Preliminary Study On The Establishment Of Callus And Hairy Root Culture System Of Apocynum Venetu

Apocynum venetum L.is a precious wild resource with economic and medicinal value in the desert region of China.It has the widest distribution and the best quality in Xinjiang.The Apocynum venetum genome contains a large number of excellent stress-resistant genes.In order to develop and utilize the excellent stress-resistant genes of Apocynum venetum,it is a prerequisite to establish a mature tissue culture system and genetic transformation system.Therefore,Apocynum venetum was used as material to establish culture system in callus induction,suspension cell culture,hairy root induction and expanded culture.The main findings are as follows:0.5 mg·L^-1 2,4-D+0.05 mg·L^-1 6-BA was added to the four mediums of MS,B5,IS and WPM,and the callus induction rate of stem segments and leaves was 100%.At the same time,it was found that the leaves inoculated on WPM were cultured under the illumination to induce the best callus state,and the texture was loose and the growth rate was fast.The culture system suitable for the initial callus induction of Apocynum venetum was obtained:the leaf explants were lightly cultured on WPM solid medium.The effects of inoculum volume from 10 g·L^-1 to 50 g·L^-1 on the growth of Apocynum venetum suspension cells were analyzed,and the maximum biomass accumulation of suspension cells was coming from inoculum volume of 30 g·L^-1.The optimal inoculation volume of 30 g·L^-1 was used to analyze the growth of the suspension cells in a growth cycle.It was found that the growth curve of suspension cells was similar to the“S”type,and the cell activity reached the maximum at the 9th day.Meanwhile,the fresh weight showed an exponential growth trend within 9～15d.It was concluded that the inoculation volume of the suspension culture was 30 g·L^-1,and the appropriate subculture cycle was about 15d.Agrobacterium rhizogenes AC1090 and AC1060 were used to infect stem explants,and the hair root induction rate was 69.17%coming from AC1090.Hair roots were verified from the morphological and molecular levels.The effect of inoculum size of 4g·L^-1～12 g·L^-1 on the growth of hairy roots was analyzed,and the maximum biomass accumulation of hair roots was coming from inoculum volume of 6 g·L^-1.The optimal inoculation volume of 6 g·L^-1 was used to analyze the growth of the hair roots in a growth cycle,and it was found that the growth amount of hairy roots in both 100 m L flask and 3L bioreactor presented an"S"shape,and the hairy roots entered the exponential growth period on the 10d and the plateau period on the 20d.It was concluded that the inoculation amount of liquid culture of the hairy root was 6 g·L^-1 and the appropriate succession period was about 20d.The total flavonoid content and antioxidant activity of field roots,hairy roots,tissue culture root and callus were analyzed.The contents of flavonoids:hairy roots>callus>field roots>tissue culture roots.The antioxidant capacity with the method of DPPH and FRAP:hairy roots>field roots>tissue culture roots>callus.