CN102301957B - 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 callus culture method take rhizoma paris rhizome as explant

Technical field

The present invention relates to a kind of Paris polyphylla method for tissue culture, particularly relate to a kind of callus culture method take rhizoma paris rhizome as explant.

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, such as Yunnan, Sichuan, Guizhou and other places.The main active ingredient of Paris polyphylla is steroid saponin, and its aglycon is mainly diosgenin and the pennogenin of different spirostan alcohols, and contains the compounds such as amino acid, sterone, moulting hormone, flavonoid glycoside.Modern pharmacological research shows, that Paris polyphylla has is antitumor, hemostasis, the 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 the important Chinese patent drugs such as Yunnan Baiyao, palace blood are peaceful, the heat poison is clear.

Along with the fast development of 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 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 lacks protection to its ecotope, causes wild Paris polyphylla resource to suffer destructive destruction, and existing oneself is endangered.

Current Paris polyphylla mainly is to breed by seed and rhizome, because the Paris polyphylla seed exists embryo development incomplete, endosperm is hard, plumular axis after-ripening etc. are " dual dormancy " characteristic obviously, therefore the Paris polyphylla seed is after planting needing experience two winters could Germination And Seedling 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 the problems such as sowing quantity is large, production cost is high.

Utilize tissue culture technique to produce medicinal plant and have the growth cycle weak point, reproduction rate is high, effectively the reduce the disaster sex climate can carry out cultivating in the anniversary and produce, and condition of culture is superior the advantages such as adverse effect of plant growth, and is very favourable to plant growth; Therefore using the Fast-propagation that method for tissue culture carries out the Paris polyphylla plant is the effective ways that solve current Paris polyphylla medicinal material shortage and protect better its wild plant resource.

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 1st phase of the 29th volume (in January, 2006) has been published " inducing and cultivating of paris plant callus " that the people such as Lie group write, the author has carried out induction of callus 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, but experimental result shows the easy fibrillatable of callus of rhizome formation, 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 callus culture method take rhizoma paris rhizome as explant, the method can realize effectively the inducing and fast breeding callus take the rhizoma paris rhizome of falling the seedling stage as explant.

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

(1) choosing of explant: the rhizome of choosing the Paris polyphylla of falling the seedling stage, downcut with the first two sections of cutter with rhizome band bud, 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, dig out the transplanting body, for 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 ~ 30 min, then the explant after will soaking takes out after with flowing water flushing 2 ~ 4 h and dries, explant after will drying changes superclean bench over to, be first mercuric chloride sterilization 6 ~ 20 min of 0. 1 % ~ 0. 2 % with concentration, then use aseptic water washing 3 ~ 6 times, blot moisture on it with sterilized filter paper again;

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

(4) propagation of callus is cultivated: the callus after step (4) is cultivated is transferred into proliferated culture medium MS+6-BA 0.5 ~ 2 mgL ^-1+ NAA 0.1 ~ 1mgL ^-1+ 2.4-D 0.5 ~ 2 mgL ^-1+ sucrose 30～50 gL ^-1+ agar 6.0～7.0 gL ^-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 1000～1800 lx.

At present the tissue of Paris polyphylla plant is cultivated and is not obtained yet promising result, and its main cause is in the selection of explant and effectively induces obvious breakthrough is not arranged in the cultivation 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 the explant of cultivating as tissue after its process preliminary treatment in early stage, by after the sterilization it being accessed in callus inducing medium, the callus that can obtain fine quality, fast growth and can breed in a large number, solved preferably current Paris polyphylla plant and organized the bottleneck problem that exists aspect the cultivation, can realize the Paris polyphylla short time, produce low-costly and in high volume.

Embodiment

Embodiment 1

(1) choosing of explant: the rhizome of choosing the Paris polyphylla of falling the seedling stage, downcut with the first two sections of sharp cutter with rhizome band bud, it is little and level and smooth that wound is made every effort to, 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, dig out the transplanting body, for 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, then the explant after will soaking washes to take out behind 2 h with flowing water and dries, explant after will drying changes superclean bench over to, it is first the mercuric chloride sterilization 2 times of 0. 1 % with concentration, then each sterilization 6min uses aseptic water washing 5 times, blots moisture on it with sterilized filter paper again;

(3) callus induce cultivation: cut the sprout of explant front portion, with the explant access callus inducing medium 1/2MS+6-BA1.0 mgL after pruning ^-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) propagation of callus is cultivated: the faster callus of growing in the selecting step (4) enters proliferated culture medium MS+6-BA 2.0mgL with it in the rear switching of size that super-clean bench is cut into about 0.5cm ^-1+ NAA 1.0mgL ^-1+ 2.4-D 0.5 mgL ^-1+ sucrose 30 gL ^-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 1500 lx.

Embodiment 2

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

Claims (1)

1. the callus culture method take rhizoma paris rhizome as explant is characterized in that comprising the steps:

(1) choosing of explant: choose the rhizome of the Paris polyphylla of falling the seedling stage, downcut with the first two sections of cutter with rhizome band bud, 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, dig out the transplanting body, for 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 ~ 30 min, then the explant after will soaking takes out after with flowing water flushing 2 ~ 4 h and dries, explant after will drying changes superclean bench over to, be first mercuric chloride sterilization 6 ~ 20 min of 0. 1 % ~ 0. 2 % with concentration, then use aseptic water washing 3 ~ 6 times, blot moisture on it with sterilized filter paper again;

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

(4) propagation of callus is cultivated: the callus after step (4) is cultivated is transferred into proliferated culture medium MS+6-BA 0.5 ~ 2 mgL ^-1+ NAA 0.1 ~ 1mgL ^-1+ 2.4-D 0.5 ~ 2 mgL ^-1+ sucrose 30～50 gL ^-1+ agar 6.0～7.0 gL ^-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 1000～1800 lx.