CN120400209A - A potato breeding method for gene editing of ALS genes - Google Patents
A potato breeding method for gene editing of ALS genesInfo
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Abstract
The invention belongs to the technical field of crop breeding, and relates to a potato breeding method for editing ALS genes. The method comprises the following steps of (1) infecting potato explants by using agrobacterium transformed with a gene editing tool, wherein an infection culture medium used for infection contains CaCl 2 with the total concentration of 9-15mM, the gene editing tool targets ALS genes of the potatoes to carry out editing mutation of the ALS genes so as to enable the potatoes to generate resistance to imidazolinone herbicides, and (2) after co-culture and recovery culture, carrying out resistance callus induction, resistance bud induction and strong seedling culture on the potato explants in sequence on a resistance callus induction culture medium, a resistance bud induction culture medium and a strong seedling culture medium to obtain regenerated seedlings, wherein the resistance callus induction culture medium, the resistance bud induction culture medium and the strong seedling culture medium all contain the imidazolinone herbicides. By utilizing the method provided by the invention, ALS gene mutated potato varieties can be obtained with high infection efficiency and high gene editing efficiency.
Description
Technical Field
The invention belongs to the technical field of crop breeding, and relates to a potato breeding method for editing ALS genes.
Background
Potato (Solanum tuberosum l.) belongs to a herb dicotyledonous perennial tuber plant of solanaceae eggplant, is an important crop for both food and vegetable, and has multiple purposes such as feed, light industry, raw material, and the like. Potatoes are the fourth largest grain crop in China, and are next to rice, wheat and corn, and the total yield is the first in the world.
The genetic transformation methods which are common at present for potatoes are a gene gun method, an ultrasonic method, a protoplast method, an agrobacterium-mediated genetic material delivery method and the like. The agrobacterium-mediated genetic transformation method is widely applied to plant transgene delivery due to convenient operation, high repeatability and lower cost. Agrobacterium-mediated genetic transformation has many advantages, such as efficient insertion of exogenous genes, simpler insertion of fragments of exogenous DNA, low copy number of exogenous genes, and wide successful application in the development of commercial events. In view of the advantages of the agrobacterium-mediated heritage transformation method, the method has important significance for further optimizing the method and improving the transformation efficiency.
The current screening systems commonly used for genetic transformation and gene editing of potatoes comprise a kanamycin screening system and a Bar screening system, and a new screening system needs to be developed.
Disclosure of Invention
The invention aims to provide a breeding method of potatoes with ALS genes edited by genes, which can obtain potato varieties with ALS gene mutation with high infection efficiency and high gene editing efficiency, so that the potatoes of the varieties generate resistance to imidazolinone herbicides.
To achieve this object, in a basic embodiment, the present invention provides a method for breeding potatoes by gene editing ALS gene, the breeding method comprising the steps of:
(1) Infecting potato explants with agrobacterium transformed with a gene editing tool that targets the ALS gene of the potato for editing mutation to render the potato resistant to imidazolinone herbicides, using an infection medium containing CaCl 2 at a total concentration of 9-15 mM;
(2) After co-culture and recovery culture, the potato explant is subjected to resistance callus induction, resistance bud induction and strong seedling culture on a resistance callus induction culture medium, a resistance bud induction culture medium and a strong seedling culture medium in sequence to obtain regenerated seedlings, wherein the resistance callus induction culture medium, the resistance bud induction culture medium and the strong seedling culture medium all contain imidazolinone herbicide.
The present invention uses imidazolinone herbicides to screen directly for potato ALS mutant genes. Acetolactate synthase (acetolactate synthase, ALS, EC 2.2.1.6) is one of key enzymes in the biosynthesis pathway of catalyzing branched-chain amino acids (valine, leucine and isoleucine) in plants and microorganisms, and ALS inhibitor herbicides have the advantages of wide herbicidal spectrum, strong selectivity, low toxicity, high efficiency, low soil residual activity and the like. ALS inhibitors herbicides that have been developed to date can be classified into more than 13 classes of 50, including sulfonylureas (sulfonylureas, SU), pyrimidyloxybenzoic acids (pyrimidinyloxy benzoic acids, PTB, also known as pyrimidinylsalicylic acids), imidazolinones (imidazolinones, IMI) and triazolopyrimidinesulfonamides (triazolopyrimidine sulfonanilides, SUT) as main classes. Their mechanism of action is to influence branched-chain amino acids and synthesis by inhibiting acetolactate synthase activity, thereby affecting protein synthesis, ultimately leading to plant growth arrest and death. There are some conserved amino acids in the ALS gene, for example Arabidopsis, ala at positions 122 and 205, pro, asp, arg, trp, ser and Gly at positions 197, 376, 377, 574, 653 and 654, respectively, and these amino acid changes may result in resistance to ALS inhibitor herbicides. Imidazolinone herbicides are ideal screening agents in genetic transformation processes due to their good absorbability, stability, and difficulty in photolysis.
In a preferred embodiment, the present invention provides a method of breeding potatoes with gene editing ALS genes, wherein in step (1), the gene editing tool carries a gene expressing Cas9 protein and carries sgrnas.
In a preferred embodiment, the present invention provides a method of breeding potatoes genetically editing the ALS gene, wherein in step (1), the Agrobacterium is Agrobacterium LBA4404.
In a preferred embodiment, the present invention provides a method of breeding potatoes in which the ALS gene is genetically edited, wherein in step (1) the stem segments or leaves of potato plants which have been subcultured for 21-28 days are precultured to obtain said potato explants for infestation.
In a preferred embodiment, the present invention provides a method of breeding potatoes genetically editing the ALS gene, wherein the variety of potato plant is Ferunorita.
In a preferred embodiment, the present invention provides a method of breeding potatoes in which the ALS gene is genetically edited, wherein the length of the stem segments is from 0.5 to 2mm.
In a preferred embodiment, the invention provides a method for breeding potatoes by gene editing ALS genes, wherein in the step (1), the coding sequence of the ALS genes is shown as SEQ ID NO.1, and the coding sequence of ALS gene mutants is shown as SEQ ID NO. 3.
The amino acid sequence of the protein encoded by the ALS gene shown in SEQ ID NO.1 is shown in SEQ ID NO. 2. The amino acid sequence of the protein encoded by the ALS gene mutant shown in SEQ ID NO. 3 is shown in SEQ ID NO. 4.
In a preferred embodiment, the present invention provides a method of breeding potatoes in which the ALS gene is genetically edited, wherein the imidazolinone herbicide is imazethapyr.
In a preferred embodiment, the present invention provides a method of breeding potatoes in which the ALS gene is genetically edited, wherein both the infection medium and the co-culture medium used for infection and co-culture contain 1 XMS.
In a preferred embodiment, the present invention provides a method of breeding potatoes with a gene editing ALS gene, wherein the breeding method comprises the steps of:
(1) Preparing explants, namely taking stem segments or leaves of potato plants subcultured in a culture flask, creating wounds by using a surgical knife, and then placing the potato plants on a preculture medium for preculture;
(2) Infecting the pre-cultured potato stems or leaves with a bacterial liquid of agrobacterium transformed with a gene editing tool, and then sucking the potato stems or leaves with filter paper;
(3) Co-culturing the infected potato stems or leaves on a co-culture medium;
(4) Carrying out recovery culture on the potato stem segments or leaves subjected to the co-culture on a recovery culture medium;
(5) Carrying out resistance callus induction on the potato stem segments subjected to recovery culture on a resistance callus induction culture medium;
(6) Carrying out resistance bud induction on the potato stem segment after the resistance callus induction is finished or the leaf blade after the recovery culture on a resistance bud induction culture medium;
(7) And (3) carrying out strong seedling culture on the regenerated buds on a strong seedling culture medium to obtain strong regenerated seedlings.
Preferably, in step (1), the preculture medium comprises 4.43g/L MS salt and vitamin+30 g/L sucrose+6.0 g/L agar, pH=5.8, preculture is an illumination culture for 48 hours.
Preferably, in step (1), the explant of the lamina is transected to the main vein of the lamina with a surgical knife.
Preferably, in step (2), the infestation medium used for the infestation comprises 4.43g/L MS salts and vitamins +20g/L sucrose +10g/L glucose +2.5 mg/L6-BA +0.5 mg/L2, 4-D +50. Mu.M AS, pH=5.6.
Preferably, in step (2), the OD 600 of the bacterial liquid is 0.3.
Preferably, in step (2), the infestation time is 15min.
Preferably, in step (2), the room temperature at the time of infestation is below 28 ℃.
Preferably, in step (3), the co-culture medium comprises 4.43g/L MS salt and vitamin+20 g/L sucrose+10 g/L glucose+2.5 mg/L6-BA+0.5 mg/L2, 4-D+50. Mu.M AS, pH=5.6.
Preferably, in step (4), the recovery medium comprises 4.43g/L MS salt and vitamin +30g/L sucrose +2.5 mg/L6-BA +0.5 mg/L2, 4-D +6.0g/L agar +250mg/L cephalosporin +100mg/L timentin, pH=5.8.
Preferably, in step (5), the resistant callus induction medium comprises 4.43g/L MS salt and vitamin+30 g/L sucrose+2.5 mg/L6-BA+0.5 mg/L2, 4-D+6.0g/L agar+250 mg/L cephalosporin+100 mg/L timentin+260. Mu.g/L imazapic acid, pH=5.8.
Preferably, in step (6), the resistant shoot induction medium comprises 4.43g/L MS salt and vitamin +30g/L sucrose +1.0mg/L trans-zeatin +0.01mg/L NAA +6.0g/L agar
+250Mg/L cephalosporin +100mg/L timentin +260. Mu.g/L imazapic, pH=5.8.
Preferably, in step (6), the resistant shoot induction medium comprises 4.43g/L MS salt and vitamin +30g/L sucrose +2.0mg/L trans-zeatin +0.02mg/L NAA +6.0g/L agar
+250Mg/L cephalosporin +100mg/L timentin +260. Mu.g/L imazapic, pH=5.8.
Preferably, in the step (7), the strong seedling medium comprises 5.688g/L MS salt and vitamin+30 g/L sucrose+250 mg/L cephalosporin+260. Mu.g/L imazethapyr+5 g/L carrageenan, and the pH=5.8.
The method has the beneficial effects that the ALS gene editing potato breeding method can obtain ALS gene mutated potato varieties with high infection efficiency and high gene editing efficiency, so that the varieties of potatoes are resistant to imidazolinone herbicides.
The method obtains the mefenamic acid-resistant gene editing seedling with gene editing and no foreign fragment insertion by optimizing the CaCl 2 total concentration, the MS culture medium concentration, the length of the stem section of the explant and the physiological state of the explant in the infection culture medium, wherein the probability of editing the regeneration seedling of the genetic transformation system is larger.
The beneficial effects of the invention are as follows:
1) The invention uses a brand new screening agent-imidazolinone herbicide in the genetic editing method of potatoes based on agrobacterium, and the screening agent has good absorptivity, is stable and is not easy to photolyze, and is an ideal genetic transformation screening agent.
2) The invention systematically optimizes the potato genetic editing method based on agrobacterium, and establishes a genetic editing method with high probability of occurrence editing of regenerated seedlings of a genetic transformation system.
Drawings
FIG. 1 is a structural diagram of a gene editing tool vector.
FIG. 2 is a graph showing the effect of total CaCl 2 concentrations on the infection efficiency of the fei-Wu-Rui stem segment.
FIG. 3 is a graph showing the results of GUS staining at various CaCl 2 total concentrations during the infection and co-cultivation stages.
FIG. 4 is a graph of the effect of different MS concentrations on the infection of the fei-WuRuita stem segment.
FIG. 5 is a graph showing the effect of different lengths of the stem on the infection efficiency of Agrobacterium.
FIG. 6 shows stem segments of different subculture days.
FIG. 7 is a comparison of GUS staining of different stem thicknesses.
FIG. 8 shows comparison of the results of sequence sequencing of ALS gene sequences corresponding to wild type and gene editing seedlings.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific conditions are not noted in the examples, and the reagents or instruments used are conventional products available through commercial purchase, and the manufacturer is not noted.
Example 1 construction of Gene editing tool vector
A gene editing tool vector having the structure shown in FIG. 1 was constructed, which had a commercial pCambia2300 plasmid as a backbone. The following five fragment sequences were synthesized artificially in gold sry biotechnology (nanjing):
1. PcUbi promoter GenBank Accession No. X64345.1,1-982bp;
2. Cas9n sequence with 3×flag tag and SV40NLS, see Xu et al.,"A design optimized prime editor with expanded scope and capability in plants,"Nat Plants,2022,8,45-52.;
3. M-MLV-RT+S40 NLS sequences, cas9n-M-MLV-RT and linker sequences, see Xu et al.,"A design optimized prime editor with expanded scope and capability in plants,"Nat Plants,2022,8,45-52.;
4. Nptii+t35S sequence, see pCambia2300 plasmid;
5. three sgRNA expression cassettes from AtU, 6-26p to polyT, target sequences were as follows (PAM sequence in brackets):
ASST1013+esgRNA(SEQ ID NO:5):AAGATCTCTTCCTCGTTAGC(AGG);
ASST1015+esgRNA(SEQ ID NO:6):CTAGACAGATAGAACACTTT(TGG);
SST1012-pegRNA(SEQ ID NO:7):CTTGGGAATGGTGGTTCAAT(GGG);
SST1014-pegRNA(SEQ ID NO:8):GTTCTACCTATGATTCCCAG(CGG),
The five fragments were ligated to the backbone of pCambia 2300T-DNA by NEBuilder HiFi DNA Assembly Master Mix (NEW ENGLAND Biolabs) to obtain a PS0967 gene editing tool vector.
EXAMPLE 2 appropriate elevation of CaCl 2 concentration in the Agrobacterium infection step and Co-cultivation stage can increase the Agrobacterium infection efficiency
Agrobacterium infection and co-cultivation are the first step in genetic transformation, and the infection efficiency directly affects the genetic transformation. In this example, the effect of CaCl 2 concentration on infection efficiency was tested in infection medium and co-medium, with total CaCl 2 concentration set at 3mM, 9mM, 15mM and 30mM for 4 different concentrations (MS medium provided 3mM CaCl 2 concentration, insufficient total CaCl 2 concentration provided by additional CaCl 2), and the best infection effect was tested as follows:
(1) Preparing explants, namely taking potato plants which are subjected to subculture in a culture flask for 21-28 days, removing the top stem segments which are the young and tender of the plants, cutting the stem segments of the rest stem segments into stem segments with 5-10mm (without axillary buds) by using a surgical knife, placing the cut stem segments into a preculture medium (MS salt and vitamin 4.43g/L (PTL company, product number is M519, the same applies below) +30g/L of sucrose+6.0 g/L of agar, pH=5.8), and culturing for 2 days at 23+/-2 ℃, wherein the photoperiod is 16 hours of illumination, 8 hours of darkness and the light intensity is 2000Lux.
(2) Preparation of the infection working fluid 4 disposable 50ml centrifuge tubes were taken and 10ml of infection medium (4.43 g/L MS salt (containing vitamins) +20g/L sucrose+10 g/L glucose+2.5 mg/L6-BA+0.5 mg/L2, 4-D+50. Mu.M AS, pH 5.6) was added thereto in sequence, with CaCl 2 total concentrations of 3mM, 9mM, 15mM and 30mM, respectively. Culturing the agrobacterium LBA4404 containing GUS gene in LB solid medium containing kanamycin, collecting the agrobacterium obtained by culturing in a 50ml centrifuge tube with a disposable inoculating loop, shaking the bacterial block with a vortex instrument, and uniformly dispersing the bacterial block in the infection medium to prepare an infection working solution with OD 600 value of about 0.3 for standby.
(3) And (3) in order to eliminate the influence of different individual physiological states and stem thickness on the infection efficiency, collecting all pre-cultured potato stems in a new culture dish, mixing, randomly dividing into 4 parts, placing into different culture dishes, adding infection working solutions with different CaCl 2 total concentrations (3 mM, 9mM, 15mM and 30 mM) into the 4 culture dishes, and carrying out infection for 15 minutes at room temperature (the room temperature is lower than 28 ℃ during infection), wherein the culture dishes are continuously rocked to enable the stems to be fully contacted with the bacterial solution.
(4) After the infection is finished, the bacterial liquid is sucked by a disposable sterile suction tube, and the residual bacterial liquid is sucked by sterile filter paper.
(5) Co-culturing, namely, placing two layers of sterile filter paper in a new culture dish, adding 2ml of infection medium (4.43 g/L MS salt (containing vitamins) +20g/L sucrose+10 g/L glucose+2.5 mg/L6-BA+0.5 mg/L2, 4-D+50 mu M AS, pH 5.6) containing CaCl 2 and 3mM, 9mM, 15mM and 30mM of CaCl 2 in total concentration respectively, ensuring no bubbles between the filter paper and the culture dish and the two layers of filter paper, placing the stem of the step (4) in the culture dish, sealing the culture dish by using a 3M sterile air-permeable adhesive tape to prevent excessive evaporation of water to cause excessive drying of the stem, and placing the culture dish in a culture box for dark culture for 3 days at 23 ℃.
(6) The stem sections after the end of co-cultivation were transferred to a recovery medium (4.43 g/L MS salt (vitamin-containing) +30g/L sucrose+2.5 mg/L6-BA+0.5 mg/L2, 4-D+6.0g/L agar+250 mg/L cephalosporin+100 mg/L timentin, pH=5.8) containing antibiotics but no selection agent for 3 days of recovery cultivation, and then GUS staining was performed on the different treated stem sections, respectively, staining was performed at 37℃overnight, decoloring with 70% ethanol (freshly prepared), and the staining results were observed under a microscope.
(7) The dyeing observation is that the potato stem is dyed in three conditions of blue-colored stem, bluish stem and undyed stem (negative), the stem numbers of different dyeing conditions are recorded during observation, and GUS dyeing efficiency is calculated.
GUS staining efficiency% = number of bluish stems/total number of stems x 100%
Experimental results:
The stem segment of the potato variety Fei-Wu-Rui is used as an explant, and GUS staining results after the infection of the agrobacterium show that the infection efficiency of the agrobacterium can be improved by properly increasing the concentration of CaCl 2 in the infection and co-culture stages (figure 2). Compared with control (3 mM), the GUS staining efficiency of the Federation ends was 74.19% and 75.86% respectively at CaCl 2 concentrations of 9mM and 15mM in the counterstain and co-culture solutions, both higher than control 68.75%, and the GUS staining efficiency was reduced to 43.33% at CaCl 2 concentrations of 30mM, lower than control. GUS staining results show that the appropriate increase of CaCl 2 concentration in infection and co-culture stage can improve GUS staining efficiency, promote the infection efficiency of agrobacterium, and thus improve conversion efficiency, and the CaCl 2 concentration reaching 30mM can reduce the infection efficiency of agrobacterium, thus affecting conversion efficiency.
Example 3 CaCl 2 increasing the Agrobacterium transformation efficiency was effected during the infection phase
The experimental results of example 2 show that CaCl 2 can improve the infection efficiency of agrobacterium. Example 2a study of this example was designed and carried out using higher concentrations of CaCl 2 during both the invasive and co-cultivation stages, in order to make clear which step CaCl 2 functions during the invasive and co-cultivation. The test variety is the stem segment of the Fabry-Perot. The specific experimental design is shown in table 1 below.
The experimental process comprises the following steps:
the medium composition, transgenic manipulation and culture conditions, staining methods and observation criteria for each stage of genetic transformation were the same as in example 2.
TABLE 1 total concentration of CaCl 2 for infection and Co-cultivation step
Experimental results:
infection experiments with the aseptic seedling stem segments of feinula decumbent as explants showed (fig. 3):
GUS staining efficiency was 22.6% and 25.8% when CaCl 2 concentration was increased to 9mM and 15mM, respectively, at the infection step, approximately 2 times (3 mM-3mM: 13.3%) the control GUS staining efficiency, and decreased to 12.9% when CaCl 2 concentration was further increased to 30mM, comparable to the control.
The co-cultivation stage increases the CaCl 2 concentration to 9mM and 15mM, and the staining efficiencies are 9.7% and 13.3%, respectively, which are equivalent to the control 13.3%, which shows that the increasing of the CaCl 2 concentration in the co-cultivation stage does not promote the infection capacity of agrobacterium.
The results of increasing the concentration of CaCl 2 only in the infection stage and in the co-culture stage show that the infection capacity of agrobacterium is promoted only by adopting the CaCl 2 with higher concentration in the infection stage, so that the GUS dyeing efficiency is improved. Therefore, when both the infection and co-cultivation stage CaCl 2 concentrations were used at 9mM and 15mM, the GUS staining efficiency observed was 25.8% as compared to the GUS staining efficiency of the control, which was 100% higher than that observed when CaCl 2 concentrations were used at 9mM and 15mM in the infection solution only during the infection stage. The treatment of the infection stage and co-cultivation stage with increased CaCl 2 concentration was also a re-validation of the results of example 2, demonstrating that the experimental results of CaCl 2 to increase the efficiency of Agrobacterium infection were reproducible. The experimental result shows that the promotion effect of CaCl 2 on the infection efficiency of the agrobacterium is effective in the infection step.
Example 4 influence of different concentrations of MS Components on the infection efficiency of Agrobacterium
It has been reported that the use of 1/4 XNB pairs in both the invasive and co-culture stages in rice genetic transformation has been found to significantly enhance rice transformation efficiency. In this example the infection step and co-cultivation stage were tested and 2 different MS component concentrations, 1/4 XS and 1 XS were set. The composition of the medium, the transgenic operation and the culture conditions at each stage of genetic transformation, and the analysis method of the results are the same as in example 2.
As a result of analysis, GUS staining results showed (FIG. 4) that the composition of 1/4 XMS was used in the infection and co-cultivation stage, the GUS staining efficiency of the Federata stem was 48.39%, and the GUS staining efficiency was 68.75% lower than that of 1 XMS, so that 1 XMS had a positive effect on the infection efficiency. Therefore, in the genetic transformation of potato stems as explants, a1×MS composition was used during the infection and co-cultivation stages. This result is contrary to that of rice, indicating that it is due to different species.
Example 5 different lengths of the Stem affect infection with Agrobacterium
Potato stems are often used as explants for genetic transformation studies of potatoes due to readily available materials and simple operation, and the stem lengths reported in the literature are mostly 5-10mm. If the length of the stem segment is shortened, more explants can be obtained from the same plant. To determine the length of the stem used for genetic transformation of potato, this example tested the effect of agrobacterium infection at both lengths of stem. The explants used were 4.43g/L MS salt and vitamin+30 g/L sucrose+10 ml CaCl 2 solution (concentration 44 mg/ml) +2ml/L MgSO 4·7H2 O solution (185 mg/ml) +2ml/L KH 2PO4 (85 mg/ml) solution+5 g/L carrageenan, pH=5.8. "long" means a stem length of 5-10mm and "short" means a stem length of 0.5-2mm, such that short stem can produce 2.5-5 times the number of long stem explants for transformation.
The composition of the medium, the transgenic operation and the culture conditions at each stage of genetic transformation, and the analysis method of the results are the same as in example 2.
The GUS staining results showed (FIG. 5) that the GUS staining efficiencies of the stem sections of 5-10mm in length of the explant and the short stem sections of 0.5-2mm in length of the explant were 17.07% and 18.75%, respectively, and the difference in GUS staining efficiency between the long stem sections and the short stem sections was not large, so that the short stem sections could also be used in the genetic transformation of potatoes. Under the same number of explant conditions or when the explant material is rarer, a shorter stem segment is more advantageous in that more explants can be produced, resulting in more transformation events.
Example 6 Effect of the physiological status of the explants on the efficiency of Agrobacterium infection
(A) Time of next generation
The experimental method of this example was the same as that of example 2.
GUS staining results show that the aseptic seedlings of potatoes can be used for genetic transformation only after at least more than 2 weeks, potato stem segments cultured only for 2 weeks after subculture are hollow in medulla (shown in the left graph of fig. 6), the transformation efficiency is affected, the development of vascular bundles is incomplete as shown by the broken line arrow of the graph, and the development of vascular bundles of stem segments cultured for 21 days is completed (shown in the right graph of fig. 6 and the solid arrow), and GUS staining is mainly concentrated on the vascular bundles. Regeneration buds from vascular bundle tissue have strong genetic transmission according to cyto-developmental principles. It is further speculated that the time of growth after the subculture and the infection efficiency of the agrobacterium will be affected by the medium composition, potato variety and culture conditions.
(B) Thickness and robustness degree of potato tissue culture seedling stalk
The experimental method of this example was the same as that of example 2.
In GUS staining experiments, GUS staining is deeper when the stem segments are thicker, and the cut ends of the stem segments are obviously enlarged in the culture process (left in figure 7), which means that more cells are transformed or the differentiation capacity of transformed cells is stronger, proliferation is fast, the transformation efficiency of the thicker stem segments is higher, on the contrary, callus induction at the cut parts of the slender stem segments is weaker (right in figure 7), which means that the transformed cells are fewer or the cell viability is weak, proliferation is slower, and the transformation efficiency is obviously reduced.
Example 7 transformation test after optimization of the parameters according to examples 2-6
(1) The stem pre-culture comprises the steps of taking potato plants which are subjected to secondary culture in a culture bottle for 21-28 days, removing the top stems which are the tender of the plants, cutting the rest stems into stem segments with the length of 0.5-2mm without axillary buds by using a surgical knife, placing the cut stem segments on a pre-culture medium (4.43 g/L MS salt (containing vitamins) +30g/L sucrose+6.0 g/L agar powder and pH value of 5.8), and culturing for 2 days, wherein the photoperiod is 16 hours of illumination, the darkness is 8 hours, and the light intensity is 2000Lux.
(2) Preparing an infection working solution, namely culturing agrobacterium LBA4404 containing a targeting potato ALS gene editing tool by using an LB solid medium containing kanamycin, collecting the agrobacterium LBA4404 into a 50ml centrifuge tube by using a disposable inoculating loop, and shaking bacterial blocks by using a vortex instrument to prepare for uniformly dispersing the bacterial blocks in different infection media respectively.
Preparing an infection medium of treatment 1, which comprises 4.43g/L MS salt, 20g/L sucrose, 10g/L glucose, 2.5 mg/L6-BA, 0.5 mg/L2, 4-D and 50 mu M AS pH=5.6;
Preparing an infection medium of treatment 2, which comprises 4.43g/L MS salt, 20g/L sucrose, 10g/L glucose, 2.5 mg/L6-BA, 0.5 mg/L2, 4-D+6mM CaCl 2 and 50 mu M AS pH=5.6;
Preparing an infection medium of treatment 3, wherein the infection medium comprises 4.43g/L MS salt, 20g/L sucrose, 10g/L glucose, 2.5 mg/L6-BA, 0.5 mg/L2, 4-D, 12mM CaCl 2 and 50 mu M AS pH=5.6;
The infection medium of treatment 4 was prepared with ingredients of 4.43g/L MS salt and vitamin+20 g/L sucrose+10 g/L glucose+2.5 mg/L6-BA+0.5 mg/L2, 4-D+27mM CaCl 2 +50. Mu.M AS pH=5.6.
The OD 600 value of the infection culture medium of each of the treatments 1 to 4 is about 0.3, and the infection culture medium is prepared for standby.
(3) And (3) infecting, namely collecting the potato stem segments after the pre-culture from a pre-culture medium into a new culture dish, adding the prepared infecting working solution, infecting for 15 minutes at room temperature, and continuously shaking the culture dish to enable the stem segments to be fully contacted with the bacterial solution (the room temperature is lower than 28 ℃ during the infecting). After the infection is finished, the bacterial liquid is sucked by a disposable sterile suction tube, and the residual bacterial liquid is sucked by sterile filter paper.
(4) Co-cultivation, namely, two layers of sterile filter paper are placed in a new culture dish, and 2ml of co-cultivation medium (4.43 g/L MS (containing vitamins) +20g/L sucrose+10g/L glucose+2.5 mg/L6-BA+0.5 mg/L2, 4-D+50 mu M AS, pH=5.6) is added to ensure that no bubbles exist between the filter paper and the culture dish and between the filter paper and the two layers of filter paper. Placing the stem segments of step (3) therein, about 30 stem segments/dish. The dishes were sealed with 3M sterile air-permeable tape to prevent excessive evaporation of water resulting in excessive drying of the stem segments. The dishes were placed in an incubator and dark cultured at 23℃for 3 days.
(5) And (3) carrying out recovery culture, namely transferring the stem segments after the co-culture is finished into a recovery culture medium (4.43 g/L MS salt (containing vitamins) +30g/L sucrose+2.5 mg/L6-BA+0.5 mg/L2, 4-D+agar 6.0g/L+250mg/L cephalosporin+100 mg/L timentin, wherein the culture condition is 23+/-2 ℃,16 hours of illumination, 8 hours of darkness and the light intensity is 2000 Lux) containing antibiotics and no screening agent, and carrying out recovery culture for 7 days.
(6) Screening and culturing the resistant callus, transferring the stem segment after the recovery culture to a resistant callus induction medium containing imazethapyr 260 μg/L (4.43 g/L MS salt (containing vitamin) +30g/L sucrose+2.5 mg/L6-BA+0.5 mg/L2, 4-D+agar 6.0g/L+250mg/L cephalosporin)
+100Mg/L timentin+260. Mu.g/L imazapic, pH=5.8) and the culture conditions were the same as in step (5). Imazethapyr may also be replaced with other imidazolinone herbicides.
(7) Regeneration of resistant shoots after 14 days of resistant callus induction the stem was transferred to resistant shoot induction medium containing imazethapyr 260. Mu.g/L (4.43 g/L MS salt (containing vitamins) +30g/L sucrose+1.0 mg/L zeatin+0.01 mg/L NAA+6.0g/L agar+250 mg/L cephalosporin
Induction of resistant shoots was performed on +100mg/L timentin +260. Mu.g/L imazapic, pH=5.8, and culture was carried out for 21 days under the same conditions as in step (5). The generation of the resistant buds is carried out for 2-3 times. Adventitious buds are typically produced at the end of the second subculture with stem segments.
(8) Rooting culture of regenerated seedling, in which the regenerated seedling is rooting cultured on rooting culture medium (5.688 g/L MS salt and vitamin+30 g/L sucrose+250 mg/L cephalosporin+260. Mu.g/L imazethapyr+5 g/L carrageenan, pH=5.8).
(9) The transformation efficiency of rooting and strengthening seedlings obtained by the method is tested, and the results are shown in the following table 2:
TABLE 2 rooting seedling detection results
| Sequence number | Treatment mode | Number of explants | Detecting the number of plants | Editing plant number | Editing efficiency |
| 1 | Treatment 1 (CaCl 2 mM) | 339 | 4 | 4 | 1.18% |
| 2 | Treatment 2 (CaCl 2 mM) | 261 | 39 | 36 | 13.8% |
| 3 | Treatment 3 (CaCl 2 mM) | 536 | 79 | 72 | 13.4% |
| 4 | Treatment 4 (CaCl 2 mM) | 972 | 15 | 14 | 1.44% |
Editing efficiency% = number of plants/total number of explants subjected to editing x 100%.
The results in the above table show that when the total concentration of CaCl 2 in the infection medium is 9-15mM, the optimal gene editing efficiency of potato with imazethapyr as a screening system can be obtained.
Further sequencing of one wild type and one gene editing seedling of treatment 2 (the sequence of the sequencing primer is shown as SEQ ID NO:9 (forward primer), SEQ ID NO:10 (reverse primer)) was performed, and the results are shown in FIG. 8.
It should be noted that the above embodiments are merely for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that the technical solution described in the above embodiments may be modified or some or all of the technical features may be equivalently replaced, and these modifications or substitutions do not deviate from the essence of the corresponding technical solution from the scope of the technical solution of the embodiments of the present invention.
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