Method for establishing cannabis sativa genetic transformation system based on mature seed bacterial liquid germination method

Abstract

The invention discloses a method for establishing a cannabis sativa genetic transformation system based on a mature seed bacterial liquid germination method. The method comprises the following steps: step 1, disinfecting and germinating seeds; step 2, preparing an engineering bacteria solution; step 3, infection co-culture; step 4, restoring culture; step 5, screening and culturing; and step 6, rooting culture. According to the method, BMET5 serves as a carrier, mature seeds are germinated through engineering bacterium liquid, then cotyledonary nodes are prepared from the germinated seeds and subjected to secondary infection through the engineering bacterium liquid, operation is easy, the transformation efficiency can reach 14.63%, and the method has important significance in targeted precise breeding of China-hemp, rapid obtaining of germplasm resources with target characters and making of a strong China-hemp seed industry chip. According to the invention, a key technical means is provided for cannabis sativa genetic transformation, a cannabis sativa genetic transformation method is expanded, ruby expression is provided to confirm stable integration of T-DNA in a cannabis sativa genome, and a foundation is laid for cannabis sativa transgenic research and commercial application.

Description

Technical Field

[0001] The present invention relates to a method for constructing a genetic transformation system for cannabis, and more particularly to a method for establishing a genetic transformation system for cannabis based on the method of germinating with bacterial liquid of mature seeds. Background Art

[0002] Cannabis sativa belongs to the Cannabaceae family of plants, and its relaxing properties have a history of over 2,700 years. In the past decade, there has been a renewed and intense interest in the medical effects of cannabinoids. Although cannabis is known for the hallucinogenic effects of its main component, tetrahydrocannabinol (THC), it also contains a variety of phytocannabinoids with therapeutic potential, which can be used to treat a variety of human diseases, from complex neurological disorders to cancer, such as cannabidiol (CBD), cannabinol (CBN), cannabigerol (CBG), and cannabichromene (CBC). In addition, its stem contains high-quality cellulose, its seeds contain high-quality oil, and its inflorescence contains valuable resin. A variety of products can be derived from the stems, seeds, and inflorescences of cannabis, from specialty pulp and paper to nutritional foods, pharmaceuticals, and cosmetics.

[0003] Tissue culture has been widely used to advance plant research through transgenic and gene editing technologies. Although successful cases of transforming seedling regeneration have been reported in some species, for Cannabis sativa, this remains a bottleneck for obtaining transgenic or gene-edited plants. Only the hairy roots of cannabis have been shown to be susceptible to infection by Aeromonas rhizogenes, which can induce the culture of transformed roots in a short time. Until recently, Deguchi et al. and Sorokin et al. detected transient GUS expression and PDS gene silencing in cannabis tissues using Agrobacterium or Agrobacterium-mediated transformation techniques. Ahmed et al. used a nanoparticle method to overexpress multiple genes simultaneously in transient transformation of cannabis, as an effective vector for introducing foreign DNA fragments. Although transient expression analysis can be used to study gene function, a stable transformation protocol remains the basis for enhancing the phenotype of cannabis through genetic engineering. Since hemp seedlings and explants are not easily transformed, and the regeneration of transgenic plants from transformed cells is a time-consuming process, usually limited by the characteristics of hemp varieties. Zhang Xiaoyu et al. found that by optimizing explants, varieties, developmental regulators, and gene editing tools, a set of Agrobacterium-mediated protocols suitable for cannabis was developed, in which the CSGRF3–CSGIF1 chimera performed best among all regulators, with a regeneration efficiency of only 13.43%. 。 Therefore, it is particularly important to develop a genetic transformation method suitable for the characteristics of hemp crops and with high regeneration efficiency. Summary of the Invention

[0004] The present invention provides a method for establishing a genetic transformation system of cannabis based on the germination method of mature seed bacterial liquid. This method uses BMET5 as a vector, germinates mature seeds with engineering bacterial liquid, then prepares cotyledon nodes with the germinated seeds and performs secondary infection on them with engineering bacterial liquid, obtaining a transformation efficiency of 14.63%. The present invention can play an important role in the functional genomics of hemp genes and release the true potential of gene editing in improving hemp varieties in production.

[0005] The object of the present invention is achieved through the following technical solutions:

[0006] A method for establishing a genetic transformation system of cannabis based on the germination method of mature seed bacterial liquid, comprising the following steps:

[0007] Step 1, seed disinfection and germination:

[0008] Select mature, plump, and shiny industrial hemp seeds, dry them and soak them in water for 30 min. After fishing them out, peel the seeds, disinfect them with hypochlorous acid, wash them with sterile water, and then place them on a germination medium. Culture them at a temperature of 24 ± 1 °C under dark conditions for 3 - 4 d. The formula of the germination medium is: MS + 100 μmol / L AS + 30 g / L sucrose, pH = 5.8;

[0009] Step 2, preparation of engineering bacterial liquid:

[0010] Coat Agrobacterium BMET5 carrying the plasmid of the target gene on an LB solid medium for activation. After culturing for 48 h, collect the bacterial cells in an infection liquid, vortex and mix well, and adjust the OD of the bacterial liquid to 0.4 - 0.6 with a spectrophotometer for standby. The formula of the infection liquid is: MS + 100 μmol / L AS + 30 g / L sucrose, pH = 5.8;

[0011] Step 3, infection and co - cultivation:

[0012] Absorb the moisture of the disinfected seeds on sterile filter paper, then pick them into the prepared engineering bacterial liquid, infect for 30 min, discard the engineering bacterial liquid, and then place the seeds on sterile filter paper added with engineering bacterial liquid for co - cultivation at 23 °C in the dark. On the 4th day, take out the co - cultivated explants to see weak light. After the explants turn yellow, remove 1 / 3 of the cotyledons from the tail, then remove half of the cotyledons from the hypocotyl connection, remove two true leaves, and scratch at the growth point. After preparation, continue to infect in the engineering bacterial liquid for 30 min, discard the engineering bacterial liquid, dry the explants on sterile filter paper, and then place the explants on a co - cultivation medium for co - cultivation at 23 °C in the dark for 4 days. The formula of the co - cultivation medium is: MS + 0.1 - 0.4 mg / L TDZ + 0.05 - 0.1 mg / L NAA + 100 μmol / L AS + 30 g / L sucrose + 7 g / L agar, pH = 5.8;

[0013] Step 4, Recovery culture:

[0014] Transfer the explants after co-culture to a recovery medium for 7 days of recovery culture under light at 25 ± 1°C until small buds grow out. The formula of the recovery medium is: MS + 0.1 - 0.4 mg / L TDZ + 0.05 - 0.1 mg / L NAA + 0.2 g / L tim (Timentin) + 30 g / L sucrose + 7 g / L agar, pH = 5.8;

[0015] Step 5, Screening culture:

[0016] Select the explants with adventitious buds grown after recovery culture, cut off the tip of the earliest-growing and middle small bud to promote the growth of lateral buds, place them on a screening medium, and culture under light at 25 ± 1°C until resistant buds grow out. The formula of the screening medium is: MS + 0.1 - 0.4 mg / L TDZ + 0.05 - 0.1 mg / L NAA + 0.2 g / L tim (Timentin) + 2 mg / L Basta + 30 g / L sucrose + 7 g / L agar, pH = 5.8;

[0017] Step 6, Rooting culture:

[0018] Cut the resistant seedlings together with a little basal callus and place them on a rooting medium for rooting culture. The formula of the rooting medium is: MS + 0.1 - 1 mg / L IBA + 30 g / L sucrose + 0.1 - 1 g / L activated carbon + 7 g / L agar, pH = 5.8.

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] 1. The operation of the present invention is simple, which can effectively improve the genetic transformation efficiency of cannabis. The transformation efficiency can reach 14.63%, and it has important significance for the targeted and precise breeding of hemp, quickly obtaining germplasm resources with target traits, and strengthening the hemp seed industry chip.

[0021] 2. The present invention provides a key technical means for the genetic transformation of cannabis, expands the genetic transformation method of cannabis, and the confirmation of ruby expression proves the stable integration of T-DNA in the cannabis genome, laying a foundation for the research and commercial application of cannabis transgenic technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a step decomposition diagram for establishing a genetic transformation system of cannabis based on the method of germinating with mature seed bacterial liquid;

[0023] Figure 2The flow chart for establishing a genetic transformation system of cannabis based on the germination method of mature seed bacterial liquid. a: Bacterial liquid germination; b: The 4th day of bacterial liquid germination; c: The 4th day of cotyledon node injection co-culture prepared from bacterial liquid germination; d: Recovery culture; e: Screening culture; f: Rooting culture;

[0024] Figure 3 The red-phenotype seedlings that appeared in the experiment of transforming industrial hemp by the cotyledon node injection method with the germinated bacterial liquid of BMET5 mature seeds. Specific implementation mode

[0025] The technical solution of the present invention will be further described below in conjunction with the accompanying drawings, but it is not limited thereto. Any modification or equivalent replacement of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention shall be covered by the protection scope of the present invention.

[0026] The present invention provides a method for establishing a genetic transformation system of cannabis based on the germination method of mature seed bacterial liquid. As Figure 1 and Figure 2 shown, the method includes the following steps:

[0027] Step 1: Seed disinfection and germination:

[0028] Select mature, plump and shiny industrial hemp seeds. Take out the seeds to be disinfected from the refrigerator and dry them in an oven at 45°C overnight (about 20 hours). Take out the seeds from the oven, soak them in water for 30 minutes, gently press them open along the side edges with your fingers after fishing them out, peel out the seeds, disinfect them with hypochlorous acid (brand: Life Mystery) for 2 hours, wash them 4 - 5 times with sterile water, and then culture them under the conditions of 24 ± 1°C and darkness for 3 - 4 days. Count the seed germination, contamination and aseptic seedling status of each treatment.

[0029] Step 2: Preparation of engineering bacterial liquid:

[0030] Coat the Agrobacterium tumefaciens BMET5 carrying the plasmid of the target gene on the LB solid medium for activation. After culturing for 48 hours, collect the bacterial cells in the infection solution, vortex and mix well, and adjust the OD of the bacterial liquid to 0.4 - 0.6 with a spectrophotometer for standby.

[0031] Step 3: Infection and co-culture:

[0032] Dry the sterilized seeds on sterile filter paper, then pick them into the prepared engineering bacterial solution, infect for 30 minutes, discard the engineering bacterial solution, and then place the sterilized seeds on sterile filter paper with engineering bacterial solution to germinate in a dark environment at 23°C. On the 4th day, take out the explants after co-cultivation and see that the explants turn yellow under weak light. Remove 1 / 3 of the cotyledons from the tail, then remove half of the cotyledons from the connection of the hypocotyl, remove two true leaves, and scratch at the growth point. After scratching, continue to infect in the engineering bacterial solution for 30 minutes, discard the engineering bacterial solution, dry the explants on sterile filter paper, and then place the explants on the co-cultivation medium (put a filter paper on the co-cultivation medium to prevent excessive proliferation of Agrobacterium), and co-cultivate at 23°C in the dark for 4 days.

[0033] Step 4: Resume culture:

[0034] After co-cultivation, the explants were transferred to the recovery medium for 7 days of recovery culture, and light cultured at 25±1°C (16 h / d light and 8 h / d) until small buds grew.

[0035] Step 5: Screening and culture:

[0036] Select the explants that grow buds after recovery culture, cut off the top of the small bud located in the middle that grows earliest, promote the growth of lateral buds, place them on the screening medium, and culture them in light at 25±1℃ (16h / d light, 8h / d), and screen until resistant buds grow.

[0037] Step 6: Rooting culture:

[0038] Cut the resistant seedlings together with a little basal callus and place them on rooting medium for rooting culture. It will take about 3 weeks for roots to form. The rooting culture is carried out in a photoculture environment at 25±1℃ (16h / d light and 8h / d).

[0039] Figure 3 These are red seedlings that appeared in the hemp transformation experiment using BMET5 mature seedlings and cotyledon node injection. Figure 3 It can be seen that the ruby ​​gene carried by the BMET5 vector has been successfully transferred into hemp and expressed.

[0040] Table 1 Medium formula for mature seed liquid germination method

[0041]

Claims

1. A method for establishing a genetic transformation system of cannabis based on the germination method of mature seed bacterial liquid, characterized in that The method comprises the following steps: Step 1: Seed disinfection and germination: Select mature, plump and shiny industrial hemp seeds, dry them and soak them in water for 30 minutes, remove them and peel the seeds, disinfect them with hypochlorous acid, wash them with sterile water, and place them on the germination medium, and culture them at a temperature of 24±1℃ and in the dark for 3-4 days; Step 2: Preparation of engineering bacterial solution: Agrobacterium BMET5 carrying the target gene plasmid was spread on LB solid medium for activation. After 48 hours of culture, the bacteria were collected in the infection solution, vortexed and mixed, and the bacterial solution OD was adjusted to 0.4-0.6 by spectrophotometer for later use; Step 3, infection and co-cultivation: Dry the sterilized seeds on sterile filter paper, then pick them into the prepared engineering bacterial solution, infect for 30 minutes, discard the engineering bacterial solution, and then place the seeds on sterile filter paper with engineering bacterial solution for co-cultivation at 23°C in the dark. On the 4th day, take out the explants after co-cultivation and see that the explants turn yellow under weak light. Remove 1 / 3 of the cotyledons from the tail, then remove half of the cotyledons from the connection of the hypocotyl, remove two true leaves, and scratch at the growth point. After preparation, continue to infect in the engineering bacterial solution for 30 minutes, discard the engineering bacterial solution, dry the explants on sterile filter paper, and then place the explants on the co-cultivation medium and co-cultivate at 23°C in the dark for 4 days. Step 4: Resume culture: After co-cultivation, the explants were transferred to the recovery medium for 7 days under light culture at 25±1℃ until small buds grew. Step 5: Screening and culture: Select the explants that grow buds after recovery culture, cut off the top of the small bud located in the middle that grows earliest, promote the growth of lateral buds, place them on the screening medium, culture them in light at 25±1℃, and screen until resistant buds grow; Step 6: Rooting culture: The resistant seedlings together with a little basal callus were cut off and placed on rooting medium for rooting culture.

2. The method for establishing a genetic transformation system of cannabis based on the germination method of mature seed bacterial liquid according to claim 1, wherein The germination medium formula is: MS+100 μmol / LAS+30 g / L sucrose, pH=5.

8.

3. The method for establishing a genetic transformation system of cannabis based on the germination method of mature seed bacterial liquid according to claim 1, characterized in that The formula of the infection solution is: MS+100 μmol / LAS+30 g / L sucrose, pH=5.

8.

4. The method for establishing a genetic transformation system of cannabis based on the germination method of mature seed bacterial liquid according to claim 1, wherein The co-culture medium formula is: MS+0.1-0.4 mg / LTDZ+0.05-0.1 mg / LNAA+100 μmol / LAS+30 g / L sucrose+7 g / L agar, pH=5.

8.

5. The method for establishing a genetic transformation system of cannabis based on the germination method of mature seed bacterial liquid according to claim 1, wherein The recovery culture medium formula is: MS+0.1-0.4 mg / LTDZ+0.05-0.1 mg / LNAA+0.2 g / Ltim+30 g / L sucrose+7 g / L agar, pH=5.

8.

6. The method for establishing a genetic transformation system of cannabis based on the germination method of mature seed bacterial liquid according to claim 1, characterized in that The screening culture medium formula is: MS+0.1-0.4 mg / LTDZ+0.05-0.1 mg / L NAA+0.2 g / L tim+2 mg / L Basta+30 g / L sucrose+7 g / L agar, pH=5.

8.

7. The method for establishing a genetic transformation system of cannabis based on the mature seed bacterial liquid germination method according to claim 1, wherein The rooting culture medium formula is: MS+0.1-1 mg / L IBA+30 g sucrose / L+0.1-1 g / L activated carbon+7 g / L agar, pH=5.8.

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