A method for inducing monkey ear ring flower filament callus and application thereof

Abstract

The application belongs to the technical field of tissue culture and relates to a kind of induction method and application of monkey ear ring flower filament callus, comprising the following steps: taking monkey ear ring flower bud, after being treated with disinfecting liquid for disinfection and sterilization, in sterile environment, take monkey ear ring flower filament as explant, it is inoculated in induction medium and dark culture, form callus, then the callus is transferred to proliferation medium and is subcultured and proliferated, and more high-quality callus is obtained.The induction method provided by the application can induce monkey ear ring flower filament to form callus quickly, and the callus induction rate is as high as 100%;The induction method of monkey ear ring flower filament callus in the application has short cultivation period, high induction efficiency and low contamination rate, the induction method is applied to the cultivation of monkey ear ring, the research path of monkey ear ring rapid propagation technology is widened, the regeneration of monkey ear ring flower filament into plant becomes possible, and high-quality plant with excellent traits can be cultivated.

Description

Technical Field

[0001] This invention belongs to the field of tissue culture technology, specifically relating to a method for inducing monkey earring filament callus and its application. Background Technology

[0002] Archidendron clypearia (Jack) Benth, a plant belonging to the subfamily Mimosoideae of the Fabaceae family, is commonly known as the monkey earring tree. It is widely distributed in tropical Asia and is cultivated in several provinces and autonomous regions of my country, including Zhejiang, Fujian, Guangdong, Guangxi, Hainan, and Yunnan. The monkey earring has a wide range of applications, encompassing medicine, food, daily chemicals, and animal husbandry. For example, in medicinal applications, the branches and leaves are rich in gallic acid, quercetin, and total polyphenols, which endow the monkey earring with multiple pharmacological effects such as antibacterial, anti-inflammatory, analgesic, and antiviral properties. Currently, single-component preparations of monkey earring extracts are widely used clinically with good therapeutic effects.

[0003] However, despite the promising application prospects of *Rhizophora stylosa*, its wild resources are relatively limited. To maintain the superior characteristics of *Rhizophora stylosa* varieties and select plants with strong resistance or high content of effective components, research on rapid propagation of *Rhizophora stylosa* seedlings through tissue culture is urgently needed. Currently, research on seedling propagation of *Rhizophora stylosa* using tissue culture technology mainly focuses on using stem segments, leaves, and petioles of aseptic seedlings grown from seeds of superior strains as explants. However, research on using *Rhizophora stylosa* filaments as explants for plant regeneration has not yet appeared in previous literature and reports. Filaments typically have strong cell division and differentiation capabilities, possessing potential regenerative capacity. Therefore, exploring tissue culture methods using *Rhizophora stylosa* filaments as explants is expected to open new avenues for rapid propagation research of *Rhizophora stylosa*, providing more efficient and reliable technical support.

[0004] Based on the above background, this invention proposes a method for inducing callus tissue from monkey earring filaments. This method uses monkey earring filaments as starting material and establishes a robust regeneration system through callus induction and organogenesis pathways, broadening the research path for rapid propagation technology of monkey earrings and laying the foundation for new variety selection and seedling propagation. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide a method and application for inducing callus tissue from monkey earring filaments. The induction method of this invention can induce rapid callus formation from monkey earring filaments with an induction rate as high as 100%. This induction method has a short cultivation cycle, high induction efficiency, and low contamination rate, successfully establishing a highly efficient callus induction system for monkey earring filaments, providing technical support for monkey earring filament cultivation and breeding. Applying this method for inducing callus tissue from monkey earring filaments to the cultivation of monkey earrings broadens the research path for rapid propagation technology of monkey earrings, making it possible for monkey earring filaments to regenerate into plants, and cultivating high-quality plants with excellent traits.

[0006] The technical solution adopted by the present invention to solve the above problems is as follows:

[0007] A method for inducing callus tissue from monkey earring filaments includes the following steps:

[0008] Monkey ear flower buds were taken, sterilized with disinfectant, and then the filaments of monkey ear flowers were taken as explants under sterile conditions and inoculated into induction medium for dark culture to form callus tissue. The callus tissue was then transferred to proliferation medium for subculture to obtain higher quality callus tissue.

[0009] Preferably, the monkey earring flower buds have good appearance quality and are free from pests and diseases.

[0010] Furthermore, the monkey earring flower buds are placed at 2-8°C for 24 hours before the disinfection and sterilization treatment.

[0011] Furthermore, the disinfectant includes at least one of the following: 75% alcohol by volume, 0.1% mercuric chloride solution by mass, and 0.5% to 5% sodium hypochlorite solution by mass.

[0012] Preferably, the disinfectant comprises 75% alcohol by volume and 0.1% mercuric chloride solution by mass.

[0013] Furthermore, the disinfection and sterilization process includes at least one of the following operations: treatment with 75% alcohol by volume for 30 seconds, treatment with 0.1% mercuric chloride by mass for 3 minutes, or treatment with 0.5% to 5% sodium hypochlorite by mass for 3 minutes.

[0014] Furthermore, the induction medium is MS + 6-BA 1.0 mg / L + NAA 0.3-0.9 mg / L + sucrose 30 g / L + agar 5 g / L, with a pH of 5.8-5.9.

[0015] Preferably, the induction medium is MS + 6-BA 1.0 mg / L + NAA 0.3 mg / L + sucrose 30 g / L + agar 5 g / L, with a pH of 5.8–5.9.

[0016] Furthermore, the proliferation medium is MS + 6-BA 0.5 mg / L + NAA 1.0 mg / L, with a pH of 5.8–5.9.

[0017] Furthermore, the dark culture time is 5–30 days. Dark culture refers to culture under conditions of no light.

[0018] Furthermore, the culture temperature is 23–27°C, and the culture humidity is 60–80% RH.

[0019] The present invention also provides the application of the above-mentioned method for inducing callus tissue of monkey earrings in the cultivation of monkey earrings.

[0020] The present invention has the following beneficial effects:

[0021] (1) The flowering period of *Rhizophora stylosa* is from February to June, which provides a relatively long period for obtaining explants. Furthermore, the flower buds of *Rhizophora stylosa* are easy to harvest, making the process simple and convenient. In addition, the stamens of *Rhizophora stylosa* are enclosed by petals, which reduces their susceptibility to environmental pollution and lowers the pollution rate. Simultaneously, the time required for disinfecting the stamens is also relatively short, minimizing harm to operators.

[0022] (2) The induction method of the present invention can induce the rapid formation of callus tissue in monkey ear ring filaments. The induction method has a short cultivation cycle, high induction efficiency, and low contamination rate, breaking through the traditional explant tissue culture method of monkey ear ring and providing technical support for the cultivation and breeding of monkey ear ring filaments.

[0023] (3) The method for inducing callus tissue of monkey earring filaments of the present invention is applied to the cultivation of monkey earrings, which broadens the research path of monkey earring rapid propagation technology, makes it possible for monkey earring filaments to regenerate into plants, and can cultivate high-quality plants with excellent traits. Attached Figure Description

[0024] Figure 1 This is a diagram of the callus tissue induced using monkey earring filaments as explants, as described in this invention.

[0025] Figure 2 This is a diagram of the callus tissue after subculture and proliferation of monkey earring filament callus tissue as described in this invention.

[0026] Figure 3 This is a diagram of callus tissue induced using leaves from a one-year-old monkey earring plant as explants in Example 4 of the present invention. Detailed Implementation

[0027] The present invention will be further described in detail below with reference to embodiments, but the present invention is not limited thereto. All instruments used in the present invention have been sterilized, and the methods involved, unless otherwise specified, are commonly used methods in the art. The reagents involved, unless otherwise specified, can be obtained from ordinary commercial channels, and no specific commercial channel is limited.

[0028] It should be noted in advance that the explants of *Mimosa pudica* used in this invention were collected from *Mimosa pudica* seedlings at the Huizhou planting base, and were identified by the South China Botanical Identification Center of the South China National Botanical Garden, Chinese Academy of Sciences as *Mimosa pudica*, a plant of the genus *Mimosa pudica* in the subfamily Mimosoideae of the Fabaceae family.

[0029] A method for inducing callus tissue from monkey earring filaments includes the following steps:

[0030] Monkey ear flower buds were taken, sterilized with disinfectant, and then the filaments of monkey ear flowers were taken as explants under sterile conditions and inoculated into induction medium for dark culture to form callus tissue. The callus tissue was then transferred to proliferation medium for subculture to obtain higher quality callus tissue.

[0031] In some preferred embodiments of the present invention, the monkey earring flower buds have good appearance quality and are free from pests and diseases.

[0032] In some specific embodiments of the present invention, the monkey earring flower buds are placed at 2-8°C for 24 hours before the disinfection and sterilization treatment.

[0033] In some specific embodiments of the present invention, the disinfectant includes at least one of the following: 75% alcohol by volume, 0.1% mercuric chloride solution by mass, and 0.5% to 5% sodium hypochlorite solution by mass.

[0034] In some preferred embodiments of the present invention, the disinfectant comprises 75% alcohol by volume and 0.1% mercuric chloride solution by mass.

[0035] In some specific embodiments of the present invention, the disinfection and sterilization treatment includes at least one of the following operations: treatment with 75% alcohol by volume for 30 seconds, treatment with 0.1% mercuric chloride by mass for 3 minutes, or treatment with 0.5% to 5% sodium hypochlorite by mass for 3 minutes.

[0036] In some more preferred embodiments of the present invention, the disinfection and sterilization process includes: first treating with 75% alcohol by volume for 30 seconds, and then treating with 0.1% mercuric chloride solution by mass for 3 minutes.

[0037] In some specific embodiments of the present invention, the induction culture medium is MS + 6-BA 1.0 mg / L + NAA 0.3-0.9 mg / L + sucrose 30 g / L + agar 5 g / L, with a pH of 5.8-5.9.

[0038] In some preferred embodiments of the present invention, the induction medium is MS + 6-BA 1.0 mg / L + NAA 0.3 mg / L + sucrose 30 g / L + agar 5 g / L, with a pH of 5.8 to 5.9.

[0039] In some specific embodiments of the present invention, the proliferation medium is MS + 6-BA 0.5 mg / L + NAA 1.0 mg / L, and the pH is 5.8 to 5.9.

[0040] In some specific embodiments of the present invention, the dark culture time is 5 to 30 days. Dark culture refers to culture under conditions of no light.

[0041] In some specific embodiments of the present invention, the culture temperature is 23-27°C and the culture humidity is 60-80%RH.

[0042] More specifically, the preferred steps of the method for inducing monkey earring filament callus tissue according to the present invention are as follows:

[0043] (1) Material collection and processing: Pick monkey ear flower buds with good appearance and free from pests and diseases. Place the picked monkey ear flower buds in a refrigerator at 2-8℃ for 24 hours. Rinse the monkey ear flower buds with water and place them in a petri dish with filter paper for later use.

[0044] (2) Disinfection and sterilization treatment: In the sterilized clean bench, soak the monkey ear flower buds treated in step (1) in 75% alcohol for 30 seconds, then rinse twice with sterile water; then soak in 0.1% mercuric chloride solution or 0.5% sodium hypochlorite solution for 3 minutes, gently shaking during the soaking process to thoroughly disinfect the flower buds. After disinfection, rinse three times with sterile water and place on sterile paper to absorb the moisture for later use.

[0045] (3) Induction of callus: Place the flower buds in an inoculation tray with sterile filter paper, gently peel the flower buds open with sterile forceps and scalpel, remove the anthers, and select the filaments with good growth and no damage to inoculate into the callus induction medium. Induction of callus is carried out at a temperature of 23-27℃, a humidity of 60-80%RH and no light. The induction medium is MS + 6-BA 1.0mg / L + NAA 0.3-0.9mg / L + sucrose 30g / L + agar 5g / L, pH 5.8-5.9.

[0046] (4) Proliferation of callus tissue: After culturing for 5 to 30 days, the callus tissue was transferred to a proliferation medium for continuous subculture according to its growth. The medium was cultured at a temperature of 23 to 27°C, a humidity of 60 to 80%RH, and in the dark. The proliferation medium was MS + 6-BA 0.5 mg / L + NAA 1.0 mg / L, with a pH of 5.8 to 5.9.

[0047] The results are as follows Figures 1-2 As shown, Figure 1 and Figure 2 This image shows the callus tissue of monkey earring filaments cultured using the induction method described in this invention. The results indicate that when monkey earring filaments are inoculated into the induction medium, the filaments begin to swell after 5 days. With prolonged culture time, callus tissue gradually forms, which is yellowish-white or pale yellow in color and has a soft texture. After subculture, the callus tissue increases in size and number, becomes yellowish-white in color, and has a soft texture. If the culture time is too long, the formed callus tissue is prone to browning and death.

[0048] The present invention also provides the application of the above-mentioned method for inducing callus tissue of monkey earrings in the cultivation of monkey earrings.

[0049] The following description, based on Examples 1-3, will provide further details. Example

[0050] A method for inducing and proliferating callus from monkey earring filaments, the specific operation of which is as follows:

[0051] (1) Material collection and processing: Pick monkey ear flower buds with good appearance and free from pests and diseases. Place the picked monkey ear flower buds in a refrigerator at 2-8℃ for 24 hours. Rinse the monkey ear flower buds with water for 2 hours. After rinsing, place them in a petri dish with filter paper for later use.

[0052] (2) Disinfection and sterilization treatment: In the sterilized clean bench, soak the monkey ear flower buds treated in step (1) in 75% alcohol for 30 seconds, rinse twice with sterile water; then soak in 0.1% mercuric chloride solution for 3 minutes, gently shake during the soaking process to thoroughly disinfect the flower buds, rinse three times with sterile water after disinfection, and place on sterile paper to absorb the moisture for later use.

[0053] (3) Induction of callus: Place the flower buds in an inoculation tray with sterile filter paper, gently peel the flower buds open with sterile forceps and scalpel, remove the anthers, and select the filaments with good growth and no damage to inoculate into the callus induction medium. Induction of callus is carried out at a temperature of 23-27℃, a humidity of 60-80%RH and no light. The induction medium is MS + 6-BA 1.0mg / L + NAA 0.3mg / L + sucrose 30g / L + agar 5g / L, and the pH is 5.8-5.9.

[0054] (4) Callus proliferation: After 5 days of culture, the filaments swelled and formed yellowish-white callus. After 30 days of culture, the callus induction rate was 100% and the contamination rate was 22.92%. The callus cultured for 30-40 days was transferred to proliferation medium and proliferated under the conditions of 23-27℃, 60-80%RH and no light. The proliferation medium was MS + 6-BA 0.5mg / L + NAA 1.0mg / L, pH 5.8-5.9. Example

[0055] The operation steps are the same as in Example 1, except that the disinfection method used in Example 2 is to soak the monkey ear flower buds in 75% alcohol for 30 seconds and then rinse them twice with sterile water; then soak them in 0.5% sodium hypochlorite solution for 3 minutes and rinse them three times with sterile water. During the disinfection process, the monkey ear flower buds showed partial browning due to the influence of the disinfectant, and the induced callus contamination rate was higher than that in Example 1, at 41.67%.

[0056] As shown in Examples 1 and 2, the disinfection effect of 75% alcohol and 0.5% sodium hypochlorite solution on monkey ear flower buds in Example 2 was not as good as that of 75% alcohol and 0.1% mercuric chloride solution in Example 1. Furthermore, the callus contamination rate induced in Example 2 was significantly higher than that induced in Example 1. Therefore, the type and concentration of disinfectant have a significant impact on the disinfection effect and subsequent callus induction during the disinfection of monkey ear flower buds. Using 75% alcohol and 0.1% mercuric chloride solution as the disinfectant for sterilization of monkey ear flower buds can significantly reduce the contamination rate of the filaments. Example

[0057] The operation steps are the same as in Example 1, except that the amount of NAA added to the induction medium used in Example 3 is 0.5 mg / L, 0.7 mg / L, and 0.9 mg / L, respectively; and the callus induction rates obtained are 86.66%, 70%, and 60%, respectively.

[0058] As shown in Examples 1 and 3, when the NAA concentration in the induction medium used in Example 3 was 0.5 mg / L, 0.7 mg / L, and 0.9 mg / L, the callus induction rate obtained was lower than that obtained in Example 1 when the NAA concentration in the induction medium was 0.3 mg / L. Therefore, the concentration of NAA in the induction method of this invention has a significant impact on the callus induction rate of monkey ear ring filaments. The induction medium of "MS + 6-BA 1.0 mg / L + NAA 0.3 mg / L + sucrose 30 g / L + agar 5 g / L, pH 5.8–5.9" is the optimal choice for achieving a 100% callus induction rate. Example

[0059] The operation steps are the same as in Example 1, except that in this Example 4, one-year-old monkey ear ring leaves are used as explants. After the leaves are picked and cleaned, they are transferred to a clean bench for sterilization (sterilization steps are the same as in Example 1). The leaves are cut and inoculated into the culture medium and cultured under dark conditions (induction medium and culture conditions are the same as in Example 1). After one week of culture, the leaves gradually swell to form callus tissue. After 30 days, the callus induction rate is 75% and the contamination rate is 56.25%. Figure 3 This is a diagram of callus tissue induced using leaves from a one-year-old monkey earring plant as explants in Example 4 of the present invention.

[0060] As shown in Examples 1 and 4, the callus induction rate of one-year-old *Auricularia auricula-judae* leaves in Example 4 was significantly lower than that of *Auricularia auricula-judae* filaments in Example 1; the contamination rate of one-year-old *Auricularia auricula-judae* leaves in Example 4 was significantly higher than that of *Auricularia auricula-judae* filaments in Example 1. Therefore, using the induction method of the present invention, under the same conditions, when inducing callus formation in *Auricularia auricula-judae* filaments and one-year-old *Auricularia auricula-judae* leaves respectively, *Auricularia auricula-judae* filaments, as explants, exhibit higher callus induction efficiency and lower contamination rate compared to one-year-old *Auricularia auricula-judae* leaves. This indicates that the induction method of the present invention is more efficient in inducing callus formation in *Auricularia auricula-judae* filaments with a lower contamination rate, providing technical support for *Auricularia auricula-judae* filament culture and breeding.

[0061] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0062] It should also be noted that the various specific technical features described in the above embodiments can be combined in any suitable manner without contradiction. To avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

[0063] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

Claims

1. A method for inducing callus tissue from monkey earring filaments, characterized in that, Includes the following steps: Monkey ear flower buds were taken, disinfected and sterilized with disinfectant, and then monkey ear flower filaments were taken as explants under sterile conditions and inoculated in induction medium for dark culture to form callus. The callus was then transferred to proliferation medium for subculture to obtain higher quality callus. The induction medium consisted of MS + 6-BA 1.0 mg / L + NAA 0.3–0.9 mg / L + sucrose 30 g / L + agar 5 g / L, with a pH of 5.8–5.9; the proliferation medium consisted of MS + 6-BA 0.5 mg / L + NAA 1.0 mg / L, with a pH of 5.8–5.

9.

2. The method for inducing callus tissue from monkey earring filaments according to claim 1, characterized in that, The monkey earring flower buds were placed at 2-8℃ for 24 hours before the disinfection and sterilization treatment.

3. The method for inducing callus tissue from monkey earring filaments according to claim 1, characterized in that, The disinfectant solution includes at least one of the following: 75% alcohol by volume, 0.1% mercuric chloride solution by mass, and 0.5% to 5% sodium hypochlorite solution by mass.

4. The method for inducing callus tissue from monkey earring filaments according to claim 1, characterized in that, The disinfection and sterilization process includes at least one of the following operations: treatment with 75% alcohol by volume for 30 seconds, treatment with 0.1% mercuric chloride by mass for 3 minutes, or treatment with 0.5% to 5% sodium hypochlorite by mass for 3 minutes.

5. The method for inducing callus tissue from monkey earring filaments according to claim 1, characterized in that, The dark culture time is 5 to 30 days.

6. The method for inducing callus tissue from monkey earring filaments according to claim 1, characterized in that, The culture temperature is 23–27°C, and the humidity is 60–80% RH.

7. The application of the method for inducing callus tissue of monkey earrings as described in any one of claims 1 to 6 in the cultivation of monkey earrings.

Images