Method for improving editing efficiency of apple CRISPR / Cas9 system by using improved leaf disc conversion method

Abstract

The invention discloses a method for improving the editing efficiency of an apple CRISPR / Cas9 system by using an improved leaf disc conversion method, and belongs to the technical field of biology. The invention provides a culture medium composition according to the requirement of woody plant tissue culture nutrients, the requirement of apple leaf regeneration can be met, calluses and regenerated buds with high differentiation capacity are induced, and the problem of low genetic transformation efficiency of apples is solved. An improved leaf disc conversion method based on the culture medium package can significantly improve the editing efficiency of an apple CRISPR / Cas9 system, can efficiently deliver a CRISPR / Cas9 knockout carrier, obtains homozygote and heterozygote plants of different mutation types including deletion, replacement, insertion and the like, and has the editing efficiency as high as 92.3%. The method provided by the invention provides a new thought for analyzing genetic mechanisms of apples, improving agronomic characters and performing germplasm innovation by utilizing a gene editing technology.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for improving the editing efficiency of an apple CRISPR / Cas9 system by using an improved leaf disc transformation method. Background Art

[0002] CRISPR / Cas9 is a widely used gene editing technology. The gene editing system dominated by CRISPR / Cas9 is developed through the mechanism of bacterial defense against exogenous nucleic acids. It consists of Cas protein, sgRNA and promoter. The vector can be delivered to the recipient plant genome through Agrobacterium tumefaciens to express Cas9 protein and sgRNA, thereby editing the target gene.

[0003] Agrobacterium tumefaciens-mediated plant transformation - leaf disc transformation is one of the most widely used genetic transformation pathways. Under natural conditions, Agrobacterium tumefaciens can infect the injured parts of dicotyledonous plants and integrate the DNA fragment with transposition function on its own Ti plasmid into the recipient cell genome for expression. The Agrobacterium tumefaciens-mediated genetic transformation method was developed based on this characteristic, and transgenic plants can be obtained with the help of Agrobacterium tumefaciens transformation.

[0004] An efficient and stable transformation method is a prerequisite for plant gene editing. However, the culture medium used in the existing leaf disc transformation method is not completely suitable for woody plant tissue culture, and the infection effect is not strong. The culture medium used cannot fully meet the needs of leaf regeneration and cannot induce callus tissue and regenerated buds with strong differentiation ability. In addition, the existing leaf disc transformation method has problems such as system instability, low transformation efficiency, and poor experimental repeatability during the genetic transformation experiment mediated by the CRISPR / Cas9 system, which makes it difficult to obtain apple transgenic materials and the apple gene editing data is single. Therefore, there is an urgent need for an improved leaf disc transformation method that 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. Summary of the invention

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

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a culture medium combination, comprising: an infection culture medium, a dedifferentiation initiation culture medium, a callus induction culture medium, a bud differentiation culture medium and a bud proliferation culture medium;

[0008] The infection culture medium contains WPM culture medium, sucrose, glucose, 4-morpholineethanesulfonic acid and magnesium sulfate as active ingredients;

[0009] The dedifferentiation initiation culture medium contains WPM culture medium, sucrose, glucose, hydrolyzed casein, hydrolyzed whey protein, 2,4-dichlorophenoxyacetic acid, amiloride, plant gel, 4-morpholineethanesulfonic acid and polyvinylpyrrolidone as active ingredients;

[0010] The callus induction medium contains WPM medium, sucrose, hydrolyzed casein, hydrolyzed whey protein, thidiazuron, 6-benzylaminopurine, naphthylacetic acid, plant gel, 4-morpholineethanesulfonic acid, timentin and polyvinyl pyrrolidone as active ingredients;

[0011] The bud differentiation medium contains modified MS medium, copper sulfate, thidiazuron, 6-benzylaminopurine, indolebutyric acid, phytagel, 4-morpholineethanesulfonic acid, timentin, polyvinyl pyrrolidone and hygromycin as active ingredients;

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

[0013] The infection culture medium is prepared as follows: 1.0-5.0 g / L WPM culture medium + 1-30 g / L sucrose + 1-20 g / L glucose + 0.5 g / L 4-morpholineethanesulfonic acid + 0.1-1 g / L magnesium sulfate;

[0014] The dedifferentiation initiation culture medium is prepared as follows: 1.0-5.0 g / L WPM culture 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 amiloride + 2.9 g / L plant gel + 0.5 g / L 4-morpholineethanesulfonic acid + 0.2-2 g / L polyvinylpyrrolidone, pH = 5.8;

[0015] The callus induction medium is prepared as follows: 1.0-5.0 g / L WPM medium + 30 g / L sucrose + 0.01-0.5 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 phytagel + 0.5 g / L 4-morpholineethanesulfonic acid + 320 mg / L timentin + 0.2-2 g / L polyvinyl pyrrolidone, pH = 5.8;

[0016] The bud differentiation medium is 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-morpholineethanesulfonic acid + 320 mg / L timentin + 0.2-2 g / L polyvinyl pyrrolidone + 3 mg / L hygromycin, pH = 5.8;

[0017] The bud proliferation culture medium is prepared as follows: 1.0-5.0 g / L MS culture 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-morpholineethanesulfonic acid + 320 mg / L timentin + 3 mg / L hygromycin, pH = 5.8.

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

[0019] The second aspect of the present invention provides the use of the above culture medium combination in the improved leaf disc transformation method.

[0020] In a third aspect, the present invention provides a method for leaf disc transformation using the above culture medium combination, comprising the following steps:

[0021] (1) Add Agrobacterium activated by YEB medium to the infection medium to prepare the infection solution, cut the leaves of the transformed recipient tissue culture seedlings and place them in an anti-browning agent, use a sterile surgical blade to cut 2-3 times vertically on the main vein, soak them in the infection solution for 5-11 minutes, and then transfer them to the dedifferentiation initiation medium for co-cultivation;

[0022] (2) After the co-cultivation is completed, the leaves are transferred to a callus induction medium, and the leaves that have been delayed in culture are transferred to a callus induction medium supplemented with hygromycin to generate resistant callus tissue, and the resistant callus tissue is transferred to a bud differentiation medium to differentiate resistant adventitious buds;

[0023] (3) When the adventitious buds grow to 1-2 cm, they are peeled off from the leaves using a surgical blade and transferred to bud proliferation medium for subculture.

[0024] The purpose of co-cultivation in the method for leaf disc transformation using the culture medium combination provided by the present invention is to allow Agrobacterium to fully infect the wound site of the leaf, so that the target gene is integrated into the cells of the wound site.

[0025] The purpose of the delayed culture is to allow the cells containing the target gene in the wound site to generate callus containing the target gene (with hygromycin resistance) on the callus induction medium.

[0026] After the infection of the agrobacterium, that is, after the co-cultivation ends, the cells containing the target gene need a period of time to divide and differentiate, so that the target gene is fully expressed, and the ability to resist hygromycin can be possessed. In addition, the physiological state of the leaves infected by agrobacterium is very weak, and it is also necessary to fully adjust, so as to be conducive to generating resistant callus in good condition. Therefore, in the method for leaf disc transformation by the culture medium combination provided by the present invention, after the co-cultivation ends, the leaves need to be buffered for a period of time on the callus induction medium without hygromycin, and then moved to the callus induction medium containing hygromycin, and while inducing callus, the callus containing the target gene screened out by the action of hygromycin.

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

[0028] The transformation recipient in step (1) is a leaf of apple 'Gala' tissue culture seedling.

[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 invention provides the application of the above method in the following (1) or (2):

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

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

[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 improved editing efficiency of the apple CRISPR / Cas9 system refers to obtaining various types of functional gene mutants and increasing apple gene editing data.

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

[0036] The beneficial effects of the present invention are:

[0037] According to the nutrient requirements of woody plant tissue culture, the present invention provides a culture medium combination that can meet the needs of apple leaf regeneration, induce callus tissue and regenerated buds with strong differentiation ability, and overcome the problem of low genetic transformation efficiency of apples. The leaf disc transformation method based on the improved culture medium package can significantly improve the editing efficiency of the apple CRISPR / Cas9 system, can efficiently deliver CRISPR / Cas9 knockout vectors, and obtain homozygous and heterozygous plants with different mutation types including deletion, replacement, and insertion, with an editing efficiency of up to 92.3%. The method provided by the present invention provides new ideas for analyzing the genetic mechanism of apples, improving agronomic traits, and using gene editing technology for germplasm innovation. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is the 'Gala' tissue culture seedling grown for 25 days, Bar=1 cm.

[0039] Figure 2 The marker vector used is MdLHP1-eGFP, RUBY map

[0040] Figure 3 This is a partial excerpt of the experimental results using the improved leaf disc transformation method to detect the expression of eGFP marker protein, where the callus indicated by the red arrow is the callus that stably expresses the eGFP marker protein, Bar=0.2 cm.

[0041] Figure 4 This is a partial excerpt of the experimental results using the original leaf disc transformation method to detect the expression of eGFP marker protein, where the callus indicated by the red arrow is the callus that stably expresses the eGFP marker protein, Bar=0.2 cm.

[0042] Figure 5 This is a partial excerpt of the experimental results using the improved leaf disc transformation method to detect the expression of RUBY marker protein, where the callus indicated by the red arrow is the callus that stably expresses the RUBY marker protein, Bar=0.5 cm.

[0043] Figure 6 This is a partial excerpt of the experimental results using the original leaf disc transformation method to detect the expression of RUBY marker protein. Bar=0.5cm.

[0044] Figure 7 This is a map of the single-target knockout vector pCas9-AtU6-lhp1sgC used.

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

[0046] Fig. 9These are resistant adventitious shoots generated on shoot differentiation medium, Bar=0.5 cm.

[0047] Fig.10 This is the electrophoresis diagram of some sequences of Hyg and Cas9.

[0048] Fig.11 Schematic diagram of the apple MdLHP1 gene structure and target site sequence.

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

[0050] Fig.13 This is the Sanger sequencing result of the Mdlhp1 mutant.

[0051] Fig.14 This is a diagram showing the editing type detection results of the Mdlhp1 mutant. DETAILED DESCRIPTION

[0052] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0053] As mentioned above, Agrobacterium tumefaciens-mediated plant transformation-leaf disc transformation is one of the most widely used genetic transformation pathways. However, the existing Agrobacterium tumefaciens-mediated apple leaf disc transformation method has problems such as system instability, low transformation efficiency, and poor experimental repeatability. Therefore, the difficulty in obtaining transgenic materials has always been a bottleneck in apple transgenic research. The single culture medium and rough transformation steps in the existing leaf disc transformation system cannot meet the needs of leaf regeneration and cannot induce callus tissue and regenerated buds with strong differentiation ability. The extremely low transformation efficiency limits the implementation and expansion of gene editing technology in apples. Compared with other plants, apple gene editing research lacks rich data and unified reference indicators.

[0054] Based on this, the purpose of the present invention is to provide a method for improving the editing efficiency of the apple CRISPR / Cas9 system using an improved leaf disc transformation method. The present invention designs a new, precise and detailed culture medium combination based on the de novo regeneration mechanism of plants in vitro, which conforms to the growth characteristics of apple leaves at different stages of regeneration, so that the transformation results are efficient and stable; based on the culture 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 utilized, a large number of functional gene deletion mutants are obtained, and the editing efficiency is as high as 92.3%, which makes up for the shortcoming of the single gene editing data of apples, and provides new ideas for analyzing the genetic mechanism of apples, improving agronomic traits, and using gene editing technology for germplasm innovation.

[0055] The present invention first improves the culture medium used in the leaf disc transformation method and provides a new culture medium combination, including: infection culture medium, dedifferentiation initiation culture medium, callus induction culture medium, bud differentiation culture medium and bud proliferation culture medium.

[0056] The infection medium component uses WPM medium suitable for woody plant tissue culture to avoid the problem of excessively high inorganic salt concentration in the infection medium of the original leaf disc transformation method using MS medium. The sucrose and glucose in the improved infection medium can provide energy for plant cells, and can also regulate osmotic pressure to protect cells from damage and death due to osmotic imbalance during the infection process. By adjusting the ratio of sucrose and glucose, it can not only maintain the osmotic balance of cells but also facilitate the infection activity of Agrobacterium with the help of osmotic potential. There is no need to add sodium citrate and betaine to avoid cell damage. 4-Morpholineethanesulfonic acid is used as a buffer solution to maintain the pH stability of the infection solution. Adding an appropriate amount of magnesium sulfate instead of acetosyringone meets the activation requirements of Agrobacterium and is conducive to Agrobacterium infection. pH 5.8 is the ideal range for the various inorganic salt components in the culture medium to play a role. Too low or too high a pH is not conducive to the absorption and utilization of inorganic salt components by plants.

[0057] The dedifferentiation initiation medium is supplemented with hydrolyzed whey protein to provide amino acid components for plant cells, which is more comprehensive and has a higher content than using hydrolyzed casein alone. The use of plant gel instead of agar overcomes the defects of traditional agar such as many impurities, pH impact, and large usage, which is beneficial to plant growth and development. The type and proportion of hormone use are re-established, and 2,4-dichlorophenoxyacetic acid and amiloride, which have better dedifferentiation initiation effects, are used to enhance the infection effect. Polyvinyl pyrrolidone is additionally added to prevent browning of wound cells during the culture process.

[0058] WPM medium is also used to replace the original MS medium in callus induction medium. The low inorganic salt content of WPM medium reduces the salt stress on woody plant cells, and the induced callus tissue is more active and in good condition. Callus formation requires a large amount of amino acid components. 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 naphthylacetic acid is beneficial to the dedifferentiation and redifferentiation process of apple cells. Timentin is an antibiotic specifically used to inhibit the proliferation of Agrobacterium tumefaciens. Compared with ceftriaxone sodium, the use of Timentin has extremely low adverse effects on plant growth and development.

[0059] Hygromycin is used in the bud differentiation medium instead of traditional kanamycin to screen resistant buds. The dosage is small and the screening intensity is high, which completely inhibits the generation of false positive escape buds and avoids interference with the statistical accuracy of the data. In the callus induction stage, WPM medium is used to meet the growth requirements of callus. In the bud differentiation stage, the modified MS medium is used, which is conducive to bud differentiation and increases the number of regenerated buds. Additional copper sulfate is added to meet the amount of copper ions required by woody plants. The purpose of the combined use of thiadiazole, 6-benzylaminopurine, and naphthylacetic acid is to obtain callus with strong differentiation ability, and the combined use of thiadiazole, 6-benzylaminopurine, and indolebutyric acid after callus formation helps to obtain regenerated buds with high proliferation coefficients.

[0060] Coconut powder added to the bud proliferation medium is used to strengthen the seedlings. Coconut powder contains a variety of amino acids, sugars and trace elements, which can improve the survival rate of regenerated buds, accelerate the growth rate of buds, and ensure the robustness of buds during proliferation. Using indolebutyric acid instead of naphthaleneacetic acid can reduce the intensity of the effect of auxin and enhance proliferation.

[0061] The present invention uses apple (Malus domestica'Gala') tissue culture seedling leaves as transformation receptors, places them in an anti-browning agent for cutting when cutting the tissue culture seedling leaves, then transfers them to an infection solution for immersion, and cuts the leaves in an anti-browning agent prepared by ascorbic acid and citric acid, completely avoiding the browning phenomenon of the wound caused by cutting and the problem of browning diffusion during the subsequent leaf culture process. During the experiment, Agrobacterium tumefaciens was used to deliver eGFP and RUBY marker proteins respectively, and the efficiency of the improved leaf disc transformation method was detected with the original leaf disc transformation method as a control. On the basis of the improved efficient leaf disc transformation method, Agrobacterium tumefaciens was used to deliver a CRISPR / Cas9 knockout vector targeting a functional gene, and the infected leaves were cultured in a dedifferentiation start-up medium, a callus induction medium, a bud differentiation medium, and a bud proliferation medium in sequence to obtain a large number of transgenic plants. On this basis, the gene editing efficiency and its editing type were statistically analyzed, the editing type of the mutant was analyzed, and the specific editing effect of the CRISPR / Cas9 system in the apple genome was analyzed, providing a reference for optimizing the editing tool and other CRISPR / Cas type tools.

[0062] The following detailed description is for illustrative purposes only and is intended to provide further explanation of the present application, rather than to limit the scope of the present invention.

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

[0064] The modified MS medium used in the embodiment is prepared by reducing 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 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] Example 1: Method for preparing a culture 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-morpholineethanesulfonic acid + 0.1 / L magnesium sulfate, pH = 5.8.

[0067] (2) Dedifferentiation 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 amiloride + 2.9 g / L plant gel + 0.5 g / L 4-morpholineethanesulfonic 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 naphthaleneacetic acid + 2.9 g / L phytagel + 0.5 g / L 4-morpholineethanesulfonic acid + 320 mg / L timentin + 0.2 g / L polyvinyl pyrrolidone, 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 thiadiol + 0.1 mg / L 6-benzylaminopurine + 0.1 mg / L indolebutyric acid + 2.9 g / L phytagel + 0.5 g / L 4-morpholineethanesulfonic acid + 320 mg / L timentin + 0.2 g / L polyvinyl pyrrolidone + 3 mg / L hygromycin, pH = 5.8.

[0070] (5) Bud proliferation medium formula: 1.0 g / L MS medium + 0.05 mg / L copper sulfate + 30 g / L sucrose + 100 mL / L coconut powder + 0.1 mg / L 6-benzylaminopurine + 0.1 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 for configuring a culture medium combination

[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) Dedifferentiation 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 amiloride + 2.9 g / L plant gel + 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) Bud 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 polyvinyl pyrrolidone + 3 mg / L hygromycin, pH = 5.8.

[0076] (5) Bud 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 a culture medium combination

[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 / 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 amiloride + 2.9 g / L plant gel + 0.5 g / L 4-morpholineethanesulfonic acid + 1.1 / 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 polyvinyl pyrrolidone, pH = 5.8.

[0081] (4) Bud 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 polyvinyl pyrrolidone + 3 mg / L hygromycin, pH = 5.8.

[0082] (5) Bud 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 culture medium combination provided in Example 3

[0084] (1) The transformation recipients used were leaves of 25-day-old 'Gala' tissue culture seedlings ( Figure 1 ), the growth conditions of tissue culture seedlings were as follows: temperature of 25±1°C, light intensity of 5000-7000Lux, and photoperiod 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 and 200 rpm shaking. 600 When the value is 0.7, centrifuge at 4000rpm for 10min to collect the bacteria; add the infection medium to adjust the bacterial solution OD 600 The value is 0.5, let it stand in the dark for 2 hours, and prepare the infection solution for use.

[0086] (3) Cut the leaves of the above tissue culture seedlings and place them in an anti-browning agent (10 mg / L ascorbic acid + 15 mg / L citric acid). Use a sterile surgical blade to cut the main veins twice vertically and soak them in the infection solution for 5 minutes. After the infection, transfer the leaves to the dedifferentiation initiation medium and culture them at 20±1°C in the dark for 2 days.

[0087] (4) After the co-cultivation, the leaves were transferred to callus induction medium and cultured at 22±1°C in the dark for 3 days.

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

[0089] (6) The leaves with resistant callus were transferred to bud differentiation medium and cultured at 22±1°C, 3000 Lux, and 16h / 8h photoperiod until the callus differentiated into resistant adventitious buds.

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

[0091] Comparative Example 1: Original leaf disc transformation method

[0092] 1. The culture medium combination configuration method used in the original leaf disc transformation method

[0093] (1) The 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 acetosyringone, without adjusting pH.

[0094] (2) Original dedifferentiation initiation medium formula: 4.43 g / L MS medium + 2 mg / L thidiazuron + 0.5 mg / L naphthaleneacetic 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 naphthaleneacetic acid + 7 g / L agar + 250 mg / L ceftriaxone sodium + 250 mg / L timentin, pH = 5.4.

[0096] (4) The original bud differentiation medium formula: 4.43 g / L MS medium + 30 g / L sucrose + 2 mg / L thidiazuron + 0.5 mg / L naphthaleneacetic acid + 7 g / L agar + 25 mg / L kanamycin + 250 mg / L ceftriaxone 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 naphthaleneacetic acid + 0.5 mg / L gibberellin + 7.0 g / L agar + 250 mg / L ceftriaxone sodium + 250 mg / L timentin + 25 mg / L kanamycin, pH = 5.8-6.0.

[0098] 2. Experimental Method of the Original Leaf Disc Transformation Method

[0099] (1) Streak the glycerol bacteria stored at -80°C and activate them on 100 mL of LB solid medium containing rifampicin + gene vector resistance for 2 days. In 100 mL of LB liquid medium containing Rif + vector resistance, shake and culture at 28°C overnight until OD600 = 1.5. Centrifuge at 5000 rpm for 5 minutes at room temperature. Resuspend in the original infection medium and dilute to OD600 = 0.5.

[0100] (2) Use leaves of 'Gala' tissue culture seedlings that are about 30 days old, and use a scalpel to make 2-3 cuts on the back of the leaves perpendicular to the main veins. After all the leaves have been cut, soak them in the bacterial solution for 8 minutes.

[0101] (3) Remove the leaves, absorb the bacterial solution on sterile filter paper, place the leaves with the back side facing up on the original dedifferentiation initiation medium, and culture in the dark for 3 days.

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

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

[0104] (6) When light-colored buds emerge from the cut of the leaf, transfer the leaf to a new bud differentiation medium and culture it under light. The resistant buds will turn green.

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

[0106] Experimental Example 1: Efficiency test of delivering eGFP and RUBY labeled proteins

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

[0108] Using pCAM1300-eGFP vector as the backbone, the vector was linearized with BamHI high-fidelity enzyme and purified and recovered. The full-length CDS sequence of MdLHP1 (gene number in the GDR database: MD07G1081800) was homologously recombined with the linearized vector pCAM1300-eGPF by In-Fusion method to obtain the pCAM1300-MdLHP1-eGFP overexpression vector, namely the marker vector MdLHP1-eGFP.

[0109] 2. Efficiency detection of delivering eGFP and RUBY marker proteins based on the improved leaf disc transformation method of the present invention:

[0110] (1) The transformation recipients used were leaves of 25-day-old 'Gala' tissue culture seedlings ( Figure 1 ), the growth conditions of tissue culture seedlings were as follows: temperature of 25±1°C, light intensity of 5000-7000Lux, and photoperiod of 16h / 8h.

[0111] (2) The marker vectors MdLHP1-eGFP, RUBY( Figure 2 ) was inoculated into YEB liquid medium supplemented with 25 mg / L rifampicin, 50 mg / L streptomycin and 50 mg / L kanamycin at a ratio of 1:1000 and cultured at 28°C with shaking at 200 rpm. 600 When the value is 0.7, centrifuge at 4000rpm for 10min, collect the bacteria, add them to the infection medium, and adjust the OD of the bacterial solution. 600 The value is 0.5, let it stand in the dark for 2 hours, and prepare the infection solution for use.

[0112] (3) Clipping step (1) Place the leaves of the tissue culture seedlings in an anti-browning agent (10 mg / L ascorbic acid + 15 mg / L citric acid), cut twice vertically along the main vein using a sterile surgical blade, and soak in the infection solution for 5 minutes. After infection, transfer the leaves to the dedifferentiation initiation medium and culture them at 20±1°C in the dark for 2 days.

[0113] (4) After the co-cultivation, the leaves were transferred to a callus induction medium and cultured at 22±1°C in the dark for 3 days.

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

[0115] (6) At this time, the callus phenotypes of MdLHP1-eGFP and RUBY transformed leaves under ultraviolet light were observed respectively, and the transformation efficiency was calculated by the formula: the number of leaves of transgenic callus generated / total number of leaves. Among them, the resistant callus stably expressing the eGFP marker protein showed green fluorescence under ultraviolet light, and the callus stably expressing the RUBY marker protein showed a red phenotype when directly observed with the naked eye. The results of the eGFP marker protein delivery test based on the improved leaf disc transformation method are shown in Figure 3 The results of the test using the modified leaf disc transformation method to deliver RUBY-tagged proteins are shown in Figure 5 .

[0116] (7) Data statistics and analysis were performed based on the phenotype of transgenic callus tissue. The improved leaf disc transformation method was used to deliver eGFP and RUBY marker proteins, with efficiencies of 83.33% (Table 1) and 86.67% (Table 2), respectively.

[0117] 3. Efficiency test of delivering eGFP and RUBY marker proteins based on the original leaf disc transformation method:

[0118] According to the original leaf disc transformation method in Comparative Example 1, callus tissue was obtained, and the callus tissue was placed under ultraviolet light to observe the phenotype. The detection results of eGFP marker protein delivered by the original leaf disc transformation method are shown in FIG. Figure 4 The results of the original leaf disc transformation method for delivering RUBY-tagged proteins are shown in Figure 6 According to the phenotype of transgenic callus, the efficiency of delivering eGFP and RUBY marker proteins by the original leaf disc transformation method was 16.67% (Table 1) and 0% (Table 2), respectively.

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

[0120]

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

[0122]

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

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

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

[0126] The target lhp1sgC nucleotide sequence 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 recipients used were leaves of 25-day-old 'Gala' tissue culture seedlings ( Figure 1 ), the growth conditions of tissue culture seedlings were as follows: temperature of 25±1°C, light intensity of 5000-7000Lux, and photoperiod of 16h / 8h.

[0129] (2) The knockout vector pCas9-AtU6-lhp1sgc ( Figure 7 ) was cultured in YEB liquid medium supplemented with 25 mg / L rifampicin, 50 mg / L streptomycin and 50 mg / L kanamycin at 28°C and 200 rpm shaking. 600 When the value is 0.7, centrifuge at 4000rpm for 10min to collect the bacteria; add the infection medium to adjust the bacterial solution OD 600 The value is 0.5, let it stand in the dark for 2 hours, and prepare the infection solution for use.

[0130] (3) Cut the leaves of the above tissue culture seedlings and place them in an anti-browning agent (10 mg / L ascorbic acid + 15 mg / L citric acid). Use a sterile surgical blade to cut the main veins twice vertically and soak them in the infection solution for 5 minutes. After the infection, transfer the leaves to the dedifferentiation initiation medium and culture them at 20±1°C in the dark for 2 days.

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

[0132] (5) The leaves after delayed culture were transferred to callus induction medium supplemented with 3 mg / L hygromycin and cultured at 22±1°C in the dark until resistant callus tissue was generated at the leaf wound ( Figure 8 ).

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

[0134] (7) When the resistant buds grow to 1-2 cm, use a surgical blade to peel them off from the leaves and transfer them to bud proliferation medium. Cultivate them at a temperature of 22±1°C, a light intensity of 3000 Lux, and a photoperiod of 16 h / 8 h, and subculture every 25 days.

[0135] (8) The genomic DNA of the resistant tissue culture seedlings was extracted by the CTAB method, and primers HygF, HygR and Cas9F, Cas9R (Table 3) were designed to perform PCR amplification on the partial sequences of Hyg and Cas9. The results of agarose gel electrophoresis showed that T1, T3-T6, T8, T10-T15 among the 16 resistant shoots were transgenic plants ( Fig.10 ); CRISPR-P 2.0 was used to analyze the full-length DNA sequence of the MdLHP1 gene online to obtain multiple target site sequences. At the selected target site sgC( Fig.11 ) were designed near the primers lhp1F and lhp1R (Table 3), PCR amplification was performed and Sanger sequencing was performed. The results of sequence comparison analysis showed that 12 of the 13 transgenic plants were MdLHP1 mutants, including 1 homozygous mutant, 2 heterozygous, and 9 chimeras ( Fig.13 ). DNA was extracted from leaves of 12 MdLHP1 mutants for testing, and all test results were summarized, showing that editing events occurred in all 12 MdLHP1 mutants ( Fig.14 ), only the L15 non-mutant target site sequence was identical to the wild type, and no editing event occurred. The editing ratio was calculated based on the ratio of edited plants to transgenic plants and was 92.3%.

[0136] Table 3: Detection primer sequences

[0137]

[0138]

[0139] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, 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 combination, characterized in that: include: Infection medium, dedifferentiation initiation medium, callus induction medium, bud differentiation medium and bud proliferation medium; The infection culture medium contains WPM culture medium, sucrose, glucose, 4-morpholineethanesulfonic acid and magnesium sulfate as active ingredients; The dedifferentiation initiation culture medium contains WPM culture medium, sucrose, glucose, hydrolyzed casein, hydrolyzed whey protein, 2,4-dichlorophenoxyacetic acid, amiloride, plant gel, 4-morpholineethanesulfonic acid and polyvinylpyrrolidone as active ingredients; The callus induction medium contains WPM medium, sucrose, hydrolyzed casein, hydrolyzed whey protein, thidiazuron, 6-benzylaminopurine, naphthylacetic acid, plant gel, 4-morpholineethanesulfonic acid, timentin and polyvinyl pyrrolidone as active ingredients; The bud differentiation medium contains modified MS medium, copper sulfate, thidiazuron, 6-benzylaminopurine, indolebutyric acid, phytagel, 4-morpholineethanesulfonic acid, timentin, polyvinyl pyrrolidone and hygromycin as active ingredients; The bud proliferation culture medium takes MS culture medium, copper sulfate, sucrose, coconut powder, 6-benzylaminopurine, indolebutyric acid, plant gel, 4-morpholineethanesulfonic acid, timentin and hygromycin as active ingredients.

2. The culture medium combination according to claim 1, characterized in that: The infection culture medium is prepared as follows: 1.0-5.0 g / L WPM culture medium + 1-30 g / L sucrose + 1-20 g / L glucose + 0.5 g / L 4-morpholineethanesulfonic acid + 0.1-1 g / L magnesium sulfate; The dedifferentiation initiation culture medium is prepared as follows: 1.0-5.0 g / L WPM culture 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 amiloride + 2.9 g / L plant gel + 0.5 g / L 4-morpholineethanesulfonic 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 + 0.01-0.5 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 phytagel + 0.5 g / L 4-morpholineethanesulfonic acid + 320 mg / L timentin + 0.2-2 g / L polyvinyl pyrrolidone, pH = 5.8; The bud differentiation medium is 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-morpholineethanesulfonic acid + 320 mg / L timentin + 0.2-2 g / L polyvinyl pyrrolidone + 3 mg / L hygromycin, pH = 5.8; The bud proliferation culture medium is prepared as follows: 1.0-5.0 g / L MS culture 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-morpholineethanesulfonic acid + 320 mg / L timentin + 3 mg / L hygromycin, pH = 5.

8.

3. The culture medium combination according to claim 2, characterized in that: The contents of ammonium nitrate and potassium nitrate in the improved MS culture medium are respectively 1 / 2 of the contents of ammonium nitrate and potassium nitrate in the MS culture medium.

4. Use of the culture medium combination according to any one of claims 1 to 3 in an improved leaf disc transformation method.

5. The use according to claim 4, characterized in that: The leaf disc transformation method is an apple leaf disc transformation method mediated by Agrobacterium tumefaciens.

6. A method for leaf disc transformation using the culture medium combination according to any one of claims 1 to 3, characterized in that: The steps include: (1) Add Agrobacterium activated by YEB medium to the infection medium to prepare the infection solution, cut the leaves of the transformed recipient tissue culture seedlings and place them in an anti-browning agent, use a sterile surgical blade to cut 2-3 times vertically on the main vein, soak them in the infection solution for 5-11 minutes, and then transfer them to the dedifferentiation initiation medium for co-cultivation; (2) After the co-cultivation is completed, the leaves are transferred to a callus induction medium, and the leaves that have been delayed in culture are transferred to a callus induction medium supplemented with hygromycin to generate resistant callus tissue, and the resistant callus tissue is transferred to a bud differentiation medium to differentiate resistant adventitious buds; (3) When the adventitious buds grow to 1-2 cm, they are peeled off from the leaves using a surgical blade and transferred to bud proliferation medium for subculture.

7. The method according to claim 6, characterized in that The Agrobacterium in step (1) is an Agrobacterium carrying a marker vector or a target knockout vector.

8. The method according to claim 6, characterized in that The transformation recipient in step (1) is a leaf of apple 'Gala' tissue culture seedling.

9. The method according to claim 6, characterized in that The anti-browning agent in step (1) is 10-100 mg / L ascorbic acid + 15-150 mg / L citric acid.

10. Use of the method according to claim 6 in the following (1) or (2): (1) Improve the efficiency of genetic transformation of apple callus; (2) Improve the editing efficiency of the apple CRISPR / Cas9 system; Preferably, the improvement of the genetic transformation efficiency of apple callus is reflected by detecting the delivery efficiency of eGFP and RUBY marker proteins.

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