Study On The Establishment Of Hairy Roots Culture System And Its Secondary Metabolites Of Pharbitis Purpurea

Pharbitis purpurea,which belongs to Convolvulaceae Pharbitis Choisy, is a herbal medicinal plants on the State pharmacopoeia. It has functions of inducing catharsis, dissolving phlegm and anthelminthic. In China, the distribution of Pharbitis purpurea which has great potential for the development, is very wide.By using R1601,1000,1025,15834,4 kinds of Agrobacterium rhizogenes,to infect the explants of Pharbitis purpurea,this article optimized the inducing conditions of growth and culture for two kinds of explants, established the culture system of Pharbitis purpurea, and made the initial tests to the polysaccharides and caffeic acids in hairy roots and natural roots which were transformed from leaves and petioles.From the research, when 4 kinds of Agrobacterium rhizogenes were used to infect Pharbitis purpurea, the most appropriate strains of leaves and petioles, were R1601, the most appropriate pre-culture time was 60 hours, the most appropriate infection time was 6～8min, the most appropriate coculture time was 48 hours and, the most appropriate concentratin of NAA was 1ml/L.In the absence of exogenous hormone,the induction rates of leaves and petioles reached 70% and 66.67%.When adding 1ml/LNAA in pre-culture and co-culture medium at the same time, the induction rates of leaves and petioles greatly increased,which reached 96.67% and 93.33%.The results of PCR showed that the obtained hairy roots of Pharbitis purpurea had bands with the same size as the rolB gene at 423bp.It showed that T-DNA of Agrobacterium Rhizogenes R1601 Ri plasmid had been successfully integrated into the genome of Pharbitis purpurea.There are many characteristics belong to Hairy roots, such as rapid growth, hormone self-support, a simple growth conditions, high levels of secondary metabolites and stable, high degree of differentiation and are not easy to variate, so that the culture of hairy roots is another important way to obtain secondary metabolites of medicinal. Therefore, according to the establishment of the culture system of Pharbitis purpurea and the effects of the factors of optimization, we do some research on its hairy roots.The two transformed hairy roots grew independently and rapidly on MS solid medium without exogenous hormone, and showed the typical features of hairy roots. In the MS liquid culture system, it also showed the typical features of hairy roots, and its growth cycle was 20 days. With adding different concentrations of JA,30μmol/L was best for the growth of the two hairy roots.Amount of the polysaccharides and caffeic acids in the hairy roots, transformed from Pharbitis purpurea, were analysised.By UV spectrophotometry, the caffeic acid content of hairy roots transformed from leaves was 1.03%, the caffeic acid content of hairy roots transformed from the petioles was 1.26%,the caffeic acid content of natural roots of original plants was 0.93%, the polysaccharides content of hairy roots transformed from the leaves was 2.13%, the polysaccharides content of hairy roots transformed from the petioles was 2.50%,the polysaccharides content of natural roots of original plants was 1.77%.The experimental data obtained that the caffeic acid and polysaccharides content of natural roots of original plants were less than the hairy roots.Compared with the hairy roots inducend by the two different explants, the caffeic acid and polysaccharides content of hairy roots transformed from the petioles were also higher than the content of hairy roots transformed from the leaves.