The Studies Of Stem Nematode Based On Hairy Root System And Construction Of Genetic Transformation System Of Sweet Potato

In this study,we investigated the possibility of stem nematode culture based on hairy root system using the technology of inducing hairy root by Agrobacterium rhizogens for sweet potato.Also,we developed the technology to identify the character of resistance to nematode in sweet potato and evaluate the effect of nematocide using this system.At last, we constructed the genetic transformation system of sweet potato.The main results are as follows:Based on the study of the system of Ditylenchus destructor in monoxenic cultures on hairy roots of carrot and sweet potato,the plant in vitro of Xushu 18 was used to induce hairy roots by Agrobacterium rhizogenes strain Acccl0060 transformed with vector RiA4b.Stem nematode was established on sweet potato hairy root.Ditylenchus destructor's reproductions were investigated.The results showed that Ditylenchus destructor can develop and reproduce normally on hairy roots with MS and MSR medium,and the nematode was able to complete its life cycle.In order to observe clear, the system of stem nematode in monoxenic culture on MSR medium was used,100 nematodes per ware for 9cm Disposable cultivation was fit at 26℃,the production rate of nematode was 4.3 fold after 30 days on sweet potato hairy roots.Egg hatching was 4 days in this system.Resistance to nematode was tested through natural infection in field for Xushul 8 and Lizhixiang which were susceptible cultivars and Lu78066 which was resistant cultivar. After inoculating nematodes on hairy root systems,the resistance to nematode for sweet potato cultivars was evaluated by observing the nematode reproduction on hairy-roots and nematode infecting root tissue in 6 weeks.The production rate of D.destructor was 8.82,0.76 and 0.70 after 6 weeks on sweet potato hairy roots Xushul8,Lizhixiang and Lu78066.Nematodes were observed at many sites in Xushul 8 root tissue,but only at few sites in Lizhixiang and Lu78066 root tissue.The results show that Xushul8 is susceptible to nematode while Lizhixiang and Lu78066 are resistant to nematode.The results of evaluating nematode resistance and evalution by natural infection in field for Xushul8 and Lu78066 are consistent,but not for Lizhixiang.It may be due to different mechanism of resistance to nematode.The testing results showed that hairy root system was a versatile,easy and reproducible system for testing nematode resistance,which will be a new system to evaluate the resistance to nematode for sweet potato cultivars under some improvement.The compositions and contents of free amino acids in Xushul8 and Lu78066 hairy roots were also investigated.There were three free amino acids in Xushul8 which was susceptible cultivar but not in Lu78066 which was resistant cultivar,which are Glu, Ala and Phe.The contents of free amino acids in these two cultivars were all reduced in different degree after 2 weeks of inoculation nematodes,and the variances of contents of free amino acids in susceptible cultivar Xushul8 were more than those in resistance cultivar Lu78066.The test of adding amino acid to medium showed that reproduction number of Ditylenchus destructor increase distinct on Lu78066 hairy roots in addition of Ala and Phe to MSR medium than on MSR medium,the production rate of nematode was 2.28 and 1.77 after 3 weeks.In this study,we used the nematocides such as 40%Isofenphos methyl oil,50% phoxime,0.2%Hongyubao and Botanical pesticide named 0.3%Azadirachtin oil,which were very effective in prevention and cure against Ditylenchus destructor in field.,They were assessed for nematicidal activity against Ditylenchus destructor using a hairy root-feeding method in vitro.40%Isofenphos methyl oil,50%phoxime,and 0.2% Hongyubao showed strong nematicidal activity against Ditylenchus destructor,according with assessing in field.Botanical pesticide 0.3%Azadirachtin oil which was friendly to environment showed strong nematicidal activity against Ditylenchus destructor too.The results showed that assessing nematicidal activity against D.destructor for nematocide using hairy root system is feasible.An Agrobacterium tumefaciens-mediated transformation system of sweet potato, Ipomoea batatas(L.)Lam.,was established by using embryogenic suspension cultures of the cultivar Lizixiang.A.turnefaciens strain C58C1 harboring a vector pCB2002H with Bar gene in the present study.An effective gene transfer system was established.After 7 days subculture of embryogenic suspension cells less than lmm in size were transferred into MS solid medium with 2mg/L 2,4-dichlorophenoxyacetic acid(2,4-D)were cocultivated with C58C1(OD_600nm=0.8)for 3 days.After cocultivation,the infected suspension cultures were transferred into MS solid medium with 2 mg/L 2,4-D,10mg/L glufosinate and 200mg/L carbencillin for the selection culture,after 4-6 weeks these formed glufosinate-resistant embryogenic calluses formed plantlets via somatic embryogenesis.Using such gene transfer system,380 Embryogenic suspension cells were transformed and 12 glufosinate-resistant embryogenic calluses were formed.Then they all formed plantlets via somatic embryogenesis in succession,which laid the groundwork for future research.