Citrus mature internode stems genetic transformation method

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...