Method for establishing cannabis sativa genetic transformation system based on cotyledonary node injection method

Abstract

The invention discloses a method for establishing a cannabis sativa genetic transformation system based on a cotyledonary node injection method. The method comprises the following steps: step 1, disinfecting and germinating seeds; step 2, preparing an explant; step 3, preparing an engineering bacteria solution; step 4, infection co-culture; step 5, restoring culture; step 6, screening and culturing; and step 7, rooting culture. According to the method, the heredity transformation efficiency of the cannabis sativa can be improved to 6.38%, the obtained positive seedlings can normally grow, the ruby expression is intuitive, the transformation efficiency can be conveniently counted, a key technical means is provided for heredity transformation of the cannabis sativa, and heredity transformation methods of the cannabis sativa are expanded. In addition, ruby expression proves that T-DNA is stably integrated in a hemp genome, and a solid foundation is laid for transgenic research and commercial application of hemp.

Description

Technical Field

[0001] The present invention relates to a method for constructing a genetic transformation system of cannabis, and particularly to a method for establishing a genetic transformation system of cannabis based on the cotyledon node injection method. Background Art

[0002] The cultivation of transgenic plants is an elusive important milestone in the breeding field of cannabis (Cannabis sativa L.). Although it has economic value, there are currently very few programs for cultivating transgenic cannabis plants. Some studies have shown that without co-cultivation with Agrobacterium, the regeneration rate of hypocotyls (53.3%) is higher than that of cotyledons (18.1%). On the other hand, under co-cultivation with 100 mg / L kanamycin, the regeneration rate of hypocotyls is 63.3%, the natural rooting rate of regenerated shoots is 12.2%, and the albino rate is 7.1%. After co-cultivation with Agrobacterium and cultivation in a selective regeneration medium containing antibiotics, the highest regeneration rate of hypocotyls is 23.1%, while the regeneration rate of cotyledons is 1.0%. In addition, new plant breeding technologies such as genome editing provide new ways for cultivating cannabis plants for medical or industrial use. One of the most important current challenges is to develop cannabis plant varieties resistant to biotic and abiotic stresses, as well as varieties with specific cannabinoid compositions. Summary of the Invention

[0003] The present invention provides a method for establishing a genetic transformation system of cannabis based on the cotyledon node injection method. This method uses the cotyledon nodes of cannabis as explants, injects the growth point with a syringe for transformation, and uses rbcs-ruby-wus-ipt for cotyledon node injection transformation. Without adding a screening agent, red phenotypes can be observed in both the red seedling stems and the bud centers, and the red seedlings can grow normally. The leaves of the red seedlings are relatively pointed and thin, and the seedlings have many branches.

[0004] The object of the present invention is achieved by the following technical solutions:

[0005] A method for establishing a genetic transformation system of cannabis based on the cotyledon node injection method, comprising the following steps:

[0006] Step 1, seed disinfection and germination:

[0007] Select mature, plump, and shiny industrial cannabis seeds, dry them and soak them in water for 30 min. After fishing them out, peel the seeds, disinfect them and place them on a germination medium, and culture them at a temperature of 24 ± 1 °C under dark conditions for 3 - 4 d. The formula of the germination medium is: MS + 30 g / L sucrose + 7 g / L agar, pH = 5.8;

[0008] Step 2, preparation of explants:

[0009] After the seeds germinate, wait until the radicle and hypocotyl grow 0.5-1 cm for explant preparation. 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.

[0010] Step 3: Preparation of engineered bacterial solution:

[0011] Agrobacterium carrying the rbcs-ruby-wus-ipt 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 OD600 was adjusted to 0.4-0.6 by spectrophotometer for standby use. The infection solution formula was: MS + 0.1-0.4 mg / L TDZ + 0.05-0.1 mg / L NAA + 100 μmol / LAS + 30 g / L sucrose, pH = 5.8;

[0012] Step 4: Infection and co-cultivation:

[0013] The prepared explants were picked up into the prepared engineering bacterial solution, and the bacterial solution was sucked up with a disposable syringe and injected at the growth point of the cotyledon node. After the injection, the infection was continued in the bacterial solution for 30 minutes, and the infection solution was discarded. The explants were dried on sterile filter paper, and then the explants were placed on the co-culture medium and co-cultured at 23°C in the dark for 4 days. The co-culture medium formula was: MS + 0.1-0.4 mg / L TDZ + 0.05-0.1 mg / L NAA + 100 μmol / LAS + 30 g / L sucrose + 7 g / L agar, pH = 5.8;

[0014] Step 5: Resume culture:

[0015] After co-cultivation, the explants were transferred to the recovery medium for 7 days of recovery culture, and light cultured at 25±1°C until small buds grew. The recovery medium formula was: MS+0.2g / Ltim (Timentin)+30g / L sucrose+7g / L agar, pH=5.8;

[0016] Step 6: Screening and culture:

[0017] After the recovery culture, the tip of the first small bud growing from the bud-producing explant was cut off to promote the growth of lateral buds, and the buds were placed on the screening medium and cultured under light at 25±1°C until resistant buds grew out. The screening medium formula was: MS+0.2g / L tim (timentin)+30g / L sucrose+7g / L agar+50mg / L Kana, pH=5.8;

[0018] Step 7: Rooting culture:

[0019] The resistant seedlings, together with a little basal callus, were cut and placed on a rooting medium for rooting culture. The formula of the rooting medium was: MS + 1 mg / L IBA + 30 g sucrose / L + 1 g / L activated carbon + 7 g / L agar, pH = 5.8.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] The present invention can increase the genetic transformation efficiency of cannabis to 6.38%, and the obtained positive seedlings can grow normally. The ruby expression is intuitive and convenient for statistical analysis of the transformation efficiency, providing a key technical means for cannabis genetic transformation and expanding the methods of cannabis genetic transformation. In addition, the ruby expression confirms the stable integration of T-DNA into the cannabis genome, laying a solid foundation for cannabis transgenic research and its commercial applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a step decomposition diagram for establishing a cannabis genetic transformation system based on the cotyledon node injection method;

[0023] Figure 2 It is a flow chart for establishing a cannabis genetic transformation system based on the cotyledon node injection method, where a: seed disinfection; b: seed germination; c: after co-culture of cotyledon node injection; d: recovery culture; e: screening; f: rooting;

[0024] Figure 3 It is the red phenotype that appears in the transformation of Cannabis sativa cv. Hanma No. 3 by the rbcs-ruby-wus-ipt cotyledon node method. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0025] The technical solutions of the present invention will be further described below in conjunction with the accompanying drawings, but are not limited thereto. Any modification or equivalent replacement of the technical solutions of the present invention without departing from the spirit and scope of the technical solutions 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 cannabis genetic transformation system based on the cotyledon node injection method, 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 cannabis seeds. Take the seeds to be disinfected out of the refrigerator and dry them in an oven at 45 °C overnight (about 20 hours). Take the seeds out of the oven and soak them in water for 30 min. After fishing them out, gently press them open along the side edges with your fingers to peel out the seeds. After disinfection, culture them at a temperature of 24 ± 1 °C in the dark for 3 - 4 d, and count the seed germination, contamination, and sterile seedling status of each treatment.

[0029] Step 2: Preparation of explants:

[0030] The seed germination time is generally 3 to 4 days. During the culture process, it is necessary to observe the seed germination and contamination in time. After the seeds germinate, wait until the radicle and hypocotyl grow about 0.5 cm before preparing the explants. 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.

[0031] Step 3: Preparation of engineered bacterial solution:

[0032] The Agrobacterium 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 OD600 was adjusted to 0.4-0.6 by spectrophotometer for later use.

[0033] Step 4: Infection and co-cultivation:

[0034] The prepared explants were picked up into the prepared engineering bacterial solution, and the engineering bacterial solution was drawn up with a disposable syringe and injected at the growth point of the cotyledon node. After the injection, the infection was continued in the engineering bacterial solution for 30 minutes, the infection solution was discarded, and the explants were dried on sterile filter paper. The explants were then placed on the co-cultivation medium (a piece of filter paper was placed on the co-cultivation medium to prevent excessive proliferation of Agrobacterium), and co-cultivated at 23°C in the dark for 4 days.

[0035] Step 5: Resume culture:

[0036] 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.

[0037] Step 6: Screening and culture:

[0038] 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.

[0039] Step 7: Rooting culture:

[0040] 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).

[0041] Table 1 Medium formula for cotyledon node injection method

[0042]

[0043] Refer to the research paper titled "Exploring an economic and highly efficient genetic transformation and genome - editing system for radish through developmental regulators and visible reporter" published by the Radish Genetic Breeding Team of the National Key Laboratory of Crop Genetics and Germplasm Enhancement / College of Horticulture, Nanjing Agricultural University. A rbcs - ruby - wus - ipt (eukaryotic kana resistance) vector was constructed. When using this vector for cotyledon node injection transformation, it was found that the obvious chimerism in the red phenotype decreased, and the red seedlings showed obvious proliferative growth, with both the stem and the growing point being red.

[0044] Table 2 Transformation of hemp by cotyledon node injection method

[0045]

[0046]

[0047] Figure 3 This is the red phenotype that appears in the cotyledon node method of rbcs - ruby - wus - ipt transformation of hemp No. 3. Figure 3 It can be seen that by transforming the cotyledon nodes of hemp with rbcs - ruby - wus - ipt and injecting and infecting them, red seedlings with a ruby phenotype can be obtained, and Figure 3 In the third picture, it can be seen that the seedlings have many red phenotypes and many branches, presumably due to the role of wus - ipt.

Claims

1. A method for establishing a genetic transformation system of cannabis based on the cotyledon node injection method, 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, sterilize them and place them on germination medium, and culture them at 24±1℃ and dark conditions for 3-4 days; Step 2: Preparation of explants: After the seeds germinate, wait until the radicle and hypocotyl grow 0.5-1 cm for explant preparation. 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. Step 3: Preparation of engineered bacterial solution: Agrobacterium carrying the rbcs-ruby-wus-ipt 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 OD600 was adjusted to 0.4-0.6 by spectrophotometer for later use; Step 4: Infection and co-cultivation: The prepared explants were placed in the prepared engineering bacterial solution, and the bacterial solution was drawn up with a disposable syringe and injected at the growth point of the cotyledon node. After the injection, the explants were infected in the bacterial solution for 30 minutes, the infection solution was discarded, the explants were dried on sterile filter paper, and then the explants were placed on the co-culture medium and co-cultured at 23°C in the dark for 4 days. Step 5: 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 6: 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 7: 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 cannabis genetic transformation system based on the cotyledon node injection method according to claim 1, wherein The germination medium formula is: MS+sucrose 30g / L+agar 7g / L, pH=5.

8.

3. The method for establishing a cannabis genetic transformation system based on the cotyledon node injection method according to claim 1, wherein The formula of the infection solution is: MS+0.1-0.4 mg / L TDZ+0.05-0.1 mg / L NAA+100 μmol / LAS+30 g / L sucrose, pH=5.

8.

4. The method for establishing a cannabis genetic transformation system based on the cotyledon node injection method according to claim 1, wherein The co-culture medium formula is: MS+0.1-0.4 mg / L TDZ+0.05-0.1 mg / L NAA+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 cotyledon node injection method according to claim 1, wherein The recovery medium formula is: MS+0.2g / L tim+30g / L sucrose+7g / L agar, pH=5.

8.

6. The method for establishing a cannabis genetic transformation system based on the cotyledon node injection method according to claim 1, wherein The screening culture medium formula is: MS+0.2g / L tim+30g / L sucrose+7g / L agar+50mg / L Kana, pH=5.

8.

7. The method for establishing a cannabis genetic transformation system based on the cotyledon node injection method according to claim 1, characterized in that The rooting medium formula is: MS+1mg / L IBA+30g sucrose / L+1g / L activated carbon+7g / L agar, pH=5.8.

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