Genetic transformation method of calluses of non-glutinous rice Chuanxiang 29B mature embryos

A method of genetic transformation and callus technology, which is applied in the field of plant tissue culture and genetic engineering, can solve the problems of difficult tissue culture, easy browning, and inapplicability, so as to improve callus induction rate, reduce pre-differentiation steps, and improve The effect of conversion efficiency

Inactive Publication Date: 2016-06-29
NANCHANG UNIV
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Problems solved by technology

However, most indica rice varieties with a wide cultivation area have poor tissue culture characteristics, which are characterized by low callus induction rate and regeneration rate, and easy browning in subsequent subculture, which makes the genetic transformation efficiency of indica rice low, and some varieties are even difficult to obtain. At present, although a few indica rice varieties have established stable tissue culture systems and transformation systems, they cannot be applied to most indica rice varieties due to the large differences in tissue culture characteristics between different indica rice varieties.
Efficient and widely applicable indica rice tissue culture system and transformation system have not yet been established, especially in indica rice varieties that have wide application value in production, there are problems such as tissue culture difficulties and low genetic transformation efficiency, which greatly hinders indica rice gene function research and gene Therefore, it is of great theoretical and practical significance to explore the most suitable medium and method for a certain type or each indica rice variety with important application value, as well as the genetic transformation method,

Method used

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  • Genetic transformation method of calluses of non-glutinous rice Chuanxiang 29B mature embryos
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  • Genetic transformation method of calluses of non-glutinous rice Chuanxiang 29B mature embryos

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Experimental program
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Effect test

Embodiment 1

[0048] Example 1. Chuanxiang 29B mature embryo tissue culture and genetic transformation.

[0049] 1) Callus induction.

[0050] Soak the mature seeds of the indica rice Chuanxiang 29B with 70-75% alcohol for 1 min after shelling, and then soak them with 0.1% HgCl 2 Sterilize for 8 minutes; rinse with sterile water for 6 times, then insert the sterilized mature embryos into the callus induction medium, and culture them for one month at 26-28°C in a dark environment to induce callus, see figure 1 a.

[0051] The formula of the induction medium is: on the basis of the basic components of the NML medium, add 2,4-D3.0mg / L; L-proline0.5g / L; Glutamine0.5g / L; hydrolyzed casein 0.6g / L; sucrose 30.0 g / L; Phytagel3.2g / L, pH5.9.

[0052] For 3 fine indica hybrid rice maintainer lines (Zhong 9B, Ⅱ-32B and Chuanxiang 29B) and 2 restorer lines (9311 and Minghui 63) in 4 rice basic media (MS, N6, NB and NML) The above callus induction was compared.

[0053] Table 1 Comparison of callus...

Embodiment 2

[0093] Example 2. In addition to the tissue culture of mature embryos of the indica rice Chuanxiang 29B, the inventors used the plant tissue culture method of the present invention to tissue culture the mature embryos of four other indica rice varieties 9311, Zhong 9B, II-32B and Minghui 63 , also achieved good results, the average induction rate was between 84% and 88%, and the state of the induced callus was better, and the proliferation rate of the subcultured callus was higher (the multiplication factor was 2.5-4 times), The differentiation rate can reach between 60-80%, which also proves the reliability and wide applicability of the invention.

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Abstract

A method for genetic transformation of mature embryo callus of indica rice Chuanxiang 29B, which includes processes such as callus induction, subculture and differentiation, and genetic transformation. The induced callus has a high induction rate and good quality, and the subculture is not easy to brown and differentiate. High efficiency, and good applicability in other four widely used indica rice varieties (9311, Minghui 63, Zhong 9B, Ⅱ-32B), the regeneration rate is between 60-80%, and its genetic transformation efficiency can be Up to 26% or more.

Description

technical field [0001] The invention belongs to the technical fields of plant tissue culture and genetic engineering. It relates to an Agrobacterium-mediated genetic transformation method suitable for the mature embryo callus of the three-line indica hybrid rice parent Chuanxiang 29B. Background technique [0002] The Agrobacterium-mediated genetic transformation system has become a routine method of genetic transformation and is widely used in rice gene function research and molecular breeding due to its advantages of simple operation, low copy number of T-DNA inserted into the genome, and ability to transform large fragments of DNA. . Rice embryogenic callus is a good recipient of Agrobacterium genetic transformation, and its tissue culture characteristics will directly determine whether Agrobacterium-mediated rice genetic transformation can be successful. Indica rice and japonica rice are two subspecies of cultivated rice. Most japonica rice varieties have good tissue c...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H4/00
CPCC12N15/8205A01H4/001A01H4/008
Inventor 廖鹏飞李绍波彭晓珏阎新朱友林
Owner NANCHANG UNIV
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