A sterile rapid propagation method of chenxiang tree based on embryo rescue and application thereof

Abstract

This invention discloses a method for aseptic rapid propagation of *Cephalotaxus fortunei* based on embryo rescue and its application, belonging to the field of plant tissue culture technology. The method includes the following steps: sterilizing the embryos of *Cephalotaxus fortunei* to obtain aseptic embryos; inoculating the aseptic embryos into a germination medium for germination culture to obtain aseptic seedlings; inoculating the cotyledons and hypocotyl regions of the aseptic seedlings into a callus induction medium to induce callus formation; transferring the callus to an adventitious bud induction medium to induce the differentiation of adventitious buds; inoculating the adventitious buds or stem segments obtained from them into an adventitious bud proliferation medium for subculture; inoculating the proliferated adventitious bud stem segments into a rooting medium to induce rooting, obtaining fully rooted seedlings; and finally, acclimatizing and transplanting the fully rooted seedlings. This invention uses embryos as starting material, which can improve the initial success rate of aseptic culture and broaden the genetic background of regenerated plants.

Description

Technical Field

[0001] This invention relates to a method for the aseptic rapid propagation of the endangered species *Cephalotaxus fortunei* based on embryo rescue and its application, belonging to the field of plant tissue culture technology. Background Technology

[0002] Hammerwood ( Sinojackia xylocarpa Hu (Hemiberleinii) is a rare and endangered plant. Under natural conditions, its sexual reproduction capacity is weak, and natural population regeneration is difficult. In order to save and restore its population, the development of artificial propagation techniques is particularly important.

[0003] In existing technologies, to overcome the shortcomings of natural propagation, methods have been attempted to use young tissues as explants for aseptic initiation culture. However, this method has significant drawbacks: firstly, the high rate of explant contamination and sensitivity to external conditions leads to a low success rate of initiation culture; secondly, the availability of young tissues for this method is limited, typically obtained from a limited number of mother plants, resulting in a relatively narrow genetic background of the obtained material. This is detrimental to maintaining and expanding intraspecific genetic diversity, thus posing a potential risk to the resource conservation of endangered species. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a rapid propagation method for *Heliotropium indicum* that can improve the success rate of initial culture and expand the genetic background of explants, in order to address the shortcomings of the existing technology in which the aseptic initiation culture using young tissues is low and the genetic background of explants is narrow.

[0005] To address the aforementioned technical problems, this invention provides a method for aseptic rapid propagation of *Hemiberlesia lataniae* based on embryo rescue, comprising the following steps: S1. Acquisition Steps: Acquire the embryo of the cycad and disinfect the surface of the embryo; S2, Embryo rescue step: The sterilized embryo is inoculated into the first culture medium for germination culture to obtain germinating seedlings; S3. Callus induction step: The germinated seedlings or tissue blocks obtained therefrom are inoculated into the second culture medium for callus induction culture to obtain callus tissue; S4. Adventitious bud induction step: The callus tissue is inoculated into the third culture medium for adventitious bud induction culture to obtain clustered adventitious buds; S5. Adventitious bud proliferation step: After dividing the clustered adventitious buds, inoculate them into the fourth culture medium for subculture proliferation culture; S6. Rooting induction step: Inoculate the adventitious bud stem segments obtained from proliferation into the fifth culture medium for rooting induction culture to obtain rooted seedlings; S7. Acclimatization and Transplanting Steps: After the rooted seedlings undergo gradual environmental acclimatization, they are transplanted into the cultivation substrate.

[0006] Furthermore, in the aforementioned method, the first culture medium comprises a basal culture medium, a carbon source, a gelling agent, and a first plant growth regulator; The first plant growth regulator is 6-benzylaminopurine; the concentration of 6-benzylaminopurine in the first culture medium is 1.0-2.0 mg / L; the basal culture medium is MS medium; the carbon source is sucrose with a concentration of 20-40 g / L; and the gelling agent is agar with a concentration of 5-9 g / L.

[0007] Furthermore, in the aforementioned method, the second culture medium comprises a basal culture medium, a carbon source, a gelling agent, and a combination of plant growth regulators, wherein the combination of plant growth regulators includes a first cytokinin and a second cytokinin. The first cytokinin is 6-benzylaminopurine, and the second cytokinin is thidiazuron; the concentration of 6-benzylaminopurine in the second culture medium is 0.5-2.0 mg / L; the concentration of thidiazuron in the second culture medium is 0.05-0.5 mg / L; the basal culture medium is WPM medium; the carbon source is sucrose with a concentration of 20-40 g / L; and the gelling agent is agar with a concentration of 5-9 g / L.

[0008] Furthermore, in the aforementioned method, the third culture medium comprises a basal culture medium, a carbon source, a gelling agent, and a combination of plant growth regulators, wherein the combination of plant growth regulators includes cytokinins and auxins; the cytokinin is 6-benzylaminopurine, and the auxin is indolebutyric acid; The concentration of 6-benzylaminopurine in the third culture medium is 0.5-3.0 mg / L; the concentration of indolebutyric acid in the third culture medium is 0.01-0.1 mg / L; the basal culture medium is WPM medium; the carbon source is sucrose with a concentration of 20-40 g / L; and the gelling agent is agar with a concentration of 5-9 g / L.

[0009] Furthermore, in the aforementioned method, the fourth culture medium has the same composition as the third culture medium; the fifth culture medium comprises a 1 / 2 strength basal medium, a carbon source, a gelling agent, an auxin, and activated carbon; the auxin is naphthaleneacetic acid, and the concentration of naphthaleneacetic acid in the fifth culture medium is 0.1-0.5 mg / L; Furthermore, in the aforementioned method, the 1 / 2 strength basal medium is MS medium; the carbon source is sucrose with a concentration of 20-40 g / L; the gelling agent is agar with a concentration of 5-9 g / L; and the activated carbon has a concentration of 0.5-2.0 g / L.

[0010] Furthermore, in the aforementioned method, the cultivation conditions in steps S2, S3, S4, S5, and S6 are independently as follows: cultivation temperature 23-27℃, and light cycle of 12-18 hours per day. In step S7, the gradual environmental acclimatization includes: cultivating the rooted seedlings in a closed or semi-closed high-humidity environment for 1-2 weeks, then gradually reducing the environmental humidity to transition to an open environment.

[0011] This invention also protects the application of an embryo-rescue-based aseptic rapid propagation method for *Cynanchum paniculatum* in the propagation of *Cynanchum paniculatum* or the protection of endangered plants.

[0012] In one implementation, the seedling substrate is a composite substrate of fine sand and peat.

[0013] Compared with the closest prior art, the beneficial effects of the present invention are as follows: This invention directly uses the embryos of *Hemiberlesia lataniae* as starting material for embryo rescue culture. As a product of plant sexual reproduction, the embryo possesses complete organ differentiation potential and high physiological activity. Germination culture under suitable aseptic conditions can effectively avoid explant contamination problems and significantly improve the initial success rate of establishing an aseptic system, thus solving the deficiency of low success rate of young tissue explants in existing technologies.

[0014] Meanwhile, since embryos originate from sexual reproduction, their genetic background is formed by the fusion of gametes from both parents. Compared to young tissues repeatedly obtained from a limited number of mother plants, embryos, as starting material, can theoretically encompass richer genetic information. By initiating culture through embryo rescue technology, followed by callus induction and redifferentiation, a population of regenerated plants with a relatively more diverse genetic background can be obtained. This effectively overcomes the problem of narrow genetic background caused by the single source of explants in existing technologies, which is beneficial for maintaining and expanding the intraspecific genetic diversity of *Cephalotaxus fortunei* during rapid propagation and is of great significance for the resource conservation of endangered species.

[0015] Furthermore, through subsequent systematic callus induction, adventitious bud differentiation and proliferation, rooting culture, and domestication and transplanting steps, a complete aseptic rapid propagation system has been formed, enabling the large-scale and rapid production of *Cephalotaxus fortunei* seedlings. Attached Figure Description

[0016] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0017] Figure 1 A schematic diagram of the rescue culture of *Hemiberlesia lataniae* embryos.

[0018] Figure 2 This is a schematic diagram of callus induction culture.

[0019] Figure 3 This is a schematic diagram of adventitious bud differentiation culture.

[0020] Figure 4 This is a schematic diagram of adventitious bud culture.

[0021] Figure 5 This is a schematic diagram of adventitious bud subculture.

[0022] Figure 6 This is a schematic diagram of rooting culture.

[0023] Figure 7 This is a schematic diagram of a rooted seedling of the *Pterocarya stenoptera* tree awaiting domestication and cultivation.

[0024] Figure 8 This is a schematic diagram of the transplanting and acclimatization process.

[0025] Figure 9 A diagram illustrating the adaptation of *Pterocarya stenoptera* seedlings to survival.

[0026] Figure 10 This is a summary chart of statistical data. A represents the diameter of callus induced by different culture media; B represents the number of adventitious buds formed by callus redifferentiation in different culture media; C represents the leaf length of adventitious buds; D represents a comparison of the proliferation rate of different culture media during subculture; E represents a comparison of the rooting rate of different rooting media after 5-8 weeks of rooting culture; and F represents a comparison of the differences in cut proliferation after rooting adventitious buds of *Heliotropium indicum*. Detailed Implementation

[0027] To enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are merely some, not all, of the embodiments of the present application. All other embodiments obtained by those skilled in the art based on the embodiments of the present application without creative effort are within the scope of protection of the present application.

[0028] As a rare and endangered plant, *Cephalotaxus fortunei* has a weak natural reproductive capacity, making population recovery difficult. This invention provides a method for the aseptic rapid propagation of the endangered species *Cephalotaxus fortunei* based on embryo rescue. This method uses embryos as starting material for embryo rescue and establishes an aseptic culture system, effectively solving the problems of low initial success rate and narrow genetic background in existing technologies using young tissues as explants. Furthermore, it enables large-scale rapid propagation of seedlings through subsequent systematic culture steps.

[0029] The aseptic rapid propagation method for *Hemiberlesia lataniae* based on embryo rescue provided in this application includes the following steps: S1: Disinfect the embryos of the cypress tree to obtain sterile embryos.

[0030] S2: Inoculate the sterile embryos into the first culture medium and carry out germination culture under sterile conditions to obtain sterile seedlings.

[0031] S3: Inoculate the cotyledons and hypocotyl regions of sterile seedlings into the second callus culture medium to induce callus formation.

[0032] S4: Transfer the callus tissue to the third culture medium to induce the differentiation of adventitious shoots.

[0033] S5: Inoculate adventitious buds or stem segments obtained from adventitious buds into the fourth culture medium for subculture and proliferation.

[0034] S6: Inoculate the proliferated adventitious bud stem segments into the fifth culture medium to induce rooting and obtain fully rooted seedlings.

[0035] S7: Acclimatize and transplant the fully rooted seedlings into the cultivation substrate; the seedling cultivation substrate is a composite substrate of fine sand and peat.

[0036] In this embodiment, the disinfection process in step S1 includes immersing the embryo in an alcohol solution and a mercuric chloride solution sequentially. Specifically, the embryo can be first immersed in a 75% alcohol solution for 0.5-1.5 minutes, then transferred to a 0.05%-0.15% mercuric chloride solution for 15-25 minutes, and finally rinsed 3-5 times with sterile water to thoroughly remove any residual disinfectant. This disinfection procedure effectively kills microorganisms on the embryo surface while minimizing the damage to embryo viability caused by the disinfectant, laying the foundation for subsequent aseptic culture. Compared to surface disinfection of delicate tissues, the embryo, due to the protection of its outer seed coat, has stronger tolerance to disinfectants, thus achieving a higher initial sterility rate.

[0037] In this embodiment, the first culture medium in step S2 comprises a basal culture medium, a carbon source, a gelling agent, and a first plant growth regulator, which is 6-benzylaminopurine. The concentration of 6-benzylaminopurine in the first culture medium is 1.0-2.0 mg / L. The basal culture medium is MS medium. The carbon source is sucrose at a concentration of 20-40 g / L. The gelling agent is agar at a concentration of 5-9 g / L. The MS medium provides the macro-elements, micro-elements, and organic components required for embryo germination. The added cytokinin 6-benzylaminopurine at a concentration range of 1.0-2.0 mg / L can effectively break embryo dormancy and promote germination. Sucrose, as a carbon source and osmotic regulator, can be at a concentration of 20-40 g / L. In one specific protocol, 1.5 mg / L of 6-benzylaminopurine and 30 g / L of sucrose were added to the germination medium. After initial dark culture at 25 ± 2 °C for 1-3 weeks, the culture was transferred to a 16-hour light / 8-hour dark photoperiod until embryo germination resulted in sterile seedlings with cotyledons and hypocotyls. Under these conditions, the embryo germination rate exceeded 90%, significantly higher than the success rate of establishing a sterile system using young tissues.

[0038] In this embodiment, the second culture medium for callus induction culture in step S3 comprises a basal culture medium, a carbon source, a gelling agent, and a combination of plant growth regulators. The plant growth regulator combination includes a first cytokinin and a second cytokinin. The first cytokinin is 6-benzylaminopurine, and the second cytokinin is thidiazuron. The concentration of 6-benzylaminopurine in the second culture medium is 0.5-2.0 mg / L; the concentration of thidiazuron in the second culture medium is 0.05-0.5 mg / L; the basal culture medium is WPM medium; the carbon source is sucrose at a concentration of 20-40 g / L; the gelling agent is agar at a concentration of 5-9 g / L; the woody plant salt formulation of WPM medium is more suitable for the induction of callus from *Pterocarya stenoptera*. The synergistic effect of 6-benzylaminopurine and thidiazuron within this concentration range can efficiently induce dedifferentiation in areas such as cotyledons and hypocotyls of sterile seedlings, forming loosely textured and vigorously growing callus. Thiidianone is a highly efficient cytokinin that can significantly promote callus formation even at very low concentrations (e.g., 0.1 mg / L). Culture conditions include a 16-hour light / 8-hour dark photoperiod and a temperature of 25±2℃. A large amount of callus can be obtained after 4-6 weeks of culture.

[0039] In this embodiment, the third culture medium for adventitious shoot induction culture in step S4 comprises a basal culture medium, a carbon source, a gelling agent, and a combination of plant growth regulators. The plant growth regulator combination includes cytokinins and auxins. The concentration of 6-benzylaminopurine in the third culture medium is 0.5-3.0 mg / L; the concentration of indolebutyric acid in the third culture medium is 0.01-0.1 mg / L; the basal culture medium is WPM medium; the carbon source is sucrose at a concentration of 20-40 g / L; and the gelling agent is agar at a concentration of 5-9 g / L. After the callus tissue is transferred to this culture medium, the higher concentration of 6-benzylaminopurine (e.g., 1.5 mg / L) combined with the lower concentration of the auxin indolebutyric acid (e.g., 0.03 mg / L) can effectively induce callus redifferentiation, forming adventitious shoots. Under these conditions, after 2-4 weeks of culture, the diameter of the adventitious shoot cluster can reach 2.0-3.0 cm, and the height of the adventitious shoots can reach 2.0-4.0 cm.

[0040] In this embodiment, the fourth culture medium in step S5 consists of WPM medium supplemented with 0.5-3.0 mg / L of 6-benzylaminopurine, 0.01-0.0 mg / L of indolebutyric acid, and sucrose. For rapid propagation, the adventitious bud clusters obtained in step S4 need to be divided. These clusters can be divided into smaller clusters with a diameter of 0.3-0.5 cm, or adventitious buds longer than 1.0 cm can be cut into stem segments approximately 1.0 cm long. These materials are inoculated into fresh adventitious bud proliferation medium (e.g., 5 clusters or stem segments per 9 cm diameter culture bottle) and cultured under the same conditions (photoperiod 16 / 8 hours, 25±2℃) for 4-5 weeks. After this subculture, the average proliferation rate of adventitious buds can exceed 7.0, thus achieving a geometric increase in the number of seedlings.

[0041] In this embodiment, the fifth culture medium in step S6 comprises 1 / 2 MS medium, a carbon source, a gelling agent, auxin, and activated carbon; the auxin is naphthaleneacetic acid (NAA), and the concentration of NAA in the fifth culture medium is 0.1-0.5 mg / L. The 1 / 2 MS medium reduces the salt concentration, which is beneficial for root induction. At a concentration of 0.1-0.5 mg / L (e.g., 0.3 mg / L), the auxin NAA can effectively stimulate the formation of root basal sites at the base of adventitious bud stem segments, thereby developing into normal roots. The addition of activated carbon (e.g., 1.0 g / L) can adsorb harmful metabolites and residual auxin in the culture medium, preventing excessive swelling of the stem segment cut and promoting the formation of long, supple taproots with abundant lateral roots. When adventitious bud stem segments with a height of 2.0-3.0 cm are vertically inserted into the rooting medium, the rooting rate can reach over 90% after 5-8 weeks of culture.

[0042] In this embodiment, the acclimatization and transplanting in step S7 includes: cultivating fully rooted seedlings indoors for a period of time (e.g., 3-6 weeks, until the root length reaches 3.0-4.0 cm), then transferring them to a greenhouse with shading conditions (e.g., 70% shading rate) for hardening off for 1-2 weeks, and then transplanting them into a seedling substrate. The seedling substrate is a composite substrate of fine sand and peat moss. The specific transplanting method is as follows: removing the rooted seedlings from the culture container, washing the root culture medium, planting them in thoroughly watered composite substrate, and covering them with a thin film to retain moisture. Then, gradually removing the film over several days allows the seedlings to gradually adapt to the external humidity, and finally completely removing the film for routine management. This gradual acclimatization process greatly improves the transplant survival rate.

[0043] Table 1 below shows a comparison of the effects of using the method of this application (Example) and using the conventional young tissue explant method (Comparative Example) in the critical culture stage.

[0044] The data above demonstrates that the embryo-rescue-based aseptic rapid propagation method provided in this application exhibits significantly higher success rates and efficiencies than traditional methods using young tissue explants at each key stage (initiation, callus induction, proliferation, and rooting). This confirms that using embryos as material for embryo rescue can fundamentally improve the initial success rate of establishing an aseptic system. Furthermore, since embryos originate from sexual reproduction, the regenerated plant populations based on them theoretically possess richer genetic diversity, which is of significant value for the germplasm resource conservation of the endangered species *Cephalotaxus fortunei*.

[0045] The above-disclosed examples are merely preferred embodiments of this application, intended to facilitate understanding and implementation by those skilled in the art. However, they cannot be used to limit the scope of this application. Therefore, equivalent variations made within the scope of this application are still within the scope of this application.

Claims

1. A method for aseptic rapid propagation of *Hylocereus undatus* based on embryo rescue, characterized in that, Includes the following steps: S1. Acquisition Steps: Acquire the embryo of the cycad and disinfect the surface of the embryo; S2, Embryo rescue step: The sterilized embryo is inoculated into the first culture medium for germination culture to obtain germinating seedlings; S3. Callus induction step: The germinated seedlings or tissue blocks obtained therefrom are inoculated into the second culture medium for callus induction culture to obtain callus tissue; S4. Adventitious bud induction step: The callus tissue is inoculated into the third culture medium for adventitious bud induction culture to obtain clustered adventitious buds; S5. Adventitious bud proliferation step: After dividing the clustered adventitious buds, inoculate them into the fourth culture medium for subculture proliferation culture; S6. Rooting induction step: Inoculate the adventitious bud stem segments obtained from proliferation into the fifth culture medium for rooting induction culture to obtain rooted seedlings; S7. Acclimatization and Transplanting Steps: After the rooted seedlings undergo gradual environmental acclimatization, they are transplanted into the cultivation substrate.

2. The method according to claim 1, characterized in that, The first culture medium comprises a basal culture medium, a carbon source, a gelling agent, and a first plant growth regulator; The first plant growth regulator is 6-benzylaminopurine; the concentration of 6-benzylaminopurine in the first culture medium is 1.0-2.0 mg / L; the basal culture medium is MS medium; the carbon source is sucrose with a concentration of 20-40 g / L; and the gelling agent is agar with a concentration of 5-9 g / L.

3. The method according to claim 1, characterized in that, The second culture medium comprises a basal culture medium, a carbon source, a gelling agent, and a combination of plant growth regulators, wherein the combination of plant growth regulators includes a first cytokinin and a second cytokinin. The first cytokinin is 6-benzylaminopurine, and the second cytokinin is thidiazuron; the concentration of 6-benzylaminopurine in the second culture medium is 0.5-2.0 mg / L; the concentration of thidiazuron in the second culture medium is 0.05-0.5 mg / L; the basal culture medium is WPM medium; the carbon source is sucrose with a concentration of 20-40 g / L; and the gelling agent is agar with a concentration of 5-9 g / L.

4. The method according to claim 1, characterized in that, The third culture medium comprises a basal culture medium, a carbon source, a gelling agent, and a combination of plant growth regulators, wherein the combination of plant growth regulators includes cytokinins and auxins; the cytokinin is 6-benzylaminopurine, and the auxin is indolebutyric acid; The concentration of 6-benzylaminopurine in the third culture medium is 0.5-3.0 mg / L; the concentration of indolebutyric acid in the third culture medium is 0.01-0.1 mg / L; the basal culture medium is WPM medium; the carbon source is sucrose with a concentration of 20-40 g / L; and the gelling agent is agar with a concentration of 5-9 g / L.

5. The method according to claim 4, characterized in that, The fourth culture medium has the same composition as the third culture medium.

6. The method according to claim 1, characterized in that, The fifth culture medium comprises a 1 / 2 strength basal medium, a carbon source, a gelling agent, an auxin, and activated carbon; the auxin is naphthaleneacetic acid, and the concentration of naphthaleneacetic acid in the fifth culture medium is 0.1-0.5 mg / L.

7. The method according to claim 6, characterized in that, The 1 / 2 strength basal medium is MS medium; the carbon source is sucrose with a concentration of 20-40 g / L; the gelling agent is agar with a concentration of 5-9 g / L; and the activated carbon has a concentration of 0.5-2.0 g / L.

8. The method according to claim 1, characterized in that, The cultivation conditions in steps S2, S3, S4, S5 and S6 are independently as follows: cultivation temperature 23-27℃, and light cycle of 12-18 hours of light per day.

9. The method according to claim 1, characterized in that, In step S7, the gradual environmental acclimatization includes: after cultivating the rooted seedlings in a closed or semi-closed high-humidity environment for 1-2 weeks, gradually reducing the environmental humidity and transitioning to an open environment.

10. The application of the method as described in any one of claims 1 to 9 in the propagation of *Hemiberlesia lataniae* or the protection of endangered plants.

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