A high-efficiency tissue culture seedling cultivation method for anthericum taiwanianum stem segments in northern zhejiang region

Abstract

This invention relates to the field of tissue culture technology, and more particularly to a method for cultivating high-efficiency tissue culture seedlings of *Anoectochilus roxburghii* stem segments from northern Zhejiang. The proliferation medium provided by this invention is based on MS medium and further includes the following components at concentrations: 0.05-0.1 mg / L naphthaleneacetic acid (NAA) and 1.8-2.5 mg / L 6-benzyladenine (6-BA). The specific combination of NAA and 6-BA in the proliferation medium of this invention can effectively induce the formation of axillary buds and explant callus, and proliferate a large number of adventitious buds, significantly increasing the propagation coefficient. This is one of the core technical points for achieving "high-efficiency" rapid propagation. In addition, the reasonable combination of rooting medium and cultivation substrate can significantly improve the survival rate of tissue culture seedlings after acclimatization, ensuring a seamless transition from in vitro culture to field planting, which is an important link in achieving a closed loop of "high-efficiency and rapid propagation." This provides a basis for the high-efficiency and rapid propagation of *Anoectochilus roxburghii*.

Description

Technical Field

[0001] This invention relates to the field of tissue culture technology, and in particular to a method for cultivating high-efficiency tissue culture seedlings of *Anoectochilus roxburghii* stem segments from northern Zhejiang. Background Technology

[0002] Golden Thread Lotus ( Anoectochilus roxburghii *Anoectochilus roxburghii*, a perennial rare medicinal plant belonging to the genus *Anoectochilus* of the Orchidaceae family, is known in folk medicine as the "King of Herbs" and "Golden Herb." It possesses anti-inflammatory, antipyretic, blood-cooling, liver-soothing, antihypertensive, cardiotonic, diuretic, and liver-protective and detoxifying effects. Its signature component, *Anoectochilus roxburghii* glycoside, exhibits significant pharmacological activity in lowering blood lipids, lowering blood sugar, and protecting the liver. Due to its demanding environmental requirements and low natural reproduction rate, wild resources of *Anoectochilus roxburghii* are increasingly endangered, and it has been listed as a national second-class protected plant. Therefore, artificial cultivation to meet medicinal needs and protect wild germplasm resources has become an important direction for industrial development. Tissue culture technology is currently the key means to achieve the industrialized propagation of *Anoectochilus roxburghii* seedlings. Existing rapid propagation systems based on tissue culture have been applied to some extent in production, but in actual promotion, they still face prominent problems such as low propagation coefficient and high production costs.

[0003] The northern Zhejiang region, encompassing Huzhou, Jiaxing, and northern Hangzhou, has a subtropical monsoon climate. The overall climate and soil conditions differ significantly from the native mountainous environment of *Anoectochilus roxburghii*, making the natural environment unsuitable for its growth. Currently, the induction, proliferation, and rooting stages of adventitious bud cultivation in northern Zhejiang are highly sensitive to the type and concentration of exogenous plant hormones, and different culture medium formulations and hormone combinations significantly affect in vitro regeneration efficiency. Studies have shown that an appropriate ratio of cytokinin to auxin is crucial for initiating explant cell totipotency expression and promoting adventitious bud formation and growth. However, a systematic optimization system for hormones in stem segment tissue culture of *Anoectochilus roxburghii* in northern Zhejiang has not yet been established. Therefore, it is urgent to establish an efficient and stable rapid propagation system for stem segment tissue culture of *Anoectochilus roxburghii* to improve the propagation coefficient, reduce production costs, provide reliable technical support for large-scale, standardized seedling production of *Anoectochilus roxburghii* in northern Zhejiang, and lay the foundation for subsequent genetic improvement and the creation of superior varieties using genetic engineering techniques. Summary of the Invention

[0004] The purpose of this invention is to provide a method for cultivating high-efficiency tissue culture seedlings of Anoectochilus roxburghii stem segments in northern Zhejiang.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a proliferation medium based on MS medium, further comprising the following components at the following concentrations: 0.05~0.1 mg / L naphthaleneacetic acid, 1.8~2.5 mg / L 6-benzyladenine.

[0006] The present invention also provides the application of the aforementioned proliferation medium in the cultivation of tissue culture seedlings of Anoectochilus roxburghii.

[0007] This invention also provides a method for cultivating high-efficiency tissue culture seedlings of *Anoectochilus roxburghii* stem segments from northern Zhejiang, comprising the following steps: (1) Inoculate the explants into the proliferation medium and culture for 10-15 days to obtain adventitious shoots; (2) Inoculate the adventitious buds into the seedling culture medium and culture for 15-20 days to obtain proliferated seedlings; (3) Inoculate the proliferated seedlings into rooting medium and culture for 18-25 days to obtain tissue culture seedlings; The proliferation medium is the proliferation medium described above.

[0008] Preferably, the temperature for cultivation in step (1) is 20~25℃ and the humidity is 70~80%.

[0009] Preferably, the seedling culture medium in step (2) is based on MS medium and further includes the following components at the following concentrations: 0.05~0.1 mg / L naphthaleneacetic acid, 1.8~2.5 mg / L 6-benzyladenine, and 80~120 g / L banana puree.

[0010] Preferably, the temperature for cultivation in step (2) is 20~25℃ and the humidity is 70~80%.

[0011] Preferably, the rooting medium in step (3) is based on 1 / 2 MS medium and further includes the following components at the following concentrations: 3~5 mg / L indolebutyric acid, 1~2 g / L activated carbon; The culture temperature in step (3) is 20~25℃ and the humidity is 70~80%.

[0012] The present invention also provides tissue culture seedlings obtained according to the cultivation method described herein, wherein the transplant survival rate of the tissue culture seedlings is ≥88%.

[0013] This invention also provides a cultivation substrate for transplanting tissue culture seedlings of *Anoectochilus roxburghii*, comprising any one of the following: (a) The mass ratio of peat moss:coconut coir:vermiculite is 1.5~2.5:0.5~1.5:0.5~1.5; (b) The mass ratio of peat moss: perlite: vermiculite is 2.5~3.5:0.5~1.5:0.5~1.5.

[0014] Preferably, the tissue culture seedlings of *Anoectochilus roxburghii* are tissue culture seedlings obtained according to the cultivation method described above.

[0015] Compared with the prior art, the present invention has the following beneficial effects: A proliferation medium was prepared by mixing 6-benzyladenine and naphthaleneacetic acid in a certain proportion. The specific combination of naphthaleneacetic acid (NAA) and 6-benzyladenine (6-BA) in the proliferation medium can effectively induce the formation of axillary buds and explant callus tissue and proliferate a large number of clustered adventitious buds, significantly improving the propagation coefficient. This is one of the core technical points for achieving "high efficiency" and rapid propagation.

[0016] When the height of the adventitious buds of the robust, rootless propagation seedlings reaches about 5.0 cm, they are transferred to rooting medium (1 / 2 MS + indolebutyric acid (IBA) + activated carbon). With proper formulation, the seedlings can be effectively promoted to grow vigorously and develop well, with a high rooting rate, thus preparing them for hardening and transplanting.

[0017] The three-component composite substrate optimizes transplanting and acclimatization. After the rooted tissue culture seedlings have fully developed their root systems, they undergo hardening-off and are finally transplanted. The cultivation substrate used during transplanting combines water retention, aeration, and appropriate nutrients, which can significantly improve the survival rate of tissue culture seedlings after acclimatization and ensure a seamless transition from in vitro culture to field planting. This is a crucial link in achieving a closed loop of "efficient and rapid propagation."

[0018] The proliferation medium of this invention can produce *Anoectochilus roxburghii* adventitious buds with a proliferation coefficient of 6.8 and an adventitious bud height of 2.3 cm. After vigorous seedling cultivation, these adventitious buds are inoculated onto a rooting medium, resulting in rapid rooting and tissue culture seedlings. After hardening off, the tissue culture seedlings are transplanted onto a cultivation substrate, achieving a survival rate of ≥88%. This provides a basis for the rapid propagation of *Anoectochilus roxburghii*. Attached Figure Description

[0019] Figure 1 Establishment of a sterile tissue culture system for *Anoectochilus roxburghii* stem segments; Figure 2 Adventitious buds obtained from stem segments of Anoectochilus roxburghii; Figure 3 Propagated seedlings obtained by cultivating robust seedlings of Anoectochilus roxburghii; Figure 4 Inducing rooting in tissue culture seedlings of Anoectochilus roxburghii; Figure 5 Image showing the rooting effect of tissue culture seedlings of Anoectochilus roxburghii; Figure 6 To successfully cultivate tissue culture seedlings of Anoectochilus roxburghii. Detailed Implementation

[0020] In this embodiment of the invention, the explant of *Anoectochilus roxburghii* is a stem segment of a sterile *Anoectochilus roxburghii* seedling.

[0021] In this invention, the method for cultivating sterile *Anoectochilus roxburghii* seedlings is as follows: Wild *Anoectochilus roxburghii* stem segments with a pair of healthy axillary buds and a length of 3 cm are cut, disinfected, and inoculated into a medium composed of MS medium + 0.08 mg / L naphthaleneacetic acid + 2.0 mg / L 6-benzyladenine. After culturing for 30 days, sterile *Anoectochilus roxburghii* seedlings are obtained. Figure 1 ).

[0022] In this invention, before transplanting the tissue culture seedlings of Anoectochilus roxburghii to the cultivation substrate, a hardening-off treatment is required. The hardening-off method is as follows: select healthy tissue culture seedlings of Anoectochilus roxburghii with well-developed root systems, open the sealing film of the culture bottle in the culture room, and harden off the seedlings for 3-5 days.

[0023] The wild Anoectochilus roxburghii in this embodiment of the invention was collected from Jiulong Mountain, Jiulong Township, Jingning She Autonomous County, Lishui City, Zhejiang Province, China (28.35°N, 118.87°E).

[0024] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0025] Example 1

[0026] Healthy, sterile seedlings of *Anoectochilus roxburghii* were selected as starting material. Stem segments with a pair of healthy axillary buds and lengths of 2 cm, 3 cm, and 4 cm were cut as explants. These explants were inoculated into proliferation medium (MS + 0.1 mg / L naphthaleneacetic acid (NAA) + 2.0 mg / L 6-benzyladenine (6-BA)) and cultured at 23°C and 75% for 15 days to obtain clustered adventitious buds.

[0027] The number of adventitious buds induced by different stem segment lengths was observed, the proliferation coefficient was calculated, and the height and callus area of ​​the adventitious buds were measured. The proliferation coefficient is the number of adventitious buds produced by a single explant. The results are shown in Table 1.

[0028] Table 1. Characteristics of clustered buds induced by different stem segment lengths (n=50)

[0029] The data were obtained from measurements taken from 50 independent explants after 15 days of culture.

[0030] Example 2

[0031] Healthy, sterile seedlings of *Anoectochilus roxburghii* were selected as starting material, and stem segments with a pair of healthy axillary buds, each 3 cm in length, were cut as explants. These explants were inoculated into proliferation media (MS+NAA+6-BA) containing different concentrations of NAA and 6-BA, and cultured at 23°C and 75% humidity for 15 days. The height of adventitious buds obtained from different proliferation media was measured, and the proliferation coefficient and callus induction rate were calculated according to Example 1. The results are shown in Table 2.

[0032] Callus induction rate = number of explants that produce callus / total number of inoculated explants × 100%.

[0033] Table 2. Effects of different concentrations of NAA and 6-BA on the proliferation of *Anoectochilus roxburghii* stem segments.

[0034] Each treatment was repeated 10 times, and the data are expressed as mean ± standard deviation. Table 2 shows that the optimal proliferation medium was MS + 2 mg / L 6-BA + 0.08 mg / L NAA, which significantly improved proliferation efficiency and shoot quality.

[0035] Example 3

[0036] Healthy, sterile seedlings of *Anoectochilus roxburghii* were selected as starting material. Stem segments with a pair of healthy axillary buds, each 3 cm in length, were cut as explants. These explants were inoculated into proliferation medium (MS + 2 mg / L 6-BA + 0.08 mg / L NAA) and cultured at 23°C for 75% for 15 days to obtain clusters of adventitious buds. Figure 2 ).

[0037] Adventitious buds were inoculated into seedling growth medium (MS + 2 mg / L 6-BA + 0.08 mg / L NAA + 100 g / L banana puree), and cultured at 23℃ and 75% for 15 days to obtain proliferating seedlings. Figure 3 ).

[0038] The proliferated seedlings were inoculated into rooting medium (1 / 2 MS + 4.0 mg / L indolebutyric acid (IBA) + 1.5 g / L activated carbon) and cultured at 23°C for 75% for 20 days to obtain tissue culture seedlings. Figures 4-6 ).

[0039] After 5 days of hardening off by opening the cover, the tissue culture seedlings were transplanted into cultivation substrates with different components. After 30 days, the number of transplanted seedlings that survived was observed, the survival rate was calculated, and the seedling growth was evaluated. The results are shown in Table 3.

[0040] Table 3. Effects of different cultivation substrates on the survival rate of tissue culture seedlings of Anoectochilus roxburghii after transplanting (n=100)

[0041] The survival standard is that the leaves still remain green and new roots have formed 28 days after transplanting.

[0042] Table 3 shows that the cultivation substrates with a peat moss:coconut coir:vermiculite ratio of 2:1:1 and peat moss:perlite:vermiculite ratio of 3:1:1 are the best for the growth of Anoectochilus roxburghii tissue culture seedlings.

[0043] As shown in the above embodiments, this invention provides a method for cultivating high-efficiency tissue culture seedlings of *Anoectochilus roxburghii* stem segments in northern Zhejiang. This invention prepares a proliferation medium by mixing 6-benzyladenine and naphthaleneacetic acid (NAA) in a certain proportion. The specific combination of NAA and 6-BA in the proliferation medium effectively induces the formation of axillary buds and explant callus tissue, resulting in the proliferation of numerous adventitious buds and significantly increasing the propagation coefficient. The rooting medium composed of IBA and activated carbon effectively promotes the abundant growth and robust development of adventitious buds, resulting in a high rooting rate and preparing the seedlings for transplanting. Furthermore, the rational configuration of the transplanting substrate significantly improves the survival rate of tissue culture seedlings after in vitro culture and ensures a seamless transition from in vitro culture to field planting, which is a crucial link in achieving a closed loop of "high-efficiency and rapid propagation." This provides a basis for the high-efficiency and rapid propagation of *Anoectochilus roxburghii*.

[0044] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A proliferation culture medium, characterized in that, Based on MS medium, it also includes the following components at the following concentrations: 0.05~0.1 mg / L naphthaleneacetic acid, 1.8~2.5 mg / L 6-benzyladenine.

2. The application of the proliferation medium described in claim 1 in the cultivation of tissue culture seedlings of Anoectochilus roxburghii.

3. A method for cultivating high-efficiency tissue culture seedlings of *Anoectochilus roxburghii* stem segments from northern Zhejiang, characterized in that... Includes the following steps: (1) Inoculate the explants into the proliferation medium and culture for 10-15 days to obtain adventitious shoots; (2) Inoculate the adventitious buds into the seedling culture medium and culture for 15-20 days to obtain proliferated seedlings; (3) Inoculate the proliferated seedlings into rooting medium and culture for 18-25 days to obtain tissue culture seedlings; The proliferation medium is the proliferation medium according to claim 1.

4. The cultivation method according to claim 3, characterized in that, The culture temperature in step (1) is 20~25℃ and the humidity is 70~80%.

5. The cultivation method according to claim 3, characterized in that, The seedling culture medium in step (2) is based on MS medium and also includes the following components at the following concentrations: 0.05~0.1 mg / L naphthaleneacetic acid, 1.8~2.5 mg / L 6-benzyladenine, and 80~120 g / L banana puree.

6. The cultivation method according to claim 3, characterized in that, The culture temperature in step (2) is 20~25℃ and the humidity is 70~80%.

7. The cultivation method according to claim 3, characterized in that, The rooting medium in step (3) is based on 1 / 2 MS medium and also includes the following components at the following concentrations: 3~5 mg / L indolebutyric acid, 1~2 g / L activated carbon; The culture temperature in step (3) is 20~25℃ and the humidity is 70~80%.

8. The tissue culture seedlings obtained by the cultivation method according to any one of claims 3 to 7, characterized in that, The transplant survival rate of the tissue culture seedlings is ≥88%.

9. A cultivation substrate for transplanting tissue culture seedlings of *Anoectochilus roxburghii*, characterized in that, Including any of the following: (a) The mass ratio of peat moss:coconut coir:vermiculite is 1.5~2.5:0.5~1.5:0.5~1.5; (b) The mass ratio of peat moss: perlite: vermiculite is 2.5~3.5:0.5~1.5:0.5~1.

5.

10. The cultivation substrate according to claim 9, characterized in that, The tissue culture seedlings of *Anoectochilus roxburghii* are tissue culture seedlings obtained according to the cultivation method described in any one of claims 3 to 7.

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