CN105594596A - Tissue culture method for strawberry virus-free and rapid propagation for large-scale production - Google Patents

Abstract

The invention relates to a tissue culture method for strawberry virus-free and rapid propagation for large-scale production. The tissue culture method includes following steps: (A), selecting materials and performing first-generation culture: selecting a strong creeping stem, using 75% alcohol to disinfect the surface of the creeping stem on a super-clean workbench for 0.5 min, using 3% sodium hypochlorite to disinfect for 10 min, using sterile water to flush for three times, cutting out a stem tip of 1cm, and inoculating the stem tip on a first-generation induced culture medium; (B), detoxifying the stem tip: after half a month, disposing a stem tip tissue culture seedling obtained from the creeping stem at 36 DEG C for culture for 10 days, detoxifying for the first time, continuing to inoculate in the first-generation induced culture medium for culture, and repeating detoxification once after two months; (C), performing regeneration multiplication culture: transferring a tissue culture seeding which grows out into a regeneration culture medium for multiplication culture; (D), performing rooting culture: after the seedling grows to 4-5 cm, transferring the seedling into a rooting culture medium for culture for about 15 days, hardening for 3 days after 4-5 roots grow out, planting the seedling in a 50-hole pan, and transplanting virus-free strawberry seedling into a large field for propagation after 2 months.

Description

A kind of tissue culture method of the strawberry detoxifying fast breeding for large-scale production

Technical field

The processing that the present invention relates to strawberry cultivates field, particularly relates to a kind of for large-scale productionThe tissue culture method of strawberry detoxifying fast breeding.

Background technology

Strawberry is rose family Fragaria herbaceos perennial, belongs to polyploid plant. Strawberry fruit is littleBerry, bright-coloured beautiful, soft and succulency, sour-sweet moderate, fragrance is strong, is one of seven large fruit in the world. MountainSun area, Dongguo is as the first export of farm produce in northern Shandong city of earning foreign exchange, and strawberry is favored deeply, in recent yearsDevelop very soon, cultivated area also constantly expands, but the Strawberry Seedlings that is used as cultivation is all vegetative propagation, and withVirus infection area and expand year by year, seriously reduced the yield and quality of strawberry. It is reported, strawberry quiltA kind of virus, cryptovirus infect, and output can decline 21～35%, two or more viral Combined Infections, outputCan decline 43～59%, and fruit is few, poor quality, acidity increases, and sugar content reduces. Use chemicalsVirosis is carried out to prevention effect very micro-, upper adopt virus-free seedling control virosis therefore produce moreSpread. The training of stem apex detoxify group is to obtain one of important channel of virus-free plant.

At present, the main flow process of Strawberry Seedlings detoxification: adopt MS culture medium, choose the healthy and strong strain stolon in fieldPoint, cleaning and sterilization sterilization is peeled off stem apex under superclean bench anatomical lens, the training of access inoculation mediumSupport, cultivate through shoot proliferation, in bottle, take root, then transplant nutritive cube and cultivate original silkworm egg seedling, field is expandedNumerous former seedling, field proliferating breeding seedling, finally for Production of Large Fields. For seedling detoxification more thorough,Part Study personnel utilize the detoxification of high temperature bond secondary.

In this process, we find, the nutritional agents mother liquor of configuration was used after a period of time, had occurredFlocculent deposit. In order not affect detoxic seedling breeding, have to outwell reprovision; On the other hand, from fieldOr the stolon material taked of greenhouse, after stem apex is peeled off under the microscope, because material is too little, approximately0.3mm, after being inoculated on culture medium, very fast brownization is dead, even add Vc antioxidant be also asThis.

In view of above situation, we have made some improvements the cultivation flow process of strawberry, to taking off for seedlingPoison technique provides useful reference, improves detoxification efficiency, reduces production costs, and meets the fast numerous life of scaleProduce requirement.

Summary of the invention

The object of the invention is to provide a kind of group training side of the strawberry detoxifying fast breeding for large-scale productionMethod, makes its detoxification efficiency that can improve strawberry, improves the utilization rate of mother liquor, cost-saving, can scaleChange and produce.

For addressing the above problem, the invention provides a kind of strawberry detoxifying fast breeding for large-scale productionTissue culture method, comprises the following steps:

(A) selection of material and just culture: select healthy and strong stolon, on superclean bench, use75% alcohol surface sterilization 0.5min, then with 3% clorox sterilization 10min, aseptic water washing threeInferior, cut 1cm stem apex, be inoculated in just on inducing culture;

(B) stem apex detoxify: after two weeks, the Shoot-tip Culture seedling that stolon is obtained is placed in 36 DEG C of cultivations10 days, carry out detoxification for the first time, continue to be inoculated in just for cultivating in inducing culture, after two months,Repeat detoxification once;

(C) shoot proliferation is cultivated: and then the group training seedling rolling bottle growing is increased in subculture mediumGrow cultivation;

(D) culture of rootage: when seedling grows to after 4-5cm, rolling bottle, in root media, is cultivated 15 days left sidesThe right side, root grows to 4-5 bar, and hardening 3 days is planted in 50 hole dishes, and after 2 months, detoxification Strawberry Seedlings canTransplant in land for growing field crops and breed.

Culture environment: except the stem apex detoxify stage, all the other culture environment are intensity of illumination and are controlled at2000-3000LX, temperature is controlled at 20-25 DEG C.

Further, describedly for inducing culture be just: improvement MSH culture medium+2mg/LBA0.1mg/LIBA+ sucrose 30g/L+ agar 6g/L+ distilled water, PH5.8;

Described subculture medium is edible for improveing MSH culture medium+0.3mg/LBA+0.1mg/LIBA+White sugar 30g/L+ agar 6g/L, PH5.8;

Described root media is: 1/2 improvement MSH culture medium+0.5mg/LIBA+ edible sugar20g/L+ agar 6g/L, PH5.8.

Further, described improvement MSH culture medium comprises MS a great number of elements and trace element, organicThing 21 Jin Weita and molysite.

Adopt after such design, the present invention at least has the following advantages:

Strawberry seedling detoxification process after improving, not only can make the making of culture medium more simple,And there is not mother liquor sedimentation problem, and increased medium nutrient content simultaneously, be more conducive to breed materialThe propagation of material; And reduced brownization of material, and in the situation that not adding any antioxidant, materialThe brown rate of material has also reduced.

The inventive method is all obviously better than group training detoxification in the past at the aspect such as method of operating, production costNumerous soon, scale commodity production stable system, the cycle of setting up are shorter, can meet scale de-The fast numerous production of poison.

Detailed description of the invention

The invention provides a kind of tissue culture method of the strawberry detoxifying fast breeding for large-scale production, comprise withLower step:

(A) selection of material and just culture:: select healthy and strong stolon, on superclean bench, use75% alcohol surface sterilization 0.5min, then with 3% clorox sterilization 10min, aseptic water washing threeInferior, cut 1cm stem apex, be inoculated in and start on culture medium;

(B) stem apex detoxify: after two weeks, the Shoot-tip Culture seedling that stolon is obtained is placed in 36 DEG C of cultivations10 days, carry out detoxification for the first time, be inoculated in just and cultivate for continuing in inducing culture, after two months,Repeat detoxification once;

(C) shoot proliferation is cultivated: and then the group training seedling rolling bottle growing is increased in subculture mediumGrow cultivation;

(D) culture of rootage: when seedling grows to after 4-5cm, rolling bottle, in root media, is cultivated 15 days left sidesThe right side, root grows to 4-5 bar, and hardening 3 days is planted in 50 hole dishes, and after 2 months, detoxification Strawberry Seedlings canTransplant in land for growing field crops and breed.

Culture environment: except the stem apex detoxify stage, all the other culture environment are intensity of illumination and are controlled at2000-3000LX, temperature is controlled at 20-25 DEG C.

Described first be improvement MSH culture medium+2mg/LBA+0.1mg/LIBA+ sugarcane for inducing cultureSugar 30g/L+ agar 6g/L+ distilled water, PH5.8;

Described subculture medium is edible for improveing MSH culture medium+0.3mg/LBA+0.1mg/LIBA+White sugar 30g/L+ agar 6g/L, PH5.8;

Described root media is: 1/2 improvement MSH culture medium+0.5mg/LIBA+ edible sugar20g/L+ agar 6g/L, PH5.8.

Described improvement MSH culture medium comprises MS a great number of elements and micro-, organic matter 21 gold medals dimensionsHe and molysite.

We utilize this technique respectively at strawberry cultivars (U.S. 13, sweet Charlie, sweet treasured, Ka Mulusha),Ipomoea batatas kind (Xu's potato 22,, cigarette potato 25, No. 6, purple potato east wind, help black No. 1) and taro have carried outTest, result shows that all kind growing ways are obviously better than cultivating system in the past, breeding coefficient has improved10%-30%; Peel off in stem apex process, stem apex did not occur that brownization was dead again, and brown rate is 0.

The above, be only preferred embodiment of the present invention, not the present invention made to any formOn restriction, those skilled in the art utilize the technology contents of above-mentioned announcement make a little simple modification,Equivalent variations or modification, all drop in protection scope of the present invention.

Claims (3)

1. for a tissue culture method for the strawberry detoxifying fast breeding of large-scale production, it is characterized in that bagDraw together following steps:

(A) selection of material and just culture: select healthy and strong stolon, on superclean bench, use75% alcohol surface sterilization 0.5min, then with 3% clorox sterilization 10min, aseptic water washing threeInferior, cut 1cm stem apex, be inoculated in just on inducing culture;

(B) stem apex detoxify: after two weeks, the Shoot-tip Culture seedling that stolon is obtained is placed in 36 DEG C of cultivations10 days, carry out detoxification for the first time, continue to be inoculated in just for cultivating in inducing culture, after two months,Repeat detoxification once;

(C) shoot proliferation is cultivated: and then the group training seedling rolling bottle growing is increased in subculture mediumGrow cultivation;

(D) culture of rootage: when seedling grows to after 4-5cm, rolling bottle, in root media, is cultivated 15 days left sidesThe right side, root grows to 4-5 bar, and hardening 3 days is planted in 50 hole dishes, and after 2 months, detoxification Strawberry Seedlings canTransplant in land for growing field crops and breed.

Culture environment: except the stem apex detoxify stage, all the other culture environment are intensity of illumination and are controlled at2000-3000LX, temperature is controlled at 20-25 DEG C.

2. the tissue culture method of the strawberry detoxifying fast breeding for large-scale production according to claim 1,It is characterized in that, described for inducing culture be just: improvement MSH culture medium+2mg/LBA+0.1mg/LIBA+ sucrose 30g/L+ agar 6g/L+ distilled water, PH5.8;

Described subculture medium is edible for improveing MSH culture medium+0.3mg/LBA+0.1mg/LIBA+White sugar 30g/L+ agar 6g/L, PH5.8;

Described root media is: 1/2 improvement MSH culture medium+0.5mg/LIBA+ edible sugar20g/L+ agar 6g/L, PH5.8.

3. the tissue culture method of the strawberry detoxifying fast breeding for large-scale production according to claim 2,It is characterized in that, described improvement MSH culture medium comprises MS a great number of elements and micro-, organic matter21 Jin Weita and molysite.