A method for improving the editing efficiency of apple CRISPR / Cas9 system by using improved leaf disc transformation method

Abstract

The application discloses a method for improving the editing efficiency of an apple CRISPR / Cas9 system by using a modified leaf disc transformation method, and belongs to the technical field of biotechnology.According to the nutrient requirements of woody plant tissue culture, a culture medium combination is provided, which can meet the requirements of apple leaf regeneration, induce strong differentiation ability of callus and regenerated shoots, and overcome the problem of low genetic transformation efficiency of apples.The leaf disc transformation method based on the culture medium kit can significantly improve the editing efficiency of the apple CRISPR / Cas9 system, can efficiently deliver the CRISPR / Cas9 knockout vector, and obtain homozygous and heterozygous plants of different mutation types including deletion, replacement, insertion and the like, and the editing efficiency is as high as 92.3%.The method provided by the application provides a new idea for analyzing the genetic mechanism of apples, improving agronomic traits and utilizing gene editing technology to carry out germplasm innovation.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biotechnology, and particularly relates to a method for improving the editing efficiency of an apple CRISPR / Cas9 system by using an improved leaf disc transformation method. BACKGROUND

[0002] CRISPR / Cas9 is a widely used gene editing technology, and is developed from the mechanism of CRISPR / Cas9 dominant gene editing system for bacterial defense of exogenous nucleic acids, and is composed of Cas protein, sgRNA and promoter. The Cas9 protein and sgRNA can be delivered into the genome of the recipient plant by Agrobacterium tumefaciens, and then the target gene is edited.

[0003] Agrobacterium tumefaciens-mediated plant transformation-leaf disc transformation method is one of the most widely used genetic transformation methods. Under natural conditions, Agrobacterium tumefaciens can infect the wounded part of dicotyledonous plants, and integrate the DNA fragment with transposition function on the Ti plasmid into the genome of the recipient cells for expression. Agrobacterium tumefaciens-mediated genetic transformation is developed based on this characteristic, and transgenic plants can be obtained by using the transformation method of Agrobacterium tumefaciens.

[0004] A high-efficiency and stable transformation method is a prerequisite for plant gene editing, and the existing leaf disc transformation method does not completely apply to woody plant tissue culture. The infection effect is not strong, and the culture medium used cannot fully meet the needs of leaf regeneration, and cannot induce strong differentiation ability of callus and regenerated shoots. And the existing leaf disc transformation method has problems such as unstable system, low transformation efficiency, poor test repeatability and the like in the process of CRISPR / Cas9 system-mediated genetic transformation experiment, which leads to difficulty in obtaining apple transgenic materials and single apple gene editing data. Therefore, an improved leaf disc transformation method which can be applied to woody plant tissue culture, enhance the genetic transformation efficiency of genes and improve the editing efficiency of the apple CRISPR / Cas9 system is urgently needed. SUMMARY

[0005] To achieve the above-mentioned purpose, the purpose of the present application is to provide a method for improving the editing efficiency of an apple CRISPR / Cas9 system by using an improved leaf disc transformation method, so as to solve the problems of low genetic transformation efficiency and low gene editing efficiency of the existing leaf disc transformation method.

[0006] To achieve the above-mentioned purpose, the present application adopts the following technical scheme:

[0007] In a first aspect of the present application, a culture medium combination is provided, comprising: an infection culture medium, a dedifferentiation start culture medium, a callus induction culture medium, a bud differentiation culture medium and a bud proliferation culture medium.

[0008] The infection medium takes WPM medium, sucrose, glucose, 4-morpholine ethanesulfonic acid and magnesium sulfate as active ingredients;

[0009] The dedifferentiation initiation medium takes WPM medium, sucrose, glucose, hydrolyzed casein, hydrolyzed whey protein, 2,4-dichlorophenoxyacetic acid, aminochlortripyridine, plant gel, 4-morpholine ethanesulfonic acid and polyvinylpyrrolidone as active ingredients;

[0010] The callus induction medium takes WPM medium, sucrose, hydrolyzed casein, hydrolyzed whey protein, thidiazuron, 6-benzylaminopurine, naphthalene acetic acid, plant gel, 4-morpholine ethanesulfonic acid, temik and polyvinylpyrrolidone as active ingredients;

[0011] The bud differentiation medium takes modified MS medium, copper sulfate, thidiazuron, 6-benzylaminopurine, indolebutyric acid, plant gel, 4-morpholine ethanesulfonic acid, temik, polyvinylpyrrolidone and hygromycin as active ingredients;

[0012] The bud proliferation medium takes MS medium, copper sulfate, sucrose, coconut powder, 6-benzylaminopurine, indolebutyric acid, plant gel, 4-morpholine ethanesulfonic acid, temik and hygromycin as active ingredients.

[0013] The infection medium configuration method is 1.0-5.0 g / L WPM medium+1-30 g / L sucrose+1-20 g / L glucose+0.5 g / L 4-morpholine ethanesulfonic acid+0.1-1 g / L magnesium sulfate;

[0014] The dedifferentiation initiation medium configuration method is 1.0-5.0 g / L WPM medium+1-30 g / L sucrose+1-20 g / L glucose+0.1-0.5 g / L hydrolyzed casein+0.1-0.5 g / L hydrolyzed whey protein+0.1-2.0 mg / L 2,4-dichlorophenoxyacetic acid+0.1-2.0 mg / L aminochlortripyridine+2.9 g / L plant gel+0.5 g / L 4-morpholine ethanesulfonic acid+0.2-2 g / L polyvinylpyrrolidone, pH=5.8;

[0015] The callus induction medium configuration method is 1.0-5.0 g / L WPM medium+30 g / L sucrose+0.1-0.5 g / L hydrolyzed casein+0.1-0.5 g / L hydrolyzed whey protein+0.1-2.0 mg / L thidiazuron+0.1-2.0 mg / L 6-benzylaminopurine+0.1-2.0 mg / L naphthalene acetic acid+2.9 g / L plant gel+0.5 g / L 4-morpholine ethanesulfonic acid+320 mg / L temik+0.2-2 g / L polyvinylpyrrolidone, pH=5.8;

[0016] The bud differentiation medium configuration method is: 1.0-5.0 g / L modified MS medium + 0.1-0.5 mg / L copper sulfate + 30 g / L sucrose + 0.1-2.0 mg / L thidiazuron + 0.1-2.0 mg / L 6-benzylaminopurine + 0.1-2.0 mg / L indole-3-butyric acid + 2.9 g / L phytagel + 0.5 g / L 4-morpholineethanesulfonic acid + 320 mg / L timentin + 0.2-2 g / L polyvinylpyrrolidone + 3 mg / L hygromycin, pH = 5.8.

[0017] The bud proliferation medium configuration method is: 1.0-5.0 g / L MS medium + 0.05-0.5 mg / L copper sulfate + 30 g / L sucrose + 100 mL / L coconut powder + 0.1-1.0 mg / L 6-benzylaminopurine + 0.1-1.0 mg / L indole-3-butyric acid + 2.9 g / L phytagel + 0.5 g / L 4-morpholineethanesulfonic acid + 320 mg / L timentin + 3 mg / L hygromycin, pH = 5.8.

[0018] The contents of ammonium nitrate and potassium nitrate in the modified MS medium are 1 / 2 of the contents of ammonium nitrate and potassium nitrate in the MS medium.

[0019] In the second aspect of the present application, the above-mentioned medium combination is applied in the modified leaf disc transformation method.

[0020] In the third aspect of the present application, a method for performing leaf disc transformation by using the above-mentioned medium combination is provided, which comprises the following steps:

[0021] (1) The agrobacterium activated by YEB medium is added into the infection medium to prepare an infection solution, the leaf of the tissue culture seedling of the transformation receptor is cut by using a sterile scalpel blade to make 2-3 cuts vertically along the main leaf vein, and then the leaf is soaked in the infection solution for 5-11 min and then transferred into the dedifferentiation start medium for co-culture;

[0022] (2) After the co-culture is completed, the leaf is transferred into the callus induction medium, the delayed culture leaf is transferred into the callus induction medium added with hygromycin to generate a resistant callus, and the resistant callus is transferred into the bud differentiation medium to differentiate into a resistant adventitious bud;

[0023] (3) When the adventitious bud grows to 1-2 cm, the adventitious bud is peeled off from the leaf by using a scalpel blade and then transferred into the bud proliferation medium for subculture.

[0024] In the method for performing leaf disc transformation by using the medium combination provided by the present application, the purpose of co-culture is to allow the agrobacterium to fully infect the wound site of the leaf, so that the target gene is integrated into the cells at the wound site.

[0025] The purpose of the delayed culture is to generate callus containing the target gene (with the property of resisting hygromycin) from the cells containing the target gene at the wound site on the wound induction medium.

[0026] After the Agrobacterium infection, i.e. after the end of co-culture, the cells containing the target gene need a period of time for division and differentiation, so that the target gene is fully expressed, and the cells can have the ability to resist hygromycin. In addition, the physiological state of the leaves infected by Agrobacterium is very weak, and it also needs to be fully adjusted to facilitate the generation of good quality resistant callus. Therefore, in the method of leaf disc transformation provided by the present application, after the end of co-culture, the leaves need to be buffered on the callus induction medium without hygromycin for a period of time, and then moved to the callus induction medium containing hygromycin. In this way, the callus containing the target gene is screened out by the action of hygromycin while inducing callus.

[0027] The Agrobacterium in step (1) is Agrobacterium carrying a marker vector or a target knockout vector.

[0028] The transformation receptor in step (1) is the leaf of apple 'Gala' tissue culture seedlings.

[0029] The anti-browning agent in step (1) is 10-100 mg / L ascorbic acid + 15-150 mg / L citric acid.

[0030] In a fourth aspect, the present application provides the above-mentioned method for use in (1) or (2) as follows:

[0031] (1) improving the genetic transformation efficiency of apple callus;

[0032] (2) improving the editing efficiency of the CRISPR / Cas9 system of apple;

[0033] Preferably, the improvement of the genetic transformation efficiency of apple callus is reflected by detecting the delivery efficiency of eGFP and RUBY marker proteins.

[0034] Preferably, the improvement of the editing efficiency of the CRISPR / Cas9 system of apple refers to obtaining various types of functional gene mutants, and increasing the data of apple gene editing.

[0035] The various types of functional gene mutants are any one of homozygous mutants, heterozygotes and chimeras.

[0036] The present application has the following beneficial effects:

[0037] The present application provides a medium combination according to the tissue culture nutrient requirement of woody plants, which can meet the demand of apple leaf regeneration, induce strong differentiation ability of callus and regenerated shoots, and overcome the problem of low genetic transformation efficiency of apple. The leaf disc transformation method improved based on the medium kit can significantly improve the editing efficiency of the CRISPR / Cas9 system of apple, can efficiently deliver the CRISPR / Cas9 knockout vector, and obtain homozygous and heterozygous plants of different mutation types including deletion, replacement and insertion, and the editing efficiency is as high as 92.3%. The method provided by the present application provides a new idea for analyzing the genetic mechanism of apple, improving agronomic traits and utilizing gene editing technology for germplasm innovation. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 For the growth of 25d 'Gala' tissue culture seedlings, Bar = 1cm.

[0039] Figure 2 For the marker vector MdLHP1-eGFP used, RUBY map

[0040] Figure 3 For the partial selection of the experimental results of using the improved leaf disc transformation method to detect the expression of eGFP marker protein, the callus indicated by the red arrow is the callus stably expressing eGFP marker protein, Bar = 0.2cm.

[0041] Figure 4 For the partial selection of the experimental results of using the original leaf disc transformation method to detect the expression of eGFP marker protein, the callus indicated by the red arrow is the callus stably expressing eGFP marker protein, Bar = 0.2cm.

[0042] Figure 5 For the partial selection of the experimental results of using the improved leaf disc transformation method to detect the expression of RUBY marker protein, the callus indicated by the red arrow is the callus stably expressing RUBY marker protein, Bar = 0.5cm.

[0043] Figure 6 For the partial selection of the experimental results of using the original leaf disc transformation method to detect the expression of RUBY marker protein, Bar = 0.5cm.

[0044] Figure 7 For the single target knockout vector pCas9-AtU6-lhp1sgC used, the map.

[0045] Figure 8 For the resistant callus generated on the callus induction medium supplemented with 3mg / L Hyg, Bar = 1cm.

[0046] Figure 9For resistant adventitious buds generated on bud differentiation medium, Bar = 0.5 cm.

[0047] Figure 10 For Hyg, Cas9 partial sequence electropherogram.

[0048] Figure 11 For apple MdLHP1 gene structure schematic diagram and target site sequence.

[0049] Figure 12 For Mdlhp1 mutant, Bar = 1 cm.

[0050] Figure 13 For Mdlhp1 mutant Sanger sequencing results.

[0051] Figure 14 For Mdlhp1 mutant editing type detection results. DETAILED DESCRIPTION

[0052] It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the present application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0053] As described previously, Agrobacterium tumefaciens-mediated plant transformation-leaf disc transformation method is one of the most widely used genetic transformation approaches, but the existing Agrobacterium tumefaciens-mediated apple leaf disc transformation method has problems such as unstable system, low transformation efficiency, poor test repeatability, and the like, and thus obtaining transgenic materials has been a bottleneck in apple transgenic research. The single medium and rough transformation steps in the existing leaf disc transformation system cannot meet the needs of leaf regeneration, and cannot induce strong differentiation ability of callus and regenerated buds. The extremely low transformation efficiency limits the implementation and expansion of gene editing technology in apples, and compared with other plants, apple gene editing research lacks rich data and unified reference indicators.

[0054] Therefore, the purpose of the present application is to provide a method for improving the editing efficiency of the apple CRISPR / Cas9 system by using an improved leaf disc transformation method. According to the mechanism of in vitro de novo regeneration of plants, a new and precise medium combination is designed, which meets the growth characteristics of different stages in the apple leaf regeneration process, so that the transformation results are efficient and stable. Based on the medium combination, the original leaf disc transformation method is improved, so that the efficiency of delivering gene editing tools mediated by the CRISPR / Cas9 system is greatly improved, the function of editing the genome is fully played, a large number of functional gene deletion mutants are obtained, the editing efficiency is as high as 92.3%, which makes up for the single disadvantage of apple gene editing data, provides a new idea for analyzing apple genetic mechanisms, improving agronomic traits, and using gene editing technology for germplasm innovation.

[0055] The application firstly improves the culture medium used in leaf disc transformation method, and provides a brand new culture medium combination, including: infection culture medium, dedifferentiation starting culture medium, callus induction culture medium, bud differentiation culture medium and bud proliferation culture medium.

[0056] The component of the infection culture medium uses WPM culture medium suitable for woody plant tissue culture, avoids the problem of too high inorganic salt concentration in the original leaf disc transformation method, and the sucrose and glucose in the improved infection culture medium can provide energy for plant cells and also can adjust the osmotic pressure to protect the cells from damage and death due to osmotic imbalance during the infection process. By adjusting the ratio of sucrose and glucose, the cell osmotic balance can be maintained and it is also conducive to the infection activity of Agrobacterium by osmotic potential. There is no need to add sodium citrate and betaine to avoid cell damage. 4-morpholine ethanesulfonic acid is used as a buffer solution to maintain the pH stability of the infection solution. A proper amount of magnesium sulfate is added to replace acetosyringone, which meets the activation needs of Agrobacterium and is conducive to the infection of Agrobacterium. pH 5.8 is the ideal range for the function of various inorganic salt components in the culture medium, and too low or too high is not conducive to the absorption and utilization of inorganic salt components by plants.

[0057] The dedifferentiation starting culture medium additionally adds hydrolyzed whey protein to provide amino acid components for plant cells, which is more comprehensive and has increased content than using hydrolyzed casein components alone. Plant gel is used instead of agar to overcome the defects of traditional agar, such as many impurities, affecting pH, and large amount of use, which is conducive to plant growth and development. The type and ratio of hormones are re-established, 2,4-dichlorophenoxyacetic acid and aminoclopyralid are used to start the dedifferentiation effect better, and the infection effect is enhanced. Polyvinylpyrrolidone is additionally added to prevent browning of wound cells during the culture process.

[0058] The callus induction culture medium also uses WPM culture medium instead of the original MS culture medium, and the low inorganic salt components of WPM culture medium reduce the salt stress on woody plant cells, and the induced callus tissue has higher activity and good condition. A large amount of amino acid components are needed for callus generation, and the addition of hydrolyzed casein and hydrolyzed whey protein can meet the growth needs of callus tissue. The combined use of thidiazuron, 6-benzylaminopurine and naphthaleneacetic acid is conducive to the dedifferentiation and redifferentiation process of apple cells. Timentin is an antibiotic specifically used to inhibit the proliferation of Agrobacterium tumefaciens, and compared with cefotaxime sodium, the use of timentin has very low adverse effects on plant growth and development.

[0059] The hygromycin is used instead of traditional kanamycin to screen resistant buds in the bud differentiation medium, the dose is small and the screening intensity is high, the generation of false positive escape buds is completely inhibited, and the interference on the data statistical accuracy is avoided. In the callus induction stage, the WPM medium is used to meet the growth needs of the callus. In the bud differentiation stage, the improved MS medium is used, which is beneficial to the differentiation of the buds and improves the number of regenerated buds. The copper sulfate is additionally added to meet the amount of copper ions required by woody plants. The thidiazuron, 6-benzylaminopurine and naphthalene acetic acid are combined to obtain callus with strong differentiation capacity, and the thidiazuron, 6-benzylaminopurine and indole butyric acid are combined after the callus is generated to help obtaining regenerated buds with high proliferation coefficient.

[0060] The coconut powder added in the bud proliferation medium is used for seedling strengthening. The coconut powder contains various amino acids, sugars and trace elements, which can improve the survival rate of regenerated buds, accelerate the growth speed of the buds and ensure the health of the buds during the proliferation period. The indole butyric acid is used instead of naphthalene acetic acid, which can reduce the action intensity of the auxin and strengthen the proliferation.

[0061] The application uses apple (Malus domestica 'Gala') tissue culture seedling leaves as transformation receptors, cuts the tissue culture seedling leaves in an anti-browning agent when cutting the leaves, and then transfers the leaves to an infection solution for soaking, thereby completely avoiding the browning phenomenon of the cut wound and the browning diffusion problem in the subsequent leaf culture process. In the experiment, Agrobacterium tumefaciens is used to deliver eGFP and RUBY marker proteins, and the original leaf disc transformation method is used as a control to detect the efficiency of the improved leaf disc transformation method. On the basis of the improved high-efficiency leaf disc transformation method, Agrobacterium tumefaciens is used to deliver a CRISPR / Cas9 knockout vector of a target functional gene, and the infected leaves are sequentially cultured in a dedifferentiation start medium, a callus induction medium, a bud differentiation medium and a bud proliferation medium to obtain a large number of transgenic plants. On this basis, the gene editing efficiency and editing types are counted and analyzed, the editing types of the mutants are analyzed, the specific editing effect of the CRISPR / Cas9 system in the apple genome is analyzed, and a reference is provided for optimizing the editing tool and other CRISPR / Cas type tools.

[0062] The specific embodiments of the application are described in further detail below in conjunction with examples. The following detailed description is exemplary in nature and is intended to provide further description of the application without limiting the scope of the application.

[0063] The plant gel in the callus induction medium, the bud differentiation medium and the bud proliferation medium used in the embodiment is purchased from Sigma-Aldrich, and the model number is Phytalgel TM .

[0064] The method for preparing the modified MS medium used in the embodiments is to reduce the content of ammonium nitrate and potassium nitrate in the MS medium by 1 / 2, specifically, the content of ammonium nitrate in the MS medium is 1650 mg / L, and the content of potassium nitrate in the MS medium is 1900 mg / L. The content of ammonium nitrate and potassium nitrate in the modified MS medium is 1 / 2 of the content of ammonium nitrate and potassium nitrate in the MS medium, that is, the content of ammonium nitrate and potassium nitrate in the modified MS medium is 825 mg / L and 950 mg / L, respectively.

[0065] Embodiment 1: Method for preparing medium combination

[0066] (1) Infection medium formula: 1.0 g / L WPM medium + 1 g / L sucrose + 1 g / L glucose + 0.5 g / L 4-morpholine ethanesulfonic acid + 0.1 / L magnesium sulfate, pH = 5.8.

[0067] (2) De-differentiation initiation medium formula: 1.0 g / L WPM medium + 1 g / L sucrose + 1 g / L glucose + 0.1 g / L hydrolyzed casein + 0.1 g / L hydrolyzed whey protein + 0.1 mg / L 2,4-dichlorophenoxyacetic acid + 0.1 mg / L aminochloropyridine acid + 2.9 g / L plant gel + 0.5 g / L 4-morpholine ethanesulfonic acid + 0.2 / L polyvinylpyrrolidone, pH = 5.8.

[0068] (3) Callus induction medium formula: 1.0 g / L WPM medium + 30 g / L sucrose + 0.01 g / L hydrolyzed casein + 0.1 g / L hydrolyzed whey protein + 0.1 mg / L thidiazuron + 0.1 mg / L 6-benzylaminopurine + 0.1 mg / L naphthalene acetic acid + 2.9 g / L plant gel + 0.5 g / L 4-morpholine ethanesulfonic acid + 320 mg / L timentin + 0.2 g / L polyvinylpyrrolidone, pH = 5.8.

[0069] (4) Bud differentiation medium formula: 1.0 g / L modified MS medium + 0.1 mg / L copper sulfate + 30 g / L sucrose + 0.1 mg / L thidiazuron + 0.1 mg / L 6-benzylaminopurine + 0.1 mg / L indolebutyric acid + 2.9 g / L plant gel + 0.5 g / L 4-morpholine ethanesulfonic acid + 320 mg / L timentin + 0.2 g / L polyvinylpyrrolidone + 3 mg / L hygromycin, pH = 5.8.

[0070] (5) Shoot proliferation medium formula: 5.0 g / L MS medium + 0.5 mg / L copper sulfate + 30 g / L sucrose + 100 mL / L coconut powder + 1.0 mg / L 6-benzylaminopurine + 1.0 mg / L indolebutyric acid + 2.9 g / L Phytagel + 0.5 g / L 4-morpholineethanesulfonic acid + 320 mg / L timentin + 3 mg / L hygromycin, pH = 5.8.

[0071] Example 2: Method of configuring medium combinations

[0072] (1) Infection medium formula: 5.0 g / L WPM medium + 30 g / L sucrose + 20 g / L glucose + 0.5 g / L 4-morpholineethanesulfonic acid + 1 / L magnesium sulfate, pH = 5.8.

[0073] (2) De-differentiation initiation medium formula: 5.0 g / L WPM medium + 30 g / L sucrose + 20 g / L glucose + 0.5 g / L hydrolyzed casein + 0.5 g / L hydrolyzed whey protein + 2.0 mg / L 2,4-dichlorophenoxyacetic acid + 2.0 mg / L aminoclopyralid + 2.9 g / L Phytagel + 0.5 g / L 4-morpholineethanesulfonic acid + 2 / L polyvinylpyrrolidone, pH = 5.8.

[0074] (3) Callus induction medium formula: 5.0 g / L WPM medium + 30 g / L sucrose + 0.5 g / L hydrolyzed casein + 0.5 g / L hydrolyzed whey protein + 2.0 mg / L thidiazuron + 2.0 mg / L 6-benzylaminopurine + 2.0 mg / L naphthaleneacetic acid + 2.9 g / L Phytagel + 0.5 g / L 4-morpholineethanesulfonic acid + 320 mg / L timentin + 2 g / L polyvinylpyrrolidone, pH = 5.8.

[0075] (4) Shoot differentiation medium formula: 5.0 g / L modified MS medium + 0.5 mg / L copper sulfate + 30 g / L sucrose + 2.0 mg / L thidiazuron + 2.0 mg / L 6-benzylaminopurine + 2.0 mg / L indolebutyric acid + 2.9 g / L Phytagel + 0.5 g / L 4-morpholineethanesulfonic acid + 320 mg / L timentin + 2 g / L polyvinylpyrrolidone + 3 mg / L hygromycin, pH = 5.8.

[0076] (5) Shoot proliferation medium formula: 5.0 g / L MS medium + 0.5 mg / L copper sulfate + 30 g / L sucrose + 100 mL / L coconut powder + 1.0 mg / L 6-benzylaminopurine + 1.0 mg / L indolebutyric acid + 2.9 g / L Phytagel + 0.5 g / L 4-morpholineethanesulfonic acid + 320 mg / L timentin + 3 mg / L hygromycin, pH = 5.8.

[0077] Example 3: Method for configuring medium combinations

[0078] (1) Infection medium formula: 2.5 g / L WPM medium + 15 g / L sucrose + 10 g / L glucose + 0.5 g / L 4-morpholineethanesulfonic acid + 0.5 g / L magnesium sulfate, pH = 5.8.

[0079] (2) Dedifferentiation initiation medium formula: 2.5 g / L WPM medium + 15 g / L sucrose + 10 g / L glucose + 0.25 g / L hydrolyzed casein + 0.25 g / L hydrolyzed whey protein + 1.0 mg / L 2,4-dichlorophenoxyacetic acid + 1.0 mg / L aminochloropyridylacetic acid + 2.9 g / L Phytagel + 0.5 g / L 4-morpholineethanesulfonic acid + 1.1 g / L polyvinylpyrrolidone, pH = 5.8.

[0080] (3) Callus induction medium formula: 2.5 g / L WPM medium + 30 g / L sucrose + 0.25 g / L hydrolyzed casein + 0.3 g / L hydrolyzed whey protein + 1.0 mg / L thidiazuron + 1.0 mg / L 6-benzylaminopurine + 1.0 mg / L naphthaleneacetic acid + 2.9 g / L Phytagel + 0.5 g / L 4-morpholineethanesulfonic acid + 320 mg / L timentin + 1.1 g / L polyvinylpyrrolidone, pH = 5.8.

[0081] (4) Shoot differentiation medium formula: 0.25 g / L modified MS medium + 0.3 mg / L copper sulfate + 30 g / L sucrose + 1.0 mg / L thidiazuron + 1.0 mg / L 6-benzylaminopurine + 1.0 mg / L indolebutyric acid + 2.9 g / L Phytagel + 0.5 g / L 4-morpholineethanesulfonic acid + 320 mg / L timentin + 1.1 g / L polyvinylpyrrolidone + 3 mg / L hygromycin, pH = 5.8.

[0082] (5) Shoot proliferation medium formula: 2.5 g / L MS medium + 0.275 mg / L copper sulfate + 30 g / L sucrose + 100 mL / L coconut powder + 0.5 mg / L 6-benzylaminopurine + 0.5 mg / L indolebutyric acid + 2.9 g / L Phytagel + 0.5 g / L 4-morpholineethanesulfonic acid + 320 mg / L timentin + 3 mg / L hygromycin, pH = 5.8.

[0083] Example 4: Method for leaf disc transformation based on the medium combinations provided in Example 3

[0084] (1) The transformation receptor used was a leaf of a 25-day-old ‘Gala’ tissue culture seedling (Fig. 1) Figure 1 ) grown under the following conditions: temperature of 25 ± 1°C, light intensity of 5000-7000 Lux, and light cycle of 16h / 8h.

[0085] (2) Agrobacterium EHA105 containing the target vector was cultured in YEB liquid medium supplemented with 25 mg / L rifampicin, 50 mg / L streptomycin and 50 mg / L kanamycin at 28°C with shaking at 200 rpm; when the bacterial culture OD 600 When the OD value is 0.7, centrifuge at 4000 rpm for 10 min and collect the bacterial cells; add infection medium and adjust the OD of the bacterial solution. 600 The value is 0.5. After standing in the dark for 2 hours, the inoculum is prepared for later use.

[0086] (3) Cut leaves from the above tissue culture seedlings and place them in an anti-browning agent (10 mg / L ascorbic acid + 15 mg / L citric acid). Make two cuts perpendicular to the main vein using a sterile scalpel and immerse them in the infection solution for 5 minutes. After infection, transfer the leaves to a dedifferentiation initiation medium and culture them for 2 days at 20±1℃ in the dark.

[0087] (4) After co-culture, the leaves were transferred to callus induction medium and cultured for 3 days at a temperature of 22±1℃ in the dark.

[0088] (5) The leaves after delayed culture were transferred to callus induction medium supplemented with 3 mg / L hygromycin and cultured in the dark at 22±1℃ until resistant callus tissue was generated at the leaf wound. Figure 8 ).

[0089] (6) Transfer the leaves with resistant callus to the bud differentiation medium and continue the process at a temperature of 22±1℃, a light intensity of 3000Lux, and a photoperiod of 16h / 8h until the callus differentiates into resistant adventitious buds.

[0090] (7) When the resistant buds grow to 1-2cm, use a scalpel to peel them off from the leaves and transfer them to the bud proliferation medium. Culture them at a temperature of 22±1℃, a light intensity of 3000Lux, and a photoperiod of 16h / 8h. Subculture once every 25 days.

[0091] Comparative Example 1: The original bladed disc conversion method

[0092] 1. Preparation method of culture medium combination used in the original leaf disc conversion method

[0093] (1) Original infection medium formula: 4.43 g / L MS medium + 25 g / L sucrose + 5 g / L glucose + 2 mg / L thidiazuron + 0.5 mg / L naphthaleneacetic acid + 0.5 g / L hydrolyzed casein + 100 Mm acetylsalicylic acid, without pH adjustment.

[0094] (2) Original dedifferentiation initiation medium formula: 4.43 g / L MS medium + 2 mg / L thidiazuron + 0.5 mg / L naphthalene acetic acid + 25 g / L sucrose + 5 g / L glucose + 0.5 g / L hydrolyzed casein + 7 g / L agar, pH = 5.2.

[0095] (3) Original callus induction medium formula: 4.43 g / L MS medium + 30 g / L sucrose + 2 mg / L thidiazuron + 0.5 mg / L naphthalene acetic acid + 7 g / L agar + 250 mg / L cephalosporin sodium + 250 mg / L timentin, pH = 5.4.

[0096] (4) Original bud differentiation medium formula: 4.43 g / L MS medium + 30 g / L sucrose + 2 mg / L thidiazuron + 0.5 mg / L naphthalene acetic acid + 7 g / L agar + 25 mg / L kanamycin + 250 mg / L cephalosporin sodium + 250 mg / L timentin, pH = 5.6.

[0097] (5) Original bud proliferation medium formula: 4.43 g / L MS medium + 30 g / L sucrose + 1 mg / L 6-benzylaminopurine + 0.2 mg / L naphthalene acetic acid + 0.5 mg / L gibberellin + 7.0 g / L agar + 250 mg / L cephalosporin sodium + 250 mg / L timentin + 25 mg / L kanamycin, pH = 5.8-6.0.

[0098] 2. Experimental method of original leaf disc transformation method

[0099] (1) The glycerol bacteria stored at -80°C were streaked and plated on 100 mL rifampicin + gene vector resistant LB solid medium for 2 days. In 100 mL Rif + vector resistant LB liquid medium, 28°C shaking culture overnight, OD600 = 1.5. 5000 rpm normal temperature centrifugal 5 min. Resuspend with original infection medium, dilute to OD600 = 0.5.

[0100] (2) Use the leaves of 'Gala' tissue culture seedlings grown for about 30 days, use a scalpel to make 2-3 cuts perpendicular to the main leaf veins on the back of the leaves, and after all the leaves are cut, soak in the bacterial solution for 8 min.

[0101] (3) Take out the leaves, absorb the bacterial solution on sterile filter paper, place the back of the leaves upwards on the original dedifferentiation initiation medium, and culture in the dark for 3 days.

[0102] (4) Place the leaves on the original callus induction medium, and culture in the dark for 3 days.

[0103] (5) Transfer the leaves to the original bud differentiation medium, and culture in the dark for 1 month.

[0104] (6) Light-colored buds grow out of the leaf cut. Transfer the leaf to a new original bud differentiation medium and culture it under light. The resistant buds turn green.

[0105] (7) After the resistant buds are exposed to light for one month, the resistant buds are cut off and transferred to the original bud proliferation medium for culture. Subculture is performed once every 30 days.

[0106] Experiment 1: Efficiency detection of eGFP, RUBY-tagged protein delivery

[0107] 1. Construction of the marker vector MdLHP1-eGFP

[0108] Using the pCAM1300-eGFP vector as a backbone, the vector was linearized and purified using BamHI high-fidelity enzyme. The full-length CDS sequence of MdLHP1 (GDR database gene ID: MD07G1081800) was homologously ligated with the linearized vector pCAM1300-eGFP using the In-Fusion method to obtain the pCAM1300-MdLHP1-eGFP overexpression vector, i.e., the marker vector MdLHP1-eGFP.

[0109] 2. Efficiency detection of eGFP and RUBY-tagged protein delivery using the improved leaf disc transformation method based on this invention:

[0110] (1) The transformation recipient used was a leaf from a 25-day-old 'Gala' tissue culture seedling. Figure 1 The growth conditions for tissue culture seedlings are a temperature of 25±1℃, a light intensity of 5000-7000 Lux, and a photoperiod of 16h / 8h.

[0111] (2) The marker vectors MdLHP1-eGFP and RUBY ( Figure 2 Agrobacterium EHA105 was inoculated at a ratio of 1:1000 into YEB liquid medium supplemented with 25 mg / L rifampin, 50 mg / L streptomycin, and 50 mg / L kanamycin, and cultured at 28°C with shaking at 200 rpm; when the bacterial culture OD... 600 When the value is 0.7, centrifuge at 4000 rpm for 10 min, collect the bacterial cells, add them to the infection medium, and adjust the OD of the bacterial solution. 600 The value is 0.5. After standing in the dark for 2 hours, the inoculum is prepared for later use.

[0112] (3) Cutting Step (1) Leaves of tissue culture seedlings were placed in an anti-browning agent (10 mg / L ascorbic acid + 15 mg / L citric acid), and two cuts were made perpendicular to the main vein using a sterile scalpel. The leaves were then immersed in the infection solution for 5 minutes. After infection, the leaves were transferred to a dedifferentiation initiation medium and cultured for 2 days in the dark at a temperature of 20±1℃.

[0113] (4) After co-cultivation, the leaves were transferred to callus induction medium containing 3 mg / L hygromycin, and cultured at 22±1℃ in dark for 3 days.

[0114] (5) The leaves after delay culture were transferred to callus induction medium containing 3 mg / L hygromycin, and cultured at 22±1℃ in dark until resistant callus tissue was generated at the leaf wound.

[0115] (6) At this time, the callus phenotype of MdLHP1-eGFP and RUBY transformed leaves was observed under ultraviolet light, and the transformation efficiency was calculated by the formula: the number of transgenic callus leaves / total leaf number. The resistant callus tissue stably expressing eGFP marker protein showed green fluorescence under ultraviolet light, and the callus tissue stably expressing RUBY marker protein showed red phenotype under direct naked eye observation. The detection results of eGFP marker protein delivered by the improved leaf disc transformation method are shown in Figure 3 , and the detection results of RUBY marker protein delivered by the improved leaf disc transformation method are shown in Figure 5 .

[0116] (7) According to the phenotype of transgenic callus tissue, the data statistics and analysis were carried out, and the efficiencies of eGFP and RUBY marker proteins delivered by the improved leaf disc transformation method were 83.33% (Table 1) and 86.67% (Table 2), respectively.

[0117] 3. Efficiency detection of eGFP and RUBY marker proteins delivered by the original leaf disc transformation method:

[0118] According to the original leaf disc transformation method in Comparative Example 1, the callus tissue was placed under ultraviolet light to observe the phenotype, and the detection results of eGFP marker protein delivered by the original leaf disc transformation method are shown in Figure 4 , and the detection results of RUBY marker protein delivered by the original leaf disc transformation method are shown in Figure 6 . According to the phenotype of transgenic callus tissue, the data statistics and analysis were carried out, and the efficiencies of eGFP and RUBY marker proteins delivered by the original leaf disc transformation method were 16.67% (Table 1) and 0% (Table 2), respectively.

[0119] Table 1: Efficiency statistics of callus tissue stably expressing eGFP obtained by using the original and improved leaf disc transformation methods

[0120]

[0121] Table 2: Efficiency statistics of callus tissue stably expressing RUBY obtained by using the original and improved leaf disc transformation methods

[0122]

[0123] Test Example 2: Detection of editing efficiency of apple CRISPR / Cas9 system

[0124] 1. Construction of knockout vector pCas9-AtU6-lhp1sgc

[0125] The pCAMBIA1300 vector was used as a backbone vector, and EcoRI and HindIII were used for double enzyme digestion to obtain a linearized vector. The AtU6::sgR expression cassette and the 35S::Cas9 expression cassette were sequentially connected to the linearized pCAMBIA1300 vector by homologous recombination to obtain pCas9-AtU6-sgR. Then, pCas9-AtU6-sgR was used as a backbone vector, and BsaI high-fidelity enzyme was used for digestion and then purified and recovered. The target lhp1sgC was connected to the linearized vector pCas9-AtU6-sgR using T4 ligase to obtain pCas9-AtU6-lhp1sgC, which was used for single-target knockout of the target gene.

[0126] The nucleotide sequence of the target lhp1sgC is shown in SEQ ID NO. 1: TACGAGAACGAGTACTC

[0127] 2. Detection of editing efficiency of apple CRISPR / Cas9 system based on improved leaf disc transformation method

[0128] (1) The transformation receptor used was the leaf of a 25-day-old ‘Gala’ tissue culture seedling ( Figure 1 ), and the tissue culture seedling growth conditions were a temperature of 25±1°C, a light intensity of 5000-7000 Lux, and a light cycle of 16h / 8h.

[0129] (2) Agrobacterium EHA105 containing the knockout vector pCas9-AtU6-lhp1sgc ( Figure 7 ) was cultured in YEB liquid medium supplemented with 25mg / L rifampicin, 50mg / L streptomycin, and 50mg / L kanamycin at 28°C with 200rpm shaking; when the OD 600 value of the bacterial solution was 0.7, the bacterial solution was centrifuged at 4000rpm for 10min, and the bacterial cells were collected; the infection medium was added, the OD 600 value of the bacterial solution was adjusted to 0.5, and the bacterial solution was placed in the dark for 2h to prepare an infection solution.

[0130] (3) The above-mentioned tissue culture seedling leaf was cut and placed in an anti-browning agent (10mg / L ascorbic acid + 15mg / L citric acid), and a sterile scalpel was used to cut the leaf vertically along the main vein twice, and the leaf was soaked in the infection solution for 5min. After the infection was completed, the leaf was transferred to the dedifferentiation initiation medium and cultured in the dark at a temperature of 20±1°C for a total of 2 days.

[0131] (4) After co-culture, the leaves were transferred to callus induction medium and cultured in dark at 22±1℃ for 3 days.

[0132] (5) The leaves after delay culture were transferred to callus induction medium with 3 mg / L hygromycin and cultured in dark at 22±1℃ until resistant callus formed at the leaf wound Figure 8 ).

[0133] (6) The leaves with resistant callus were transferred to bud differentiation medium and cultured at 22±1℃, 3000 Lux light intensity and 16h / 8h photoperiod until resistant adventitious buds differentiated from the callus Figure 9 ).

[0134] (7) When the resistant buds grew to 1-2 cm, they were peeled off from the leaves using a scalpel and transferred to bud proliferation medium and cultured at 22±1℃, 3000 Lux light intensity and 16h / 8h photoperiod, subcultured every 25 days;

[0135] (8) The genomic DNA of the resistant plantlets was extracted using CTAB method, and primers HygF, HygR and Cas9F, Cas9R (Table 3) were designed to amplify the Hyg and Cas9 partial sequences by PCR, and the results of agarose gel electrophoresis showed that T1, T3-T6, T8, T10-T15 among the 16 resistant buds were transgenic plants Figure 10 ); The DNA full-length sequence of MdLHP1 gene was analyzed using CRISPR-P 2.0 online, and multiple target site sequences were obtained. Primers lhp1F, lhp1R (Table 3) were designed near the selected target site sgC Figure 11 ) and used for PCR amplification and Sanger sequencing. Sequence alignment analysis showed that 12 out of 13 transgenic plants were MdLHP1 mutants, including 1 homozygous mutant, 2 heterozygotes and 9 chimeras Figure 13 ). The DNA of the leaves of the 12 MdLHP1 mutants was extracted for detection, and the results showed that all 12 MdLHP1 mutants had editing events Figure 14 ), and the editing ratio was 92.3% according to the proportion of edited plants to transgenic plants.

[0136] Table 3: Sequences of detection primers

[0137]

[0138]

[0139] The above descriptions are only the preferred embodiments of the present application, and are not intended to limit the present application. The present application can have various modifications and changes for those skilled in the art. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A culture medium composition, characterized in that, include: Infection medium, dedifferentiation initiation medium, callus induction medium, bud differentiation medium, and bud proliferation medium; explants were apple tissue culture seedling leaves. The infection culture medium is prepared as follows: 1.0-5.0 g / L WPM medium + 1-30 g / L sucrose + 1-20 g / L glucose + 0.5 g / L 4-morpholine ethanesulfonic acid + 0.1-1 g / L magnesium sulfate; The dedifferentiation initiation medium is prepared as follows: 1.0-5.0 g / L WPM medium + 1-30 g / L sucrose + 1-20 g / L glucose + 0.1-0.5 g / L hydrolyzed casein + 0.1-0.5 g / L hydrolyzed whey protein + 0.1-2.0 mg / L 2,4-dichlorophenoxyacetic acid + 0.1-2.0 mg / L aminopyridine acid + 2.9 g / L plant gel + 0.5 g / L 4-morpholinoethanesulfonic acid + 0.2-2 g / L polyvinylpyrrolidone, pH=5.8; The callus induction medium is prepared as follows: 1.0-5.0 g / L WPM medium + 30 g / L sucrose + 0.1-0.5 g / L hydrolyzed casein + 0.1-0.5 g / L hydrolyzed whey protein + 0.1-2.0 mg / L thidiazuron + 0.1-2.0 mg / L 6-benzylaminopurine + 0.1-2.0 mg / L naphthaleneacetic acid + 2.9 g / L plant gel + 0.5 g / L 4-morpholinoethanesulfonic acid + 320 mg / L termetidine + 0.2-2 g / L polyvinylpyrrolidone, pH=5.8; The bud differentiation medium was prepared as follows: 1.0-5.0 g / L modified MS medium + 0.1-0.5 mg / L copper sulfate + 30 g / L sucrose + 0.1-2.0 mg / L thidiazuron + 0.1-2.0 mg / L 6-benzylaminopurine + 0.1-2.0 mg / L indolebutyric acid + 2.9 g / L plant gel + 0.5 g / L 4-morpholinoethanesulfonic acid + 320 mg / L termethin + 0.2-2 g / L polyvinylpyrrolidone + 3 mg / L hygromycin, pH=5.8; The bud proliferation medium was prepared as follows: 1.0-5.0 g / L MS medium + 0.05-0.5 mg / L copper sulfate + 30 g / L sucrose + 100 mL / L coconut powder + 0.1-1.0 mg / L 6-benzylaminopurine + 0.1-1.0 mg / L indolebutyric acid + 2.9 g / L plant gel + 0.5 g / L 4-morpholinoethanesulfonic acid + 320 mg / L termethin + 3 mg / L hygromycin, pH=5.8; The modified MS medium contains half the ammonium nitrate and potassium nitrate content of the original MS medium.

2. The application of the culture medium combination according to claim 1 in the improved leaf disc transformation method, characterized in that, The leaf disc transformation method is an apple leaf disc transformation method mediated by Agrobacterium tumefaciens.

3. A method for leaf disc transformation using the culture medium combination described in claim 1, characterized in that, Includes the following steps: (1) Add Agrobacterium activated by YEB medium to the infection medium to prepare an infection solution. Cut apple tissue culture seedling leaves and place them in an anti-browning agent. Use a sterile scalpel to make 2-3 cuts perpendicular to the main vein. Soak the leaves in the infection solution for 5-11 minutes and then transfer them to the dedifferentiation start-up medium for co-culture. (2) After co-culture, the leaves were transferred to callus induction medium, and the leaves with delayed culture were transferred to callus induction medium supplemented with hygromycin to generate resistant callus tissue. The resistant callus tissue was then transferred to bud differentiation medium to differentiate resistant adventitious buds. (3) When the adventitious buds grow to 1-2 cm, use a scalpel to peel them off from the leaves and transfer them to the bud proliferation medium for subculture.

4. The method according to claim 3, characterized in that, The Agrobacterium mentioned in step (1) is an Agrobacterium carrying a marker vector or a target knockout vector.

5. The method according to claim 3, characterized in that, The anti-browning agent mentioned in step (1) is 10-100 mg / L ascorbic acid + 15-150 mg / L citric acid.

6. The application of the method of claim 3 in either (1) or (2): (1) Improve the genetic transformation efficiency of apple callus; (2) Improve the editing efficiency of Apple's CRISPR / Cas9 system.

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