Gene editing method for octaploid cultivation of strawberries

Abstract

The gene editing method comprises the following steps: (1) designing a target gene double-target editing primer based on an octaploid cultivated strawberry genome, integrating the target gene double-target editing primer to a target gene editing vector, and completing agrobacterium transformation; (2) co-culturing the activated agrobacterium tumefaciens containing the target gene editing vector and strawberry tissue culture seedlings on sterile vermiculite, and inducing positive calluses; (3) carrying out induced differentiation on the positive callus on a positive callus regeneration induction culture medium until positive buds are differentiated; (4) cutting off positive single buds induced by different positive calluses, and respectively transferring the positive single buds to a positive bud subculture medium for subculture proliferation until a strain is subcultured; (5) extracting DNA of different strains, and performing genotype identification and off-target site detection; and (6) cutting the positive strain into single plants, transferring the single plants to a positive bud rooting culture medium to induce rooting, and acclimatizing and transplanting after rooting. According to the method, the T0 positive rate is increased to 60% or above; and the stem positive callus regeneration time is shortened to 3 months.

Description

Technical Field

[0001] The invention belongs to the field of plant molecular breeding, and particularly relates to a gene editing method for octoploid cultivated strawberries. Background Art

[0002] The information disclosed in this background technology section is only intended to increase some understanding of the overall background of the invention, and should not be necessarily regarded as an admission or any form of suggestion that the information constitutes the prior art already known to ordinary technicians in this field.

[0003] Octoploid cultivated strawberry (Fragaria×ananassa) is a nutritious herbaceous berry. According to data from the Food and Agriculture Organization of the United Nations, the global strawberry planting area in 2020 was about 3.8 billion hectares, with a production of about 8.66 million tons. As the planting area expands, the demand for new strawberry varieties is vigorous. At present, the selection and breeding of strawberry varieties mainly relies on traditional hybridization, backcrossing, and self-pollination techniques, which are severely restricted by the biological traits of the germplasm. In recent years, with the release of the octoploid cultivated strawberry genome, it has become possible to discover key genes and then use gene editing to create new germplasm.

[0004] Since Zhou et al. used CRISPR-Cas9 to mutate the auxin synthesis gene TAA1 and the auxin response gene ARF8 of diploid forest strawberry in 2018, the genetic improvement of octoploid cultivated strawberry using CRISPR-Cas9 gene editing has developed rapidly. So far, Wilson and Martín-Pizarro et al. used CRISPR-Cas9 gene editing technology to mutate the FaPDS gene and FaTM6 gene of octoploid cultivated strawberry in 2019, Gao et al. used CRISPR-Cas9 gene editing technology to mutate the FaRAP gene of octoploid cultivated strawberry in 2020, and López-Casado et al. used CRISPR-Cas9 gene editing technology to mutate the FaPG1 gene of octoploid cultivated strawberry in 2023. There have been reports of gene editing in octoploid cultivated strawberry.

[0005] Although CRISPR-Cas9 gene editing technology has been applied to the creation of new germplasm of octoploid strawberry, the following problems still exist: 1. Low transformation efficiency (nearly 10%); 2. Long regeneration time (8 to 16 months). The above shortcomings seriously limit the application of gene editing technology in strawberry breeding. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention provides a gene editing method for octoploid cultivated strawberry, which specifically involves the construction of gene editing vectors for octoploid cultivated strawberry, the acquisition of gene editing positive callus, positive bud regeneration, positive bud subculture, strain genotype identification, and rooting, hardening and transplanting of strains. The present invention includes six technical links: target gene editing vector construction and Agrobacterium transformation, co-cultivation of Agrobacterium and octoploid cultivated strawberry tissue culture seedlings to obtain positive callus, positive callus regeneration induction, positive bud subculture proliferation to strains, regeneration strain genotype identification, and positive strain rooting, hardening and transplanting.

[0007] Specifically, the technical solution adopted by the present invention is as follows:

[0008] The present invention provides a gene editing method for octoploid cultivated strawberry, the method comprising the following steps:

[0009] (1) Designed target gene dual-target editing primers based on the octoploid cultivated strawberry genome, integrated them into the target gene editing vector pKSE401G, and completed GV3101 Agrobacterium tumefaciens transformation;

[0010] (2) The activated GV3101 Agrobacterium tumefaciens containing the target gene editing vector was injected into the base of the strawberry tissue culture seedling stem and co-cultured on sterile vermiculite for 10 days to induce positive callus;

[0011] (3) placing the positive callus in a positive callus regeneration induction medium to induce differentiation until positive buds are differentiated;

[0012] (4) cutting the positive buds differentiated from different positive calli into single buds, and transferring them to positive bud subculture medium for subculture and proliferation, until a strain is generated through subculture and proliferation;

[0013] (5) DNA was extracted from each strain, followed by genotyping and off-target site detection;

[0014] (6) The identified positive strains were cut into individual plants, transferred to positive bud rooting medium to induce rooting, and then hardened and transplanted.

[0015] The explant used in the present invention is a tissue culture seedling. The traditional strawberry gene editing method uses chopped leaf discs as explants (refer to Xing Si Nian, etc.). The explant itself does not have photosynthetic ability and requires MS culture medium to provide nutrients for the growth of positive callus. The present invention uses tissue culture seedlings as explants, and uses the photosynthetic products of the tissue culture seedlings themselves to supply nutrients for the growth of positive callus at the base of the stem, which is conducive to prolonging the co-cultivation time of Agrobacterium and explants. The traditional leaf disc method is carried out on MS culture medium for Agrobacterium and explant co-culture infection. The infection efficiency of this method is subject to the concentration of Agrobacterium (0.5-1OD600) and the infection time (30 minutes to 3 days). The positive rate is low when the concentration of Agrobacterium is low and the infection time is short. High Agrobacterium concentration and long infection time may increase the positive rate, but high concentration of Agrobacterium and long infection time will cause Agrobacterium to grow explosively on the nutrient-rich MS culture medium, causing the death of the explant, but unable to obtain positive callus. Therefore, in the traditional leaf disc transformation method, after balancing the concentration of Agrobacterium and the infection time, the T0 generation positive rate is nearly 10%. The present invention adopts autologous photosynthetic tissue culture seedlings, and only uses sterilized vermiculite as a fixed object, and vermiculite does not provide nutrition for Agrobacterium to explode. Therefore, the restrictions on Agrobacterium concentration and infection time are relaxed. The present invention adopts 1OD600 concentration GV3101 tumefaciens Agrobacterium and octoploid strawberry tissue culture seedlings to co-cultivate on sterilized vermiculite for 10 days, which can make the positive rate reach more than 60%, and the positive rate is increased by 6 times. At the same time, the leaf disc in the traditional leaf disc transformation method does not have the ability to directly regenerate buds. The positive callus regeneration obtained by the leaf disc transformation method requires dedifferentiation and redifferentiation, and the regeneration cycle is about 8 to 16 months. The positive callus obtained by the present invention occurs at the base of the strawberry stem. In production, the base of the strawberry stem will form root tiller seedlings, and the base of the stem has the ability to directly regenerate buds. Using this biological characteristic, the regeneration time of the stem base positive callus is shortened to 3 months, and the time to hardening and transplanting is shortened to 4 months, and the regeneration time is shortened by at least 1 times.

[0016] In one or some embodiments of the present invention, in step (1), the specific method includes: designing target genes sgRNA1 and sgRNA2, integrating the double target sites (sgRNA1 and sgRNA2) into pCBC-DT1T2 by four-primer PCR, and then assembling the double target sites into pKSE401G by enzyme digestion and ligation, and transforming DH5α Escherichia coli, and then shaking the bacteria, extracting the plasmid and transforming GV3101 Agrobacterium tumefaciens after sequencing and identification;

[0017] In one or some embodiments of the present invention, in step (2), the activated GV3101 tumefaciens Agrobacterium containing the target gene editing vector is obtained by the following method: the Agrobacterium containing the target gene editing vector is activated by shaking in a LB medium containing three antibiotics in a dark environment, and then the cells are collected, dissolved in an Agrobacterium infection induction medium, and then acetyl syringone is added to activate the Agrobacterium.

[0018] The activation method specifically includes: adding 1.0% (v / v) acetyl syringone (50 mg / mL) to the GV3101 Agrobacterium tumefaciens culture containing the target gene editing vector, and then shaking the culture at 100 rpm and 25°C for 3 hours to activate the Agrobacterium.

[0019] Preferably, the contents of the components of the Agrobacterium infection induction medium are as follows: the concentration of MS liquid medium (without sucrose and agar) is 4.43 g / L, sucrose is 20.00 g / L, and the pH value of the medium is 5.80-6.00.

[0020] In one or some embodiments of the present invention, in step (2), the co-cultivation method is: first, the vermiculite is placed in a tissue culture bottle and sterilized under high pressure, and after the vermiculite is completely cooled, a single sterile tissue culture strawberry seedling is inserted into the clean bench, and then the activated GV3101 Agrobacterium tumefaciens solution containing the target gene editing vector is injected into the base of the stem of the tissue culture seedling.

[0021] In one or some embodiments of the present invention, in step (3), the specific method includes: under the irradiation of a blue light, cutting off the positive callus emitting green fluorescence at the base of the stem of the tissue culture seedling, washing it with sterile water, and then placing the positive callus in a positive callus regeneration induction medium, replacing the medium every 25 days until positive buds are differentiated.

[0022] Preferably, the contents of the components of the positive callus regeneration induction medium are as follows: MS medium (containing 30 g / L sucrose and 7 g / L agar) at a concentration of 41.4 g / L, TDZ at a concentration of 3 mg / L, IBA at a concentration of 0.2 mg / L, carbenicillin at a concentration of 250 mg / L, and kanapencillin at a concentration of 10 mg / L, and the pH value of the culture medium is 5.80-6.00.

[0023] In one or some embodiments of the present invention, in step (4), the specific method includes: cutting off the positive buds differentiated from different positive calli, transferring them to positive bud subculture medium respectively, and replacing the medium every 25 days until a strain is proliferated by subculture.

[0024] Preferably, the contents of the components of the positive bud subculture medium are as follows: MS medium (containing 30 g / L sucrose and 7 g / L agar) at a concentration of 41.4 g / L, IBA at a concentration of 0.05 mg / L, 6-BA at a concentration of 0.5 mg / L, carbenicillin at a concentration of 250 mg / L, and kanapencillin at a concentration of 10 mg / L, and the pH value of the culture medium is 5.80-6.00.

[0025] In one or some embodiments of the present invention, in step (5), the specific method includes: confirming the specific positions of the target gene, the target site on the target gene allele, and the predicted off-target site based on the GDR website, and then obtaining the before and after sequences of the target site and the off-target site, designing primers based on the before and after sequences of the target site, and analyzing the genotype of each strain.

[0026] In one or some embodiments of the present invention, in step (6), the contents of the components of the positive bud rooting medium are as follows: 1 / 2MS medium (containing sucrose 30 g / L, agar 7 g / L) with a concentration of 39.3 g / L, IBA concentration of 0.2 mg / L, and the pH value of the medium is 5.80-6.00.

[0027] In one or some embodiments of the present invention, in step (6), the seedlings are hardened and transplanted two weeks after rooting is induced.

[0028] In one or some embodiments of the present invention, in steps (2), (3), (4) and (6), the culture conditions include: temperature 25°C, light intensity 1500-2000 Lux, and light / darkness duration (16h / 8h).

[0029] Compared with the related art known to the inventor, one of the technical solutions of the present invention has the following beneficial effects:

[0030] The method of the present invention designs two sgRNAs of target genes and their alleles based on the genome of octoploid cultivated strawberry, integrates the two sgRNAs with pCBC-DT1T2 and assembles them into pKSE401G, and then transforms the vector into GV3101 Agrobacterium tumefaciens; by co-cultivating GV3101 Agrobacterium tumefaciens and strawberry tissue culture seedlings on sterile vermiculite for 10 days, a positive callus rate of more than 60% is achieved, and the positive callus rate is increased by 6 times; the positive callus is cultured in a positive callus regeneration induction medium for 80 days, 3 to 5 positive buds are induced, and the regeneration time is shortened by at least 1 times; the positive buds obtained from different calli are subcultured respectively, and a proliferation rate of more than 5 times is achieved in 30 days, forming 3 to 5 strains; the upstream and downstream primers of the sgRNA site are designed based on the strawberry genome, and the genotype identification of the regenerated strain is realized through PCR and Top0 cloning sequencing. The identified mutant genotype is induced to root and transplanted to obtain the gene-edited strain of octoploid cultivated strawberry. The entire technical method can complete the regeneration of positive buds in about 3 months. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their description are used to explain the present invention and do not constitute improper limitations on the present invention.

[0032] Figure 1: Flow chart of the gene editing method for octoploid cultivated strawberry of the present invention.

[0033] Figure 2 : Obtaining positive callus at the stem base.

[0034] Figure 3 : Induction of bud regeneration from positive callus at stem base.

[0035] Figure 4 : Peak diagram of sgRNA sequence and positive strain genotype identification.

[0036] Figure 5 : Genotype identification results of positive strains.

[0037] Figure 6 : Detection results of off-target sites in positive strains.

[0038] Figure 7 : Positive strains are hardened and transplanted. DETAILED DESCRIPTION

[0039] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. 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 invention belongs.

[0040] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations and / or combinations thereof.

[0041] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0042] In this embodiment, the content of each component of the Agrobacterium infection induction medium used is: MS liquid medium (without sucrose and agar) with a concentration of 4.43 g / L, sucrose 20.00 g / L, and the pH value of the medium is 5.80-6.00.

[0043] In this embodiment, the positive callus regeneration induction medium used has the following components: MS medium (containing 30 g / L sucrose and 7 g / L agar) at a concentration of 41.4 g / L, TDZ at a concentration of 3 mg / L, IBA at a concentration of 0.2 mg / L, carbenicillin at a concentration of 250 mg / L, and kanacillin at a concentration of 10 mg / L, and the pH value of the medium is 5.80-6.00.

[0044] In this embodiment, the positive bud subculture medium used has the following components: MS medium (containing 30 g / L sucrose and 7 g / L agar) at a concentration of 41.4 g / L, IBA at a concentration of 0.05 mg / L, 6-BA at a concentration of 0.5 mg / L, carbenicillin at a concentration of 250 mg / L, and kanacillin at a concentration of 10 mg / L, and the pH value of the culture medium is 5.80-6.00.

[0045] In this embodiment, the positive bud rooting medium used has the following components: 1 / 2MS medium (containing 30 g / L sucrose and 7 g / L agar) with a concentration of 39.3 g / L, IBA with a concentration of 0.2 mg / L, and a medium pH of 5.80-6.00.

[0046] The gene editing process of the octoploid cultivated strawberry of the present invention is as follows Figure 1 , specifically including the following steps and methods:

[0047] 1. Construction of gene editing vector and Agrobacterium transformation

[0048] Submit the target gene CDS sequence to the CRISPR online gene editing primer design website (if the target gene CDS length is greater than 2300bp, directly split it into multiple sequences of nearly 2300bp and upload it), select the octoploid cultivated strawberry genome for the genome, select the Cas9 option of NGG for PAM, and then submit it. Determine the two sgRNAs of the target gene and allele and the off-target sites based on the editing primer score. The primer design is shown in Table 1. Replace the N in primer F0 / BsF with one target sequence; replace the N in -R0 / BsR with the reverse complementary sequence of the other target. Complete the design of the four primers of dual-target DT1-BsF, DT1-F0, DT2-R0, and DT2-BsR.

[0049] Table 1. Design of primers for gene editing

[0050] Primers for gene editing Sequences(5'-3') DT1-BsF ATATATGGTCTCGATTGNNNNNNNNNNNNNNNNNGTT DT1-F0 TGNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGC DT2-R0 AACNNNNNNNNNNNNNNNNNNNCAATCTCTTAGTCGACTCTAC DT2-BsR ATTATTGGTCTCGAAACNNNNNNNNNNNNNNNNNNNCAA

[0051] The double target sequence was integrated with pCBC-DT1T2 by four-primer PCR, and the specific PCR reaction system and procedure are shown in Table 2. Then, the PCR product was recovered according to the instructions of the gel recovery kit of Shanghai Bioengineering Company, and the PCR product and pKSE401G vector were digested by Bsa I of NEB Company, and the target gene sequence of NEB Company T4 ligase was connected to pKSE401G, and the restriction enzyme ligation was completed in one step (the specific reaction system and restriction enzyme ligation procedure are shown in Table 3), and the ligation product was transformed into DH5α Escherichia coli, and PCR was performed with U626-IDF and U629-IDF primers, and then the PCR product was recovered and cloned and sequenced for verification (the specific reaction system and PCR procedure are shown in Table 4). After successful verification, the Escherichia coli plasmid was extracted and transformed into GV3101 Agrobacterium tumefaciens, and then identified again (the specific reaction system and PCR procedure are shown in Table 4).

[0052] Table 2 Four-primer PCR reaction system

[0053] composition volume <![CDATA[ddH 2 The]]> 17.5μl 10×KOD buffer 5μl <![CDATA[Mg SO 4 ]]> 3.5μl dNTP 5μl DT1-BsF 5μl DT1-F0 0.25μl DT2-R0 0.25μl DT2-BsR 5μl DMSO 2.5μl Template(pCBC-DT1T2) 5μl KOD plus 1μl

[0054] PCR reaction conditions: 95°C for 5 min, 95°C for 30 s, 55°C for 30 s, 72°C for 3 min, 72°C for 7 min, 4°C for 5 min.

[0055] Table 3 Reaction system for enzyme-linked vector construction

[0056] composition volume PCR fragment (626 bp) 2μl pKSE401G 2μl 10xNEB T4 Buffer 1.5μl 10xBSA 1.5μl BnS 1μl T4 Ligase(NEB) / High concentration 1μl ddH2O 6μl

[0057] The reaction conditions for enzyme-linked vector construction were as follows: 37°C for 3 min, 16°C for 200 min, 50°C for 5 min, and 80°C for 10 min.

[0058] Table 4 Carrier detection reaction system

[0059] composition volume Monoclonal bacterial suspension 1μl 2×ES Taq Master Mix 12.5μl U626-IDF 1.25μl U629-IDF 1.25μl <![CDATA[H 2 The]]> 9μl

[0060] Vector detection reaction conditions: 95°C for 5 min, 95°C for 30 s, 55°C for 30 s, 72°C for 3 min, 72°C for 10 min, and 4°C for 5 min.

[0061] 2. Co-culture to obtain positive callus

[0062] 5 μL of GV3101 Agrobacterium tumefaciens culture containing the target gene editing vector was added to 4 mL of LB medium containing 50 μg / mL gentamicin, 50 μg / mL rifampicin, and 50 μg / mL canadian penicillin for the first activation at 28°C and 200 rpm. Then 100 μL of the culture solution was added to 100 mL of LB medium containing 50 μg / mL gentamicin, 50 μg / mL rifampicin, and 50 μg / mL canadian penicillin, and activated again at 28°C and 200 rpm. The above 100 mL of bacterial solution was centrifuged at 8000 rpm for 2 minutes to collect the bacterial solution, and then diluted with Agrobacterium infection induction medium to an OD600 of about 1.00, and then 1% (v / v) volume of 50 mg / L

[0063] Acetosyringone was added and the incubation was carried out at 25°C and 100 rpm in the dark for 3 h to activate Agrobacterium and complete the preparation of the infection solution.

[0064] A single sterile tissue culture strawberry seedling was inserted into a 240 mL tissue culture bottle pre-filled with sterilized vermiculite, and the above-mentioned activated Agrobacterium solution was injected into the base of the stem of the tissue culture seedling using a pipette, 1 mL was injected for each sterile tissue culture strawberry seedling, and then cultured for 10 days at 25°C, 1500-2000 Lux of light, and a light / darkness duration of 16h / 8h to induce positive callus.

[0065] 3. Positive callus regeneration induction

[0066] The strawberry seedlings co-cultured in the tissue culture bottle were transferred to the clean bench, and the positive callus emitting green fluorescence at the base of the stem of the tissue culture seedlings was cut off under the irradiation of blue light. Figure 2 . Wash with sterile water several times and dry with sterile filter paper. Transfer the cut positive callus to the positive callus regeneration induction medium. Induce the positive callus to differentiate and sprout under the conditions of 25℃, 1500-2000Lux, and light / darkness duration (16h / 8h). Replace the medium every 25 days until positive buds are differentiated. Figure 3 .

[0067] 4. Subculture of positive buds

[0068] The positive buds induced from different callus blocks were cut off as single buds, transferred to the positive bud subculture medium respectively, and marked. Subculture was carried out at 25℃, light intensity of 1500-2000Lux, and light / darkness duration (16h / 8h). The culture medium was changed every 25 days until cluster buds were subcultured. Cluster buds were derived from single buds and had the same genotype, which was called a strain.

[0069] 5. Genotype identification of regenerated lines

[0070] A single bud was selected from each strain to extract DNA, and then genotype identification and off-target site detection were performed (based on the GDR website, the target gene, the target site on the target gene allele, and the specific location of the predicted off-target site were confirmed, and then the target sequence before and after was obtained. Based on the target sequence before and after, and the off-target site sequence before and after, the target gene and allele target and off-target site detection primers were designed (the allele sequences were consistent, but the adjacent sequences were different, so each allele could design its own specific primer). Using single bud DNA as a template, target site, off-target site detection primers and 2×ES Taq Master Mix for PCR. The specific PCR reaction system and procedure are shown in Table 5. The PCR products were then recovered using the Shanghai Biotech Glue Recovery Kit, and cloned using the top0 cloning kit. Ten single clones were then picked for each pair of primers and sequenced using shake bacteria. For an allele of the same strain, if the sequencing results of 10 single clones are the same, then the positive and negative chains of the allele are considered to be completely complementary and homozygous. If the sequencing results of 10 single clones have two types of sequence information, then the positive and negative chains of the allele are considered to be incompletely complementary and heterozygous. The positive and negative chains of the allele may be of three types: unedited, homozygous mutation, and heterozygous mutation. At the same time, the target mutation types include deletion, insertion, substitution, etc., which are random and diverse. The sequencing results of the experimental targets of the present invention are shown as follows: Figure 4 Verify the target gene and allele target sequence of octoploid strawberry to determine the genotype of each strain. Check the sequence of each allele of 1#, 2#, and 3# and find that at least one of the two target sites of each allele is mutated, ensuring that the CDS of the gene is mutated. However, the sequence of each allele after mutation is mostly different, such as Figure 5 The off-target site sequences mostly differed by 3-4 bases from the sgRNA1 and sgRNA2 sequences. The predicted off-target sites were verified by primer PCR, top0 cloning, and sequencing, showing that the off-target site sequences were consistent with the wild-type sequences, confirming that the sgRNA of each strain had no off-target effects. Figure 6 Three strains with different genotypes were selected for subculture and the subculture was referred to (IV. Subculture of positive buds).

[0071] Table 5 PCR detection reaction system

[0072] composition volume 2×ES Taq Master Mix 12.5μl Primer 1 1.25μl Primer 2 1.25μl Template DNA 1μl <![CDATA[H 2 The]]> 9μl

[0073] PCR reaction conditions: 95°C for 5 min, 95°C for 30 s, 55°C for 30 s, 72°C for 3 min, 72°C for 7 min, 4°C for 5 min.

[0074] 6. Rooting and hardening of regenerated strains

[0075] Cut the positive strains after subculture into single buds, transfer them to the positive bud rooting medium for rooting, and induce rooting at 25°C, light intensity of 1500-2000Lux, and light / darkness (16h / 8h). Two weeks later, each single bud will induce about 5 1-2cm long fibrous roots. Open the cap of the tissue culture bottle to ventilate for one day to harden the seedlings, then use tweezers to pick up the rooted strawberry seedlings, wash the culture medium and transplant them to the plug tray, cultivate the matrix, water it and turn upside down the disposable transparent plastic cup to keep it moist, and cultivate it at 25°C, light intensity of 1500-2000Lux, and light / darkness (16h / 8h). Check the survival of the seedlings after one week and remove the plastic cup. If Figure 7 .

[0076] Compared with the traditional strawberry gene editing method, this embodiment has the following advantages:

[0077] 1. The infected explants are different. The traditional strawberry gene editing method uses chopped leaf discs as explants (refer to Xing Si Nian et al.). The explants themselves do not have photosynthetic ability and need nutrients for positive callus growth on MS medium. This example uses tissue culture seedlings as explants, and uses the photosynthetic products of the tissue culture seedlings themselves to provide nutrients for positive callus at the base of the stem, avoiding the MS medium supplying nutrients for leaf disc callus growth.

[0078] 2. The traditional leaf disc method is used for co-culture infection on MS culture medium, which is subject to the Agrobacterium concentration (0.5-1OD600) and infection time (30 minutes-3 days). The positive rate is low when the Agrobacterium infection time is short, and the positive rate may be increased when the infection time is long. However, Agrobacterium is prone to explosive growth on the nutrient-rich MS culture medium, causing explant death. The positive rate of the T0 generation is nearly 10% when the balance is reached. This embodiment uses autophotosynthetic tissue culture seedlings and sterilized vermiculite as a fixed material. Vermiculite has no nutrition and allows Agrobacterium to explode. Therefore, the restrictions on Agrobacterium concentration (1OD600) and infection time (10 days) are relaxed, and the positive rate can reach more than 60% (65 / 105, 65 / 105). Figure 1 .

[0079] 3. In the traditional leaf disc transformation method, the regeneration of leaf disc callus requires dedifferentiation and redifferentiation, and the cycle is about 8 to 16 months. In production, when cultivating octoploid strawberries, root tillers are often formed at the base of the stem. In this embodiment, the positive callus is generated at the base of the stem. By utilizing the biological characteristics of root tillers at the base of the stem, the regeneration time is shortened to 3 months. Figure 1 and Figure 2 .

[0080] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A gene editing method for octoploid strawberry cultivation, characterized in that: The method comprises the following steps: (1) Designed target gene dual-target editing primers based on the octoploid cultivated strawberry genome, integrated them into the target gene editing vector pKSE401G, and completed GV3101 Agrobacterium tumefaciens transformation; (2) The activated GV3101 Agrobacterium tumefaciens containing the target gene editing vector was injected into the base of the strawberry tissue culture seedling stem and co-cultured on sterile vermiculite for 10 days to induce positive callus; (3) Placing the positive callus on a positive callus regeneration induction medium to induce differentiation until positive buds are differentiated; (4) cutting the positive buds differentiated from different positive calli into single buds, and transferring them to positive bud subculture medium for subculture and proliferation, until a strain is generated through subculture and proliferation; (5) DNA was extracted from each strain, followed by genotyping and off-target site detection; (6) The identified positive strains were cut into individual plants, transferred to positive bud rooting medium to induce rooting, and then hardened and transplanted.

2. The gene editing method for octoploid cultivated strawberry according to claim 1, characterized in that: In step (1), the specific method includes: designing target genes sgRNA1 and sgRNA2, integrating the dual target sites (sgRNA1 and sgRNA2) into pCBC-DT1T2 by four-primer PCR, and then assembling the dual target sites into pKSE401G by enzyme digestion and ligation, and transforming DH5α Escherichia coli, and then shaking the bacteria, extracting the plasmid after sequencing and identification, and transforming GV3101 Agrobacterium tumefaciens.

3. The gene editing method for octoploid cultivated strawberry according to claim 1, characterized in that: In step (2), the activated GV3101 Agrobacterium tumefaciens containing the target gene editing vector is obtained by the following method: the Agrobacterium containing the target gene editing vector is activated by shaking in a LB medium containing three antibiotics in a dark environment, and then the cells are collected and dissolved in an Agrobacterium infection induction medium, and then acetyl syringone is added to activate the Agrobacterium; Preferably, the contents of the components of the culture medium for inducing Agrobacterium infection are as follows: the concentration of MS liquid culture medium is 4.43 g / L, sucrose is 20.00 g / L, and the pH value of the culture medium is 5.80-6.

00.

4. The gene editing method for octoploid cultivated strawberry according to claim 1, characterized in that: In step (2), the co-cultivation method is: first, the vermiculite is placed in a tissue culture bottle and sterilized under high pressure, and after the vermiculite is completely cooled, a single sterile tissue culture strawberry seedling is inserted, and then the activated GV3101 Agrobacterium tumefaciens solution containing the target gene editing vector is injected into the base of the stem of the tissue culture seedling for co-cultivation.

5. The gene editing method for octoploid cultivated strawberry according to claim 1, characterized in that: In step (3), the specific method includes: under the irradiation of a blue light, cutting off the positive callus emitting green fluorescence at the base of the tissue culture seedling, washing it with sterile water, placing it in a positive callus regeneration induction medium, and replacing the medium every 25 days until positive buds are differentiated; Preferably, the contents of the components of the positive callus regeneration induction medium are as follows: MS medium concentration is 41.4 g / L, TDZ concentration is 3 mg / L, IBA concentration is 0.2 mg / L, carbenicillin concentration is 250 mg / L, kanapencillin concentration is 10 mg / L, and the pH value of the medium is 5.80-6.

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6. The gene editing method for octoploid cultivated strawberry according to claim 1, characterized in that: In step (4), the specific method includes: cutting off the positive buds differentiated from different positive calli, transferring them to positive bud subculture medium respectively, and replacing the medium every 25 days until a strain is subcultured and proliferated; Preferably, the contents of the components of the positive bud subculture medium are as follows: MS medium concentration is 41.4 g / L, IBA concentration is 0.05 mg / L, 6-BA concentration is 0.5 mg / L, carbenicillin concentration is 250 mg / L, kanapencillin concentration is 10 mg / L, and the pH value of the culture medium is 5.80-6.

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7. The gene editing method for octoploid cultivated strawberry according to claim 1, characterized in that: In step (5), the specific method includes: confirming the specific positions of the target gene, the target site on the target gene allele, and the predicted off-target site based on the GDR website, and then obtaining the sequence before and after the target site, designing primers based on the sequence before and after the target site, and analyzing the genotype of each strain.

8. The gene editing method for octoploid cultivated strawberry according to claim 1, characterized in that: In step (6), the contents of the components of the positive bud rooting medium are as follows: the concentration of 1 / 2MS medium is 39.3 g / L, the concentration of IBA is 0.2 mg / L, and the pH value of the medium is 5.80-6.

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9. The gene editing method for octoploid cultivated strawberry according to claim 1, characterized in that: In step (6), the seedlings are hardened and transplanted after two weeks of rooting.

10. The gene editing method for octoploid cultivated strawberry according to claim 1, characterized in that: In steps (2), (3), (4) and (6), the culture conditions include: a temperature of 25°C, a light intensity of 1500 to 2000 Lux, and a light / darkness duration of (16h / 8h).

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