Establishment Of Culture System Of Impatiens Balsamina L. Hairy Roots And Determination Of The Quercetin

Impatiens balsamina L, a.k.a. known as Impatientis Semen and henna, is an annual herb native to China, India and Malaysia. The whole herb, seed(Impatientis Semen), and stem(tougucao), have been used for clinical application as well as an ornamental plant in our country. In this research, Four Agrobacterium rhizogenes strains R15834, R1000, A4, and R1 were used to induce hairy root produced from leaf, stalk and root in order to establish the hair roots culture system of Impatiens balsamina L and optimize the proliferation culture. The PCR examination result showed rolB genes in Agrobacterium rhizogenes could be inserted into the genome of the hairy root. The content of quercetin was detected by HPLC in hair roots culturing system. The contents of quercetin in stalk, root, hair roots adding elicitors and Impatientis Semen were compared. The main conclusions are shown as following in this research:1. The best conditions of establishing hairy root system were disinfected for 5 minutes by 0.2% mercuric chloride for seeds and the seedlings growed 10 d in the MS0 solid medium culture. Four Agrobacterium rhizogenes strains R15834, R1000, A4 and R1 were used to induce hairy roots produced from leaf, stalk androot. The average induction rates are 92% and 79.33% from the seedlings of stalk, root by A4 strain.The best explents are stalk, root which produce hairy root with taproot stout, milky white fluff. In conclution, The culture methods are for 24 h pre-culture, 48 h co-culture;8min and 6 min for stalk, root are the best infecting time.The OD600 of A4 strain is 0.6 of stalk;is 0.8of root. The most appropriate pH value in the medium is 6.0.2. The PCR examination result showed that the T-DNA genes in Agrobacterium rhizogenes strain A4 have been inserted into the genome of the transformed roots.3. The best proliferation culture conditions were described as below. The 1/2MS liquid culture medium is more suitable for the explants to induce hairy root growth. After the stalk-induced hairy roots and the root-induced hairy roots grow 28 d and 21 d, the average fresh weight is 9.53 times than beginning growth. At the same time, the content of quercetin in the hairy root is the highest.When the 30g·L-1 sugars are in the 1/2MS liquid culture medium, The transformed roots which are from stalk, root proliferation culture times are 9.23 and 9.85. Added sugar culture medium is the most appropriate growth of hairy root.4. The HPLC method was used to detecte the content of quercetin in hair roots culturing system that the mass fraction of quercetin is 0.292% in the seeds, 0.537% in the stalk-induced hairy roots, 1.22% in the root-induced hairy roots. It is determined that the quercetin contents in Impatiens balsamina L.hairy roots of stalk and root are 1.84 times and 4.20 times that in the seed. The content of quercetin in the root-induced hairy roots is 2.27 times that the stalk-induced hairy roots.5. The mass fraction of quercetin is 2.75% in the culture medium adding salicylic acid whose content is 9.43 times that of Impatientis Semen and is 2.25 times without adding elicitor induced hairy root.