Water spinach tissue culture medium and application thereof in water spinach transgenosis

Abstract

The invention belongs to the technical field of plant transgenosis and tissue culture, and particularly relates to a water spinach tissue culture medium and application thereof in water spinach transgenosis. The culture medium suitable for water spinach transgenosis or tissue culture is formed by adjusting the hormone type and proportion in the water spinach tissue culture culture medium. On the basis, the water spinach transgenosis method is established by limiting the conditions of water spinach transgenosis, such as explant selection, co-culture time and infection time, and the transgenosis efficiency of water spinach is improved. The successful implementation of the water spinach transgenic method provides a new way for genetic improvement of water spinach, promotes industrial development of water spinach, and meets the requirements of the market for high-quality water spinach.

Description

Technical Field

[0001] The invention belongs to the technical field of plant transgenic and tissue culture, and particularly relates to a water spinach tissue culture medium and application thereof in water spinach transgenic. Background Art

[0002] Water spinach (Ipomoea aquatica) is loved by consumers for its rich nutrition and delicious taste. Water spinach is particularly important in Southeast Asia and southern China, and is one of the indispensable vegetables in the daily diet of local residents. However, the breeding and improvement of water spinach faces many challenges, including poor stress resistance and difficulty in controlling pests and diseases. These factors limit the yield and quality of water spinach and affect its market competitiveness.

[0003] In recent years, with the development of molecular biology technology, the application of transgenic technology in plant breeding has gradually become a hot spot for innovation. Through genetic engineering, foreign genes can be effectively introduced to improve the stress resistance, disease and pest resistance and nutritional value of plants. Although some transgenic technologies have been successful in other vegetable crops, efficient transgenic technology for water spinach is still relatively lagging behind, and there is an urgent need to develop more efficient and reliable transgenic methods. Summary of the invention

[0004] The purpose of the present invention is to provide a water spinach tissue culture medium and application thereof in water spinach transgenic. Based on the water spinach tissue culture medium, water spinach transgenic can be achieved and the transgenic efficiency of water spinach can be improved.

[0005] The present invention provides a water spinach tissue culture medium, comprising one or more of a co-culture medium, a callus induction medium, an adventitious bud induction medium and a rooting medium;

[0006] The co-culture medium is based on MS culture medium and further includes the following components in concentrations: 1.8-2.0 mg / L kinetin (KT), 0.2-0.4 mg / L naphthaleneacetic acid (NAA), 150-180 μmol / L acetosyringone (AS), 20-25 g / L glucose and 6-8 g / L agar;

[0007] The callus induction medium is based on MS medium and also includes the following components in concentrations: 1.8-2.0 mg / L kinetin, 0.2-0.4 mg / L naphthaleneacetic acid, 0.5-1.0 mg / L silver nitrate, 150-180 μmol / L acetosyringone, 250-260 mg / L carbenicillin disodium, 20-25 g / L glucose and 6-8 g / L agar;

[0008] The adventitious bud induction medium is based on MS medium and also includes the following components in concentrations: 1.8-2.0 mg / L kinetin, 0.2-0.4 mg / L naphthaleneacetic acid, 0.5-1.0 mg / L silver nitrate, 250-260 mg / L carbenicillin disodium, 40-50 mg / L hygromycin B, 20-25 g / L glucose and 6-8 g / L agar;

[0009] The rooting culture medium uses 1 / 2MS culture medium as the basic culture medium and further comprises components in the following concentrations: 0.8-1.0 mg / L naphthaleneacetic acid, 20-25 g / L glucose, 6-8 g / L agar and 250-260 mg / L carbenicillin disodium.

[0010] Preferably, the water spinach tissue culture medium also includes MS solid culture medium and / or LB liquid culture medium.

[0011] The present invention also provides the use of the kiwifruit tissue culture medium described in the above technical solution in kiwifruit transgenic and / or kiwifruit tissue culture.

[0012] The present invention also provides a method for genetically modifying kiwifruit based on the kiwifruit tissue culture medium described in the above technical solution, comprising the following steps:

[0013] The sterilized water spinach seeds are germinated and cultured to obtain sterile water spinach seedlings;

[0014] Infecting the petioles of the sterile water spinach seedlings with Agrobacterium containing the target gene to obtain petioles of water spinach infected with Agrobacterium;

[0015] inoculating the water spinach petiole infected by the Agrobacterium into a co-culture medium for co-cultivation to obtain co-cultivated water spinach petiole;

[0016] inoculating the co-cultured water spinach petioles into a callus induction medium for callus induction culture to obtain water spinach callus;

[0017] Inoculating the water spinach callus into an adventitious bud induction medium for bud induction culture to obtain water spinach adventitious buds;

[0018] The water spinach adventitious buds are inoculated into a rooting medium for rooting culture to obtain water spinach transgenic seedlings.

[0019] Preferably, the length of the petiole of the sterile water spinach seedling is 0.3 to 0.6 cm.

[0020] Preferably, the infection method comprises: soaking the water spinach explants with an Agrobacterium infection solution containing the target gene; the OD of the Agrobacterium infection solution is 600 is 0.5-0.7; and the soaking time is 35-40 minutes.

[0021] Preferably, the co-cultivation is dark culture at a temperature of 25 to 28° C. for 3 to 4 days;

[0022] The callus induction culture is dark culture at a temperature of 25 to 28° C. for 20 to 25 days;

[0023] The bud induction culture temperature is 25-28°C, the light intensity is 2000-2500lx, and the light duration is 12-14h / day; the bud induction culture ends when the water spinach adventitious buds grow to 3-5cm;

[0024] The light intensity of the rooting culture is 2000-2500 lx, and the light duration is 12-14 h / day.

[0025] Preferably, the transgenic water spinach seedlings contain 5 to 6 roots, and the root length is 3 to 5 cm.

[0026] Preferably, the water spinach transgenic method further comprises hardening and transplanting the water spinach transgenic seedlings to obtain water spinach transgenic plants.

[0027] Preferably, the method of hardening the seedlings comprises: irradiating the transgenic water spinach seedlings under scattered light for 4 to 5 days;

[0028] The transplanting culture comprises: planting the hardened water spinach transgenic seedlings in a sterilized plant matrix, and culturing them under the condition of 25-30° C. to obtain the water spinach transgenic plants.

[0029] Beneficial effects:

[0030] The present invention provides a water spinach tissue culture medium, including one or more of a co-culture medium, a callus induction medium, an adventitious bud induction medium and a rooting medium. The present invention forms a culture medium suitable for water spinach transgenic or tissue culture by adjusting the type and ratio of hormones in the water spinach tissue culture medium. On this basis, the present invention establishes a water spinach transgenic method by limiting the conditions for water spinach transgenic, such as explant selection, and improves the transgenic efficiency of water spinach. The successful implementation of the water spinach transgenic method of the present invention provides a new way for the genetic improvement of water spinach, promotes the industrial development of water spinach, and meets the market demand for high-quality water spinach. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0032] Figure 1 The figure is a result of callus induction, resistance bud induction and rooting culture phenotype after Agrobacterium infection of water spinach petiole in Example 2;

[0033] Figure 2 This is a diagram showing the PCR detection results of the transgenic water spinach VvPL1 gene in Example 2;

[0034] Figure 3 The effects of different Agrobacterium infection solution concentrations on the differentiation rate of adventitious buds of Water spinach in Examples 13 to 14 and Comparative Examples 42 to 45 are shown;

[0035] Figure 4 The effects of different Agrobacterium infection times on the differentiation rate of adventitious buds of Water spinach in Examples 15 to 16 and Comparative Examples 46 to 49 are shown;

[0036] Figure 5 The effect of acetosyringone concentration on the differentiation rate of adventitious buds of Water spinach in Example 17 and Comparative Examples 50-52;

[0037] Figure 6 The effects of different co-cultivation times on the differentiation rate of adventitious buds of Water spinach in Example 18 and Comparative Examples 53-55 are shown;

[0038] in, Figures 3 to 6 Different letters indicate significant differences (p<0.05). DETAILED DESCRIPTION

[0039] The present invention provides a water spinach tissue culture medium, comprising one or more of a co-culture medium, a callus induction medium, an adventitious bud induction medium and a rooting medium;

[0040] The co-culture medium uses MS culture medium as a basic culture medium, and further includes the following components in concentrations: 1.8-2.0 mg / L kinetin, 0.2-0.4 mg / L naphthaleneacetic acid, 150-180 μmol / L acetosyringone, 20-25 g / L glucose and 6-8 g / L agar;

[0041] The callus induction medium is based on MS medium and also includes the following components in concentrations: 1.8-2.0 mg / L kinetin, 0.2-0.4 mg / L naphthaleneacetic acid, 0.5-1.0 mg / L silver nitrate, 150-180 μmol / L acetosyringone, 250-260 mg / L carbenicillin disodium, 20-25 g / L glucose and 6-8 g / L agar;

[0042] The adventitious bud induction medium is based on MS medium and also includes the following components in concentrations: 1.8-2.0 mg / L kinetin, 0.2-0.4 mg / L naphthaleneacetic acid, 0.5-1.0 mg / L silver nitrate, 250-260 mg / L carbenicillin disodium, 40-50 mg / L hygromycin B, 20-25 g / L glucose and 6-8 g / L agar;

[0043] The rooting culture medium uses 1 / 2MS culture medium as the basic culture medium and further comprises components in the following concentrations: 0.8-1.0 mg / L naphthaleneacetic acid, 20-25 g / L glucose, 6-8 g / L agar and 250-260 mg / L carbenicillin disodium.

[0044] As an embodiment, the water spinach tissue culture medium of the present invention includes a co-culture medium, a callus induction medium, an adventitious bud induction medium and a rooting medium.

[0045] As an embodiment, the concentration of kinetin in the co-culture medium of the present invention is 1.9 mg / L, the concentration of naphthylacetic acid is 0.3 mg / L, the concentration of acetosyringone is 180 μmol / L, the concentration of glucose is 21-22 g / L, and the concentration of agar is 7 g / L.

[0046] As an embodiment, the concentration of kinetin in the callus induction medium of the present invention is 1.9 mg / L kinetin, the concentration of naphthaleneacetic acid is 0.3 mg / L, the concentration of silver nitrate is 0.7-0.8 mg / L, the concentration of acetosyringone is 180 μmol / L, the concentration of glucose is 22-24 g / L, and the concentration of agar is 7 g / L.

[0047] As an embodiment, the concentration of kinetin in the adventitious bud induction medium of the present invention is 1.9 mg / L, the concentration of naphthaleneacetic acid is 0.3 mg / L, the concentration of silver nitrate is 0.6-0.9 mg / L, the concentration of carbenicillin disodium is 252-258 mg / L, the concentration of hygromycin B is 42-48 mg / L, the concentration of glucose is 22-24 g / L, and the concentration of agar is 7 g / L.

[0048] As an embodiment, the concentration of naphthylacetic acid in the rooting medium of the present invention is 0.9 mg / L, the concentration of glucose is 22-24 g / L, the concentration of agar is 7 g / L, and the concentration of carbenicillin disodium is 254-258 mg / L.

[0049] In the present invention, the kinetin and naphthylacetic acid are beneficial to the differentiation of callus and buds; and acetosyringone can induce the activation of Agrobacterium infection fluid, thereby promoting the integration of exogenous genes, making it easier to transform into plant genomes, and improving the transformation efficiency of plants.

[0050] As an embodiment, the water spinach tissue culture medium of the present invention further comprises MS solid culture medium and / or LB liquid culture medium; the MS solid culture medium can be used for germination culture of water spinach; the LB liquid culture medium can be used for preparation of Agrobacterium infection solution.

[0051] The present invention also provides the use of the water spinach tissue culture medium described in the above technical solution in water spinach transgenic and / or water spinach tissue culture. As an embodiment, the application of the present invention is the use of the water spinach tissue culture medium in water spinach transgenic and water spinach tissue culture.

[0052] The present invention also provides a method for genetically modifying water spinach based on the water spinach tissue culture medium described in the above technical solution, comprising the following steps:

[0053] The sterilized water spinach seeds are germinated and cultured to obtain sterile water spinach seedlings;

[0054] Infecting the petioles of the sterile water spinach seedlings with Agrobacterium containing the target gene to obtain petioles of water spinach infected with Agrobacterium;

[0055] inoculating the water spinach petiole infected by the Agrobacterium into a co-culture medium for co-cultivation to obtain co-cultivated water spinach petiole;

[0056] inoculating the co-cultured water spinach petioles into a callus induction medium for callus induction culture to obtain water spinach callus;

[0057] Inoculating the water spinach callus into an adventitious bud induction medium for bud induction culture to obtain water spinach adventitious buds;

[0058] The water spinach adventitious buds are inoculated into a rooting medium for rooting culture to obtain water spinach transgenic seedlings.

[0059] As an embodiment, the present invention disinfects water spinach seeds to obtain disinfected water spinach seeds. As an embodiment, the water spinach seed disinfection method comprises: soaking the water spinach seeds in warm water for 30 minutes, and then soaking them in clean water for 22 to 24 hours to obtain soaked water spinach seeds; soaking the soaked water spinach seeds in an ethanol solution with a volume concentration of 75% for 1 to 2 minutes, and then soaking the water spinach seeds in a sodium hypochlorite solution with a mass fraction of 3% for 8 to 10 minutes, and rinsing them with sterile water for 6 to 8 times to obtain the disinfected water spinach seeds.

[0060] After obtaining the sterilized water spinach seeds, the present invention performs germination culture on the sterilized water spinach seeds to obtain sterile water spinach seedlings. As an embodiment, the temperature of the germination culture is 25-28°C; as another embodiment, the temperature of the germination culture is 26-27°C. As an embodiment, the light intensity of the germination culture is 2000-2500lx, and the light time is 12-14h / d; as another embodiment, the light intensity of the germination culture is 2200-2400lx, and the light time is 13h / d. As an embodiment, when the sterile water spinach seedlings grow to 6-8cm, the germination culture is stopped.

[0061] After obtaining the sterile water spinach seedlings, the present invention infects the petioles of the sterile water spinach seedlings with Agrobacterium containing the target gene to obtain the water spinach petioles infected with Agrobacterium. As an embodiment, the infection method comprises: soaking the water spinach explants with an Agrobacterium infection solution containing the target gene. As an embodiment, the OD of the Agrobacterium infection solution is 600 is 0.5-0.7. As one embodiment, the soaking time is 35-40 minutes; as another embodiment, the soaking time is 36-38 minutes. As one embodiment, the length of the petiole of the sterile water spinach seedlings can be 0.3-0.6 cm; as another embodiment, the length of the petiole of the sterile water spinach seedlings can be 0.4-0.5 cm. The present invention does not specifically limit the type of the target gene in the Agrobacterium infection solution containing the target gene, and it can be conventionally selected according to needs. The present invention does not specifically limit the preparation method of the Agrobacterium infection solution, and the conventional preparation method of the Agrobacterium infection solution in the field can be used.

[0062] After obtaining the petiole of water spinach infected by Agrobacterium, the present invention inoculates the petiole of water spinach infected by Agrobacterium into a co-culture medium for co-culture to obtain the co-cultured petiole of water spinach. As an embodiment, the co-culture of the present invention is dark culture at a temperature of 25 to 28° C. for 3 to 4 days. As another embodiment, the temperature of the co-culture is 26 to 27° C.

[0063] After obtaining the co-cultured water spinach petiole, the present invention inoculates the co-cultured water spinach petiole into a callus induction culture medium for callus induction culture to obtain water spinach callus. As an embodiment, the callus induction culture of the present invention is dark culture at a temperature of 25 to 28° C. for 20 to 25 days; as another embodiment, the callus induction culture is at a temperature of 26 to 27° C. for 22 to 24 days.

[0064] After obtaining the water spinach callus, the present invention inoculates the water spinach callus into an adventitious bud induction medium for bud induction culture to obtain water spinach adventitious buds. As an embodiment, the temperature of the bud induction culture of the present invention is 25-28°C, the light intensity is 2000-2500lx, and the light duration is 12-14h / day; as another embodiment, the temperature of the bud induction culture is 26-27°C, the light intensity is 2200-2400lx, and the light duration is 13h / day. As an embodiment, the bud induction culture ends when the water spinach adventitious buds grow to 3-5cm.

[0065] After obtaining the water spinach adventitious buds, the present invention inoculates the water spinach adventitious buds into a rooting medium for rooting culture to obtain water spinach transgenic seedlings. As an embodiment, the light intensity of the rooting culture is 2000-2500 lx, and the light duration is 12-14 h / day; as another embodiment, the light intensity is 2200-2400 lx, and the light duration is 13 h / day. As an embodiment, the water spinach transgenic seedlings contain 5-6 roots, and the root length is 3-5 cm.

[0066] After obtaining the transgenic water spinach seedlings, as an embodiment, the present invention hardens and transplants the transgenic water spinach seedlings to obtain transgenic water spinach plants. As an embodiment, the hardening method is: irradiating the transgenic water spinach seedlings under scattered light for 4 to 5 days. As an embodiment, the source and type of the scattered light can be sunlight. As an embodiment, the transplanting culture of the present invention includes: planting the transgenic water spinach seedlings after hardening in a sterilized plant matrix, culturing at 25 to 30° C., and obtaining the transgenic water spinach plants. The present invention does not specifically limit the type of the plant matrix, and a conventional plant matrix in the art can be used.

[0067] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0068] Example 1

[0069] A water spinach tissue culture medium, which consists of a co-culture medium, a callus induction medium, an adventitious bud induction medium and a rooting medium;

[0070] The co-culture medium was MS medium + 1.9 mg / L kinetin + 0.3 mg / L naphthaleneacetic acid + 180 μmol / L acetosyringone + 23 g / L glucose + 7 g / L agar;

[0071] The callus induction medium was MS medium + 1.9 mg / L kinetin + 0.3 mg / L naphthaleneacetic acid + 0.8 mg / L silver nitrate + 180 μmol / L acetosyringone + 250 mg / L carbenicillin disodium + 23 g / L glucose + 7 g / L agar;

[0072] The adventitious bud induction medium is MS medium + 1.9 mg / L kinetin + 0.3 mg / L naphthaleneacetic acid 0.8 mg / L silver nitrate + 260 mg / L carbenicillin disodium + 45 mg / L hygromycin B + 23 g / L glucose + 7 g / L agar;

[0073] The rooting medium was 1 / 2MS medium + 0.9 mg / L naphthaleneacetic acid + 23 g / L glucose + 7 g / L agar + 260 mg / L carbenicillin disodium.

[0074] The preparation steps of the co-culture medium, callus induction medium, adventitious bud induction medium and rooting medium are as follows: adding corresponding amounts of other components to MS medium or 1 / 2MS medium and then sterilizing to obtain the corresponding culture medium.

[0075] Example 2

[0076] A method for genetically modifying water spinach, comprising the following steps:

[0077] 1) Cultivation of sterile seedlings

[0078] First, soak the water spinach seeds in 55℃ warm water for 30min, then soak them in clean water for 23h, pick them up, wash them, and soak them in 75% ethanol solution by volume for 2min, then soak them in 3% sodium hypochlorite (NaClO) solution by mass for 9min, then rinse them with sterile water for 8 times, dry the surface moisture of the seeds with sterile filter paper, inoculate them in germination medium (MS solid medium), and place them in the culture room for germination culture. The conditions of germination culture are: temperature 26℃, light intensity 2300lx, and light time 13h / d. When the sterile seedlings grow to 8cm, they are ready for use.

[0079] 2) Agrobacterium infection

[0080] The petioles of the sterile seedlings obtained in step 1) were cut into 0.5 cm lengths using sterile scissors, placed in a culture dish, and the Agrobacterium infection solution was added and infected for 38 minutes.

[0081] Construction of PC1300s-VvPL1 expression vector: VvPL1 (a gene on grapes, gene ID: VIT_05s0051g00590 in the grape reference genome database (http: / / plants.ensembl.org / Vitis_vinifera / Info / Index)), the detailed steps of the construction of the recombinant expression vector refer to Yu Jun's doctoral thesis (Yu Jun. Research on grape fruit cracking and its exogenous calcium regulation mechanism [D]. Hunan Agricultural University, 2020.).

[0082] Agrobacterium infection solution was prepared by the following method: EHA105 Agrobacterium culture containing PC1300s-VvPL1 expression vector was stored in this laboratory. PC1300s-VvPL1-EHA105 was inoculated into LB solid medium (containing 50 mg / L kanamycin sulfate + 20 mg / L rifampicin) by streaking method, incubated at 28°C, and single colonies that were positive in PCR test were picked and inoculated into LB liquid medium (50 mg / L kanamycin sulfate + 20 mg / L rifampicin) after 2 days, and cultured at 28°C, 200 r / min shaking until OD 600 =0.8, then centrifuge to collect the cells, add suspension (MS medium + 180 μmol / L acetosyringone + 23 g / L glucose + 0.02% Tween-20) to resuspend the cells, and adjust the OD 600 =0.6 to obtain the Agrobacterium infection solution.

[0083] 3) Co-culture

[0084] Use tweezers to pick out the petiole infected with Agrobacterium solution in step 3), place it on sterile filter paper to absorb the bacterial solution, inoculate it into co-culture medium (MS medium + 1.9 mg / L KT + 0.3 mg / L NAA + 180 μmol / LAS + 23 g / L glucose + 7 g / L agar), and culture it in the dark at 26°C for 3 days to obtain the co-cultured petiole.

[0085] 4) Callus induction

[0086] After the co-cultivation, the co-cultivated petioles in step 3) were transferred to callus induction medium (MS medium + 1.9 mg / L KT + 0.3 mg / L NAA + 0.8 mg / L silver nitrate + 180 μmol / L acetosyringone + 250 mg / L carbenicillin disodium + 23 g / L glucose + 7 g / L agar), and cultured in the dark at 26°C for 23 days to obtain callus tissue. Figure 1 Shown in the middle left picture.

[0087] 5) Resistance bud induction screening and culture

[0088] The callus obtained in step 4) was inoculated into adventitious bud induction medium (MS medium + 1.9 mg / L KT + 0.3 mg / L NAA + 0.8 mg / L silver nitrate + 260 mg / L carbenicillin disodium + 45 mg / L hygromycin B + 23 g / L glucose + 7 g / L agar), at 25-28°C, light time 13 h / day, light intensity 2300 lx, to obtain adventitious buds, such as Figure 1 As shown in the middle figure.

[0089] 6) Rooting culture

[0090] When the adventitious buds in step 5) grow to 4 cm, they are transferred to rooting medium (1 / 2MS medium + 0.9 mg / L NAA + 23 g / L glucose + 7 g / agar + 260 mg / L carbenicillin disodium). They are cultured under the conditions of 13 h / day of light, 26°C, and light intensity of 2300 lx to obtain transgenic seedlings. Figure 1 As shown in the middle right picture.

[0091] 7) Hardening and transplanting of tissue culture seedlings

[0092] When the transgenic seedlings grew 6 roots with a root length of about 5 cm, the bottle cap was opened and the seedlings were placed under scattered light for 5 days for hardening. The root culture medium was washed and planted in a sterilized substrate and cultured in a greenhouse at 26°C to obtain transgenic plants.

[0093] 8) PCR identification of transgenic plants

[0094] The genomic DNA of the transgenic plant obtained in step 7) was extracted, and the vector plasmid carrying the VvPL1 gene was used as a positive control, and the wild type was used as a negative control. PCR amplification was performed using specific primers of the VvPL1 gene (F: 5'-ACAACTCATCAATGGCAGCC-3' (SEQ ID NO: 1); R: 5'-CGTGACAAATTGG ACGGTGA-3' (SEQ ID NO: 2)). The amplified product was then identified by agarose gel electrophoresis. A band of about 470 bp could be detected in the strains successfully transformed with the VvPL1 gene, and no band was detected in the strains that were not successfully transformed, such as Figure 2 As shown, in Figure 2 In the figure, M: Marker; -: negative control; +: positive control; 1, 2, 3, 4: plants containing VvPL1 disease resistance gene; 7: plant without VvPL1 gene.

[0095] In this example, 150 petioles were infected using this technology, and 68 transgenic water spinach seedlings containing the VvP L1 gene were obtained, with a genetic transformation efficiency of 45.33%.

[0096] Examples 3 to 10 and Comparative Examples 1 to 7

[0097] The water spinach tissue culture medium in Example 1 was used to carry out genetic modification of water spinach according to the method in Example 2, except that the concentrations of kinetin (KT) and naphthaleneacetic acid (NAA) in the co-culture medium, callus induction medium and adventitious bud induction medium were adjusted at the same time, and the explants used were petioles of about 0.5 cm in length (0.4-0.6 cm), as shown in Table 1. The effects of different hormone concentration combinations in the water spinach tissue culture medium on the adventitious bud induction rate of water spinach were detected, and the results are shown in Table 1.

[0098] Table 1 Effects of different hormone concentration combinations on adventitious bud induction rate of water spinach tissue culture medium in Examples 3 to 10 and Comparative Examples 1 to 7

[0099]

[0100]

[0101] Note: Different letters in Table 1 indicate significant differences (p < 0.05), the same as in Tables 2 to 4.

[0102] Comparative Examples 8 to 23

[0103] The water spinach tissue culture medium in Example 1 was used to carry out transgenic water spinach according to the method in Example 2, except that the concentrations of kinetin (KT) and naphthaleneacetic acid (NAA) in the co-culture medium, callus induction medium and adventitious bud induction medium were adjusted at the same time, and the explants used were 0.5 cm 2 The leaves of different sizes are shown in Table 2, and the effects of different hormone concentration combinations in the water spinach tissue culture medium on the induction rate of water spinach adventitious buds are detected. The results are shown in Table 2.

[0104] Table 2 Effects of different hormone concentration combinations on adventitious bud induction rate of water spinach tissue culture medium in comparative examples 8 to 23

[0105]

[0106] Comparative Examples 24 to 39

[0107] The water spinach tissue culture medium in Example 1 was used to carry out genetic modification of water spinach according to the method in Example 2, except that the concentrations of kinetin (KT) and naphthaleneacetic acid (NAA) in the co-culture medium, callus induction medium and adventitious bud induction medium were adjusted at the same time, and the explant used was a hypocotyl of about 0.5 cm, as shown in Table 3. The effects of different hormone concentration combinations in the water spinach tissue culture medium on the adventitious bud induction rate of water spinach were detected, and the results are shown in Table 3.

[0108] Table 3 Effect of different hormone concentration combinations on adventitious bud induction rate of water spinach tissue culture medium in comparative examples 24 to 39

[0109]

[0110] According to the results in Tables 1 to 3, it can be concluded that the induction rate of adventitious buds of Water spinach can be significantly improved by using the KT and NAA concentrations in the Water spinach tissue culture medium of the present invention and using petioles as explants.

[0111] Examples 11 to 12 and Comparative Examples 40 to 41

[0112] The water spinach tissue culture medium in Example 1 was used to perform genetic modification of water spinach according to the method in Example 2, except that the concentration of naphthaleneacetic acid (NAA) in the rooting medium was adjusted, as shown in Table 4, and the effects of different concentrations of NAA in the rooting medium on the rooting rate of water spinach were detected. The results are shown in Table 4.

[0113] Table 4 Effect of rooting medium on rooting rate of water spinach in Examples 11-12 and Comparative Examples 40-41

[0114] serial number NAA concentration (mg / L) Rooting rate (%) Comparative Example 40 0.7 76.82b Embodiment 11 0.8 93.46a Example 1 0.9 96.28a Example 12 1.0 95.19a Comparative Example 41 1.1 79.39b

[0115] It can be concluded from Table 4 that the setting of the naphthaleneacetic acid concentration in the rooting medium of the present invention can effectively improve the rooting rate of water spinach.

[0116] Examples 13 to 14 and Comparative Examples 42 to 45

[0117] The water spinach tissue culture medium in Example 1 was used to carry out the genetic modification of water spinach according to the method in Example 2, except that the concentration of the Agrobacterium infection solution (OD 600 OD of Agrobacterium infection solution in Examples 13 to 14 and Comparative Examples 42 to 45 600 The values ​​were 0.5, 0.7, 0.1, 0.3, 0.9 and 1.1, respectively. The differentiation rate of adventitious buds of Ipomoea aquatica was tested. The results were as follows Figure 3 shown.

[0118] Depend on Figure 3 It can be concluded that the concentration of the Agrobacterium infection solution of the present invention can significantly increase the differentiation rate of adventitious buds of Water spinach.

[0119] Examples 15 to 16 and Comparative Examples 46 to 49

[0120] The water spinach tissue culture medium in Example 1 was used to perform genetic modification of water spinach according to the method in Example 2, except that the infection time of the Agrobacterium infection solution was adjusted. The infection time of the Agrobacterium infection solution in Examples 15 to 16 and Comparative Examples 46 to 49 was 35, 40, 20, 25, 30 and 45 minutes, respectively, and the differentiation rate of the water spinach adventitious buds was detected. The results are as follows: Figure 4 shown.

[0121] Depend on Figure 4 It can be concluded that the infection time of the Agrobacterium infection solution of the present invention can significantly increase the differentiation rate of adventitious buds of Water spinach.

[0122] Example 17 and Comparative Examples 50 to 53

[0123] The water spinach tissue culture medium in Example 1 was used to perform genetic modification of water spinach according to the method in Example 2, except that the concentrations of acetosyringone in the co-culture medium and the callus induction medium were adjusted at the same time. The concentrations of acetosyringone in the co-culture medium and the callus induction medium in Example 17, Example 1 and Comparative Examples 50 to 52 were 150, 180, 90, 120, 210 and 230 μmol / L, respectively, and the differentiation rate of water spinach adventitious buds was detected. The results are as follows: Figure 5 shown.

[0124] Depend on Figure 5 It can be concluded that the concentration of acetosyringone in the co-culture medium and callus induction culture medium of the present invention can significantly improve the differentiation rate of adventitious buds of Ipomoea aquatica.

[0125] Example 18 and Comparative Examples 54 to 56

[0126] The water spinach tissue culture medium in Example 1 was used to perform genetic modification of water spinach according to the method in Example 2, except that the dark culture time in the co-culture step 3) was adjusted. The concentrations of acetosyringone in the co-culture medium and the callus induction medium in Example 18, Example 1, and Comparative Examples 54 to 56 were 4, 3, 2, 5, and 6 days, respectively, and the differentiation rate of water spinach adventitious buds was detected. The results are as follows: Figure 6 shown.

[0127] Depend on Figure 6 It can be concluded that the co-cultivation time set in the present invention can significantly increase the differentiation rate of adventitious buds of Ipomoea aquatica.

[0128] It can be concluded from the above embodiments that the water spinach tissue culture medium and the transgenic method of the present invention can realize the transgenic of water spinach and improve the transgenic efficiency of water spinach.

[0129] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A water spinach tissue culture medium, characterized in that: comprising one or more of a co-culture medium, a callus induction medium, an adventitious bud induction medium and a rooting medium; The co-culture medium uses MS culture medium as a basic culture medium, and further includes the following components in concentrations: 1.8-2.0 mg / L kinetin, 0.2-0.4 mg / L naphthaleneacetic acid, 150-180 μmol / L acetosyringone, 20-25 g / L glucose and 6-8 g / L agar; The callus induction medium is based on MS medium and also includes the following components in concentrations: 1.8-2.0 mg / L kinetin, 0.2-0.4 mg / L naphthaleneacetic acid, 0.5-1.0 mg / L silver nitrate, 150-180 μmol / L acetosyringone, 250-260 mg / L carbenicillin disodium, 20-25 g / L glucose and 6-8 g / L agar; The adventitious bud induction medium is based on MS medium and also includes the following components in concentrations: 1.8-2.0 mg / L kinetin, 0.2-0.4 mg / L naphthaleneacetic acid, 0.5-1.0 mg / L silver nitrate, 250-260 mg / L carbenicillin disodium, 40-50 mg / L hygromycin B, 20-25 g / L glucose and 6-8 g / L agar; The rooting culture medium uses 1 / 2MS culture medium as the basic culture medium and further comprises components in the following concentrations: 0.8-1.0 mg / L naphthaleneacetic acid, 20-25 g / L glucose, 6-8 g / L agar and 250-260 mg / L carbenicillin disodium.

2. The water spinach tissue culture medium according to claim 1, characterized in that: The water spinach tissue culture medium also includes MS solid culture medium and / or LB liquid culture medium.

3. Use of the water spinach tissue culture medium according to claim 1 or 2 in water spinach transgenic and / or water spinach tissue culture.

4. A method for genetically modifying kiwifruit based on the kiwifruit tissue culture medium according to claim 1 or 2, characterized in that: The steps include: The sterilized water spinach seeds are germinated and cultured to obtain sterile water spinach seedlings; Infecting the petioles of the sterile water spinach seedlings with Agrobacterium containing the target gene to obtain petioles of water spinach infected with Agrobacterium; inoculating the water spinach petiole infected by the Agrobacterium into a co-culture medium for co-cultivation to obtain co-cultivated water spinach petiole; Inoculating the co-cultured water spinach petioles into a callus induction medium for callus induction culture to obtain water spinach callus; Inoculating the water spinach callus into an adventitious bud induction medium for bud induction culture to obtain water spinach adventitious buds; The water spinach adventitious buds are inoculated into a rooting medium for rooting culture to obtain water spinach transgenic seedlings.

5. The method for genetically modifying water spinach according to claim 4, characterized in that: The length of the petiole of the water spinach sterile seedling is 0.3-0.6 cm.

6. The method for genetically modifying water spinach according to claim 4, characterized in that: The infection method comprises: soaking the water spinach explant with an Agrobacterium infection solution containing the target gene; the OD of the Agrobacterium infection solution is 600 is 0.5-0.7; and the soaking time is 35-40 minutes.

7. The method for genetically modifying water spinach according to claim 4, characterized in that: The co-cultivation is dark cultivation at a temperature of 25-28° C. for 3-4 days; The callus induction culture is dark culture at a temperature of 25 to 28° C. for 20 to 25 days; The bud induction culture temperature is 25-28°C, the light intensity is 2000-2500lx, and the light duration is 12-14h / day; the bud induction culture ends when the water spinach adventitious buds grow to 3-5cm; The light intensity of the rooting culture is 2000-2500 lx, and the light duration is 12-14 h / day.

8. The method for genetically modifying water spinach according to claim 4, characterized in that: The transgenic water spinach seedlings contain 5 to 6 roots, and the root length is 3 to 5 cm.

9. The method for genetically modifying water spinach according to claim 4 or 8, characterized in that: The water spinach transgenic method further comprises hardening and transplanting the water spinach transgenic seedlings to obtain water spinach transgenic plants.

10. The method for genetically modifying water spinach according to claim 9, characterized in that: The method of hardening the seedlings comprises: irradiating the transgenic water spinach seedlings under scattered light for 4 to 5 days; The transplanting culture comprises: planting the hardened water spinach transgenic seedlings in a sterilized plant matrix, and culturing them under the condition of 25-30° C. to obtain the water spinach transgenic plants.

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