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Mature barley embryo tissue culture method for inhibiting sprout and rooting and culture medium used

A technology for inhibiting germination and tissue culture, which is applied in the field of plant tissue culture, and can solve problems such as easy germination/root growth, high pollution rate of tissue culture, and low green seedling differentiation rate

Inactive Publication Date: 2013-11-20
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The technical problem to be solved by the present invention is to provide a method for effectively inhibiting germination / rooting of mature barley embryo tissue culture and the culture medium used, and adopting this method can solve the problem that the current isolated mature embryo culture of barley is very easy to germinate / root and inhibit callus The phenomenon of tissue growth and the high contamination rate of tissue culture, low green seedling differentiation rate and other problems, using this method can obtain a higher green seedling differentiation rate

Method used

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  • Mature barley embryo tissue culture method for inhibiting sprout and rooting and culture medium used

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Embodiment 1, barley mature embryo in vitro culture medium, is made up of these 3 types of culture medium of callus induction medium, differentiation medium and rooting strong seedling medium:

[0054] 1), induction medium for callus induction:

[0055] First prepare the induction base solution:

[0056] The induction base fluid is composed of the following components:

[0057] KNO 3 2830mg / L, (NH 4 ) 2 SO 4 460mg / L, KH 2 PO 4 400mg / L, MgSO 4 ·7H 2 O 185mg / L, CaCl 2 2H 2 O 165mg / L, MnSO 4 4H 2 O 22.3mg / L, ZnSO 4 ·7H 2 O 8.6mg / L, H 3 BO 3 6.2mg / L, Na 2 MoO 4 2H 2 O 0.25mg / L, CuSO 4 ·5H 2 O 1.25mg / L, CoCl 2 ·6H 2 O 0.025mg / L, KI 0.83mg / L, Na 2 -EDTA·2H2 O 37.3mg / L, FeSO 4 ·7H 2 O 27.8mg / L, proline 600mg / L, glutamine 250mg / L, hydrolyzed casein 400mg / L, inositol 100mg / L and sucrose 30g / L, the rest is distilled water.

[0058] The preparation method of the induction base solution is as follows:

[0059] ①. Weigh KNO separately 3 28.3g, (NH ...

Embodiment 2

[0082] Embodiment 2, a method for in vitro culture of mature barley embryos, using the corresponding culture medium in Example 1, using the barley variety "Zhepi No. 8" as the experimental material and proceeding as follows:

[0083] 1), after cleaning the mature seeds with full grains of "Zhepi No. 8", peel off part of the shell by hand to expose the embryo, then soak the seeds in 50% (mass concentration) sodium hypochlorite solution for 30 minutes, discard Add sodium hypochlorite solution and add 70% (v / v) alcohol to soak for 5 minutes, then discard 70% alcohol and add 3% (v / v) hydrogen peroxide to soak for 5 minutes, and finally wash with sterilized water for 5-6 times. On a sterile operating table, use a sterilized scalpel to slowly scrape the embryos from the front of the embryo to the back (it may contain part of the endosperm), and inoculate the scraped embryos on the medium for inducing callus (such as figure 1 shown in A).

[0084] 2), put the isolated embryos inocul...

Embodiment 3

[0090] Example 3, according to the method of Example 2, the mature embryo of the barley variety "Golden Promise" was cultured in vitro, the callus induction rate was 87.42%, the pollution rate was 0, and the germination / rooting rate was 0; the green shoot differentiation The rate is 80.95%.

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Abstract

The invention discloses a mature barley embryo in-vitro culture medium comprising a callus inducing culture medium, a differential medium and a rooting and strengthening culture medium. The invention further discloses a mature barley embryo tissue culture method for inhibiting sprout and rooting effectively by using the mature barley embryo in-vitro culture medium, wherein the mature barley embryo tissue culture method comprises the following steps: firstly, an embryo is scraped as an in-vitro embryo so as to be inoculated on the callus inducing culture medium after seeds are disinfected; secondly, the in-vitro embryo inoculated on the callus inducing culture medium is put in a culture box at the temperature of 22 to 26 DEG C for culture in the dark; thirdly, the obtained callus is transferred to the differential medium and is subjected to illumination culture at the temperature of 22 to 26 DEG C until 2 to 3 leaves are grown; fourthly, the green plant is transferred to the rooting and strengthening culture medium for illumination culture at the temperature of 22 to 26 DEG C until roots of more than or equal to 2 cm are grown; and fifthly, the green plant is placed indoor for 1-2 days, the culture medium on the roots is washed, and then a plant capable of being transplanted is obtained.

Description

technical field [0001] The invention belongs to the field of plant tissue culture, and in particular relates to a method for tissue culture of mature barley embryos that can effectively inhibit germination and a corresponding tissue culture system. Background technique [0002] Barley tissue culture is not only an effective way to improve barley varieties, but also an important part of barley genetic transformation. Currently, barley tissue-cultured explants include apical meristems, anthers, immature embryos, and mature embryos. Among them, immature embryos are considered to be the most ideal explant material, and the callus induction rate and plant regeneration rate are relatively high. The reports of obtaining transgenic barley basically use immature embryos as recipients. However, the selection of barley immature embryos is limited by time, space, and seasons, and it is difficult to grasp the appropriate time for sampling, which restricts the effective development and a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/04A01H4/00
Inventor 华为杨建明汪军妹朱靖环贾巧君尚毅林峰顾玉坤
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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