Method for cultivating gene-edited pomegranate by two-step somatic embryogenesis technique

Abstract

The application discloses a method for cultivating and obtaining gene edited Punica granatum by a two-step somatic embryogenesis technique, which comprises the following steps: material obtaining, obtaining of embryogenic callus, preparation of Agrobacterium tumefaciens liquid, pre-culturing of callus, infection, co-culturing, washing, selection culture, subculture and ex vitro rooting, etc. The application firstly takes Punica granatum stem segments as explants to establish a Punica granatum gene editing plant system and obtain Punica granatum gene editing plants, avoids the separation of offspring traits when taking Punica granatum seed hypocotyls as explants, and lays a good foundation for subsequent researches on Punica granatum gene function and fine variety cultivation; in addition, the application further refines the somatic embryogenesis conditions under the selection pressure of Kan and TMT on the basis of the established Punica granatum somatic embryogenesis, directly transforms embryogenic cells by using the bipolar characteristics of somatic embryos, and thus complete and stable Punica granatum gene editing plants are obtained, and the transformation rate is high.

Description

Technical Field

[0001] This invention relates to the field of pomegranate cultivation technology, and in particular to a method for cultivating gene-edited pomegranates using a two-step method of somatic embryogenesis. Background Technology

[0002] Gene editing technology, which emerged in this century, can target and modify DNA fragments to alter specific genes. This technology allows for the directional improvement of target traits and shows great promise for applications in crop genetic improvement.

[0003] Pomegranate( Punicagranatum Pomegranate (L.) is a healthy fruit with high economic, nutritional, and medicinal value, and its economic benefits are significant. However, there are still certain problems in gene function research and improved variety breeding of pomegranates, mainly because a genetic transformation system and gene editing system have not been established. The establishment of such systems is crucial for the continued high-quality development of the pomegranate industry.

[0004] Currently, the most common gene editing system is the CRISPR / Cas9 system, which can edit target DNA sequences in organisms. However, gene editing often causes some growth defects in plants, including weak plants or difficulty in rooting, and it is impossible to obtain the complete gene-edited plants needed for subsequent research through subsequent hardening and transplanting.

[0005] Pomegranates are woody fruit trees, and gene editing processes can lead to problems such as browning, difficulty in cell differentiation into seedlings, difficulty in plant rooting, and low survival rates after transplanting. This has resulted in the slow establishment of a gene editing system, hindering progress in pomegranate gene function research and impeding the development of the pomegranate industry. Summary of the Invention

[0006] Based on the technical problems existing in the background technology, this invention proposes a two-step method for cultivating gene-edited pomegranates using somatic embryogenesis technology. For the first time, a pomegranate gene-edited plant system is established using pomegranate stem segments as explants, and gene-edited pomegranate plants are obtained. This avoids the phenomenon of phenotypic segregation in offspring that occurs when using pomegranate seed hypocotyls as explants, and can lay a good foundation for subsequent research on pomegranate gene function and breeding of superior varieties.

[0007] The present invention proposes a two-step method for cultivating gene-edited pomegranates using somatic embryogenesis technology. The method steps are as follows:

[0008] S1: Inoculate the shoot tip growth point of pomegranate into the primary culture medium and culture for 20-25 days;

[0009] S2: Remove the growing point from the stem segments of the young plants cultured in S1 and inoculate them in callus induction medium for 20-25 days.

[0010] S3: Agrobacterium liquid culture in liquid medium, after the bacteria liquid OD 600 =0.6-0.8, centrifugation, collection of bacteria, resuspension of bacteria to bacteria liquid state with infection liquid, incubation at room temperature and standby;

[0011] S4: The callus cultured in S2 is inoculated in pre-culture medium for 2-3d;

[0012] S5: The resuspended Agrobacterium liquid of S3 is added to the infection liquid, and then the pre-cultured recipient material in S4 is placed in the infection liquid for infection;

[0013] S6: The infected recipient material is cultured in co-culture medium;

[0014] S7: The recipient material after co-culture is washed and cultured in selection medium;

[0015] S8: The somatic embryo of the recipient material after selection culture is inoculated into subculture medium for 10-15d;

[0016] S9: The plant after subculture of S8 is rooted outside the test tube, and after survival, it is transplanted to obtain gene edited pomegranate.

[0017] Preferably, the primary culture medium in S1 is WPM+0.7-0.9mg / L 6-BA+5.5g / L agar+20-25g / L sucrose+0.4-0.6g / L activated carbon.

[0018] Preferably, the callus induction medium in S2 is MS+0.5mg / L 6-BA+1.0-1.5mg / L NAA+5.5g / L agar+20-30g / L sucrose.

[0019] Preferably, the liquid medium in S3 is LB+25mg / L Rif+50mg / L Kan.

[0020] Preferably, the pre-culture medium in S4 is MS+0.5mg / L 6-BA+0.5mg / L NAA+5.5g / L agar+20-30g / L sucrose.

[0021] Preferably, the infection liquid in S5 is MS+0.45-0.55mg / L 6-BA+0.5-0.6mg / L NAA+200μmol / L AS+20-30g / L sucrose.

[0022] Preferably, the volume ratio of Agrobacterium liquid and infection liquid in S5 is 1:1000; the infection time is 5-10min.

[0023] Preferably, the co-culture medium in S6 is MS+ 0.45-0.55 mg / L 6-BA+0.5-0.6 mg / L NAA+200 μmol / L AS+5.5 g / L agar+20-30 g / L sucrose; the culture condition is temperature 25±2℃, dark culture for 2d.

[0024] Preferably, the selection medium in S7 is MS+ 0.45-0.55 mg / L 6-BA+0.5-0.6 mg / L NAA+50 mg / L Kan+200 mg / L TMT+5.5 g / L agar+20-30 g / L sucrose; the culture condition is temperature 25±2℃, illumination 1500-2000 Lx, time 30-35d.

[0025] Preferably, the subculture medium in S8 is WPM+50 mg / L Kan+200 mg / L TMT+6 g / L agar+20-30 g / L sucrose.

[0026] The beneficial technical effects of the present application are:

[0027] The present application first establishes a pomegranate gene editing plant system with pomegranate stem segments as explants, and obtains pomegranate gene editing plants, which avoids the phenomenon of offspring trait separation when using pomegranate hypocotyls as explants, and lays a good foundation for subsequent research on pomegranate gene function, elite breeding, etc.

[0028] Since pomegranate is a woody plant, it is prone to problems such as difficulty in differentiating seedlings (both buds and roots are difficult to differentiate) and easy browning during Agrobacterium transformation. Therefore, based on the established pomegranate somatic embryogenesis, the somatic embryogenesis conditions under the selection pressure of Kan and TMT are further refined, and the bipolarity of somatic embryos (without hormones and external conditions, they can grow buds and roots by themselves) is used to directly transform embryonic cells to obtain complete and stable pomegranate gene editing plants with high transformation efficiency.

[0029] The present application combines some of the previous research contents of the pomegranate tissue rapid propagation system (Jingjing Qian et al., 2020), the pomegranate genetic transformation system (Qian Jingjing et al., ZL202210361786.4), the pomegranate somatic embryogenesis system (Jingjing Qian et al., 2023) and the pomegranate test tube exogenous root system (Qian Jingjing et al., 2022, ZL202210650792.1), and establishes a pomegranate gene editing system based on somatic embryogenesis, which breaks the technical bottleneck of pomegranate gene editing. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1Flow chart of cultivating pomegranate by gene editing proposed in the present application; (a) young stem section inoculation (culture time 20-25d), (b) embryogenic callus acquisition (culture time 20-25d), (c) pre-culture (culture 2-3d), (d) infection, (e) co-culture (2d), (f) selection culture (culture 30-35d), (g) acquisition of gene editing tissue culture seedlings, (h) in vitro rooting (10-15d), (i) transplanting and obtaining gene editing seedlings.

[0031] Figure 2 Growth states of inoculated stem tip growth points (a) 7d, (b) 14d and (c) 21d proposed in the present application.

[0032] Figure 3 Screening of different concentrations of 6-BA and NAA combinations for embryogenic callus induction proposed in the present application.

[0033] Figure 4 (a) callus growth state and (b) callus local observation chart after 20d of embryogenic callus induction proposed in the present application.

[0034] Figure 5 Morphological observation of somatic embryogenesis at each period during the selection culture process proposed in the present application; (a) 7d, (b) 14d, (c) 21d, (d) 28d, (e) 35d.

[0035] Figure 6 Wild type pomegranate WT (left) and gene edited pomegranate (right) 60d growth morphology proposed in the present application.

[0036] Figure 7 (a) wild type pomegranate WT (left) and gene edited pomegranate (right) 120d growth morphology and (b) comparison of sequencing results proposed in the present application. DETAILED DESCRIPTION

[0037] The present application is further illustrated below in conjunction with specific embodiments.

[0038] EMBODIMENT

[0039] REFERENCE Figure 1 The method for cultivating and obtaining gene edited pomegranate by somatic embryogenesis technology two-step method proposed in the present application has the following method steps:

[0040] (1) Material acquisition

[0041] Select healthy, disease-free and pest-free plants as materials in the established provincial pomegranate germplasm nursery, and select vigorous young stems.

[0042] The collected tender stem tips of Punica granatum are placed in a beaker and washed with clean water for 15-30 min, then washed with 75% alcohol for 20-30 s, and then washed with sterile water for 2-3 times to remove surface dust and other impurities. The washed material is placed in a clean bench, 0.1% HgCl2 is selected as the disinfectant, and the material is disinfected for 8-12 min, and then washed with sterile water for 3-5 times. Under a microscope, the stem tip explants are separated with a dissecting needle, and only the leaf primordium growth points are left, with a diameter of about 0.3-0.5 mm. The growth points of the stem tips are cut off with a surgical knife and inoculated into the prepared culture medium (WPM+0.7-0.9 mg / L 6-BA+5.5 g / L agar+20-25 g / L sucrose+0.4-0.6 g / L activated carbon) for 20-25 d. The growth state of the stem tip growth points in the culture medium is shown in Figure 2 .

[0043] (2) Obtaining embryogenic callus

[0044] After the above material is cultured for 20-25 d, the tender plants with good growth and no contamination and growing to 0.8-1.2 cm are used as the material. The tender stem segments are cut into 0.50 cm small sections after removing the growth points, and are horizontally inoculated into the culture medium (MS+0.5 mg / L 6-BA+1.0 mg / L NAA+5.5 g / L agar+25-30 g / L sucrose) for 20-25 d.

[0045] (3) Preparation of Agrobacterium liquid

[0046] The Agrobacterium liquid (GV3101) stored at -80°C is added to 50 mL LB liquid medium (containing 50 mg / L Kan, 25 mg / L Rif) and placed in a 28°C constant temperature shaker for 150 r / min shaking culture for 20-24 h. When the OD 600 value is 0.60-0.80, it is transferred into a 50 mL centrifuge tube on a clean bench, centrifuged at 4000 r / min and 28°C for 10 min, the supernatant is discarded, the bacterial body is resuspended with an infection liquid (MS+0.51 mg / L 6-BA+0.55 mg / L NAA+25-30 g / L sucrose) containing 200 μmol / L AS, and is incubated at room temperature for 2 h for standby.

[0047] (4) Pre-culture

[0048] The light green granular and vigorous growth callus in step (2) is inoculated into the culture medium (MS+0.5 mg / L 6-BA+0.5 mg / L NAA+5.5 g / L agar+25-30 g / L sucrose) for 2-3 d.

[0049] (5) Infection

[0050] The prepared Agrobacterium solution (50 μL) in step (3) was added to the infection solution (MS+0.51 mg / L 6-BA+0.55 mg / L NAA+25-30 g / L sucrose) on the super-clean workbench, and then the recipient material in step (4) was placed in the infection solution for 8 min of infection, and then placed on sterile filter paper to dry.

[0051] (6) Co-culture

[0052] After the recipient material was taken out of the infection solution and placed on sterile filter paper to absorb the excess surface water, it was inoculated in the co-culture medium (MS+0.51 mg / L 6-BA+0.55 mg / L NAA+200 μmol / L AS+5.5 g / L agar+25-30 g / L sucrose) for growth, sealed with PARAFILM sealing film, and placed in a light incubator (GXZ-500D) with a temperature of 25±2°C for 2 days of dark culture.

[0053] (7) Washing

[0054] After co-culture, the recipient material was washed 4 times with sterile water to remove the remaining Agrobacterium on the surface of the tissue, and then soaked in sterile water containing 200 mg / L TMT for 30 min; after soaking, the material was taken out of the sterile water and placed on sterile filter paper to dry.

[0055] (8) Selection culture

[0056] The dried callus was inoculated in the selection medium (MS+0.55 mg / L 6-BA+0.52 mg / L NAA+50 mg / L Kan+200 mg / L TMT+5.5 g / L agar+25-30 g / L sucrose) for growth, and placed in a culture room with a temperature of 25±2°C and light intensity of 1500-2000 Lx for 30-35 days of growth.

[0057] (9) Subculture

[0058] The somatic embryos in step (8) were taken out and inoculated into the blank medium (WPM+50 mg / L Kan+200 mg / L TMT+6 g / L agar+30 g / L sucrose) for culture for 10-15 days.

[0059] (10) Obtaining of gene-edited pomegranate

[0060] The young plants cultured in step (9) were promoted to grow roots by the method of pomegranate in vitro rooting (Qian Jingjing et al., 2022, ZL2022 1 0650792.1), and after survival, they were transplanted to obtain gene-edited pomegranate.

[0061] The results of induction of embryogenic callus by different concentrations of 6-BA and NAA combinations are shown in Table 1 and Figure 3 As shown in Table 1 and Figure 3 It can be seen that when the concentration of NAA is 1.5 mg / L, the induction rate of embryogenic callus is low, and the embryogenic callus is mostly brown or even partially browned, making it difficult to induce embryogenic callus. When 0.5 mg / L 6-BA and 1.0 mg / L NAA are added, the induced embryogenic callus is light yellow to light green granular. Figure 4 Further, it can be seen that the embryogenic callus induced by 0.5 mg / L 6-BA and 1.0 mg / L NAA grows rapidly and is not dense.

[0062] Table 1 Effect of different concentrations of 6-BA and NAA combinations on induction of pomegranate embryogenic callus

[0063] Note: Different lowercase letters in the table indicate significant differences at the 0.05 level.

[0064] During the selection culture, the morphological observation of each period of somatic embryogenesis is shown in Figure 5 Embryogenic callus was observed after 7 days of induction; spherical embryos were observed after 14 days of induction; torpedo-shaped embryos were observed after 21 days of induction; heart-shaped embryos were observed after 28 days of induction; cotyledon stage somatic embryos (mature embryos) were observed after 35 days of induction.

[0065] In addition, during the selection culture, the effect of different concentrations of 6-BA and NAA combinations on the somatic embryo differentiation rate under the selection pressure of 50 mg / L Kan and 200 mg / L TMT on embryogenic callus was studied, and the test results are shown in Table 2. When the concentration of 6-BA is the same, the somatic embryo differentiation rate increases first and then decreases with the increase of NAA concentration, and when the concentration of NAA is 0.52 mg / L, the somatic embryo differentiation rate of each combination is the highest. When the concentration of NAA is 0.52 mg / L, the somatic embryo differentiation rate reaches 53.11% ± 0.13% when 0.55 mg / L 6-BA is added. However, when the concentrations of 6-BA (0.60 mg / L) and NAA (0.54 mg / L) are too high, the somatic embryo differentiation rate is the lowest, which is 42.25% ± 0.06%. Therefore, the optimal hormone combination for inducing pomegranate somatic embryo differentiation is 0.55 mg / L 6-BA and 0.52 mg / L NAA.

[0066] Table 2 Test results of somatic embryo differentiation rate

[0067] Application examples

[0068] The 'Red Agate' pomegranate variety was selected for the experiment to dwarf the gene. PgCYP85A1 To achieve the editing goal, a gene-edited pomegranate was constructed.

[0069] Constructing a dual-target pC1300-35S::Cas9- using the CRISPR / Cas9 system sgPgCYP85A1 Vector. Plant phenotype was observed after 60 days. Gene-edited pomegranates showed a clear phenotype with the PgCYP85A1 protein deletion, thus morphologically proving that this method can effectively obtain gene-edited pomegranate plants. Morphologically, wild-type pomegranates (WT) and gene-edited pomegranates showed elongated stem nodes and curled leaves (see...). Figure 6 After 120 days, DNA was extracted and sequenced from the obtained plants, revealing a deletion of the target sequence bases. This demonstrates at the gene level that this method can effectively obtain stably heritable gene-edited pomegranates with stable morphological phenotypes (e.g., Figure 7 (As shown). Similar to the PgCYP85A1 deletion, with 30 replicates, a total of 12 gene-edited plants were obtained, with a success rate of 40%, which is extremely high, successfully verifying the feasibility of this technology.

Claims

1. A method for obtaining genetically edited pomegranate by somatic embryogenesis technique in two steps, characterized in that, The method steps are as follows: S1: the stem tip growth point of Punica granatum is inoculated in the primary culture medium and cultured for 20-25 days; S2: the stem segment of the young plant after S1 culture is removed from the growth point and inoculated in the callus induction culture medium and cultured for 20-25 days; S3: Agrobacterium liquid culture, after the bacteria liquid OD = 0.6-0.8, centrifugation, collection of bacteria, resuspended bacteria with infection liquid to bacteria liquid state, room temperature incubation after standby; 600 =0.6-0.8, centrifugation, collection of bacteria, resuspended bacteria with infection liquid to bacteria liquid state, room temperature incubation after standby; S4: the light green granular and vigorous growth callus in S2 is selected and inoculated in the pre-culture medium and cultured for 2-3 days; S5: the resuspended Agrobacterium liquid in S3 is added into the infection liquid, and then the pre-cultured receptor material in S4 is placed in the infection liquid for infection; S6: the infected receptor material is cultured in the co-culture medium; S7: the co-cultured receptor material is washed and cultured in the selection medium; S8: the somatic embryo of the receptor material after selection culture is inoculated in the subculture medium and cultured for 10-15 days; S9: the plant after subculture in S8 is rooted outside the test tube, and transplanted after survival to obtain the gene edited Punica granatum; The primary culture medium in S1 is WPM+0.7-0.9 mg / L 6-BA+5.5 g / L agar+20-25 g / L sucrose+0.4-0.6 g / L activated carbon; The callus induction culture medium in S2 is MS+0.5 mg / L 6-BA+1.0-1.5 mg / L NAA+5.5 g / L agar+20-30 g / L sucrose; The pre-culture medium in S4 is MS+0.45-0.55 mg / L 6-BA+0.5-0.6 mg / L NAA+5.5 g / L agar+20-30 g / L sucrose; The infection liquid in S5 is MS+0.45-0.55 mg / L 6-BA+0.5-0.6 mg / L NAA+200 μmol / L AS+20-30 g / L sucrose; The co-culture medium in S6 is MS+0.45-0.55 mg / L 6-BA+0.5-0.6 mg / L NAA+200 μmol / L AS+5.5 g / L agar+20-30 g / L sucrose; The selection medium in S7 is MS+0.45-0.55 mg / L 6-BA+0.5-0.6 mg / L NAA+50 mg / L Kan+200 mg / L TMT+5.5 g / L agar+20-30 g / L sucrose; The subculture medium in S8 is WPM+50 mg / L Kan+200 mg / L TMT+6 g / L agar+20-30 g / L sucrose.

2. The method of claim 1, wherein the genetically edited pomegranate is obtained by two-step method through somatic embryogenesis technique, characterized in that, The liquid medium in S3 is LB+25 mg / L Rif+50 mg / L Kan.

3. The method of claim 1, wherein the genetically edited pomegranate is obtained by two-step method of somatic embryogenesis, characterized in that, The volume ratio of the Agrobacterium liquid to the infection liquid in S5 is 1:1000; the infection time is 5-10 min.

4. The method of claim 1, wherein the genetically edited pomegranate is obtained by two-step method through somatic embryogenesis technique, characterized in that, The culture condition in S6 is temperature 25±2℃, dark culture for 2 days.

5. The method of claim 1, wherein the genetically edited pomegranate is obtained by two-step method of somatic embryogenesis, characterized in that, The culture condition in S7 is temperature 25±2℃, illumination 1500-2000 Lx, time 30-35 days.

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