Ramie highly effective genetic transformation method

A technology of genetic transformation and ramie, which is applied in the field of new plants and plant genetic engineering, and can solve problems such as genetic transformation of ramie cotyledons that have not been seen

Inactive Publication Date: 2008-08-27
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

And, also do not see the report that utilizes ramie cotyledon to carry out genetic transformation at present

Method used

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  • Ramie highly effective genetic transformation method
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  • Ramie highly effective genetic transformation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] The specific method of the present invention using ramie cotyledons as explants for transformation and regeneration is: select mature ramie seed variety 5041-3 seeds (Wang et al., 2007), and the seeds are harvested from the ramie germplasm resource garden of Huazhong Agricultural University. Treat the seeds with 75% alcohol for 30 seconds, then soak them in 10% sodium hypochlorite solution for 12 minutes, rinse them with sterile water for 3 to 4 times, and inoculate them on 1 / 2 MS basic medium (the MS medium used comes from Publicly reported MS basal medium, which includes its inorganic components, organic components and iron salts. The prepared medium was sterilized at 121° C. by autoclaving for 20 minutes). Ramie seeds were cultured into sterile seedlings in vitro. The present invention selects cotyledons of seedlings with a seedling age of 4 to 6 days as the material for transformation (see the "Summary of the Invention" section of this manual for specific steps and ...

Embodiment 2

[0060] On the basis of Example 1, when the resistant bud of Example 1 grows to about 2 centimeters, it is cut off, transferred to selective rooting medium (1 / 2 MS basic medium+0~0.1mg / L naphthalene Acetic acid + 3% glucose + 0.8% agar powder + 100mg / L cephalosporin + 30mg / L kanamycin, add water to 1L, adjust the pH to 5.8) for rooting culture, and place it under the condition of 25±3°C under light Culture (3000lux). Plenty of normal roots can be produced after about 2 weeks. As can be seen from the results in Table 7, the optimal concentration of NAA in the rooting medium is 0.025mg / L.

[0061] Table 7 Effect of concentration of naphthaleneacetic acid (NAA) on rooting of ramie cotyledon-transformed regenerated seedlings

[0062] NAA concentration (mg / L)

Rooting rate

Rooting number

state of the root

0 (CK)

0.001

0.005

0.025

0.05

0.1

100％

100％

100％

100％

100％

100％

6.4±0.7e

9.7±0...

Embodiment 3

[0074] In order to verify the effectiveness of the operation process described in Example 2, the ramie cotyledons of 4 different genotypes of ramie varieties or lines have been used to carry out genetic transformation comparative studies, and the results of table 8 show that: utilize the present invention, tested 4 All genotype ramie varieties (lines) can obtain resistant plants, although the difference in the regeneration frequency of resistant buds is significant between the genotypes, the regeneration frequency is all above 10%, thus proving the broad effectiveness of the transformation method of the present invention (i.e. the present invention) The method of invention is applicable to ramie varieties or strains of different origins).

[0075] Table 8 Transformation efficiency of different genotypes of ramie by transformation method of the present invention

[0076]

[0077] Variety sources: Luzhuqing (from the local variety of Hunan Province), Nianhongxuanma (fr...

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Abstract

The invention discloses a method for constructing a ramie genetic transformation system, which belongs to the agricultural biological technical field or the plant genetic engineering field. The method mainly comprises following steps: taking a ramie seedling cotyledon as a transformation explant to be cocultured with agrobacterium, then, picking up the cotyledon which is cocultured, transferring the ramie seedling cotyledon on selective medium to eliminate bacteria and to culture selectively, and transferring a resistant plant which is produced in the selective medium in selective elongation medium and selective rooting medium to carry out elongation culture and rooting culture successively until a resistant bud is regenerated into a complete plant. The resistant bud which is obtained is respectively checked through GUS, PCR and Southern hybridization and the result shows that a foreign gene is integrated in a ramie genome. The method of the invention has simple operation, materials are conveniently obtained, the transformation efficiency is high, and the method can be widely used in genetic engineering operations of the ramie.

Description

technical field [0001] The invention belongs to the technical field of new plants and plant genetic engineering. In particular, it relates to a high-efficiency genetic transformation method of ramie and its application. Background technique [0002] After more than 20 years of research, people have established a variety of plant genetic transformation systems (methods), which can generally be divided into the following three categories: (1) DNA direct introduction method. Including chemically induced direct DNA transformation (PEG-mediated method, liposome-mediated method) and physical method-induced direct DNA transformation (electric shock method, ultrasound-mediated, microinjection, laser microbeam-mediated, gene gun method-mediated) . (2) Germplasm system mediated gene transformation (germ line transformation). Including pollen tube passage method, germ cell soaking method, embryo sac, ovary injection method. (3) Vector-mediated gene transformation. Including Agroba...

Claims

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Application Information

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IPC IPC(8): C12N15/09C12N15/82C12N15/87C12N5/10A01H4/00C12N15/84
Inventor 汪波彭定祥孙珍夏高世梅张娜邢秀龙刘立军
Owner HUAZHONG AGRI UNIV
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