Agrobacterium Tumefaciens-mediated Genetic Transformation Of Taraxacum Antungense Kitag.

Dandelion contains terpenoids,phenols and polysaccharides.In addition,it has been found that chlorogenic acids in phenols has antibacterial,antioxidative and other effects.At present,the metabolic regulation of plant secondary metabolite biosynthesis is a possible means,and can improve the production of high-value secondary metabolites.It is known that hydroxycinnamoyl-CoA:quinihydroxy-cinnamoyltransferase（HQT）is a key enzyme in the plant chlorogenic acid synthesis pathway.In this experiment,the regeneration system of Taraxacum antungense Kitag.was established by using leaf explants of plant sterile seedlings,and a genetic transformation system was established using Agrobacterium-mediated methods.The HQT gene was introduced into the genome of Taraxacum antungense Kitag.plant genome.The test results were as follows:1.In this experiment,Taraxacum antungense Kitag.seedlings were used as the source of leaf explants to establish the Taraxacum antungense Kitag.regeneration system.The best medium for directly inducing shoot regeneration from leaf explants was MS+2.0 mg/L 6-BA+0.2 mg/L NAA+3%sucrose+0.7%agar powder,and the maximum regeneration frequency was 95.2±2.58%.Then,by selecting adventitious buds for rooting culture conditions,the best medium for rooting was 1/2 MS+0.1 mg/L NAA+3%sucrose+0.7%agar powder,and the maximum rooting rate was 99.0±0.58%.2.This experiment established the Agrobacterium-mediated Taraxacum antungense Kitag.genetic transformation protocol:the preculture time of leaf explants was 48 h,OD₆₀₀concentration of Agrobacterium tumefaciens GV3101 was about 0.6,duration of explants infection 10 min,AS concentration was 50 mg/L,and co-culture time was 48 h.These parameters were used for transformation in this experiment.The initially infected explants were cultured on adventitious shoot induction medium containing 30 mg/L kanamycin and500 mg/L cefalexin.The concentration of cefalexin in the late stage of induction of resistant shoots was 250 mg/L.Finally,the concentration of kanamycin selective sprouting roots was15 mg/L.3.In this experiment,the resistant plants were first analyzed by PCR,and showed that the HQT gene was successfully poured into the genome of of Taraxacum antungense Kitag.with a transformation efficiency of 5.7%.Then,through the qRT-PCR and HPLC method of analysis of transgenic plants and wild type plants,the relative expression of HQT gene and the chlorogenic acid content in the leaves of the three transgenic plants were higher than that of the wild plant.Finally,the chlorogenic acid content in the three transgenic plants was 1.12,1.34,and 0.94 mg/g,respectively,and the chlorogenic acid content in the wild plant was 0.74mg/g.