CN-121992025-A - Establishment method of apple tissue culture seedling transgenic system

Abstract

The invention discloses a method for establishing an apple tissue culture seedling transgenic system, which comprises the steps of obtaining tissue culture seedlings, taking an in-vitro culture regeneration system as a basis for genetic transformation technology of the apple tissue culture seedlings, and carrying out transformation by agrobacterium mediation by using a tissue culture method. The target gene is introduced into the apple tissue culture seedling by utilizing a transgenic technology, DNA recombination is mainly applied, an exogenous gene is inserted into a genome through gene fusion, and a transgenic plant of the apple tissue culture seedling is obtained through tissue culture, so that a new variety is obtained. And (3) after degerming, obtaining resistant buds, carrying out PCR and RT-PCR detection on regenerated plants, selecting positive plants, and repeating the operation for 3 times to finish the process. The invention can realize the rapid cultivation of transgenic apple tissue culture seedlings, and along with the rapid development of genetic transformation technology, plants can obtain excellent characters which are not possessed by themselves by introducing exogenous genes.

Inventors

• ZHANG JIE
• LIU JIAWEI
• QIN XIAOXIAO
• TIAN JI
• SONG TINGTING
• YAO YUNCONG

Assignees

• 北京农学院

Dates

Publication Date

20260508

Application Date

20230130

Claims (1)

1. 1. The establishment method of the transgenic system of the apple tissue culture seedling is characterized by comprising the following steps of: (1) Cutting second and third young leaves at the upper end of morphology from the apple tissue culture seedlings subjected to subculture for 21-28 days, and carrying out overnight culture on a preculture MS culture medium for genetic transformation; (2) Picking single colony of agrobacterium containing target gene from LB solid culture medium, adding the single colony into LB liquid culture medium containing Spe and Rif, shaking overnight at 28 deg.C and 200rpm, adding shaking bacterial liquid into LB liquid culture medium containing Spe and Rif, mixing thoroughly according to the ratio of 1:4, continuing shaking culture until OD600 is 0.4-0.7, centrifuging at 5000rpm at room temperature for 5min, collecting bacterial body, suspending bacterial liquid to OD600 of 0.5 with liquid culture medium, and adding 10 μl of acetosyringone into 25mL of re-suspended bacterial liquid; (3) Taking out the leaves treated in the step (1), shearing the edges of the leaves and uniformly shearing the leaves into small leaf blocks with the length of 0.3 multiplied by 0.5 cm, transversely cutting 3-4 knives at the veins, placing the sheared leaf blocks in a liquid culture medium for soaking for 5min, then placing the cut leaf blocks in the resuspension bacteria liquid obtained in the step (2) for infection, shaking the bacteria liquid slightly every 3min for 40S each time, sucking the agrobacterium bacteria liquid on the small leaf blocks by using sterile filter paper and rapidly moving the agrobacterium bacteria liquid into a co-culture medium, placing the leaf back downwards, and performing dark culture for 3-4 days at the temperature of 25 ℃; (4) Transferring the small leaf blocks onto a regeneration culture medium after dark culture is finished, placing the leaf backs upwards, continuing dark culture until callus and adventitious buds appear, transferring the small leaf blocks under light for culture, and transferring the small leaf blocks into a subculture medium for culture after green adventitious buds grow up gradually; (5) Taking leaves of regenerated plants obtained by subculture in the step (4), extracting genome DNA by using a CTAB method, taking plasmid DNA containing a target gene as a positive control, taking untransformed plant DNA and ddH 2 O as a negative control, carrying out PCR amplification, selecting positive apple tissue culture seedling transgenic regenerated plants, extracting RNA of the positive apple tissue culture seedling gene regenerated plants by using an improved SDS method, carrying out RT-PCR detection, and selecting positive apple tissue culture seedling gene regenerated plants to finish establishment of an apple tissue culture seedling transgenic system; in the step (1), the secondary culture is specifically carried out for 26 days; in the step (2), the overnight culture means culture for 14 hours; in the step (3), the dark culture is performed for 3 to 4 days, which means dark culture is performed for 4 days.

Description

Establishment method of apple tissue culture seedling transgenic system The application relates to a method for establishing a transgenic system of apple tissue culture seedlings, which is applied separately, and the application date is 2023, 01, 30 and 202310045599.X. Technical Field The invention belongs to the field of genetic engineering, and particularly relates to a method for establishing an apple tissue culture seedling transgenic system. Background The genetic transformation technology of apple tissue culture seedlings is based on the establishment of an in-vitro culture regeneration system, and transformation is carried out by agrobacterium mediation by utilizing a tissue culture method. The target gene is introduced into the apple tissue culture seedling by utilizing a transgenic technology, DNA recombination is mainly applied, an exogenous gene is inserted into a genome through gene fusion, and a transgenic plant of the apple tissue culture seedling is obtained through tissue culture, so that a new variety is obtained. There is an increasing search for genetic transformation on apple tissue culture seedlings using agrobacterium tumefaciens-mediated methods. And adopting a leaf disc method to co-transform agrobacterium tumefaciens and apple tissue culture seedling green sleeve leaves to obtain a transgenic strain. As an important method for genetic improvement of apple tissue culture seedlings, the genetic engineering can overcome a plurality of problems existing in traditional breeding. With the rapid development of genetic transformation technology, plants acquire excellent traits that are not possessed by themselves by being introduced with exogenous genes. Disclosure of Invention The invention aims to provide a method for establishing an apple tissue culture seedling transgenic system. The technical scheme of the invention is that the method for establishing the transgenic system of the apple tissue culture seedling comprises the following steps: (1) Cutting second and third young leaves at the upper end of morphology from the tissue culture seedlings of apples subjected to subculture for 3-4 weeks, and carrying out overnight culture on a preculture MS culture medium for genetic transformation; (2) Picking single colony of agrobacterium containing target gene from LB solid culture medium, adding the single colony into LB liquid culture medium containing Spe and Rif, shaking overnight at 28 deg.C and 200rpm, adding shaking bacterial liquid into LB liquid culture medium containing Spe and Rif, mixing thoroughly according to the ratio of 1:4, continuing shaking culture until OD600 is 0.4-0.7, centrifuging at 5000rpm at room temperature for 5min, collecting bacterial body, suspending bacterial liquid to OD600 of 0.5 with liquid culture medium, and adding 10 μl of acetosyringone into 25mL of re-suspended bacterial liquid; (3) Taking out the leaves treated in the step (1), shearing the edges of the leaves and uniformly shearing the leaves into small leaf blocks with the length of 0.3 multiplied by 0.5cm, transversely cutting 3-4 knives at the veins, placing the sheared leaf blocks in a liquid culture medium for soaking for 5min, then placing the cut leaf blocks in the resuspension bacteria liquid obtained in the step (2) for infection, shaking the bacteria liquid slightly every 3min for 40S each time, sucking the agrobacterium bacteria liquid on the small leaf blocks by using sterile filter paper and rapidly moving the agrobacterium bacteria liquid into a co-culture medium, placing the leaf back downwards, and performing dark culture for 3-4 days at the temperature of 25 ℃; (4) Transferring the small leaf blocks onto a regeneration culture medium after dark culture is finished, placing the leaf backs upwards, continuing dark culture until callus and adventitious buds appear, transferring the small leaf blocks under light for culture, and transferring the small leaf blocks into a subculture medium for culture after green adventitious buds grow up gradually; (5) And (3) taking leaves of the regenerated plants obtained by the secondary culture in the step (4), extracting genome DNA by using a CTAB method, taking plasmid DNA containing a target gene as a positive control, taking untransformed plant DNA and ddH 2 O as a negative control, performing PCR amplification, selecting positive apple tissue culture seedling transgenic regenerated plants, extracting RNA of the positive apple tissue culture seedling gene regenerated plants by using an improved SDS method, performing RT-PCR detection, and selecting the positive apple tissue culture seedling gene regenerated plants to finish establishment of an apple tissue culture seedling transgenic system. Further, the composition of the preculture MS culture medium is 4.43g/L MS+2mg/L TDZ+0.2mg/L IBA + 30G/L sucrose+7 g/L agar, and the pH value is adjusted to 5.88. Further, the LB liquid medium containing Spe and Rif had a composition of 4.43 g/L MS+15g/L sucros