Gordon euryale seed genetic transformation culture medium and application thereof in genetic transformation of gordon euryale seeds

Abstract

The invention belongs to the technical field of plant genetic transformation, and particularly relates to a gordon euryale seed genetic transformation culture medium and application thereof in gordon euryale seed genetic transformation. The gordon euryale seed genetic transformation culture medium comprises a callus induction culture medium, a subculture multiplication culture medium, a co-culture medium and a screening culture medium. According to the present invention, the composition and the concentration of each component (such as acetosyringone, kanamycin and other hormones) in each culture medium in the Gorgon fruit genetic transformation culture medium are adjusted to form the culture medium suitable for Gorgon fruit genetic transformation; on the basis of the gordon euryale seed genetic transformation culture medium, a standard gordon euryale seed genetic transformation system, especially a system suitable for gordon euryale seed callus genetic transformation, is successfully constructed by limiting genetic transformation conditions such as types of explants, so that the gordon euryale seed genetic transformation efficiency is effectively improved; and a technical support is provided for research and explanation of Gorgon fruit gene functions.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic transformation, and particularly relates to an Euryale ferox genetic transformation medium and its application in the genetic transformation of Euryale ferox. Background Art

[0002] Euryale ferox is an aquatic plant with extremely high economic and medicinal values. However, due to the special planting conditions, long growth cycle of Euryale ferox, its genetic improvement is relatively lagging behind.

[0003] As an aquatic plant, the efficiency of tissue genetic transformation of Euryale ferox is much lower than that of some model plants, and the immaturity of the transformation system greatly limits the functional elucidation of Euryale ferox genes and the process of molecular breeding. Taking the callus of Euryale ferox as the infection object and finally obtaining transgenic Euryale ferox callus can greatly shorten the time to obtain transgenic Euryale ferox materials. However, there is currently no perfect genetic transformation method targeting the callus of Euryale ferox as the explant in this field. Summary of the Invention

[0004] The purpose of the present invention is to provide an Euryale ferox genetic transformation medium and its application in the genetic transformation of Euryale ferox. Based on the Euryale ferox genetic transformation medium, the genetic transformation of Euryale ferox can be realized, and the genetic transformation efficiency of Euryale ferox can be improved.

[0005] The present invention provides an Euryale ferox genetic transformation medium, which includes a callus induction medium, a subculture proliferation medium, a co-culture medium and a screening medium;

[0006] The callus induction medium uses the MS medium as the basic medium, and also includes 0.3 - 0.5 mg / L naphthaleneacetic acid (NAA), 1.8 - 1.9 mg / L 6-benzylaminopurine (6-BA), 25 - 30 g / L glucose and 7 - 8 g / L agar powder, and the pH value is 5.5 - 6.5;

[0007] The subculture proliferation medium uses the MS medium as the basic medium, and also includes 0.3 - 0.5 mg / L naphthaleneacetic acid, 1.8 - 1.9 mg / L 6-benzylaminopurine, 25 - 30 g / L glucose and 7 - 8 g / L agar powder, and the pH value is 5.5 - 6.5;

[0008] The co-culture medium uses the MS medium as the basic medium, and also includes 0.3 - 0.5 mg / L naphthaleneacetic acid, 1.8 - 1.9 mg / L 6-benzylaminopurine, 60 - 90 μmol / L acetosyringone (AS), 25 - 30 g / L glucose and 7 - 8 g / L agar powder, and the pH value is 5.5 - 6.5;

[0009] The screening medium is based on MS medium and further includes 0.3 - 0.5 mg / L naphthaleneacetic acid, 1.8 - 1.9 mg / L 6-benzylaminopurine, 10 - 15 mg / L kanamycin (Kan), 25 - 30 g / L glucose, and 7 - 8 g / L agar powder, with a pH value of 5.5 - 6.5.

[0010] The present invention also provides the application of the Euryale ferox genetic transformation medium described in the above technical solution in Euryale ferox tissue culture and / or Euryale ferox genetic transformation.

[0011] Preferably, the Euryale ferox genetic transformation includes the genetic transformation of Euryale ferox callus.

[0012] Preferably, the transformation of Euryale ferox callus includes the genetic transformation of Euryale ferox embryogenic callus.

[0013] The present invention also provides a method for genetic transformation of Euryale ferox embryogenic callus based on the Euryale ferox genetic transformation medium described in the above technical solution, which includes the following steps:

[0014] Inoculate the Euryale ferox seed embryo into the callus induction medium for callus induction culture to obtain Euryale ferox embryogenic callus;

[0015] Inoculate the Euryale ferox embryogenic callus into the subculture proliferation medium for subculture to obtain subcultured Euryale ferox embryogenic callus;

[0016] Immerse the subcultured Euryale ferox embryogenic callus into the Agrobacterium infection solution containing the target gene for infection, and inoculate the infected Euryale ferox embryogenic callus into the co-culture medium for co-culture to obtain co-cultured Euryale ferox callus;

[0017] Inoculate the co-cultured Euryale ferox callus into the screening medium for screening culture to obtain genetically transformed callus.

[0018] Preferably, the OD 600 value of the Agrobacterium infection solution is 0.4 - 0.6.

[0019] Preferably, the Agrobacterium infection solution is obtained by resuspending Agrobacterium cells with a suspension; the suspension is based on MS medium and further includes 60 - 90 μmol / L acetosyringone, 25 - 30 g / L glucose, and 0.02 wt.% - 0.03 wt.% Tween-20.

[0020] Preferably, the callus induction culture is dark culture at a temperature of 25 - 26 °C for 20 - 25 days;

[0021] The subculture is dark culture at a temperature of 25 - 26 °C, and subculture is carried out every 10 - 12 days.

[0022] Preferably, the milky white and brittle callus in the subcultured Euryale ferox embryogenic callus is infected; the infection time is 12 - 16 min.

[0023] Preferably, the co - culture is carried out in the dark, the temperature is 25 - 26 °C, and the time is 2 - 3 d;

[0024] The screening culture is carried out in the dark, the temperature is 25 - 26 °C, and the time is 20 - 25 d.

[0025] Beneficial effects:

[0026] The present invention provides a genetic transformation medium for Euryale ferox, including a callus induction medium, a subculture proliferation medium, a co - culture medium and a screening medium. By adjusting the composition and concentration of each component (such as hormones like acetosyringone and kanamycin) in each medium of the genetic transformation medium for Euryale ferox, a medium suitable for the genetic transformation of Euryale ferox is formed. On the basis of the genetic transformation medium for Euryale ferox, by defining genetic transformation conditions such as the type of explant, a standard genetic transformation system for Euryale ferox is successfully constructed, especially a system suitable for the genetic transformation of Euryale ferox callus, effectively improving the genetic transformation efficiency of Euryale ferox and providing technical support for the research and interpretation of Euryale ferox gene functions. Brief description of the drawings

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments.

[0028] Figure 1 It is the GUS detection result of the genetic transformation of Euryale ferox embryogenic callus in Example 2, where A: control, untransformed callus; B: callus stained by GUS after transformation. Detailed implementation manners

[0029] The present invention provides a genetic transformation medium for Euryale ferox, including a callus induction medium, a subculture proliferation medium, a co - culture medium and a screening medium;

[0030] The callus induction medium is based on the MS medium, and also includes 0.3 - 0.5 mg / L naphthylacetic acid, 1.8 - 1.9 mg / L 6 - benzylaminopurine, 25 - 30 g / L glucose and 7 - 8 g / L agar powder, and the pH value is 5.5 - 6.5;

[0031] The subculture proliferation medium is based on the MS medium, and also includes 0.3 - 0.5 mg / L naphthylacetic acid, 1.8 - 1.9 mg / L 6 - benzylaminopurine, 25 - 30 g / L glucose and 7 - 8 g / L agar powder, and the pH value is 5.5 - 6.5;

[0032] The co-culture medium is based on MS medium and further includes 0.3 - 0.5 mg / L naphthylacetic acid, 1.8 - 1.9 mg / L 6-benzylaminopurine, 60 - 90 μmol / L acetosyringone, 25 - 30 g / L glucose and 7 - 8 g / L agar powder, with a pH value of 5.5 - 6.5;

[0033] The screening medium is based on MS medium and further includes 0.3 - 0.5 mg / L naphthylacetic acid, 1.8 - 1.9 mg / L 6-benzylaminopurine, 10 - 15 mg / L kanamycin, 25 - 30 g / L glucose and 7 - 8 g / L agar powder, with a pH value of 5.5 - 6.5.

[0034] As an embodiment, the callus induction medium of the present invention is based on MS medium and further includes 0.5 mg / L naphthylacetic acid, 1.9 mg / L 6-benzylaminopurine, 30 g / L glucose and 8 g / L agar powder, with a pH value of 6.5.

[0035] As an embodiment, the subculture proliferation medium of the present invention is based on MS medium and further includes 0.5 mg / L naphthylacetic acid, 1.9 mg / L 6-benzylaminopurine, 30 g / L glucose and 8 g / L agar powder, with a pH value of 6.5.

[0036] As an embodiment, the co-culture medium of the present invention is based on MS medium and further includes 0.5 mg / L naphthylacetic acid, 1.9 mg / L 6-benzylaminopurine, 90 μmol / L acetosyringone, 30 g / L glucose and 8 g / L agar powder, with a pH value of 6.5.

[0037] As an embodiment, the screening medium of the present invention is based on MS medium and further includes 0.5 mg / L naphthylacetic acid, 1.9 mg / L 6-benzylaminopurine, 10 mg / L kanamycin, 30 g / L glucose and 8 g / L agar powder, with a pH value of 6.5.

[0038] The present invention forms a medium suitable for the genetic transformation of Euryale ferox by defining the composition and concentration of each component (such as hormones like acetosyringone and kanamycin) in each medium of the Euryale ferox genetic transformation medium. The concentrations of acetosyringone and kanamycin in the Euryale ferox genetic transformation medium of the present invention respectively have the advantages of improving the transformation efficiency and high accuracy in screening transformants.

[0039] The present invention also provides the application of the Euryale ferox genetic transformation medium described in the above technical solution in the tissue culture of Euryale ferox and / or the genetic transformation of Euryale ferox. As an implementation manner, the genetic transformation of Euryale ferox in the present invention can be the genetic transformation of Euryale ferox callus; as another implementation manner, the callus transformation of Euryale ferox in the present invention can be the genetic transformation of Euryale ferox embryogenic callus. The present invention has the advantage of high transformation efficiency by using Euryale ferox embryogenic callus as the explant.

[0040] The present invention also provides a method for the genetic transformation of Euryale ferox embryogenic callus based on the Euryale ferox genetic transformation medium described in the above technical solution, comprising the following steps:

[0041] Inoculate the Euryale ferox seed embryo into a callus induction medium for callus induction culture to obtain Euryale ferox embryogenic callus;

[0042] Inoculate the Euryale ferox embryogenic callus into a subculture proliferation medium for subculture to obtain subcultured Euryale ferox embryogenic callus;

[0043] Immerse the subcultured Euryale ferox embryogenic callus in an Agrobacterium infection solution containing a target gene for infection, and inoculate the infected Euryale ferox embryogenic callus into a co-culture medium for co-culture to obtain co-cultured Euryale ferox callus;

[0044] Inoculate the co-cultured Euryale ferox callus into a screening medium for screening culture to obtain genetically transformed callus.

[0045] As an implementation manner, the present invention cultures the Euryale ferox seed embryo. As an implementation manner, the method for culturing the Euryale ferox seed embryo in the present invention comprises the following steps: break the dormancy of the Euryale ferox seeds and then perform disinfection and sterilization, and cut the disinfected and sterilized Euryale ferox seeds to take the seed embryo to obtain the Euryale ferox seed embryo. As an implementation manner, the way to break the dormancy can be to store the Euryale ferox seeds at 2-4°C for 100-120 days to break the dormancy; as another implementation manner, the way to break the dormancy can be to store the Euryale ferox seeds at 4°C for 100 days. As an implementation manner, the method of disinfection and sterilization can be: soak the Euryale ferox seeds with broken dormancy in an ethanol solution with a volume concentration of 75% for 1 minute, and then soak the soaked Euryale ferox seeds in a sodium hypochlorite solution with a volume concentration of 3% for 10 minutes to obtain disinfected and sterilized Euryale ferox seeds. The present invention has no special limitation on the process of cutting the Euryale ferox seeds to take the seed embryo, and the operation process well-known to those skilled in the art can be adopted.

[0046] After obtaining the Euryale ferox embryo, the present invention inoculates the Euryale ferox embryo into a callus induction medium for callus induction culture to obtain Euryale ferox embryogenic callus. As an implementation method, the callus induction culture of the present invention is dark culture, the temperature is 25-26 °C, and the time is 20-25 d; as another implementation method, the time of the callus induction culture is 20 d.

[0047] After obtaining the Euryale ferox embryogenic callus, the present invention inoculates the Euryale ferox embryogenic callus into a subculture proliferation medium for subculture to obtain subcultured Euryale ferox embryogenic callus. As an implementation method, the subculture of the present invention is dark culture, the temperature is 25-26 °C, and subculture is carried out every 10-12 d; as another implementation method, the subculture of the present invention is carried out every 10 d.

[0048] After obtaining the subcultured Euryale ferox embryogenic callus, the present invention immerses the subcultured Euryale ferox embryogenic callus into an Agrobacterium infection solution containing the target gene for infection, and inoculates the infected Euryale ferox embryogenic callus into a co-culture medium for co-culture to obtain co-cultured Euryale ferox callus. As an implementation method, the present invention infects the milky white and brittle-textured callus in the subcultured Euryale ferox embryogenic callus; the Euryale ferox embryogenic callus of this morphology has the advantage of high transformation efficiency. As an implementation method, the OD 600 value of the Agrobacterium infection solution is 0.4-0.6; as another implementation method, the OD 600 value of the Agrobacterium infection solution is 0.4; the Agrobacterium infection solution of this concentration has the advantages of high infection efficiency and little damage to explant cells. As an implementation method, the Agrobacterium infection solution of the present invention is obtained by resuspending Agrobacterium cells with a suspension. As an implementation method; the suspension is based on MS medium and also includes 60-90 μmol / L acetosyringone, 25-30 g / L glucose and 0.02 wt%-0.03 wt% Tween-20; as another implementation method, the suspension is based on MS medium and also includes 90 μmol / L acetosyringone, 30 g / L glucose and 0.02 wt% Tween-20; the concentration of acetosyringone in the suspension has the advantage of high transformation efficiency. The present invention has no special limitation on the type of the target gene, and it can be selected according to research needs.

[0049] As an implementation method, the infection time of the present invention is 12-16 min; as another implementation method, the infection time can be 14 min; the infection time has the advantages of high infection efficiency and little damage to explant cells.

[0050] As an implementation manner, the co-culture in the present invention is carried out under shading, the temperature is 25-26 °C, and the time is 2-3 days.

[0051] After obtaining the co-cultured Euryale ferox callus, the present invention inoculates the co-cultured Euryale ferox callus into a screening medium for screening culture to obtain genetically transformed callus. As an implementation manner, the screening culture is carried out under shading, the temperature is 25-26 °C, and the time is 20-25 days; as another implementation manner, the time of the screening culture is 20 days.

[0052] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below in conjunction with the drawings and embodiments, but they cannot be understood as limiting the protection scope of the present invention.

[0053] Example 1

[0054] A genetic transformation medium for Euryale ferox consists of a callus induction medium, a subculture proliferation medium, a co-culture medium and a screening medium;

[0055] Callus induction medium: MS medium + 0.5 mg / L NAA + 1.9 mg / L 6-BA + 30 g / L glucose + 8 g / L agar powder, pH value: 6.5;

[0056] Subculture proliferation medium: MS medium + 0.5 mg / L NAA + 1.9 mg / L 6-BA + 30 g / L glucose + 8 g / L agar powder, pH value: 6.5;

[0057] Co-culture medium: MS medium + 0.5 mg / L NAA + 1.9 mg / L 6-BA + 90 μmol / L AS + 30 g / L glucose + 8 g / L agar powder, pH value: 6.5;

[0058] Screening medium: MS medium + 0.5 mg / L NAA + 1.9 mg / L 6-BA + 10 mg / L Kan + 30 g / L glucose + 8 g / L agar powder, pH value: 6.5;

[0059] The callus induction medium, the subculture proliferation medium, the co-culture medium and the screening medium are all based on the MS medium, adding the corresponding amounts of other components, adjusting the pH value with 10% HCl or 10% NaOH, and then sterilizing the medium with adjusted pH value to obtain the corresponding medium.

[0060] Example 2

[0061] A method for genetic transformation of embryogenic callus of Euryale ferox based on the genetic transformation medium in Example 1, the steps are as follows:

[0062] (1) Construction of pCAMBIA2301 + GUS vector: The construction of the pCAMBIA2301 + GUS vector refers to the method in the following literature [Li Fei, Cao Yongqiong, Wang Gang, et al. Establishment and optimization of an Agrobacterium tumefaciens-mediated genetic transformation system for Zantedeschia hybrida [J]. Journal of Southern Agriculture, 2024, 55(06): 1692 - 1699.].

[0063] Preparation of Agrobacterium infection solution: The pCAMBIA2301 + GUS vector was transferred into Agrobacterium EHA105 competent cells by the freeze - thaw method to obtain pCAMBIA2301 + GUS + EHA105. pCAMBIA2301 + GUS + EHA105 was inoculated on LB solid medium (containing 50 mg / L kanamycin (Kan) + 20 mg / L rifampicin (Rif)) by the streaking method and incubated upside - down at 28 °C for 2 - 3 d; then a single colony was picked and inoculated into LB liquid medium (containing 50 mg / L Kan + 20 mg / L Rif), and cultured with shaking at 200 rpm in a 28 °C constant - temperature shaker for 16 h to make the OD of the bacterial solution 600 = 0.6; 1 mL of the above - mentioned bacterial solution was added to 50 mL of LB liquid medium (containing 50 mg / L Kan + 20 mg / L Rif) and cultured again until OD 600 = 0.6; then the above - mentioned Agrobacterium suspension was centrifuged at room temperature for 10 min (4000 rpm), the supernatant was discarded, and the cells were resuspended with a suspension (MS medium + 90 μmol / L AS + 30 g / L glucose + 0.02 wt% Tween - 20), and the OD 600 was adjusted to 0.4 for standby.

[0064] (2) Select plump and pest - and - disease - free Euryale ferox seeds, store them at 4 °C for 100 d to break dormancy, take them out and soak them in 75% (v / v) alcohol for 1 min in a laminar flow bench, rinse them 3 times with sterile water, then soak them in 3% (w / v) sodium hypochlorite solution for 10 min, and rinse them 3 times with sterile water again. Dry the moisture with sterile filter paper, cut the seeds with a sterile scalpel to take out the embryo in the center part, and inoculate it on the callus induction medium (MS medium + 0.5 mg / L NAA + 1.9 mg / L 6 - BA + 30 g / L glucose + 8 g / L agar powder, pH value: 6.5), and place it in the dark at 25 °C for 3 weeks.

[0065] (3) Transfer the embryogenic callus of Euryale ferox induced in step (2) to the subculture and proliferation medium (MS medium + 0.5 mg / L NAA + 1.9 mg / L 6 - BA + 30 g / L glucose + 8 g / L agar powder, pH value: 6.5), and place it in the dark at 25 °C for subculture every 10 d.

[0066] (4) Select the milky white and friable callus from the subcultured callus in step (3), soak it in a sterile bottle containing the Agrobacterium infection solution for 16 min, blot the excess bacterial liquid with sterile filter paper, and inoculate it on the co-culture medium (MS medium + 0.5 mg / L NAA + 1.9 mg / L 6-BA + 90 μmol / L AS + 30 g / L glucose + 8 g / L agar powder), and co-culture it in the dark at 25 °C for 2 d.

[0067] (5) Rinse the co-cultured callus in step (4) 3 times with sterile water for 3 min each time, then blot the surface moisture with sterile filter paper and transfer it to the selection medium (MS medium + 0.5 mg / L NAA + 1.9 mg / L 6-BA + 10 mg / L Kan + 30 g / L glucose + 8 g / L agar powder, pH value: 6.5), and culture it in the dark at 25-26 °C for 20 d to obtain the transformed callus.

[0068] (6) GUS staining to detect the transformed callus

[0069] Put the transformed callus obtained in step (5) into a 5 mL centrifuge tube, add the GUS staining working solution (GUS staining kit purchased from Beijing Huayueyang Company, and the GUS staining working solution is prepared according to the kit operation manual) until it completely covers the callus, incubate it at 37 °C in the dark for 12 h, then take out the callus and wash it 3 times with 70% (v / v) alcohol solution, and then observe the stained situation of the callus under a microscope. Among them, the callus showing blue is positive, indicating successful transformation. If the callus does not show blue, it means the transformation is not successful. The control is the non-infected callus, which is not affected by the GUS staining solution and does not change color. The detection results are as Figure 1 shown.

[0070] (7) Callus transformation rate

[0071] The inventor used the above method for genetic transformation of Euryale ferox callus. A total of 90 embryos were inoculated. After GUS detection of the obtained genetically transformed callus, a total of 38 positive calli were obtained. According to the callus transformation rate formula (callus transformation rate = number of calli showing blue in GUS detection / number of explants × 100%), the genetic transformation rate of Euryale ferox callus obtained by this method is 42.22%.

[0072] Example 1, Examples 3-8 and Comparative Examples 1-53

[0073] Using the callus induction medium in the Euryale ferox genetic transformation medium in Example 1, the embryogenic callus of Euryale ferox was induced according to the method in Example 2. The difference is that the concentrations of 6-BA and NAA in the callus induction medium and the type of explants were adjusted simultaneously, as shown in Table 1 specifically, and the effects of different hormone concentration combinations in the callus induction medium on the callus induction rates of different explants of Euryale ferox were detected (leaf size 0.5 cm 2 , hypocotyl length 0.5 cm, embryo length 0.5 cm, all taken from sterile seedlings of Euryale ferox), and the results are shown in Table 1.

[0074] Table 1 Effects of different hormone concentration combinations in the callus induction medium in Example 1, Examples 3-8 and Comparative Examples 1-53 on the callus induction rates of different explants of Euryale ferox

[0075]

[0076]

[0077] Note: Different letters in Table 1 indicate significant differences (p < 0.05), the same as in Table 2 and Table 3.

[0078] It can be concluded from Table 1 that by using the concentrations of 6-BA and NAA in the callus induction medium of the present invention and using the embryo as the explant for callus induction, the callus induction rate of Euryale ferox can be significantly improved.

[0079] Example 1, Example 9 and Comparative Examples 54-55

[0080] Using the Euryale ferox genetic transformation medium in Example 1, the genetic transformation of Euryale ferox was carried out according to the method in Example 2. The difference is that the concentration of kanamycin (Kan) in the screening medium was adjusted, as shown in Table 2 specifically, and the callus survival rate and the proliferation status of the callus were detected, and the results are shown in Table 2.

[0081] Table 2 Screening of kanamycin concentration in the screening medium in Example 1, Example 9 and Comparative Examples 54-55

[0082] Number Kanamycin concentration (mg / L) Survival rate of callus (%) Comparative Example 54 0 100a Example 1 10 75.19b Example 9 15 71.75b Comparative Example 55 20 43.76c

[0083] It can be concluded from Table 2 that when there is no Kan, the wild-type callus does not die; when containing Kan 10 and 15 mg / L, the survival rate of the wild-type callus is relatively high, and when containing Kan 20 mg / L, most of the wild-type callus dies; therefore, when Kan is 10-15 mg / L, the growth and proliferation of the callus can be basically inhibited, and at the same time, the survival rate of the explants is relatively high. Therefore, 10-15 mg / L Kan is used as the screening mass concentration of transgenic Euryale ferox callus.

[0084] Examples 10 to 13 and Comparative Examples 56 to 67

[0085] The genetic transformation of Euryale ferox was carried out according to the method in Example 2 using the Euryale ferox genetic transformation medium in Example 1, except that the concentration of the Agrobacterium infection solution (i.e., OD 600 value), the infection time, the co-culture time, and the concentration of acetosyringone (AS) in the co-culture medium were as shown in Table 3 specifically, and the genetic transformation rate of the callus was detected, and the results are shown in Table 3.

[0086] Table 3 Genetic transformation rates of callus in the genetic transformation of Euryale ferox in Examples 10 to 13 and Comparative Examples 56 to 67

[0087]

[0088] It can be seen from Table 3 that: when the concentration of the Agrobacterium infection solution (OD 600 value) is between 0.4 and 0.6, the AS concentration is 60 to 90 mg / L, the infection time is 14 to 16 min, and the co-culture time is 2 to 3 d, the genetic transformation efficiency of the Euryale ferox callus is the highest.

[0089] It can be concluded from the above examples that: the present invention has established a standard genetic transformation system for Euryale ferox callus, which can successfully achieve the genetic transformation of Euryale ferox genes and improve the genetic transformation rate of callus.

[0090] Although the above examples have described the present invention in detail, they are only a part of the embodiments of the present invention, not all embodiments. People can also obtain other embodiments according to these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A genetic transformation medium for Euryale ferox, characterized in that: It includes callus induction medium, subculture proliferation medium, co-culture medium and screening medium; The callus induction medium is based on MS medium and further includes 0.3-0.5 mg / L naphthaleneacetic acid, 1.8-1.9 mg / L 6-benzylaminopurine, 25-30 g / L glucose and 7-8 g / L agar powder, and the pH value is 5.5-6.5; The secondary proliferation culture medium is based on MS culture medium, and further includes 0.3-0.5 mg / L naphthaleneacetic acid, 1.8-1.9 mg / L 6-benzylaminopurine, 25-30 g / L glucose and 7-8 g / L agar powder, and the pH value is 5.5-6.5; The co-culture medium is based on MS culture medium, and further includes 0.3-0.5 mg / L naphthaleneacetic acid, 1.8-1.9 mg / L 6-benzylaminopurine, 60-90 μmol / L acetosyringone, 25-30 g / L glucose and 7-8 g / L agar powder, with a pH value of 5.5-6.5; The screening culture medium uses MS culture medium as a basic culture medium and further comprises 0.3-0.5 mg / L naphthaleneacetic acid, 1.8-1.9 mg / L 6-benzylaminopurine, 10-15 mg / L kanamycin, 25-30 g / L glucose and 7-8 g / L agar powder, and the pH value is 5.5-6.

5.

2. Application of the Euryale ferox genetic transformation medium according to claim 1 in Euryale ferox tissue culture and / or Euryale ferox genetic transformation.

3. The use according to claim 2, characterized in that: The genetic transformation of Euryale ferox includes genetic transformation of Euryale ferox callus.

4. The use according to claim 3, characterized in that: The transformation of Euryale ferox callus includes genetic transformation of Euryale ferox embryonic callus.

5. A method for genetic transformation of Euryale ferox embryonic callus based on the Euryale ferox genetic transformation medium according to claim 1, characterized in that: The steps include: Inoculating the Euryale ferox seed embryos into a callus induction medium for callus induction culture to obtain Euryale ferox embryonic callus; Inoculating the Euryale ferox embryonic callus into a subculture proliferation medium for subculture to obtain subculture Euryale ferox embryonic callus; The subcultured Euryale ferox embryonic callus is immersed in an Agrobacterium infection solution containing a target gene for infection, and the infected Euryale ferox embryonic callus is inoculated into a co-culture medium for co-cultivation to obtain a co-cultured Euryale ferox callus; The co-cultured Euryale ferox callus is inoculated into a screening medium for screening and culture to obtain genetically transformed callus.

6. The method according to claim 5, characterized in that The OD of the Agrobacterium infection solution 600 The value is 0.4~0.

6.

7. The method according to claim 5 or 6, characterized in that: The Agrobacterium infection solution is obtained by resuspending Agrobacterium cells in a suspension; the suspension uses MS culture medium as a basic culture medium and further includes 60-90 μmol / L acetosyringone, 25-30 g / L glucose and 0.02 wt%-0.03 wt% Tween-20.

8. The method according to claim 5, characterized in that The callus induction culture is dark culture at a temperature of 25-26° C. for 20-25 days; The subculture is dark culture at a temperature of 25-26° C., and the subculture is performed every 10-12 days.

9. The method according to claim 5, characterized in that The milky white and crispy callus tissue in the subcultured Euryale ferox embryonic callus tissue is infected; the infection time is 12 to 16 minutes.

10. The method according to claim 5, characterized in that The co-cultivation is performed in a light-shielding manner at a temperature of 25 to 26° C. for 2 to 3 days; The screening culture is a light-shielding culture at a temperature of 25 to 26° C. for 20 to 25 days.

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