CN110800611A - Tissue culture method and device for plant, and method for producing metabolites - Google Patents

Abstract

The invention discloses a plant tissue culture method, a device and a production method of metabolites, and belongs to the field of plant tissue culture and metabolites. The plant tissue culture method and its device and the production method of metabolites, by clarifying the plant tissue culture method, optimizing the callus culture process, speeding up the callus culture process, and by optimizing the auxin, it plays a role in reducing the callus tissue. Due to the damage of the current ecological environment and the blind collection of wild resources, many wild plants tend to be endangered, resulting in the reduction of many sources of biological natural active substances, and The chemical synthesis method has complicated process and high cost, and at the same time is easy to cause new environmental pollution. The present invention can effectively increase the production amount of plant natural active substances.

Description

Tissue culture method and device for plant, and method for producing metabolites

technical field

The present invention relates to the field of plant tissue culture and metabolites, in particular to a plant tissue culture method and its device and a method for producing metabolites.

Background technique

There are a wide variety of plants on the earth and the vast number of plants are valuable resources for human survival. The organic matter from plants includes two categories: primary metabolites and secondary metabolites. Primary metabolites are directly related to plant growth, development and reproduction. They are the metabolism of energy obtained by plants and provide energy and intermediate products for plant survival, growth, development and reproduction. Compared with primary metabolites, secondary metabolites use some intermediate products of primary metabolism as raw materials or precursors for further metabolic processes. This secondary metabolite of plants is a substance produced in the process of releasing energy and has no direct relationship with plant growth, development and reproduction. However, these secondary metabolites often have strong biological activity and economic value, and are natural active substances (ie, medicinal active ingredients) for human disease prevention or treatment or natural products with unique economic value. The mechanism of secondary metabolism is still obscure and recognized as difficult to study, but it is also a research field with great application prospects.

The existing plant tissue culture method and its device and the production method of metabolites can not meet the existing needs because the yield of metabolites is disclosed.

SUMMARY OF THE INVENTION

The purpose of the present invention is to solve the problem that the existing metabolite yields can not meet the existing needs, and the proposed plant tissue culture method and its device and the production method of the metabolites.

In order to achieve the above object, the present invention adopts the following technical solutions:

Plant tissue culture method and its device and production method of metabolite, comprising the following steps:

S1. Collect the young stems and leaves of the current year, cut the stems into 3-4cm long pieces, and cut the leaves into 1cm wide strips, wash them with hydrogen peroxide with a volume fraction of 2%, and wash them repeatedly with tap water, then rinse them with running water for 1.5 hours. And disinfected with 65% ethanol by volume for 40s, rinsed with sterile water for 3-4 times, then disinfected with 1g/L HgCl ₂ for 3-7min, and then rinsed with sterile water for 4-5 times for use;

S2. Select the full seeds of the current year, soak them for 22 hours, peel off the seed coats, rinse with water repeatedly, then use alcohol with a volume fraction of 70%, disinfect for 1 minute, rinse with sterile water 3-4 times, 1g/L HgCl ₂ , and disinfect for 15 minutes , rinse 4-5 times with sterile water, insert into 1/2MS medium, keep the temperature at about 22-27℃ and cultivate under natural light;

S3, cut the young stems developed in S2 into 0.7cm long sections, cut the young leaves into 0.5cm*0.5cm squares, inoculate on the spare material collected in S1, and then place them in the callus medium for callus Tissue culture;

S4, the callus induced in S3 is cultivated on the first-generation medium, and after the cultivation is completed, the callus is subcultured on the subculture medium;

S5, get the third-generation callus of the stem subculture in S4, add 30ml B ₅ medium in a 100ml tripod flask and carry out callus extraction and culture;

S6. Collect the callus cultured in S5 and place it in an oven at 80°C to dry to constant weight, crush it, put it into a flask, add alcohol with a volume fraction of 60%, condense and reflux in a 75°C water bath and extract twice, each time For 1 h, the combined filtrate was suction filtered to obtain callus metabolites.

Preferably, the sucrose in the callus medium in S3 is 35g/L, solidified with agar 8g/L, the pH is adjusted to 6.3 before the callus medium is autoclaved, and the temperature of the culture room is controlled at about 22-27°C , When cultivating in dark light, place the culture bottle on a non-illuminated culture rack and cover it with black cloth. When cultivating in light, the light is 12h/d, and the light intensity is 1,000-1,500lx.

Preferably, the first generation medium is B5 medium supplemented with _0.5 mg/LNAA and 0.5 mg/LBA.

Preferably, the subculture medium is B5 medium supplemented with 0.5mg/L 2,4 _- D and 0.5mg/LNAA.

Preferably, the callus powder in the S6 is mixed with alcohol with a volume fraction of 60% in a mass ratio of 1:16.

Preferably, the S3 medium is cultured for 30 days after germination and seedlings are used for later use.

Preferably, the S5 is harvested after culturing for 20 days.

Compared with the prior art, the present invention provides a plant tissue culture method and device thereof and a production method for metabolites, which have the following beneficial effects:

1. The present invention accelerates the callus culture process by clarifying the tissue culture method of the plant, optimizes the callus culture process, and plays a role in the induction and growth of the callus by optimizing the auxin, increasing its growth metabolism. Due to the destruction of the current ecological environment and the blind collection of wild resources, many wild plants tend to be endangered, resulting in the reduction of many sources of biological natural active substances, and the chemical synthesis method is complicated and expensive due to the process. It is easy to cause new environmental pollution, and the present invention can effectively increase the production of natural active substances of plants.

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments.

In the description of the present invention, it should be understood that the terms "upper", "lower", "front", "rear", "left", "right", "top", "bottom", "inside", " The orientation or positional relationship indicated by "outside" is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the indicated device or element must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as Limitations of the present invention.

Example 1:

Plant tissue culture method and its device and production method of metabolite, comprising the following steps:

S1. Collect the young stems and leaves of the current year, cut the stems into 3-4cm long pieces, and cut the leaves into 1cm wide strips, wash them with hydrogen peroxide with a volume fraction of 2%, and wash them repeatedly with tap water, then rinse them with running water for 1.5 hours. And disinfected with 65% ethanol by volume for 40s, rinsed with sterile water for 3-4 times, then disinfected with 1g/L HgCl ₂ for 3-7min, and then rinsed with sterile water for 4-5 times for use;

S2. Select the full seeds of the current year, soak them for 22 hours, peel off the seed coats, rinse with water repeatedly, then use alcohol with a volume fraction of 70%, disinfect for 1 minute, rinse with sterile water 3-4 times, 1g/L HgCl ₂ , and disinfect for 15 minutes , rinse 4-5 times with sterile water, insert into 1/2MS medium, keep the temperature at about 22-27℃ and cultivate under natural light;

S3, cut the young stems developed in S2 into 0.7cm long sections, cut the young leaves into 0.5cm*0.5cm squares, inoculate on the spare material collected in S1, and then place them in the callus medium for callus Tissue culture;

S4, the callus induced in S3 is cultivated on the first-generation medium, and after the cultivation is completed, the callus is subcultured on the subculture medium;

S5, get the third-generation callus of the stem subculture in S4, add 30ml B ₅ medium in a 100ml tripod flask and carry out callus extraction and culture;

S6. Collect the callus cultured in S5 and place it in an oven at 80°C to dry to constant weight, crush it, put it into a flask, add alcohol with a volume fraction of 60%, condense and reflux in a 75°C water bath and extract twice, each time For 1 h, the combined filtrate was suction filtered to obtain callus metabolites.

Further, preferably, the sucrose 35g/L in the callus medium in S3 is solidified with agar 8g/L, the pH will be adjusted to 6.3 before the callus medium is autoclaved, and the temperature of the culture room is controlled at about 22-27°C , When cultivating in dark light, place the culture bottle on a non-illuminated culture rack and cover it with black cloth. When cultivating in light, the light is 12h/d, and the light intensity is 1,000-1,500lx.

Further, preferably, the first-generation medium is B5 medium, and _0.5 mg/LNAA and 0.5 mg/LBA are added.

Further, preferably, the subculture medium is B ₅ medium, and 0.5mg/L 2,4-D and 0.5mg/LNAA are added.

Further, preferably, the callus powder in S6 and the alcohol with a volume fraction of 60% are mixed in a mass ratio of 1:16.

Further, preferably, the culture in S3 is used for 30 days after germination into seedlings.

Further, preferably, it is harvested after culturing in S5 for 20 days.

Example 2: Based on Example 1, but with the following differences:

Table 1 Effects of different concentrations of 2,4-D on callus induction

Table 1 shows that callus was inoculated on MS medium with 2,4-D of 0.0, 0.1, 03, 0.5, 1.0, 2.0 and 4.0, respectively, and the callus induction experiment by plant growth regulator was carried out. After the young stems were cultured for 3 days, both ends of the young stems of each treatment began to swell and turn white. At 6 days, most of the explants began to grow callus from both ends. There was no obvious change in the control, and no callus was produced. Among the different concentrations of 2,4-D, the callus rate of 0.5 mg/L was the highest, which was 100%, and the callus growth was also the best. Too high or too low is not conducive to callus induction and growth. When the concentration of 2,4-D was 4.0 mg/L, the explants were completely inhibited from producing callus.

Example 3: Based on Examples 1 and 2, but with the following differences:

Table 2 Effects of different concentrations of NAA on callus induction

When the NAA concentration was below 2.0mg/L, the callus rate was above 90%, and there was no significant difference among the treatments, but the callus time, callus color, size and growth status after subculture were significantly different when NAA was 0.1 At -0.5mg/L, although the callus grows well, the color is dull and dull, and the growth is slow after subculture. When NAA was 1.0 mg/L, the callus grew better, and it was cream-colored, shiny and shiny, and the growth was better after transfer. When NAA is below 1.0mg/L, the time for inducing callus is basically the same, which is 7d; when NAA is 2.0mg/L, the induction rate and growth rate are good, and the growth after subculture is normal, but the callus induced is less The time required is 2-3d later than that of the concentration below 1.0mg/L; when the NAA is 4.0mg/L, the healing time is 2d later than that of 2.0mg/L. After only a small amount of callus grows, it gradually turns brown. die. The above description is only a preferred embodiment of the present invention, but the protection scope of the present invention is not limited to this. The equivalent replacement or change of the inventive concept thereof shall be included within the protection scope of the present invention.

Claims (7)

1. the tissue culture method of plant and its device and the production method of metabolite, is characterized in that, comprises the following steps: S1. Collect the young stems and leaves of the current year, cut the stems into 3-4cm long pieces, and cut the leaves into 1cm wide strips, wash them with hydrogen peroxide with a volume fraction of 2%, and wash them repeatedly with tap water, then rinse them with running water for 1.5 hours. And disinfected with 65% ethanol by volume for 40s, rinsed with sterile water for 3-4 times, then disinfected with 1g/L HgCl ₂ for 3-7min, and then rinsed with sterile water for 4-5 times for use; S2. Select the full seeds of the current year, soak them for 22 hours, peel off the seed coats, rinse with water repeatedly, then use alcohol with a volume fraction of 70%, disinfect for 1 minute, rinse with sterile water 3-4 times, 1g/L HgCl ₂ , and disinfect for 15 minutes , rinse 4-5 times with sterile water, insert into 1/2MS medium, keep the temperature at about 22-27℃ and cultivate under natural light; S3, cut the young stems developed in S2 into 0.7cm long sections, cut the young leaves into 0.5cm*0.5cm squares, inoculate on the spare material collected in S1, and then place them in the callus medium for callus Tissue culture; S4, the callus induced in S3 is cultivated on the first-generation medium, and after the cultivation is completed, the callus is subcultured on the subculture medium; S5, get the third-generation callus of the stem subculture in S4, add 30ml B ₅ medium in a 100ml tripod flask and carry out callus extraction and culture; S6. Collect the callus cultured in S5 and place it in an oven at 80°C to dry to constant weight, crush it, put it into a flask, add alcohol with a volume fraction of 60%, condense and reflux in a 75°C water bath and extract twice, each time For 1 h, the combined filtrate was suction filtered to obtain callus metabolites. 2. the tissue culture method of plant according to claim 1 and the production method of its device and metabolite, is characterized in that: in the callus medium in described S3, sucrose 35g/L is solidified with agar 8g/L, The pH of the callus medium was adjusted to 6.3 before autoclaving, and the temperature of the culture room was controlled at about 22-27°C. When cultivating in dark light, place the culture bottle on a non-illuminated culture rack and cover it with black cloth. 12h/d, light intensity 1,000-1,500lx. 3. the tissue culture method of plant according to claim 1 and its device and the production method of metabolite, it is characterized in that: described first generation medium is B ₅ medium, and add 0.5mg/LNAA and 0.5mg/LBA . 4. the tissue culture method of plant according to claim 1 and the production method of its device and metabolite, it is characterized in that: in described subculture medium is B ₅ medium, and add 0.5mg/L 2,4- D and 0.5 mg/LNAA. 5. The method for plant tissue culture and its device and the method for producing metabolites according to claim 1, wherein the callus powder in the S6 and the alcohol with a volume fraction of 60% are in a ratio of 1:16 The mass ratio is matched. 6 . The tissue culture method and device thereof according to claim 1 , and the method for producing metabolites, characterized in that: in the S3, germination and seedlings are cultivated for 30 d for later use. 7 . 7 . The method for tissue culture of plants, the device thereof, and the method for producing metabolites according to claim 1 , wherein: the S5 is cultured for 20 days and then harvested. 8 .