Study On Regeneration In Vitro And Hairy Root Inducement Of The Glycyrrhiza

The Glycyrrhiza is one of the most common Chinese herbal medicine which has many drug action, such as cleaning up heat detoxification, moistening lung to arrest cough, relieving pain, coordinating the drug actions of a prescription. Due to the ruinously spading of the grow wide Glycyrrhiza, now the quantity of the Glycyrrhiza is very cut down, so it's very urgent to protect the Glycyrrhiza. The establishments of vitro plant regeneration system and hairy root cultivation have a very important significance in protection of the Glycyrrhiza resource, the researching of genetic engineering and the utilization of the active constitutents.Glycyrrhiza uralensis Fisch, Glycyrrhiza inflata Bat. Glycyrrhiza glabra L, Glycyrrhiza korshinski G. Grig, grow wild Glycyrrhiza glabra L are used as the test materials, this research contains Glycyrrhiza vitro plant regeneration and hairy root induction, the main results are as follows:1.Establish the Glycyrrhiza hypocotyl vitro plant regeneration, provide the foundation of the Glycyrrhiza breed improvement and good breed intermediate propagation.Use the Glycyrrhiza hypocotyl as the explant, research the factor which effects hypocotyl vitro regeneration adventive bud, the results show that KT has a predominance effect when combine with 6-BA, IBA, which can induce adventive bud from the hypocotyl, the optimize medium is consist of MS, 6-BA 2.0 mg/L , KT 0.5 mg/L , IBA 0.5 mg/L. the average optimize rate is 41.5%, the G. inflate has the highest optimize rate, which is as high as 44.7%, preincubated for four days, using 6mg/L AgNO3 can promote the growth Glycyrrhiza hypocotyl regeneration adventive bud.2. establish the Glycyrrhiza intermediate propagation technology syetem, prepare for the deeper development and utilization of Glycyrrhiza resource. using the stem apex of cotyledon and the stem apex of lateral bud as the explants, the results show: the induced G. uralensis regrowth is best among the three breeds, the stem apex of cotyledon is suitable for explant, the medium which suits the Glycyrrhiza intermediate propagation system are as follows:induction medium: MS + 6-BA 0.5 mg/L + KT 0.5 mg/L + IBA0.1 mg/L rejuvenescence medium: MS + 6-BA 2.0 mg/L + KT 0.5 mg/L + IBA0.5 mg/L root medium: MS + IBA 0.5 mg/L3. Induction of Glycyrrhiza callus and somatic embryogenesis provide materials for transgenic and artificial seeds.when callus was induced, then the embryogenic callus and the embryoid were produced, we analysis the factors which have effect on the process of formation and development from genotype, explants, hormones, accessory, etc. Then make a further analysis to the morphous and structure of embryonic callus and non-embryonic callus with paraffin section and Semi-thin Sectioning.The results show that there are three different ways of Glycyrrhiza somatic embryos formation: the first is the formation from the hypocotyls superior extremity in morphology without callus phase, but embryoid were few induced; Second, the somatic embryoid was formatted through callus stage, and the embryoid were more induced; Third, the embryoid were formated from suspension cells, and they were more induced. It is difficult to formatted to the regeneration plants from Glycyrrhiza embryoid in these three ways.Several suitable medium are :Callus induction medium MS + 6-BA 2.0 mg/L + 2,4-D 0.5 mg/LCallus subculture medium MS + 6-BA 0.5 mg/L + NAA 0.2 mg/L + 2,4-D 0.2 mg/LEmbryogenic callus induction medium MS + 6-BA 0.5 mg/L + KT 0.5 mg/L + IBA 0.1 mg/LEmbryogenic callus subculture medium and embryoid differentiation medium MS + 6-BA 0.5 mg/L + KT 0.5 mg/L + IBA 0.1 mg/L + ME 500 mg/L Embryoid proliferation medium MS + 6-BA 0.5 mg/L + KT 0.5 mg/L + IBA 0.1 mg/L + ME 500 mg/L4. Established a technology platform for Glycyrrhiza hairy root culture by Agrobacterium rhizogenes LBA9402, which lay a solid foundation for Glycyrrhiza hairy Root industrial production of medicina1 plant secondary metabolite.Give a systemic expatiation to the factors that effects the Induction of hairy root factors from different strains, different explants, different concentrations of bacteria, and Infection different time, the results showed: the strain LBA9402 induced hairy root easily, which is a sensitive strain to Glycyrrhiza.the hairy root can be induced from Hypocotyls, cotyledons, leafs, the hypocotyls can induced the highest transform rate. Using the diluted concentration of Agrobacterium rhizogenes LBA9402 to induce the Glycyrrhiza embryoid, it will be the best at the time of 5~10 minutes. Induced hairy root are tender, white,thin,and rapid speed, have many branchs, many root hairs, submit cluster and lose geotropism.