CN102301957A - Callus culture method by taking Rhizoma Paridis roots and stems as explants - Google Patents

Abstract

The invention discloses a callus culture method by taking Rhizoma Paridis roots and stems as explants. The method provided by the invention takes specially pre-treated Rhizoma Paridis roots and stems as the explants; and the method finally realizes an effective induction and a rapid propagation on the Rhizoma Paridis plant calluses by inoculating the explants into a callus induction culture medium and a propagation culture medium to be cultured after disinfecting the explants. The method provided by the invention better solves the bottleneck problem of the traditional Rhizoma Paridis plant on the aspect of tissue culture and lays a substantial theoretical foundation on carrying out all biotechnology researches on the Rhizoma Paridis plant in a deep-going way in future.

Description

A kind of is the callus culture method of explant with the rhizoma paris rhizome

Technical field

The present invention relates to a kind of Paris polyphylla method for tissue culture, particularly relating to a kind of is the callus culture method of explant with the rhizoma paris rhizome.

Background technology

Paris polyphylla is one of rare traditional Chinese medicine, and the whole world has 24 kinds, and China has 19 kinds, and wherein kind and the resource with the each province, southwest is particularly abundant again, like Yunnan, Sichuan, Guizhou and other places.The main active ingredient of Paris polyphylla is steroid saponin, and its aglycon is mainly the diosgenin and the pennogenin of different spirostan alcohols, and contains compounds such as amino acid, sterone, moulting hormone, flavonoid glycoside.Modern pharmacological research shows; That Paris polyphylla has is antitumor, hemostasis, effect such as relieving cough and asthma, antibiotic, antiviral; Its good curative effect has been used for the use of medicament by numerous pharmaceuticals industries, be the main component of important Chinese patent drugs such as Yunnan Baiyao, palace blood are peaceful, the heat poison is clear.

Along with the fast development of traditional Chinese medicine industry, the demand of Paris polyphylla medicinal material just increases progressively with the amplitude in every year 20%, and its wild resource reduces year by year, causes the Paris polyphylla price to escalate, and has seriously restricted the output and the quality of pharmacy corporation.Current, Paris polyphylla is mainly by excavating wild resource, owing to excessively excavate for a long time, and lack protection to its ecotope, and cause wild Paris polyphylla resource to suffer destructive destruction, endangered at present.

Current Paris polyphylla mainly is to lean on seed and rhizome to breed; Because there is the embryo ateliosis in the Paris polyphylla seed, endosperm is hard, and plumular axis after-ripening etc. are " dual dormancy " characteristic obviously; Therefore the Paris polyphylla seed is after planting needing experience could sprout into seedling two winters usually; Be typical " biennial seed ", and the Paris polyphylla seeding ratio is very low, growth cycle is long under nature, from emerging to becoming a useful person, needs the time in 8～10 years; And use rhizoma paris rhizome to nourish and generate, because rhizoma paris rhizome itself is exactly its medicinal active component, therefore utilizes the rhizome stripping and slicing to produce the Paris polyphylla seedling and have problems such as sowing quantity is big, production cost height.

Utilizing tissue culture technique to produce medicinal plant, to have a growth cycle short, and reproduction rate is high, can effectively reduce the advantages such as adverse effect of disastrous weather to plant growing, can carry out the anniversary cultivating and producing, and condition of culture is superior, and is very favourable to plant growing; Therefore to carry out the quick breeding of Paris polyphylla plant be to solve current Paris polyphylla medicinal material shortage and the effective ways of protecting its wild plant resource better to the application organizes cultural method.

Up to the present, the Study on tissue culture of Paris polyphylla plant does not also achieve satisfactory results, and minority achievement report is only arranged.Sichuan Teachers University journal (natural science edition) the 29th volume the 1st phase (in January, 2006) has been published " inducing and cultivating of paris plant callus " that people such as Lie group write; The author has carried out the callus induction cultivation with the different parts of Paris polyphylla plant as explant in the literary composition; Find that Paris polyphylla root and blade can not induce callus; And rhizome, ovary and young shoot can induce callus to some extent, are prone to fibrillatable but experimental result shows the callus that rhizome forms, and do not possess multiplication capacity; In addition, they find that the rhizome in seedling stage is difficult to induce the generation callus.

Summary of the invention

The object of the present invention is to provide a kind of is the callus culture method of explant with the rhizoma paris rhizome, and this method can realize that with the rhizoma paris rhizome of falling the seedling stage be explant effectively inducing and fast breeding callus.

For achieving the above object, the solution that the present invention adopts comprises the following steps:

(1) explant selection: the rhizome of choosing the Paris polyphylla of falling the seedling stage; Downcut with preceding two sections of cutter, behind the otch with ash processing cutting-out section the section of cutting-out is transplanted in detritus soil, when treating that it has new root to grow rhizome band bud; Dig out the transplanting body, subsequent use as explant;

(2) sterilization of explant: earth and the dirt of cleaning the explant surface with flowing water; Explant after will cleaning is again put into Efficacious Disinfeitant solution and is soaked 10～30min; Explant after will soaking then takes out after with flowing water flushing 2～4h and dries, and the explant after will drying changes superclean bench over to, and using concentration earlier is 0.1%～0.2% mercuric chloride, the 6～20min that sterilizes; Use aseptic water washing then 3～6 times, blot the moisture on it with sterilized filter paper again;

(3) inducing culture of callus: cut the sprout of explant front end, the explant after pruning is inserted callus inducing medium 1/2MS+6-BA0.5～2mgL ^-1+ NAA 0～1.0mgL ^-1+ IAA 0～3mgL ^-1+ sucrose 30～50gL ^-1+ agar 6.0～7.0gL ^-1In cultivate;

(4) enrichment culture of callus: the callus after step (4) cultivation is transferred into proliferated culture medium MS+6-BA 0.5～2mgL ^-1+ NAA 0.1～1mgL ^-1+ 2.4-D 0～2mgL ^-1+ sucrose 30～50gL ^-1+ agar 6.0～7.0gL ^-1In cultivate;

The pH value of above-mentioned all medium is 5.6～6.5,18～28 ℃ of cultivation temperature, illumination every day 8～16 hours, intensity of illumination are 1000～1800lx.

The tissue culture of Paris polyphylla plant does not obtain promising result yet at present, and its main cause is in the selection of explant and carries out that obvious breakthrough is not arranged on effective inducing culture yet.

The present invention is directed to the Paris polyphylla medicinal material and generally falling the characteristics of gathering seedling stage; The special rhizoma paris rhizome that adopts seedling stage; With its through preliminary treatment in early stage after as the explant of tissue culture, through after the sterilization it being inserted in callus inducing medium, the callus that can obtain fine quality, fast growth and can breed in a large number; Solved the bottleneck problem that current Paris polyphylla plant exists preferably aspect tissue culture, can realize the Paris polyphylla short time, produce low-costly and in high volume.

Embodiment

Embodiment 1

(1) explant selection: the rhizome of choosing the Paris polyphylla of falling the seedling stage; Downcut with preceding two sections of sharp cutter rhizome band bud; It is little and level and smooth that wound is made every effort to, and behind the otch with ash processing cutting-out section the section of cutting-out transplanted in detritus soil, when treating that it has new root to grow; Dig out the transplanting body, subsequent use as explant;

(2) sterilization of explant: earth and the dirt of cleaning the explant surface with flowing water; Explant after will cleaning is again put into Efficacious Disinfeitant solution and is soaked 15min, and the explant after will soaking then takes out after with flowing water flushing 2h and dries, and the explant after will drying changes superclean bench over to; Using earlier concentration is 0.1% mercuric chloride sterilization 2 times; Each sterilization 6min uses aseptic water washing 5 times then, blots the moisture on it with sterilized filter paper again;

(3) inducing culture of callus: cut the anterior sprout of explant, the explant after pruning is inserted callus inducing medium 1/2MS+6-BA1.0mgL ^-1+ IAA 2mgL ^-1+ sucrose 30gL ^-1+ agar 6.5gL ^-1In cultivate, cultivate after 60 days, the inductivity of adding up its callus is 31.3%;

(4) enrichment culture of callus: choose the callus faster of growing in the step (4), it is gone into proliferated culture medium MS+6-BA2.0mgL in the size back switching that is cut on the super-clean bench about 0.5cm ^-1+ NAA 1.0mgL ^-1+ 2.4-D 0.5mgL ^-1+ sucrose 30gL ^-1+ agar 6.5gL ^-1In cultivate, cultivate after 60 days, its callus propagation multiple is 2.15 times;

The pH value of above-mentioned all medium is 6.5,25 ℃ of cultivation temperature, illumination every day 12 hours, intensity of illumination are 1500lx.

Embodiment 2

Change the callus inducing medium in embodiment 1 step (3) into 1/2MS+6-BA1.0mgL ^-1+ IAA2.0mgL ^-1+ NAA 0.5mgL ^-1+ sucrose 30gL ^-1+ agar 6.5gL ^-1, other step is with embodiment 1, and inducing culture is after 60 days, and the inductivity of its callus is 33.4%.

Embodiment 3

Change the callus proliferated culture medium in embodiment 1 step (4) into MS+6-BA2.0mgL ^-1+ NAA0.3mgL ^-1+ sucrose 30～50gL ^-1+ agar 6.0～7.0gL ^-1, other step is with embodiment 1, and enrichment culture is after 60 days, and callus propagation multiple is 1.75 times.

Claims (1)

1. one kind is the callus culture method of explant with the rhizoma paris rhizome, it is characterized in that comprising the steps:

(1) explant selection: choose the rhizome of the Paris polyphylla of falling the seedling stage, downcut with preceding two sections of cutter, behind the otch with ash processing cutting-out section with rhizome band bud; The section of cutting-out is transplanted in detritus soil; When treating that it has new root to grow, dig out the transplanting body, subsequent use as explant;

(2) sterilization of explant: earth and the dirt of cleaning the explant surface with flowing water; Explant after will cleaning is again put into Efficacious Disinfeitant solution and is soaked 10～30min; Explant after will soaking then takes out after with flowing water flushing 2～4h and dries, and the explant after will drying changes superclean bench over to, and using concentration earlier is 0.1%～0.2% mercuric chloride, the 6～20min that sterilizes; Use aseptic water washing then 3～6 times, blot the moisture on it with sterilized filter paper again;

(3) inducing culture of callus: cut the sprout of explant front end, the explant after pruning is inserted callus inducing medium 1/2MS+6-BA0.5～2mgL ^-1+ NAA 0～1.0mgL ^-1+ IAA 0～3mgL ^-1+ sucrose 30～50gL ^-1+ agar 6.0～7.0gL ^-1In cultivate;

(4) enrichment culture of callus: the callus after step (4) cultivation is transferred into proliferated culture medium MS+6-BA 0.5～2mgL ^-1+ NAA 0.1～1mgL ^-1+ 2.4-D 0～2mgL ^-1+ sucrose 30～50gL ^-1+ agar 6.0～7.0gL ^-1In cultivate;

The pH value of above-mentioned all medium is 5.6～6.5,18～28 ℃ of cultivation temperature, illumination every day 8～16 hours, intensity of illumination are 1000～1800lx.