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Method and medium formula capable of increasing sprouting ratio of immature wheat embryos

A technology for inducing culture medium and immature embryos, applied in the field of improving germination rate of wheat immature embryos, methods and medium formulations, can solve problems such as difficulty in achieving scale, low transformation efficiency, and low frequency of DNA introduction, and achieve embryonic healing Effect of injury occurrence rate improvement

Active Publication Date: 2017-03-15
BEIJING NEXT GENERATION HYBRID WHEAT BIOTECHNOLOGY CO LTD
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

[0002] Wheat is the last important cereal crop successfully transformed. On the one hand, wheat is a hexaploid crop with a complex genetic background and a relatively large genome (about 17Gb), which is 7 times that of corn and 40 times that of rice; , during the process of wheat genetic transformation, the frequency of DNA introduction is low and the regenerative ability after transformation is not high, which has a strong genotype dependence
Since the first case of transgenic wheat was successfully obtained in 1992, people have tried to transform wheat using various transformation methods and various explants, including Agrobacterium, gene gun, pollen tube passage, ultrasonic method, ion beam implantation, laser Microbeam puncture method and PEG method, etc. Explants include immature embryos, mature embryos, anther callus and young panicles, etc., and some progress has been made in reducing the genotype dependence of transformation and improving transformation efficiency, but generally The problem is that the conversion efficiency is still very low and it is difficult to achieve scale

Method used

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  • Method and medium formula capable of increasing sprouting ratio of immature wheat embryos
  • Method and medium formula capable of increasing sprouting ratio of immature wheat embryos
  • Method and medium formula capable of increasing sprouting ratio of immature wheat embryos

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1. Preparation of culture medium

[0020] The medium formulations used in the present invention are shown below.

[0021] Induction medium MSV4: CaCl 2 2.99mM, KNO 3 18.79mM, NH 4 NO 3 20.61mM, KH 2 PO 4 1.25mM, MgSO 4 1.50mM, MnSO 4 1mM, ZnSO 4 30 μM, H 3 BO 3 100 μM, KI 5 μM, NaMoO 4 1 μM, CuSO 4 0.1 μM, CoCl 6 0.1 μM, FeSO 4 100 μM, Na 2-EDTA100μM, Niacin 8μM, Inositol 0.56mM, Thiamine Hydrochloride 30μM, Pyridoxine Hydrochloride 4.9μM, Glycine 0.027mM, Glutamine 0. 2 mM, hydrolyzed casein 250 mg / L, sucrose 30g / L, pH5.8, phytagel 4g / L, 121°C, 20min, add Dicamba after sterilization 3 mg / L.

[0022] Induction medium MSV5: CaCl 2 2.99mM, KNO 3 18.79mM, NH 4 NO 3 20.61mM, KH 2 PO 4 1.25mM, MgSO 4 1.50mM, MnSO 4 1mM, ZnSO 4 26 μM, H 3 BO 3 81 μM, KI 4.5 μM, NaMoO 4 1 μM, CuSO 4 0.1 μM, CoCl 6 0.1 μM, FeSO 4 100 μM, Na 2 -EDTA 100μM, Niacin 4.1μM, Inositol 0.56mM, Thiamine Hydrochloride 0.3μM, Pyridoxi...

Embodiment 2

[0023] Example 2. Pre-cultivation of immature embryos

[0024] Young wheat embryos with a diameter of 0.8-1.5 mm 12-16 days after wheat pollination are collected for pre-cultivation. The purpose of pre-culture is to dedifferentiate the immature embryos under the action of hormones to produce callus, and the resulting callus can be used as the real recipient of gene gun bombardment. The callus produced by the dedifferentiation of immature embryos is divided into embryogenic callus and non-embryogenic callus. Only embryogenic callus can re-differentiate into complete plants under the action of hormones. Therefore, the high rate of embryogenic callus is The basis for high conversion efficiency.

[0025] Specifically, 12-16 days after wheat pollination, put the immature seeds on the ears of wheat into a sterilized Erlenmeyer flask, add 75% ethanol to shake and wash for 1 min, and wash with sterilized water three times; add 1% silver nitrate and put them in a shaker Shake at 170r...

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Abstract

The invention specifically relates to a method and medium formula capable of increasing the sprouting ratio of immature wheat embryos, belonging to the field of plant transformation. The invention provides an induction medium MSV4 for the embryonic calluses of the immature wheat embryos. The induction medium MSV4 can improve the induction rate of the embryonic calluses of the immature wheat embryos and is characterized by comprising glycine with a final concentration of 0.027 mM, glutamine with a concentration of 0.1 to 2.57 mM, casein hydrolysate with a concentration of 50 to 500 mg / L and Dicamba with a concentration of 0.5 to 52 mg / L. A high-efficiency wheat embryo cultivation system is established in the invention and is of great significance to subsequent functional genomics research on wheat and molecular breeding of wheat.

Description

technical field [0001] The invention belongs to the field of plant transformation, and in particular relates to a method for improving the germination rate of young wheat embryos and a medium formula thereof. Background technique [0002] Wheat is the last important cereal crop successfully transformed. On the one hand, wheat is a hexaploid crop with a complex genetic background and a relatively large genome (about 17Gb), which is 7 times that of corn and 40 times that of rice; , during the process of genetic transformation of wheat, the frequency of DNA introduction is low and the regenerative ability after transformation is not high, which has a strong genotype dependence. Since the first case of transgenic wheat was successfully obtained in 1992, people have tried to transform wheat using various transformation methods and various explants, including Agrobacterium, gene gun, pollen tube passage, ultrasonic method, ion beam implantation, laser Microbeam puncture method an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01H4/00
CPCA01H4/001A01H4/008
Inventor 李健王峥马力耕邓兴旺
Owner BEIJING NEXT GENERATION HYBRID WHEAT BIOTECHNOLOGY CO LTD
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