Induction method and application of pithecellobium clypearia filament callus

Abstract

The invention belongs to the technical field of tissue culture, and relates to a pithecellobium clypearia filament callus induction method and application, and the method comprises the following steps: taking pithecellobium clypearia buds, disinfecting and sterilizing with a disinfectant, taking pithecellobium clypearia filaments as explants in a sterile environment, inoculating the explants into an induction culture medium for dark culture to form calluses, and carrying out tissue culture to obtain the pithecellobium clypearia filament callus. And then transferring the callus to a proliferation culture medium for subculture proliferation to obtain the callus with higher quality. The induction method provided by the invention can induce pithecellobium clypearia filaments to quickly form calluses, and the induction rate of the calluses is as high as 100%; the induction method of the pithecellobium clypearia filament callus is short in cultivation period, high in induction efficiency and low in pollution rate, the research path of a pithecellobium clypearia rapid propagation technology is widened when the induction method is applied to pithecellobium clypearia cultivation, it is possible that pithecellobium clypearia filaments are regenerated into plants, and high-quality plants with excellent characters can be cultivated.

Description

Technical Field

[0001] The invention belongs to the technical field of tissue culture, and particularly relates to an induction method and application of monkey earring filament callus tissue. Background Art

[0002] Archidendron clypearia (Jack) Benth is a plant of the subfamily Mimosinoideae of the Leguminosae family, commonly known as the spit tree. It is widely distributed in tropical Asia and is cultivated in many provinces and autonomous regions in my country, including Zhejiang, Fujian, Taiwan, Guangdong, Guangxi, Hainan, Yunnan, etc. The application value of Archidendron clypearia is extremely wide, covering many fields such as medicine, food, daily chemicals, and animal husbandry. For example, in terms of medicine, the branches and leaves of Archidendron clypearia are rich in gallic acid, quercetin, and total polyphenols. These ingredients give Archidendron clypearia multiple pharmacological effects such as antibacterial, anti-inflammatory, analgesic, and antiviral. At present, the single preparation of Archidendron clypearia extract has been widely used in clinical practice and has achieved good therapeutic effects.

[0003] However, despite the broad application prospects of monkey earrings, their wild resources are relatively limited. In order to maintain the excellent variety characteristics of monkey earrings and select plants with strong stress resistance or high content of effective ingredients, it is urgent to carry out research on monkey earring seedling propagation through tissue culture rapid propagation technology. At present, the research on monkey earring seedling propagation using tissue culture technology is mainly based on the stem segments, leaves, petioles and other parts of sterile seedlings cultivated from seeds or seeds of excellent strains as explants to carry out tissue culture research. However, the research on the regeneration of monkey earring plants using monkey earring filaments as explants has not appeared in previous literature and reports. The cell division and differentiation ability of filaments is usually strong and has potential regeneration ability. Therefore, exploring the tissue culture method using monkey earring filaments as explants is expected to open up new paths for the rapid propagation technology research of monkey earrings and provide more efficient and reliable technical support.

[0004] Based on the above background, the present invention proposes a method for inducing callus tissue of monkey earring filaments. The method uses monkey earring filaments as starting materials, establishes a good regeneration system through callus induction and organogenesis pathways, broadens the research path of monkey earring rapid propagation technology, and lays a foundation for new variety breeding and seedling breeding. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for inducing monkey earring filament callus and its application. The induction method of the present invention can induce monkey earring filament to quickly form callus, and the induction rate is as high as 100%; the induction method has a short cultivation cycle, high induction efficiency, and low pollution rate, and successfully establishes an efficient callus induction system for monkey earring filament, providing technical support for monkey earring filament cultivation and breeding; the method for inducing monkey earring filament callus is applied to the cultivation of monkey earrings, broadening the research path of monkey earring rapid propagation technology, 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 monkey earring filament callus tissue comprises the following steps:

[0008] The monkey earring flower buds are disinfected and sterilized with a disinfectant, and then the monkey earring flower filaments are taken as explants in a sterile environment, inoculated into an induction medium for dark culture to form callus tissue, and then the callus tissue is transferred to a proliferation medium for subculture proliferation to obtain better quality callus tissue.

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

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

[0011] Furthermore, the disinfectant includes at least one of 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 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.

[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, and the pH is 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, and the pH is 5.8-5.9.

[0016] Furthermore, the proliferation culture medium is MS+6-BA0.5mg / L+NAA1.0mg / L, and the pH is 5.8-5.9.

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

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

[0019] The invention also provides application of the induction method of monkey earring filament callus in cultivating monkey earrings.

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

[0021] (1) The flowering period of monkey earrings is from February to June, so the explants can be obtained for a relatively long time, and the flower buds of monkey earrings are easy to pick, and the process is simple and convenient. In addition, the filaments of monkey earrings are wrapped by petals, which makes them less polluted by the external environment and has a lower contamination rate. At the same time, the time required to disinfect the filaments of monkey earrings is also shorter, which causes less harm to the operators.

[0022] (2) The induction method of the present invention can induce monkey earring filaments to quickly form callus tissue. The induction method has a short cultivation cycle, high induction efficiency, and low pollution rate. It breaks through the traditional explant tissue culture method of monkey earrings and provides technical support for the cultivation and breeding of monkey earring 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 to regenerate monkey earring filaments into plants, and can cultivate high-quality plants with excellent traits. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a diagram of callus tissue induced by using monkey earring filaments as explants as described in the present invention;

[0025] Figure 2 This is a callus diagram of the monkey earring filament callus after subculture and proliferation according to the present invention;

[0026] Figure 3 This is a diagram of callus tissue induced using annual monkey earring leaves as explants in Example 4 of the present invention. DETAILED DESCRIPTION

[0027] The present invention is further described in detail below with reference to the examples, but the present invention is not limited thereto. All instruments used in the present invention are sterilized, the methods involved are commonly used methods in the art unless otherwise specified, and the reagents involved can be obtained from common commercial channels unless otherwise specified, and the specific commercial channels are not limited.

[0028] It should be noted in advance that the monkey earring explants used in the present invention were collected from monkey earring seedlings from the Huizhou planting base and were identified as monkey earrings of the genus Monkey Earring of the subfamily Mimosoideae of the Fabaceae by the South China Plant Identification Center of the South China National Botanical Garden, Chinese Academy of Sciences.

[0029] A method for inducing monkey earring filament callus tissue comprises the following steps:

[0030] The monkey earring flower buds are disinfected and sterilized with a disinfectant, and then the monkey earring flower filaments are taken as explants in a sterile environment, inoculated into an induction medium for dark culture to form callus tissue, and then the callus tissue is transferred to a proliferation medium for subculture proliferation to obtain better quality callus tissue.

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

[0032] In some specific embodiments of the present invention, the monkey earring flower buds are refrigerated 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 75% alcohol by volume, 0.1% mercuric chloride solution by mass, and 0.5% to 5% sodium hypochlorite solution by mass.

[0034] In certain 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 treatment comprises: 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 medium is MS+6-BA 1.0 mg / L+NAA 0.3-0.9 mg / L+sucrose 30 g / L+agar 5 g / L, and the pH is 5.8-5.9.

[0038] In certain 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, and the pH is 5.8-5.9.

[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 5.8-5.9.

[0040] In some specific embodiments of the present invention, the dark culture time is 5 to 30 days. The dark culture refers to culture under the condition 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 method for inducing callus of monkey earring filaments of the present invention preferably comprises the following steps:

[0043] (1) Material collection and processing: Pick monkey earring flower buds with good appearance quality and no pests and diseases, put the picked monkey earring flower buds in a refrigerator at 2-8°C for 24 hours, rinse the monkey earring flower buds with water, and place them in a culture dish with filter paper for later use.

[0044] (2) Disinfection and sterilization: In a sterilized clean bench, soak the monkey earring flower buds treated in step (1) in 75% alcohol for 30 seconds, and then rinse them twice with sterile water; then soak them 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 them with sterile water three times, place them on sterile paper to absorb the moisture for later use.

[0045] (3) Induction of callus: Place flower buds on an inoculation plate with sterile filter paper, gently peel open the flower buds with sterilized tweezers and a scalpel, remove the anthers, select filaments with good growth and no damage and inoculate them into callus induction medium, and induce callus at a temperature of 23-27°C, a humidity of 60-80% RH, and no light. 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, and the pH is 5.8-5.9.

[0046] (4) Proliferation of callus tissue: After culturing for 5 to 30 days, the callus tissue is transferred to a proliferation medium for continuous subculture according to its growth condition, and cultured at a temperature of 23 to 27°C, a humidity of 60 to 80% RH, and no light; wherein 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.

[0047] The results are as follows Figures 1-2 As shown, Figure 1 and Figure 2 The figure is a callus of monkey earring filaments cultured by the induction method of the present invention. The results in the figure show that when the monkey earring filaments are inoculated into the induction medium, the filaments begin to swell 5 days after inoculation, and gradually form callus with the extension of the culture time. The color is yellowish white or light yellow, and the texture is soft; after subculture and proliferation, the volume of the callus increases, the number increases, the color is yellowish white, and the texture is soft. If the culture time is too long, the formed callus is easy to brown and die.

[0048] The invention also provides application of the induction method of monkey earring filament callus in cultivating monkey earrings.

[0049] This will be further described below with reference to Examples 1-3.

[0050] Example 1

[0051] A method for inducing and proliferating monkey earring filament callus, the specific operations are as follows:

[0052] (1) Material collection and processing: Pick monkey earring flower buds with good appearance quality and no diseases and insect pests, put the picked monkey earring flower buds into a refrigerator at 2-8°C for 24 hours, rinse the monkey earring flower buds with water for 2 hours, and place them in a culture dish with filter paper for later use after rinsing.

[0053] (2) Disinfection and sterilization: In a sterilized clean bench, soak the monkey earring flower buds treated in step (1) in 75% alcohol for 30 seconds, and then rinse them twice with sterile water; then soak them in 0.1% mercuric chloride solution for 3 minutes, gently shaking during the soaking process to thoroughly disinfect the flower buds. After disinfection, rinse them three times with sterile water, place them on sterile paper to absorb the moisture for later use.

[0054] (3) Induction of callus: Place flower buds on an inoculation plate with sterile filter paper, gently peel open the flower buds with sterilized tweezers and a scalpel, remove the anthers, select filaments with good growth and no damage and inoculate them into callus induction medium, and induce callus at a temperature of 23-27°C, a humidity of 60-80% RH, and no light, wherein the induction medium is MS+6-BA1.0 mg / L+NAA 0.3 mg / L+sucrose 30 g / L+agar 5 g / L, and the pH is 5.8-5.9.

[0055] (4) Proliferation of callus: After 5 days of culture, the filaments gradually expanded and formed yellow-white callus. After 30 days of culture, the callus induction rate was 100% and the contamination was 22.92%. The callus cultured for 30 to 40 days was transferred to the proliferation medium and the callus was proliferated at a temperature of 23 to 27°C, a humidity of 60 to 80% RH, and no light; wherein the proliferation medium was MS + 6-BA 0.5 mg / L + NAA 1.0 mg / L, and the pH was 5.8 to 5.9.

[0056] Example 2

[0057] The operation steps are the same as those in Example 1, except that the disinfection method adopted in Example 2 is to soak the monkey earring flower buds in 75% alcohol for 30 seconds, and then rinse them with sterile water twice; then soak them in 0.5% sodium hypochlorite solution for 3 minutes, and rinse them with sterile water three times; during the disinfection process, the monkey earring flower buds are partially browned due to the influence of the disinfectant, and the induced callus contamination rate is higher than that in Example 1, which is 41.67%.

[0058] It can be seen from Example 1 and Example 2 that the disinfection effect of monkey earring flower buds using 75% alcohol and 0.5% sodium hypochlorite solution in Example 2 is not as good as the disinfection effect of monkey earring flower buds using 75% alcohol and 0.1% mercuric chloride solution in Example 1; at the same time, the callus contamination rate induced by Example 2 is significantly higher than the callus contamination rate induced by Example 1. It can be seen that in the disinfection process of monkey earring flower buds, the type and concentration of disinfectants have an important influence on the disinfection effect and subsequent callus induction. Selecting 75% alcohol and 0.1% mercuric chloride solution as disinfectants to disinfect and sterilize monkey earring flower buds can significantly reduce the contamination rate of monkey earring filaments.

[0059] Example 3

[0060] The operation steps are the same as those 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; the callus induction rates obtained are 86.66%, 70%, and 60%, respectively.

[0061] It can be seen from Example 1 and Example 3 that when the amount of NAA added in the induction medium used in Example 3 was 0.5 mg / L, 0.7 mg / L, and 0.9 mg / L, respectively, the callus induction rate obtained was not as good as the amount of NAA added in the induction medium used in Example 1 was 0.3 mg / L. It can be seen that the concentration of NAA in the induction method of the present invention has a significant effect on the callus induction rate of monkey earring filaments, and the induction medium of "MS+6-BA 1.0 mg / L+NAA0.3 mg / L+sucrose 30 g / L+agar 5 g / L, pH 5.8-5.9" is the best choice to achieve 100% callus induction rate.

[0062] Example 4

[0063] The operating steps are the same as those in Example 1, except that in this Example 4, annual monkey earring leaves are used as explants. The picked leaves are cleaned and transferred to a clean bench for disinfection and sterilization (the disinfection step is the same as in Example 1). The leaves are cut and inoculated into a culture medium and transferred to culture under no light conditions (the induction culture medium and culture conditions are the same as in Example 1). After culturing for one week, the leaves gradually swell to form callus. 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 annual monkey earring leaves as explants in Example 4 of the present invention.

[0064] It can be seen from Example 1 and Example 4 that the callus induction rate of the annual monkey earring leaves in Example 4 is significantly lower than the callus induction rate of the monkey earring filaments in Example 1; the contamination rate of the annual monkey earring leaves in Example 4 is significantly higher than the contamination rate of the monkey earring filaments in Example 1. It can be seen that when the induction method of the present invention is used to induce monkey earring filaments and annual monkey earring leaves to form callus under the same conditions, the monkey earring filaments as explants show higher callus induction efficiency and lower contamination rate than the annual monkey earring leaves. This shows that the induction method of the present invention is more efficient in inducing monkey earring filament callus and has a low contamination rate, providing technical support for the cultivation and breeding of monkey earring filaments.

[0065] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0066] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. To avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0067] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A method for inducing callus tissue of monkey earring filaments, characterized in that: The following steps are involved: The monkey earring flower buds are disinfected and sterilized with a disinfectant, and then the monkey earring flower filaments are taken as explants in a sterile environment, inoculated into an induction medium for dark culture to form callus tissue, and then the callus tissue is transferred to a proliferation medium for subculture proliferation to obtain better quality callus tissue.

2. The method for inducing callus of monkey earring filaments according to claim 1, characterized in that: The monkey earring flower buds are placed in a refrigerator at 2-8° C. for 24 hours before the disinfection and sterilization treatment.

3. The method for inducing callus of monkey earring filaments according to claim 1, characterized in that: The disinfectant comprises at least one of alcohol with a volume fraction of 75%, mercuric chloride solution with a mass fraction of 0.1%, and sodium hypochlorite solution with a mass fraction of 0.5% to 5%.

4. The method for inducing callus of monkey earring filaments according to claim 1, characterized in that: 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.

5. The method for inducing callus of monkey earring filaments according to claim 1, characterized in that: 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, and the pH value is 5.8-5.

9.

6. The method for inducing callus of monkey earring filaments according to claim 1, characterized in that: The proliferation culture medium is MS+6-BA0.5mg / L+NAA1.0mg / L, and the pH value is 5.8-5.

9.

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

8. The method for inducing callus of monkey earring filaments according to claim 1, characterized in that: The culture temperature is 23-27° C., and the culture humidity is 60-80% RH.

9. Use of the method for inducing monkey earring filament callus as claimed in any one of claims 1 to 9 in cultivating monkey earrings.

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