| Morus alba L. is a perennial woody plant with pair of cotyledons. China as one of the world's most resource-rich countries of Morus alba L.. Morus alba L. has a long history as a medical plant. Morus alba L. is a treasure body, and its Fruits, roots, stems, leaves and processing by-products have a high medicinal value and health value. Mulberry leaves are rich in the human body needs 18 amino acids, in which 8 kinds are necessary for the human body, Morus alba L. leaves also contains quercetin, superoxide dismutase, polysaccharide, trigonelline and other special functional ingredients; Morus alba L. stalk is a good Chinese herbal medicines, it Contains flavonoids which have anti-bacterial, anti-virus, anti-oxidation, anti-aging, anti-tumor and other functions, and has an important application value in agriculture, medicine, chemical industry.The roots of Morus alba L. play a significant role in lowering blood pressure, and also has certain anti-HIV activity. By the time, there's no report on orthogonal design in establishing sterile rapid propagation system of Morus alba L.The main medical Compound in Mulberry is flavonoids, in which quercetin has functions with anti-tumor, anti-inflammatory, anti-platelet aggregation and expanding the role of coronary artery.and so on. Agrobacterium tumefaciens contains Ri plasmid, with the Vir gene under duress, its T-DNA can integrated into the plant nuclear genome, to induce the host plant produce hairy root. There has been many reports on the use of hairy roots distinctive feature such as rapid growth, multi-branch and sustained synthesis of secondary metabolites, to produce medicinal active ingredient in plant secondary metabolites.This paper aims to discuss the effection of different hormones and different media on the inducement of mulberry's budding stem and bud proliferation as well as its roots culturing, with a view to filter out the best portfolio of medium and hormone. Then lay a foundation for the fulture reserch on the genetic transformation. Afterwards, hairy roots of Mulberry will be obtained through infecting etiolated seedlings with Agrobacterium tumefaciens strain C58C1, to establish the induce and culture system of hairy roots, then determin the contain of quercetin, aiming to increase the flavonoid content in kuwabara plant. So lay the foundation for industrial production on active ingredients in medicinal plant for the fulture use of genetic transformation.In this study, Morus alba L. seeds germination was used as material, the sterile rapid propagation system was established by design of orthogonal, with transformed Agrobacterium tumefaciens strain, Morus alba L. hairy roots was induced successfully. And by High-performance liquid chromatography(HPLC), the quercetin in hairy root was measured analyzed.The main studies and results of this paper are as follows:1. Research on the Control of Contamination Rate in Tissue culture of Mulberry:By design of orthogonal, use MS as basical culture medium, effects on exterior sterilization methods of Mulberry explants in the tissue culture was researched. The lateral branches of Mulberry were taken as explants, effects of various medium and different plant hormones on caespitose buds induction was studied by L 9(34) orthogonal design, and the roots induction was researched. The results showed that, using 70% alcohol sterilized for 30 s and 0.1% corrosive sublimate sterilized for 5 min and 6 min in 2 steps, besides, adding Amp100 mg/L or Cef100 mg/L could effectively control the contamination rate at zero. The optimal medium for buds induction was WPM+1.0 mg/L 6-BA+0.1 mg/L NAA, meanwhile the breed up of casespitose buds was realized; the cultural medium for growth of roots was WPM+1.0 mg/L IB A.2. Establishment of transformation system in Mulberry and its optimization:Hairy roots of Mulberry were obtained through infecting etiolated seedlings with Agrobacterium tumefaciens strain C58Cl; The culture conditions of hairy roots was optimized. The transformation of T-DNA was examined by PCR assay. When infecting stem cutting of etiolated seedlings via C58C1 strain, the optimal transformation conditions were as follows:10 minutes' infection, two-days pre-culture and co-culture, aditional hydroxyace-tosyringone(As) 100 mg·L-1. The PCR examination result showed that rolB and rolC genes could be inserted into the hairy roots of Mulberry. Hairy roots appeared after infecting for 10 days, the frequency of stems explants was up to 92% after culturing 30 days.3. Determination of quercetin in the hairy roots of Morus alba L.:After culturing for 50 days in 1/2MS+0.05 mg/L IBA liquid medium, by RP-HPLC we know that the content of quercetin was increased 8.5 times.
|
PDF Full Text Request