Agrobacterium tumefaciens-mediated spinach genetic transformation method

Abstract

The invention relates to the technical field of plant genetic transformation, in particular to an agrobacterium tumefaciens-mediated spinach genetic transformation method, which takes spinach stems as explants, takes pBI121-GUS as a plant expression vector, and takes agrobacterium GV3101 (pSoop-p19) as receptor agrobacterium. According to the invention, each step of the spinach genetic transformation system is optimized and selected, and an agrobacterium-mediated spinach stable genetic transformation system is established, so that not only can the transformation efficiency be improved, but also the functional research of spinach agronomic trait key regulatory genes and the cultivation of excellent spinach varieties can be carried out by utilizing the system disclosed by the invention.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic transformation and relates to a method for genetic transformation of spinach mediated by Agrobacterium tumefaciens. Background Art

[0002] Spinach (Spinacia oleracea L.) is an annual or biennial dicotyledonous plant of the Amaranthaceae family. It is an important vegetable cash crop in my country and is rich in vitamins, minerals and antioxidants. In order to meet the urgent demand for high-quality spinach, breeding cultivars that are resistant to pests and diseases, tolerant to high temperatures, late flowering, and low in nitrate and oxalate concentrations is the main goal of spinach breeders.

[0003] Compared with traditional breeding methods, molecular design breeding can accelerate the breeding process to a certain extent and is one of the effective breeding strategies for cultivating new spinach varieties with high resistance and high quality. The establishment of an efficient and stable spinach genetic transformation system is an important prerequisite for studying the gene function and genetic improvement of spinach. By utilizing the natural characteristics of Agrobacterium to integrate T-DNA into the plant genome, the stable genetic transformation and expression of exogenous target genes in the target plant can be achieved, thereby giving the plant new traits. However, the transformation efficiency of the Agrobacterium-mediated genetic transformation system is often affected by species and experimental conditions. Among them, explants of different sources and developmental states have different degrees of damage and ability to form callus tissue after Agrobacterium infection; different Agrobacterium strains and bacterial concentrations often have different mediated transformation efficiencies; the selection and addition ratio of different plant growth regulators determine whether regenerated plants can be induced, which is a key factor in each stage of plant tissue culture.

[0004] In 2009, Bao et al. used the leaves of the spinach cultivar 'Glory' as explants and infected them with Agrobacterium EHA105. They successfully transferred the synthetic insect-resistant gene cry1Ac into spinach and obtained T1 transgenic plants. The callus induction rate of this system was about 24.24%, and the adventitious bud induction rate was only 18.75%. In 2013, Nguyen et al. established a genetic transformation system with spinach taproots and lateral roots as explants by screening spinach varieties, Agrobacterium strains and concentrations, co-cultivation time and other conditions. However, the experimental cycle of this system was long, and the positive transformation rate of callus regeneration buds was only 24.96%.

[0005] In the process of plant tissue culture, spinach regeneration efficiency is low, and callus browning and regenerated plant vitrification are prone to occur. Therefore, it is urgent to re-establish an efficient and stable genetic transformation system. How to determine the optimal conditions of the genetic transformation system to further improve the genetic transformation efficiency and shorten the genetic transformation cycle is a technical problem that needs to be solved urgently by those skilled in the art. Summary of the invention

[0006] The invention provides a method for genetic transformation of spinach mediated by Agrobacterium tumefaciens. Based on the characteristics of spinach itself, experimental conditions are optimized and the most suitable genetic transformation conditions for spinach are determined. The method has the advantages of simple operation and high transformation efficiency, and lays a foundation for subsequent research on the gene function of spinach and breeding of excellent spinach varieties.

[0007] The present invention provides a method for genetic transformation of spinach mediated by Agrobacterium, comprising the following steps:

[0008] Step 1, obtaining stem explants: selecting spinach seeds with full grains, peeling off the seed coat, and after disinfection, sowing them on a culture medium plate and culturing them for 5 days, transferring the sterile seedlings with good growth to a tissue culture bottle and continuing to culture them for 30 to 35 days, and cutting out 2 mm stem segments as explants for subsequent infection;

[0009] Step 2, constructing a plant expression vector: constructing the target gene into a plant expression vector;

[0010] Step 3, prepare Agrobacterium infection solution: transform the plant expression vector containing the target gene into Agrobacterium GV3101 (pSoup-p19), and prepare Agrobacterium infection solution;

[0011] Step 4, infecting explants: immerse the stem segments in step 1 in the Agrobacterium infection solution in step 3, and culture at room temperature with shaking for 30 minutes;

[0012] Step 5, co-cultivation: Place the explants treated in step 4 on sterile filter paper to dry the surface moisture, transfer to co-cultivation medium, and co-cultivate for 2 days;

[0013] Step 6, callus induction: rinse the explants in step 5 with sterile water, place them on sterile filter paper to dry the surface moisture, transfer them to callus induction medium, and culture them for 4 weeks;

[0014] Step 7, adventitious bud induction: peeling the callus from the explant, transferring it to an adventitious bud induction medium, and inducing adventitious buds;

[0015] Step 8, rooting induction: when the adventitious buds in step 7 grow to 2-3 cm, they are transferred to the culture medium SHT to induce rooting and obtain complete plants;

[0016] Step 9, transplanting: Transplant the transgenic positive plants in step 8 into the soil, cover with film for one week, and remove the film after the plants adapt to the external environment. After transplanting, the culture conditions are restored to long-day conditions.

[0017] Furthermore, the spinach seeds in step 1 are seeds of the heat-resistant spinach material Sp75.

[0018] Furthermore, the disinfection treatment in step 1 is: first disinfect with 75% ethanol for 5 minutes, repeat once, wash with sterile water 2 to 3 times, then disinfect with 20% sodium hypochlorite solution for 12 minutes, and wash with sterile water 5 to 6 times.

[0019] Furthermore, the culture medium in the culture dish and tissue culture bottle in step 1 is 1 / 2MS culture medium, the formula is 2.215g / L M519, 10g / L sucrose, 4g / L plant gel, pH=5.8-6.0.

[0020] Furthermore, in step 1, the sterile spinach tissue culture seedlings are cultured in an RDN type artificial climate chamber, and the artificial climate chamber is operated alternately in a light 16h / dark 8h mode, and the light temperature is controlled at 24°C, the dark temperature is controlled at 18°C, and the relative humidity is controlled at 50%.

[0021] Furthermore, the plant expression vector in step 2 is pBI121-GUS.

[0022] Further, in step 3, the plant expression vector containing the target gene is transformed into Agrobacterium GV3101 (pSoup-p19), and an Agrobacterium infection solution is prepared, which specifically includes the following steps:

[0023] The plant expression vector containing the target gene was transformed into Agrobacterium GV3101 (pSoup-p19) by heat shock method, and a single positive colony was obtained by culture;

[0024] The single positive colony was cultured and the OD of the bacterial solution was 600 When the pH value is 1.0-1.2, the culture is stopped to obtain an activated bacterial suspension;

[0025] Take 1 ml of the activated bacterial suspension for mass culture. 600 When the concentration is 0.6-0.8, collect the bacteria by centrifugation;

[0026] The bacteria were resuspended in the infection solution and diluted to OD 600 is about 0.28, and the preliminary Agrobacterium infection solution is obtained;

[0027] Place the initial Agrobacterium infection solution in a 28°C constant temperature incubator in the dark for 2 hours and adjust the infection solution OD 600 is 0.3, and the final Agrobacterium infection solution is obtained;

[0028] The infection solution includes SH basic culture medium (see Table 1), 20 g / L sucrose and 100 μM acetosyringone, and pH=5.8-6.0.

[0029] Furthermore, the culture medium formula for the co-cultivation in step 5 is SH basal medium, 100 μM acetosyringone, 20 g / L sucrose, 4 g / L plant gel, pH=6.0.

[0030] Furthermore, in step 6, the sterile water is added with 600 mg / L of timentin.

[0031] Furthermore, in step 6, the callus induction medium formula is SH basal medium, 3.5 mg / L 6-BA, 0.5 mg / L NAA, 0.1 μM GA3, 300 mg / L timentin, 20 g / L sucrose, 4 g / L plant gel, pH = 6.0, and is replaced every two weeks during the culture process.

[0032] Furthermore, in step 6, the callus induction stage does not only induce callus. At this stage, some callus will further differentiate into adventitious buds, and when they grow to 2 to 3 cm, they can be directly induced to root.

[0033] Furthermore, both step 5 and step 6 are cultured in an RDN type artificial climate chamber at 25° C. in the dark.

[0034] Furthermore, in step 7, the adventitious bud induction medium formula is SH basal medium, 5 μM GA3, 107.4 μM NAA, 300 mg / L timentin, 20 g / L sucrose, 4 g / L plant gel, pH = 5.5, and is replaced every two weeks during the culture process.

[0035] Furthermore, in step 7, the induction of adventitious buds is cultured in an artificial climate chamber, wherein the artificial climate chamber is operated in an alternating manner of 16 h of light / 8 h of darkness, a temperature of 22° C., and a relative humidity of 50%.

[0036] Furthermore, in step 8, the rooting culture is carried out in an RDN type artificial climate chamber, and the artificial climate chamber is alternately operated in a light 8h / dark 16h mode, and the light temperature is controlled at 20°C, the dark temperature is controlled at 18°C, and the relative humidity is controlled at 50%.

[0037] Furthermore, in step 8, the formula of the culture medium SHT is: 10 g / L sucrose, 300 mg / L timentin, and 4 g / L plant gel are added to the SH basic culture medium, and the pH is 5.8-6.0.

[0038] Furthermore, the whole plants obtained in step 8 are tested for transgenic positivity by extracting leaf DNA from the regenerated spinach plants for PCR identification, and the spinach plants that are positive for PCR identification are then tested for GUS staining.

[0039] Furthermore, the genetic transformation efficiency = the number of positive plants identified by PCR / the total number of regenerated plants*100%.

[0040] Furthermore, in step 9, when transplanting, the regenerated plants are taken out of the culture medium and the culture medium remaining at the roots is gently washed off with running water. After transplanting into the soil, the plants are covered with plastic wrap to maintain humidity. After one week, the film is partially opened until the plants adapt to the external environment, and then the plastic wrap can be completely removed.

[0041] Furthermore, in step 9, the transplanting soil includes perlite and peat soil, and the volume ratio of perlite to peat soil is 1:2, and the soil is wetted with SH basic culture medium before transplanting.

[0042] By implementing the above technical solution, the present invention has the following advantages:

[0043] The invention establishes an Agrobacterium-mediated stable genetic transformation system for spinach, which can not only improve the transformation efficiency, but also utilize the system to carry out functional research on key regulatory genes of spinach agronomic traits and cultivate excellent spinach varieties.

[0044] The invention optimizes and selects each step of the spinach genetic transformation system, and the genetic transformation efficiency can reach more than 50%, which far exceeds the genetic transformation efficiency of spinach disclosed in the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 A schematic diagram of the process of the spinach genetic transformation system mediated by Agrobacterium tumefaciens provided by the present invention;

[0046] Figure 2 The effects of Agrobacterium strains and infection solution concentration on callus induction rate and transformation efficiency;

[0047] Figure 3 The statistics of adventitious bud morphology and adventitious bud induction rate formed under different bud induction media;

[0048] Figure 4 PCR identification of GUS gene in transgenic regenerated spinach plants. DETAILED DESCRIPTION

[0049] The technical solutions in the embodiments of the present invention are described clearly and completely below through the accompanying drawings. The described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0050] Example 1

[0051] Screening of callus induction medium for different explants

[0052] Inducing callus is the basis for establishing a genetic transformation system. The type of explant and the combination of different plant growth regulators directly affect the ability of callus formation. Therefore, we first screened the optimal callus induction medium for different explants.

[0053] The specific analysis steps are as follows:

[0054] 1. Prepare spinach materials: The heat-resistant spinach material Sp75 was provided by the Germplasm Resources Development Center of Shanghai Normal University.

[0055] The seeds of Sp75 with full grains were peeled off the seed coat, disinfected with 75% ethanol for 5 minutes, repeated once, washed with sterile water 2-3 times, disinfected with 20% sodium hypochlorite solution for 12 minutes, washed with sterile water 5-6 times, sown in 1 / 2MS medium (2.215g / L M519, 10g / L sucrose, 4g / L plant gel, pH=5.8-6.0) and cultured under long-day conditions for 5 days (24°C / 18°C, 16h light / 8h dark, relative humidity of 50%, light intensity of 16000LX). The sterile seedlings with good growth were transferred to the tissue culture bottle containing 1 / 2MS medium in the clean bench and continued to be cultured for 30-35 days, at which time the spinach stems were in the continuous elongation stage.

[0056] 2. Prepare explants: Take sterile spinach seedlings cultured for 30 to 35 days, cut their petioles into small sections (about 1 cm), stems into stem sections (about 2 mm), and leaves into small pieces (about 1 cm × 1 cm) with a scalpel in an ultra-clean workbench, and place them separately on 1 / 2 MS culture medium (2.215 g / L M519, 10 g / L sucrose, 4 g / L phytagel, pH = 5.8-6.0) for later use.

[0057] 3. Callus induction: petiole, leaf blade and stem explants were placed in S1 medium (SH basic medium (formula as shown in Table 1), 1.5 mg / L 6-BA, 0.1 mg / L NAA, 20 g / L sucrose, 4 g / L phytagel, pH = 6.0), S2 medium (SH basic medium, 1.5 mg / L 6-BA, 0.3 mg / L NAA, 20 g / L sucrose, 4 g / L phytagel, pH = 6.0), S3 medium (SH basic medium, 1.5 mg / L 6-BA, 0.5 mg / L NAA, 20 g / L sucrose, 4 g / L phytagel, pH = 6.0), S4 medium (SH basic medium, 3.5 mg / L 6-BA, 0.1 mg / L NAA, 20 g / L sucrose, 4 g / L phytagel, pH = 6.0), S5 medium (SH basic medium, 3.5 mg / L Callus was induced on S6 medium (SH basal medium, 3.5 mg / L 6-BA, 0.5 mg / L NAA, 20 g / L sucrose, 4 g / L phytagel, pH = 6.0) and cultured in the dark at 25°C. The culture medium was replaced every two weeks during this process.

[0058] 4. Statistics of callus induction rate: After 4 weeks, the induction rates of the three explants in each callus induction medium were counted. Callus induction rate (%) = number of explants induced with callus / total number of explants * 100%. The statistical results are shown in Table 2. The callus induction rates of explant leaves and petioles on S2 medium were the highest, 81.11% and 85.55% respectively; the callus induction rate of explant stems on S6 medium was the highest, 91.11%. Compared with the three explants, the callus induction rate of stems was the highest, which determined that the explant stems were the best explants for inducing callus, so the explant stems were selected for subsequent research.

[0059] Table 1 SH basal medium formula

[0060]

[0061] Table 2 Callus induction rates of three explants on different callus induction media

[0062]

[0063] Example 2

[0064] Effects of Agrobacterium strains and infection solution concentration on callus induction and infection efficiency

[0065] The specific steps are as follows:

[0066] 1. Prepare spinach materials: Peel off the seed coat of the full-grained Sp75 seeds, sterilize with 75% ethanol for 5 minutes, repeat once, wash with sterile water 2-3 times, sterilize with 20% sodium hypochlorite solution for 12 minutes, wash with sterile water 5-6 times, sow in 1 / 2MS medium (2.215g / L M519, 10g / L sucrose, 4g / L plant gel, pH=5.8-6.0) and culture under long-day conditions for 5 days (24°C / 18°C, 16h light / 8h dark, relative humidity of 50%, light intensity of 16000LX). Transfer the sterile seedlings with good growth to the tissue culture bottle containing 1 / 2MS medium in the clean bench and continue to culture for 30-35 days.

[0067] 2. Prepare explants: Take sterile spinach seedlings cultured for 30 to 35 days, cut stem segments (about 2 mm) with a scalpel in an ultra-clean workbench, and place them on co-cultivation culture medium (SH basal medium, 100 μM acetosyringone, 20 g / L sucrose, 4 g / L phytagel, pH = 6.0) for later use.

[0068] 3. Prepare Agrobacterium infection solution:

[0069] (1) The plant expression vector pBI121-GUS was transformed into EHA105 and GV3101 (pSoup-p19) Agrobacterium by heat shock method, and the transformed Agrobacterium was stored in 50% (v / v) glycerol and placed in a -80°C refrigerator. The frozen Agrobacterium was streaked on a YEP solid plate (25 μg / mL rifampicin + 50 μg / mL kanamycin) for three zones and cultured at 28°C for 48 h to obtain a single positive colony.

[0070] (2) Day 1: Agrobacterium activation. A single positive colony obtained in the above step was inoculated into 1 mL of YEP liquid medium (25 μg / mL rifampicin + 50 μg / mL kanamycin) and cultured in a shaking incubator at 28°C and 220 rpm for 12 to 18 h until the OD reached 0. 600 =1.0~1.2.

[0071] (3) Day 2: Inoculate 1 mL of the Agrobacterium culture medium in the above step into 30 mL of YEP liquid medium and culture in a shaking incubator at 28°C and 200 rpm for nearly 4 h until the logarithmic growth phase (OD 600 =0.6~0.8). Centrifuge at 5000 rpm for 7 min at room temperature and discard the supernatant. Then, resuspend the collected bacteria in infection solution (SH basic medium, 20 g / L sucrose, 100 μM acetosyringone, pH=5.8~6.0) and dilute to obtain the initial infection solution:

[0072] If the final infection fluid OD 600=0.1, then the initial infection solution OD 600 Dilute to about 0.08;

[0073] If the final infection fluid OD 600 =0.2, then the initial infection fluid OD 600 Dilute to about 0.18;

[0074] If the final infection fluid OD 600 =0.3, then the initial infection fluid OD 600 Dilute to about 0.28;

[0075] After the preliminary infection solution was left to stand for 2 hours, the final infection solution OD was adjusted. 600 The values ​​are 0.1, 0.2, 0.3, ready to infect.

[0076] 4. Infection: The stem segments were placed in the corresponding OD of GV3101 (pSoup-p19) and EHA105 Agrobacterium strains. 600 The explants were placed in the infection solution with a value of 1:1 and 1:1, and oscillated at room temperature for 30 minutes using a gyratory oscillator. Then, the stem segments were removed with tweezers, placed on sterile filter paper to absorb the residual liquid in the explants, and the surface moisture was blown dry (about 10 minutes) to ensure that there was no obvious residual bacterial solution (note that if the drying time is too long, it will damage the explants and affect the callus induction rate; if the infection solution remains on the surface of the explants, it will cause the Agrobacterium to be unable to inhibit and erode the explants, further affecting the callus induction effect). Subsequently, the explants were placed on the co-culture medium and cultured in the dark at 25°C for 2 days. The number of explants under each treatment was ≥60.

[0077] 5. Callus induction: Wash the co-cultivated stem explants with sterile water containing 600 mg / L timentin, place them on sterile filter paper to dry the surface moisture, the degree of drying is as described in 4, and place the stems randomly and orderly on callus induction medium S6T (SH basal medium, 3.5 mg / L 6-BA, 0.5 mg / L NAA, 300 mg / L timentin, 20 g / L sucrose, 4 g / L phytagel, pH = 6.0) and continue to culture at 25°C in the dark. During this period, the culture medium was replaced every two weeks.

[0078] 6. Statistics of callus induction rate: After culturing the infected stem explants for 4 weeks, the callus induction rate was calculated. Callus induction rate (%) = number of explants induced with callus / total number of infected explants * 100%. The results are as follows Figure 2 As shown, for Agrobacterium EHA105, when the infection solution OD 600 =0.1, the callus induction rate was the highest, 37.22%, but its callus transformation efficiency was the lowest, 24.44%. 600=0.3, the callus induction rate was the lowest, 16.67%, while the callus transformation efficiency was the highest, 46.67%. For Agrobacterium GV3101 (pSoup-p19), when the infection solution OD 600 =0.2, the callus induction rate was the highest, 81.67%, and the callus transformation efficiency was 52.22%; 600 =0.3, although the callus induction rate was the lowest, only 44.44%, but the callus transformation efficiency was the highest, 82.22%. Under the same infection solution concentration, the callus induction rate and transformation efficiency of Agrobacterium GV3101 (pSoup-p19) were significantly higher than those of EHA105, which determined that Agrobacterium GV3101 (pSoup-p19) was more suitable for the infection of spinach explants. Based on the callus transformation efficiency, the optimal infection concentration of GV3101 (pSoup-p19) was preliminarily determined to be OD 600 =0.3.

[0079] 7. GUS staining: In order to further determine the appropriate Agrobacterium strain and infection solution concentration, the callus tissue was immersed in a working solution (1M Na2HPO4·12H2O, 1M NaH2PO4·2H2O, 50mM K3Fe(CN)6, 50mM K4Fe(CN)6·3H2O, 0.5M EDTA·Na2, 10% Triton X-100 and 100μg / mL X-Gluc) and vacuumed for 3h, then reacted at 37°C in the dark for 36h, and the transformation efficiency of Agrobacterium was determined by the degree of GUS staining. The GUS staining effect is shown in Figure 2. Figure 2 As shown, when the concentration of Agrobacterium GV3101 (pSoup-p19) infection solution OD 600 =0.3, the GUS staining effect of callus tissue was the strongest, so the optimal infection concentration of GV3101 (pSoup-p19) was determined to be OD 600 =0.3.

[0080] Example 3

[0081] It can be seen from Example 2 that Agrobacterium infection will affect callus formation to a certain extent. The higher the concentration of the infection solution, the lower the induction rate of callus.

[0082] Gibberellic acid GA3 can improve the embryonic quality of callus tissue. Increasing the concentration of auxin analog NAA within a certain range can quickly induce callus tissue from explants, thereby improving the state of callus tissue.

[0083] The optimal explant stem determined in Example 1, the most suitable strain GV3101 (pSoup-p19) determined in Example 2, and the concentration OD of the Agrobacterium infection solution were600 =0.3, the effects of NAA and GA3 on callus induction of explants were studied, and the callus induction medium was further optimized to improve the callus induction rate after infection.

[0084] The specific analysis method is as follows:

[0085] 1. Prepare spinach materials: peel off the seed coat of the full-grained Sp75 seeds, disinfect with 75% ethanol for 5 minutes, repeat once, wash with sterile water 2-3 times, disinfect with 20% sodium hypochlorite solution for 12 minutes, wash with sterile water 5-6 times, sow in 1 / 2MS medium (2.215g / L M519, 10g / L sucrose, 4g / L plant gel, pH=5.8-6.0) and culture for 5 days under long daylight (24℃ / 18℃, 16h light / 8h dark, relative humidity of 50%, light intensity of 16000LX), transplant the well-growing sterile seedlings into tissue culture bottles containing 1 / 2MS medium in the clean bench and continue to culture for 30-35 days.

[0086] 2. Prepare explants: Take sterile spinach seedlings cultured for 30 to 35 days, cut stem segments (about 2 mm) with a scalpel on a clean bench, and place them on co-cultivation medium (SH basal medium, 100 μM acetosyringone, 20 g / L sucrose, 4 g / L phytagel, pH = 6.0) for later use.

[0087] 3. Prepare Agrobacterium infection solution:

[0088] (1) The plant expression vector pBI121-GUS was transformed into GV3101 (pSoup-p19) Agrobacterium by heat shock method, and the transformed Agrobacterium was stored in 50% (v / v) glycerol and placed in a -80°C refrigerator. The frozen Agrobacterium was streaked on a YEP solid plate (25 μg / mL rifampicin + 50 μg / mL kanamycin) for 48 h at 28°C to obtain a single positive colony.

[0089] (2) Day 1: Agrobacterium activation. A single positive colony obtained in the above step was inoculated into 1 mL of YEP liquid medium (25 μg / mL rifampicin + 50 μg / mL kanamycin) and cultured in a shaking incubator at 28°C and 220 rpm for 12 to 18 h until the OD 600 =1.0~1.2.

[0090] (3) Day 2: Transfer 1 mL of the Agrobacterium culture medium from the above step into 30 mL of YEP liquid medium and culture in a shaking incubator at 28°C and 200 rpm for nearly 4 h until the logarithmic growth phase (OD 600=0.6-0.8). Centrifuge at 5000 rpm for 7 min at room temperature and discard the supernatant. Then, resuspend the collected bacteria and dilute with infection solution (SH basal medium, 20 g / L sucrose, 100 μM acetosyringone, pH = 5.8-6.0) to obtain a preliminary infection solution OD 600 =0.28, let it stand for 2 hours, and adjust the final infection concentration OD 600 =0.3, ready to infect.

[0091] 4. Infection: Immerse the explant stems in OD 200 μL of the expression vector pBI121-GUS. 600 =0.3 Agrobacterium infection solution, oscillate for 30 min at room temperature using a gyratory oscillator, place on sterile filter paper to dry surface moisture, and place in order on co-culture medium (SH basal medium, 100 μM acetosyringone, 20 g / L sucrose, 4 g / L plant gel, pH=6.0) and culture at 25°C in the dark for 2 days.

[0092] 5. Callus induction: The explants were washed with sterile water containing 600 mg / L timentin, placed on sterile filter paper to dry the surface moisture, and randomly placed on S6T medium (SH medium, 3.5 mg / L 6-BA, 0.5 mg / L NAA, 300 mg / L timentin, 20 g / L sucrose, 4 g / L phytagel, pH = 6.0), N1 medium (SH basal medium, 3.5 mg / L 6-BA, 0.5 mg / L NAA, 0.1 μM GA3, 300 mg / L timentin, 20 g / L sucrose, 4 g / L phytagel, pH = 6.0), N2 medium (SH basal medium, 3.5 mg / L 6-BA, 0.7 mg / L NAA, 0.1 μM GA3, 300 mg / L timentin, 20 g / L sucrose, 4 g / L phytagel, pH = 6.0) and N3 medium (SH basal medium, 3.5 mg / L 6-BA, 0.9 mg / L NAA, 0.1 μM GA3, 300 mg / L timentin, 20 g / L sucrose, 4 g / L phytagel, pH = 6.0), continued to culture in the dark at 25°C, the number of explants in each treatment was ≥ 60, repeated three times. The medium was changed every two weeks, and statistics were collected after four weeks.

[0093] The statistical results are shown in Table 3. The callus induction rate of the explant stems in N1, N2, and N3 mediums all reached more than 90%, which was significantly higher than the callus induction rate in S6T medium. In addition, we also observed that on N1, N2, and N3 mediums, the callus induction of the explant stems was accompanied by the generation of preliminary adventitious buds, which could significantly shorten the genetic transformation cycle. Among them, the induction rate of preliminary adventitious buds of the explant stems on N1 medium was the highest at 11.61%.

[0094] Table 3 Effects of different plant growth regulator ratios on stem callus induction rate and initial adventitious bud induction rate

[0095]

[0096] Based on the results of Examples 1-3, under the conditions of this experiment, a method was established with stems as explants, GV3101 (pSoup-p19) as Agrobacterium strain, and infection fluid OD 600 The genetic transformation system of spinach Sp75 was 0.3 and the callus induction medium was N1 medium.

[0097] Example 4

[0098] Effects of different NAA concentrations on adventitious bud induction from callus

[0099] 1 to 4 are the same as the process of Example 3.

[0100] 5. Callus induction: Wash the co-cultivated explants with sterile water containing 600 mg / L timentin, place them on sterile filter paper to dry the surface moisture, and place them randomly and orderly on callus induction N1 medium (SH basal medium, 3.5 mg / L 6-BA, 0.5 mg / L NAA, 0.1 μM GA3, 300 mg / L timentin, 20 g / L sucrose, 4 g / L phytogel, pH = 6.0), continue to culture in the dark at 25°C, and replace the medium every two weeks.

[0101] 6. Adventitious bud induction: The callus cultured for 4 weeks was peeled off from the explants and placed in adventitious bud induction medium Gm (SH basic medium, 5 μM GA3, 20 μM NAA, 300 mg / L timentin, 20 g / L sucrose, 4 g / L plant gel, pH = 5.5) and G1 (SH basic medium, 5 μM GA3, 107.4 μM NAA, 300 mg / L timentin, 20 g / L sucrose, 4 g / L plant gel, pH = 5.5), respectively, and cultured in an artificial climate chamber with 16 h of light / 8 h of darkness, a temperature of 22° C., and a relative humidity of 50%. After one week, adventitious buds began to appear in the callus, and the medium was replaced every two weeks. The number of callus in each treatment was ≥ 60.

[0102] 7. Statistics of adventitious bud induction rate and morphological observation: After 4 weeks of callus culture, the adventitious bud induction rate was calculated. Adventitious bud induction rate (%) = number of induced adventitious buds / number of callus * 100%. The morphology of adventitious buds induced on the two bud induction media is as follows: Figure 3As shown in A, the adventitious buds induced on Gm medium were deformed and grew in clusters; the adventitious buds induced on G1 medium had good morphology and grew individually. Figure 3 As shown in B, the adventitious bud induction rate of G1 medium (23.28%) was higher than that of Gm medium (19.05%); in addition, the time for inducing adventitious buds in the two mediums was different, Gm medium induced adventitious buds in about three weeks, while G1 medium could form adventitious buds in about one week;

[0103] According to the time and state of inducing adventitious buds, G1 medium was determined to be the best medium for inducing adventitious buds.

[0104] Example 5

[0105] Screening of rooting medium and identification of regenerated plants of spinach

[0106] The analysis method is as follows:

[0107] 1 to 5 are the same as the process of Example 4.

[0108] 6. Adventitious bud induction: The callus cultured for 4 weeks was peeled off from the explant, placed in adventitious bud induction medium G1 (SH basic medium, 5μM GA3, 107.4μM NAA, 300mg / L timentin, 20g / L sucrose, 4g / L phytogel, pH=5.5), and cultured in an artificial climate chamber with 16h light / 8h dark, 22°C, and 50% relative humidity. After one week, morphologically differentiated adventitious buds began to appear in the callus, and the culture medium was replaced every two weeks. The number of callus in each treatment was ≥60.

[0109] 7. Rooting induction of adventitious buds: When the adventitious buds grow to 2-3 cm, they are transferred to tissue culture bottles containing rooting medium 1 / 2MS (2.215 g / L M519, 10 g / L sucrose, 300 mg / L Timentin, 4 g / L plant gel, pH = 5.8-6.0) and medium SHT (SH basal medium, 10 g / L sucrose, 300 mg / L Timentin, 4 g / L plant gel, pH = 5.8-6.0) to induce rooting. The culture conditions are adjusted from the original long day to short day (20°C / 18°C, 8 h light / 16 h dark, light intensity 16000 LX). Rooting can be induced by culturing in the rooting medium for 15-45 days to obtain regenerated plants.

[0110] 8. Rooting rate statistics: The rooting rate of adventitious buds was counted after the adventitious buds were cultured in the rooting induction medium for 2 months. Rooting rate of adventitious buds (%) = number of rooted adventitious buds / total number of adventitious buds*100%. The statistical results of the rooting rate of adventitious buds are shown in Table 4. The rooting efficiency of the rooting SHT medium is 48.84%, which is significantly higher than the rooting efficiency of 1 / 2MS medium. Therefore, SHT medium is used as the best adventitious bud induction rooting medium.

[0111] Table 4 Effects of different rooting media on rooting of adventitious buds

[0112]

[0113] 9. Transplanting soil: After the green adventitious buds grow roots (3 to 5 roots) in the rooting medium, remove the plant from the medium and gently wash the residual medium on the roots with running water. After washing, transplant it to a sterile mixed soil containing peat soil and perlite (2:1) that has been wetted with SH basic medium in advance, cover with plastic wrap to maintain humidity, and partially open the film after one week. The plastic wrap can be completely removed until the plant adapts to the external environment. After transplanting, the culture conditions are restored to long-day conditions.

[0114] 10.PCR identification:

[0115] (1) The CTAB method was used to extract leaf genomic DNA.

[0116] The specific steps include:

[0117] ① Take 0.1g of leaves from regenerated spinach plants and grind them;

[0118] ② Add 600 μL of CTAB solution to each sample and place in a 65°C oven for 30 min (invert every 10 min);

[0119] ③ Add 650 μL of chloroform precooled at -20°C, shake vigorously and let stand for 2 minutes, and centrifuge at 12000 rpm at room temperature for 10 minutes;

[0120] ④ Take 500 μL of supernatant, add 1 mL of anhydrous ethanol precooled at -20°C, and place in a -20°C refrigerator for 1 hour;

[0121] ⑤ Centrifuge at 12000rpm for 2min at room temperature, discard the supernatant, and centrifuge at 12000rpm for 2min at room temperature.

[0122] Aspirate the residual liquid, and there should be a white DNA precipitate;

[0123] ⑥ Blow dry the residual water in the EP tube and add 20-50 μL ddH2O to dissolve it again.

[0124] (2) PCR identification

[0125] The extracted leaf genomic DNA was used as a template and specific primers (GUS-F: 5'-AACCGACGAACTAGTATGTACC-3', GUS-R: 5'-TCACACGTGATGGTGATGGTG-3') were used to amplify the GUS reporter gene. The band sizes were compared after agarose gel electrophoresis to determine the transformed positive plants and calculate the genetic transformation efficiency of spinach. Genetic transformation efficiency (%) = number of positive plants identified by PCR / total number of regenerated plants * 100%. Some PCR results are shown in Figure 2. Figure 4 As shown, according to the PCR results, the transformation efficiency of this genetic transformation system is 50.83%.

[0126] In summary, the present invention mainly optimizes the conditions in four aspects, namely, the explant type, Agrobacterium strain, infection solution concentration, and the effects of different plant growth regulator combinations on various stages of plant tissue culture in the spinach genetic transformation system, so as to improve the transformation efficiency of the genetic transformation system, and verifies the feasibility and stability of the genetic transformation system through GUS staining and PCR identification. Therefore, the present invention establishes an Agrobacterium-mediated stable genetic transformation system for spinach, laying a foundation for subsequent research on spinach gene function and breeding of excellent spinach varieties.

[0127] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for genetic transformation of spinach mediated by Agrobacterium tumefaciens, characterized in that: Spinach stem segments were used as explants, pBI121-GUS was used as a plant expression vector, and Agrobacterium GV3101 (pSoup-p19) was used as a recipient Agrobacterium.

2. The method for genetic transformation of spinach mediated by Agrobacterium tumefaciens according to claim 1, characterized in that: The steps include: Step 1, obtaining stem explants: selecting spinach seeds with full grains, peeling off the seed coat, and sowing them on a culture medium plate after disinfection. The sterile seedlings with good growth are transferred to a tissue culture bottle and cultured for 30 to 35 days. The stem segments are cut and used as explants for subsequent infection. Step 2, constructing a plant expression vector: constructing the target gene into the plant expression vector pBI121-GUS; Step 3, prepare Agrobacterium infection solution: transform the plant expression vector containing the target gene into Agrobacterium GV3101 (pSoup-p19), and prepare Agrobacterium infection solution; Step 4, infecting explants: immerse the stem segments in step 1 in the Agrobacterium infection solution in step 3, and culture at room temperature with shaking; Step 5, co-cultivation: placing the explants treated in step 4 on sterile filter paper to dry the surface moisture, transferring them to co-cultivation medium, and co-cultivating them; Step 6, callus induction: rinse the explants in step 5 with sterile water, place them on sterile filter paper to dry the surface moisture, transfer them to callus induction medium, and culture them; Step 7, adventitious bud induction: peeling the callus from the explant, transferring it to an adventitious bud induction medium, and inducing adventitious buds; Step 8, rooting induction: when the adventitious buds in step 7 grow to 2-3 cm, they are transferred to the culture medium SHT to induce rooting, obtain complete plants, and verify transgenic positive plants; Step 9, transplanting: Transplant the transgenic positive plants in step 8 into the soil, cover with film for one week, and remove the film after the plants adapt to the external environment. After transplanting, the culture conditions are restored to long-day conditions.

3. The method for genetic transformation of spinach mediated by Agrobacterium tumefaciens according to claim 2, characterized in that: The callus induction medium is supplemented with 6-BA, NAA and GA3, and the adventitious bud induction medium is supplemented with NAA and GA3.

4. The method for genetic transformation of spinach mediated by Agrobacterium tumefaciens according to claim 3, characterized in that: The callus induction medium formula is SH basic medium, 3.5 mg / L 6-BA, 0.5 mg / L NAA, 0.1 μM GA3, 300 mg / L timentin, 20 g / L sucrose, and 4 g / L plant gel. pH = 6.

0.

5. The method for genetic transformation of spinach mediated by Agrobacterium tumefaciens according to claim 3, characterized in that: The adventitious bud induction culture medium formula is SH basic culture medium, 5 μM GA3, 107.4 μM NAA, 300 mg / L timentin, 20 g / L sucrose, 4 g / L plant gel, and pH=5.

5.

6. The method for genetic transformation of spinach mediated by Agrobacterium tumefaciens according to claim 4, characterized in that: The culture medium formula of the co-cultivation is SH basic culture medium, 100 μM acetosyringone, 20 g / L sucrose, 4 g / L plant gel, and pH=6.

0.

7. The method for genetic transformation of spinach mediated by Agrobacterium tumefaciens according to claim 4, characterized in that: In step 6, the sterile water is added with 600 mg / L of timentin.

8. The method for genetic transformation of spinach mediated by Agrobacterium tumefaciens according to claim 2, characterized in that: The OD of the Agrobacterium infection solution 600 is 0.

3.

9. The method for genetic transformation of spinach mediated by Agrobacterium tumefaciens according to claim 8, characterized in that: Step 3 specifically includes the following steps: S1. The plant expression vector containing the target gene was transformed into Agrobacterium GV3101 (pSoup-p19) by heat shock method, and a single positive colony was obtained by culture; S2. Cultivate the single positive colony and wait until the OD of the bacterial solution reaches 600 When the pH value is 1.0-1.2, the culture is stopped to obtain an activated bacterial suspension; S3. Take 1 ml of the activated bacterial suspension described in S2 for mass culture. 600 When the concentration is 0.6-0.8, collect the bacteria by centrifugation; S4. Resuspend the bacteria in the infection solution and dilute to OD 600 is about 0.28, and the preliminary Agrobacterium infection solution is obtained; S5. Place the initial Agrobacterium infection solution in a 28°C constant temperature incubator in the dark for 2 hours and adjust the infection solution OD 600 is 0.3, and the final Agrobacterium infection solution is obtained.

10. The method for genetic transformation of spinach mediated by Agrobacterium tumefaciens according to claim 9, characterized in that: The infection solution includes SH basic culture medium, 20 g / L sucrose and 100 μM acetosyringone, and the pH value is 5.8-6.0.

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