Study On The Establishment Of Tissue Culture Mircropropagation System And The Differentiation Mechanism On Amor Phophallus Konjac

Amorphophallus konjac is a perennial herb of Araceae,rich in glucomannan within the tubers.Glucomannan has the characteristics of rubber with in soluble,gel,film,etc.It is widely used in food and health care,with lowering blood pressure,lowering blood sugar,fighting cancer,losing weight,beauty and other effects.In recent years,the cultivation area of konjac are expanding and seed quantity are rising constantly,but the low reproductive rate naturally and long growth cycle, which greatly limits the development of konjac. The experiment studied the problems of the establishment of micropropagation system for tissue culture,and discussed the mechanism of antioxidants in controling browning and the mechanism of cell differentiation in the tissue culture.The main results are as follows:1. The test optimized the culture conditions of the konjac tissue culture. A comparative study of the explant sterilization methods of the konjac tissue culture improved the sterilization effect.The better explants disinfection methods as follows:75%alcohol for30S,then washed3times with sterile water,each time1min,and then0.1%mercuric chloride for12min,washed five times with sterile water,each time1min,The highest disinfection index was0.24.A comparative study of the effect of different antioxidants in controling the explant browning,the better methods as follows:the sterilized of konjac explants in the100mg·L-1citric acid cut into small pieces after inoculation, this method can effectively control konjac explant browning,the browning rate was8.7%.On MS+4mg·L-1TDZ+3mg·L-1NAA medium,the test studied the induced effect of top heart block, tuber parenchyma, the main bud, lateral bud, coleoptile, rhizome tip and rhizome segment.It showed that the combined effect of main buds was best and the culture index was0.85.2. The test optimized the hormone ratio of the medium in konjac tissue culture. Appropriate induction medium for the top center of konjac was MS+8mg·L-1TDZ+1mg· L-1NAA, callus induction rate was100%.The suitable subculture medium for callus was MS+4mg·L-1TDZ+7mg·L-1NAA.In this medium,the callus grow fastly and the growth rate was0.292gd·d-1. The structure of inducted callus was dense compact, the color was light red. The suitable medium for adventitious buds differentiation from callus was MS+8mg-L-1TDZ+lmg·L-1IBA.The differentiation rate was137.50%,and adventitious buds was more robust and growing well.The suitable rooting medium was1/2MS+5mg·L-1NAA+0.2%activated carbon, rooting rate was100%and the root was more and sturdy.3. The test studied the browning mechanism of controling konjac explant for different antioxidants.100mg·L-1citric acid could inhibit browning of konjac explant significantly, and reduce browning degree,polyphenol content, PPO and POD activity.It showed that the citric acid may reduce PPO and POD activity, and polyphenol content to lower BD.4. The test explored the cell differentiation mechanism of the Konjac tissue culture. In the process of cell differentiation,A high level of endogenous ZR,ZR/IAA ratio and the amount of IAA,GA3content could conductive callus cell differentiation,but ABA was not conducive to the differentiation of callus cells. The increase of the POD,alpha-amylase and PAL activity was in favor of redifferentiation of callus cells,these changes of enzyme activity could promote cell differentiation by the way of improving the cell metabolism level and providing the matter and energy of cells differentiation. Reduction of the soluble sugar content was also conducive to cell differentiation, probably due to it could provide energy and material basis for cell differentiation.5. The studies established amicropropagation system of the konjac tissue culture.The konjac tissue culture of the main route was as follows:the explants of konjac was main bud; The disinfection methods was75%alcohol soaked30S,washed3times with sterile water,1min each time,and then0.1%mercuric chloride for12min,sterile water washed five times,each1min;The methods of control explant browning was cuting explant in100mg·L-1citric acid solution;The primary culture medium was MS+8mg·L-1TDZ+1mg·L-1NAA;The subculture medium was MS+4mg·L-1TDZ+7mg·L-1NAA;The adventitious buds differentiation medium was MS+8mg·L-1TDZ+1mg·L1IBA; The rooting medium was1/2MS+5mg·L-1NAA+0.2%activated carbon;The transplanting matrix was vermiculite and perlite2:1.The micropropagation system of konjac tissue culture was established finally through the above measures.