A method for constructing a direct induction of adventitious root regeneration pathway of Idesia polycarpa Maxim. leaf

Abstract

The application discloses a kind of construction methods of Idesia leaf direct induction adventitious root regeneration pathway, comprising the following steps: obtaining explant, cutting treatment aseptic leaf;Leaf base is inserted or leaf block is laid on adventitious root induction medium, first dark culture 3~7 days are induced to adventitious root under light transfer;After treatment, leaf without petiole, internal incision or unincision leaf block is laid on adventitious root induction medium, first dark culture 3~7 days are transferred to light culture induction to generate adventitious root.The application directly uses leaf as explant to directly induce adventitious root, without passing through callus induction stage, can shorten Idesia regeneration culture period, simultaneously reduce the mutation probability generated in somatic mutation during callus subculture process, improve the genetic stability of regenerated plant, can provide technical support for gene function research, genetic improvement and excellent seedling scale production of Idesia.

Description

Technical Field

[0001] This invention relates to the field of plant cell engineering technology, and in particular to a method for constructing a pathway for the direct induction of adventitious root regeneration from leaves of the Chinese tallow tree. Background Technology

[0002] Shantongzi( Idesia polycarpa Maxim., a high-yield woody oilseed tree species unique to my country, is known as the "oil depot on the tree." It has multiple values, including ecological restoration and edible oil development. It can be planted on a large scale on barren mountains and slopes without occupying arable land. It is a key strategic woody oilseed tree species in the national development plan, and there is an urgent need for industrial scale.

[0003] Currently, the propagation of *Vernicia fordii* relies heavily on seed sowing, which presents challenges such as difficulty in distinguishing male and female seedlings during the seedling stage and the need for 4-5 years after fruiting to differentiate between male and female plants. This can easily lead to an imbalance in the male-to-female ratio after afforestation, seriously hindering the efficient development of the *Vernicia fordii* industry.

[0004] Therefore, establishing a stable and efficient in vitro regeneration system is the foundation for the asexual propagation of superior seedlings of *Vernicia fordii*, the early fixation of sex, and the conduct of gene function research.

[0005] Existing regeneration pathways for *Vernicia fordii* mainly involve callus induction and redifferentiation. This pathway requires first inducing callus formation, followed by callus subculture and adventitious shoot induction, which has problems such as long culture cycles and high probability of somatic cell mutation.

[0006] Regarding the construction of regeneration systems through direct organogenesis pathways, there are currently only reports of inducing adventitious buds directly through petioles, and no reports of directly inducing adventitious roots from leaves and then inducing adventitious buds to achieve complete plant regeneration.

[0007] Therefore, in view of the problems in the prior art, a method is urgently needed to solve the technical problem that requires the art to provide a method that can directly induce adventitious root redifferentiation from leaf explants to obtain adventitious shoots without the need for a callus induction stage, which can effectively shorten the culture cycle and reduce the probability of mutation. Summary of the Invention

[0008] In view of this, the present invention provides a method for constructing a direct induced adventitious root regeneration pathway for *Vernicia fordii* leaves, which can establish an efficient and stable regeneration system for *Vernicia fordii*.

[0009] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: A method for constructing a pathway for direct induction of adventitious root regeneration from *Vernicia fordii* leaves includes the following steps: (1) Obtaining explants There are three ways to obtain it; choose one of them: Method 1: Select branches from superior male and female *Vernicia fordii* plants and bring them back to the laboratory for hydroponics, aeroponics, substrate culture, aseptic culture, etc. The newly sprouted leaves are used as explants. Method 2: After removing the surface wax from *Vernicia fordii* seeds and performing low-temperature stratification, disinfect them with 75% alcohol for 2 minutes, rinse 2-3 times with sterile water, then disinfect them with 0.1% mercuric chloride solution for 15 minutes, rinse 4-6 times with sterile water, and then inoculate them onto blank MS medium plates for aseptic germination. After culturing at 25℃ for 15-30 days, use leaves from sterile seedlings as explants. The blank MS medium plates contain MS medium powder and agar, with a pH of 5.8. Method 3: Aseptic seedlings of *Vernicia fordii* need to be subcultured regularly. During the subculture process, excess leaves need to be pruned. The conventional practice is to discard the excess leaves directly. Therefore, the excess sterile leaves pruned during the subculture process can be used as explants. (2) Pretreatment of explants: After obtaining sterile leaves, the sterile leaves are cut. (3) Induction of adventitious roots: The leaves were inserted at the base and the leaf pieces were laid flat on the adventitious root induction medium. They were first cultured in the dark for 3-7 days and then transferred to light for culture to induce the production of adventitious roots. (4) Induction of adventitious buds: Leaves without petioles, or leaf pieces with internal scratches or no scratches, after the above treatment, were laid flat on an adventitious bud induction medium and cultured in the dark for 3-7 days before being transferred to light to induce the generation of adventitious buds.

[0010] Preferably, in step (2), the cutting process is as follows: a complete leaf without petiole, a complete leaf with a petiole of 0.5 cm in length, a complete leaf with a petiole of 1.0 cm in length, or a leaf piece of 0.5~1.0 × 0.5~1.0 cm without internal scoring. 2 Leaves with 2-4 incisions (0.5-1.0 x 0.5-1.0 cm) without damaging the main vein are cut. 2 .

[0011] Preferably, in steps (3) and (4), the conditions for light-induced culture are: a photoperiod of 12 h of light and 12 h of darkness, and a light intensity of 1500~3000 lux.

[0012] Preferably, in step (3), the adventitious root induction medium is: 1 / 4 MS medium + 0.01~4.0 mg / L IBA + 5~30 g / L sucrose + 4~8 g / L carrageenan, pH of the culture medium is 5.4~6.2.

[0013] Preferably, step (4) further includes: when the adventitious bud grows to 0.5-1.0 cm, cutting off a single adventitious bud and inoculating it into a proliferation medium for propagation; after the adventitious bud grows out, cutting off an adventitious bud block with 1-2 growth points and inoculating it into an adventitious bud proliferation medium.

[0014] Preferably, in step (4), the adventitious bud induction medium is: MS + 0.1-2.5 mg / L 6-BA + 0-0.3 mg / L IBA + 20-40 g / L sucrose + 4-10 g / L agar, and the pH value of the medium is 5.4-6.2.

[0015] Preferably, the adventitious bud proliferation medium is: MS + 0.1-5.0 mg / L 6-BA + 0.1-2.0 mg / L IBA + 20-40 g / L sucrose + 4-10 g / L agar, with a pH of 5.4-6.2.

[0016] The present invention achieves the following technical effects compared to the prior art: (1) Technological breakthrough: For the first time, a one-step direct regeneration system for adventitious roots from leaves of *Vernicia fordii* has been established. No callus induction is required. Leaf explants of different sizes and cutting methods can be directly induced to produce adventitious roots. This completely solves the common problems in the industry of existing *Vernicia fordii* tissue culture technology, which relies on callus regeneration, has a long culture cycle, high somatic cell mutation rate, and poor genetic stability. This fills the technological gap in the field of direct organogenesis of *Vernicia fordii*.

[0017] (2) Reduce production costs: The overall operation process is simple, no complicated pretreatment is required, explants are easy to obtain and the material utilization rate is high, which can greatly reduce the reagent input and labor costs of tissue culture production.

[0018] (3) Solving industry pain points: It can quickly and directionally breed high-quality female tung tree seedlings with stable traits, solving the industrial development problem that existing seedlings cannot be identified as male or female in the early stage and the supply gap of more than 10 million high-quality seedlings, supporting the high-quality and large-scale development of the tung tree industry.

[0019] (4) Expanding application scenarios: It can be widely used for the large-scale asexual propagation of superior individual plants of *Vernicia fordii*, and at the same time, it provides core technical support for the construction of molecular breeding systems such as CRISPR editing and Agrobacterium-mediated genetic transformation of *Vernicia fordii*, and can support research work such as gene function verification and targeted genetic improvement of *Vernicia fordii*.

[0020] (5) Meets strategic needs: Taking the tung oil crop, which has multiple values ​​including ecological restoration, oil production and landscape utilization, and is a new type of oil crop that ensures national food and oil security by "not competing with grain for land", as the research object, this technology provides key underlying technical support for the development of the tung oil industry under the national food and oil security layout. Attached Figure Description

[0021] Figure 1 This is a diagram of the pre-cultivation of branches of the superior variety of *Vernicia fordii* in the embodiment; Where a represents hydroponics and b represents substrate culture; Figure 2 This is a diagram illustrating the induction of adventitious buds using stem segments of *Vernicia fordii* as explants in the embodiments. Among them, a, b, and c are all side views of explants 20 days after being inoculated into adventitious shoot induction medium; Figure 3 This is a diagram showing the growth status of the aseptic tissue culture seedlings of *Vernicia fordii* during the subculture and propagation process in the embodiment. Figure 4 This is a pre-processed image of the explants in the embodiment; Figure 5 This is a diagram showing the growth status of the leaves 10-15 days after inoculation in the example (the veins locally lighten in color and swell). Where a represents 10 days after vaccination and b represents 15 days after vaccination; Figure 6 This is a growth diagram of the leaf (1×1 cm, internally punctured, with the front or back facing up) in the example 20 days after inoculation; Among them, a, b, d, e, and f are all top views 20 days after inoculation with the leaf face up, and c is a top view 20 days after inoculation with the leaf face down. Figure 7 This is a growth diagram of the leaves 23 days after inoculation in the example; Where a is a top view of the leaves 23 days after inoculation, and b is a side view of the leaves 23 days after inoculation; Figure 8 This is a growth chart of a leaf with a 0.5 cm petiole in the example, after 10 days of inoculation with a culture medium. Among them, a is a side view of a leaf with a 0.5 cm petiole after being inserted into the culture medium and cultured for 10 days, and b is a bottom view of a leaf with a 0.5 cm petiole after being inserted into the culture medium and cultured for 10 days. Figure 9 This is a growth diagram of a leaf with a 0.5 cm petiole in the embodiment, after 15 days of inoculation using a culture medium. Among them, a, b, c, d, and e are all side views of leaves with petioles of 0.5 cm taken after being inserted into the culture medium and cultured for 15 days. Figure 10 This is a growth diagram of a leaf with a 1.0 cm petiole 10 days after inoculation in the embodiment. In this paper, a and b are side views of leaves with 1.0 cm petioles 10 days after inoculation. Figure 11This is a growth diagram of a leaf with a 1.0 cm petiole 15 days after inoculation in the embodiment. Among them, a, b, and c are all side views of leaves without petioles after 15 days of inoculation by inserting them into the culture medium. Figure 12 This is a growth diagram of the leaf without petiole in the embodiment after 15 days of inoculation by inserting it into the culture medium. Figure 13 This is a diagram showing the growth status of *Vernicia fordii* leaves that have developed adventitious roots 3 days after transplanting, as described in the example. Detailed Implementation

[0022] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] This invention discloses a method for constructing a pathway for direct induction of adventitious root regeneration from *Vernicia fordii* leaves, comprising the following steps: (1) Obtaining explants There are three ways to obtain it; choose one of them: Method 1: Select branches from superior male and female *Vernicia fordii* plants and bring them back to the laboratory for hydroponics, aeroponics, substrate culture, aseptic culture, etc. The newly sprouted leaves are used as explants. Method 2: After removing the surface wax from *Vernicia fordii* seeds and performing low-temperature stratification, disinfect them with 75% alcohol for 2 minutes, rinse 2-3 times with sterile water, then disinfect them with 0.1% mercuric chloride solution for 15 minutes, rinse 4-6 times with sterile water, and then inoculate them onto blank MS medium plates for aseptic germination. After culturing at 25℃ for 15-30 days, use leaves from sterile seedlings as explants. The blank MS medium plates contain MS medium powder and agar, with a pH of 5.8. Method 3: Aseptic seedlings of *Vernicia fordii* need to be subcultured regularly. During the subculture process, excess leaves need to be pruned. The conventional practice is to discard the excess leaves directly. Therefore, the excess sterile leaves pruned during the subculture process can be used as explants. (2) Pretreatment of explants: After obtaining sterile leaves, the sterile leaves are cut. (3) Induction of adventitious roots: The leaves were inserted at the base and the leaf pieces were laid flat on the adventitious root induction medium. They were first cultured in the dark for 3-7 days and then transferred to light for culture to induce the production of adventitious roots. (4) Induction of adventitious buds: Leaves without petioles, or leaf pieces with internal scratches or no scratches, after the above treatment, were laid flat on an adventitious bud induction medium and cultured in the dark for 3-7 days before being transferred to light to induce the generation of adventitious buds.

[0024] In step (2), the cutting methods are as follows: complete leaf blade without petiole, complete leaf blade with petiole of 0.5 cm length, complete leaf blade with petiole of 1.0 cm length, and leaf pieces of 0.5~1.0 × 0.5~1.0 cm without internal scoring. 2 Leaves with 2-4 incisions (0.5-1.0 x 0.5-1.0 cm) without damaging the main vein are cut. 2 .

[0025] In steps (3) and (4), the conditions for light-induced culture are: a photoperiod of 12 h light, 12 h darkness, and a light intensity of 1500~3000 lux.

[0026] In step (3), the adventitious root induction medium is: 1 / 4 MS medium + 0.01~4.0 mg / L IBA + 5~30 g / L sucrose + 4~8 g / L carrageenan, pH of the culture medium is 5.4~6.2.

[0027] Step (4) also includes: when the adventitious bud grows to 0.5-1.0 cm, cut off a single adventitious bud and inoculate it into the proliferation medium for propagation. After the adventitious bud grows out, cut off an adventitious bud block with 1-2 growth points and inoculate it into the adventitious bud proliferation medium.

[0028] In step (4), the adventitious bud induction medium is: MS + 0.1-2.5 mg / L 6-BA + 0-0.3 mg / L IBA + 20-40 g / L sucrose + 4~10 g / L agar, and the pH value of the medium is 5.4~6.2.

[0029] The adventitious bud proliferation medium is: MS + 0.1-5.0 mg / L 6-BA + 0.1-2.0 mg / L IBA + 20-40 g / L sucrose + 4-10 g / L agar, with a pH of 5.4-6.2. Example 1:

[0030] A method for constructing a pathway for direct induction of adventitious root regeneration from *Vernicia fordii* leaves includes the following steps: (1) Obtaining explants: Branches from superior male and female *Vernicia fordii* plants were selected and brought back to the laboratory for hydroponics, aeroponics, and substrate culture. Newly sprouted leaves were used as explants. Specific culture methods are detailed in the appendix. Figure 1 ; (2) Pretreatment of explants: After obtaining newly sprouted leaves through hydroponics, aeroponics, or substrate culture, the leaf surfaces are disinfected. The disinfection process is as follows: Soak in saturated detergent water for 5-10 minutes, rinse with running water for 10 minutes, rinse with sterile water 2-3 times, soak in 75% alcohol for 10-30 seconds, rinse with sterile water 2-3 times, soak in 2% NaClO or 0.1% mercuric chloride solution for 3-6 minutes, rinse with sterile water 5-6 times.

[0031] Then, absorb the surface moisture and follow... Figure 2 The cutting methods include whole leaf blades without petioles, whole leaf blades with 0.5 cm long petioles, whole leaf blades with 1.0 cm long petioles, and 1.0 × 1.0 cm leaf blades. 2 Leaves with internal slits or no slits are prepared. Intact leaves are inserted into the culture medium, while leaf pieces are laid flat on the culture medium with the back facing up or down.

[0032] (3) Induction of adventitious roots: The leaves were inserted at the base and laid flat on the culture medium (with the back of the leaves facing up and the back of the leaves facing down). They were first cultured in the dark for 3-7 days and then transferred to a light environment with a photoperiod of 12 h light, 12 h dark and a light intensity of 1500-3000 lux to induce the formation of adventitious roots.

[0033] The adventitious root induction medium is as follows: 1 / 4 MS medium + 0.01~4.0 mg / L IBA + 5~30 g / L sucrose + 4~8 g / L carrageenan, pH of the culture medium is 5.4~6.2; In this embodiment, the adventitious root regeneration rate of *Vernicia fordii* leaves reached as high as 90%. Implementation: 2:

[0034] A method for constructing a pathway for direct induction of adventitious root regeneration from *Vernicia fordii* leaves includes the following specific steps: (1) Obtaining explants: After removing the surface wax from the seeds of the *Vernicia fordii* and performing low-temperature stratification, the seeds are first disinfected with 75% alcohol for 2 minutes, rinsed with sterile water 2-3 times, and then disinfected with 0.1% mercuric chloride solution for 15 minutes, and rinsed with sterile water 4-6 times. Subsequently, the seedlings were inoculated onto blank MS medium plates for aseptic germination. After culturing at 25°C for 15-30 days, leaves from the aseptic seedlings were used as explants. The blank MS medium plate contains MS medium powder and agar, with a pH of 5.8; (2) Pretreatment of explants: According to the aseptic seedling leaves Figure 2The cutting methods include whole leaf blades without petioles, whole leaf blades with 0.5 cm long petioles, whole leaf blades with 1.0 cm long petioles, and 1.0 × 1.0 cm leaf blades. 2 The leaf pieces were scored with a few cuts or left unmarked. Intact leaves were inserted into the culture medium, while leaf pieces were laid flat on the culture medium with the back facing up or down.

[0035] (3) Induction of adventitious roots: The leaves were inserted at the base and laid flat on the culture medium (with the back of the leaves facing up and the back of the leaves facing down). They were first cultured in the dark for 3-7 days and then transferred to a light environment with a photoperiod of 12 h light, 12 h dark and a light intensity of 1500-3000 lux to induce the formation of adventitious roots.

[0036] The adventitious root induction medium is as follows: 1 / 4 MS medium + 0.01~4.0 mg / L IBA + 5~30 g / L sucrose + 4~8 g / L carrageenan, pH of the culture medium is 5.4~6.2; In this embodiment, the adventitious root regeneration rate of *Vernicia fordii* leaves reached as high as 90%. Implementation: 3:

[0037] A method for constructing a pathway for direct induction of adventitious root regeneration from *Vernicia fordii* leaves includes the following steps: (1) Obtaining explants: Aseptic seedlings of *Vernicia fordii* need to be subcultured regularly. During the subculture process, excess leaves need to be pruned. In this example, excess sterile leaves pruned during the subculture process of conventional *Vernicia fordii* tissue culture seedlings with leaf age of 15-30 days were selected as explants. (2) Pretreatment of explants: According to the aseptic seedling leaves Figure 2 The cutting methods include whole leaf blades without petioles, whole leaf blades with 0.5 cm long petioles, whole leaf blades with 1.0 cm long petioles, and 1.0 × 1.0 cm leaf blades. 2 Leaves with internal slits or no slits are prepared. Intact leaves are inserted into the culture medium, while leaf pieces are laid flat on the culture medium with the back facing up or down.

[0038] (3) Induction of adventitious roots: The leaves were inserted at the base and laid flat on the culture medium (with the back of the leaves facing up and the back of the leaves facing down). They were first cultured in the dark for 3-7 days and then transferred to a light environment with a photoperiod of 12 h light, 12 h dark and a light intensity of 1500-3000 lux to induce the formation of adventitious roots.

[0039] The adventitious root induction medium is as follows: 1 / 4 MS medium + 0.01~4.0 mg / L IBA + 5~30 g / L sucrose + 4~8 g / L carrageenan, pH of the culture medium is 5.4~6.2; In this embodiment, the adventitious root regeneration rate of *Vernicia fordii* leaves reached as high as 90%.

[0040] Therefore, it can be seen that adventitious roots can be directly induced from leaves of *Vernicia fordii* using explants obtained from different sources, and the induction regeneration rate is stable at a high level. Leaf explants of different sizes and cutting methods can directly generate adventitious roots without going through the callus induction stage. The culture process is simple and the induction efficiency is stable, which meets the needs of large-scale asexual propagation of *Vernicia fordii* and molecular breeding research.

[0041] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the technical scope of the present invention. Therefore, any minor modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. A method for constructing a pathway for direct induction of adventitious root regeneration from *Vernicia fordii* leaves, characterized in that, Includes the following steps: (1) Obtaining explants There are three ways to obtain it; choose one of them: Method 1: Select branches from superior male and female *Vernicia fordii* plants and bring them back to the laboratory for hydroponics, aeroponics, substrate culture, aseptic culture, etc. The newly sprouted leaves are used as explants. Method 2: After removing the surface wax from *Vernicia fordii* seeds and performing low-temperature stratification, disinfect them with 75% alcohol for 2 minutes, rinse 2-3 times with sterile water, then disinfect them with 0.1% mercuric chloride solution for 15 minutes, rinse 4-6 times with sterile water, and then inoculate them onto blank MS medium plates for aseptic germination. After culturing at 25℃ for 15-30 days, use leaves from sterile seedlings as explants. The blank MS medium plates contain MS medium powder and agar, with a pH of 5.

8. Method 3: Aseptic seedlings of *Vernicia fordii* need to be subcultured regularly. During the subculture process, excess leaves need to be pruned. The conventional practice is to discard the excess leaves directly. Therefore, the excess sterile leaves pruned during the subculture process can be used as explants. (2) Pretreatment of explants: After obtaining sterile leaves, the sterile leaves are cut. (3) Induction of adventitious roots: The leaves were inserted at the base and the leaf pieces were laid flat on the adventitious root induction medium. They were first cultured in the dark for 3-7 days and then transferred to light for culture to induce the production of adventitious roots. (4) Induction of adventitious buds: Leaves without petioles, or leaf pieces with internal scratches or no scratches, after the above treatment, were laid flat on an adventitious bud induction medium and cultured in the dark for 3-7 days before being transferred to light to induce the generation of adventitious buds.

2. The method for constructing a pathway for direct induction of adventitious root regeneration from *Vernicia fordii* leaves according to claim 1, characterized in that, In step (2), the cutting process is as follows: a complete leaf without a petiole, a complete leaf with a petiole of 0.5 cm in length, a complete leaf with a petiole of 1.0 cm in length, and a leaf piece of 0.5~1.0 × 0.5~1.0 cm without internal scoring. 2 Leaves with 2-4 incisions (0.5-1.0 x 0.5-1.0 cm) without damaging the main vein are cut. 2 .

3. The method for constructing a pathway for direct induction of adventitious root regeneration from *Vernicia fordii* leaves according to claim 1, characterized in that, In steps (3) and (4), the conditions for light-induced culture are: a photoperiod of 12 h of light and 12 h of darkness, and a light intensity of 1500~3000 lux.

4. The method for constructing a pathway for direct induction of adventitious root regeneration from *Vernicia fordii* leaves according to claim 1, characterized in that, In step (3), the adventitious root induction medium is: 1 / 4 MS medium + 0.01~4.0 mg / L IBA + 5~30 g / L sucrose + 4~8 g / L carrageenan, pH of the culture medium is 5.4~6.

2.

5. The method for constructing a pathway for direct induction of adventitious root regeneration from *Vernicia fordii* leaves according to claim 1, characterized in that, Step (4) further includes: when the adventitious bud grows to 0.5-1.0 cm, cut off a single adventitious bud and inoculate it into the proliferation medium for propagation; after the adventitious bud grows out, cut off an adventitious bud block with 1-2 growth points and inoculate it into the adventitious bud proliferation medium.

6. The method for constructing a pathway for direct induction of adventitious root regeneration from *Vernicia fordii* leaves according to claim 1, characterized in that, In step (4), the adventitious bud induction medium is: MS + 0.1-2.5 mg / L 6-BA + 0-0.3 mg / L IBA + 20-40 g / L sucrose + 4-10 g / L agar, with a pH of 5.4-6.

2.

7. The method for constructing a pathway for direct induction of adventitious root regeneration from *Vernicia fordii* leaves according to claim 5, characterized in that, The adventitious bud proliferation medium is: MS + 0.1-5.0 mg / L 6-BA + 0.1-2.0 mg / L IBA + 20-40 g / L sucrose + 4-10 g / L agar, with a pH of 5.4-6.2.

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