Tissue culture and rapid propagation method for potted anthurium andraeanum seedlings

Abstract

The invention provides a tissue culture and rapid propagation method for potted anthurium andraeanum seedlings, and relates to the technical field of tissue culture of potted anthurium andraeanum seedlings. The tissue culture and rapid propagation method for the potted anthurium andraeanum seedlings comprises the following tissue culture methods: step 1, explant selection and treatment: (1) taking stem segments as explants, and selecting tender and non-lignified parts; (2) washing the explant stem with tap water for 10 minutes to remove surface impurities, sequentially soaking the explant stem with 75% alcohol for 30-60 seconds, soaking the explant stem with 0.1% mercury bichloride liquid for 8 minutes, rinsing the explant stem with sterile water for 4-6 times, and soaking the explant stem into the sterile water for later use; and (3) in an ultra-clean workbench, using sterilized filter paper to absorb moisture, cutting the stem segments into small segments of 0.5-1cm, keeping 1-2 stem nodes in each segment, and adding an anti-browning agent when the cut of the stem segments is easy to brown. The anti-browning agent with the concentration of 50-100 mg / L and the anti-browning agent activated carbon with the concentration of 0.5%-2.5% are added, browning products are adsorbed, the permeability of the culture medium is adjusted, high-efficiency oxidation resistance can be achieved through the operation method, the activated carbon adsorbs phenolic substances, ascorbic acid neutralizes free radicals, and browning is synergistically reduced.

Description

Technical Field

[0001] This invention relates to the field of tissue culture technology for potted Anthurium seedlings, specifically a method for rapid propagation of potted Anthurium seedlings through tissue culture. Background Technology

[0002] Taihong No. 1 potted Anthurium seedlings are a premium horticultural variety belonging to the Anthurium genus of the Araceae family. Its signature heart-shaped spathe is a soft pink and emerald green with a waxy, satin-like sheen, paired with a green spadix. The plant grows to approximately 30-40cm tall, with leathery, glossy leaves. It blooms for 8-10 months, with each flower lasting over 2 months. It tolerates shade well, thriving in indoor environments with diffused light, and grows vigorously at 18-28℃ and 60% humidity. As an excellent air purifier, it absorbs harmful substances such as formaldehyde and benzene, making it a high-end flowering plant that combines ornamental and functional qualities.

[0003] Currently, a single potted Anthurium seedling can only produce 3-4 new plants per year, a low propagation rate that falls far short of market demand. Therefore, tissue culture is currently the most common method for propagating potted Anthuriums. This method utilizes the totipotency of plant cells to culture small pieces of plant tissue in vitro for rapid, industrialized asexual propagation. The specific process involves cutting young leaves from the Anthurium, inducing callus tissue, differentiating adventitious buds, and then cultivating them into robust seedlings suitable for transplanting and acclimatization. However, this method primarily uses young leaves, which are quite fragile. Damage to these young leaves can occur during processing, cleaning, inoculation, and cultivation, leading to a high failure rate and impacting tissue culture efficiency.

[0004] When applying for this invention, the applicant, through a search, discovered a Chinese patent disclosing a "rapid propagation method for potted Anthurium seedlings via tissue culture," application number "200410052480.2." This patent primarily utilizes the vigorous proliferation of adventitious buds on both culture media, and the formation of aerial roots in aseptic seedlings during propagation, enabling absorption and direct use for domestication and transplanting, thus achieving rapid propagation. This invention, by shortening the induction and propagation time and simplifying culture conditions or methods, can reduce production costs and improve the efficiency and profit of commercial production. Therefore, the rapid propagation method for potted Anthurium seedlings via tissue culture of this invention offers fast propagation speed and low cost. However, this method mostly uses young leaves, which are relatively fragile and may be damaged during processing, cleaning, inoculation, and cultivation. Therefore, based on the applicant's invention, a rapid propagation method for potted Anthurium seedlings via tissue culture has been developed, solving the problem of young leaves being fragile and potentially damaged during processing, cleaning, inoculation, and cultivation. Summary of the Invention

[0005] (a) Technical problems to be solved To address the shortcomings of existing technologies, this invention provides a method for rapid propagation of potted Anthurium seedlings through tissue culture. This method solves the problem of young, tender leaves being fragile and potentially damaged during each stage of processing, cleaning, inoculation, and cultivation.

[0006] (2) Technical solution To achieve the above objectives, the present invention provides the following technical solution: a method for rapid propagation of potted Anthurium seedlings via tissue culture, comprising the following tissue culture methods. Step 1: Selection and treatment of exogenitals (1) When taking stem segments as explants, the tender and non-lignified parts should be selected; (2) Rinse the explant stem segments with tap water for 10 minutes to remove surface impurities, then soak them in 75% alcohol for 30-60 seconds, followed by soaking in 0.1% mercuric chloride solution for 8 minutes. Rinse with sterile water 4-6 times, and then immerse in sterile water for later use. (3) In a clean bench, after blotting off the moisture with sterile filter paper, cut the stem segments into 0.5-1cm segments, retaining 1-2 nodes in each segment. Since the cut surfaces of the stem segments are prone to browning, an anti-browning agent should be added. Step 2: Inoculation and Culture (1) Inside the laminar flow hood, use sterile forceps to vertically insert the stem segment into the culture medium, ensuring full contact between the base and the medium; (2) Immediately after inoculation, seal the culture dish or Erlenmeyer flask with sealing film to prevent contamination; Step 3: Cultivation Conditions (1) Temperature control: constant temperature 26±2℃; (2) Dark culture for 10 days in the early stage, then switch to light 1500-3000 Lux, with 8-10 hours of light per day; (3) Humidity 70%-85% to avoid condensation buildup; (4) After 4-6 weeks, transfer to a culture medium containing a low concentration of auxin to promote lateral bud differentiation.

[0007] As a preferred technical solution of the present invention: in step one, the stem segment length is 1-2cm, and 1-2 stem segments and 1-2 tender leaves at the top are retained.

[0008] As a preferred technical solution of the present invention: in step one, the stem tip or internode is preferred, as it has vigorous metabolism and strong regeneration ability.

[0009] As a preferred technical solution of the present invention: the anti-browning agent in step one needs to be added at a concentration of 50-100 mg / L to directly inhibit the oxidation reaction.

[0010] As a preferred technical solution of the present invention: in step one, the anti-browning agent activated carbon is 0.5%-2.5%, which adsorbs browning products and adjusts the permeability of the culture medium.

[0011] As a preferred technical solution of the present invention: after the stem segment is cut in step one, it is immediately immersed in cold sterile water containing ascorbic acid (100mg / L) for 10 minutes.

[0012] As a preferred technical solution of the present invention: in step two, ensure that the base is in full contact with the culture medium, and inoculate 3-5 segments per bottle.

[0013] As a preferred technical solution of the present invention: the hard material of the stem segment in step one can be lightly brushed with soft bristles.

[0014] (III) Beneficial Effects This invention provides a method for rapid propagation of potted Anthurium seedlings via tissue culture. It has the following beneficial effects: 1. This invention utilizes stem segments as explants, which offers several advantages: First, the material is readily available: stem segments can be directly cut from the mother plant or subculture seedlings, without seasonal restrictions, and the operation is simple; Second, regeneration is highly efficient: stem segments contain meristematic cells, easily inducing adventitious buds and callus tissue, resulting in rapid proliferation; Third, the traits are stable: the genetic variation rate is low, and tissue-cultured seedlings can better maintain the excellent characteristics of the mother plant; Fourth, the cost is controllable: it is suitable for large-scale production, and the browning problem can be effectively solved by optimizing the culture medium.

[0015] 2. This invention addresses the issue of stem segments as explants, which are prone to browning after incision. By adding an anti-browning agent, the survival rate can be improved: effectively inhibiting the oxidation of phenolic substances, reducing browning at the incision site, and maintaining the activity of the explants; it can also shorten the culture cycle: avoiding metabolic disorders caused by browning, accelerating callus formation and bud differentiation; and it can reduce costs: reducing material waste caused by browning, and improving the uniformity and yield of tissue culture seedlings; the operation is simple: the anti-browning agent can be directly added to the culture medium, making it easy to apply on a large scale.

[0016] 3. This invention adds an anti-browning agent at a concentration of 50-100 mg / L and activated carbon as an anti-browning agent at 0.5%-2.5%, adsorbing browning products and adjusting the permeability of the culture medium. This method can achieve highly efficient antioxidant effects: activated carbon adsorbs phenolic substances, ascorbic acid neutralizes free radicals, and synergistically reduces browning. Detailed Implementation

[0017] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0018] Example 1: This invention provides a method for rapid propagation of potted Anthurium seedlings via tissue culture, including the following tissue culture methods. Step 1: Selection and treatment of exogenitals (1) When taking stem segments as explants, the tender and non-lignified parts should be selected; (2) Rinse the explant stem segments with tap water for 10 minutes to remove surface impurities, then soak them in 75% alcohol for 60 seconds, 0.1% mercuric chloride solution for 8 minutes, rinse them 6 times with sterile water, and then immerse them in sterile water for later use; (3) In a clean bench, after blotting off the moisture with sterile filter paper, cut the stem segments into 1cm segments, retaining 2 nodes in each segment. Since the cut surfaces of the stem segments are prone to browning, an anti-browning agent needs to be added. The anti-browning agent used is cysteine ​​and its hydrochloride: by inhibiting the activity of polyphenol oxidase, it effectively blocks the oxidation of phenolic substances, with a concentration of 50-200mg / L; Step 2: Inoculation and Culture (1) Inside the laminar flow hood, use sterile forceps to vertically insert the stem segment into the culture medium, ensuring full contact between the base and the medium; (2) Immediately after inoculation, seal the culture dish or Erlenmeyer flask with sealing film to prevent contamination; Step 3: Cultivation Conditions (1) Temperature control: constant temperature 28℃; (2) The culture was carried out in the dark for 10 days in the early stage, and then switched to light with 3000 Lux and 10 hours of light per day. (3) Humidity 85% to avoid condensation buildup; (4) After 6 weeks, transfer to a culture medium containing a low concentration of auxin to promote lateral bud differentiation.

[0019] In step one, the stem segment length is 2cm, retaining 2 stem segments and 2 tender leaves at the top. The 2cm length optimizes the tissue culture operation of the stem segment, improving ease of handling. In step one, the stem tip or internode is preferred due to its vigorous metabolism and strong regeneration ability. The anti-browning agent in step one needs to be added at a concentration of 100mg / L to directly inhibit the oxidation reaction. The activated carbon anti-browning agent in step one is 2.5%, which adsorbs browning products and adjusts the permeability of the culture medium. Simultaneously, the activated carbon anti-browning agent physically adsorbs quinones produced by browning through its porous structure, and can also bind chemically... The adsorption surface oxygen-containing functional groups bind with phenols to inhibit polyphenol oxidase activity. In step one, after the stem segments are cut, they are immediately immersed in cold sterile water containing ascorbic acid (100 mg / L) for 10 minutes. Ascorbic acid, as a strong antioxidant, can directly remove reactive oxygen species generated by oxidative stress at the cut, and block the reaction of polyphenol oxidase catalyzing the oxidation of phenolic substances to quinone substances, thereby effectively preventing browning of the stem segment cut. In step two, ensure that the base is in full contact with the culture medium, and inoculate 5 segments per bottle. The hard material of the stem segments in step one can be gently brushed with a soft bristle to improve the cleaning of dirt on the stem segment surface.

[0020] Example 2: The difference between this embodiment and Embodiment 1 is that: a method for rapid propagation of potted Anthurium seedlings via tissue culture includes the following tissue culture methods. Step 1: Selection and treatment of exogenitals (1) When taking stem segments as explants, the tender and non-lignified parts should be selected; (2) Rinse the explant stem segments with tap water for 10 minutes to remove surface impurities, then soak them in 75% alcohol for 30 seconds, 0.1% mercuric chloride solution for 8 minutes, rinse four times with sterile water, and then immerse them in sterile water for later use; (3) In a clean bench, after blotting off the moisture with sterile filter paper, cut the stem segments into 0.5cm segments, retaining one node in each segment. Since the cut surfaces of the stem segments are prone to browning, an anti-browning agent should be added. Step 2: Inoculation and Culture (1) Inside the laminar flow hood, use sterile forceps to vertically insert the stem segment into the culture medium, ensuring full contact between the base and the medium; (2) Immediately after inoculation, seal the culture dish or Erlenmeyer flask with sealing film to prevent contamination; Step 3: Cultivation Conditions (1) Temperature control: constant temperature 26℃; (2) The culture was carried out in the dark for 10 days in the early stage, and then switched to light with 1500 Lux and 8 hours of light per day; (3) Humidity 70% to avoid condensation buildup; (4) After 4 weeks, transfer to a culture medium containing a low concentration of auxin to promote lateral bud differentiation.

[0021] In step one, the stem segment length is 2cm, retaining one stem segment and one tender leaf at the top. In step one, the stem tip or internode is preferred because it has vigorous metabolism and strong regeneration ability. In step one, the anti-browning agent needs to be added at a concentration of 50mg / L to directly inhibit the oxidation reaction. In step one, the anti-browning agent activated carbon is 0.5% to adsorb browning products and adjust the permeability of the culture medium. After the stem segment is cut in step one, it should be immediately immersed in cold sterile water containing ascorbic acid (100mg / L) for 10 minutes. In step two, ensure that the base is in full contact with the culture medium. Inoculate 3 segments per bottle. The hard material of the stem segment in step one can be gently brushed with a soft bristle.

[0022] Example 3: The difference between this embodiment and Embodiment 1 is that: a method for rapid propagation of potted Anthurium seedlings via tissue culture includes the following tissue culture methods. Step 1: Selection and treatment of exogenitals (1) When taking stem segments as explants, the tender and non-lignified parts should be selected; (2) Rinse the explant stem segments with tap water for 10 minutes to remove surface impurities, then soak them in 75% alcohol for 40 seconds, 0.1% mercuric chloride solution for 8 minutes, rinse five times with sterile water, and immerse them in sterile water for later use; (3) In a clean bench, after blotting off the moisture with sterile filter paper, cut the stem segments into 1cm segments, retaining 2 nodes in each segment. The cut surfaces of the stem segments are prone to browning, so an anti-browning agent should be added. Step 2: Inoculation and Culture (1) Inside the laminar flow hood, use sterile forceps to vertically insert the stem segment into the culture medium, ensuring full contact between the base and the medium; (2) Immediately after inoculation, seal the culture dish or Erlenmeyer flask with sealing film to prevent contamination; Step 3: Cultivation Conditions (1) Temperature control: constant temperature 27℃; (2) The culture was carried out in the dark for 10 days in the early stage, and then switched to light with 2500 Lux and 9 hours of light per day; (3) Humidity 80% to avoid condensation buildup; (4) After 5 weeks, transfer to a culture medium containing a low concentration of auxin to promote lateral bud differentiation.

[0023] In step one, the stem segment length is 2cm, retaining 2 stem segments and 2 tender leaves at the top. The 2cm length optimizes the tissue culture operation of the stem segments, improving ease of handling. In step one, the stem tip or internode is preferred due to its vigorous metabolism and strong regeneration ability. The anti-browning agent in step one needs to be added at a concentration of 100mg / L to directly inhibit the oxidation reaction. The activated carbon in step one is 2.5% to adsorb browning products and adjust the permeability of the culture medium. Immediately after cutting the stem segments in step one, immerse them in cold sterile water containing ascorbic acid (100mg / L) for 10 minutes. Ascorbic acid, as a strong antioxidant, can directly remove reactive oxygen species generated by oxidative stress at the cut, blocking the oxidation of phenolic substances to quinones catalyzed by polyphenol oxidase, thus effectively preventing browning of the stem segment cut. In step two, ensure full contact between the base and the culture medium, inoculating 4 segments per bottle. For the hard material of the stem segments in step one, a soft brush can be used to gently clean the surface dirt.

[0024] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for rapid propagation of potted Anthurium seedlings via tissue culture, characterized in that: Including the following tissue culture methods, Step 1: Selection and treatment of exogenitals (1) When taking stem segments as explants, the tender and non-lignified parts should be selected; (2) Rinse the explant stem segments with tap water for 10 minutes to remove surface impurities, then soak them in 75% alcohol for 30-60 seconds, followed by soaking in 0.1% mercuric chloride solution for 8 minutes. Rinse with sterile water 4-6 times, and then immerse in sterile water for later use. (3) In a clean bench, after blotting off the moisture with sterile filter paper, cut the stem segments into 0.5-1cm segments, retaining 1-2 nodes in each segment. Since the cut surfaces of the stem segments are prone to browning, an anti-browning agent should be added. Step 2: Inoculation and Culture (1) Inside the laminar flow hood, use sterile forceps to vertically insert the stem segment into the culture medium, ensuring full contact between the base and the medium; (2) Immediately after inoculation, seal the culture dish or Erlenmeyer flask with sealing film to prevent contamination; Step 3: Cultivation Conditions (1) Temperature control: constant temperature 26±2℃; (2) The culture was carried out in the dark for 10 days in the early stage, and then switched to light with 1500-3000 Lux, with 8-10 hours of light per day; (3) Humidity 70%-85% to avoid condensation buildup; (4) After 4-6 weeks, transfer to a culture medium containing low concentration of auxin to promote lateral bud differentiation.

2. The method for rapid propagation of potted Anthurium seedlings via tissue culture according to claim 1, characterized in that: In step one, the stem segment length is 1-2cm, and 1-2 stem segments and 1-2 tender leaves at the top are retained.

3. The method for rapid propagation of potted Anthurium seedlings via tissue culture according to claim 1, characterized in that: In step one, the stem tip or internode is preferred because it has vigorous metabolism and strong regeneration ability.

4. The method for rapid propagation of potted Anthurium seedlings via tissue culture according to claim 1, characterized in that: In step one, the anti-browning agent needs to be added at a concentration of 50-100 mg / L to directly inhibit the oxidation reaction.

5. The method for rapid propagation of potted Anthurium seedlings via tissue culture according to claim 1, characterized in that: In step one, the anti-browning agent activated carbon is 0.5%-2.5%, which adsorbs browning products and adjusts the permeability of the culture medium.

6. The method for rapid propagation of potted Anthurium seedlings via tissue culture according to claim 1, characterized in that: In step one, the stem segments are immediately immersed in cold, sterile water containing ascorbic acid (100 mg / L) for 10 minutes after being cut.

7. The method for rapid propagation of potted Anthurium seedlings via tissue culture according to claim 1, characterized in that: In step two, ensure that the base is in full contact with the culture medium, and inoculate 3-5 segments per bottle.

8. The method for rapid propagation of potted Anthurium seedlings via tissue culture according to claim 1, characterized in that: In step one, the hard material of the stem segment can be gently brushed with a soft bristle.