop Sciences, 2017, 39(6): 861-868. (in Chinese)
［4］ TANG JM, WEI X, ZOU R, et al. Liposoluble nutrients in hemp seed oils from different production area［J］. Journal of Guangxi Academy of Sciences, 2019, 35(1): 1-5, 83. (in Chinese)
［5］ WU JF. Application studies on deep processing of hemp (Cannabis sativa L.) resources byproducts［D］. Guangzhou: South China University of Technology, 2016. (in Chinese)
［6］ WEI YY, LI L. Advance on functional ingredients and safety on hemp seed food［J］. Food Industry, 2015, 36(7): 256-260. (in Chinese)
［7］ YU JQ, YU XZ, CHEN XY, et al. Physicochemical properties of hemp seed and its oils［J］. China Oils and Fats, 2012, 37(4): 84-87. (in Chinese)
［8］ XIAO JJ, YE N, MA DW. Characteristics and uses of hemp ‘Bamahuoma’［J］. Journal of Green Science and Technology, 2012(3): 145-146. (in Chinese)
［9］ LI GJ, LI X, WANG Q, et al. Promoting effect of soaking with different exogenous substances on seed germination of hemp ‘Bamahuoma’ under drought stress［J］. Journal of Yunnan University: Natural Sciences Edition, 2018, 40(5): 1034-1041. (in Chinese)
［10］ LI GJ, WANG Q, LI X, et al. Effects of gibberellin and Vc soaking on seedling physiology during initial germination of hemp seeds under drought stress［J］. Seed, 2018, 37(6): 67-71. (in Chinese)
［11］ LYU YM. Effects of different cultivation conditions on hemp traits［J］. Chinas Fiber Crops, 1985(3): 42-45, 18. (in Chinese)
［Methods］ Using hemp seeds as explants, a regeneration system was established through explant sterilization, callus induction, callus differentiation, and rooting culture.
［Results］ The results showed that the best sterilization effect was achieved when sterilizing with 75% ethanol for 30 s, followed by 0.1% HgCl2 solution for 9 min, with a contamination rate as low as 11.4%. In presence of 3 mg/L 2,4D and 0.1 mg/L6BA, the callus induction effect from hemp seeds was better. The formula for better differentiation of callus was MS+2.0 mg/L 6BA+0.2 mg/L NAA. IBA had a promoting effect on the rooting of hemp aseptic plantlets. The highest rooting rate reached 80% when MS+0.3 mg/L IBA were used.
［Conclusions］ This study established a hemp seed regeneration system to provide technical support for the conservation and breeding of hemp germplasm resources.
Key words Hemp seeds; Tissue culture; Regeneration system
Received: July 17, 2019Accepted: October 11, 2019
Supported by Guangxi Natural Science Foundation (2017JJB130027); The Basic Ability Enhancement Program for Young and Middleaged Teachers of Guangxi (2017KY0724); Masters Degree Authorization Unit Construction Authorization Point (GXW［2018］7).
Yanfeng NONG (1988-), female, P. R. China, lecturer, master, devoted to research about plant tissue culture and plant resources development research.
*Corresponding author. Email: nongyan889@126.com.
Hemp (Cannabis sativa L.) is an annual herb in Cannabis of Cannabaceae, which is dioecious. Its fruit is called hemp seed. Hemp is one of the two special crops used as both medicine and food in Guangxi. Hemp seed is a national geographical indication agricultural product, mainly distributed in the Bama Yao Autonomous County of Hechi City, Guangxi and surrounding ethnic minority settlements［1］. Hemp seed is rich in oil, and has an oil content of 30%, higher than the fatty oil content of soybean, so hemp oil is the main source of edible oil in the region［2-3］. In addition, local people have also produced edible products such as hemp sprouts, hemp soup and hemp cake, which have good economic value［4-5］. Hemp oil contains 70%-80% of unsaturated fatty acids, which has antioxidation, antitumor and immuneenhancing effects. It can significantly reduce the content of highdensity protein cholesterol, thereby reducing the morbidity of diseases such as heart disease, stroke and hypertension. Linolenic acid contained in hemp oil also has a good preventive effect on cardiovascular diseases such as hypertension and atherosclerosis［6-7］. The traditional propagation method of hemp is seed propagation, but due to its dioecism, low pollination efficiency and other factors, the reproduction rate is not high, and the risk of artificial cultivation is higher［8-11］. In order to protect the germplasm resources of hemp while meeting the food consumption needs of the market, artificial propagation should be intensified. The use of tissue culture technology can speed up the propagation of hemp and make up for the lack of traditional propagation. In this study, the tissue culture technique of Bama hemp seeds was used as the material to determine the best sterilization time, select the best induction medium, differentiation medium and rooting medium, and establish an efficient and rapid propagation technology system, so as to fill the technical blank of the hemp tissue culture. This study will provide technical support for the preservation and breeding of hemp variety germplasm resources.
Materials and Methods
Experimental materials
Hemp seeds were collected from Bama Yao Autonomous County, Hechi City, Guangxi.
Experimental methods
Sterilization of experimental materials
The hemp seeds were soaked in tap water for 2 to 3 h. After laying wet gauze in culture dishes, the soaked hemp seeds were added and then covered with a layer of wet gauze for germination. The next day, the seeds germinated. The germinated hemp seeds were stripped off and added into a small beaker, soaked in a 1% potassium permanganate solution for 15 min, and rinsed with running water for 10 min. The pretreated seeds were treated with 75% ethanol (15 s, 30 s) and 0.1% HgCl2 (7, 8, 9, 10 min). Each treatment was inoculated with 35 seeds and was done in triplicate. After 7 d, the contamination rate was calculated according to according to following equation: Contamination rate=Total number of contaminated seeds/Number of inoculated seeds*100%.
Callus induction
MS was used as the basic induction medium, and the mixed media with different concentrations of 2,4D (0.5-4.0 mg/L) was set to directly induce calli from hemp seeds when fixing 6BA at 0.1 mg/L. The growth of induced calli on these media was observed and compared. Each treatment was inoculated with 35 seeds and was done in triplicate. The callus induction rate was calculated according to following equation: Callus induction rate=Number of growing calli/Total number of inoculated seeds*100%.
Differentiation culture With MS as the basic medium, the calli were differentiated in different concentrations of 6BA (0.5-3.0 mg/L) when fixing NAA at 0.2 mg/L, and the callus differentiation of hemp seeds on these media was compared. Each treatment was inoculated with 35 calli and was done in triplicate. The callus differentiation rate and average number of buds were calculated according to following equations: Callus differentiation rate=Number of differentiated callus blocks/Total number of inoculated calli*100%, and Average number of buds=Total number of adventitious buds differentiated /Total number of inoculated calli.
Rooting culture
MS was used as the basic medium, and different concentrations of IBA (0.1-0.9 mg/L) were set to induce rooting of healthy aseptic plantlets. Each treatment was inoculated with 35 calli and was done in triplicate. The rooting rate was calculated according to following equation: Rooting rate=Number of roots/Total number of aseptic buds*100%.
Culture condition
The culture conditions were 12-14 h/d for light and 25-27 ℃ for temperature.
Results and Analysis
Effect of different sterilization treatments on explants
As shown in Table 1, the treatment with 75% ethanol was carried out for 15 and 30 s, respectively, and the treatment with 0.1% HgCl2 was carried out for 7, 8, 9 and 10 min, respectively. It can be seen from the table that in the case of the same ethanol concentration (75%) and different sterilization time, the contamination rate decreased over the time of sterilization with 0.1% HgCl2. The survival rate of explants was observed to be 100% in the experiment, and the differences between various treatments were extremely significant. The comprehensive results showed that the sterilization effect was best when the seeds were sterilized with 75% ethanol for 30 s and then sterilized with 0.1% HgCl2 for 9 min, and the contamination rate was as low as 11.4%.
Callus induction
As shown in Table 2, explants inoculated with five different 2,4D concentration gradients all showed callus growth at a fixed 6BA concentration of 0.1 mg/L. The callus induction rate obtained with 0.5 mg/L 2,4D concentration was 42.9%, which was the lowest, while the callus induction rate obtained with 3 mg/L 2,4D concentration was 80.0%, which was the highest. The calli showed a size constantly increasing, and were fluffy and pale yellow. It can be seen from Table 2 that with the increase of 2,4D concentration, the callus rate of hemp seeds increased with the increase of the 2,4D concentration at first and then showed a downward trend after the 2,4D concentration increased to a certain extent. The differences between various treatments reached the extremely significant level. The study showed that the induction effect of callus from hemp seeds was the best at the 2,4D concentration of 3 mg/L and the 6BA concentration of 0.1 mg/L. Differentiation culture
As shown in Table 3, the inoculated calli were able to induce adventitious bud growth in the four treatments, and most of them were 3 to 4 cm long after 30 d of culture and had distinct stems and leaves. In Table 3, the 6BA concentration of 0.5 mg/L produced a differentiation rate of 22.9%, which was the lowest, and the average number of buds was only 0.4; the 6BA concentration of 2 mg/L gave a differentiation rate of 82.9%, which was the highest, and the average number of buds was 2.0. The differences in the differentiation rate reached the extremely significant level. The treatment of 0.5 mg/L 6BA plus 0.2 mg/L NAA was significantly different in the average number of buds from other three treatments, while there were no significant differences between any two of other three treatments. It can be concluded from the table that with the increase of the 6BA concentration, the callus differentiation rate and the average number of buds increased, and then showed a decreasing trend after the 6BA concentration increased to a certain extent. Considering the comprehensive differentiation rate and the average number of buds, the effect of 2.0 mg/L 6BA combined with 0.2 mg/L NAA was better on callus differentiation.
Rooting culture
Adventitious buds obtained from callus differentiation grow into robust aseptic plantlets after strong seedling culture, and the aseptic plantlets can grow into complete plants after rooting induction. IBA is a common plant growth regulator that induces rooting. In this study, IBA had a promoting effect on the rooting of hemp aseptic plantlets, and the treatment effect was obvious. The rooting rate was the highest at 80%, and the lowest at 40%, and the difference between 0.6 mg/L IBA and 0.9 mg/L IBA treatment was not significant. In comparison, MS + 0.3 mg/L IBA was the best. It was found that the roots of the aseptic plants were whiter and thicker, and the length was about 2 cm.
Discussion and Conclusions
In this study, the hemp seeds sprouted from the shell were used as the explants for sterilization. The test results showed that sterilizing with 75% ethanol for 30 s and then with 0.1% HgCl2 for 9 min was the best. In the sterilization test, dried hemp seeds were directly sterilized as explants, but the sterilization effect was not satisfactory. The initial contamination rate after sterilization was extremely low, but the contamination rate gradually increased at the beginning of seed germination, which might be caused by more endophytic bacteria in the seeds. Therefore, in this study, the seeds that had just sprouted from the shell were used as explants to reduce the source of endophytic bacteria. In this study, calli were induced with different concentrations of 2,4D and 6BA. The results showed that the medium containing 2,4D 3.0 mg/L+6BA 0.1 mg/L had the best effect on callus induction of hemp seeds, and the induced calli were larger and denser. When inducing calli with 2,4D alone, the induction rate was low, and the calli were white and loose. 6BA combined with NAA promoted the differentiation of callus, achieving a high differentiation rate, but the average number of buds differentiated was smaller. In the next stage, different cytokinins and auxins can be used to induce differentiation. In rooting culture, lower concentrations of auxins could induce rooting and achieved a rooting rate higher than 40%; and the roots were whiter, and there were 2 to 5 new roots in each aseptic plantlets. Prospects
Bama hemp seed is one of the national geographical indication agricultural products. Hemp is one of the crops used both as medicine and food, and has important economic value and medicinal value in the ethnic food culture resources in northwest Guangxi, but the seedling resources are lacking. The use of tissue culture biotechnology can solve current problems. This study established a regeneration system through hemp seeds, which provides a technical basis for seed production, and provides technical support for the development and utilization of hemp germplasm resources. On the basis of this study, cultivation and breeding can be further explored in the next step.
References
［1］ QIN HY. Analysis on the tourism development of ethnic food culture resource in northwest of Guangxi［J］. Journal of Huaihua University, 2016, 35(2): 16-18. (in Chinese)
［2］ GAO Z, ZHANG ZJ, LI HZ, et al. Research progress of the comprehensive processing and product development of hemp resources［J］. Journal of The Chinese Cereals and Oils Association, 2019, 34(3): 141-146. (in Chinese)
［3］ CONG RH, XU CF, ZHENG MM, et al. Liposoluble nutrients in hemp seed oils from different production area［J］. Chinese Journal of Oil Crop Sciences, 2017, 39(6): 861-868. (in Chinese)
［4］ TANG JM, WEI X, ZOU R, et al. Liposoluble nutrients in hemp seed oils from different production area［J］. Journal of Guangxi Academy of Sciences, 2019, 35(1): 1-5, 83. (in Chinese)
［5］ WU JF. Application studies on deep processing of hemp (Cannabis sativa L.) resources byproducts［D］. Guangzhou: South China University of Technology, 2016. (in Chinese)
［6］ WEI YY, LI L. Advance on functional ingredients and safety on hemp seed food［J］. Food Industry, 2015, 36(7): 256-260. (in Chinese)
［7］ YU JQ, YU XZ, CHEN XY, et al. Physicochemical properties of hemp seed and its oils［J］. China Oils and Fats, 2012, 37(4): 84-87. (in Chinese)
［8］ XIAO JJ, YE N, MA DW. Characteristics and uses of hemp ‘Bamahuoma’［J］. Journal of Green Science and Technology, 2012(3): 145-146. (in Chinese)
［9］ LI GJ, LI X, WANG Q, et al. Promoting effect of soaking with different exogenous substances on seed germination of hemp ‘Bamahuoma’ under drought stress［J］. Journal of Yunnan University: Natural Sciences Edition, 2018, 40(5): 1034-1041. (in Chinese)
［10］ LI GJ, WANG Q, LI X, et al. Effects of gibberellin and Vc soaking on seedling physiology during initial germination of hemp seeds under drought stress［J］. Seed, 2018, 37(6): 67-71. (in Chinese)
［11］ LYU YM. Effects of different cultivation conditions on hemp traits［J］. Chinas Fiber Crops, 1985(3): 42-45, 18. (in Chinese)