Dangshan pear genetic transformation method

A genetic transformation method and technology for genetic transformation, applied in the field of genetic engineering of horticultural crops, can solve the problems of low survival rate of transplanting, insufficient robustness of roots, rooting rate of callus, etc., and ensure the efficiency of genetic transformation, browning rate and pollution. The effect of low rate and small amount of bacteria carried

Active Publication Date: 2016-05-04
ANHUI AGRICULTURAL UNIVERSITY
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Problems solved by technology

[0005] At present, the types of explants used in pear tissue culture include stem tips, cotyledons, anthers, petioles, leaves, and protoplasts. These explants can be used to obtain regeneration test-tube plantlets, but pear test-tube plantlets tend to produce a large number of callus when they take root. Injury to the tissue and low rooting rate, and the transplanting survival rate is low because the root is not strong enough

Method used

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  • Dangshan pear genetic transformation method
  • Dangshan pear genetic transformation method
  • Dangshan pear genetic transformation method

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Experimental program
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Effect test

Embodiment 1

[0041] Establishment of genetically transformed receptors

[0042] From March to April, cut Dangshan crispy pears from the field as explants, soak them in washing powder water for 20 minutes, then rinse them with running water for 1 hour, and place the explants in 75% alcohol on a super-clean bench Soak for 30s, rinse with sterile water once or twice, then soak in 0.1% mercuric acid solution for 5-6 minutes, in which 1 to 2 drops of Tween-80 should be added to the mercuric solution, and rinse with sterile water for 4-5 times , blot the excess water with sterile filter paper, inoculate horizontally on the surface of the callus induction medium, place in a 16-hour light, 8-hour dark photoperiod culture room for cultivation, light intensity 1500-2000lx, temperature 25°C; 20 days Callus can be induced by left and right, such as figure 2As shown, the callus can be directly used as a genetic transformation recipient; the formula of the callus induction medium is: MS+6-BA1.0~2.0mg / ...

Embodiment 2

[0044] The cultivation of Agrobacterium and the configuration of infection solution

[0045] The selected Agrobacterium EHA105 carries the recombinant plasmid PCAMBIA-1304, and contains the marker gene NptII and the reporter gene GUS. The vector map of PCAMBIA-1304 is as follows: figure 1 As shown, the inserted target gene is the ferulic acid 5-hydroxylase gene (F5H) cloned in our laboratory (Genbank accession number: KC852907), the insertion sites are NcoI and BglII, and the ferulic acid 5-hydroxylase The gene (F5H) is used to regulate the enzyme that converts G-lignin to S-lignin in the process of lignin synthesis. The higher the content of S-lignin, the lower the polymerization degree of stone cells formed, and the smaller the stone cell clusters. The quality of pear fruit will be better; the preserved Agrobacterium will be activated by streaking on the double-antibody LB solid medium. When the OD value reaches 0.8-1.0, transfer the bacterial solution to 100 ml double-anti...

Embodiment 3

[0047] Infection and co-cultivation

[0048] Soak the callus in the infection solution, infect at 28°C and 50r / min for 10 minutes, take it out, blot the excess bacteria with sterile filter paper, and inoculate it on the co-cultivation medium; The basic formula is: 1 / 2MS+6-BA2.5~3.0mg / L+IBA0.2~0.4mg / L+10~20g / L sorbitol+AgNO 3 0.5mg / L+AS0.15mmol / L, the co-cultivation time is 48-60 hours, the temperature is 25 ℃, dark culture.

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Abstract

The invention provides a Dangshan pear genetic transformation method. The Dangshan pear genetic transformation method comprises the following steps: (1) constructing a genetic transformation receptor; (2) culturing agrobacterium tumefaciens and preparing infection solution; (3) carrying out infection and coculture; (4) carrying out sterilization culture and screening culture; and (5) carrying out resistant callus GUS (glucuronidase) staining identification. The Dangshan pear genetic transformation method has the advantages that Dangshan pear callus is taken as the receptor, NptII is taken as a marker gene, GUS gene is taken as a reporter gene, Dangshan pear genetic transformation is carried out by adopting an agrobacterium EHA105 mediated process for obtaining positive resistant callus, and then redifferentiation is carried out by virtue of the positive resistant callus for obtaining test-tube plantlets; meanwhile, a method for carrying out genetic transformation by firstly inducing resistant callus by virtue of fresh treetop, taken as an explant, grown the same year of Dangshan pear is firstly established, content of phenolic substances in fresh treetops of the Dangshan pear in spring is low, less germs are carried, both inoculation browning rate and contamination rate are low, materials are available, callus induction ratio is high, and demand of genetic transformation can be met.

Description

technical field [0001] The invention relates to the field of genetic engineering of horticultural crops, in particular to an Agrobacterium-mediated genetic transformation method using Dangshansu pear callus as a receptor. Background technique [0002] Pear belongs to the plant of the genus Pyrus L. of the subfamily Pomaeeae of the Rosaceae family and is one of the most important deciduous fruit trees in the world. Pear varieties can be divided into two categories: Oriental pears and Western pears. Oriental pears are mainly cultivated in Asian countries such as China, Japan, and Korea, including sand pears (Pyrus pyrifolia), white pears (Pyrusbretschneideri) and autumn pears (Pyrusus suriensis). The cultivated area and yield of pears in China are second only to citrus and apples, ranking third. Among them, the Dangshansu pear, which is originally produced in Dangshan County, Anhui Province, belongs to the white pear system and is one of the main varieties of pears exported in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84A01H4/00A01H5/00
CPCA01H4/005C12N15/8205
Inventor 蔡永萍程曦金青李姝妹张金云闫冲冲马晨辉程俊孙宁
Owner ANHUI AGRICULTURAL UNIVERSITY
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