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Efficient agrobacterium tumefaciens-mediated melon genetic transformation method

A genetic transformation method, Agrobacterium-mediated technology, is applied in the field of highly efficient Agrobacterium-mediated genetic transformation of melons, which can solve the problems of no melons, and the low ratio of regenerated shoots with clustered buds to take root to form complete plants, so as to increase the number and reduce the number of yellow melons. The effect of conversion rate

Pending Publication Date: 2022-08-02
WUHAN ACADEMY OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Although previous research on muskmelon tissue culture has lasted for more than 30 years, there are still many problems to be studied. For example, although scholars at home and abroad have obtained a high frequency of adventitious bud regeneration through cotyledon explant culture, the extension and regeneration of clustered buds cannot be guaranteed. The rate of seedling rooting and complete plant formation is not high, and so far there is no fixed regeneration system suitable for all melon germplasm resources

Method used

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  • Efficient agrobacterium tumefaciens-mediated melon genetic transformation method
  • Efficient agrobacterium tumefaciens-mediated melon genetic transformation method
  • Efficient agrobacterium tumefaciens-mediated melon genetic transformation method

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Experimental program
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Effect test

preparation example Construction

[0052] The preparation process of bud extension medium MeM4 is (for example, the amount of 100 mL): take finished powder 0.44g, sucrose 3g, agar powder 0.9g, and the composition of plant growth regulator 0.05~0.1mg·L -1 6-BA+0.5~1.0mg·L -1 GA3, make up to 100 mL with double distilled water, adjust the pH to 5.80-5.85, and after sterilization, when the temperature is lowered to 55 °C, add 100 μl of 200 mg / ml inhibitor, 100 μl to 125 μl of 20 to 25 mM silver thiosulfate.

[0053] Apply different types and concentrations of screening agents such as kanamycin (Kan15-25mg / L), hygromycin (Hpt 5-10mg / L), glufosinate-ammonium (Basta 8-10mg / L) according to the selection marker on the genetic transformation vector ), etc., mix evenly and dispense into sterile petri dishes for later use.

[0054] Wherein the method for preparing 20mM silver thiosulfate mother liquor is divided into three steps:

[0055] (1) 100mM sodium thiosulfate preparation method: 158mg sodium thiosulfate is disso...

Embodiment 1

[0074] 1. Regeneration efficiency of different types of melon germplasm on MeM3 medium

[0075] Seed selection: thick-skinned melon types, Dumi No. 5, Jade Mushroom, Jiucuimei, Snow Honey, Lemon Honey, Thin-skinned Melon Melon, Green Honey, and Wu Nong Qingyu;

[0076] S1, inoculate on seed germination medium MeM1 after seed selection and sterilization and cultivate in dark, for subsequent use;

[0077] Specifically: selected plump melon seeds were soaked in water at 50°C for 15 minutes, then soaked in tap water for 1 hour, and then rinsed the seeds twice with tap water. Gently cut the side surface of the seeds with a knife, and place the peeled seeds in a sterilized conical flask.

[0078] Seed disinfection steps: soak in 70% ethanol for 30s, and shake gently, then soak the seeds in 0.1% mercuric chloride for 8 minutes, shake gently during the period, rinse the seeds with sterile water 3 times, 5 minutes each time, end Then use sterile filter paper to absorb water for later...

Embodiment 2

[0094] The aforementioned S1-S3 steps in this example are the same as those in Example 1, and 0.5 mg·L was selected in MeM3 medium -1 6-BA+0.5mg·L -1 Zeatin+0.08mg·L -1 IAA as a plant growth regulator in induction regeneration medium;

[0095] S4, the bud cluster regenerated on the step S3 medium is cut off and placed in the bud extension medium MeM4 and continues to screen and cultivate, until the bud is extended to 2cm and then transferred to the rooting medium (such as Figure 1D );

[0096] 1. The bud elongation rate of different types of melon germplasm on MeM4 medium (the bud length is 1cm-1.5cm is the statistical value), as shown in the following table 3-6:

[0097] Among them, the preparation process of bud extension medium MeM4 is (for example, the amount of 100mL): take finished product powder 0.44g, sucrose 3g, agar powder 0.9g, and the composition of plant growth regulator 0.05~0.1mg·L -1 6-BA+0.5~1.0mg·L - 1 GA3, make up to 100 mL with double distilled wate...

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Abstract

The efficient agrobacterium tumefaciens-mediated melon genetic transformation method comprises the following steps: S1, selecting seeds, sterilizing, inoculating the seeds to a seed germination culture medium MeM1, and carrying out dark culture for later use; s2, performing dip-dyeing on cotyledon explants cut off from the robust melon seedlings growing in the step S1 by using an agrobacterium dip-dyeing culture medium IM suspension bacterium solution, and finally, putting the dip-dyed cotyledon explants into an agrobacterium co-culture medium MeM2 for co-culture for later use; s3, placing the cotyledon explant subjected to co-culture in the step S2 on an induced bud regeneration culture medium MeM3 for culture, and subculturing once every two weeks for later use; s4, cutting off bud clusters regenerated on the culture medium in the step S3, putting the bud clusters into a bud extension culture medium MeM4, continuously screening and culturing, and transferring the buds into a rooting culture medium until the buds extend to 2cm; s5, transferring the buds obtained in the step S4 to a rooting culture medium MeM5 for growth, and then transferring the buds into a nutrition pot for culture; the method has the advantages that the method is suitable for both thin-peel muskmelons and thick-peel muskmelons, and the yellowing rate of regenerated buds is as low as 1.6%.

Description

technical field [0001] The invention relates to the technical field of melon biotechnology breeding, in particular to an efficient method for genetic transformation of melon mediated by Agrobacterium. Background technique [0002] Melon (Cucumis melo L.) is an important melon crop of the genus Cucurbitaceae. It is rich in many vitamins, diverse in color, fresh and sweet in taste, rich in nutrition and high in economic value. It is a fruit favored by people. Melon has poor disease resistance, and the main diseases are powdery mildew, fusarium wilt, downy mildew and virus disease. Breeding and utilization of disease-resistant variety resources is an effective way to improve the disease-resistant traits of melon, but the conventional breeding cycle of melon is long, and it is difficult to quickly Obtain varieties with improved target traits. With the rapid development of plant cell engineering and genetic engineering technology, the research and application of genetic engineer...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84A01H5/00A01H6/34A01H4/00
CPCC12N15/8205A01H4/002A01H4/008Y02A40/10
Inventor 万丽丽王转茸汤谧张学军任俭张娜曾红霞熊建顺李煜华
Owner WUHAN ACADEMY OF AGRI SCI
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