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Spinach transgenosis method

A technology of transgenic and spinach, applied in the field of plant genetic transformation, can solve the problems of easy to produce deformed regenerated seedlings, long culture period, large genotype dependence, etc., to improve ergonomic problems, reduce spinach genotype dependence, reduce effect of risk

Pending Publication Date: 2022-06-24
SYNGENTA BIO TECH CHINA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the disadvantages of obtaining transgenic plants through embryogenic callus are that the culture period is long, the genotype dependence is large, and deformed regenerated seedlings are easily produced.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Agrobacterium transformation method using mature seeds as explants

[0035] 1.1. Construct spinach transformation vector p24545 and transfer it into Agrobacterium EHA101 for use

[0036] p24545 is a binary transformation vector, including two gene expression cassettes, namely, the first expression cassette is GFP gene driven by tobacco mosaic virus 35S promoter; the second expression cassette is Arabidopsis thaliana EF promoter driven aadA gene.

[0037] 1.2. Obtainment of transgenic spinach plants

[0038] figure 1 The transformation workflow using mature seeds as explants is shown.

[0039] Spinach seed disinfection: Select the seeds of spinach variety 1 with agronomic properties, 70% alcohol surface disinfection for 3 minutes, deionized water containing 0.1% Tween-20 at 30-60 ° C at 160-200 rpm Treated on a shaker for 40-80 minutes; after that, in a 1% sodium hypochlorite solution containing 0.1% Tween-20 at 30-60 °C on a shaker at 160-200 rpm for 40-80...

Embodiment 2

[0080] Example 2: Agrobacterium transformation method using spinach seedlings as explants

[0081] 2.1. The spinach transformation vector p24545 was constructed and transformed into Agrobacterium EHA101 for use.

[0082] 2.2. Obtainment of transgenic spinach plants

[0083] Spinach seed sterilization: (see section 1.2 in Example 1).

[0084] Spinach seed germination: The seeds were placed on the seed germination medium SIM14 (Table 27) for germination and growth for 7 days at a temperature of 21±1°C and dark conditions.

[0085] Isolation of explants: After 7 days of germination, hypocotyls containing meristems were obtained, cotyledons and true leaves and the bottom of epicotyls were excised, and the remaining approximately 3 mm long tissue was used as explants for transformation.

[0086] Agrobacterium infection: Resuspend Agrobacterium using liquid medium spCMliq (Table 13) with 40 mg / L AS and adjust OD660 to 0.5 for later use. Infection: Place 30-60 explants in a 50ml c...

Embodiment 3

[0118] Example 3: Biolistic Transformation Method Using Mature Seeds as Explants

[0119] 3.1. The spinach transformation vector p24545 was constructed and linearized at the AatII site.

[0120] 3.2. Obtainment of transgenic spinach plants

[0121] Spinach seed sterilization: (see section 1.2 in Example 1).

[0122] Spinach seed germination: (see section 1.2 in Example 1).

[0123] Pretreatment of explants: One day after germination, the seeds were dissected, the seed coats, cotyledons and true leaves were removed, and the plant tissue was placed on the hypertonic medium SposmT1 (Table 25) for 4-6 hours in the dark at 21°C , the explants were placed in the center of the medium.

[0124] Preparation of ammunition: Mix an appropriate amount of gold powder, calcium chloride, spermidine and DNA, and wrap the DNA on the surface of the gold powder.

[0125] Gold powder carries DNA to bombard explants: After the explants are pretreated for 4-6 hours, the treated materials are pla...

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Abstract

The invention relates to the field of plant genetic transformation, in particular to genetic transformation of spinach. Specifically, the invention establishes a novel spinach transgenic method which can be carried out by adopting agrobacterium tumefaciens or a gene gun.

Description

technical field [0001] The present application relates to the field of plant genetic transformation, particularly to the genetic transformation of spinach. Specifically, the present invention establishes a new spinach transgenic method, which can be carried out using Agrobacterium or gene gun. Background technique [0002] Spinach (Spinacia oleracea L.) is one of the most important green leafy vegetables in the world, it is rich in many valuable nutrients and antioxidant factors and minerals [1] , such as vitamins, carotenoids, flavonoids, and wheat phenolics [2] . [0003] At present, there have been some successful reports of regeneration systems. As early as 1996, Japanese scientists Komai et al. used 6 basal media to induce callus on cotyledons, epicotyls, roots and leaves of spinach seedlings from 8 varieties, of which 3 varieties showed 60% embryogenic callus. injury induction rate. In 1997, Knoll et al. took the root tips and the middle of the roots of spinach se...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/84A01H4/00A01H5/00A01H6/02
CPCC12N15/8205C12N15/8207A01H4/001A01H4/008
Inventor 王艳丽文琴许建平但颖晖
Owner SYNGENTA BIO TECH CHINA
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