Induction medium for culturing callus of barley microspore

A technology for inducing culture medium and callus, applied in the field of plant biology, can solve the problems of poor effect and low callus induction rate

Active Publication Date: 2014-02-19
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, microspore culture of cereal crops has been poorly performed, where low callus induction rate is the main limiting factor

Method used

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  • Induction medium for culturing callus of barley microspore
  • Induction medium for culturing callus of barley microspore
  • Induction medium for culturing callus of barley microspore

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Embodiment 1: the acquisition of callus

[0073]Select middle part floret microspore development to be in the tassel of uninucleate early stage, middle stage from Daejeon, put into refrigerator and refrigerate for 15 days. When inoculating, the tassels are sterilized with saturated bleach solution for 15 minutes, and rinsed with sterile water 3-4 times. Each test tube received 10 tassels, poured 15ml of mannitol 60g / L, CaCl 2 1.1g / L, MES0.976g / L extract, use a 3000rpm high-speed disperser to spin at a high speed, filter with a 150-mesh screen, and centrifuge the filtrate at a low speed of 800rpm, repeat 3 times, and collect microspores. The microspores were pretreated with the extract at 25°C in the dark for 2 days. Induction medium as shown in Table 1 N 6 The medium is basic medium, but Na 2 The concentration of EDTA is 74.6mg / L, FeSO 4 ·7H 2 The concentration of O is up to 55.6mg / L, which also added 2,4,5-T1.0mg / L, KT0.5mg / L, maltose 90g / L, inositol 200mg / L, glu...

Embodiment 2

[0074] Embodiment 2: the acquisition of regeneration plant

[0075] After 21 days of induction culture, the calli were transferred to the differentiation medium. The differentiation medium used 2 / 3MS as the basic medium, added 6-BA0.5mg / L, KT1.5mg / L, NAA0.05mg / L and maltose 30g / L, and the pH was 5.8. Differentiate and cultivate for 25-30 days at 25±1° C. under the condition of 10-12 hours of light per day until green regenerated plants are differentiated.

Embodiment 3

[0077] Prepare different KNO 3 and (NH 4 ) 2 SO 4 The concentration of the modified N 6 Induction medium (except KNO 3 , (NH 4 ) 2 SO 4 , Glu and CH are shown below, the concentration of the rest of the components is the same as in Example 1), according to Example 1, collect 4 parts of barley material microspore-induced callus, culture 28d when the culture medium is blotted and weighed, the results are shown in the following table .

[0078] The yield of callus induced on medium with different CH and Glu concentrations (mg / dish)

[0079] Inorganic nitrogen concentration in induction medium

[0080]

[0081] N 6 -Total Nitrogen: KNO 3 and (NH 4 ) 2 SO 4 The concentrations of Glu and CH were 2830mg / L and 463mg / L respectively, and Glu and CH were 800mg / L each.

[0082] N 6 -3 / 4 Nitrogen: KNO 3 and (NH 4 ) 2 SO 4 The concentration of nitrogen is 3 / 4 of total nitrogen, that is, 2122.5mg / L and 347.3mg / L respectively, Glu and CH are 600mg / L each.

[0083] N 6...

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Abstract

The invention provides an induction medium for culturing a callus of a barley microspore. The induction medium is prepared on the basis of a formula of an N6 culture medium, but the callus induction medium has the KNO3 concentration of 0-2500mg/ L, the (NH4) SO4 concentration of 0-400mg/ L, and is added with 400-2500mg/ L of casein hydrolyzate and/ or 400-2500mg/ L of glutamine. The invention also provides a method for culturing the callus of the barley microspore and obtaining regenerated plants, and a kit containing the induction medium, an optional differential medium and a rooting and seedling-strengthening medium. The whole process is carried out under controlled conditions in a laboratory; results are real and reliable; after differentiation of the obtained callus, plenty of homozygous regenerated plants can be obtained.

Description

technical field [0001] The invention belongs to the field of plant biotechnology, and relates to a method for improving the induction medium of cereal crop microspores to obtain a large amount of callus and doubling haploid (DH) regeneration plants. Background technique [0002] Plant cells are totipotent, that is, each cell contains all the genetic information of the species, thus having the genetic ability to develop into a complete plant. Under the right conditions, any cell can develop into a new individual. Plant cell totipotency is the theoretical basis of plant tissue culture. Plant microspores are a kind of cells in a special developmental stage, which have the characteristics of haploid and single cells. Therefore, the microspore culture system can be used to study the mechanism of cell dedifferentiation and redifferentiation, and to construct doubled haploid (DH) mapping. Populations, mutant screening, haploid protoplast culture and fusion, and haploid transgenic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01H4/00A01H5/00C12N5/04
Inventor 陆瑞菊黄剑华王亦菲陈志伟刘成洪杜志钊何婷高润红徐红卫郭桂梅
Owner SHANGHAI ACAD OF AGRI SCI
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