Establishment Of Soybean Hairy Root Mediated By Agrobacterium And Its Application

Hairy roots are disease manifestations developed by plants that are wounded and infected byAgrobacterium rhizogenes. Specifically, root genes harbored by the root inducing (Ri) plasmid of A.rhizogenes are incorporated into the nuclear genome of the host plant, ultimately causing root hairproliferation. The production of hairy roots in axenic cultures is a more rapid, stable procedure. Hairyroots are highly differentiated, abundant of roots and can proliferate on phytohormone-free media, thatprovide lots of infested sites. The soybean cyst nematode could complete its life cycle on soybean hairyroots cultures. Hairy roots are exploring interactions with nematodes.This character demonstrates the successful transformation of soybean cultures by injecting colonies ofAgrobacterium into the hypocotyl. Sterilized soybean seeds were cultured in the soil. Soybean seedlingswere inoculated by an injection for three times parallel to the vascular bundle of hypocotyl. Afterinoculation, soybean seedlings were co-cultivated to induce hairy roots under humidy conditions. GUSassay was done for the chimeric seedlings. The results showed all of the three soybean cultivars(zhonghuang13、zigongdongdou、Heidou51) could produce hairy roots, the number of hairy root has nosignificent difference between three cultivars（p＞0.05）. The concentration of Agrobacterium culturesand inoculated site affect the hairy root number and transformation effenciency: number andtransformation effenciency were higher by inoculating colonies of Agrobacterium than that by cultures（p＜0.05）.Inoculated hypocotyl round natural root had a higher number than that of cotyledon node,while its transformation ratio was lower. soybean seeds were cultured in the medium. Soybean seedlingswere inoculated by an injection for three times parallel to the vascular bundle of hypocotyl. Afterinoculation, soybean seedlings were co-cultivated on co-cultivation medium. The roots together withcotyledons were transferred onto selection medium (1/2MS agar medium with100mg/L kanamycin).The results demonstrate that the cultured medium has no significant effect on hairy root number.Number of hairy root between soil and medium has no significant effect, while the transformation ofhairy root in soil is higher than that on medium.This character shows an successful transformation of soybean cultures by co-culturing with strainsof Agrobacterium. Soybean seeds were cultured in the agar medium. Explants of cotyledon of soybeanwere inoculated with freshly grown A6000.6Agrobacterium cultures. After inoculation, the explantswere transferred to the1/10MS agar medium, and were supplemented with0.02mg/L acetosyringone(co-cultivation medium) and cultured in darkless for3～5days. Explants were soaked in1/2MSsolution complemented with250mg/L carbenicillin sodium+250mg/L cephalothin to remove theexcessed bacterium. The explant was transferred onto1/2MS agar medium without hormone under25℃,12h/d light conditions. After30days, hairy roots were produced from the wounded surface of thecotyledon node explants. There are abandunt of negative gravity hairy roots instead of significant root.GUS assay is done for the chimeric roots. Tips of transformed root were transferred to1/2MS solution for subculturing, and roots were challenged with M. incognita. The results showed that Heidou44produced the most number and transformation ratio of hairy roots. There is no significant effect on lightor darkness condition for zhonghuang13, but the number of hairy root is highly more under darknessthan that under light condition. GUS assay of tansformed and subcultured roots was positive. PCRtested the specific lines in chimeric root. These results demonstrated the T-DNA incorporated into thenuclear genome of the host plant. The results showed that both of the pathway could make explantproduce hairy roots, there is a significant difference between soybean cultivars.The optimized system of nematode infestion: The J2s were surface-sterilized in0.1%HgCl2+0.5%,500J2s per plant is the optimized concentration for J2s infestion.The higher concentration of J2s, thelower infested ratio. J2s can not infest the root in the liquid medium, the infestion ration is the highestunder8%agar. Soybean cyst neamtode J2s could efficiently infest hairy roots of three cultivars, andinfested sites were the same. The infested ratio of Heidou51is higher than that of Heidou44andzhonghuang13.Transgenic roots have shown promises towards plant resistance against parasitic nematodes. Thisstudy also showed significant foundation of studying parasitic genes and approaches.