Method for inducing transgenic hairy root formation of lagenaria vulgaris by non-tissue culture

Abstract

The application provides a method for inducing transgenic hairy root formation of Lagenaria vulgaris by non-tissue culture, comprising the following steps: (1) Lagenaria vulgaris culture: sowing Lagenaria vulgaris seeds in a seedling substrate, and culturing in a light incubator until the Lagenaria vulgaris plant grows to a stage between one heart and one leaf and one heart; (2) infection by a hole-punching and smearing method: taking the cotyledon node to 1 cm below the cotyledon node of the Lagenaria vulgaris plant as an infection site, punching a hole around the infection site, and smearing the hole with Agrobacterium rhizogenes; (3) nodule induction: continuing to culture the Lagenaria vulgaris plant in the light incubator, and observing that the infection site is swollen; and (4) inducing hairy root formation by cutting and culturing: obliquely cutting the swollen position of the infection site of the Lagenaria vulgaris plant, and inserting the lower end of the obtained upper plant, i.e. the obliquely cut end, into a growth carrier for culture to induce hairy root formation. The application can induce transgenic hairy root formation of Lagenaria vulgaris, has a high induction rate, and can be applied to promoter function research, functional gene analysis, secondary metabolite research and the like.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and more specifically, to the field of transgenic hairy root formation technology of common loofah, particularly to a method for inducing transgenic hairy root formation in common loofah without tissue culture. Background Technology

[0002] Common loofah (Luffa cylindrica L.) belongs to the Cucurbitaceae family and is widely cultivated in temperate and tropical regions worldwide, including throughout my country, where it is one of the main gourd vegetables supplied to the market in summer and autumn. Loofah is not only edible, but the entire plant can be used medicinally, possessing properties such as clearing heat and detoxifying, promoting blood circulation, and reducing inflammation and swelling. The loofah sponge is a major component of many traditional Chinese medicines. Furthermore, loofah sponge, as an environmentally friendly material, can be processed into over 100 different household items, including organic cloth shoes and high-end bath products, greatly enhancing the economic benefits of loofah. In recent years, with the publication of loofah genome sequencing results, research on loofah has deepened. However, the difficulty in genetic transformation of loofah severely restricts the research on its functional genes.

[0003] Agrobacterium rhizogenes-mediated plant transformation is a genetic transformation system developed by utilizing the ability of Agrobacterium rhizogenes Ri to induce the formation of a large number of hairy roots in infected plants. Compared with Agrobacterium tumefaciens Ti plasmid-mediated genetic transformation, this technology has the advantages of short cycle, high transformation efficiency and less tendency to produce mutations. It has been widely used in legumes, solanaceae, cruciferous and other crops.

[0004] Although the technology of inducing transgenic hairy roots using Agrobacterium rhizogenes has been applied in cucurbit crops such as pumpkin, bottle gourd and cucumber, there are no reports of non-tissue culture-induced hairy roots in common loofah.

[0005] Therefore, developing a method for inducing the formation of transgenic hairy roots in common loofah without tissue culture is of great practical significance. Summary of the Invention

[0006] In order to overcome the shortcomings of the prior art, one object of the present invention is to provide a method for non-tissue culture induction of transgenic hairy roots of common loofah, which can induce the formation of transgenic hairy roots of common loofah with a high induction rate. It can be applied to promoter function research, functional gene analysis and secondary metabolite research, and is suitable for large-scale promotion and application.

[0007] Another objective of this invention is to provide a method for non-tissue culture induction of transgenic hairy roots in common loofah. This method is unique in concept and ingenious in design. It uses a puncture and smear method to infect root nodules, induce root growth, and then obliquely cut and culture the roots, which can form a large number of hairy roots and is suitable for large-scale promotion and application.

[0008] To achieve the above objectives, the present invention provides a method for inducing the formation of transgenic hairy roots in common loofah without tissue culture, characterized by comprising:

[0009] (1) Cultivation of ordinary loofah: Sow ordinary loofah seeds in seedling substrate and cultivate them in a light incubator until the loofah plants grow to the point between the one-heart stage and the one-leaf-one-heart stage.

[0010] (2) Infection by puncture and smear method: The infection site is from the cotyledon node of the loofah plant to 1 cm below the cotyledon node. Puncture a hole around the infection site and apply Agrobacterium rhizogenes to the puncture site.

[0011] (3) Nodule induction: The loofah plants were further cultured in a light incubator, and swelling of the infected sites was observed.

[0012] (4) Cutting culture to induce hair roots: obliquely cut the swollen part of the infected part of the loofah plant, insert the lower end of the upper plant, i.e. the oblique cut end, into the growth carrier for culture to induce the formation of hair roots.

[0013] Preferably, in step (1), the seedling substrate is a bud seedling substrate.

[0014] Preferably, in step (1), the incubation conditions of the light incubator are: alternating between 15000 lx light at 28℃ for 12h and light avoidance at 22℃ for 12h, with a relative humidity of 60%.

[0015] Preferably, in step (1), before the seeding of the open sowing, the ordinary loofah seeds are disinfected and rinsed.

[0016] Preferably, in step (2), the Agrobacterium rhizogenes is Agrobacterium rhizogenes Ar1193.

[0017] More preferably, in step (2), the Agrobacterium rhizogenes Ar1193 carries a binary expression vector pSuper1300FLAG-RUBY containing the RUBY reporter system.

[0018] Preferably, in step (2), the Agrobacterium rhizogenes is streak-activated before the dip.

[0019] Preferably, in step (3), the continued culture includes: culture at 21°C and 95% relative humidity in the dark for 2 days; culture at 21°C, 95% relative humidity, 10000 lx light intensity, and 12 h photoperiod for 3 days; and culture at 26°C, 60% relative humidity, 10000 lx light intensity, and 12 h photoperiod for 3 days.

[0020] Preferably, in step (4), the growth carrier is water-soaked rock wool.

[0021] Preferably, in step (4), the cultivation includes: cultivating for 1 day at 26°C, 60% relative humidity, 10000 lx light intensity, and 12h photoperiod until slight wilting; cultivating for 2 days at 26°C, 95% relative humidity, 10000 lx light intensity, and 12h photoperiod; and cultivating normally for 7 days at 26°C, 60% relative humidity, 10000 lx light intensity, and 12h photoperiod.

[0022] The beneficial effects of this invention are as follows:

[0023] 1. The method for non-tissue culture induction of transgenic hairy roots in common loofah of the present invention includes: (1) Common loofah culture: common loofah seeds are sown in seedling substrate and cultured in a light incubator until the loofah plant grows to the point between the one-heart stage and the one-leaf-one-heart stage; (2) Puncture-and-smear infection: the infection site is the area from the cotyledon node to 1 cm below the cotyledon node of the loofah plant. A hole is punched around the infection site, and Agrobacterium tumefaciens is dipped in the hole and smeared on the puncture site; (3) Nodule induction: the loofah plant is cultured in a light incubator and the swelling of the infection site is observed; (4) Cutting culture to induce hairy roots: the swollen part of the infection site of the loofah plant is obliquely cut, and the lower end of the upper plant, i.e. the oblique cut end, is inserted into the growth carrier for culture to induce hairy root formation. Therefore, it can induce the formation of transgenic hairy roots in common loofah and has a high induction rate. It can be applied to promoter function research, functional gene analysis and secondary metabolite research, etc., and is suitable for large-scale promotion and application.

[0024] 2. The method for non-tissue culture induction of transgenic hairy roots in common loofah of the present invention includes: (1) Common loofah culture: common loofah seeds are sown in seedling substrate and cultured in a light incubator until the loofah plant grows to the point between the one-heart stage and the one-leaf-one-heart stage; (2) Puncture-and-smear infection: the infection site is the area from the cotyledon node to 1 cm below the cotyledon node of the loofah plant. A hole is punched around the infection site, and Agrobacterium tumefaciens is dipped in the hole and smeared on the puncture site; (3) Nodule induction: the loofah plant is cultured in a light incubator and the swelling of the infection site is observed; (4) Cutting culture to induce hairy roots: the swollen part of the infection site of the loofah plant is obliquely cut, and the lower end of the upper plant, i.e. the oblique cut end, is inserted into the growth carrier for culture to induce hairy root formation. Therefore, its concept is unique and its design is ingenious. After infection by puncture-and-smear method, root nodule induction and then oblique cutting culture can form a large number of hairy roots, which is suitable for large-scale promotion and application.

[0025] These and other objects, features and advantages of the present invention will be fully apparent from the following detailed description and drawings, and can be achieved by the methods, means and combinations thereof specifically pointed out in the specification. Attached Figure Description

[0026] Figure 1 These are photographs of the infected sites and root nodules formed in the loofah in Example 1. A is the site of Agrobacterium infection 1 cm below the cotyledon node of the loofah (indicated by the red arrow); B is the root nodule formed after infection with Ar1193 Agrobacterium 1 cm below the cotyledon node of the loofah (indicated by the red arrow).

[0027] Figure 2 These are photos of the induction and culture results of hairy roots of loofah in Example 1. A is a schematic diagram of a slanted cut of the root nodules at the infection site of the loofah seedling, with red lines indicating the cut area; B is a schematic diagram of the loofah seedling placed in rock wool culture after cutting; C is a schematic diagram of the purple-red transgenic hairy roots after 7 days of rock wool culture.

[0028] Figure 3 This is a photograph of the hair roots of Control 1 set up in Example 1.

[0029] Figure 4 This is a photograph of the hair roots of Control 2 set up in Example 1.

[0030] Figure 5 These are photographs of the infected parts and root nodules formed in the loofah in Example 2. In Example 2, A is the cotyledon node of the loofah (indicated by the red arrow), which is the site of Agrobacterium infection; B is the root nodule formed after the cotyledon node of the loofah (indicated by the red arrow) is infected with Agrobacterium ar1193.

[0031] Figure 6 These are photos of the induction and culture results of hairy roots of loofah in Example 2. A is a schematic diagram of a slanted cut of root nodules at the infection site of the loofah seedling, with red lines indicating the cut area; B is a schematic diagram of the loofah seedling placed in rock wool culture after cutting; C is a schematic diagram of the purple-red transgenic hairy roots cultured in rock wool for 7 days.

[0032] Figure 7 This is a photograph of the hair roots of Control 1 set up in Example 2.

[0033] Figure 8 This is a photograph of the hair roots of Control 2 set up in Example 2.

[0034] Figure 9 This is a schematic diagram of the RUBY Reporting System insertion site in the pSuper1300 FLAG-RUBY recombinant vector, where the insertion site EcoRI is displayed in blue font. Detailed Implementation

[0035] In order to induce the formation of transgenic hairy roots in common loofah without tissue culture, so as to facilitate promoter function research, functional gene analysis, and secondary metabolite research, the inventors conducted extensive research and proposed a method for inducing the formation of transgenic hairy roots in common loofah without tissue culture. This method can induce the formation of transgenic hairy roots in common loofah with a high induction rate and can be applied to promoter function research, functional gene analysis, and secondary metabolite research. Based on this, the present invention was completed.

[0036] The method for non-tissue culture induction of transgenic hairy roots in common loofah of the present invention includes:

[0037] (1) Cultivation of ordinary loofah: Sow ordinary loofah seeds in seedling substrate and cultivate them in a light incubator until the loofah plants grow to the point between the one-heart stage and the one-leaf-one-heart stage.

[0038] (2) Infection by puncture and smear method: The infection site is from the cotyledon node of the loofah plant to 1 cm below the cotyledon node. Puncture a hole around the infection site and apply Agrobacterium rhizogenes to the puncture site.

[0039] (3) Nodule induction: The loofah plants were further cultured in a light incubator, and swelling of the infected sites was observed.

[0040] (4) Cutting culture to induce hair roots: obliquely cut the swollen part of the infected part of the loofah plant, insert the lower end of the upper plant, i.e. the oblique cut end, into the growth carrier for culture to induce the formation of hair roots.

[0041] In step (1), the seedling substrate can be any suitable seedling substrate, preferably, in step (1), the seedling substrate is a bud seedling substrate.

[0042] In step (1), the culture conditions of the light incubator can be determined as needed. Preferably, in step (1), the culture conditions of the light incubator are: alternating between 15000 lx light at 28℃ for 12h and light avoidance at 22℃ for 12h, with a relative humidity of 60%.

[0043] In step (1), before the sowing at the opening, any suitable steps may be included. Preferably, in step (1), before the sowing at the opening, the common loofah seeds are disinfected and rinsed.

[0044] In step (2), the Agrobacterium rhizogenes can be any suitable Agrobacterium rhizogenes, preferably, in step (2), the Agrobacterium rhizogenes is Agrobacterium rhizogenes Ar1193.

[0045] In step (2), the Agrobacterium rhizogenes Ar1193 may carry any suitable expression vector, more preferably, in step (2), the Agrobacterium rhizogenes Ar1193 carries the binary expression vector pSuper1300FLAG-RUBY containing the RUBY reporter system.

[0046] In step (2), prior to the dip, the Agrobacterium rhizogenes may be subjected to any suitable treatment. Preferably, in step (2), prior to the dip, the Agrobacterium rhizogenes is subjected to streak activation culture.

[0047] In step (3), the specific steps of the continued cultivation can be determined as needed. Preferably, in step (3), the continued cultivation includes: cultivation at 21°C and 95% relative humidity in the dark for 2 days; cultivation at 21°C, 95% relative humidity, 10000 lx light intensity, and 12 h photoperiod for 3 days; and cultivation at 26°C, 10000 lx light intensity, 60% relative humidity, and 12 h photoperiod for 3 days.

[0048] In step (4), the growth carrier can be any suitable growth carrier, preferably, in step (4), the growth carrier is water-soaked rock wool.

[0049] In step (4), the specific steps of the cultivation can be determined as needed. Preferably, in step (4), the cultivation includes: cultivating for 1 day at 26°C, 60% relative humidity, 10000 lx light intensity, and 12h photoperiod until slight wilting; cultivating for 2 days at 26°C, 95% relative humidity, 10000 lx light intensity, and 12h photoperiod; and normally cultivating for 7 days at 26°C, 60% relative humidity, 10000 lx light intensity, and 12h photoperiod.

[0050] To provide a clearer understanding of the technical content of this invention, the following embodiments are provided for detailed description. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: ColdSpring Harbor Laboratory Press, 1989), or as recommended by the manufacturer.

[0051] Example 1: Induction of hairy roots in "Qusi No. 2" loofah

[0052] (1) Plant materials

[0053] The common loofah "Qusi No. 2" (the loofah seeds were bred and provided by Quzhou Municipal Academy of Agricultural and Forestry Sciences).

[0054] (2) Plant culture

[0055] First, select mature, undamaged "Qusi No. 2" loofah seeds. Disinfect the seeds with a 1 / 1000 concentration of potassium permanganate for 15 minutes, rinse 2-3 times with sterile water, and then make a cut on one side of the seed with scissors sterilized with 75% alcohol, taking care not to damage the embryo. Sow the seeds in 50-cell seedling trays filled with Beilei seedling substrate (Jiangsu Beilei Substrate Technology Development Co., Ltd.), cover with a layer of vermiculite, and water thoroughly. Place the sown trays in a light incubator with the following conditions: 28℃ for 12 hours of light at 15000 lx / 22℃ for 12 hours in darkness, relative humidity 60%. Once the seedlings have germinated and reached the first heart (8 days after sowing), they are ready for use.

[0056] (3) Activation culture of Agrobacterium rhizogenes

[0057] Agrobacterium rhizogenes Ar1193 (purchased from Shanghai Weidi Biotechnology Co., Ltd.) was used. The binary expression vector pSuper1300 FLAG-RUBY containing the RUBY reporter system (a gift from Professor Luo Chen of the Chengdu Urban Agriculture Research Institute; this binary expression vector constructs the RUBY reporter system (He, Y., Zhang, T., Sun, H. et al. (2020). A reporter for noninvasively monitoring gene expression and plant transformation. Horticulture Research 7:152) reported in the literature into the EcoRI site of the pSuper1300 FLAG vector (Xu, C., Chang P., Guo S. et al. (2024). Transcriptional activation by WRKY23 and derepression by removal of bHLH041 coordinately establish callus pluripotency in Arabidopsis regeneration. The Plant Cell 36:158=173). The vector map can be found in [link to vector map]. Figure 9(As shown) Transformed into Ar1193, positive clones were selected and stored in 50% glycerol at -80℃ for later use. Before the experiment, glycerol-containing bacteria carrying the binary expression vector pSuper1300 FLAG-RUBY containing the RUBY reporter system, stored at -80℃, were streaked on LB solid medium (containing 50 mg / L Kana) for activation. After 2 days, the activated strains were substrated on LB solid medium (containing 50 mg / L Kana) for propagation and culture for 2 days.

[0058] (4) Infection by Agrobacterium rhizogenes

[0059] First, use the needle of a 1ml syringe to make several holes around the cotyledon node, about 1cm below it. Then, use a cotton swab to apply Ar1193 bacteria carrying the binary expression vector pSuper1300FLAG-RUBY containing the RUBY reporter system from the culture medium to the holes.

[0060] (5) Nodule induction

[0061] Infected loofah seedlings were placed in a light incubator at 21°C and 95% relative humidity for 2 days in the dark. Then, they were cultured at 21°C and 95% relative humidity for 3 days with 10,000 lx light and a 12-hour photoperiod. Finally, they were cultured at 26°C and 60% relative humidity for 3 days with 10,000 lx light and a 12-hour photoperiod to induce root nodules to form at the infected sites.

[0062] (6) Root induction

[0063] Oblique cuts were made at the root nodules, and hairy roots were induced using rock wool as a growth medium. The cut upper part of the loofah plant was inserted into water-soaked rock wool (3cm x 3cm x 4cm) and cultured for 1 day at 26℃, 60% relative humidity, 10000 lx light, and 12h photoperiod until slight wilting occurred. Then, it was cultured for 2 days at 26℃, 95% relative humidity, 10000 lx light, and 12h photoperiod. After that, it was cultured normally for 7 days at 26℃, 60% relative humidity, 10000 lx light, and 12h photoperiod before the positive rate of transgenic hairy roots was counted.

[0064] (7) Control treatment

[0065] Two controls were set up. Control 1 involved direct oblique incision and treatment to induce hair roots. Control 2 involved applying the treatment through punctures without oblique incision to induce hair roots. Specific procedures are described below:

[0066] Control 1: The "Qusi No. 2" loofah seedlings cultivated to the 1-heart stage in step (2) were obliquely cut 1 cm below the cotyledon node for infection. The cut upper part of the loofah plant was inserted into rock wool (3cm x 3cm x 4cm) saturated with the infection solution and cultured for 1 day under the conditions of 26℃, 60% relative humidity, 10000 lx light and 12h photoperiod until slight wilting. Then, it was cultured for 2 days under the conditions of 26℃, 95% relative humidity, 10000 lx light and 12h photoperiod. After that, it was cultured normally for 7 days under the conditions of 26℃, 60% relative humidity, 10000 lx light and 12h photoperiod. The positive rate of transgenic hair roots was counted. The infection solution was activated Ar1193 bacteria (carrying the binary expression vector pSuper1300FLAG-RUBY containing the RUBY reporter system) resuspended in MES and 0.3mM acetylsyringone, with an OD value of 1.0.

[0067] Control 2: The loofah seedlings induced by root nodules in step (5) were not cut obliquely and were cultured as whole plants. The culture conditions at this stage were constant temperature of 26℃, light intensity of 10000lx, photoperiod of 12h, and environmental humidity of 60%. The positive rate of transgenic hairy roots was counted after 7 days.

[0068] (8) Statistics on the positive rate of transgenic hair roots

[0069] The RUBY reporter system couples CYP76AD1, DODA, and GT to an open reading frame. After expression, these genes catalyze the formation of betaine from tyrosine in plants, resulting in a red color under visible light (He, Y., Zhang, T., Sun, H. et al. (2020). A reporter for noninvasively monitoring gene expression and plant transformation. Horticulture Research 7:152). Therefore, visible hair roots are counted as transgenic hair roots. Positive rate % = (Percentage of transgenic hair roots / Total hair roots) × 100.

[0070] (9) Test Results

[0071] In this embodiment, the infection site and the resulting root nodules are shown by puncturing a common loofah. Figure 1 As shown, the oblique cutting induction of hair roots and the results of hair root induction are shown in the figure. Figure 2 As shown. See the comparison results for reference 1. Figure 3 As shown, the results of root development in control 2 are as follows. Figure 4As shown. These results indicate that control 1 treatment can induce root formation, but the positive root rate is low, only 20%, and control 2 did not show any root formation during the observation period. The positive rate of transgenic hairy roots induced in the experimental group "Qusi 2" loofah seedlings was 52.9%, which is 2.3 times the transformation rate reported in pumpkin (Ilina EL, Logachov AA, Laurent L, et al. (2012) Composite Cucurbita pepo plants with transgenic roots as a tool to study root development. Annals of Botany 2:479), and 1.43 times the transformation rate reported in gourd (Wang Ying, Li Yanwei, Huang Lijuan et al., (2020), A method for efficiently inducing transgenic hairy root formation in gourd. Chinese invention patent application CN110885852A).

[0072] Example 2: Induction of hairy roots in "SS-22" loofah

[0073] (1) Plant materials

[0074] The common loofah “SS-22” (the loofah seeds were bred and provided by Quzhou Municipal Academy of Agricultural and Forestry Sciences).

[0075] (2) Plant culture

[0076] First, select mature, undamaged "SS-22" loofah seeds. Disinfect the seeds with a 1 / 1000 concentration of potassium permanganate for 15 minutes, rinse 2-3 times with sterile water, and then make a cut on one side of the seed with scissors sterilized with 75% alcohol, taking care not to damage the embryo. Sow the seeds in 50-cell seedling trays filled with Beilei seedling substrate (Jiangsu Beilei Substrate Technology Development Co., Ltd.), cover with a layer of vermiculite, and water thoroughly. Place the sown trays in a light incubator with the following conditions: 28℃ for 12 hours of light at 15000 lx / 22℃ for 12 hours in darkness, relative humidity 60%. Once the seedlings have germinated and reached the first heart stage (8 days after sowing), they are ready for use.

[0077] (3) Activation culture of Agrobacterium rhizogenes

[0078] Agrobacterium rhizogenes was selected using Ar1193 (purchased from Shanghai Weidi Biotechnology Co., Ltd.). The binary expression vector pSuper1300FLAG-RUBY containing the RUBY reporter system (same as in Example 1) was transformed into Ar1193 via electroporation. Positive clones were selected and stored in 50% glycerol at -80°C for later use. Before the experiment, the glycerol-containing bacteria carrying the binary expression vector pSuper1300FLAG-RUBY, stored at -80°C, were streaked onto LB agar (containing 50 mg / L Kana) for activation. After 2 days, the activated strain was substrated onto LB agar (containing 50 mg / L Kana) for propagation and culture for another 2 days.

[0079] (4) Infection by Agrobacterium rhizogenes

[0080] First, use the needle of a 1ml syringe to make several holes around the cotyledon node. Then, use a cotton swab to apply Ar1193 bacteria carrying the binary expression vector pSuper1300FLAG-RUBY containing the RUBY reporter system from the culture medium to the holes.

[0081] (5) Nodule induction

[0082] Infected loofah seedlings were placed in a light incubator and cultured in the dark at 21°C and 95% relative humidity for 2 days. Then, they were cultured at 21°C and 95% relative humidity, with a light intensity of 10,000 lx and a photoperiod of 12 h for 3 days. Finally, they were cultured at 26°C and 60% relative humidity, with a light intensity of 10,000 lx and a photoperiod of 12 h for 3 days to induce the formation of root nodules at the infected sites.

[0083] (6) Root induction

[0084] Oblique cuts were made at the root nodules, and hairy roots were induced using rock wool as a growth medium. The cut upper part of the loofah plant was inserted into water-soaked rock wool (3cm x 3cm x 4cm) and cultured for 1 day at 26℃, 60% relative humidity, 10000 lx light, and 12h photoperiod until slight wilting occurred. Then, it was cultured for 2 days at 26℃, 95% relative humidity, 10000 lx light, and 12h photoperiod. After that, it was cultured normally for 7 days at 26℃, 60% relative humidity, 10000 lx light, and 12h photoperiod before the positive rate of transgenic hairy roots was counted.

[0085] (7) Control treatment

[0086] Two controls were set up. Control 1 involved direct oblique incision and treatment to induce hair roots. Control 2 involved applying the treatment through punctures without oblique incision to induce hair roots. Specific procedures are described below:

[0087] Control 1: “SS-22” loofah seedlings cultured to the 1-heart stage in step (2) were obliquely cut at the cotyledon node for infection. The cut upper part of the loofah plant was inserted into rock wool (3cm x 3cm x 4cm) saturated with infection solution and cultured for 1 day at 26℃, 60% relative humidity, 10000 lx light, and 12h photoperiod until slight wilting. Then, it was cultured for 2 days at 26℃, 95% relative humidity, 10000 lx light, and 12h photoperiod. After that, it was cultured normally for 7 days at 26℃, 60% relative humidity, 10000 lx light, and 12h photoperiod. The positive rate of transgenic hair roots was then counted. The infection solution was activated Ar1193 bacteria (carrying the binary expression vector pSuper1300FLAG-RUBY containing the RUBY reporter system) resuspended in MES and 0.3mM acetylsyringone, with an OD value of 1.0.

[0088] Control 2: The loofah seedlings induced by root nodules in step (5) were not cut obliquely and were cultured as whole plants. The culture conditions at this stage were constant temperature of 26℃, light intensity of 10000lx, photoperiod of 12h, and environmental humidity of 60%. The positive rate of transgenic hairy roots was counted after 7 days.

[0089] (8) Statistics on the positive rate of transgenic hair roots

[0090] The RUBY reporter system couples CYP76AD1, DODA, and GT to an open reading frame. After expression, these genes catalyze the formation of betalains from tyrosine in plants, resulting in a red color under visible light (He, Y., Zhang, T., Sun, H. et al. (2020). A reporter for noninvasively monitoring gene expression and plant transformation. Horticulture Research 7:152). Therefore, red, visible hair roots are counted as transgenic hair roots. Positive rate % = percentage of transgenic hair roots / total hair roots × 100.

[0091] (9) Test Results

[0092] In this embodiment, the infection site and the resulting root nodules are shown by puncturing a common loofah. Figure 5 As shown, the oblique cutting induction of hair roots and the results of hair root induction are shown in the figure. Figure 6 As shown. See the comparison results for reference 1. Figure 7 As shown, the results of root development in control 2 are as follows. Figure 8As shown. These results indicate that control 1 treatment can induce root formation, but no positive roots were observed, while control 2 did not show any root formation during the observation period. The positive rate of transgenic hairy roots induced in the experimental group "SS-22" loofah seedlings was 58.6%, which is 2.54 times the transformation rate reported in pumpkin (Ilina EL, Logachov AA, Laurent L, et al. (2012) Composite Cucurbita pepoplants with transgenic roots as a tool to study root development. Annals of Botany 2:479), and 1.59 times the transformation rate reported in bottle gourd (Wang Ying, Li Yanwei, Huang Lijuan et al. (2020), A method for efficiently inducing transgenic hairy root formation in bottle gourd. Chinese invention patent application CN110885852A).

[0093] Therefore, this invention cultivates common loofah seeds to a stage between the one-heart and one-leaf-one-heart stages, using the cotyledon node to 1 cm below it as the infection site. Infection is achieved through a puncture-and-smear method, followed by root nodule induction. Hairy roots are induced by cutting and culturing at the nodule location. This process can induce the formation of numerous hairy roots, making it applicable to promoter function research, functional gene analysis, and secondary metabolite research.

[0094] In summary, the non-tissue culture-induced hairy root formation method of the present invention can induce the formation of hairy roots of transgenic common loofah with a high induction rate. It can be applied to promoter function research, functional gene analysis and secondary metabolite research. The method is unique in conception and ingenious in design. After infection by puncturing and smearing, the root nodules are induced and then obliquely cut to culture the hairy roots, which can form a large number of hairy roots and is suitable for large-scale promotion and application.

[0095] In this specification, the invention has been described with reference to specific embodiments thereof. However, it will be apparent that various modifications and variations can be made without departing from the spirit and scope of the invention. Therefore, the specification and drawings should be considered illustrative rather than restrictive.

Claims

1. A method for inducing transgenic hairy root formation in Lagenaria vulgaris by non-tissue culture, characterized in that, The method comprises the following steps: (1) common lagenaria siceraria culture: the common lagenaria siceraria seeds are sowed in a seedling substrate, and are cultured in an illumination incubator until the lagenaria siceraria plant grows to a stage between one heart stage and one leaf one heart stage; (2) hole-punching and smearing method infection: the cotyledon node of the lagenaria siceraria plant to 1cm below the cotyledon node is taken as an infection site, a hole is punched around the infection site, and the Agrobacterium rhizogenes is smeared on the hole-punching position; (3) nodule induction: the lagenaria siceraria plant is continuously cultured in the illumination incubator, and it is observed that the infection site is swollen; (4) cutting culture to induce hairy roots: the swollen position of the infection site of the lagenaria siceraria plant is obliquely cut, the lower end of the obtained upper plant, i.e. the obliquely cut end, is inserted into a growth carrier for culture to induce the formation of hairy roots. In the step (2), the Agrobacterium rhizogenes is Agrobacterium rhizogenes Ar1193, and the Agrobacterium rhizogenes Ar1193 carries a binary expression vector pSuper1300FLAG-RUBY containing a RUBY reporter system; In the step (3), the continuous culture comprises the following steps: 21℃, 95% relative humidity, and dark culture for 2d; 21℃, 95% relative humidity, 10000 lx illumination, and 12h light cycle culture for 3d; 26℃, 60% relative humidity, 10000 lx illumination, and 12h light cycle culture for 3d; In the step (4), the culture comprises the following steps: 26℃, 60% relative humidity, 10000 lx illumination, and 12h light cycle culture for 1d to light wilting; 26℃, 95% relative humidity, 10000 lx illumination, and 12h light cycle culture for 2d; 26℃, 60% relative humidity, 10000 lx illumination, and 12h light cycle normal culture for 7d.

2. The method for inducing the formation of transgenic hairy roots in common loofah without tissue culture according to claim 1, characterized in that, In the step (1), the seedling substrate is a bud seedling substrate.

3. The method for inducing the formation of transgenic hairy roots in common loofah without tissue culture according to claim 1, characterized in that, In the step (1), the culture condition of the illumination incubator is that 28℃, 15000 lx illumination for 12h and 22℃, dark for 12h are alternately performed, and the relative humidity is 60%.

4. The method of claim 1, wherein the non- tissue culture induction of hairy roots formation in Lagenaria vulgaris is characterized by, In the step (1), before the sowing, the common lagenaria siceraria seeds are subjected to disinfection and washing.

5. The method of claim 1, wherein the non- tissue culture induction of hairy roots formation in Lagenaria vulgaris is characterized by, In the step (2), before the smearing, the Agrobacterium rhizogenes is subjected to streak activation culture.

6. The method of claim 1, wherein the non- tissue culture induction of hairy roots formation in Lagenaria vulgaris is characterized by, In the step (4), the growth carrier is water-soaked rock wool.

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