Tissue culture and rapid propagation method taking dendrobium devonianum paxt stem as starting material

Abstract

The invention discloses a tissue culture and rapid propagation method taking a dendrobium devonianum paxt stem segment as a starting material, and belongs to the technical field of plant tissue culture. The method comprises the following steps: after branches are collected in winter and dried in the shade, regularly spraying a hydrogen peroxide silver ion disinfectant with a specific concentration to induce axillary buds to germinate; taking germinated axillary buds as explants, disinfecting, and inoculating to a pre-culture medium; if pollution occurs, soaking with an original disinfectant and transferring into a rescue culture medium containing potassium sorbate for rescue; inoculating the sterile material to a starting culture medium, and performing one-step induction to form multiple shoots with roots; and finally, strong tissue culture seedlings are obtained through seedling strengthening and rooting culture. By means of unique explant pretreatment and induction, an efficient pollution rescue mechanism and an optimized culture medium system, stable and efficient rapid propagation of excellent characters of dendrobium devonianum is achieved, the propagation period is greatly shortened, and reliable technical support is provided for industrialization.

Description

Technical Field

[0001] This application relates to the field of plant tissue culture technology, and in particular to a method for rapid tissue culture propagation using Dendrobium officinale stem segments as starting material. Background Technology

[0002] Purple Dendrobium ( Dendrobium devonianum Dendrobium, belonging to the genus Dendrobium in the family Orchidaceae, is also known as Dendrobium dentata. It is an important economic crop with both medicinal and ornamental value.

[0003] In terms of propagation, the seeds of *Dendrobium nobile* are tiny and powdery, lacking endosperm, making germination extremely difficult under natural conditions. Although aseptic germination can be achieved through tissue culture, the resulting seedlings are prone to individual differentiation, making it difficult to stably maintain the superior traits of the parent plant. Currently, asexual propagation mainly relies on division, cuttings, and top-bud propagation. In Longling County, cuttings are commonly used for seedling cultivation because they root easily: robust branches are typically selected as cuttings during the harvest season, dried in the shade, and stored until the following spring, then cut into stem segments with 2-3 nodes for further cultivation. However, this traditional asexual propagation method has a low propagation coefficient and a long cycle, generally requiring 8-10 years to achieve large-scale application of superior individual plants, which is insufficient to meet the industry's rapid demand for high-quality seedlings.

[0004] In contrast, using superior single-plant stem segments as explants for in vitro rapid propagation through tissue culture not only preserves the superior genetic traits of the parent plant but also enables large-scale seedling supply within approximately two years, significantly shortening the cycle from superior variety selection to industrial application. Therefore, systematically conducting research on the rapid propagation technology of *Dendrobium nobile* stem segments through tissue culture is of significant practical importance for accelerating the industrialization of superior single plants, ensuring a stable supply of high-quality seedlings, and promoting the sustainable development of the *Dendrobium nobile* industry in Longling County. Summary of the Invention

[0005] To address or partially address the problems existing in related technologies, this application provides a method for rapid propagation of Dendrobium nobile stem segments as starting material through tissue culture. While stabilizing the superior traits of the parent plant, this method effectively enhances the efficient propagation of superior Dendrobium nobile individual plants, thereby significantly shortening the cycle from breeding to industrial application and realizing the economic benefits of tissue culture.

[0006] This application discloses a method for rapid tissue culture propagation using Dendrobium nobile stem segments as starting material, comprising the following steps: S1. Preparation of axillary bud explants: Fresh stems of superior Dendrobium officinale plants were collected in winter and placed in a cool and ventilated place to dry for 30-50 days; then, hydrogen peroxide silver ion disinfectant solution was sprayed 1-2 times per month to induce axillary bud germination, and the germinating tender axillary buds were used as explants for rapid tissue culture propagation. The hydrogen peroxide silver ion disinfectant solution contains 4-6% hydrogen peroxide and 0.01% silver ions. S2. Explant disinfection: Young axillary buds sprouting from S1 were cut off, disinfected, and then cultured in a pre-culture medium to obtain sterile explants. S3. Primary culture: The sterile explants are inoculated into the starter culture medium and cultured until rooting occurs to obtain rooted shoot clusters; The formulation of the start-up medium is as follows: MS + 6-BA 4 mg / L + NAA 0.8 mg / L + 30 g / L sucrose + 4.2 g / L agar, pH 5.8; S4. Proliferation Culture: The obtained rooted shoot clusters are inoculated into the proliferation culture medium for proliferation culture to obtain a large number of rooted shoot clusters; S5. Seedling rooting culture: The large number of rooted shoots obtained in S4 are inoculated into seedling rooting culture medium for seedling rooting culture, thus obtaining tissue culture seedlings.

[0007] Furthermore, in S2, if contamination occurs during the culture process after the explant is disinfected, the explant is soaked in the hydrogen peroxide silver ion disinfectant solution in S1 for 8-10 minutes for disinfection, without rinsing, and then inoculated into the rescue culture medium for rescue. The rescue medium is formulated as follows: 50 mg / L of potassium sorbate is added to the start-up medium.

[0008] Furthermore, in S1, lateral young axillary buds with a bud height of 3-5cm are selected as explants for rapid tissue culture propagation.

[0009] Furthermore, the formulation of the proliferation medium is as follows: MS + 6-BA 2mg / L + NAA 1.5mg / L + activated carbon 1g / L + potato starch 100 g / L + sucrose 30g / L + agar 4.2g / L, pH 5.8.

[0010] Furthermore, the formulation of the seedling rooting medium is MS + 6-BA 0.3 mg / L + NAA 0.5 mg / L + KT 0.4 mg / L + activated carbon 1 g / L + sucrose 26 g / L + agar 4.2 g / L + potato starch 60 g / L, with a pH of 5.8.

[0011] Further, the disinfection process in step S2 is as follows: first, the explant is soaked in 75% ethanol for 1-2 minutes, then rinsed with sterile water, then disinfected by shaking with 0.1% HgCl2 solution for 6-10 minutes, and finally rinsed with sterile water and dried with filter paper.

[0012] Furthermore, the pre-culture medium is formulated as follows: MS + 1.0-2.0 mg / L 6-BA + 0.8 mg / L NAA + 30 g / L sucrose + 4.2 g / L agar, with a pH of 5.8.

[0013] Furthermore, in step S4, before inoculating the obtained rooted shoot clusters into the proliferation medium, the excessively long parts of the roots are cut off, leaving roots with a length of 1-2 cm.

[0014] Furthermore, in step S5, before inoculating the obtained large number of rooted shoots into the seedling rooting medium for seedling rooting culture, the excessively long parts of the roots are cut off, retaining roots with a length of 1-3 cm. The operation of cutting off the excessively long parts of the roots in S4 and S5 can stimulate the germination of new roots, forming a more vigorous absorbing root group, while facilitating the inoculation operation in the culture medium, which is beneficial to nutrient absorption and growth.

[0015] The beneficial effects of this application are: 1. Achieve efficient, stable, and rapid propagation of superior traits, significantly shortening the industrialization cycle.

[0016] This invention uses stem segments from superior individual plants and induced axillary buds as explants, falling under the category of asexual reproduction. It completely preserves the superior genetic traits of the parent plant, overcoming the phenotypic differentiation problems caused by seed propagation. Through a systematic "first generation-proliferation-seedling strengthening" tissue culture rapid propagation process, large-scale supply of superior individual plant seedlings can be achieved in approximately two years. Compared to the 8-10 years required by traditional asexual propagation methods such as division and cuttings, the industrialization cycle is shortened by more than 75%. This greatly accelerates the process from superior variety selection to industrial application, enabling a rapid response to market demand for high-quality Dendrobium officinale seedlings.

[0017] 2. The unique "explant pretreatment" mechanism effectively ensures the supply of high-quality starting materials.

[0018] This application innovatively uses a hydrogen peroxide silver ion disinfectant solution (H2O2 4-6%, Ag) + (0.01%) This treatment was applied periodically to branches collected in winter and dried in the shade. This treatment combines physical bud-promoting and chemical antiseptic effects: on the one hand, the decomposition of hydrogen peroxide slowly releases oxygen, breaking the dormancy of axillary buds and stimulating bud sprouting; on the other hand, the slow-release bactericidal effect of silver ions effectively prevents mold growth during long-term shade drying, significantly improving the survival rate and sprouting quality of axillary buds. Example data confirms that branches treated with this specific concentration solution (Group C1) sprouted significantly more lateral buds than the untreated group and other concentration treatment groups, providing a large number of tender, robust, and relatively sterile high-quality explants for subsequent tissue culture, laying the first foundation for successful rapid propagation.

[0019] 3. Establish a "contamination rescue" mechanism to significantly improve the utilization rate and culture success rate of explants.

[0020] This application presents a dedicated rescue procedure for explants that become contaminated during the early stages of culture. After a short soaking in the original induction disinfectant, the explants are directly transferred to a rescue medium supplemented with 50 mg / L potassium sorbate. Potassium sorbate, a food-grade preservative, effectively inhibits the growth of common molds and bacteria at this concentration, and its toxicity to plant tissues is far lower than that of conventional antibiotics. This provides a "secondary purification" and growth recovery opportunity for slightly contaminated explants, reducing the loss rate of precious explants and improving overall experimental efficiency and cost-effectiveness.

[0021] 4. Root-based propagation enables stable and efficient propagation of Dendrobium nobile.

[0022] Using rooted seedlings for Dendrobium propagation culture allows the already functional root system to enable the tissue-cultured seedlings to quickly adapt to the new environment after transfer, eliminating the need for the rooting induction period required for rootless seedlings. This allows the seedlings to immediately and efficiently absorb mineral nutrients and sugars from the culture medium, providing a direct material and energy basis for the rapid proliferation of clustered shoots. During this process, the existing root system continuously stimulates the development of new roots, forming a virtuous cycle of "absorption-growth," thus achieving stable and efficient in vitro rapid propagation of Dendrobium. Attached Figure Description

[0023] The above and other objects, features and advantages of this application will become more apparent from the more detailed description of exemplary embodiments thereof in conjunction with the accompanying drawings, wherein the same reference numerals generally represent the same components in the exemplary embodiments thereof.

[0024] Figure 1 This is a schematic diagram showing the axillary bud germination of Dendrobium officinale branches treated with hydrogen peroxide silver ion disinfectant solution in Example 1 of the present invention and its control. Figure 2 A schematic diagram of the lateral bud explants used for rapid tissue culture propagation in Example 1 of this invention; Figure 3 This is a schematic diagram of rooted shoot clusters induced in the shoot cluster rooting induction medium in Example 1 of the present invention; Figure 4 This is a schematic diagram of the proliferation culture of rooted shoot clusters in Example 1 of the present invention; Figure 5 This is a schematic diagram of tissue culture seedlings after rooting in Embodiment 1 of the present invention. Detailed Implementation

[0025] The embodiments of this application will now be described in more detail with reference to the examples. While embodiments of this application are shown in the examples, it should be understood that this application can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to make this application more thorough and complete, and to fully convey the scope of this application to those skilled in the art.

[0026] Example 1 This embodiment describes a method for rapid tissue culture propagation using Dendrobium nobile stem segments as starting material, comprising the following steps: (1) Axillary bud induction of detached branches: In December 2023, mature fresh stems were collected from the Dendrobium officinale planting base in Longling County, the leaves were removed, and the stems were brought back to the laboratory of the College of Forestry of Southwest Forestry University and left for about 40 days. Disinfectant solutions of different concentrations of hydrogen peroxide and silver ion content were sprayed once or twice a month on nearly equal-length Dendrobium officinale branches. The lateral bud sprouting was counted at the end of April of the following year. The details are shown in Table 1 below:

[0027] As shown in Table 1 above, branches treated with group C1 hydrogen peroxide silver ion disinfectant solution (5% hydrogen peroxide and 0.01% silver ion) developed a large number of lateral buds, while branches not treated with hydrogen peroxide silver ion disinfectant solution or treated with other concentrations of hydrogen peroxide silver ion disinfectant solution only had a small number of lateral buds sprouting. The results are as follows: Figure 1 As shown.

[0028] (2) Explant disinfection: Select lateral tender axillary buds with a bud height of 3-5cm as explants for rapid tissue culture propagation. After disinfection, place them in pre-culture medium (culture conditions: light intensity 1500-2000lx, light time 8h / d, temperature controlled at 25±2℃) to obtain sterile explants; The disinfection process specifically involves: first, immersing the sample in 75% alcohol for 1 or 2 minutes, rinsing it four times with sterile water, and then disinfecting it with 0.1% HgCl2 solution by shaking for 6, 8, 10, and 12 minutes respectively. The sample is then rinsed six times with sterile water, and the surface moisture is absorbed with filter paper.

[0029] The pre-medium formulation was: MS + 2 mg / L 6-BA + 0.8 mg / L NAA, pH 5.8. After inoculation, the contamination rate and germination rate were recorded. After 20 days, the contamination status and germination rate were also recorded. Uncontaminated material was then transferred to the start-up medium. The contamination rate was determined by the presence of colonies, hyphae, slime, and other signs of microbial growth on and around the explant surface. Specific results are shown in Table 2 below.

[0030] As can be seen from Table 2, explants treated with 75% alcohol for 1 min and 0.1% HgCl2 for 8 min had a lower contamination rate and a higher survival rate. Overall, the T2 disinfection method was the most effective.

[0031] (3) Primary culture: Aseptic explants were inoculated into starter culture medium, the formulation of which was: MS + 6-BA 4 mg / L + NAA 0.8 mg / L + 30 g / L sucrose + 4.2 g / L agar, pH 5.8. After 45 days of culture (culture conditions: 23±2°C, light time 12 h / d), new lateral buds grew from the explants and adventitious roots formed at the base, such as Figure 3 As shown; (4) Proliferation Culture: The obtained rooted shoot clusters were transferred to a proliferation medium. The proliferation medium formula was: MS + 6-BA 2mg / L + NAA 1.5mg / L + activated carbon 1g / L + 100 g / L potato + 30g / L sucrose + 4.2g / L agar, pH 5.8; (culture conditions: 23±2°C, light time 12h / d). The root length was retained to about 1 cm. Subculture was carried out once every 60 days, and the average proliferation ratio reached 5.8 times. The proliferation effect was as follows. Figure 4 As shown; a large number of rooted shoots were obtained; (5) Seedling rooting culture: The obtained rooted clumps of shoots were inoculated in groups of 3-5, with the root length retained to be about 2 cm. They were inoculated into seedling rooting culture medium, the formula of which was: MS + 6-BA 0.3 mg / L + NAA 0.5 mg / L + KT 0.4 mg / L + 60 g / L potato (culture conditions: 23±2°C, light time 12h / d). The rooting rate was 100% after 60 days. The growth of the tissue culture seedlings was as follows. Figure 5 As shown.

[0032] Example 2 (This example specifically focuses on the rescue of explants if the disinfection is incomplete during the explant disinfection process) Experimental steps: During the disinfection process of explants, some explants may not be thoroughly disinfected, resulting in incompletely disinfected explants, i.e., contaminated explants. The contamination criterion is as follows: After culturing the disinfected explants for 20 days, observation is performed. If signs of microbial growth, such as colonies, hyphae, or mucus, appear on and around the explant surface, it is considered contaminated.

[0033] Contaminated explants were rescued by soaking them in a disinfectant solution containing 5% hydrogen peroxide and 0.01% silver ions for 8-10 minutes, and then inoculating them into the rescue medium. The rescue medium was prepared by adding 50 mg / L potassium sorbate to the budding rooting induction medium.

[0034] Statistics show that the rescue rate reached 50%. Rescue rate = Number of uncontaminated explants after disinfection and rescue / Total number of contaminated explants used for disinfection and rescue.

[0035] The various embodiments of this application have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or improvement of the technology in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A method for rapid tissue culture propagation using Dendrobium nobile stem segments as starting material, characterized in that, Includes the following steps: S1. Preparation of axillary bud explants: Fresh stems of superior Dendrobium officinale plants were collected in winter and placed in a cool and ventilated place to dry for 30-50 days; then, hydrogen peroxide silver ion disinfectant solution was sprayed 1-2 times per month to induce axillary bud germination, and the germinating tender axillary buds were used as explants for rapid tissue culture propagation. The hydrogen peroxide silver ion disinfectant solution contains 4-6% hydrogen peroxide and 0.01% silver ions. S2. Explant disinfection: Young axillary buds sprouting from S1 were cut off, disinfected, and then cultured in a pre-culture medium to obtain sterile explants. S3. Primary culture: The sterile explants are inoculated into the starter culture medium and cultured until rooting occurs to obtain rooted shoot clusters; The formulation of the start-up medium is as follows: MS + 6-BA 4 mg / L + NAA 0.8 mg / L + 30 g / L sucrose + 4.2 g / L agar, pH 5.8; S4. Proliferation Culture: The obtained rooted shoot clusters are inoculated into the proliferation culture medium for proliferation culture to obtain a large number of rooted shoot clusters; S5. Seedling rooting culture: The large number of rooted shoots obtained in S4 are inoculated into seedling rooting culture medium for seedling rooting culture, thus obtaining tissue culture seedlings.

2. The method for rapid tissue culture propagation using Dendrobium nobile stem segments as starting material as described in claim 1, characterized in that, In S2, if contamination occurs during the culture process after the explant is disinfected, the explant is soaked in the hydrogen peroxide silver ion disinfectant solution in S1 for 8-10 minutes for disinfection. No washing is required. Then, it is inoculated into the rescue culture medium for rescue. The rescue medium is formulated as follows: 50 mg / L of potassium sorbate is added to the start-up medium.

3. The method for rapid tissue culture propagation using Dendrobium nobile stem segments as starting material as described in claim 1, characterized in that, In S1, tender lateral axillary buds with a bud height of 3-5cm are selected as explants for rapid tissue culture propagation.

4. The method for rapid tissue culture propagation using Dendrobium nobile stem segments as starting material as described in claim 1, characterized in that, The culture medium formula is: MS + 6-BA 2mg / L + NAA 1.5mg / L + activated carbon 1g / L + potato starch 100 g / L + sucrose 30g / L + agar 4.2g / L, pH 5.

8.

5. The method for rapid tissue culture propagation of Dendrobium nobile stem segments as starting material according to claim 1, characterized in that, The formula for the seedling rooting medium is MS + 6-BA 0.3 mg / L + NAA 0.5 mg / L + KT 0.4 mg / L + activated carbon 1 g / L + sucrose 26 g / L + agar 4.2 g / L + potato starch 60 g / L, with a pH of 5.

8.

6. The method for rapid tissue culture propagation of Dendrobium nobile stem segments as starting material according to claim 1, characterized in that, The disinfection process in step S2 is as follows: first, soak the explants in 75% ethanol for 1-2 minutes, then rinse with sterile water, then disinfect with 0.1% HgCl2 solution by shaking for 6-10 minutes, and finally rinse with sterile water and blot dry with filter paper.

7. The method for rapid tissue culture propagation of Dendrobium nobile stem segments as starting material according to claim 1, characterized in that, The pre-culture medium was formulated as follows: MS + 1.0-2.0 mg / L 6-BA + 0.8 mg / L NAA + 30 g / L sucrose + 4.2 g / L agar, with a pH of 5.

8.

8. The method for rapid tissue culture propagation using Dendrobium nobile stem segments as starting material as described in claim 1, characterized in that, In step S4, before inoculating the obtained rooted shoot clusters into the proliferation medium, the excessively long parts of the roots are cut off, leaving roots with a length of 1-2 cm.

9. A method for rapid tissue culture propagation using Dendrobium nobile stem segments as starting material, as described in claim 1, characterized in that... In step S5, before inoculating the obtained large number of rooted shoots into the seedling rooting culture medium for seedling rooting culture, the excessively long parts of the root system are cut off, and the root system with a length of 1-3 cm is retained.

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