A method for inducing callus tissue from aseptic seedlings of monkey earrings and its application

Abstract

The present invention belongs to the field of plant tissue culture technology, and specifically relates to a method and application for inducing callus tissue of aseptic monkey earring seedlings. The method comprises the following steps: taking the root system of the aseptic monkey earring seedlings as an explant and inoculating it in an induction medium for cultivation; the induction medium is an MS medium containing sucrose and agar, to which 0.4-0.6 mg / L of 6-benzylaminopurine, 0.1-0.5 mg / L of naphthaleneacetic acid, and 0-0.3 mg / L of gibberellins are added, and the pH is 5.8-5.9. The aseptic monkey earring seedlings cultivated by the present invention have lush branches and leaves, vigorous root growth, and have the advantages of convenient material collection and low contamination rate. The method can efficiently cultivate monkey earring root callus tissue, provide a reference for the asexual breeding of monkey earrings, and lay the foundation for inducing complete plants and realizing large-scale production of monkey earrings in the future.

Description

Technical Field

[0001] The invention belongs to the technical field of plant tissue culture, and particularly relates to an induction method and application of callus tissue of aseptic seedling roots of monkey earrings. Background Art

[0002] Archidendron clypearia (Jack) Benth, an evergreen tree of the genus Archidendron (Mimosoideae), belongs to the Fabaceae family. It thrives in sunny, humid climates. As a medicinal tree, Archidendron clypearia has high medicinal value, boasting anti-inflammatory, antibacterial, antiviral, and anti-tumor properties. Its processed products are widely used in medicine, food and health care, daily chemical products, and animal husbandry.

[0003] With the increasing market demand for monkey earrings, its wild resources are decreasing. Monkey earrings mainly use traditional seedling breeding technology, which is difficult to meet the needs of large-scale planting and genetic improvement. In addition, domestic and foreign workers currently have little research on the rapid propagation technology of monkey earrings, especially the method of inducing monkey earring plant regeneration through callus tissue. Summary of the Invention

[0004] The technical problem addressed by the present invention is to provide a method for inducing callus from sterile root seedlings of monkey earrings and its application. Using sterile root seedlings of monkey earrings as explants, callus induction and proliferation are achieved, effectively increasing the callus induction rate of monkey earrings. This provides technical support for the efficient breeding of superior monkey earrings varieties and lays the foundation for future research on monkey earrings' variety improvement and regeneration mechanisms.

[0005] The present invention solves the above problems through the following technical solutions:

[0006] The invention provides a method for inducing root callus tissue of sterile monkey earring seedlings. The method comprises the following steps: taking the root system of the sterile monkey earring seedlings as explants, and inoculating the root system into an induction culture medium for culturing; the induction culture medium is an MS culture medium containing sucrose and agar, to which 0.4-0.6 mg / L of 6-benzylaminopurine, 0.1-0.5 mg / L of naphthaleneacetic acid, and 0-0.3 mg / L of gibberellin are added, and the pH value is 5.8-5.9.

[0007] Furthermore, the induction culture conditions include: culture temperature of 24-26° C., humidity of 60-80% RH, light intensity of 2000-3000 Lx, and light cycle of 10-12 h / d.

[0008] Furthermore, the method for obtaining the sterile monkey earring seedlings includes: disinfecting the pretreated monkey earring seeds, and then culturing them in a blank culture medium for 50-60 days to obtain the sterile monkey earring seedlings; preferably, the culture conditions for cultivating the sterile seedlings include: culture temperature of 24-26°C, humidity of 60-80% RH, light intensity of 2000-3000Lx, and light cycle of 10-12h / d.

[0009] Furthermore, the pretreatment step includes: peeling off the seed coat of the monkey earring seeds to obtain peeled seed embryos, and washing them with clean water.

[0010] Furthermore, the disinfection step includes: sequentially treating with 75% alcohol for 30 seconds, treating with 0.1% mercuric chloride solution for 10 to 12 minutes, and treating with 0.5% sodium hypochlorite for 20 minutes.

[0011] Furthermore, the blank culture medium is one of MS, 1 / 2MS or WPM culture medium.

[0012] Furthermore, the blank culture medium is a WPM culture medium without sucrose and agar, to which 0-1.0 g / L of activated carbon, 30 g / L of sucrose, and 5 g / L of agar are added.

[0013] Furthermore, the method for inducing callus tissue of the root of the sterile monkey earring seedlings also includes the step of transferring the callus tissue cultured in the induction culture medium to the proliferation culture medium for subculture and proliferation.

[0014] Furthermore, the proliferation culture medium is an MS culture medium containing sucrose and agar, to which 1.0 mg / L of 6-benzylaminopurine and 2.0 mg / L of naphthaleneacetic acid are added, and the pH is 5.8-5.9.

[0015] According to another aspect of the present invention, the present invention also provides an application of a method for inducing callus tissue of sterile monkey earring seedlings in cultivating monkey earrings. By using the above method to induce callus tissue of sterile monkey earring seedlings and establish a rapid propagation technology system, high-quality plants with excellent traits can be cultivated.

[0016] The beneficial effects of the present invention are as follows: the monkey earring sterile seedlings cultivated by the present invention have luxuriant branches and leaves, and vigorous root growth; the efficiency of inducing callus tissue by using the roots of the monkey earring sterile seedlings is high, and the induction rate is as high as 100%, which lays the foundation for inducing complete plants and realizing large-scale mass production of monkey earrings in the future, and also provides technical support for the rapid propagation of excellent monkey earring varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 : The monkey earring aseptic seedlings that the cultivation method of the present invention cultivates.

[0018] Figure 2: The monkey earring root callus diagram cultivated by the induction method of the present invention.

[0019] Figure 3 : Diagram of the callus of the monkey earring root cultivated by the proliferation method of the present invention. DETAILED DESCRIPTION

[0020] The present invention will be 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, and the methods involved are commonly used in the art unless otherwise specified. The reagents involved are all available from common commercial sources unless otherwise specified, and the specific commercial sources are not limited.

[0021] The method for inducing root callus tissue of sterile monkey earring seedlings of the present invention comprises the following steps: taking the root system of the sterile monkey earring seedlings as explants, and inoculating the root system into an induction culture medium for cultivation; the induction culture medium is an MS culture medium containing sucrose and agar, to which 0.4-0.6 mg / L of 6-benzylaminopurine, 0.1-0.5 mg / L of naphthaleneacetic acid, and 0-0.3 mg / L of gibberellin are added, and the pH value is 5.8-5.9.

[0022] In one embodiment of the present invention, the induction culture conditions include: culture temperature 24-26°C, humidity 60-80% RH, light intensity 2000-3000 Lx and photoperiod 10-12 h / d.

[0023] In one embodiment of the present invention, the method for obtaining the sterile monkey earring seedlings includes: sterilizing the pretreated monkey earring seeds, and then culturing them in a blank culture medium for 50-60 days to obtain the sterile monkey earring seedlings; preferably, the culture conditions for cultivating the sterile seedlings include: culture temperature 24-26°C, humidity 60-80% RH, light intensity 2000-3000Lx and light cycle 10-12h / d.

[0024] In one embodiment of the present invention, the pretreatment step comprises: peeling off the seed coat of the monkey earring seeds to obtain peeled seed embryos, and washing them with clean water.

[0025] In one embodiment of the present invention, the disinfection step comprises: sequentially treating with 75% alcohol for 30 seconds, treating with 0.1% mercuric chloride solution for 10 to 12 minutes, and treating with 0.5% sodium hypochlorite for 20 minutes.

[0026] In one embodiment of the present invention, the blank culture medium is one of MS, 1 / 2MS or WPM culture medium.

[0027] In one embodiment of the present invention, the blank culture medium is a WPM culture medium without sucrose and agar, to which 0-1.0 g / L of activated carbon is added; preferably, it contains 30 g / L of sucrose and 5 g / L of agar.

[0028] In one embodiment of the present invention, the method further comprises the step of transferring the callus cultured in the induction medium to a proliferation medium for subculture and proliferation.

[0029] In one embodiment of the present invention, the proliferation medium is an MS medium containing sucrose and agar, to which 1.0 mg / L of 6-benzylaminopurine and 2.0 mg / L of naphthaleneacetic acid are added, and the pH is 5.8-5.9.

[0030] More specifically, the method for inducing callus tissue from the aseptic seedlings of the monkey earring of the present invention preferably comprises the following steps:

[0031] (1) Obtaining sterile seedlings of monkey earrings: Peel off the seed coat of monkey earrings seeds to obtain peeled embryos, rinse with running water for 2 hours, move to a clean bench, soak in 75% alcohol for 30 seconds, rinse with sterile water 2 to 3 times, then transfer to 0.1% mercuric chloride solution for sterilization for 12 minutes, rinse with sterile water 3 to 5 times, soak in 0.5% sodium hypochlorite for 20 minutes, rinse with sterile water 3 to 5 times, and place in an inoculation tray with sterile paper. WPM medium without sucrose and agar was added with 1.0 g / L activated carbon, 30 g / L sucrose, and 5 g / L agar as a blank medium. The peeled embryos were inoculated into the blank medium and cultured under light for 50-60 days to obtain sterile monkey earring seedlings, wherein the light intensity was 2000-3000 Lx, the light duration was 10-12 h / d, the culture room temperature was 24-26°C, and the humidity was 60-80% RH; the culture results were as follows: Figure 1 shown.

[0032] (2) Induction of root callus: using MS containing sucrose and agar as a basal medium, adding 0.5 mg / L of 6-benzylaminopurine (hereinafter referred to as 6-BA), 0.5 mg / L of naphthaleneacetic acid (hereinafter referred to as NAA), and 0.1 mg / L of gibberellin (hereinafter referred to as GA3), adjusting the pH to 5.8-5.9, and obtaining an induction medium. Taking the roots of the monkey earring sterile seedlings cultivated in step (1) as explant materials, cutting them into 0.8-1.6 cm segments, inoculating them into the above-mentioned induction medium and carrying out light culture for 5-30 days to form monkey earring sterile seedling root callus, wherein the light intensity is 2000-3000 Lx, the light time is 10-12 h / d, the culture room temperature is 24-26 ° C, and the humidity is 60-80% RH; the induction results are as follows: Figure 2 As shown, after inoculation, the root segments of the monkey earring gradually swelled to form callus tissues of varying degrees and visible to the naked eye, which were yellowish white or light yellow in color and had a loose and hard texture.

[0033] (3) Proliferation of root callus: using MS medium containing sucrose and agar, adding 6-BA 1.0 mg / L and NAA 2.0 mg / L, adjusting the pH to 5.8-5.9, to obtain a proliferation medium, then transferring the root callus induced in step (2) to a proliferation culture for callus proliferation, and performing subculture transfer according to the subsequent growth of the callus; Figure 3 It is the callus tissue after subculture and proliferation. It has increased in size and quantity, is white in color, and has a soft texture. However, if the culture time is too long, the formed callus tissue will easily turn brown and die.

[0034] This will be further described below with reference to Examples 1-3.

[0035] Example 1:

[0036] Effects of different disinfection methods on the cultivation of sterile seedlings of monkey earring seeds:

[0037] The seed coat of the monkey earring seeds was peeled to obtain peeled embryos, which were rinsed with running water for 2 hours and moved into a clean bench. The peeled embryos were disinfected according to the disinfection method shown in Table 1. After each treatment, they were cleaned with sterile water and placed in an inoculation tray with sterile paper. WPM medium without sucrose and agar was used, and 1.0 g / L activated carbon, 30 g / L sucrose, and 5 g / L agar were added as blank medium. The peeled embryos were inoculated into the blank medium and cultured under light for 50-60 days to obtain monkey earring sterile seedlings, wherein the light intensity was 2000-3000 Lx, the light exposure time was 10-12 h / d, the culture room temperature was 24-26° C., and the humidity was 60-80% RH; the contamination rate results are shown in Table 1 below:

[0038] Table 1: Effects of different disinfection methods on the cultivation of sterile seedlings of monkey earring seeds

[0039]

[0040]

[0041] As shown in Table 1, different disinfection treatment methods have significant effects on monkey earring seeds; among them, the seed contamination rate after soaking in 75% alcohol for 30s, 0.1% mercuric chloride for 12min, and 0.5% sodium hypochlorite for 20min is the lowest, which is 3.23%. This shows that this disinfection method is the most suitable disinfection method for monkey earring seeds.

[0042] Example 2:

[0043] Effects of different blank culture media on the cultivation of sterile seedlings of monkey earring seeds:

[0044] The seed coat of the monkey earring seeds was peeled to obtain peeled embryos, which were rinsed with running water for 2 hours, moved to a clean bench, soaked in 75% alcohol for 30 seconds, washed 2 to 3 times with sterile water, and then sterilized in 0.1% mercuric chloride solution for 12 minutes, washed 3 to 5 times with sterile water, soaked in 0.5% sodium hypochlorite for 20 minutes, and washed 3 to 5 times with sterile water. The seeds were placed in an inoculation tray with sterile paper, and a blank culture medium was prepared as shown in Table 2 below. The peeled embryos were inoculated into the blank culture medium described in the table below and cultured under light for 50-60 days to obtain monkey earring sterile seedlings, wherein the light intensity was 2000 to 3000 Lx, the light duration was 10 to 12 hours / day, the culture room temperature was 24 to 26° C., and the humidity was 60 to 80% RH. The cultivation results are shown in Table 2 below:

[0045] Table 2: Effects of different blank culture media on the cultivation of sterile seedlings of monkey earring seeds

[0046]

[0047] As shown in Table 2, different blank culture media can cultivate and form sterile seedlings, among which WPM has the highest formation rate, followed by 1 / 2MS; in addition, according to the state of sterile seedlings, the sterile seedlings cultivated in the culture medium with 1.0 g / L activated carbon grew faster, had more luxuriant branches and leaves, thicker main roots and developed root systems, and their growth state was significantly better than that of the culture medium without activated carbon; among them, WPM culture medium containing 30 g / L sucrose and 5 g / L agar and added with 1.0 g / L activated carbon was the most suitable culture medium for sterile seedlings of monkey earring seeds.

[0048] Example 3:

[0049] Effects of different induction media on callus induction from sterile seedlings of Psoralea corylifolia:

[0050] Using MS containing sucrose and agar as a basal medium, different concentrations of hormones were added as shown in Table 3 below, and the pH was adjusted to 5.8-5.9 to obtain an induction medium. The roots of the aseptic monkey earring seedlings cultivated in Group 6 of Example 2 were taken as explant materials, cut into 0.8-1.6 cm segments, and inoculated into the above-mentioned induction medium for light culture for 5-30 days to form aseptic monkey earring seedling root callus, wherein the light intensity was 2000-3000 Lx, the light duration was 10-12 h / d, the culture room temperature was 24-26° C., and the humidity was 60-80% RH; the induction results are shown in Table 3 below:

[0051] Table 3: Effects of different induction media on callus induction from sterile seedlings of Psoralea corylifolia

[0052]

[0053] As shown in Table 3, the root segments of the sterile monkey earring seedlings can form callus tissue in different hormone ratios, but the induction rates of different hormone ratios are significantly different. When the NAA content is 0.1 mg / L, the callus induction rate of the monkey earring root increases with the increase of GA3 concentration; when the concentration of GA3 is constant, the increase of NAA concentration will also promote the induction rate of the monkey earring root callus tissue. When the addition amount of NAA is 0.5 mg / L and the addition amount of GA3 is 0.1 mg / L, the induction rate of the root callus tissue can reach 100%.

[0054] For those skilled in the art, all other modifications and alterations obtained without creative work are within the scope of protection of the present invention.

Claims

1. A method for inducing callus tissue of aseptic seedlings of monkey earrings, characterized in that: The following steps are involved: The root system of the sterile monkey earring seedling is taken as an explant and inoculated into an induction medium for cultivation; the induction medium is an MS medium containing sucrose and agar, 0.4-0.6 mg / L of 6-benzylaminopurine, 0.1-0.5 mg / L of naphthaleneacetic acid, and 0.1-0.3 mg / L of gibberellin, and the pH is 5.8-5.9; The method for obtaining the sterile monkey earring seedlings comprises: sterilizing the pretreated monkey earring seeds and then culturing them in a blank culture medium for 50-60 days to obtain the sterile monkey earring seedlings; The blank medium is WPM medium without sucrose and agar + activated carbon 1.0 g / L + sucrose 30 g / L + agar 5 g / L; The disinfection step comprises: sequentially treating with 75% alcohol for 30 seconds, treating with 0.1% mercuric chloride solution for 10 to 12 minutes, and treating with 0.5% sodium hypochlorite for 20 minutes.

2. The method for inducing callus of aseptic seedling root of monkey earring according to claim 1, wherein The induction culture conditions include: culture temperature of 24-26° C., humidity of 60-80% RH, light intensity of 2000-3000 Lx, and light cycle of 10-12 h / d.

3. The induction method of the aseptic seedling root callus of monkey earring according to claim 1, wherein The pretreatment step comprises: peeling off the seed coat of the monkey earring seeds to obtain peeled seed embryos, and washing them with clean water.

4. The method for inducing callus of aseptic seedling root of monkey earring according to claim 1, wherein The method comprises the steps of transferring the callus cultured in the induction medium to the proliferation medium for subculture and proliferation.

5. The method for inducing callus of aseptic seedling root of monkey earring according to claim 4, characterized in that, The proliferation culture medium is an MS culture medium containing sucrose and agar, to which 1.0 mg / L of 6-benzylaminopurine and 2.0 mg / L of naphthaleneacetic acid are added, and the pH value is 5.8-5.

9.

6. Use of the method for inducing callus tissue of sterile seedling roots of monkey earrings according to any one of claims 1 to 5 in cultivating monkey earrings.

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