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Abstract [Objectives] This study aimed to determine the optimal medium components and culture conditions for the induction and proliferation of Dioscorea nipponica Makino axillary buds in vitro. [Methods] The effects of basic medium （MS， WPM， B5 and N6）， cytokinins type and concentration （0， 1.0， 1.5， 2.0， 2.5 and 3.0 mg/L 6??BA； 0， 1.0， 1.5， 2.0， 2.5 and 3.0 mg/L KT）， auxins type and concentration （0， 0.25， 0.50， 0.75， 1.00 mg/L IBA， 2，4??D and NAA） on the induction of D. nipponica axillary buds were respectively measured and compared. Then， the effects of sugar source （30 g/L sucrose， fructose， white sugar， maltose and glucose） and light intensity （0， 800， 1 600， 2 400 and 3 200 lx） on the proliferation coefficient of D. nipponica axillary buds were evaluated. [Results] Using stem segments with leaf axils as the explants， the highest induction rate （90.8%） of axillary buds was achieved in MS supplemented with 2.5 mg/L 6??BA and 0.5 mg/L 2，4??D， and the fresh and dry weights of tissue culture seedlings in this medium were also the highest， up to 1.5 g and 160.9 mg， respectively. Then， the highest proliferation coefficient of was D. nipponica axillary buds noted when sucrose was used as the sugar source in medium， and the optimal light intensity for the proliferation of D. nipponica axillary buds was 2 400 lx. [Conclusions] The results provide an experimental evidence for rapid propagation of D. nipponica.
Key words Dioscorea nipponica； Axillary bud； Induction； Proliferation
Dioscorea nipponica Makino， commonly known as yam， is a dioecious， twining， herbaceous perennial vine in the family Dioscoreaceae[1-2]. It is widely distributed in most parts of China， among which， Changbai Mountain is one of the major D. nipponica??growing areas[3-4]. D. nipponica is a medicinal plant of high commercial value. Its root is rich in diosgenin， which is an important raw material for the synthesis of various steroid hormones[5-6]. Diosgenin also has anti??tumor， anti??aging， anti??platelet coagulation and hypolipidemic effects[7-8]. D. nipponica is considered as one of the best choices for the production of diosgenin by considering the reserve of Dioscoreaceae resources， diosgenin content of each species and processing techniques[9-10].
D. nipponica is mainly propagated by rhizome division and seed at present[11-12]. Although propagation by rhizome is the simpler way， there are still some disadvantages， such as high requirements for rhizomes and long propagation cycle[13-14]. It is more difficult to propagate from seed due to the low germination percentage and growth rate， etc.[15-16]. Tissue culture is the most effective way for rapid propagation and improvement of yam species[17-18]. Viana et al.[19] found that somatic tissues do not respond readily to tissue culture manipulations designed to achieve regeneration of plantlets from single cells. It takes a long time to induce calluses and then adventitious buds from young plant organs， and there is great variation between the plantlets differentiated from calluses[20-21]. In contrast， the plantlets regenerated from buds are usually genetically stable and identical because， therefore buds are the best choice as explants in tissue culture for multiplication of yams[22].
This study was conducted to determine the optimal medium components and tissue culture conditions for the induction and proliferation of D. nipponica axillary buds， and to provide an experimental evidence for rapid propagation and conservation of D. nipponica.
Materials and Methods
Materials
Wild D. nipponica plants were collected from Dongshengyong Town， Longjing City， and the young stems with leaf axils but no axillary buds were used as initial explants， which were rinsed with water repeatedly at first， and then washed with 75% ethanol for 30 s， sterile water 5 times， 0.2% HgCl2 for 2 min， and sterile water 5 times again under aseptic condition.
Optimization of the factors influencing the induction of D. nipponica axillary buds
Basic medium Under aseptic condition the stem segments were inoculated to MS， WPM， B5 or N6 medium （pH 5.8）， supplemented with 2.0 mg/L 6??BA ， 0.75 mg/L 2，4??D， 30 g/L sucrose and 5 g/L agar， cultured at temperature （25??2）??， relative humidity 70%， light intensity 1 600 lx and a photoperiod of 16??h light. There were three replicates for each treatment. The induction rate， height， fresh weight and dry weight of the tissue culture seedlings were measured 30 d later.
Induction rate = Number of axillary buds/Total number of surviving explants ?? 100%
Cytokinin type and concentration The explants were inoculated into the medium MS supplemented with 0.75 mg/L 2， 4??D， and 0， 1.0， 1.5， 2.0， 2.5， 3.0 mg/L 6??benzylaminopurine （6??BA） or kinetin （KT）， or 0， 0.05， 0.10， 0.15， 0.20， 0.25 mg/L thiadiazuron （TDZ）， respectively. Then， they were cultured under the same conditions described above.
Auxin type and concentration The explants were inoculated into the medium MS +2.5 mg/L6??BA + 30g/L sucrose + 5 g/agar， with pH 5.8， supplemented with 0. 0.25， 0.50， 0.75， 1.00 mg/L IBA， 2，4??D or NAA. Then， they were cultured under the same conditions described above. Optimization of the factors influencing the proliferation of D. nipponica axillary buds
Sugar source The stem segments with axillary buds were inoculated into medium MS + 3.0 mg/L 6??BA + 0.75 mg/L NAA+ 5 g/L agar， with pH 5.8， supplemented with 30 g/L sucrose， fructose， white sugar， maltose or glucose. Then， they were cultured under the same conditions described above for 40 d， before the proliferation coefficient， height， fresh weight and dry weight of regenerated plantlets were measured.
Proliferation coefficient = Number of proliferated buds/Number of inoculated buds
Light intensity The stem segments with axillary buds were inoculated into medium MS + 3.0 mg/L6??BA + 0.75 mg/L NAA +30 g/L sucrose + 5 g/L agar， with pH 5.8. Then， they were cultured under the same conditions described above except that the light intensity was 0， 800， 1 600， 2 400 or 3 200 lx.
Results and Analysis
Factors influencing the induction of D. nipponica axillary buds
Effect of basic medium type on the induction of D. nipponica axillary buds Medium type had a certain impact on the induction of D. nipponica axillary buds. As shown in Table 1， the highest induction rate （78.7%） of D. nipponica axillary buds appeared on MS medium， which was significantly higher than that on other media. The largest number of axillary buds was observed on N6 medium， but the plantlets were shorter. Fewer axillary buds were induced and the regenerated tissue culture seedlings grew slowly on WPM and B5 medium （Fig. 1）. In summary， MS was the optimal basic medium for the induction of D. nipponica axillary buds.
Effects of cytokinin type and concentration on the induction of D. nipponica axillary buds The cytokinin type and concentration also had a certain impact on the induction of D. nipponica axillary buds. As shown in Table 2， 6??BA showed much better effect than other cytokinins in inducing axillary buds of D. nipponica. Among all concentrations of 6??AB， the induction rate at 2.5 mg/L was the highest， up to 84.4%. The highest induction rate among KT concentrations （42.8%） was observed at 2.0 mg/L， and that among TDZ concentrations （78.0%） was observed at 0.2 mg/L. The degree of browning of explants increased with increasing KT concentration， and some of the explants even died. The number of deformed plants increased with increasing TDZ concentration. In contrast， the highest induction rate， plantlet fresh weight and dry weight were obtained at 2.5 mg/L 6??BA. Effects of auxin type and concentration on the induction of D. nipponica axillary buds The type and concentration of auxins also had a certain impact on the induction of axillary buds of D. nipponica. As shown in Table 3， all the three auxins promoted axillary bud induction at low concentrations， and inhibited the induction at high concentrations. The highest induction rate among all 2，4??D treatments was observed at 0.5 mg/L， and that among all IBA treatments was also at 0.5 mg/L， while the highest induction rate among all NAA treatments appeared at 0.75 mg/L. Among all auxin treatments， the highest induction rate 90.8% was obtained when 0.5 mg/L 2，4??D was supplemented to medium. The results revealed that 2，4??D was the optimal auxin， and 0.5 mg/L was its optimal concentration for the induction of axillary buds of D. nipponica.
Factors influencing the proliferation of D. nipponica axillary buds
Effects of sugar source on the proliferation of D. nipponica axillary buds On the medium using glucose or maltose as the sugar source， fewer plantlets appeared， and they were yellow??green. The highest proliferation coefficient （8.0）， fresh weight （1.3 g）， and dry weight （143.0 mg） of tissue culture seedlings were all achieved in the medium using sucrose as the sugar source， all of which were significantly higher than those obtained using other sugar sources （Table 4）.
Effect of light intensity on the proliferation of D. nipponica axillary buds Light intensity also influenced the proliferation of axillary buds. At light intensity of 0 or 800 lx， fewer tissue culture seedlings grew， and they were yellow??green. At light intensity of 3 200 lx， the tissue culture seedlings grew to an average height of 5.1 cm， but the number of proliferated axillary buds reduced. At a light intensity of 2 400 lx， the fresh weight of the plantlets was 1.3 g， the dry weight was 143.2 mg， and the proliferation coefficient was 8.4， which were significantly higher than those of other treatments （Table 5）.
Discussion and Conclusions
MS， B5， N6 and WPM are commonly used basic media in plant tissue culture. The requirement for medium nutrients and components is different among plant species[23]. Our results showed that MS is the optimal basic medium for the induction of D. nipponica axillary buds. Plant growth regulators have the functions of regulating and controlling the growth and differentiation of cells and organogenesis， etc. in plants. Thidiazuron （TDZ）， a synthetic phenylurea derivative， can promote the regeneration of plants difficult to regenerate， but also can lead to the malformation of adventitious buds[24-25]. By studying the effects of different concentrations of cytokinins 6??BA， KT， TDZ and different concentrations of auxins IBA， NAA， 2，4??D on the induction of D. nipponica axillary buds， we found that at a high concentration of TDZ the plantlets are likely to expand and deform， and a high concentration of KT may lead to the browning tissue culture seedlings， which greatly reduces their survival rate. The combined use of 6??BA and 2，4??D is conducive to the induction of axillary buds of D. nipponica. Sugar not only provides energy to tissue culture seedlings， but also is the main osmotic regulator in medium[6]. The type and concentration of sugar in medium affect not only the growth rate， but also the metabolic rate， secondary metabolite synthesis， cellogenesis and cell morphology of tissue culture seedlings， so it is one of the key factors determining the success of plant tissue culture[27]. Our results showed that sucrose is the optimal sugar source for the proliferation of axillary buds of D. nipponica. Light is an indispensable condition for plant growth， and light intensity has significant effects on growth rate， the number of leaves and height yam plants[28]. In summary， stem segments with leaf axils can be used as the explants for the rapid propagation of D. nipponica in vitro. MS supplemented with 2.5 mg/L 6??BA and 0.5 mg/L 2，4??D is the optimal medium for the induction of D. nipponica axillary buds， and the induction rate was increased to 90.8%， and the fresh and dry weights of tissue culture seedlings in this medium were increased to 1.5 g and 160.9 mg， respectively. Sucrose is the optimal sugar source in medium for the proliferation of D. nipponica axillary buds， and the optimal light intensity is 2 400 lx.
References
[1] Editorial Committee of the Flora of China. Flora of China， Volume 16[M]. Beijing： Science Press， 1985.
[2] MENG XC， ZOU YF， LI QF， et al. Development and resources of Dioscorea nipponica[J]. Modern Chinese Medicine， 2011， 13（12）： 15-17.
[3] XU WY. Pharmaceutical botany[M]. Beijing： China Medical Science Press， 2005.
[4] LI JL， ZHANG CX， LI PD， et al. Studies on diversity of diosgenin in Dioscorea nipponica mak. from different places and growth years[J]. Chinese Pharmaceutical Journal， 2010， 45（4）： 245-247.
[5] XU L， AN W. Study on the medicine??property of Rhizoma dioscoreae Nipp and prospect in Xinzhou[J]. Science & Technology for China Rural Prosperity， 2011（1）： 23-25.
[6] ZHANG N， KANG TG， YIN HB. Chemical composition and pharmacological effects of traditional Chinese medicine Dioscorea nipponica[J]. Research and Practice on Chinese Medicines， 2010， 24（6）： 87-90.
[7] MA HY， ZHAO ZT. Comparative study on anti??hypercholesterolemia activity of diosgenin and total saponin of Dioscorea panthaica[J]. China Journal of Chinese Materia Medica， 2002， 27： 528-530.
[8] NING KY， LI YK. Effects of methyl protodioscin on in??vivo and in??vitro thrombosis and blood viscosity in rats[J]. Traditional Chinese Drug Research and Clinical Pharmacology， 2008， 19（1）： 3-5.
[9] CHEN S. Study on chemical constituents and pharmacological activities of the stems and leaves of Dioscorea nipponica Makino[D]. Changchun： Jilin University， 2008.
[10] ZHANG R. Rapid propagation of Dioscorea zingiberensis and Dioscorea composita[D]. Xi??an： Shaanxi Normal University， 2010.
[11] ZHANG SX， ZHOU LY， YU YJ. Advances in research of Dioscorea nipponica[J]. Molecular Plant Breeding， 2005， 3（1）： 107-111.
[12] SONG B， HAN M， LIN CX， et al. Study on the optimum condition of new and old seed germination of Dioscorea nipponica Makino[J]. Seed， 2012， 31（3）： 46-49. [13] LIU WH， WANG X， LI JG. Propagation and processing of Dioscorea nipponica[J]. Special Economic Animal and Plant， 2009， 12（4）： 41.
[14] WANG ZK， WANG YX， FU H， et al. Decision on rhizome age class of Dioscorea nipponica and its propagation[J]. Gansu Agricultural Science and Technology， 2011， （11）： 7-9.
[15] TSCHANNEN A B， ESCHER F， STAMP P. Post??harvest treatment of seed tubers with gibberellic acid and field performance of yam （Dioscorea cayenensis??rotundata） in Ivory Coast[J]. Expl Agric， 2005， 41： 175-186.
[16] ZHENG Y. Cultivation of Dioscorea nipponica in East Liaoning[J]. Practical Forestry Technology， 2017， （7）： 75-76.
[17] XU XL. Advances of on plant tissue culture of yams[J]. Hunan Forestry Science & Technology， 2000， 1（27）： 5-8.
[18] LUO FX， ZHU PF， ZHOU GG， et al. Tissue culture and rapid propagation of Dioscorea nipponica[J]. Plant Physiology Communications， 2004（3）： 329.
[19] VIANA AM， MANTELL SH. Callus induction and plant regeneration from excised zygotic embryos of the seed??propagated yams Dioscorea composite Hemsl and D. cayenensis Lam[J]. Plant Cell Tiss Org Cult， 1989， 16： 113-122.
[20] LIN GM， MOU HF， LI B， et al. Studies on tissue culture of stem segment of Dicorea composita[J]. Guangxi Agricultural Sciences， 2006（6）： 710-712.
[21] ZHANG SX. Tissue culture of traditional Chinese herb Dioscorea nipponica Makino[D]. Qingdao： Shandong Agricultural University， 2006.
[22] WANG HQ. In vitro rapid propagation and forest farming of Dioscorea nipponica[D]. Changchun： Northeast Normal University， 2009.
[23] QIANG XL. Tissue culture of Dioscorea zingiberensis[D]. Xi??an： Shaaxi Normal University， 2004.
[24] HUETTEMAN C A， PREECE J E. Thidiazuron： a potent cytokinin for woody plant tissue culture[J]. Plant cell， Tissue and Organ Culture，1993， 33： 105-119.
[25] HUANG J， CHEN XR， WU WX， et al. Propagation and regeneration of Dioscorea composita using[J]. Journal of Northwest A&F University （Natural Science Edition）， 2008（6）： 126-130.
[26] LI S， WU RZ. Study on proliferation of virus??free strawberry seeding[J] Journal of Anhui Agricultural Sciences， 2015， 43（7）： 25-27， 46.
[27] YU FK. Rapid propagation of rose[D]. Yangling： Northwest A&F University， 2003.
[28] SUN J. Effect of light intensity on growth and physiological properties of Dioscorea zingiberensis[D]. Xi??an： Shaaxi Normal University， 2011.
Key words Dioscorea nipponica； Axillary bud； Induction； Proliferation
Dioscorea nipponica Makino， commonly known as yam， is a dioecious， twining， herbaceous perennial vine in the family Dioscoreaceae[1-2]. It is widely distributed in most parts of China， among which， Changbai Mountain is one of the major D. nipponica??growing areas[3-4]. D. nipponica is a medicinal plant of high commercial value. Its root is rich in diosgenin， which is an important raw material for the synthesis of various steroid hormones[5-6]. Diosgenin also has anti??tumor， anti??aging， anti??platelet coagulation and hypolipidemic effects[7-8]. D. nipponica is considered as one of the best choices for the production of diosgenin by considering the reserve of Dioscoreaceae resources， diosgenin content of each species and processing techniques[9-10].
D. nipponica is mainly propagated by rhizome division and seed at present[11-12]. Although propagation by rhizome is the simpler way， there are still some disadvantages， such as high requirements for rhizomes and long propagation cycle[13-14]. It is more difficult to propagate from seed due to the low germination percentage and growth rate， etc.[15-16]. Tissue culture is the most effective way for rapid propagation and improvement of yam species[17-18]. Viana et al.[19] found that somatic tissues do not respond readily to tissue culture manipulations designed to achieve regeneration of plantlets from single cells. It takes a long time to induce calluses and then adventitious buds from young plant organs， and there is great variation between the plantlets differentiated from calluses[20-21]. In contrast， the plantlets regenerated from buds are usually genetically stable and identical because， therefore buds are the best choice as explants in tissue culture for multiplication of yams[22].
This study was conducted to determine the optimal medium components and tissue culture conditions for the induction and proliferation of D. nipponica axillary buds， and to provide an experimental evidence for rapid propagation and conservation of D. nipponica.
Materials and Methods
Materials
Wild D. nipponica plants were collected from Dongshengyong Town， Longjing City， and the young stems with leaf axils but no axillary buds were used as initial explants， which were rinsed with water repeatedly at first， and then washed with 75% ethanol for 30 s， sterile water 5 times， 0.2% HgCl2 for 2 min， and sterile water 5 times again under aseptic condition.
Optimization of the factors influencing the induction of D. nipponica axillary buds
Basic medium Under aseptic condition the stem segments were inoculated to MS， WPM， B5 or N6 medium （pH 5.8）， supplemented with 2.0 mg/L 6??BA ， 0.75 mg/L 2，4??D， 30 g/L sucrose and 5 g/L agar， cultured at temperature （25??2）??， relative humidity 70%， light intensity 1 600 lx and a photoperiod of 16??h light. There were three replicates for each treatment. The induction rate， height， fresh weight and dry weight of the tissue culture seedlings were measured 30 d later.
Induction rate = Number of axillary buds/Total number of surviving explants ?? 100%
Cytokinin type and concentration The explants were inoculated into the medium MS supplemented with 0.75 mg/L 2， 4??D， and 0， 1.0， 1.5， 2.0， 2.5， 3.0 mg/L 6??benzylaminopurine （6??BA） or kinetin （KT）， or 0， 0.05， 0.10， 0.15， 0.20， 0.25 mg/L thiadiazuron （TDZ）， respectively. Then， they were cultured under the same conditions described above.
Auxin type and concentration The explants were inoculated into the medium MS +2.5 mg/L6??BA + 30g/L sucrose + 5 g/agar， with pH 5.8， supplemented with 0. 0.25， 0.50， 0.75， 1.00 mg/L IBA， 2，4??D or NAA. Then， they were cultured under the same conditions described above. Optimization of the factors influencing the proliferation of D. nipponica axillary buds
Sugar source The stem segments with axillary buds were inoculated into medium MS + 3.0 mg/L 6??BA + 0.75 mg/L NAA+ 5 g/L agar， with pH 5.8， supplemented with 30 g/L sucrose， fructose， white sugar， maltose or glucose. Then， they were cultured under the same conditions described above for 40 d， before the proliferation coefficient， height， fresh weight and dry weight of regenerated plantlets were measured.
Proliferation coefficient = Number of proliferated buds/Number of inoculated buds
Light intensity The stem segments with axillary buds were inoculated into medium MS + 3.0 mg/L6??BA + 0.75 mg/L NAA +30 g/L sucrose + 5 g/L agar， with pH 5.8. Then， they were cultured under the same conditions described above except that the light intensity was 0， 800， 1 600， 2 400 or 3 200 lx.
Results and Analysis
Factors influencing the induction of D. nipponica axillary buds
Effect of basic medium type on the induction of D. nipponica axillary buds Medium type had a certain impact on the induction of D. nipponica axillary buds. As shown in Table 1， the highest induction rate （78.7%） of D. nipponica axillary buds appeared on MS medium， which was significantly higher than that on other media. The largest number of axillary buds was observed on N6 medium， but the plantlets were shorter. Fewer axillary buds were induced and the regenerated tissue culture seedlings grew slowly on WPM and B5 medium （Fig. 1）. In summary， MS was the optimal basic medium for the induction of D. nipponica axillary buds.
Effects of cytokinin type and concentration on the induction of D. nipponica axillary buds The cytokinin type and concentration also had a certain impact on the induction of D. nipponica axillary buds. As shown in Table 2， 6??BA showed much better effect than other cytokinins in inducing axillary buds of D. nipponica. Among all concentrations of 6??AB， the induction rate at 2.5 mg/L was the highest， up to 84.4%. The highest induction rate among KT concentrations （42.8%） was observed at 2.0 mg/L， and that among TDZ concentrations （78.0%） was observed at 0.2 mg/L. The degree of browning of explants increased with increasing KT concentration， and some of the explants even died. The number of deformed plants increased with increasing TDZ concentration. In contrast， the highest induction rate， plantlet fresh weight and dry weight were obtained at 2.5 mg/L 6??BA. Effects of auxin type and concentration on the induction of D. nipponica axillary buds The type and concentration of auxins also had a certain impact on the induction of axillary buds of D. nipponica. As shown in Table 3， all the three auxins promoted axillary bud induction at low concentrations， and inhibited the induction at high concentrations. The highest induction rate among all 2，4??D treatments was observed at 0.5 mg/L， and that among all IBA treatments was also at 0.5 mg/L， while the highest induction rate among all NAA treatments appeared at 0.75 mg/L. Among all auxin treatments， the highest induction rate 90.8% was obtained when 0.5 mg/L 2，4??D was supplemented to medium. The results revealed that 2，4??D was the optimal auxin， and 0.5 mg/L was its optimal concentration for the induction of axillary buds of D. nipponica.
Factors influencing the proliferation of D. nipponica axillary buds
Effects of sugar source on the proliferation of D. nipponica axillary buds On the medium using glucose or maltose as the sugar source， fewer plantlets appeared， and they were yellow??green. The highest proliferation coefficient （8.0）， fresh weight （1.3 g）， and dry weight （143.0 mg） of tissue culture seedlings were all achieved in the medium using sucrose as the sugar source， all of which were significantly higher than those obtained using other sugar sources （Table 4）.
Effect of light intensity on the proliferation of D. nipponica axillary buds Light intensity also influenced the proliferation of axillary buds. At light intensity of 0 or 800 lx， fewer tissue culture seedlings grew， and they were yellow??green. At light intensity of 3 200 lx， the tissue culture seedlings grew to an average height of 5.1 cm， but the number of proliferated axillary buds reduced. At a light intensity of 2 400 lx， the fresh weight of the plantlets was 1.3 g， the dry weight was 143.2 mg， and the proliferation coefficient was 8.4， which were significantly higher than those of other treatments （Table 5）.
Discussion and Conclusions
MS， B5， N6 and WPM are commonly used basic media in plant tissue culture. The requirement for medium nutrients and components is different among plant species[23]. Our results showed that MS is the optimal basic medium for the induction of D. nipponica axillary buds. Plant growth regulators have the functions of regulating and controlling the growth and differentiation of cells and organogenesis， etc. in plants. Thidiazuron （TDZ）， a synthetic phenylurea derivative， can promote the regeneration of plants difficult to regenerate， but also can lead to the malformation of adventitious buds[24-25]. By studying the effects of different concentrations of cytokinins 6??BA， KT， TDZ and different concentrations of auxins IBA， NAA， 2，4??D on the induction of D. nipponica axillary buds， we found that at a high concentration of TDZ the plantlets are likely to expand and deform， and a high concentration of KT may lead to the browning tissue culture seedlings， which greatly reduces their survival rate. The combined use of 6??BA and 2，4??D is conducive to the induction of axillary buds of D. nipponica. Sugar not only provides energy to tissue culture seedlings， but also is the main osmotic regulator in medium[6]. The type and concentration of sugar in medium affect not only the growth rate， but also the metabolic rate， secondary metabolite synthesis， cellogenesis and cell morphology of tissue culture seedlings， so it is one of the key factors determining the success of plant tissue culture[27]. Our results showed that sucrose is the optimal sugar source for the proliferation of axillary buds of D. nipponica. Light is an indispensable condition for plant growth， and light intensity has significant effects on growth rate， the number of leaves and height yam plants[28]. In summary， stem segments with leaf axils can be used as the explants for the rapid propagation of D. nipponica in vitro. MS supplemented with 2.5 mg/L 6??BA and 0.5 mg/L 2，4??D is the optimal medium for the induction of D. nipponica axillary buds， and the induction rate was increased to 90.8%， and the fresh and dry weights of tissue culture seedlings in this medium were increased to 1.5 g and 160.9 mg， respectively. Sucrose is the optimal sugar source in medium for the proliferation of D. nipponica axillary buds， and the optimal light intensity is 2 400 lx.
References
[1] Editorial Committee of the Flora of China. Flora of China， Volume 16[M]. Beijing： Science Press， 1985.
[2] MENG XC， ZOU YF， LI QF， et al. Development and resources of Dioscorea nipponica[J]. Modern Chinese Medicine， 2011， 13（12）： 15-17.
[3] XU WY. Pharmaceutical botany[M]. Beijing： China Medical Science Press， 2005.
[4] LI JL， ZHANG CX， LI PD， et al. Studies on diversity of diosgenin in Dioscorea nipponica mak. from different places and growth years[J]. Chinese Pharmaceutical Journal， 2010， 45（4）： 245-247.
[5] XU L， AN W. Study on the medicine??property of Rhizoma dioscoreae Nipp and prospect in Xinzhou[J]. Science & Technology for China Rural Prosperity， 2011（1）： 23-25.
[6] ZHANG N， KANG TG， YIN HB. Chemical composition and pharmacological effects of traditional Chinese medicine Dioscorea nipponica[J]. Research and Practice on Chinese Medicines， 2010， 24（6）： 87-90.
[7] MA HY， ZHAO ZT. Comparative study on anti??hypercholesterolemia activity of diosgenin and total saponin of Dioscorea panthaica[J]. China Journal of Chinese Materia Medica， 2002， 27： 528-530.
[8] NING KY， LI YK. Effects of methyl protodioscin on in??vivo and in??vitro thrombosis and blood viscosity in rats[J]. Traditional Chinese Drug Research and Clinical Pharmacology， 2008， 19（1）： 3-5.
[9] CHEN S. Study on chemical constituents and pharmacological activities of the stems and leaves of Dioscorea nipponica Makino[D]. Changchun： Jilin University， 2008.
[10] ZHANG R. Rapid propagation of Dioscorea zingiberensis and Dioscorea composita[D]. Xi??an： Shaanxi Normal University， 2010.
[11] ZHANG SX， ZHOU LY， YU YJ. Advances in research of Dioscorea nipponica[J]. Molecular Plant Breeding， 2005， 3（1）： 107-111.
[12] SONG B， HAN M， LIN CX， et al. Study on the optimum condition of new and old seed germination of Dioscorea nipponica Makino[J]. Seed， 2012， 31（3）： 46-49. [13] LIU WH， WANG X， LI JG. Propagation and processing of Dioscorea nipponica[J]. Special Economic Animal and Plant， 2009， 12（4）： 41.
[14] WANG ZK， WANG YX， FU H， et al. Decision on rhizome age class of Dioscorea nipponica and its propagation[J]. Gansu Agricultural Science and Technology， 2011， （11）： 7-9.
[15] TSCHANNEN A B， ESCHER F， STAMP P. Post??harvest treatment of seed tubers with gibberellic acid and field performance of yam （Dioscorea cayenensis??rotundata） in Ivory Coast[J]. Expl Agric， 2005， 41： 175-186.
[16] ZHENG Y. Cultivation of Dioscorea nipponica in East Liaoning[J]. Practical Forestry Technology， 2017， （7）： 75-76.
[17] XU XL. Advances of on plant tissue culture of yams[J]. Hunan Forestry Science & Technology， 2000， 1（27）： 5-8.
[18] LUO FX， ZHU PF， ZHOU GG， et al. Tissue culture and rapid propagation of Dioscorea nipponica[J]. Plant Physiology Communications， 2004（3）： 329.
[19] VIANA AM， MANTELL SH. Callus induction and plant regeneration from excised zygotic embryos of the seed??propagated yams Dioscorea composite Hemsl and D. cayenensis Lam[J]. Plant Cell Tiss Org Cult， 1989， 16： 113-122.
[20] LIN GM， MOU HF， LI B， et al. Studies on tissue culture of stem segment of Dicorea composita[J]. Guangxi Agricultural Sciences， 2006（6）： 710-712.
[21] ZHANG SX. Tissue culture of traditional Chinese herb Dioscorea nipponica Makino[D]. Qingdao： Shandong Agricultural University， 2006.
[22] WANG HQ. In vitro rapid propagation and forest farming of Dioscorea nipponica[D]. Changchun： Northeast Normal University， 2009.
[23] QIANG XL. Tissue culture of Dioscorea zingiberensis[D]. Xi??an： Shaaxi Normal University， 2004.
[24] HUETTEMAN C A， PREECE J E. Thidiazuron： a potent cytokinin for woody plant tissue culture[J]. Plant cell， Tissue and Organ Culture，1993， 33： 105-119.
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