A tissue culture method for ornamental aquatic plant *Cymbidium goeringii*

Abstract

This invention provides a tissue culture method for the ornamental aquatic plant *Cephalotaxus fortunei*, including explant preparation: selecting healthy plants free from diseases and pests, cutting stem segments with strong regeneration ability containing 1-2 leaf buds, washing with soapy water, rinsing with tap water for 15 minutes, placing the washed stem segments in a clean bench, soaking in 75% alcohol for 30 seconds, rinsing with sterile water 1-2 times, soaking in 0.1% mercuric chloride solution for 5 minutes, and rinsing with sterile water 3-5 times. Explant inoculation: inoculating the sterilized stem segments into a basic medium formulated with MS + 0.5 mg / L 6-BA + 0.1 mg / L NAA for induction culture. After 30-35 days of culture, differentiated seedlings are obtained. The tissue culture seedlings obtained by the method of this invention have a high proliferation coefficient, are sterile and free from pests and diseases, are uniform and robust, have well-developed root systems, have a long shelf life, and a survival rate of 100%. The overall tissue culture process is simple and easy to operate, shortening the time by more than 20 days compared with traditional methods, which is of great significance to the development of the ornamental aquatic plant industry.

Description

Technical Field

[0001] This invention relates to the field of plant production technology, specifically to a tissue culture method for an ornamental herbaceous plant called *Hedyotis diffusa*. Background Technology

[0002] Ornamental aquatic plants boast rich colors and elegant appearances, beautifying the environment and purifying the air, making them highly valuable for their aesthetic appeal. *Cephalotaxus fortunei* (also known as *Cephalotaxus fortunei*) is an easy-to-cultivate ornamental aquatic plant, belonging to the dicotyledonous family. It is highly adaptable, grows rapidly, has a beautiful shape, and its leaves spread out gracefully. It is easy to establish and, especially, absorbs a large amount of nutrients in the early stages of a new aquarium setup, effectively inhibiting algae growth. Therefore, it is very popular among aquarists. Furthermore, it has significant ecological value, maintaining biodiversity, providing abundant food sources, habitats, and breeding grounds for aquatic organisms, and also purifying water.

[0003] Plant tissue culture is a technique that separates a portion of a plant's cells or tissues from the parent plant and cultivates them under appropriate conditions to enable them to grow, develop, differentiate, and proliferate. The principle stems from the totipotency of plant cells, meaning that a certain type of cell within the plant is inoculated onto a specialized culture medium under sterile conditions to obtain regenerated, complete plants. This not only preserves the inherent traits and characteristics of the original variety but also saves propagation material, accelerates reproduction, and conserves land. It is suitable for removing pathogens, enabling factory-scale seedling production, and using tissue culture methods to rejuvenate varieties and promote the transition of individual plants to a younger stage of development. Traditionally, *Aquatic Plants* (specifically, the *Aquatic Plants of the Palace* variety) are primarily propagated through greenhouse division. If the temperature is between 22-25℃, propagation can be carried out year-round. However, due to the significant difference between greenhouse cultivation and aquarium environments, *Aquatic Plants of the Palace* are very prone to death during the transition to aquatic environments. Furthermore, greenhouse-grown *Aquatic Plants of the Palace* easily carry various pathogens, weeds, snails, etc., which can easily introduce contamination into the aquarium, affecting its aesthetics, diminishing its landscaping effect, and causing fish and insect diseases and even death. Therefore, we have made improvements and proposed a tissue culture method for ornamental aquatic plant *Cymbidium goeringii*. Summary of the Invention

[0004] The purpose of this invention is to address the problems raised in the existing background technology. To achieve the above-mentioned objective, this invention provides the following technical solution: a method for tissue culture of the ornamental aquatic plant *Cymbidium goeringii*, comprising the following steps:

[0005] Step 1: Explant preparation. Select healthy plants free from pests and diseases. Cut stem segments with strong regeneration ability containing 1-2 leaf buds. Wash with soapy water, then rinse with tap water. Place the cleaned stem segments in a clean bench and soak in 75% alcohol for 30 seconds. Then rinse with sterile water 1-2 times. Soak in 0.1% mercuric chloride solution for 15 minutes, and then rinse with sterile water 3 times.

[0006] Step 2: Inoculation of explants. MS basal medium was used with added hormones 6-BA and NAA. The medium was dispensed, autoclaved, and cooled before use. In a clean bench, the cut stem segments were inoculated into the sterilized MS medium. After inoculation, the plants were placed in a culture room with a light intensity of 2000-2500 Lux, a light intensity of 10 h, and a room temperature of 23-25℃. Differentiated seedlings were obtained after 30-35 days of culture.

[0007] Step 3: Subculture. The culture medium used for subculture is MS + 2 mg / L 6-BA + 0.1 mg / L NAA. Sterile seedling stem segments are cut into 1-2 cm lengths and inoculated into the subculture medium in clusters. Propagation is carried out once every 40-45 days. After one cycle of subculture, sterile *Cyperus rotundus* is obtained.

[0008] Step 4: Rooting. Transfer individual propagated seedlings to rooting medium. The rooting medium used is MS + 0.25 mg / L 6-BA + 0.1 mg / L NAA. Induce rooting to obtain tissue culture seedlings. Repeat steps 3 to 4 to continuously obtain tissue culture seedlings.

[0009] As a preferred technical solution of the present invention, the explant surface sterilization in step 1 is performed by soaking in 75% alcohol for 30 seconds, rinsing with sterile water 1-2 times, then soaking in 0.1% mercuric chloride solution for 5 minutes, and finally rinsing with sterile water 3 times.

[0010] As a preferred technical solution of the present invention, the experimental material used for differentiation culture in step 1 is a stem segment of a healthy plant free from diseases and pests, containing 1-2 leaf buds.

[0011] As a preferred technical solution of the present invention, the composition of the differentiation medium in step 2 is: MS + 0.5 mg / L 6-BA + 0.1 mg / L NAA; the culture conditions are: photoperiod 10 h / d, temperature 25 ± 2 ℃, and light intensity 2000-2500 Lux.

[0012] As a preferred technical solution of the present invention, the composition of the proliferation medium in step 3 is: MS + 2.0 mg / L 6-BA + 0.1 mg / L NAA; the culture conditions are: photoperiod 10 h / d, temperature 25 ± 2 ℃, and light intensity 2000-2500 Lux.

[0013] As a preferred technical solution of the present invention, the experimental material used for rooting culture in step 4 is a single propagated seedling, and the rooting medium is MS + 0.25 mg / L 6-BA + 0.1 mg NAA. The culture conditions are: photoperiod 10 h / d, temperature 25 ± 2 ℃, and light intensity 2000-2500 Lux.

[0014] As a preferred technical solution of the present invention, during the sterilization process of the explant surface, the soaking time of 75% alcohol must be strictly controlled within 30 seconds ± 2 seconds to ensure that the alcohol can effectively disinfect without damaging the explant.

[0015] As a preferred technical solution of the present invention, the experimental material used for differentiation culture is a stem segment containing 1-2 leaf buds, which is rinsed with sterile water at least three times before being inoculated into the differentiation culture medium to ensure that there is no residual alcohol and mercuric chloride solution on the surface.

[0016] As a preferred technical solution of the present invention, in the differentiation medium in step 2, the concentration of 6-BA must be precisely controlled at 0.5±0.05mg / L, and the concentration of NAA must be precisely controlled at 0.1±0.01mg / L, in order to ensure the best differentiation effect.

[0017] As a preferred technical solution of the present invention, the proliferation medium in step 3 is subcultured 3-4 times in each cycle to achieve the best balance between the quality and quantity of seedlings.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: In the solution of the present invention, the technical solution of this application provides a tissue culture method for ornamental aquatic plant *Cymbidium goeringii*, including the steps of obtaining explants, differentiating, proliferating, and rooting, and simultaneously obtaining tissue culture seedlings and production seedlings of *Cymbidium goeringii*, which can be used for expanded reproduction and can also be used directly for aquatic plant cultivation. The explants are widely available, the process is simple, the culture process is convenient, there are few steps, and the reproduction coefficient is high. It can rapidly obtain a large number of seedlings with consistent genetic traits, that are sterile, free from diseases, pests, and weeds, and is not affected by seasonal climate changes or natural disasters, making it suitable for large-scale industrial production. Attached Figure Description

[0019] Figure 1 The method flowchart provided by the present invention. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0021] Therefore, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely illustrates some embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. It should be noted that, unless otherwise specified, the embodiments and features and technical solutions in the embodiments of the present invention can be combined with each other. It should be noted that similar reference numerals and letters in the following figures indicate similar items; therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.

[0022] Example 1: Please refer to Figure 1 A tissue culture method for ornamental aquatic plant *Cymbidium goeringii* includes the following steps: Step 1: Explant preparation. Select healthy plants free from pests and diseases. Cut stem segments with strong regeneration ability containing 1-2 leaf buds. Wash with soapy water and rinse with tap water. Place the cleaned stem segments in a clean bench and soak in 75% alcohol for 30 seconds. Rinse with sterile water 1-2 times. Soak in 0.1% mercuric chloride solution for 15 minutes. Then rinse with sterile water 3 times.

[0023] Step 2: Inoculation of explants. MS basal medium was used with added hormones 6-BA and NAA. The medium was dispensed, autoclaved, and cooled before use. In a clean bench, the cut stem segments were inoculated into the sterilized MS medium. After inoculation, the plants were placed in a culture room with a light intensity of 2000-2500 Lux, a light intensity of 10 h, and a room temperature of 23-25℃. Differentiated seedlings were obtained after 30-35 days of culture.

[0024] Step 3: Subculture. The culture medium used for subculture is MS + 2 mg / L 6-BA + 0.1 mg / L NAA. Sterile seedling stem segments are cut into 1-2 cm lengths and inoculated into the subculture medium in clusters. Propagation is carried out once every 40-45 days. After one cycle of subculture, sterile *Cyperus rotundus* is obtained.

[0025] Step 4: Rooting. Transfer individual propagated seedlings to rooting medium. The rooting medium used is MS + 0.25 mg / L 6-BA + 0.1 mg / L NAA. Induce rooting to obtain tissue culture seedlings. Repeat steps 3 to 4 to continuously obtain tissue culture seedlings.

[0026] The explant surface sterilization step 1 involves soaking in 75% alcohol for 30 seconds, rinsing with sterile water 1-2 times, then soaking in 0.1% mercuric chloride solution for 5 minutes, and finally rinsing with sterile water 3 times.

[0027] The experimental material used for differentiation culture in step 1 is a stem segment of a healthy plant free from disease and pests, containing 1-2 leaf buds.

[0028] The differentiation medium in step 2 consists of MS + 0.5 mg / L 6-BA + 0.1 mg / L NAA; the culture conditions are: 10 h / d photoperiod, 25 ± 2 ℃, and 2000-2500 Lux light intensity.

[0029] The composition of the proliferation medium in step 3 is: MS + 2.0 mg / L 6-BA + 0.1 mg / L NAA; the culture conditions are: photoperiod 10 h / d, temperature 25 ± 2 ℃, and light intensity 2000-2500 Lux.

[0030] The experimental material used for rooting culture in step 4 is a single propagated seedling. The rooting medium is MS + 0.25 mg / L 6-BA + 0.1 mg NAA. The culture conditions are: photoperiod 10 h / d, temperature 25 ± 2 ℃, and light intensity 2000-2500 Lux.

[0031] During the sterilization process of explant surface, the immersion time in 75% alcohol must be strictly controlled within 30 seconds ± 2 seconds to ensure that the alcohol can effectively disinfect without damaging the explant.

[0032] The experimental material used for differentiation culture, stem segments containing 1-2 leaf buds, should be rinsed with sterile water at least three times before inoculation into the differentiation medium to ensure that there is no residual alcohol or mercuric chloride solution on the surface.

[0033] In the differentiation medium of step 2, the concentration of 6-BA must be precisely controlled at 0.5±0.05 mg / L, and the concentration of NAA must be precisely controlled at 0.1±0.01 mg / L to ensure the best differentiation effect.

[0034] In step 3, the number of subcultures in the proliferation medium is controlled to be 3-4 times per cycle to achieve the best balance between the quality and quantity of seedlings.

[0035] Specifically, step 1 involves obtaining the explants. The explants mentioned in step 1 are derived from healthy seedlings free from diseases and pests used in production.

[0036] Step 2: Differentiation. After removing the culture medium, roots, and miscellaneous leaves, cut off the stem containing 1-2 leaf buds and transfer it to the differentiation medium to obtain differentiated seedlings. The differentiation medium consists of MS + 0.5 mg / L 6-BA + 0.1 mg / L NAA. The culture conditions are: 10 h / d photoperiod, 25 ± 2℃, and 2000-2500 Lux light intensity.

[0037] Step 3: Proliferation. Differentiated seedlings were transferred to proliferation medium and cultured for 40-45 days to obtain proliferated seedlings, with a 20-30 fold increase in seedling number. The proliferation medium consisted of MS medium + 2.0 mg / L 6-BA + 0.1 mg / L NAA. Culture conditions were: 10 h / d photoperiod, 25 ± 2℃, and 2000-2500 Lux light intensity.

[0038] Step 4: Rooting. Transfer the proliferated seedlings to rooting medium to induce rooting and obtain tissue culture seedlings. The composition of the rooting medium is: MS + 0.25 mg / L 6-BA + 0.1 mg / L NAA; the culture conditions are: photoperiod 10 h / d, temperature 25 ± 2℃, and light intensity 2000-2500 Lux.

[0039] Example 2

[0040] In this embodiment, the difference between Embodiment 2 and Embodiment 1 is as follows:

[0041] Sterilization of explants: Soak in 75% alcohol for 30 seconds, then rinse with sterile water, then soak in 0.1% mercuric chloride solution for 5 minutes, and finally rinse with sterile water 3 times.

[0042] The optimal light duration for cultivating *Crassula ovata* is 10 hours, with a light intensity of 2000-2500 Lux and a temperature of 25℃±2℃. Temperatures below 15℃ or above 35℃ are detrimental to growth; maintain a relative humidity of 70%-80%. Differentiated seedlings will be obtained after 30-35 days of cultivation.

[0043] Step 4: Subculture propagation: After a period of cultivation, a sterile line of *Gymnocalycium mihanovichii* was established, followed by subculture propagation. The culture medium used for subculture was MS medium supplemented with 2.0 mg / L 6-BA and 0.1 mg / L NAA. Axillary buds germinating from the sterile line were cut off, and 3-4 axillary buds were inoculated into each bottle. After sealing, the bottles were placed in a culture room for subculture. The culture room was disinfected weekly, and the culture medium was changed every 25-28 days. After one cycle of subculture, sterile *Gymnocalycium mihanovichii* was obtained. The propagation coefficient of *Gymnocalycium mihanovichii* reached 9.3.

[0044] Example 3

[0045] The difference between Example 3 and Example 1 is as follows:

[0046] In step 2, the explants are sterilized: the leaves on the tender stems are cut off, the stem segments are washed with laundry detergent, and then rinsed with tap water. The washed stem segments are cut into 2cm lengths. The cut stem segments are placed in a clean bench and soaked in 75% alcohol for 30 seconds. The alcohol is then rinsed off with sterile distilled water. The segments are then soaked in 0.1% mercuric chloride solution for 20 minutes and then rinsed 4 times with sterile water.

[0047] In step 3, aseptic system establishment: The basic culture medium used to establish the aseptic system is MS medium, and the hormones used are 6BA and NAA. The culture medium is poured into wide-mouth bottles, with 40 ml of culture medium in each bottle. After sealing, the bottles are autoclaved and cooled for later use. In a laminar flow hood, the cut stem segments are then inserted into the sterilized MS medium, one per bottle.

[0048] Five stem segments were inserted into the culture medium, sealed, and then placed in a culture chamber for culture.

[0049] In step 4, subculture propagation: After a period of cultivation, a sterile line of *Hedyotis diffusa* was established, followed by subculture propagation. The culture medium used for subculture was MS medium + 2 mg / L 6-BA + 0.1 mg / L NAA, with each cup containing 40 ml of medium. Axillary buds germinating from the sterile line were cut off, and 3-4 axillary buds were inoculated into each cup. After sealing, the cups were placed in a culture room for subculture. The culture room was disinfected weekly, and the culture medium was changed every 25-28 days. After one cycle of subculture, sterile *Hedyotis diffusa* was obtained. The propagation coefficient of *Hedyotis diffusa* reached 9.6.

[0050] The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described herein. Although the present invention has been described in detail with reference to the above embodiments, the present invention is not limited to the specific embodiments described above. Therefore, any modifications or equivalent substitutions to the present invention, as well as all technical solutions and improvements that do not depart from the spirit and scope of the invention, are covered within the scope of the claims of the present invention.

Claims

1. A method for tissue culture of the ornamental aquatic plant *Crassula ovata*, characterized in that, Includes the following steps: Step 1: Explant preparation. Select healthy plants free from pests and diseases. Cut stem segments with strong regeneration ability containing 1-2 leaf buds. Wash with soapy water, then rinse with tap water. Place the cleaned stem segments in a clean bench and soak in 75% alcohol for 30 seconds. Then rinse with sterile water 1-2 times. Soak in 0.1% mercuric chloride solution for 15 minutes, and then rinse with sterile water 3 times. Step 2: Inoculation of explants. The differentiation medium consists of MS medium + 0.5 mg / L 6-BA + 0.1 mg / L NAA. The medium is dispensed, autoclaved, and cooled before use. In a clean bench, the cut stem segments are inoculated into the sterilized MS medium. After inoculation, the plants are placed in a culture room with a light intensity of 2000-2500 Lux, a light intensity of 10 h, and a room temperature of 23-25℃. After 30-35 days of culture, differentiated seedlings are obtained. Step 3: Subculture. The culture medium used for subculture was MS + 2 mg / L 6-BA + 0.1 mg / L NAA. Sterile seedling stem segments were cut into 1-2 cm lengths and inoculated into the subculture medium in clusters. Propagation was carried out once every 40-45 days. After one cycle of subculture, sterile *Cymbidium goeringii* was obtained. The culture conditions were: photoperiod 10 h / d, temperature 25 ± 2℃, and light intensity 2000-2500 Lux. Step 4: Rooting. The proliferated seedlings are transferred to the rooting medium, which is MS + 0.25 mg / L 6-BA + 0.1 mg / L NAA. Rooting is induced to obtain tissue culture seedlings. Steps 3 and 4 are repeated to continuously obtain tissue culture seedlings. The culture conditions for step 4 are: photoperiod 10 h / d, temperature 25 ± 2 ℃, and light intensity 2000-2500 Lux.

2. The method for tissue culture of the ornamental aquatic plant *Crassula ovata* according to claim 1, characterized in that, In step 3, the number of subcultures in the proliferation medium is controlled to be 3-4 times per cycle to achieve the best balance between the quality and quantity of seedlings.

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