Establishment Of Genetic Transformation System Mediated By Agrobacterium Tumefaciens Of Glycyrrhiza Uralensis Fisch

Glycyrrhiza uralensis Fisch is a plant of leguminous Glycyrrhiza shrubby herb, whosedried root and rhizome are used as medicine. It is used as drugs for a long history andusually used in China. In recent years, over-dug makes the wild resources tends to beexhausted. Thus the cultivated licorice has become the main way to alleviate thecontradiction between market supply and demand. But the liquiritin content in cultivatedplants is usually lower than that in wild ones and can’t meet the requirement of the People’sRepublic of China Pharmacopoeia Specification. So it’s important to explore the functionalmechanism of liquiritin biosynthesis genes to improve licorice quality. But till now lessprogress has been reported due to the poor regeneration system and lack of efficientgenetic transformation system in licorice.This paper studied the effect of culture medium component and explant types onregeneration results to set the high efficient regeneration system of Glycyrrhiza uralens.Taking Agrobacterium tumefaciens strain EHA105carrying pB7WG2D-CHI to infect thecotyledon-excised embryo, this paper studied several factors such as engineering strainconcentration and co-culture days to set the high efficient genetic transformation system.The main results were as follows：1. As far as adventitious buds differentiation concerned, there were significant differencesamong three types of explants. No adventitious buds were induced for cotyledon.Cotyledon node could differentiate1-2buds averagely with low ratio (8%only).Cotyledon-excised embryo was the suitable explants, each embryo could produce3-5buds on the average with the differentiation ratio high to90%.2. During the differentiation process on MS+6-BA1.0mg/L+NAA0.2mg/L, there weresevere vitrification for cotyledon-excised embryo, which could not be recovered bysubculturing on6-BA-decreased medium. Soak the seeds with6-BA2.0mg/L overnightfollowed by culturing the cotyledon-excised embryo on MS+6-BA0.5mg/L couldsynchronously improve the differentiation ratio and reduce the vitrification ratio from13%to7%.3. Adventious roots could be induced at the7thday after the plantlets were transferred to rooting medium. Root ratio on MS and1/2MS+IAA0.5mg/L was77%and60%separately. On1/2MS the root ratio could reach93%and the plantlets developed well.4. The sensitivity test of PPT were performed according to the principle of screeningpositive transformants and the lowest rates of plant injury. The results indicated that thesuitable PPT concentration in differentiation and rooting medium was2.0and1.0mg/L,separately.5. The effects of engineering bacterial concentration, infection duration, AS concentration,co-culture time on the positive plant percentage were studied. The results showed thatOD600is0.5, infection duration was30min, co-cultivation medium with AS50mg/Land the co-culturing for4days were optimum for licorice genetic transformation.Under these conditions, the positive transformation ratio was up to10%.6. The concentration of Cef was selected to inhibit the bacterial growth effectively. Thesurvival ratio of cotyledon-excised embryo reached83%when Cef concertration was300mg/L in differentiation medium. The contamination rate and rooting rate was0and90%, respectively, when Cef concentration was200mg/L in rooting medium.This paper has established high efficient regeneration and genetic transformationsystem of Glycyrrhiza uralensis, which laid a foundation for gene cloning and functionalverification of licorice medicinal ingredients biosynthesis-related genes.