CN115380823A - Method for delaying subculture growth rate of tissue culture seedlings of black-bone vegetables - Google Patents
Method for delaying subculture growth rate of tissue culture seedlings of black-bone vegetables Download PDFInfo
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Abstract
The invention provides a method for delaying the subculture growth rate of tissue culture seedlings of black-boned vegetables, which comprises the following steps: shearing tender leaf stalks of the black-bone mustard into leaf stalk sections and cleaning; sterilizing the petiole segment in a sterile environment; cutting the stem segments of tender leaves and stems of the aseptic lindera aggregate into Cheng She stem blocks, and then inoculating the stem blocks on an induction culture medium for induction culture; transferring the callus to a subculture medium for subculture; meanwhile, 50-200 mg/kg of TIBA is added into a subculture medium, the culture temperature is controlled to be 10-18 ℃, the light quality is adjusted to be red and blue composite light, the red/blue light ratio is 1:3 and 1:1, and the illumination intensity is controlled to be 10-70 mu mol/(m 2 S) controlling the illumination time to be 1 day of illumination/1 to 3 days of darkness. The invention combines the red and blue light intermittent illumination, low-temperature culture and the TIBA adding method to obtain a novel method capable of effectively delaying the subculture growth rate of the tissue culture seedlings of the lindera aggregata.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for delaying the subculture growth rate of tissue culture seedlings of lindera aggregata.
Background
The black-boned vegetable is also called as the mustard, the Chinese cabbage, the collapsed pine and the black vegetable, and is a variant of brassica subspecies of brassicaceae; it has few basal leaves, dense, short and wide, and is remarkably shriveled, round oval or inverted oval; it is fed by leaves, and native China is mainly distributed in Yangtze river basin. The black-bone dish has fat and tender leaves, can be fried to eat, used as soup and cold mixed, has beautiful color, delicious taste and rich nutrition.
Plant tissue culture, also called as in vitro plant culture, refers to a technique of culturing in vitro plant organs, tissues, cells and protoplasts in a proper culture medium under sterile and artificially controlled environmental conditions to regenerate the cells or complete plants. The method for cultivating the black-boned vegetable by utilizing the plant tissue culture technology not only can ensure the purity of the black-boned vegetable strain, but also can greatly improve the propagation coefficient and shorten the outplanting period, and is more favorable for the popularization and the application of the good breed of the black-boned vegetable. Therefore, the plant tissue culture technology becomes the best choice for the commercial production of the black-bone cabbage.
However, in the industrial tissue culture process of the black cabbage, the following are found: when the bottle needs to be rotated and seedlings are transferred, due to the fact that manual labor is insufficient or the number of fields is limited, all black vegetable seedlings cannot be transplanted into the fields in a short time, waste of the seedlings with proper age due to the fact that the seedlings cannot land on the fields is caused, and economic loss is brought. Accordingly, it is desirable to provide a method for effectively delaying the subculture growth rate of the tissue culture seedlings of lindera aggregata, so as to effectively control the time of planting the seedlings with the proper age.
Light quality, light intensity, illumination time and the like are important environmental factors influencing the growth and development of plants. Research shows that different light quality conditions play an important role in regulating and controlling various stages of plant growth and development. Light Emitting Diodes (LEDs) can emit monochromatic light in a precise spectrum, with a narrow wavelength range, and can be controlled for light intensity by a control device. The red light can promote the radial growth of plant stems and leaves, is favorable for the accumulation of soluble sugar and starch, and reduces the pigment content. The blue light can reverse the effect, promote the synthesis of pigment and soluble protein, facilitate the transverse growth of stem and leaf, and make the plant incapable of growing normally only under the condition of full red light. The combined red and blue light is beneficial to the growth of plants, and the openness degree and the light energy conversion efficiency of a photosystem reaction center are enhanced. However, the current research focuses on the species with relatively mature rapid propagation system, and the application of delaying tissue culture subculture by using the ratio of red to blue, especially illumination and time, in the tissue culture of the brassica oleracea is not reported.
The leaves are important places for plants to utilize light energy and synthesize organic compounds, and play an important role in the growth and development process of the plants. The aging of leaves is an extremely complex process, and influences the delay of the shelf life of leaf vegetables, the reduction of the artificial pressure for transplanting tissue culture seedlings and the maintenance of the excellent properties of single plants of the tissue culture seedlings. The low temperature can reduce the photosynthetic rate and the respiratory rate, so that the growth of the plants is delayed.
Triiodobenzoic acid (TIBA) is a growth inhibitor, also known as a growth retardant, which is a type of plant growth regulator that is a synthetic or natural substance that blocks the growth of the entire plant or a particular organ of the plant. The chemical structure of TIBA is similar to that of auxin, and the TIBA mainly generates an effect opposite to that of the auxin through competitive inhibition, is beneficial to reducing the plant height and lodging and also has the effect of inhibiting the apical growth.
Therefore, if the combined method of red and blue light intermittent illumination, moderate low temperature and TIBA added in the culture medium is tried to be used for tissue culture of the lindera aggregata, a new method capable of effectively delaying the subculture growth rate of the tissue culture seedlings of the lindera aggregata is expected to be obtained.
Disclosure of Invention
The invention aims to provide a method for delaying the subculture growth rate of tissue culture seedlings of lindera aggregata by simultaneously using red and blue light intermittent illumination, moderate low temperature and a culture medium to add TIBA.
The invention adopts the following technical scheme to solve the technical problems:
a method for delaying the subculture growth rate of tissue culture seedlings of black-bone vegetables comprises the following steps:
(1) Selection of explants
Shearing tender leaf stalks of the black-bone mustard into leaf stalk sections and cleaning;
(2) Disinfection of explants
Placing the cleaned tender leaf stem segments of the black-bone cabbage in a sterile environment for disinfection;
(3) Induced culture
Cutting the tender leaf and stem segments of the aseptic lindera aggregata into Cheng She stem blocks, and then inoculating the stem blocks to an induction culture medium to perform induction culture of callus;
(4) Subculture
Transferring the cultured callus to a subculture medium for subculture; meanwhile, 50-200 mg/kg of TIBA is added into the subculture medium, the culture temperature is controlled to be 10-18 ℃, the light quality is adjusted to be red and blue composite light, the red/blue light ratio is 1:3 and 1:1, and the illumination intensity is controlled to be 10-70 mu mol/(m 2 S) controlling the illumination time to be 1 day of illumination/1 to 3 days of darkness.
In the step (1), the tender leaf stalks of the lindera aggregate collected from the field are cut into leaf stalk sections, then the leaf stalk sections are put into a wide-mouth bottle, and the wide-mouth bottle is washed for 20min by using running water, then washed for 10min by using 0.05% anli washing liquid in a shaking way, and washed by using tap water until no foam exists, and then the wide-mouth bottle is regarded as being cleaned.
In a preferred embodiment of the present invention, in the step (2), the washing is performedAfter the clean tender leaf stem segments of the black-bone cabbage are moved to a super clean workbench, the leaves and stem segments are firstly washed by sterile water for 4 times, then the leaves and stem segments are poured into 70 to 75 percent ethanol for sterilization for 10s, the leaves and stem segments are washed by sterile water for 2 to 3 times, and the content of HgCl is 0.1 percent 2 The solution was sterilized by shaking for 30s, followed by washing with sterile water 6 times by shaking.
In a preferred embodiment of the present invention, in the step (3), the sterile tender leaf and stem of lindera aggregata is cut into She Bingkuai with a size of 0.2cm, and then inoculated into an induction medium to induce and culture callus.
In a preferred embodiment of the present invention, in the step (3), the formulation of the induction medium is: MS +30g/L sucrose +5.5g/L agar, pH 5.8-6.0; the induction culture conditions are as follows: the culture temperature is 25 ℃, the light quality is a fluorescent lamp, the illumination time is 14h day/10 h night, and the illumination intensity is 70 mu mol/(m) 2 ·s)。
In a preferred embodiment of the present invention, in the step (4), the formulation of the subculture medium without TIBA addition is: MS +30g/L sucrose +5.5g/L agar, pH 5.8-6.0.
In a preferred embodiment of the present invention, in the step (4), the concentration of the added TIBA in the secondary culture medium is specifically 100mg/kg.
In a preferred embodiment of the present invention, in the step (4), the subculture temperature is specifically 18 ℃.
In a preferred embodiment of the present invention, in the step (4), the red/blue light ratio of the red-blue composite light is 1:3, and the illumination intensity is 70 μmol/(m) 2 ·s)。
In a preferred embodiment of the present invention, in the step (4), the light time of the red and blue composite lights is specifically 1 day of light/2 days of dark.
The technical principle is as follows:
light quality, light intensity, illumination time and the like are important environmental factors influencing the growth and development of plants. The red light can promote the radial growth of plant stems and leaves, is beneficial to the accumulation of soluble sugar and starch, reduces the pigment content, and the blue light can reverse the effect, promote the synthesis of pigment and soluble protein and is beneficial to the transverse growth of the stems and leaves. The low temperature can reduce the photosynthetic rate and the respiratory rate, so that the growth of the plants is delayed. Triiodobenzoic acid (TIBA) is similar to auxin in chemical structure, and through competitive inhibition, produces the action opposite to auxin, is favorable to reducing plant height and lodging, and also has the action of inhibiting tip growth.
Compared with the prior art, the invention has the advantages that:
(1) The invention combines the red and blue light intermittent illumination, moderate low temperature and the method of adding TIBA in the culture medium, and is used for the tissue culture of the black-boned vegetable, thus obtaining a new method which can effectively delay the subculture growth rate of the tissue culture seedling of the black-boned vegetable; the method can prolong the subculture growth time of the tissue culture seedlings of the black-bone mustard by 142% to the maximum;
(2) The method uses trace amount of reagents, is efficient and is simple to operate.
Detailed Description
The following examples are given for the detailed implementation and the specific operation procedures, but the scope of the present invention is not limited to the following examples.
Example 1
The method for delaying the subculture growth rate of the tissue culture seedlings of the lindera aggregate comprises the following steps:
(1) Selection of explants
The tender leaf stalks of the black-boned cabbage collected from the field are cut into leaf stalk sections and then placed into a wide-mouth bottle, and the leaves are washed for 20min by using running water, then washed for 10min by using 0.05% anli washing liquid in a vibration mode and washed by using tap water until no foam exists.
(2) Disinfection of explants
Transferring cleaned tender leaf and stem of caulis et folium Brassicae campestris to super clean bench, washing with sterile water for 4 times, adding 70% ethanol, sterilizing for 10s, washing with sterile water for 2 times, and processing with 0.1% of HgCl 2 The solution was sterilized by shaking for 30s, followed by washing 6 times with sterile water.
(3) Induced culture
Cutting the tender leaf and stem segments of the aseptic lindera aggregata into She Bingkuai with the size of 0.2cm, and then inoculating the tender leaf and stem segments of the aseptic lindera aggregata to an induction culture medium to perform induction culture of callus. Induction cultureThe formula of the nutrient medium is as follows: MS +30g/L sucrose +5.5g/L agar, pH 5.8; the induction culture conditions are as follows: the culture temperature is 25 ℃, the light quality is a fluorescent lamp, the illumination time is 14h day/10 h night, and the illumination intensity is 70 mu mol/(m) 2 ·s)。
(4) Subculture
Transferring the cultured callus onto a subculture medium for subculture, wherein the subculture medium comprises the following formula: MS +30g/L sucrose +5.5g/L agar, pH 5.8; meanwhile, 50mg/kg of TIBA is added into a subculture medium, the culture temperature is controlled to be 10 ℃, the light quality is adjusted to be red-blue composite light, the red-blue light ratio is 1:3, and the illumination intensity is controlled to be 10 mu mol/(m) 2 S), controlling the illumination time to be 1 day of illumination/1 day of darkness.
Example 2
The method for delaying the subculture growth rate of the tissue culture seedlings of the lindera aggregate comprises the following steps:
(1) Selection of explants
The tender leaf stalks of the black-boned cabbage collected from the field are cut into leaf stalk sections and then placed into a wide-mouth bottle, and the leaves are washed for 20min by using running water, then washed for 10min by using 0.05% anli washing liquid in a vibration mode and washed by using tap water until no foam exists.
(2) Disinfection of explants
Transferring cleaned tender leaf stem of caulis et folium Brassicae Capitatae to superclean bench, washing with sterile water for 4 times, adding 72% ethanol for sterilization for 10s, washing with sterile water for 3 times, and processing with 0.1% of HgCl 2 The solution was sterilized by shaking for 30s, followed by washing 6 times with sterile water.
(3) Induced culture
Cutting the tender leaf and stem segments of the aseptic lindera aggregata into She Bingkuai with the size of 0.2cm, and then inoculating the tender leaf and stem segments of the aseptic lindera aggregata to an induction culture medium to perform induction culture of callus. The formula of the induction culture medium is as follows: MS +30g/L sucrose +5.5g/L agar, pH5.9; the induction culture conditions are as follows: the culture temperature is 25 ℃, the light quality is a fluorescent lamp, the illumination time is 14h day/10 h night, and the illumination intensity is 70 mu mol/(m) 2 ·s)。
(4) Subculture
Transferring the cultured callus onto subculture medium for subcultureThe formula of the subculture medium is as follows: MS +30g/L sucrose +5.5g/L agar, pH5.9; meanwhile, 200mg/kg of TIBA is added into the subculture medium, the culture temperature is controlled to be 15 ℃, the light quality is adjusted to be red-blue composite light, the red-blue light ratio is 1:1, and the illumination intensity is controlled to be 50 mu mol/(m) 2 S), controlling the illumination time to be 1 day of illumination/3 days of darkness.
Example 3
The method for delaying the subculture growth rate of the tissue culture seedlings of the lindera aggregate comprises the following steps:
(1) Selection of explants
The tender leaf stalks of the black-boned cabbage collected from the field are cut into leaf stalk sections and then placed into a wide-mouth bottle, and the leaves are washed for 20min by using running water, then washed for 10min by using 0.05% anli washing liquid in a vibration mode and washed by using tap water until no foam exists.
(2) Disinfection of explants
Transferring cleaned tender leaf stem of caulis et folium Brassicae Capitatae to superclean bench, washing with sterile water for 4 times, adding 75% ethanol for sterilization for 10s, washing with sterile water for 2 times, and processing with 0.1% of HgCl 2 The solution was sterilized by shaking for 30s, followed by washing 6 times with sterile water.
(3) Induced culture
Cutting the tender leaf and stem segments of the aseptic lindera aggregata into She Bingkuai with the size of 0.2cm, and then inoculating the tender leaf and stem segments of the aseptic lindera aggregata to an induction culture medium to perform induction culture of callus. The formula of the induction culture medium is as follows: MS +30g/L sucrose +5.5g/L agar, pH 6.0; the induction culture conditions are as follows: the culture temperature is 25 ℃, the light quality is a fluorescent lamp, the illumination time is 14h day/10 h night, and the illumination intensity is 70 mu mol/(m) 2 ·s)。
(4) Subculture
Transferring the cultured callus onto a subculture medium for subculture, wherein the subculture medium comprises the following formula: MS +30g/L sucrose +5.5g/L agar, pH 6.0; meanwhile, 100mg/kg of TIBA is added into the subculture medium, the culture temperature is controlled to be 18 ℃, the light quality is adjusted to be red-blue composite light, the red-blue light ratio is 1:3, and the illumination intensity is controlled to be 70 mu mol/(m) 2 S), controlling the illumination time to be 1 day of illumination/2 days of darkness.
Example 4
This example was used to verify the effect of different culture temperatures, light quality, illumination time, illumination intensity and different TIBA concentrations in the medium on the subculture time of the brassica campestris:
the experimental method comprises the following steps:
(1) The tender leaf stalks of the black-boned cabbage collected from the field are cut into leaf stalk sections and then placed into a wide-mouth bottle, and the leaves are washed for 20min by using running water, then washed for 10min by using 0.05% anli washing liquid in a vibration mode and washed by using tap water until no foam exists.
(2) Transferring cleaned tender leaf stem of caulis et folium Brassicae Capitatae to superclean bench, washing with sterile water for 4 times, adding 75% ethanol for sterilization for 10s, washing with sterile water for 2 times, and processing with 0.1% of HgCl 2 The solution was sterilized by shaking for 30s, followed by washing with sterile water 6 times by shaking.
(3) Cutting the tender leaf and stem segments of the aseptic lindera aggregata into She Bingkuai with the size of 0.2cm, and then inoculating the tender leaf and stem segments of the aseptic lindera aggregata to an induction culture medium to perform induction culture of callus. The formula of the induction culture medium is as follows: MS +30g/L sucrose +5.5g/L agar, pH 6.0; the induction culture conditions are as follows: the culture temperature is 25 ℃, the light quality is fluorescent lamp, the illumination time is 14h day/10 h night, and the illumination intensity is 70 mu mol/(m) 2 ·s)。
(4) Subculture
Transferring the cultured callus to a subculture medium (the formula of the subculture medium is MS +30g/L sucrose +5.5g/L agar, and the pH value is 6.0) for subculture. The step is divided into 5 experimental groups for respective cultivation, and the grouping conditions are as follows:
experimental group 1: 50, 100, 150 and 200mg/kg of TIBA were added to the subculture medium, respectively, but the other culture conditions were consistent: the culture temperature is 25 deg.C, the light quality is fluorescent lamp, the illumination time is 14h/10h (day/night), and the illumination intensity is 70 μmol/(m) 2 ·s)。
Experimental group 2: the culture temperatures were set to 10, 14, 18, 22, 26 ℃ respectively, but other culture conditions were identical: the culture medium is not added with TIBA, the light quality is fluorescent lamp, the illumination time is 14h/10h (day/night), and the illumination intensity is 70 mu mol/(m) 2 ·s)。
Experimental group 3: time division of illuminationThe culture medium is respectively 14h/10h (day/night), 2 days of illumination + 1 day of darkness, 3 days of illumination + 1 day of darkness, 1 day of illumination + 2 days of darkness, and 1 day of illumination +3 days of darkness, but other culture conditions are consistent: no TIBA was added to the culture medium, the culture temperature was 25 deg.C, the light quality was fluorescent lamp, and the illumination intensity was 70. Mu. Mol/(m) 2 ·s)。
Experimental group 4: the light quality is fluorescent lamp or red and blue light with the proportion of 1:3, 3:7, 1:1, 7:3 and 3:1 respectively, and the illumination intensity is 10, 40, 70 and 100 mu mol/(m mol) 2 S), but other culture conditions were consistent: no TIBA was added to the medium, and the culture temperature was 25 ℃ and the light irradiation time was 14h/10h (day/night).
Experimental group 5: the culture temperature is 18 or 25 ℃, the light quality is fluorescent lamp, 1:3 red blue light, red light or blue light, the concentration of TIBA is 0 or 100, but other culture conditions are consistent: the illumination intensity is 70 mu mol/(m) 2 S) and a light time of 14h/10h (day/night).
The experimental results are as follows:
experimental group 1: as a result, as shown in Table 1, the number of subculture days was delayed by 42.07% in the medium supplemented with 100mg/kg of TIBA as compared with the medium supplemented with no TIBA; the secondary culture media of 150, 200 and 50mg/kg TIBA were delayed by 29.91%, 22.05% and 16.11% of the subculture days, respectively.
TABLE 1 Effect of different TIBA concentrations on the duration of subculture
Experimental group 2: the results are shown in table 2, the tissue culture seedlings are cultured at the culture temperature within the range of 10-26 ℃, the subculture time is shorter when the temperature is higher, and the subculture delaying effect is better when the temperature is lower; however, compared with the tissue culture seedling cultured under the condition of 22 ℃, the dry matter content of five true leaves of the tissue culture seedling cultured under the temperature of 18 ℃ is the highest, which is improved by 5.74 percent, and the dry matter content is reduced at the temperature of 10 ℃, 14 and 26 ℃. Overall, the best results were obtained with prolonged subculture days at 18 ℃ and better growth status.
TABLE 2 Effect of different temperatures on the duration of subculture
| Temperature (. Degree. C.) | Subculture time (sky) | Dry matter/g |
| 10 | 53.63 | 1.79 |
| 14 | 42.48 | 1.81 |
| 18 | 32.79 | 2.09 |
| 22 | 20.15 | 1.97 |
| 26 | 19.13 | 2.14 |
Experimental group 3: the results are shown in table 3, and compared with 14h/10h (day/night), the effect of delaying the subculture time in 1 day of light + 2 days of darkness is best, the time is prolonged by 29.35%, and then in 1 day of light +3 days of darkness, 1 day of light + 1 day of darkness are delayed by 16.75% and 15.85%, respectively, and the subculture time in 2 days of light + 1 day of darkness and in 3 days of light + 1 day of darkness is shortened by 11.70% and 15.95%. For dry matter accumulation, there were increases of 3.14%, 5.66% and 1.26% in light for 3 days + dark for 1 day, light for 1 day + dark for 1 day, and light for 1 day + dark for 3 days, respectively. In conclusion, the subculture time delay effect is the best when the plants are illuminated for 1 day and dark for 2 days, and at the moment, more dry matters are accumulated on the plants, so that the growth and development of the plants are promoted.
TABLE 3 Effect of different illumination times on the duration of subculture
Experimental group 4: the results are given in Table 4, and 70. Mu. Mol/(m) 2 S) light intensity of 10, 40. Mu. Mol/(m) 2 S) can be extended by 47.60% and 17.80% of the subculture cycle, respectively, 100. Mu. Mol/(m) 2 S) can shorten the subculture period by 13.60%. In the same way, the concentration is 70 [ mu ] mol/(m) 2 S) light quality of light intensity, compared with fluorescent lamp irradiation, the red and blue composite light proportion is 1:3, the delay effect is the best, the delay effect is prolonged by 25.70%, 1:1 is prolonged by 11.70%, and 3:1 is shortened by 4.55% of the relay period. As a result, 70. Mu. Mol/(m) 2 S) the red and blue composite light with the light intensity ratio of 1:3 has the best successive transfer time delay effect.
TABLE 4 Effect of different illumination intensities and light qualities on the subculture time
Experimental group 5: as shown in Table 5, the "proper low temperature + red and blue intermittent light + addition of TIBA at proper concentration" extended the subculture period compared to "culture at 25 ℃ + fluorescent light + without addition of TIBA". Wherein, under the temperature of 18 ℃, the red and blue compound light proportion is 1:3, and the TIBA concentration in the culture medium is 100mg/kg, the subculture period prolonging effect is the best, and the average subculture time is 47.92 days, which is 142% longer than the control time of 19.84 days.
TABLE 5 Effect of different temperatures, light quality and TIBA concentrations on the subculture time
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (10)
1. A method for delaying the subculture growth rate of tissue culture seedlings of black-bone vegetables is characterized by comprising the following steps:
(1) Selection of explants
Shearing tender leaf stalks of the black-bone mustard into leaf stalk sections and cleaning;
(2) Disinfection of explants
Placing the cleaned tender leaf stem segments of the black-bone cabbage in a sterile environment for disinfection;
(3) Induced culture
Cutting the tender leaf and stem segments of the aseptic lindera aggregata into Cheng She stem blocks, and then inoculating the stem blocks to an induction culture medium to perform induction culture of callus;
(4) Subculture
Transferring the cultured callus to a subculture medium for subculture; meanwhile, 50-200 mg/kg of TIBA is added into the subculture medium, the culture temperature is controlled to be 10-18 ℃, the light quality is adjusted to be red and blue composite light, the red/blue light ratio is 1:3 and 1:1, and the illumination intensity is controlled to be 10-70 mu mol/(m 2 S) controlling the illumination time to be 1 day of illumination/1 to 3 days of darkness.
2. The method for delaying the subculture growth rate of tissue culture seedlings of lindera aggregata according to claim 1, wherein in the step (1), tender leaf stalks of the lindera aggregata collected from the field are cut into leaf stalk sections and then placed in a wide-mouth bottle, and the leaves are washed for 20min by using running water, then washed for 10min by using 0.05% anli washing liquid in a shaking way, and then washed by using tap water until no foam exists, and then the leaves are regarded as clean.
3. The method for delaying the subculture growth rate of tissue culture seedlings of lindera aggregata according to claim 1, wherein in step (2), the cleaned tender leaf stem segments of lindera aggregata are moved to an ultra-clean bench, washed with sterile water for 4 times, poured into 70% -75% ethanol for sterilization for 10s, washed with sterile water for 2-3 times, and subjected to 0.1% HgCl 2 The solution was sterilized by shaking for 30s, followed by washing with sterile water 6 times by shaking.
4. The method for delaying the subculture growth rate of tissue culture seedlings of lindera aggregata according to claim 1, wherein in step (3), the sterile tender leaf stem sections of the lindera aggregata are cut into She Bingkuai with the size of 0.2cm, and then inoculated onto an induction medium for induction culture of callus.
5. The method for delaying the subculture growth rate of tissue culture seedlings of lindera aggregata according to claim 1, wherein in the step (3), the formula of the induction medium is as follows: MS +30g/L sucrose +5.5g/L agar, pH5.8-6.0; the induction culture conditions are as follows: the culture temperature is 25 ℃, the light quality is fluorescent lamp, the illumination time is 14h day/10 h night, and the illumination intensity is 70 mu mol/(m) 2 ·s)。
6. The method for delaying the subculture growth rate of tissue culture seedlings of lindera aggregata according to claim 1, wherein in the step (4), the formula of the subculture medium without adding TIBA is as follows: MS +30g/L sucrose +5.5g/L agar, pH 5.8-6.0.
7. The method for delaying the subculture growth rate of tissue culture seedlings of lindera aggregate according to claim 1, wherein in the step (4), the concentration of TIBA added to the subculture medium is specifically 100mg/kg.
8. The method for delaying the subculture growth rate of tissue culture seedlings of lindera aggregata according to claim 1, wherein the subculture temperature in the step (4) is specifically 18 ℃.
9. The method for delaying the subculture growth rate of tissue culture seedlings of lindera aggregata according to claim 1, wherein in the step (4), the red-blue light ratio of the red-blue composite light is 1:3, and the illumination intensity is 70 μmol/(m) 2 ·s)。
10. The method for delaying the subculture growth rate of tissue culture seedlings of lindera aggregata according to claim 1, wherein in the step (4), the illumination time of red and blue compound light is specifically illumination 1 day/darkness 2 days.
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