| ‘Huai Yam’（Disocorea opposita Thunb.）is regarded as one of the famous‘four major Huai medicines’in China.As an excellent variety in‘Huai Yam’,D.opposita cv.Tiegun is a medicinal and edible plant,and has important economic value.However,due to the long-term vegetative propagation in the production of D.opposita cv.Tiegun,the virus infection is a serious problem,resulting in a decrease in yield and degradation of the variety.To solve the above problems,we carried systematic researches on callus induction,transgenerational seedling,cell suspension culture,mini tuber inducing formation,storage and germination,seedling field application and yield and quality of D.opposita cv.Tiegun.These researches aim to establish a technology system for tissue culture and detoxification of mini tubers,and to lay a technical foundation for the industrial production and application of effective medicinal ingredients and detoxification mini tubers in‘Huai Yam’.The results of the study are as follows:1.Induction and succession of embryonic callus（1）The suitable medium for the induction of embryogenic callus of D.opposita cv.Tiegun was MS+Pic 5 mg·L-1+Cu SO4 2 mol·L-1+sucrose 30 g·L-1and MS+6-BA 0.5 mg·L-1+Pic 1mg·L-1+CH 0.6g·L-1+Pro 1 g·L-1+sucrose 30 g·L-1.（2）suitable for embryonic callus of successive transfer culture medium for MS+6-BA 0.5 mg·L-1+Pic 1mg·L-1+CH 0.6 g·L-1+Pro 1 g·L-1+banana puree 50 g·L-1+sugar 30 g·L-1,under the condition of the dark,after 3 to 5 generations successive transfer culture,according to the color and texture,callus can be divided intoⅠ,Ⅱ,Ⅲ,Ⅳfour types.2.Establishment of cell suspension culture system and study of physiological and biochemical characteristics（1）Establishment of cell suspension culture systemAmong the four types of callus of iron stick yam,typeⅠhas the best effect.The suitable conditions for the good growth of callus cells in suspension culture were MS+6-BA 0.5 mg L-1+Pic 1mg L-1+CH 0.6 g L-1+Pro 1g L-1,30 g L-1white granulated sugar was used as carbon source,p H was adjusted to 6-7,30 g L-1was inoculated,and suspension culture lasted for 18 d.The optimal growth curve of suspension culture callus cells showed"S"type,which could be divided into delayed period（0-8 d）,logarithmic period（8-18d）and stationary period（18-22 d）.The callus cells grew significantly in the logarithmic phase,with a maximum proliferation of 3-4 times,and the color changed from light yellow in the logarithmic phase to reddish brown in the stationary phase.（2）Physiological,physiological and biochemical characteristics in cell suspension cultureDuring the cell suspension culture,the p H of the culture fluid decreased first and then increased,and stabilized at about 5.7 after 18 days;the conductivity of the culture fluid decreased at the first 3 days,remained at a low level for 3-9 days,and gradually increased 15 days after 9 days to reach the peak,and then gradually decreased.POD of cultured cells remained at a low level at 3-6 d,began to increase after 6d,peaked at 12 d,then began to decrease,and stabilized at a lower level after 18 d;the soluble protein content of cultured cells showed an increase before decreasing Trend,peaked at 12 d and then decreased,tended to be stable after 18 d.3.Establishment of virus-free micro-tuber breeding technology system（1）Induction of virus-free microtubersThree methods of tuber induction were studied in this paper.Method 1 was the combination of batch submerged bioreactor and static culture.When the sucrose concentration in the culture medium was 60g·L-1,the number of induced tubers was 3.84/plant and the average mass was 132.9 mg/plant.Method 2was induced by two-step static treatment.The number of induced tubers was 3.09/plant and the average mass was 145.38 mg/plant.Method 3 was one-step shock induction,the number of induction was 1.13/plant,and the average mass was74.16/plant.Three methods have their advantages and disadvantages,method 1 form of mini tuber,most larger average quality but for a long time（90 d）,2 of the large number of mini tuber formation method,average quality but the largest longest（140-145 d）,method 3 form but at least the number of mini tuber induction time（45 d）according to the quality and the shortest seedling formation of mini tuber size can be divided intoⅠ,Ⅱ,Ⅲlevel.（2）Germination and storage time of virus-free microtubesUnder 4℃condition,the best time for improvingⅠ,Ⅱ,Ⅲof mini tuber germination rate is 2 months,and the highest germination rate forⅠtype is 63.2%,followed byⅡtype（54.5%）,typeⅢ（34.3%）.（3）Virus-free microtubes form virus-free seedTypeⅠ,Ⅱ,andⅢtype virus-free mini-tubers were planted in the field to form typeⅠ,Ⅱ,andⅢtype virus-free seeds.Among them,the average quality and average underground rhizomes of typeⅡvirus-free seeds were 80 g/piece and 4.25,respectively./Plant;Type III virus-free seeding has the lowest average quality and average length,respectively 27 g/piece and 13.77 cm/（piece）;Type I has the longest average length,22.5 cm/piece.（4）Virus-free seeds were planted to form virus-free yamsThe typeⅠ,ⅡandⅢdetoxified seeds were planted in the field to formⅠ,ⅡandⅢdetoxified yam.The average weight of each plant was 0.282 kg,0.272 kg and 0.26 kg respectively,compared with non-detoxified iron rods.The yield of yam per acre has increased by 26%,21.63%and 16.33%respectively.4.Quality of virus-free yamCompared with non-detoxification yam,there are no significant differences of the polysaccharide,protein,starch and polyphenol content in I,ⅡandⅢtype yam.However,the flavonoids content in detoxification yam is higher than the non-detoxification yam,and the flavonoids content in type III yam is higher than type I and II yam.In total,there is little difference in quality between virus-free and non-virus-free D.opposita cv.Tiegun. |
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