A culture medium for dwarfing Cibotium barometz

Abstract

This invention discloses a culture medium for dwarfing *Cibotium barometz*, belonging to the field of plant tissue culture technology. The culture medium comprises the following components: 1 / 6-1 / 3 MS, sucrose 2-6 g / L, 6-benzylaminopurine 0.03-0.15 mg / L, agar 4-8 g / L, indolebutyric acid 0.01-0.08 mg / L, paclobutrazol 0.25-0.8 mg / L, and activated carbon 0.3-1.0 g / L. This culture medium can synergistically inhibit excessive petiole growth and delay leaf senescence, thus solving the technical problem that *Cibotium barometz* cannot maintain its miniature and graceful morphology and require no maintenance in a closed microenvironment for a long period.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, and more specifically to a culture medium for dwarfing Cibotium barometz. Background Technology

[0002] Cibotium barometz, also known as the golden-haired dog fern, belongs to the genus Cibotium in the family Ceratophyllaceae. It is an ancient relict plant originating from the Jurassic period, possessing extremely high medicinal and ornamental value. However, due to habitat destruction and over-harvesting, its wild population has drastically declined, and it has been listed in the "National Key Protected Wild Plants List" (Class II) and Appendix II of the "Convention on International Trade in Endangered Species of Wild Fauna and Flora" (CITES). In today's world, where biodiversity conservation has become a global consensus, how to resolve the contradiction between strict protection and rational utilization of endangered plant resources—that is, meeting the public's needs for science education, medicinal use, and ornamental purposes through artificial propagation techniques without relying on wild harvesting—is a crucial issue facing the field of plant biotechnology.

[0003] To address the seed source problem of Cibotium barometz, existing technologies primarily employ plant tissue culture (in vitro rapid propagation). Typical technical solutions (such as Chinese patent documents CN105230492B and CN107736248A) typically use full-strength MS medium or high-salt modified medium, combined with high concentrations of sucrose and conventional concentrations of cytokinins and auxins. The core objective of these existing technologies is rapid proliferation and biomass acquisition, i.e., obtaining a large number of seedlings in a short period of time for subsequent transplanting to fields or greenhouses.

[0004] However, when attempting to directly apply the aforementioned conventional rapid propagation techniques to the emerging application scenario of desktop micro-landscapes, serious technical drawbacks exist: First, excessive growth and a short viewing period occur. The high nitrogen sources and high concentrations of hormones in conventional culture media cause tissue culture seedlings to grow rapidly in sealed containers, with petioles elongating beyond the container height within 30-45 days, leading to leaf curling, morphological degradation, and loss of ornamental value. Second, physiological instability and susceptibility to vitrification occur. High humidity and high nutrient environments make seedlings highly susceptible to vitrification, manifesting as water-soaked, translucent leaves that lose normal physiological function and leaf color. Furthermore, maintenance costs are high, making zero-care-requirement impossible. Existing technologies rely on frequent subculturing (typically requiring opening and changing the culture medium every 30-60 days), which not only increases the risk of contamination but also prevents non-professional users from maintaining and appreciating the plants in home or office environments for extended periods. Summary of the Invention

[0005] The main objective of this invention is to provide a culture medium for dwarfing *Cibotium barometz*, which can synergistically inhibit excessive petiole growth and delay leaf senescence, thereby solving the technical problem that *Cibotium barometz* cannot maintain its miniature and beautiful shape and require no maintenance for a long time in a closed microenvironment.

[0006] To achieve the above objectives, the present invention provides a culture medium for dwarfing Cibotium barometz, comprising the following components: 1 / 6-1 / 3 MS, sucrose 2-6 g / L, 6-benzylaminopurine 0.03-0.15 mg / L, agar 4-8 g / L, indolebutyric acid 0.01-0.08 mg / L, paclobutrazol 0.25-0.8 mg / L, activated charcoal 0.3-1.0 g / L.

[0007] Based on the above technical solutions, preferably, the culture medium for dwarfing Cibotium barometz further includes 0.1-0.5 mg / L of sucrose octaacetate.

[0008] Based on the above technical solutions, preferably, the culture medium for dwarfing Cibotium barometz also includes ascorbic acid 5-20 mg / L.

[0009] Based on the above technical solutions, preferably, the ratio of 6-benzylaminopurine to indolebutyric acid is 2-10:1.

[0010] The present invention also provides a method for cultivating Cibotium barometz using the aforementioned culture medium, comprising the following steps: (1) Culture medium preparation: After the culture medium is mixed evenly, it is dispensed into transparent containers and allowed to solidify. (2) Inoculation: Inoculate the sterile young sporophytes or sterile seedlings of the golden-haired dog fern into the culture medium; (3) Sealing: The transparent container is sealed with a breathable seal; (4) Cultivation: Place the sealed container in a controlled environment for static cultivation.

[0011] Based on the above technical solutions, preferably, the height of the sterile larval sporophytes or sterile seedlings inoculated in step (2) is 2-4 cm; and in the cultivation process of step (4), there is no need to change the culture medium or open the bottle, and the continuous cultivation time is ≥200 days.

[0012] Based on the above technical solutions, preferably, in step (3), the gas exchange rate between the inside and outside of the transparent container is controlled to be 0.15-0.25 mL / d.

[0013] Based on the above technical solutions, preferably, the breathable sealing treatment in step (3) is as follows: a polytetrafluoroethylene breathable membrane with a pore size of 0.15-0.3μm is used for sealing, and a sealing film is used for secondary reinforcement.

[0014] Based on the above technical solutions, preferably, the controlled environmental conditions in step (4) are: temperature 16-20℃, light intensity 10-25μmol·m -2 ·s -1 The photoperiod is 6-8 hours per day.

[0015] The present invention also provides the use of culture medium for dwarfing *Cibotium barometz* in the long-term in vitro preservation of endangered germplasm of *Cibotium barometz*.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: 1) This invention provides a culture medium for dwarfing Cibotium barometz. By utilizing the synergistic effect of paclobutrazol and sucrose octaacetate, it inhibits gibberellin biosynthesis and cell wall relaxation and stretching through a dual pathway, achieving low hormone-high dwarfing efficiency and effectively avoiding the plant toxicity and vitrification risks caused by high concentrations of hormones. Meanwhile, this invention introduces an antioxidant-ethylene adsorption system composed of activated carbon and ascorbic acid. This system can continuously remove endogenous ethylene and reactive oxygen species in a closed environment, ensuring that the leaves are free of yellow tips for up to 200 days, thus solving the problems of yellow tips and premature aging. By controlling the ratio of 6-benzylaminopurine to indolebutyric acid to 2-10:1, it can ensure the micro-proliferation of lateral buds while inhibiting excessive petiole growth. At the same time, this invention deeply couples the above chemical system with the physical environment of low temperature (16-20℃) + short day (6-8h) to form a positive feedback regulation, further compressing the elongation potential of petiole cells. With the special microporous membrane sealing structure, the gas exchange rate is precisely locked in the range of 0.15-0.25mL / d, which not only ensures weak aerobic respiration but also prevents external pollution and internal ethylene accumulation, thereby establishing a highly stable micro-ecological balance in the closed system. 2) This invention pioneers a new application scenario for the micro-landscape of *Cibotium barometz*, significantly enhancing the ornamental value and compliant utilization of endangered germplasm resources. It achieves long-term preservation with zero maintenance. Compared to conventional rapid-propagation seedlings with a viewing period of only 30 days, prone to excessive petiole elongation (elongation rate >100%), and requiring frequent transplanting and maintenance, the *Cibotium barometz* micro-landscape obtained using this invention exhibits significantly improved performance: its viewing period is greatly extended, petiole elongation is strictly controlled to ≤15%, and there is no vitrification phenomenon throughout the process, with leaves remaining healthy and vibrant green. This technological breakthrough allows *Cibotium barometz*, a national second-class protected plant, to enter the public eye for the first time in the form of a micro-landscape, successfully solving the long-standing pain points of this endangered species being visible but not harvestable, difficult to maintain, and difficult to promote. The product of this invention requires no opening of the bottle, no watering or fertilization, and no professional maintenance throughout its entire life cycle, greatly reducing maintenance costs and technical barriers. Its miniaturized, maintenance-free, and long shelf-life characteristics make it extremely commercially promising. It can be widely used not only in ecological science education and the development of high-end cultural and creative gifts, but also in providing a new, low-cost, and highly safe solution for the in vitro preservation of rare and endangered fern germplasm resources, achieving a balance of ecological, social, and economic benefits. Detailed Implementation

[0017] To avoid unnecessary details, unless otherwise specified, all items used in the following examples are commercially available products, and all methods used are conventional methods unless otherwise specified.

[0018] The sources of some of the raw materials used in this invention are as follows: Sucrose octaacetate, purchased from Maclean's Reagents, CAS 126-14-7, purity ≥98%.

[0019] Activated carbon, 300 mesh, purchased from Chengdu Zhongbang Activated Carbon Technology Co., Ltd. Example 1

[0020] A method for cultivating dwarf dog fern includes the following steps: (1) Culture medium preparation: Prepare 1 / 4 MS medium and make up to 1L; add 3g sucrose, 0.5g activated carbon, 10mg ascorbic acid and 6g agar; adjust the pH to 5.5 and sterilize at 121℃ for 15min; when the culture medium cools to 45℃, filter and sterilize and add 0.08mg 6-BA, 0.02mg IBA, 0.4mg paclobutrazol and 0.2mg sucrose octaacetate; mix well and dispense into 150mL transparent borosilicate glass bottles, 40mL per bottle, and let stand horizontally to solidify; (2) Inoculation: Select sterile young sporophytes of *Ceratophyllum demersum* with a height of 3 cm and uniform growth, and vertically insert them into the center of the culture medium prepared in step (1) under sterile conditions. Inoculate one plant per bottle. (3) Sealing and cultivation: The vent hole of the bottle was sealed with a PTFE breathable membrane with a pore size of 0.22 μm, and then reinforced with Parafilm sealing film to form a closed microenvironment with a gas exchange rate of 0.20 mL / d; the inoculated container was placed in an artificial climate chamber with a temperature of 18℃ and a light intensity of 20 μmol·m -2 ·s -1 Photoperiod 7h / d (short day), static culture. Example 2

[0021] A method for cultivating dwarf dog fern includes the following steps: (1) Culture medium preparation: Prepare 1 / 6 MS medium and bring the volume to 1L; add 2g sucrose, 0.3g activated carbon, 5mg ascorbic acid and 6g agar; adjust the pH to 5.4, sterilize and cool to 45℃, aseptically add 0.05mg 6-BA, 0.01mg IBA, 0.25mg paclobutrazol and 0.1mg sucrose octaacetate (SA); mix well and dispense; (2) Inoculation: Same as in Example 1; (3) Sealing and cultivation: The sealing method is the same as in Example 1; the cultivation environment is set at a temperature of 16℃ and a light intensity of 15μmol·m. -2 ·s -1 Photoperiod 6h / d. Example 3

[0022] A method for cultivating dwarf dog fern includes the following steps: (1) Culture medium preparation: Prepare 1 / 3 MS medium and bring the volume to 1L; add 6g sucrose, 1.0g activated carbon, 20mg ascorbic acid and 6g agar; adjust the pH to 5.8, sterilize and cool to 45℃, aseptically add 0.15mg 6-BA, 0.05mg IBA, 0.8mg paclobutrazol and 0.5mg sucrose octaacetate, mix well and dispense; (2) Inoculation: Same as in Example 1; (3) Sealing and cultivation: The sealing method is the same as in Example 1; the cultivation environment is set at a temperature of 20°C and a light intensity of 25 μmol·m. -2 ·s -1 The photoperiod is 8 hours per day.

[0023] Comparative Example 1 A method for cultivating Cibotium barometz, simulating existing conventional rapid propagation techniques, is similar in steps to Example 1, except for the different culture medium formulation and cultivation conditions, specifically including: (1) Culture medium preparation: Use full-strength MS medium, add 30g sucrose and 6g agar; hormone is 1.5mg 6-BA + 0.3mg NAA; do not add paclobutrazol, SA, activated charcoal and ascorbic acid; (2) Inoculation: Same as in Example 1; (3) Sealing and cultivation: Use a conventional polypropylene breathable cap (without parafilm), with a gas exchange rate >1.0 mL / d; the cultivation environment is a temperature of 25℃ and a light intensity of 40 μmol‧m. -2 ·s -1 The photoperiod is 14h / d (long day); the petiole elongates by 120% on the 30th day, and the yellowing rate of the leaves is 80% on the 60th day. The plant must be opened and transplanted. The culture medium of the roots of the plant should be washed off and transplanted into a substrate of perlite and peat moss (1:1). The plant should be hardened off in a high-humidity greenhouse environment.

[0024] Comparative Example 2 A method for cultivating dwarf dogwood fern, the steps of which are similar to those in Example 1, except that sucrose octaacetate is not added, specifically including: (1) Culture medium preparation: The basic components and hormones are the same as in Example 1, but sucrose octacetate (SA) is not added, and 0.6 mg / L paclobutrazol is used as a dwarfing agent; (2) Inoculation: Same as in Example 1; (3) Sealing and cultivation: Same as in Example 1.

[0025] Comparative Example 3 A method for cultivating dwarf dogwood fern, the steps of which are similar to those in Example 1, except that paclobutrazol is not added, specifically including: (1) Culture medium preparation: The basic components and hormones are the same as in Example 1, but without the addition of paclobutrazol, and 0.6 mg / L sucrose octaacetate is used as a dwarfing agent; (2) Inoculation: Same as in Example 1; (3) Sealing and cultivation: Same as in Example 1.

[0026] Comparative Example 4 A method for cultivating dwarf dogwood fern, the steps of which are similar to those in Example 1, except that the ratio of 6-BA to IBA is 1:1, specifically including: (1) Culture medium preparation: The basic components and additives are the same as in Example 1, except that 0.08 mg 6-BA + 0.08 mg IBA are added; (2) Inoculation: Same as in Example 1; (3) Sealing and cultivation: Same as in Example 1.

[0027] Comparative Example 5 A method for cultivating dwarf dog fern, the steps of which are similar to those in Example 1, except that activated carbon and ascorbic acid are not added to the culture medium.

[0028] Comparative Example 6 A method for cultivating Cibotium barometz, the steps of which are similar to those in Example 1, differs in the sealing structure and environmental conditions, specifically including: (1) Culture medium preparation: The formula is exactly the same as in Example 1; (2) Inoculation: Same as in Example 1; (3) Sealing and cultivation: Use conventional polypropylene breathable caps (without Parafilm secondary sealing, high breathability); the cultivation environment is 25℃ temperature and 14h / d photoperiod.

[0029] The *Cibotium barometz* micro-landscape bottles (30 bottles per group) prepared in Examples 1-3 and Comparative Examples 2-4 were continuously cultured without opening the bottles, changing the medium, or performing any human intervention. On day 200 of cultivation, various indicators were statistically analyzed. Among these, petiole elongation rate = (petiole length on day 200 - initial petiole length) / initial petiole length × 100%; a lower value indicates a better dwarfing effect. SPAD retention rate = chlorophyll SPAD value on day 200 / initial value × 100%; the higher the value, the greener and healthier the leaves; Vitrification rate = Number of plants with water-soaked, translucent leaves / Total number of plants × 100%.

[0030] The results are shown in Table 1: Table 1 Plant growth performance table As can be seen from the results in Table 1, the present invention, through the coupling of a specific formula with the microenvironment, enables the petiole elongation rate of the golden dog fern to be <15% and the chlorophyll SPAD value to decrease by ≤8% under 200 days of closed culture, with zero vitrification and evergreen, which is significantly better than the excessive growth and death caused by existing technologies, and achieves long-term maintenance-free operation.

[0031] The above are merely preferred embodiments of the present invention and do not limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the patent protection scope of the present invention.

Claims

1. A culture medium for dwarfing Cibotium barometz, characterized in that, Includes the following components: 1 / 6-1 / 3 MS, sucrose 2-6 g / L, 6-benzylaminopurine 0.03-0.15 mg / L, agar 4-8 g / L, indolebutyric acid 0.01-0.08 mg / L, paclobutrazol 0.25-0.8 mg / L, activated charcoal 0.3-1.0 g / L.

2. The culture medium according to claim 1, characterized in that: It also includes sucrose octacetate at 0.1-0.5 mg / L.

3. The culture medium according to claim 1 or 2, characterized in that: It also includes ascorbic acid 5-20 mg / L.

4. The culture medium according to any one of claims 1-3, characterized in that: The ratio of 6-benzylaminopurine to indolebutyric acid is 2-10:

1.

5. A method for cultivating Cibotium barometz using the culture medium according to any one of claims 1-3, characterized in that, Includes the following steps: (1) Culture medium preparation: After the culture medium is mixed evenly, it is dispensed into transparent containers and allowed to solidify. (2) Inoculation: Inoculate the sterile young sporophytes or sterile seedlings of the golden-haired dog fern into the culture medium; (3) Sealing: The transparent container is sealed with a breathable seal; (4) Cultivation: Place the sealed container in a controlled environment for static cultivation.

6. The method according to claim 5, characterized in that: The height of the sterile larval sporophytes or sterile seedlings inoculated in step (2) is 2-4 cm; and in the culture process of step (4), there is no need to change the culture medium or open the bottle, and the continuous culture time is ≥200 days.

7. The method according to claim 5, characterized in that: In step (3), the gas exchange rate between the inside and outside of the transparent container is controlled to be 0.15-0.25 mL / d.

8. The method according to claim 5, characterized in that, The specific steps of step (3) involve sealing the opening with a polytetrafluoroethylene breathable membrane with a pore size of 0.15-0.3μm, and then reinforcing it with a sealing film.

9. The method according to claim 5, characterized in that: The controlled environmental conditions in step (4) are: temperature 16-20℃, light intensity 10-25 μmol·m -2 ·s -1 The photoperiod is 6-8 hours per day.

10. The use of the culture medium according to any one of claims 1-4 in the long-term in vitro preservation of endangered germplasm of Cibotium barometz.

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