Cucumber Flamingo-bill explant transgenic method

An explant and transgene technology, applied in the field of transgene, achieves the effect of short regeneration time, high survival rate and high transformation frequency

Inactive Publication Date: 2009-05-13
HENAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

On tomato, someone once used this explant as the recipient of genetic transformation to obtain better results (Pozueta-Romero J, Houlne G, Cafias L, et al.Enhanced regeneration of tomato and pepper seedling explants for Agrobacterium-mediatedtransformation[ J]. Plant Cell Tiss Org Cul, 2001, 67: 173-180), but there is no report on cucumber

Method used

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  • Cucumber Flamingo-bill explant transgenic method
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  • Cucumber Flamingo-bill explant transgenic method

Examples

Experimental program
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Effect test

Embodiment 1

[0082] Follow the steps below

[0083] a. The cucumber inbred line 'IL 69' was used as the material. The inbred line 'IL 69' was isolated from the offspring of Jinza 2. It has the characteristics of strong growth vigor, high resistance to downy mildew, long fruit shape, and ribs. . Disinfect the surface of 50 inbred seeds, inoculate them into MS medium, and cultivate them to form seedlings. The culture conditions are light intensity 54 μmol·m -2 ·s -1 , photoperiod 14h / d, temperature 25°C. A cotyledon and growth point were excised from sterile seedlings with a seedling age of 2 days. The depth of the incision was 1 / 2 of the diameter of the hypocotyl, and the length of the slope of the incision was 0.4 cm to obtain 50 Flamingo-bill explants. And the Flamingo-bill explants were placed in MS liquid medium containing 0.1 mg / L 6-benzyl adenine and 30 g / L sucrose for pre-culture for 2 days.

[0084] b. Pick the colony of Agrobacterium LBA4404 containing the target gene, add it t...

Embodiment 2

[0090] Follow the steps below

[0091] a. The cucumber inbred line 'IL 103' was used as the material. The inbred line 'IL 103' was isolated from the offspring of Jinchun 4. It has strong growth vigor, high resistance to downy mildew, medium fruit shape, no ribs, Prickly and other characteristics. Disinfect the surface of 50 inbred seeds, inoculate them into MS medium, and cultivate them to form seedlings. The culture conditions are light intensity 56 μmol·m -2 ·s -1 , photoperiod 16h / d, temperature 26°C. A cotyledon and growth point were excised from sterile seedlings with a seedling age of 3 days. The depth of the incision was 1 / 2 of the diameter of the hypocotyl, and the length of the slope of the incision was 0.5 cm to obtain 49 Flamingo-bill explants. And the Flamingo-bill explants were placed in MS liquid medium containing 0.3mg / L 6-benzyl adenine and 32g / L sucrose for pre-culture for 3 days.

[0092] b. Pick the Agrobacterium LBA4404 colony containing the target gene...

Embodiment 3

[0098] Follow the steps below

[0099] a. The cucumber inbred line 'IL 67' was used as the material. The inbred line 'IL 67' was isolated from the offspring of Yuanfengyuan No. 6. It has strong growth potential, long growth period, high resistance to downy mildew, and short fruit shape. , No edges and other features. Disinfect the surface of 52 inbred seeds, inoculate them into MS medium, and cultivate them to form seedlings. The culture conditions are light intensity 55 μmol·m -2 ·s -1 , photoperiod 15h / d, temperature 28°C. A cotyledon and growth point were excised from sterile seedlings with a seedling age of 2 days. The depth of the incision was 1 / 2 of the diameter of the hypocotyl, and the length of the slope of the incision was 0.5 cm to obtain 50 Flamingo-bill explants. And the Flamingo-bill explants were placed in MS liquid medium containing 0.5mg / L 6-benzyl adenine and 35g / L sucrose for pre-culture for 2 days.

[0100] b. Pick the colony of Agrobacterium LBA4404 co...

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Abstract

The invention discloses a cucumber Flamingo-bill explant transgene method. In the method, the cucumber Flamingo-bill explant is used as an acceptor for gene transformation, the reported gene GUS gene is transferred in the acceptor by mediating agricultural bacillus, the transformed plant is preliminarily screened by kanamycin to obtain the resistance plant, and the resistance plant is subjected to PCR molecular identification and GUS dyeing to obtain the transgene plant. The method has the following advantages 1. at home and abroad, the Flamingo-bill explant is used as the acceptor to perform the genetic transformation of the cucumber for the first time; 2. the gene transformation frequency is as high as more than 30 percent; 3. the regeneration time of the transgene plant is short, and only 40 to 45 days are needed from selection culture to rooting; 4. the survival rate of the transgene plant is as high as 95 percent; and 5. the method is applied to the genetic transformation of multiple genotypes of the cucumber.

Description

technical field [0001] The invention relates to a transgenic method using cucumber Flamingo-bill explants as receptors, and belongs to the field of plant genetic engineering. Background technique [0002] Cucumber is one of the ten most important vegetable crops in the world, and it is also the largest vegetable crop produced in protected areas in my country (FAO, http: / / faostat.fao.org / faostat, 2005). pivotal role. However, the genetic background of cucumber is narrow, and it is difficult to achieve a breakthrough of useful new varieties through sexual cross breeding (Dijkuizen A, Kennnard WC, Havey MJ, et al. RFLP variability and genetic relationships in cultivated cucumber [J]. Euphytica, 1996, 90: 79- 87). Through gene transfer technology, not only the transfer of genes between different species can be realized, but also the directional improvement of individual traits can be carried out. It is an important technical guarantee for crops with narrow genetic background to...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12Q1/68A01H4/00
Inventor 李建吾胡建斌秦智伟周秀艳
Owner HENAN AGRICULTURAL UNIVERSITY
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