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Preparation method and transformation method of papaya hypocotyledonary axis agrobacterium transformation receptor

A technology of Agrobacterium transformation and papaya, which is applied in the field of plant genetic engineering, can solve the problems of not particularly stable system, long callus induction period, and restricting the development of papaya breeding industry, etc., to achieve simple and efficient medium and simple operation Ease of operation, avoiding the effect of multiplication time

Pending Publication Date: 2020-08-07
FUJIAN AGRI & FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the existing transgenic methods, the callus induction period of papaya is very long, and the system is not particularly stable, which seriously restricts the development of papaya breeding industry.

Method used

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  • Preparation method and transformation method of papaya hypocotyledonary axis agrobacterium transformation receptor
  • Preparation method and transformation method of papaya hypocotyledonary axis agrobacterium transformation receptor
  • Preparation method and transformation method of papaya hypocotyledonary axis agrobacterium transformation receptor

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] A kind of preparation method of papaya Agrobacterium transformation receptor

[0038] Include the following steps:

[0039] (1) Seed disinfection: take 150-200 papaya seeds and place them in a sterilized tissue culture bottle, ①, disinfect with alcohol with a volume concentration of 75% for 1 min, and wash with sterile water for 3-5 times, ②, enter a volume concentration of Disinfect with 3.5% NaClO solution for 20 minutes, wash with sterile water for 3 to 5 times, ③, transfer to mercuric chloride with a mass concentration of 0.1% for 10 minutes, wash with sterile water for 3 to 5 times, ④, use sterile tweezers Put it into a sterilized Erlenmeyer flask, add 100ml of NaClO solution with a volume concentration of 20%, and put it in a shaker at 28°C and 180rpm for 4h; this process is all performed on a clean bench.

[0040] (2) 1M KNO3 treatment: take the seeds obtained in step (1), take them out from the 20% NaClO solution, wash them with sterile water for 3 to 5 times, re...

Embodiment 2

[0050] Target gene vector construction

[0051] The recombinant vector containing the 35s-CpYh-1 target gene is constructed according to the method comprising the following steps: using the cDNA of SunUp variety papaya as a template, using primers 35s-CpYh-1F and 35s-CpYh-1R to carry out PCR amplification, and The 735bp fragment of the amplified product (such as Figure 6 shown) was inserted between the M13 universal primers of the plasmid pMC202 (purchased from the ABRC official website) vector by homologous recombination to obtain the recombinant vector pMC202-35s-CpYh-1, and the primers HYG-F / R and primer 35s for the recombinant vector -CpYh-1F / R amplification confirms that the vector is constructed successfully (such as Figure 7 shown).

[0052] The nucleotide sequence of the target gene 35s-CpYh-1 is shown in SEQ ID NO.01:

[0053] ATGGAGTTTGAGGATCAAGATGAGCAAGATGAAGAGATGGGAATGGGAACTGGTTGTGGGTCACTCCGTAACTCGACCGGGGTCAAACTGGGCGGTCCGAAGGCTGTGGGGACGGGGACAGGGACGGATCATCGGCAG...

Embodiment 3

[0061] An Agrobacterium-mediated transformation method using the above-mentioned papaya hypocotyl Agrobacterium transformation receptor

[0062] Include the following steps:

[0063] 1. Infection: adjust the OD value of the Agrobacterium infection solution containing the target gene suspended in 1 / 2 MS liquid medium to 0.5-0.8, and then dilute it 10 times to obtain the prepared infection solution. The papaya hypocotyl section obtained by the above preparation method can be soaked in the infection solution for about 8 minutes, the supernatant is poured off, blotted dry with filter paper, and blown until the surface of the hypocotyl section is dry, and can not be blown for too long. (Cell division is more severe after five days of pre-cultivation, so the infection is done directly in a sterile petri dish).

[0064] 2. Co-cultivation: place the hypocotyls infected in step (1) on k4 medium and add sterile filter paper to co-culture for 24 hours, and dry the co-cultured hypocotyls...

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Abstract

The invention provides a preparation and transformation method of a papaya hypocotyledonary axis agrobacterium transformation receptor. The method comprises the following steps: after disinfecting papaya seeds, soaking papaya in a NaClO solution and a KNO3 solution in sequence, transferring the soaked papaya into sterile water containing antibiotics to germinate and grow buds, transplanting the buds into an agar culture medium after the buds grow out, performing dark culture until seedlings grow, and inducing explants to grow calluses on an optimized culture medium M13, thereby obtaining the papaya hypocotyledonary axis agrobacterium transformation receptor. Compared with traditional papaya transformation, the transformation method has the advantages that the pre-cultured hypocotyls are directly used as the explants, so that 4-5 months of propagation after callus induction are avoided, and the transformation period is remarkably shortened. Equipment required by the method is simple, the operation technology is easy to master, and the method has wide development and application prospects.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering, in particular to a method for preparing and transforming papaya hypocotyl Agrobacterium transformation receptors. Background technique [0002] Papaya is a major tropical fruit and the main supply fruit in the fresh fruit market. Papaya is also processed into beverages, jams, dried fruits and desserts, and its green fruits, leaves and flowers can also be used as vegetables (Watson, 1997). At present, the breeding of papaya mainly adopts traditional breeding. Since papaya ringspot mosaic virus is the main factor that restricts the production of papaya, it is difficult for us to endow papaya cultivars with the resistance characteristics of wild papaya by conventional hybridization methods. It is still an important task to increase the yield of papaya and improve the quality of papaya. Through transgenic technology, we can directional change plant resistance, yield, quality and o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H4/00A01H5/00A01H6/00
CPCC12N15/8205A01H4/00
Inventor 岳晶晶刘娟明瑞光
Owner FUJIAN AGRI & FORESTRY UNIV
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