Method for obtaining transgenic Malus hupehensis rehd plant without selectable marker genes

A selection marker-free, Pingyi sweet tea technology, applied in the fields of botanical equipment and methods, horticultural methods, genetic engineering, etc.

Active Publication Date: 2011-09-21
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, cultivating unmarked transgenic Pingyi sweet tea has become an important goal of molecular br

Method used

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  • Method for obtaining transgenic Malus hupehensis rehd plant without selectable marker genes
  • Method for obtaining transgenic Malus hupehensis rehd plant without selectable marker genes
  • Method for obtaining transgenic Malus hupehensis rehd plant without selectable marker genes

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Embodiment 1

[0042] 1. Acquisition of transgenic Pingyi sweet tea plants without selection marker gene

[0043]Obtain the tissue culture seedlings (tissue culture seedlings) of Pingyi sweet tea according to the following steps: pick the annual branches of Pingyi sweet tea in early spring, cultivate them in water for about 10 days, take the full buds on the branches, wash them with running water for 3 hours, and use 0.1% mercury chloride (HgCl 2 ) for 6 minutes, rinsed with sterile water for 6 times, blotted the residual water with sterile absorbent paper, and inoculated the buds on the primary culture medium. The primary culture medium is the addition of 30 mg·L MS basic medium -1 Sucrose, 6.5mg·L -1 Agar powder, 1.0mg·L -1 6-benzyl adenine and 0.2mg·L -1 Indolebutyric acid, pH 5.6 medium. On the second day after inoculation, some materials began to brown. Once browned materials were found, they were immediately replaced with new primary medium until the browning was eliminated. 10 d...

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Abstract

The invention relates to a method for obtaining a transgenic Malus hupehensis rehd plant without selectable marker genes. The method comprises the following steps of: culturing Agrobactrium tumefaciens carrying expression vectors in kanamycin-containing LB liquid culture medium for 16 hours for later use; culturing Malus hupehensis rehd tissue culture seedlings on a subculture medium for 35 days; cutting stretched laminae into leaf disks; placing the leaf disks into the differential culture medium to culture for 5 minutes; inoculating the leaf disks on a regeneration culture medium to culture in the dark for 1 day; connecting the leaf disks on a cephalosporin-containing differential culture medium to culture at the temperature of (25+/-1) DEG C in the dark for 14 days; culturing the leaf disks for 30 days under the conditions of photoperiod of 16-hour lighting/8-hour dark and illumination intensity of 30 umol.m<-2>.s<-1>; cutting off the obtained adventitious buds; inoculating the adventitious buds on a rooting culture medium to culture for 40 days under the conditions of photoperiod of 16-hour lighting/8-hour dark and illumination intensity of 30 umol.m<-2>.s<-1> so that the buds root; performing thermal treatment at the temperature of 42 DEG C for 15 minutes; and further identifying to obtain the transgenic Malus hupehensis rehd plant without selectable marker genes.

Description

technical field [0001] The invention relates to a method for producing transgenic Pingyi sweet tea plants, in particular, a method for producing transgenic Pingyi sweet tea plants without a selection marker gene. Background technique [0002] Pingyi Sweet Tea [Malus hupehensis (Pamp.) Rehd.var.pingyiensis Jiang] is a variety of Malus hupehensis (Pamp.) Rehd.var.pingyiensis Jiang. Arborization, strong shade tolerance, waterlogging resistance, and strong affinity for grafting apples, is one of the rootstocks that have been widely used in apple production. Due to the characteristics of apomixis, the traditional breeding of Pingyi sweet tea is more difficult, and the traits of Pingyi sweet tea can be improved through transgenic technology. In the transgenic process, a selection marker gene is often used, which is often co-transformed with the target gene. Under the action of selection pressure, non-transformed cells are killed, while transformed cells survive due to the resista...

Claims

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Application Information

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IPC IPC(8): C12N15/84A01H4/00A01H5/00
Inventor 金万梅王媛花张强刘松忠
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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