Agrobacterium Rhizogenes-Mediated Hairyroot Culture And Flavonoid Biosynthesis Of Ginkgo Biloba L.

Ginkgo biloba L.is a perennial tree of the genus Ginkgo,which has high ornamental value.It is often used as a landscape tree species for street shading or landscape gardening,and has high edible value and medical value.However,the content of flavonoids in leaves is low,and a large number of leaves need to be collected to meet the needs of pharmaceutical industry.In order to provide a new method of collecting raw materials for Ginkgo flavonoids,which is suitable for industrial production rather than low-efficiency collection,the genetic transformation system of Ginkgo biloba with Agrobacterium rhizogenes was established in order to improve the yield of Ginkgo flavonoids and provide a new method for Ginkgo flavonoids production.And it also provides a theoretical basis of industrial production of hairy roots.The specific conclusions of this study are as follows:1)The hairy root induction rate can be improved by screening and optimizing the genetic transformation procedures through single factor experiments.The experiment found that the mortality of explants under pre-culture for 3 days was significantly lower than 0d,1d and 2d;the hairy root induction rate of strain R1601 was significantly higher than that of A4,ATCC15834 and LBA9402,with an increase of 153%,121% and 144% respectively;explant mortality in White medium was significantly lower than 1/2MS medium with a decrease of 10%,while hairy root induction rate was significantly higher than 1/2MS medium,and induction rate of hairy root in 1/2MS was up-regulated in 364%;the hairy root induction rate of explants treated for 30 min was significantly higher than that of 15 min and 25 min,with an increase of 68% and 79%,respectively.Through the above single factor tests,we found that the operation suitable for inducing hairy roots of Ginkgo biloba was: after pre-incubation of Ginkgo biloba leaves for three days,inoculation in R1601 Agrobacterium for 30 min and blotting the bacterial solution,and transferring into White medium for co-cultivation and sterilization.to cultivate.The optimized genetic transformation system operation can increase the hairy root induction rate to 2 times before the optimization.2)The hairy root elongation in 1.0 mg/L IBA treatment was significantly higher than 0.1 mg/L IBA and hormone-free control,with an increase of 429% and 363%,respectively,but after 30 days’ subculture,the callus were induced from hairy root of Ginkgo biloba;the elongation of hairy roots under 0.5 mg/L IBA treatment was significantly higher than that of 0.1 mg/L IBA and control,with an increase of 357% and 300%,respectively,and no callus appeared within 30 days of subculture,the degree of browning of hairy roots was small,so we choose 0.5 mg/L IBA as the most suitable concentration for hairy roots subculture.During the subculture period from 25 d to 35 d,the roots of some hairy roots began to change from milky white to yellow,the expressions of PAL,C4 H,4CL and POD genes in the 35 th day were significantly lower than those of 25 d in 32%,56%,50% and 48%;During the subculture period from 35 d to 45 d,most of the hairy roots turned from yellow to brown,and the expression of PAL and C4 H genes was significantly lower than that of 35 d,the declines were 81% and 25% respectively,while the POD expression was up 5% from 35 d.The content of polyphenols in hairy roots was significantly higher than that in the next generation of 35 d,which was 19.6% higher than that of 45 d.3)The expressions of PAL,4CL and C4 H genes in hairy roots were significantly higher than those in tissue cultured plants,with an increase of 185%,576% and 72%,respectively.The expression of PAL,4CL and C4 H genes in tissue cultured seedlings were also significantly higher than that of seedlings,the increase rates were 712%,633% and 1038%,respectively.The expressions of CHS,CHI,F3 H and FLS in the leaves of tissue cultured seedlings were significantly higher than those of hairy roots,with an increase of 186%,233%,96% and 60% respectively.The expression levels of CHS,CHI and F3 H in hairy roots were also significantly higher than those in seedlings,with an increase of 106%,881% and 440%,respectively.However,the expression of FLS gene in leaves of seedlings was significantly higher than that of hairy roots,which was 71% higher than that of tissue cultured plants;the total flavonol content of seedlings was significantly higher than that of tissue cultured plants and hairy roots,with an increase of 155% and 486%,and the content of tissue culture seedlings was also significantly higher than that of hairy roots,and tissue culture seedlings were 130% higher than hairy roots.