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Citrus mature internode stems genetic transformation method

A technology of genetic transformation and stem segments, applied in the field of genetic transformation systems, can solve the problems of difficult genetic transformation of citrus adult internode stem segments, restrictions on the practical process of citrus transgenics, and difficulty in differentiation, so as to achieve good disinfection effect and regeneration The effect of high efficiency and high survival rate

Inactive Publication Date: 2008-08-27
HUNAN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current problem is that, due to factors such as serious pollution and difficulty in differentiation during the in vitro culture of citrus adult materials, it is difficult to achieve successful genetic transformation of citrus adult internode stem segments, which also limits the practical application of citrus transgenics.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0008] Example 1 Transformation example of Bingtang orange adult state internode stem segment

[0009] The test material is rock sugar orange. (1) Select semi-lignified new shoots in the greenhouse. One week before picking the shoots, wipe the shoots with 5% sodium hypochlorite solution every other day for a total of 4 times. Cut off the branches wiped by sodium hypochlorite solution, put them in a clean plastic bag, bring them back indoors, cut them into 5cm stem segments, soak them in 10% sodium hypochlorite solution plus 2-3 drops of Tween for 30 minutes, and rinse them with sterile water 4 times , cut into 100 internode stem segments of about 1 cm, and set aside; (2) Agrobacterium (gene bank registration number: AF027497) carrying the target gene chit42 was streak-cultured in YEB medium containing 100 mg / l kanamycin Above, cultivate in the dark at 28°C. After the colonies grow, pick a single colony in YEB liquid medium, culture with shaking at 28°C for 22 hours, and reach ...

Embodiment 2

[0010] Embodiment 2 Newhall navel orange adult state internode stem segment transformation example

[0011] The test material is Newhall navel orange, and the target gene to be transformed is chit42. The method is the same as in Example 1. By this method, 100 internode stem segments are used to obtain 38 resistant buds, and 10 transgenic Newhall navel oranges are obtained. .

Embodiment 3

[0012] Embodiment 3 Ponkan adult state internode stem section transformation example,

[0013] The test material is ponkan, and the disinfection and transformation methods are the same as in Example 1 (1), (2). The co-cultivation method is the same as that in Example 1 (3), but the selection medium is MS+6BA 1 mg / l+NAA 0.5 mg / l+Cef 500 mg / l+Km 30 mg / l. PCR identification is the same as in Example 1 (4)-(6). The target gene to be transformed is the phyB gene (gene bank registration number: AF443799). By this method, 100 internode stem segments were used to obtain 12 resistant buds, and a total of 3 transgenic ponkans were obtained.

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Abstract

The invention relates to a method for genetically transforming citrus mature internode stems, which comprises following steps: firstly, disinfecting mature materials, selecting semi lignified new shoots in a greenhouse, using sodium hypochlorite solution 5wt% to wipe the shoots once every two days for a week before the shoots are sampled, wiping for 3-4 times together, then, cutting the shoots which are wiped with the sodium hypochlorite solution, cutting the shoots into stems of 4-5cm under the aseptic condition, using the sodium hypochlorite solution 10wt% which is added with tween 2-3 drops to dip for 20-35min, washing with sterilized water for 3-4 times, cutting the shoots into the internode stems of 1-1.5cm to preserve for use, secondly, transforming, thirdly, regenerating resistant buds, fifthly, grafting for the second time in the greenhouse, sixthly, identifying the resistant buds and resistant plants, and adopting a public-known PCR analysis method to identify. The citrus mature materials of the invention have good disinfection effect and low contamination rate, the adventitious bud induction frequency reaches 48%, the grafting survival rate of the resistant buds reaches more than 80%, and regenerated transformed plants do not have chimerism. The method is in particular suitable for genetically transforming mature internode stems of main cultivars of citrus such as crystal sugar oranges, navel orange, Ponkan and the like.

Description

technical field [0001] The invention relates to a method for genetic transformation of adult internode stems of citrus, in particular to a genetic transformation system suitable for adult internode stems of rock sugar oranges, navel oranges, ponkans and other varieties. Background technique [0002] Citrus is the largest fruit in the world, with an annual output of more than 110 million tons of citrus; the annual trade volume of citrus and its products reaches nearly 10 billion US dollars, ranking third in the trade volume of agricultural products. In 2006, the area of ​​citrus in my country has exceeded 1.7 million hectares, ranking first in the world, and the output is nearly 18 million tons, ranking second in the world. However, our country lacks high-quality citrus varieties, and its international competitiveness is relatively weak. Due to the characteristics of citrus itself, the progress of conventional breeding has been slow. The use of transgenic technology to impr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/11A01H1/00A01H5/00A01G1/06
Inventor 邓子牛谢玉明熊兴耀张家银杨莉胡新喜袁飞荣罗赛男戴素明
Owner HUNAN AGRICULTURAL UNIV
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