Breeding and Evaluation of New lron Yam（Dioscorea opposita Thunb）Varieties

Sufang DUAN，Jianjun Ll，Meiling REN，Jun WANG，Guolun JlA，Shuwu SUN

1.People's Park of Xinxiang，Xinxiang 453007，China；

2.Engineering Technology Research Center of Nursing and Utilization of Genuine Chinese Crude Drugs，College of Life Science，Henan Normal University，Xinxiang 453007，China；

3.Henan Bai Liao Huai Medicine Science and Technology Development Co.，Ltd.，Jiaozuo 454950，China

Breeding and Evaluation of New lron Yam（Dioscorea opposita Thunb）Varieties

Sufang DUAN1，Jianjun Ll2*，Meiling REN2，Jun WANG2，Guolun JlA2，Shuwu SUN3

1.People's Park of Xinxiang，Xinxiang 453007，China；

2.Engineering Technology Research Center of Nursing and Utilization of Genuine Chinese Crude Drugs，College of Life Science，Henan Normal University，Xinxiang 453007，China；

3.Henan Bai Liao Huai Medicine Science and Technology Development Co.，Ltd.，Jiaozuo 454950，China

［Objective］This study was conducted to develop some new iron yam（Dioscorea opposita）varieties with excellent cooking，eating and nutritional qualities.［Method］The fresh weight per plant，drying rate，dry weight per plant，yield，allan-toin content，water soluble extract content and disease resistance of seven new iron yam cultivars （No.1，No.2，No.4，No.6，No.8，No.9 and No.10）obtained through spaceflight mutation breeding were measured and compared with control（No.11）. Then，the nutrition quality of two elite cultivars No.6 and No.10 was determined by comparing with the control.［Result］The eight iron yam cultivars ranked in descend-ing order of their fresh weight per plant as follows：No.6（0.255 kg）＞No.10（0.254 kg）＞No.4（0.242 kg）＞No.9（0.237 kg）＞No.1（0.233 kg）＞No.11（0.206 kg）＞No.2（0.191 kg）＞No.8（0.157 kg）.There was no significant difference in fresh weight per plant between No.6 and No.10，but extremely significant difference between No.6 and the control，No.10 and the control.The eight iron yam cultivars ranked in descending order of their drying rate as follows：No.2（32.641%）＞No.10（32.230%）＞No.9（28.223%）＞No.6（25.174%）＞No.8（25.122%）＞No.11（25.043%）＞No.1（24.291%）＞No.4（20.234%）.The dry-ing rate of both No.6 and No.10 was higher than that of the control.The eight iron yam cultivars ranked in descending order of their dry weight per plant as follows：No.10（0.082 kg）＞No.9（0.067 kg）＞No.6（0.064 kg）＞No.2（0.062 kg）＞No.1（0.056 kg）＞No.11（0.052 kg）＞No.4（0.049 kg）＞No.8（0.039 kg）.The dry weight per plant of both No.6 and No.10 is higher than that of the control.The eight iron yam cultivars ranked in descending order of their allantoin content as follows No.6（0.484%）＞No.4（0.467%）＞No.10（0.399%）＞No.11（0.386%）＞No.9（0.378%）＞No.8（0.350%）＞No.2（0.342）＞No.1（0.325%）.The allantoin content of No.6 was significantly higher than that of No.10 and the control.There was significant difference in allantoin content between No.6 and No.10，but no significant difference between No 10 and the control.The eight iron yam cultivars ranked in descending order of their extract content as follows No. 6（20.49%）＞No.2（16.01%）＞No.4（15.54%）＞No.10（15.35%）＞No.1（14.48%）＞No.11（14.10%）＞No.9（13.88%）＞No.8（11.87%）.The extract content of No.6 exhibited extremely sig-nificant difference with that of No.10 and the control，and the extract content of No. 10 was also significantly different from that of the control.The taste of No.10 was dry，soft，floury，sweet and fragrant，better than that of the other seven cultivars. No.6 had the strongestresistance to Gloeosporium pestis and Cylindrosporium dioscoreae.No.10 had a middle resistance to G.pestis and a strong resistance to C.dioscoreae.No.6 and No.10 had higher contents of starch，reducing sugar，pro-tein and ash，lower water content and better nutrition quality than the control.［Conclusion］No.10 can be popularized as a new variety of edible iron yam，and No.6 as a new variety of medicinal iron yam.

Iron yam；New cultivar；Breeding；Production；Medicinal ingredients；Nu-trition quality

Y am is the dried rhizome of Dioscorea opposita Thunb，which belongs to the family Dioscoreaceae.It has the efficacy of nourishing spleen and stomach and in-vigorating lung and kidney［1］.Yam with strong adaptability is widely planted in China，mostly produced in Henan［2］. The yam produced in Jiaozuo（the ancient Huaiqing Prefecture）has the best quality，which is called Huai Shanyao in Chinese.At present，iron yam and Taigu yam are two main va-rieties planted in this region.Iron yam has high medicinal value but low yield，while Taigu yam has high yield but low medicinal value［3］.

The long-term asexual reproduc-tion in yam-producing area has result-ed in degradation of yam variety，seri-ous plant diseases and insect pests，but also has reduced yam yield and quality.The original varieties can not meet the increasing demands of Chi-nese medicinal materials enterprises and farmers any longer，so it is urgent to develop some new varieties with high yield，good quality and strong re-sistance.Because of the difficulties in hybridization breeding of iron yam，re-searchers collected iron yam bulbils and sent them into space with"Prac-tice No.8"satellite in 2006.After spaceflight，the iron yam bulbils were planted in Wuzhi.Seven new cultivars with good quality were generated after six years'breeding and propagation. Theiryield，quality and resistance were measured and compared with control cultivar in the present study to screen new varieties with the best properties，and thus to provide theo-retical and technological basis for the identification and promoting of new yam varieties.

Materials and Methods

Materials and instruments

Plant materials Seven new iron yam cultivars（No.1，No.2，No.4，No.6，No.8，No.9andNo.10）developed through spaceflight mutation，and the control（No.11）which is a main iron yam variety planted Jiaozuo，were all grown in the same experimental filed on April 4，2013 through conventional cultivation techniques.Allofthem grew well and were then identified as Dioscorea opposita Thunb by associ-ate professor Faqi Li from Henan Nor-mal University.

The rhizomes of the eight culti-vars were harvested on November 25，2013.Mold-and mildew-free samples were selected，rinsed with running wa-ter，peeled and sliced into pieces about 3 mm，dried at 50℃ for 18 h and finally crushed for later analysis.

Instruments and reagents Agilent 1200 series HPLC System，equipped with GI311 quaternary pump，auto-matic sampler，VWD detector，Agi-lent1100 LC chemical workstation was purchased from Agilent Technologies Co.，Ltd.，KH-250DE CNC ultrasonic cleaner from Kunshan Hechuang Ul-trasonic Instrument Co.， Ltd.，FA2204B electronic analytical balance from Shanghai Jinghai Instrument Co.，Ltd.，DHY-300 pulverizer from Beijing Donghua Medical Equipment Co.，Ltd.，ZRD-A5110 electric oven thermostat blast from Shanghai Zhicheng Analyti-cal Instruments Co.，Ltd.，JP Rober-val's balance from Shanghai Lineng Electronic Instruments Co.，Ltd.The detailed description of instruments for nutrition quality analysis can be seen in reference［4］.

Allantoin standard （Batch No. 111501-200202）was purchased from Chinese Food and Drug Inspection Institute.Methanolwas chromato-graphic grade.Ultra-pure water was used for regent preparation in this study.The detailed description of reagents for nutrition quality analysis can be seen in reference［4］.

Methods

Measurement of yam yield Thirty plants of each cultivar were collected from three sites，10 plants from each site，and the fresh weight of each plant was measured with a balance.Then part of the yam samples was dried at 50℃to constant weight and weighed to calculate drying rate using the for-mula as follows：Drying rate=Weight of dried yam/Weight of fresh yam× 100%.Finally，yam yield of each culti-var was calculated according to plant-ing density.

Evaluationofyam taste Yam samples were rinsed with running wa-ter to remove soil attached to their sur-face，sliced into10-cm pieces，before being steamed in a steamer for 25 min.Finally，the steamed samples of the seven new yam cultivars and the control were tasted by eight experts and evaluated using dry，floury，crisp，numb，sweet，fragrant，soft，hard and etc.

Determination of medicinal ingredi-ents in yam

Determination of extract content

According to the method described in Pharmacopoeia of the People's Republic of China［1］，4 g of powder of each yam cultivar was weighed，sieved through 20 mesh，put into a 250 ml conical flask，added with 100 ml water.The flask was closed with a stopper，constantly shaken within the first 6 h，and then allowed to stand for another 18 h.After that，the solution was filtered，and 20 ml of the filtrate was transferred into a evaporating dish which had been dried to constant weight，evaporated to dryness on a water bath，before being dried at 105℃for 3 h.After cooling for 30 min in a dryer，the extract powder was weighed precisely.

Determination of allantoin content The chromatographic conditions were set as follows：chromatographic col-umn filled with octadecylsilane chemi-cally bonded silica，Agilent ODS（4.6 mm×250 mm，5 μm）；mobile phase：methanol-water （5∶95）；flow rate of 0.8 ml/min；detection wavelength of 224 nm；column temperature of 30℃；elution time of 7 min；and sample vol-ume of 10 μl.The HPLC was per-formed according to the methods de-scribed in Pharmacopoeia of the People's Republic of China［1］.

Allantoin standard solutions were prepared by following steps.Allantoin standard （18.5 mg）which had been dried to constant weight was accu-rately weighed，put into a 10 ml volu-metric flask，dissolved with ultra-pure water to the mark，mixed by shaking to obtain 1.85 mg/ml stock solution of al-lantoin standard.Then，0.5，1，1.5，2 and 2.5 ml of the stock solution was re- spectively transferred into five 25 ml flasks.To each of the flask ultra-pure water was added to marker，mixed by shaking to obtain allantoin standard solutions of 0.037，0.074，0.111，0.148 and 0.185 mg/ml.

Iron yam sample solutions were prepared byfollowing steps.Yam powder prepared above was sieved through 80 mesh，dried at 50℃for 24 h.Then，0.5 g of the sample of each yam cultivar was precisely weighed and dissolved in 25 ml of methanol，transferred into a flask，and treated by ultrasound twice（30 min every time，with an interval of 30 min）.The solu-tion was centrifuged at 10 000 r/min for 15 min.The supernatant was fil-tered through 0.45 μm organic micro-porous membrane and collected for later analysis［5-6］.

To plot the standard curve of al-lantoin，10 μl of each of the allantoin standard solutions at 0.037，0.074，0.111，0.148 and 0.185 mg/ml pre-pared above was loaded for HPLC. The standard curve of allantoin was obtained by plotting peak area（mAU）on Y-axis and the concentration of standard allantoin （μg）on X-axis.The regressionequationwas Y=3 585.7X-1.819 9，R2=0.999 8，which confirmed the good linear relationship between peak area and the concentration of al-lantoin standard ranging from 0.037 and 0.185 mg/ml.Allantoin content in yam extract was then calculated ac-cording to the standard curve.

Identification of yam resistance to GloeosporiumpestisandCylindrosporium dioscoreae The resis-tance of the seven new iron yam culti-vars to G.pestis and C.dioscoreae was surveyed in field in September when leaf diseases were prevalent. Ten plants of each variety were se-lected，and all of their leaves 1 m above the ground were observed to record the infection ofG.pestis and C.dioscoreae.The field exper-imentwasrepeatedtwice.Finally the disease index was calculated to identify their resistance to G.pestis and C.dioscoreae.

Analysis of yam nutrition quality Yam sampleswerepreparedand starch content was measured through acid hydrolysis according to GB/T 5009.9-2008 Determination of Starchin Foods.

Reducing sugar content in yam samples was measured with direct titration according to GB/T 5009.7-2008 Determination of Reducing Sugar in Foods.

Protein content in yam samples was measured with kjeldahl nitrogen method according to GB/5009.5-2010 Determination of Protein Content in Foods.

Ash content in yam samples was measured by combustion method according to GB 5009.4-2010 Determination of Ash Content in Foods.

Water content in yam samples was measured by drying according to GB 5009.3-2010 Determination of Water Content in Foods.

Table 1 Comparison of fresh weight per plant and taste between new iron yam cultivars and control

Results and Analysis

Comparison of yield and taste be-tween new iron yam cultivars and the control

SPSS software was employed for variance analysis on the fresh weight per plant of the seven new iron yam cultivars and the control.Then the dry weight per plant was calculated according to drying rate.The yam yield for each cultivar was calculated from planting density.As shown in Table 1，The eight iron yam cultivars ranked in descending order of their fresh weight per plant as follows：No.6（0.255 kg）＞No.10（0.254 kg）＞No.4（0.242 kg）＞No.9 （0.237 kg）＞No.1 （0.233 kg）＞No.11（0.206 kg）＞No.2（0.191 kg）＞No.8 （0.157 kg）.Among them，the fresh weight per plant of No.6（0.255 kg）and No.10（0.254 kg）were greater than that of other cultivars，while that of No.8 was the smallest.The fresh weight per plant had insignificant difference between No.6 and No.10，significant difference between No.6 and No.4，and extremely significant difference between No.6 and other cultivars.The results proved that No.6 and No.10 was much better than the control in fresh weight per plant.

The eightiron yam cultivars ranked in descending order of their dryingrateasfollows：No.2（32.641%）＞No.10（32.230%）＞No.9（28.223%）＞No.6（25.174%）＞No.8（25.122%）＞No.11 （control，25.043% ）＞No.1（24.291%）＞No.4（20.234%），in descending order of their dry weight per plant as：No.10 （0.082 kg）＞No.9（0.067 kg）＞No.6 （0.064 kg）＞No.2（0.062 kg）＞No.1 （0.056 kg）＞No.11（control，0.052 kg）＞No.4（0.049 kg）＞No.8 （0.039 kg）.The results suggested that the dry weight per plant of No. 10，No.9，No.6，No.2 and No.1 were greater than that of the control，and No.10，No.9 and No.6 were more competitive in dry weight per plant.

The results of taste evaluation revealed that No.10 which tasted dry，floury，sweet and fragrant had the best eating quality；both No.6，which tested dry，floury and hard，and No.9，which tasted dry and crisp were not outstanding in eating quality.

Comparison of medicinal ingredi-ents between new iron yam culti-vars and control

The extract content of the seven new iron yam cultivars and the control were measured，and the data were analyzed with SPSS.The HPLC fingerprints of allantoin standard and yam samples were shown in Fig.1 and Fig.2，the allantoin content for each cultivar was calculated based on the peak area in HPLC fingerprints，and the data were analyzed using SPSS（Table 2）.

As shown in Table 2，the extract content of the eight yam cultivars ranged from 11.87%to 20.49%，all exceeding the standard described in 2010 version of Pharmacopoeia of the People's Republic of China［1］.No.6 has the highest extract content，up to 20.49%，while No.8 has the lowest extract content，only 11.87%.Significance test revealed that there was extremely significant difference in extract content between No.6 and other cultivars.The extract content of No.10 was higher than No.1，No.11（control），No.9 and No.8，showing insignificant difference with that of No.1，but significant difference with that of No.11（control）and No.9，extremely significant difference with that of No.8.The extract content of No.9 was lower than of the control，and the difference between them was not significant.The results revealed that No.6 had extremely significant advantage，and No.10 had significant advantage in extract content over the control.

It also could be concluded from Table 2 that the eight iron yam cultivars ranked in descending order of their allantoin content as follows No.6（0.484%）＞No.4 （0.467%）＞No.10（0.399%）＞No.11 （0.386%）＞No.9（0.378%）＞No.8 （0.350%）＞No.2（0.342）＞No.1 （0.325%）.Among them，No.6 had the the highest allantoin content 0.484%，while No.1 had the lowest allantoin content 0.325%. There was no significant difference in allantoin content between No.6 and No.4，significant difference between No.6 and No.10，No.9，No.11（control），extremely significant difference between No.6 and the others.The allantoin content of No.10 was higher than that of No.11（control），No.1，No.2 andNo.8，but the difference was not signifi-cant.In summary，No.6 had significant advantage in allantoin content over control，but No.10 did not.

Disease resistance of the iron yam cultivars

As shown in Table 3，No.6 was highly resistant to G.pestis，while No.1，No.2，No.4，No.9 and No.10 had mid-dle resistance to G.pestis；the control No.11 was moderately susceptible to G.pestis and No.8 was highly suscep-tible to G.pestis.Both No.6 and No.10 were highly resistant to C.dioscoreae；No.1，No.2，No.4，No.9 and No.11（control）had middle resistance to C.dioscoreae and No.8 was highly susceptible to C.dioscoreae.It could be inferred that the new variety No.6 had high resistance to G.pestis and C.dioscoreae，indicating it was highly resistant to leaf diseases，while No.10 was moderately resistant to G.pestis and highly resistant to C.dioscoreae，suggesting it also had good resistance to leaf diseases.

Comparison of nutrition quality be-tween two new iron yam cultivars and the control It could be conclud- ed from Table 4 that the contents of nutritional ingredients in the three yam samples met the national standards of People's Republic of China for geographical indication product-Chinese yam （GB/T20351-2006）.In detail，the contents of starch，reducing sugar，protein and ash in No.6 and No.10 were higher than that of the control，while the water content of No.6 and No.10 was lower than that of control. The results proved that No.6 and No. 10 had better nutrition quality than the control.Analysis of variance revealed thatthe starch contentofNo.10 showed extremely significant differ-ence with that of No.6 and the control；the starch content of No.6 was higher than that of the control，and the differ-ence between them was extremely significant.The reducing sugar content of No.10 was higher than that of No.6 and the control，with insignificant dif-ference between No.10 and No.6，ex-tremely significant difference between No.10 and the control；the reducing sugar content of No.6 was higher than that of the control，with extremely sig-nificant difference between them.The protein content of No.6 was higher than that of No.10 and the control，with extremely significant difference be-tween No.6 and No.10，control；the protein content of No.10 was higher than that of the control，and the differ-ence between them was extremely significant.The ash content of No.10 was higher than that of No.6 and the control，with insignificantdifference between No.10 and No.6，significant difference between No.10 and the con-trol；the ash content of No.6 was high-er than that of the control，but the dif-ference was not significant.The water content of No.6 and No.10 was lower than that of the control，and the differ-ence between No.6，No.10 and control was significant；the water content of No.6 was higher than that of No.10，and the difference was extremely sig-nificant.In summary，both the two new cultivars No.6，especially No.10 had advantages in nutrition quality over control.

Table 2 Comparison of extract content and allantoin content between the seven new yam cultivars and the control %

Table 3 Resistance evaluation of the eight iron yam cultivars（n=20）

Conclusion and Discussion

As one of the medicinal and edi-ble herbs，yam is consumed much more as food than as medicine.Yield，taste，medicinal ingredients，disease resistance and nutrition quality are im-portant indices to evaluate yam vari-eties［7］.Extract content is one of the test items of yam by pharmacopoeia，which reflects the contents of medici-nal ingredients in general［8］.Allantoin，one of the main active ingredient of yam can soften cutin， promote wound healing，accelerate the growth of epithelial cells，and it also has some other physiological effects such as anesthesia，analgesia，bacterio-static，antiphlogistic and anti-corro-sion.So，it is often used as a basis for evaluating the quality of yam or yam preparations［9］.

Table 4 Comparison of nutrition quality between new cultivars No.6，No.10 and the control %

Through the comparative analysis on various indices between the seven new iron yam cultivars and the control，we found that the difference in fresh weight per plant was not significant between No.6 and No.10，but ex-tremely significant between them and control，indicating that the two new cultivars No.6 and No.10 had obvious advantage in fresh weight per plant. The dry weight per plant of No.6 was lower than that of No.10，but higher than that of the control，so No.10 has obvious advantage in dry weight per plant.The taste assessment result re-vealed that No.10 was the best in eat-ing quality，while No.6 was not out-standing.

The extract content of No.6 was the highest among the eight iron yam cultivars tested，significantly different from that of No.11（control），indicating that No.6 had obvious advantage in extract content.The extract content of No.10 was also higher than that of the control，and the difference between them was significant.The allantoin content of No.6 was the highest，sig-nificantly different from that of No.11（control）；while the extract content of No.10 was higher than that of the con-trol，but the difference between was not significant，suggesting No.6 had high contents of medicinal ingredients，compared with control and No.10.

The results of resistance identifi-cation revealed that No.6 was highly resistant to G.pestis and C.dioscoreae，indicating that it had high resis-tance to leaf diseases.No.10 was moderately resistant to G.pestis，but highly resistant to C.dioscoreae，indi-cating it also had good resistance to leaf diseases.

Analysis on nutrition quality of No. 6 and No.10 revealed that their con-tents of starch，reducing sugar，protein and ash were higher than that of the control，while their water content was less than that of the control，indicating that both No.10 and No.6 had better nutrition quality than the control.

To sum up，the mutation induced by spaceflight greatly improved the yield and quality of the new iron yam cultivars.No.10 which has high fresh weightperplant，drying rate，dry weight per plant，good taste，nutrition quality and disease resistance，can be popularized as a new variety of edible iron yam.Now，No.10 has been ap-proved as a new yam variety by Henan Province Professional Pharmacy Committee，and named Iron 06-1（Iron No.1）.No.6 which has high fresh weight per plant，dry weight per plant，allantoin content and extract content，good disease resistance and nutrition quality，can be promoted as a new va-riety of medicinal iron yam.Overall，the new iron yam varieties we developed have significance for the healthy and sustainable development of iron yam industry.

［1］Committee of Pharmacopeia in Ministry of Health of the People's Republic of China.Pharmacopeia of the People's Republic of China［M］.Beijing：China Medical Science Press，2010.

［2］Institute of Botany of Chinese Academy of Sciences.Higher plants of Chins（Volume five）［M］.Beijing：Science Press，1983.

［3］HAN SY，ZHANG XY，WANG SX，et al. The status of yam production in Henan Province［J］.Journal of Henan Agricul-tural Sciences，2011，（09）：109-111.

［4］FU XY.Comparison of nutrition quality andpharmacologicalcomponentsin different yam varieties ［D］.Wuhan：Wuhan University of Technology，2012.

［5］YI GQ，JIANG ML，LU YB，et al.Opti-mization of extraction method of allan-toininyam［J］.ModernChineseMedicine，2013，（09）：786-788.

［6］BAI Y，ZHU FY，WANG D，et al.Deter-mination of allantoin content in Rhizoma dioscoreae collected from different re-gions by HPlC［J］.Chinese Herbal Medi-cines，2003，34（2）：179-180.

［7］ZHANG WM，SHAN CY，HE HL，et al. Quality comparison between two culti-vated Chinese yam Kunming Y am and Tiegun Yam［J］.Journal of Chinese Me-dicinal Materials，2007，（05）：513-515.

［8］LI YT，WU XY，WANG X，et al.Study on methodology for determination of ex-tracts of the crude and processed rhi-zoma of Coptidis［J］.China Journal of Traditional Chinese Medicine and Ph-armacy，2011，（03）：538-541.

［9］GU WZ，QIN WZ.The effect and clinical application of allantoin［J］.Chinese Jour-nal of New Drugs and Clinical Reme-dies，1990，（04）：232-234.

Responsible editor：Qingqing YlN

Responsible proofreader：Xiaoyan WU

Supported by Special Fund for Public Health from State Administration of Traditional Chinese Medicine（Financial Institution［2011］76）；Special Fund for Scientific Research of Traditional Chinese Medicine Industry（201207002）；Henan Social Sciences Planning Project （2013BJJ084）；Henan Industry-University-Research Institute Cooperation Project（142107000078）.

*Corresponding author.E-mail：043081@htu.cn

Received：August 2，2015 Accepted：September 19，2015