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CN-120648742-B - Genetic transformation method and application of callus of Hanfu apple seeds

CN120648742BCN 120648742 BCN120648742 BCN 120648742BCN-120648742-B

Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a genetic transformation method and application of callus of Hanfu apple seeds. The callus which is successfully transformed is obtained by the explant through induction culture, proliferation culture, dip-dyeing culture, co-culture and screening culture, the explant is 'Hanfu' apple seeds, and the fluorescence signal of the callus which is not infected and is induced by the 'Hanfu' apple seeds obtained by the method is weaker under ultraviolet excitation light waves. When the callus is subjected to agrobacterium-mediated genetic transformation, the fluorescence signal intensity of the callus is obviously increased after the callus is introduced into pRI101 GFP vector, which indicates that the 'Hanfu' apple callus can adopt a genetic transformation method containing GFP tag.

Inventors

  • WANG FENG
  • MA YUE
  • YAN JUNYI
  • ZHANG SHUYUAN

Assignees

  • 沈阳农业大学

Dates

Publication Date
20260512
Application Date
20250711

Claims (6)

  1. 1. The genetic transformation method of the apple seed callus containing GFP tag is characterized in that the explant is the apple seed with the 'Hanfu' which is successfully transformed through induction culture, proliferation culture, dip-dyeing culture, co-culture and screening culture; The formula of the induction culture medium used for the induction culture is 4 g~5g MS+0.1 mg~0.4 mg NAA+1.0 mg~2.0 mg 2,4-D+0.5 mg-1.0 mg 6-BA+10 g-30 g sucrose+7 g-9 g agar, and water is added to 1L; The formula of the proliferation culture medium used in the proliferation culture is 4 g~5g MS+1.5 mg6-BA+0.4 mg~0.8 mg ZT+10 g~30 g sucrose+7g-9g agar, and water is added to 1L; the dip-dyeing culture is to use agrobacterium tumefaciens carrying a target gene vector pRI101-GFP to dip-culture the callus of the Hanfu apple seeds; The Hanfu apple seeds are selected from 30-40 days after flowers, and the Hanfu apple seed sample is a wound embryo.
  2. 2. The genetic transformation method according to claim 1, wherein the co-culture medium is prepared from 4 g~5g MS+1.0 mg~2.0 mg 2,4-D+0.5-1.0 mg 6-BA+10-30 g sucrose+7-9 g agar and water to 1L.
  3. 3. The genetic transformation method according to claim 1, wherein the composition of the screening medium used in the screening culture is 4 g~5g MS+1.0 mg~2.0 mg2,4-D+0.5-1.0 mg 6-BA+20-40 mg kanamicin+150-250 mg cephalosporin+10-30 g sucrose+8-10 g agar, and water is added to 1L.
  4. 4. The genetic transformation method according to claim 1, wherein the induction culture time is 60 days to 90 days.
  5. 5. The genetic transformation method according to claim 1, wherein the proliferation culture time is 14 days.
  6. 6. The genetic transformation method according to claim 1, wherein the medium is adjusted to pH 5.6 with 1mol/LNaOH solution.

Description

Genetic transformation method and application of callus of Hanfu apple seeds Technical Field The invention belongs to the technical field of plant genetic engineering, and particularly relates to a genetic transformation method and application of callus of Hanfu apple seeds. Background In the field of botanics, when plant cells are subjected to a wound stimulus, a specific cell population, callus, is formed on the wound surface. The callus is composed of thin-walled cells which have increased in volume and have been dedifferentiated, plays an important role in plant molecular research and genetic engineering research, and is a commonly used test material. For genetic transformation of genes, a stable regeneration system is an indispensable prerequisite. However, in the field of apple genetic transformation, there are challenges in that the transformation difficulty is high, and the probability of successfully obtaining a transformed plant with stable genetic characteristics is low. However, in contrast, it is relatively easy to obtain stable callus genetic transformation material. Therefore, the apple can successfully cultivate the callus of the apple, and a perfect apple callus transformation system is established, so that the callus with consistent genotypes and stable growth is obtained, and a rapid and stable genetic transformation method is explored, so that a brand-new transgene receptor material and test path are provided for the molecular biology and genetic engineering research of the apple, and the method has great research significance and application value. At present, in the research field of apple callus, most of the adopted materials are callus obtained through induction of 'Wang Lin' apple fruits. It is notable that the 'Wang Lin' callus without any transformation exhibited a distinct green fluorescent signal at the 488nm uv excitation wavelength. This property makes the 'Wang Lin' apple callus unsuitable for transformation studies with Green Fluorescent Protein (GFP) tags. Because the fluorescence signal carried by the fluorescent probe can cause serious interference to the observation of the test effect, thereby affecting the accuracy and reliability of the research result. Disclosure of Invention In order to solve the problems, the invention provides a genetic transformation method and application of callus of Hanfu apple seeds. The genetic transformation method of the callus of the 'Hanfu' apple seeds comprises the steps of carrying out induction culture, multiplication culture, dip-dyeing culture, co-culture and screening culture on an explant to obtain the callus which is successfully transformed, wherein the explant is the 'Hanfu' apple seeds; The formula of the induction culture medium used for the induction culture is 4 g~5g MS+0.1 mg~0.4 mg NAA+1.0 mg~2.0 mg 2,4-D+0.5 mg-1.0 mg 6-BA+10 g-30 g sucrose+7 g-9 g agar, and water is added to 1L; The formula of the proliferation culture medium used in the proliferation culture is 4 g~5g MS+1.5 mg6-BA+0.4 mg~0.8 mg ZT+10 g~30 g sucrose+7g-9g agar, and water is added to 1L; the dip-dyeing culture is to use MdPHV-GFP agrobacterium carrying a target gene vector to dip-culture the callus of the Hanfu apple seeds. Preferably, the co-culture medium used for co-culture has a formula of 4 g~5g MS+1.0 mg~2.0 mg 2,4-D+0.5 mg-1.0 mg 6-BA+10 g-30 g sucrose+7 g-9 g agar, and water is supplemented to 1L. Preferably, the composition of the screening culture medium used for screening culture is 4 g~5g MS+1.0 mg~2.0 mg2,4-D+0.5-1.0 mg 6-BA+20-40 mg calicheamicin+150-250 mg cephalosporin+10-30 g sucrose+8-10 g agar, and water is added to 1L. Preferably, the induction culture time is 60-90 days. Preferably, the proliferation culture time is 14 days. Preferably, the Hanfu apple seeds are selected from 30-40 days after flowers. Preferably, the 'Hanfu' apple seed sample is a wounded embryo. Preferably, the medium is adjusted to pH 5.6 with 1mol/LNaOH solution. The genetic transformation method is applied to genetic transformation. Preferably, the genetic transformation is a genetic transformation method comprising a GFP tag. Compared with the prior art, the invention has the following advantages: The invention provides a genetic transformation method of callus of 'Hanfu' apple seeds, and the callus induced by uninfected 'Hanfu' apple seeds obtained by using a culture medium in the method has weaker fluorescent signals under ultraviolet excitation light waves. When the callus is subjected to agrobacterium-mediated genetic transformation, the fluorescence signal intensity of the callus is obviously increased after the callus is introduced into pRI101 GFP vector. The characteristic shows that the callus of the Hanfu apples can adopt a genetic transformation method containing GFP (green fluorescent protein) tag, thereby effectively improving the identification efficiency of transgenic materials and providing a more convenient and accurate means for related research