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Preparation and transformation method of papaya callus agrobacterium transformation receptor

A technology of Agrobacterium transformation and callus, applied in the field of transgenics, can solve the problems of lack of papaya, offspring separation, complex papaya strain, etc., and achieve the effect of simple operation, stable transformation system, simple and efficient formula

Pending Publication Date: 2020-07-28
FUJIAN AGRI & FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Yet at present in the production of papaya has run into practical problems that urgently need to be solved: papaya plants are complex (male strains, female strains and hermaphrodites are arranged), and papaya is a cross-pollinated fruit tree, even if it is successfully selected by hybridization means For disease-resistant varieties, the traits of their offspring must be severely separated, and it is difficult to keep their excellent disease-resistant traits unchanged.
However, at present, there is a relatively lack of mature papaya transgenic technology in the prior art, the transformation 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 and transformation method of papaya callus agrobacterium transformation receptor
  • Preparation and transformation method of papaya callus agrobacterium transformation receptor
  • Preparation and transformation method of papaya callus agrobacterium transformation receptor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0038] Include the following steps:

[0039] (1) Seed disinfection: Take 250 papaya seeds and place them in sterilized tissue culture bottles, ①, 75% ethanol for 1 min, and sterile water for 3 to 5 times, ②, 3.5% NaClO solution for 20 min, sterile water Wash for 3 to 5 times, ③, 0.1% mercury liter disinfection for 10 minutes, and wash with sterile water for 3 to 5 times, ④, put the seeds into a sterilized Erlenmeyer flask with sterile tweezers, and add 100ml of 25% NaClO solution , put into 28, 180rpm shaker for 4h;

[0040] (2) 1M KNO3 treatment: take the seeds obtained in step (1), take them out from the 25% NaClO solution, wash them with sterile water for 3 to 5 times, replace them with 1mol / L KNO3 solution, and place them at 28°C, 180rpm Shaker 24h.

[0041] (3) Change water for germination: take the seeds obtained in step (2), take them out from the 1mol / L KNO3 solution, wash t...

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 8 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 9 shown).

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

[0053]

[0054]

[0055] The target gene 35s-CpYh-1 belongs to the transcription factor ZF-HD (Zinc-finger homeodomain), which plays an imp...

Embodiment 3

[0062] Agrobacterium-mediated papaya transformation method of papaya callus

[0063] Include the following steps:

[0064] 1. Infection: the OD value of the Agrobacterium infection solution containing the target gene prepared by the embodiment 2 suspended in 1 / 2 MS liquid medium is adjusted to 0.5~0.8, and the papaya prepared in Example 1 is healed with the infection solution Wounded tissue (ie papaya callus Agrobacterium transformed receptor) was shaken and soaked for 15-20 minutes (shaking infection for 3-4 minutes, resting for 11-17 minutes). Settle the callus upright, pour off the supernatant, blot the callus dry with filter paper, and blow it for 10 minutes, but don't blow it for too long.

[0065] 2. Co-cultivation: Put the infected callus on Ci medium and add sterile filter paper to co-cultivate in the dark room for 24 hours. If Agrobacterium visible to the naked eye is seen, it needs to be washed. The cultured callus was blow-dried in an ultra-clean bench.

[0066] ...

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Abstract

The invention provides a preparation method of a papaya callus agrobacterium transformation receptor. The preparation method of the papaya callus agrobacterium transformation receptor is characterizedin that sterilized papaya seeds are sequentially soaked in a NaClO solution and a KNO3 solution, transferred to sterile water containing antibiotics to germinate and grow buds, and planted into an agar culture medium after the buds grow out until seedlings are formed, hypocotyls of the seedlings are cut off for callus induction, propagation is performed four or five times after embryogenic calluses grow out, and the papaya callus agrobacterium transformation receptor is obtained. Compared with a traditional papaya callus agrobacterium transformation method, the method had the advantages thatthe callus induction time is remarkably shortened by improving the callus induction culture medium, the callus state is good and facilitates the transformation, and the papaya transformation efficiency is high. Moreover, equipment required by the method is simple, the operation technology is easy to grasp, and the method has wide development and application prospects.

Description

technical field [0001] The invention relates to a method for preparing and transforming papaya callus Agrobacterium transformation receptors, and belongs to the technical field of transgenes. Background technique [0002] Papaya (Carica papaya L.), also known as papaya, milk melon, longevity fruit, is a tropical and subtropical evergreen cork large papaya perennial herb, evergreen cork small tree. Papaya, banana, and pineapple are also known as the three major herbal fruit trees in the tropics. They are popular fruits and vegetables and medicinal tropical fruits. They enjoy the reputation of "Longevity Fruit" and "Lingnan Best Fruit" in southern my country. In addition to being eaten fresh, papaya can also be processed into fruit juice, jam, candied fruit, pickled; papaya has a strong ability to decompose protein, can be used to make stomach medicine, insect repellant, and can also be used for clarification of wine and fruit juice agent and meat softener. Papaya is a plant...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H5/00A01H6/00A01H4/00
CPCC12N15/8205A01H4/001
Inventor 岳晶晶刘娟明瑞光
Owner FUJIAN AGRI & FORESTRY UNIV
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