Composition used for plants direct gene conversion

A technology of gene transformation and composition, applied in the field of bioengineering, can solve the problems of low protoplast regeneration frequency, popularization restrictions, safety issues, etc., and achieve the effect of avoiding the establishment of bottlenecks, expanding the scope, and having biological safety

Inactive Publication Date: 2007-08-15
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the low frequency of protoplast regeneration, the difficulty of cultivating and the long cycle, the popularization of this method is limited.
[0007] At the same time, the widely used gene gun and Agrobacterium-mediated plant transformation methods always have potential safety problems caused by exogenous DNA fragments of non-target genes such as selection marker genes and vector backbone sequences.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Rapid and efficient transient expression of GUS gene in onion epidermal cells

[0027] Take the lower epidermis of the onion and use a blade to divide it into tissue pieces of appropriate size, sterilize in 70% ethanol for 1 minute, rinse with MS medium once, sterilize with mercury chloride for 5 minutes, rinse with MS several times, and then culture in hypertonic buffer MS liquid Base + 47g / L mannitol + 47g / L sorbitol for 10-20 minutes.

[0028] The Agrobacterium containing the Ti plasmid was inoculated in the YEB medium containing antibiotics, and cultured with shaking at 28°C and 250rpm until the logarithmic phase of growth (the absorbance of the bacterial solution OD 550 1.8-2.0); ③Centrifuge the cultured bacterial solution at 5000rpm for 10min, and then resuspend the bacterial cell in 1 / 2MS liquid medium (with 20mg / L acetosyringone) + transformation element + 0.01% Dow corning Q2-5211+10mmol / L CaCl 2 +0.1mmol / L arginine, the concentration of the dilute...

Embodiment 2

[0029] Example 2: Efficient and Stable Transformation of a Linear Fragment Containing the GUS Gene Through the Pollen Tube Passage Method

[0030] Preparation of the buffer solution with the Agrobacterium toxin protein virD2 / E2: Agrobacterium LBA4404 containing the Ti plasmid was inoculated in the YEB medium containing 50 mg / L rifampicin and 25 mg / L streptomycin, at 28 ° C and Under the condition of 250rpm, shake culture to the logarithmic phase of growth (absorbance of bacterial solution OD 550 1.8-2.0); Centrifuge the cultured bacterial liquid at 5000rpm for 10min, resuspend the bacterial cells in 1 / 2MS liquid medium to make the bacterial liquid, the concentration of the diluted bacterial liquid is about 2×10 8 cfu / ml, then add acetosyringone to the bacterial solution to a final concentration of 20mg / L, continue shaking culture at 28°C and 250rpm for 3-4 hours, centrifuge at 3000rpm for 10min to remove the bacteria, supernatant is the buffer with virD2 and virE2. Then, the...

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PUM

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Abstract

The invention discloses a composition of plant direct gene transformation, transforming method and appliance, which is characterized by the following: making the component include goal gene and free nucleic acid component of adjusted and controlled element, transforming buffer liquid and transforming assistance component; co-culturing; soaking; surface-daubing; injecting; dripping; spraying; transforming with dip flower method or through genetic gun bombardment, supersonic wave and electric shock method. This component can be used to receptor plant cell direct transformation of fertilized ovum, suspended cell, protoplast, single-layer cell and diachyma cell and can be used to seed, receptor plant pollen, pistil, flower pillar, pollen pipe and ovary, which possesses impotent meaning for plant genetic seeding, variety improvement and gene functional analysis.

Description

technical field [0001] The invention relates to a composition for direct gene transformation of plants and its application, belonging to the field of bioengineering. Background technique [0002] Plant genetic engineering is developed on the basis of the in-depth development of molecular biology and the improvement of recombinant DNA technology. Many transgenic plants have been obtained in terms of plant disease resistance, insect resistance, weed resistance, and changes in certain plant components, and some have formed strains. In order to improve crop yield, stress resistance, and quality improvement, fast, high-quality, and stable yield varieties Breeding offers a whole new way. In the genetic engineering research of higher plants, the transfer of exogenous genes is an extremely important part. Due to the particularity of plants and their cells, there are also some special requirements for gene transfer methods. Looking at many exogenous gene transfer methods, they can...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82
Inventor 杨君程云清安利佳
Owner DALIAN UNIV OF TECH
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