A rapid propagation method for *Hymenoptera* leaves using axillary buds as explants through tissue culture.

Abstract

This invention discloses a rapid propagation method for *Hylocereus undatus* leaves using axillary buds as explants, comprising the following steps: selecting axillary buds of *Hylocereus undatus* leaves as explants and disinfecting them; removing bracts from the disinfected explants and inoculating them into an induction medium for bud induction culture; inoculating the induced *Hylocereus undatus* leaf bud clusters into a proliferation medium for proliferation culture; removing leaves and fibrous roots from the proliferated *Hylocereus undatus* leaf tissue culture seedlings and inoculating them into a rooting medium for rooting culture to obtain rooted seedlings. This invention uses axillary buds of *Hylocereus undatus* leaves as explants and employs an in vitro rapid propagation method for *Hylocereus undatus* leaf seedling production, enabling rapid and efficient mass production of high-quality *Hylocereus undatus* leaf seedlings. It is suitable for industrialized seedling production, achieving large-scale rapid propagation of *Hylocereus undatus* leaves, breaking through the traditional division propagation method, and solving the problem of *Hylocereus undatus* leaf proliferation rate. This is of great significance for the large-scale planting and widespread utilization of *Hylocereus undatus* leaves.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture technology, and specifically relates to a rapid propagation method of *Hymenochloa crus-galli* leaf tissue culture using axillary buds as explants. Background Technology

[0002] Phrynium capitatum Willd. is a perennial herbaceous plant belonging to the Marantaceae family and the Phrynium genus. It can grow up to 1 meter tall, with basal leaves that are oblong or oblong-lanceolate and petioles that can reach 60 centimeters in length.

[0003] The leaves of the holly tree (Platycladus orientalis) can clear heat and promote diuresis, treat hoarseness, sore throat, oral ulcers, and detoxify alcohol. According to *Nanfang Caomu Zhuang* (by Ji Han, 304 AD), "The south is mostly humid and hot, and things easily spoil; only by wrapping them in holly leaves can they be preserved for a long time." Research results also show that the volatile oil and ethanol extract of holly leaves have good antioxidant functions. Furthermore, the volatile oil of holly leaves has a significant inhibitory effect on common foodborne spoilage bacteria and pathogenic bacteria. Therefore, holly leaves can also be used as packaging materials with preservative properties. Natural, biodegradable, and environmentally friendly packaging materials have been increasingly sought after in recent years, thus holly leaves have promising applications in packaging, pharmaceuticals, and the food industry.

[0004] Currently, *Hylocereus undatus* is typically propagated by division. For example, CN108739146A discloses a method for cultivating *Hylocereus undatus* that involves dividing the mother plant during a specific season, usually separating robust new shoots from the mother plant as seedlings. However, this propagation method is not only limited by seasonal climate but also has a very low propagation rate, severely hindering the large-scale, rapid propagation of *Hylocereus undatus* and failing to guarantee the stability of its horticultural traits. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a rapid propagation method for *Hymenochloa crus-galli* leaf tissue culture using axillary buds as explants. This method utilizes axillary buds for rapid in vitro propagation, overcoming the problem of low proliferation rates associated with traditional division propagation.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A rapid propagation method for *Hylocereus undatus* leaf tissue culture using axillary buds as explants includes the following steps:

[0008] (1) Select axillary buds of *Hylocereus undatus* leaves as explants and disinfect them;

[0009] (2) After detoxification, the bracts of the explants were removed and inoculated into the induction medium for the induction of shoot clusters.

[0010] (3) The induced clustered shoots of *Hylocereus undatus* were inoculated into a proliferation medium for proliferation culture;

[0011] (4) The buds of the holly leaf obtained by proliferation culture are inoculated into the rooting medium for rooting culture to obtain rooted seedlings.

[0012] Preferably, the holly leaf axillary bud is a tender holly leaf axillary bud that has sprouted for 15-20 days.

[0013] Preferably, the disinfection process includes: first washing the mud and sand off the axillary buds of the holly leaves with water, then soaking them in a diluted laundry detergent solution, then rinsing them with running water, then disinfecting them with alcohol on a clean bench, then disinfecting them with mercuric chloride solution, and finally rinsing them with sterile water.

[0014] Preferably, the induction medium is: MS + (2.5-3.0) mg / L 6-BA + (1.5-2.0) mg / L LTDZ + 0.1 mg / L NAA + (25-30) g / L sugar + (7-9) g / L carrageenan.

[0015] Preferably, the culture temperature for inducing the growth of clustered shoots is 24-26℃.

[0016] Preferably, the proliferation medium is: MS + (1.5-2.0) mg / L 6-BA + (1.0-1.5) mg / L TDZ + 0.1 mg / L NAA + (25-30) g / L sugar + (7-9) g / L carrageenan.

[0017] Preferably, the conditions for the proliferation culture are: culture for 20-25 days, culture temperature 24-26℃, light duration 8-10h / day, and light intensity 2500-4000lx.

[0018] Preferably, the proliferation culture is performed 3-4 times.

[0019] Preferably, the rooting medium is: MS + 0.1 mg / L NAA + (25-30) g / L sugar + (7-9) g / L carrageenan + 0.5 g / L activated carbon.

[0020] Preferably, the conditions for rooting culture are: culture for 25-30 days, culture temperature 26-30℃, light duration 8-10h / day, and light intensity 2500-4000lx.

[0021] Compared with existing technologies, the beneficial effects of this invention are as follows: This invention uses axillary buds of *Polygonum aviculare* as explants and produces *Polygonum aviculare* seedlings through a rapid in vitro propagation method. By optimizing and screening the induction culture medium for explants, the proliferation culture medium for clustered buds, and the rooting culture medium for tissue culture seedlings, the induction rate of explants reaches over 76%, the proliferation coefficient of clustered buds reaches over 2.3, and the rooting rate of tissue culture seedlings reaches over 99%. This invention can rapidly and effectively produce high-quality *Polygonum aviculare* seedlings in large quantities, making it suitable for industrialized seedling production. It achieves large-scale rapid propagation of *Polygonum aviculare*, breaking through the traditional division propagation method and solving the problem of *Polygonum aviculare* proliferation rate. This is of great significance for the large-scale planting and widespread utilization of *Polygonum aviculare*. Attached Figure Description

[0022] Figure 1 Image of an explant from an axillary bud of *Polygonum aviculare*.

[0023] Figure 2 Image of induced clustered buds in *Hylocereus undatus* leaves;

[0024] Figure 3 Image of clustered buds of *Hylocereus undatus* during propagation culture;

[0025] Figure 4 Image of rooted seedlings of *Hylocereus undatus* (a type of shrub) during rooting culture. Detailed Implementation

[0026] 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. 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.

[0027] Example 1

[0028] A rapid propagation method for *Hylocereus undatus* leaf tissue culture using axillary buds as explants includes the following steps:

[0029] (1) Selection and disinfection of explants: Select tender axillary buds of holly leaves that have sprouted for 15 days as explants. Wash the mud and sand off the buds with tap water, then soak them in a diluted laundry detergent solution for 30 minutes, then rinse them with running water for 30 minutes, then disinfect them with 75% alcohol for 30 seconds on a clean bench, then disinfect them with 0.1% mercuric chloride for 9 minutes, and finally rinse them with sterile water 3-4 times.

[0030] (2) Induction of shoot clusters: After detoxification, the bracts of the explants were removed and inoculated into the induction medium for shoot cluster induction culture at a temperature of 24-26℃. The induction medium was: MS + 3.0 mg / L 6-BA + 2.0 mg / L TDZ + 0.1 mg / L NAA + 30 g / L sugar + 8 g / L carrageenan; the induction rate was 79%.

[0031] (3) Proliferation culture of clustered shoots: The induced clustered shoots of *Hylocereus undatus* were inoculated into a proliferation medium for 20 days. The culture temperature was 24-26℃, the photoperiod was 10h / day, and the light intensity was 2500lx. The proliferation medium was: MS + 2.0mg / L 6-BA + 1.0mg / L TDZ + 0.1mg / L NAA + 30g / L sugar + 8g / L carrageenan. The proliferation coefficient reached 2.5.

[0032] (4) Rooting culture: The buds of *Hylocereus undatus* obtained from four proliferation cultures were inoculated into a rooting medium for 25 days of rooting culture at a temperature of 26-30℃, a light duration of 10 h / day, and a light intensity of 2500 lx to obtain rooted seedlings. The rooting medium was: MS + 0.1 mg / L NAA + 30 g / L sugar + 8 g / L carrageenan + 0.5 g / L activated carbon. The rooting rate reached 100%.

[0033] Example 2

[0034] A rapid propagation method for *Hylocereus undatus* leaf tissue culture using axillary buds as explants includes the following steps:

[0035] (1) Selection and disinfection of explants: Select tender axillary buds of holly leaves that have sprouted for 18 days as explants. Wash the mud and sand off the buds with tap water, then soak them in a diluted laundry detergent solution for 30 minutes, then rinse them with running water for 30 minutes, then disinfect them with 75% alcohol for 30 seconds on a clean bench, then disinfect them with 0.1% mercuric chloride for 9 minutes, and finally rinse them with sterile water 3-4 times.

[0036] (2) Induction of shoot clusters: After detoxification, the bracts of the explants were removed and inoculated into the induction medium for shoot cluster induction culture at a temperature of 24-26℃. The induction medium was: MS + 3.0 mg / L 6-BA + 2.0 mg / L TDZ + 0.1 mg / L NAA + 30 g / L sugar + 9 g / L carrageenan; the induction rate was 80%.

[0037] (3) Proliferation culture of clustered shoots: The induced clustered shoots of *Hylocereus undatus* were inoculated into a proliferation medium and cultured for 23 days at a temperature of 24-26℃, a light duration of 9 h / day, and a light intensity of 3000 lx. The proliferation medium was: MS + 2 mg / L 6-BA + 1.0 mg / L TDZ + 0.1 mg / L NAA + 30 g / L sugar + 9 g / L carrageenan. The proliferation coefficient reached 2.5.

[0038] (4) Rooting culture: The buds of *Hylocereus undatus* obtained from four proliferation cultures were inoculated into a rooting medium for 28 days of rooting culture at a temperature of 26-30℃, a light duration of 9 h / day, and a light intensity of 3000 lx to obtain rooted seedlings. The rooting medium was: MS + 0.1 mg / L NAA + 30 g / L sugar + 9 g / L carrageenan + 0.5 g / L activated carbon. The rooting rate reached 100%.

[0039] Example 3

[0040] A rapid propagation method for *Hylocereus undatus* leaf tissue culture using axillary buds as explants includes the following steps:

[0041] (1) Selection and disinfection of explants: Select tender axillary buds of holly leaves that have sprouted for 20 days as explants. Wash the mud and sand off the buds with tap water, then soak them in a diluted laundry detergent solution for 30 minutes, then rinse them with running water for 30 minutes, then disinfect them with 75% alcohol for 30 seconds on a clean bench, then disinfect them with 0.1% mercuric chloride for 9 minutes, and finally rinse them with sterile water 3-4 times.

[0042] (2) Induction of shoot clusters: After detoxification, the bracts of the explants were removed and they were inoculated into the induction medium for shoot cluster induction culture at a temperature of 24-26℃. The induction medium was: MS + 3.0 mg / L 6-BA + 2.0 mg / L TDZ + 0.1 mg / L NAA + 30 g / L sugar + 8 g / L carrageenan; the induction rate was 82%.

[0043] (3) Proliferation culture of clustered shoots: The induced clustered shoots of *Hylocereus undatus* were inoculated into a proliferation medium for 25 days. The culture temperature was 24-26℃, the photoperiod was 8h / day, and the light intensity was 4000lx. The proliferation medium was: MS + 2mg / L 6-BA + 1.0mg / L TDZ + 0.1mg / L NAA + 30g / L sugar + 8g / L carrageenan. The proliferation coefficient reached 2.6.

[0044] (4) Rooting culture: The buds of *Hylocereus undatus* obtained from the three proliferation cultures were inoculated into the rooting medium for 30 days of rooting culture at a temperature of 26-30℃, a light duration of 8 hours / day, and a light intensity of 4000 lx to obtain rooted seedlings. The rooting medium was: MS + 0.1 mg / L NAA + 30 g / L sugar + 8 g / L carrageenan + 0.5 g / L activated carbon. The rooting rate reached 100%.

[0045] Example 4

[0046] A rapid propagation method for *Hylocereus undatus* leaf tissue culture using axillary buds as explants includes the following steps:

[0047] (1) Selection and disinfection of explants: Select tender axillary buds of holly leaves that have sprouted for 15 days as explants. Wash the mud and sand off the buds with tap water, then soak them in a diluted laundry detergent solution for 30 minutes, then rinse them with running water for 30 minutes, then disinfect them with 75% alcohol for 30 seconds on a clean bench, then disinfect them with 0.1% mercuric chloride for 9 minutes, and finally rinse them with sterile water 3-4 times.

[0048] (2) Induction of shoot clusters: After detoxification, the bracts of the explants were removed and inoculated into the induction medium for shoot cluster induction culture at a temperature of 24-26℃. The induction medium was: MS + 2.5 mg / L 6-BA + 1.5 mg / L TDZ + 0.1 mg / L NAA + 25 g / L sugar + 7 g / L carrageenan; the induction rate was 76%.

[0049] (3) Proliferation culture of clustered shoots: The induced clustered shoots of *Hylocereus undatus* were inoculated into a proliferation medium for 20 days. The culture temperature was 24-26℃, the photoperiod was 10h / day, and the light intensity was 2500lx. The proliferation medium was: MS + 1.5mg / L 6-BA + 1.5mg / L TDZ + 0.1mg / L NAA + 25g / L sugar + 7g / L carrageenan. The proliferation coefficient reached 2.3.

[0050] (4) Rooting culture: The buds of *Hylocereus undatus* obtained from four proliferation cultures were inoculated into a rooting medium for 25 days of rooting culture at a temperature of 26-30℃, a light duration of 10 h / day, and a light intensity of 2500 lx to obtain rooted seedlings. The rooting medium was: MS + 0.1 mg / L NAA + 25 g / L sugar + 7 g / L carrageenan + 0.5 g / L activated carbon. The rooting rate reached 99%.

[0051] Comparative Example 1

[0052] The difference from Example 1 is that the induction medium used is different, but everything else is the same.

[0053] The induction medium for Comparative Example 1 was: MS medium + 2.5 mg / L 6-BA + 0.1 mg / L NAA + 25 g / L sugar + 7 g / L carrageenan; the induction rate was 70%.

[0054] Comparative Example 2

[0055] The difference from Example 1 is that the proliferation medium used is different, but everything else is the same.

[0056] The proliferation medium for Comparative Example 2 was: MS + 1.5 mg / L TDZ + 0.1 mg / L NAA + 25 g / L sugar + 7 g / L carrageenan; the proliferation coefficient reached 1.8.

[0057] Comparative Example 3

[0058] The difference from Example 1 is that the rooting medium used is different, but everything else is the same.

[0059] The rooting medium for Comparative Example 3 was MS + 0.1 mg / L NAA; the rooting rate reached 80%.

[0060] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A rapid propagation method for *Hylocereus undatus* leaf tissue culture using axillary buds as explants, characterized in that... Includes the following steps: (1) Select tender axillary buds of *Hylocereus undatus* leaves that have sprouted for 15-20 days as explants and disinfect them; (2) After the explants were detoxified, the bracts were removed and the explants were inoculated into an induction medium for the induction of shoot clusters. (3) The induced clustered shoots of *Hylocereus undatus* were inoculated into a proliferation medium for proliferation culture; (4) The buds of the leaf proliferating culture obtained by the proliferation culture were inoculated into the rooting medium for rooting culture to obtain rooted seedlings; The induction medium was: MS + (2.5-3.0) mg / L 6-BA + (1.5-2.0) mg / L LTDZ + 0.1 mg / L NAA + (25-30) g / L sugar + (7-9) g / L carrageenan; The proliferation medium was: MS + (1.5-2.0) mg / L 6-BA + (1.0-1.5) mg / L LTDZ + 0.1 mg / L NAA + (25-30) g / L sugar + (7-9) g / L carrageenan; The rooting medium is: MS + 0.1 mg / L NAA + (25-30) g / L sugar + (7-9) g / L carrageenan + 0.5 g / L activated carbon.

2. The rapid propagation method of *Hylocereus undatus* leaf tissue culture using axillary buds as explants according to claim 1, characterized in that, The culture temperature for inducing the growth of clustered shoots is 24-26℃.

3. The rapid propagation method of *Hylocereus undatus* leaf tissue culture using axillary buds as explants according to claim 1, characterized in that, The conditions for the proliferation culture are: culture for 20-25 days, culture temperature 24-26℃, light duration 8-10h / day, and light intensity 2500-4000lx.

4. The rapid propagation method of *Hylocereus undatus* leaf tissue culture using axillary buds as explants according to claim 1, characterized in that, The proliferation culture is performed 3-4 times.

5. The rapid propagation method of *Hylocereus undatus* leaf tissue culture using axillary buds as explants according to claim 1, characterized in that, The conditions for rooting culture are: culture for 25-30 days, culture temperature 26-30℃, light duration 8-10h / day, and light intensity 2500-4000lx.

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