Method for regeration of citrus internode stem and genetic transformation

A genetic transformation and internode technology, which is applied in the field of citrus internode stem segment regeneration and genetic transformation, can solve the problems of citrus genetic transformation limitations, citrus breeding difficulties, receptor system variety specificity and low regeneration efficiency, and achieve regeneration efficiency High, good disinfection effect

Inactive Publication Date: 2006-09-20
邓子牛 +2
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  • Summary
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AI Technical Summary

Problems solved by technology

[0003] The genetic transformation of citrus is based on the development of the receptor system. Having a good tissue culture regeneration system is often the key to success. Citrus is a perennial woody plant with many varieties, and the variety specificity and regeneration efficiency of the receptor system are low. The genetic transformation of citrus is greatly restricted
These limitations have brought great difficulties to the use of genetic engineering in citrus breeding

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] (1) Seed disinfection and germination After the common Xupu sweet orange seeds are taken out from the fruit, the outer testa and about 2 / 3 of the inner testa are peeled off, soaked in 70% alcohol for 30s, and then soaked in 0.1% mercuric chloride solution Soak in water for 10 minutes, and finally rinse with sterile water for 4 times, sow on the medium of 1 / 2MS + 3% sucrose + 0.8% agar, cultivate in the dark at 25°C for 20 days, and then transfer to light for 7 days to obtain sterile seedlings (2) Induction of buds of control materials Cut the internode stems (epicotyls) of seedlings about 30 days old into lengths of about 1-1.5 cm, and directly induce adventitious buds without infecting with Agrobacterium. Use MS medium as the basic medium, add growth regulator 6-BA 1~5mg·L -1 (The preferred solution is 3mg·L -1 ) is cultivated, the culture temperature is 27 DEG C, the light intensity is 1400lx, and the light time is 12h every day. After two weeks, the induction rate o...

Embodiment 2

[0033] (1) Get lemon seeds, disinfect and germinate with the same method as in Example 1 (1), (2)-(6) Agrobacterium with pBin19 plasmid, T-DNA with chit42 gene and selection gene nptII. Co-cultivation medium (GM) is MS+BA3mg·L -1 , screening medium MS+BA 3mg / L+Cef (cefotaxime) 500mg L -1 +Km 100mg·L -1 . Others are the same as in Embodiment 1 (2) to (6).

[0034] The specific primers of known nptII and chit42 genes were used for analysis, and 5 transformed shoots were obtained from 350 transformed explants, and 3 transformed lines were obtained. Through propagation, 100 transgenic seedlings were obtained in total. The regenerated transformed plants showed no chimerism.

Embodiment 3

[0036] (1) Take ordinary Xupu sweet orange seeds, disinfect and germinate them in the same way as in Example 1 (1), (2)-(6) Agrobacterium with pBvin19 plasmid, T-DNA with chit42 gene and selection gene nptII. Co-cultivation medium (GM) MS+BA3mg·L -1 , screening medium MS+BA 3mg / L+Cef300mg·L -1 +Km 50mg·L -1 , other with embodiment 1 (2)~(6).

[0037] DNA was extracted from leaves of resistant buds and resistant plants, and analyzed by PCR. PCR amplification was performed with specific primers of chit42, and 3 transformed lines and 43 transformed plants were obtained. Transformed plants showed no chimerism.

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Abstract

A regeneration and genetic transformation method for citrus includes immersing seeds in the solution of alcohol or mercuric chloride for disinfection, flushing with aseptic water, culturing in culture medium to obtain aseptic seedling, inducing the bud of check material, culturing in culture medium MS, adding growth regulator, transforming explant, using Agrobacterium to dipdys the intersegemental stem of said aseptic seedling, inducing resistant bud, adventitious bud grafting, and secondary grafting in greenhouse.

Description

technical field [0001] The invention relates to a method for regeneration and genetic transformation of citrus internode stem segments, in particular to a method for efficient regeneration and genetic transformation of internode stem segments (epicotyls) suitable for varieties such as sweet orange, lemon, pomelo, and citrange system. Background technique [0002] Citrus is the number one fruit in the world. The annual output of citrus in the world exceeds 100 million tons, ranking first among all kinds of fruits; the annual trade volume of citrus and its products reaches nearly 10 billion US dollars, ranking third in the trade volume of agricultural products. my country's citrus area exceeds 1.6 million hectares, accounting for 23% of the world's total citrus area, ranking first in the world. [0003] The genetic transformation of citrus is based on the development of the receptor system. Having a good tissue culture regeneration system is often th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01H4/00A01H3/02A01H3/04
Inventor 邓子牛谢玉明敖小平郭琛焦徕胡春华
Owner 邓子牛
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