CN113016620B - Method for rapid propagation of ardisia crenata tissue - Google Patents
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- 241000722824 Ardisia crenata Species 0.000 title claims abstract description 56
- 238000000034 method Methods 0.000 title claims abstract description 26
- 239000001963 growth medium Substances 0.000 claims abstract description 39
- 235000013399 edible fruits Nutrition 0.000 claims abstract description 25
- 210000001161 mammalian embryo Anatomy 0.000 claims abstract description 23
- 230000001954 sterilising effect Effects 0.000 claims abstract description 21
- 230000012010 growth Effects 0.000 claims abstract description 17
- FAIXYKHYOGVFKA-UHFFFAOYSA-N Kinetin Natural products N=1C=NC=2N=CNC=2C=1N(C)C1=CC=CO1 FAIXYKHYOGVFKA-UHFFFAOYSA-N 0.000 claims abstract description 16
- QANMHLXAZMSUEX-UHFFFAOYSA-N kinetin Chemical compound N=1C=NC=2N=CNC=2C=1NCC1=CC=CO1 QANMHLXAZMSUEX-UHFFFAOYSA-N 0.000 claims abstract description 16
- 229960001669 kinetin Drugs 0.000 claims abstract description 16
- 229920001817 Agar Polymers 0.000 claims abstract description 12
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims abstract description 12
- 229930006000 Sucrose Natural products 0.000 claims abstract description 12
- 239000008272 agar Substances 0.000 claims abstract description 12
- 239000003864 humus Substances 0.000 claims abstract description 12
- 239000005720 sucrose Substances 0.000 claims abstract description 12
- 235000013575 mashed potatoes Nutrition 0.000 claims abstract description 11
- 239000000203 mixture Substances 0.000 claims abstract description 11
- 239000002689 soil Substances 0.000 claims abstract description 11
- 239000005631 2,4-Dichlorophenoxyacetic acid Substances 0.000 claims abstract description 9
- 238000012258 culturing Methods 0.000 claims abstract description 9
- HXKWSTRRCHTUEC-UHFFFAOYSA-N 2,4-Dichlorophenoxyaceticacid Chemical compound OC(=O)C(Cl)OC1=CC=C(Cl)C=C1 HXKWSTRRCHTUEC-UHFFFAOYSA-N 0.000 claims abstract description 4
- 238000004659 sterilization and disinfection Methods 0.000 claims description 29
- 238000005286 illumination Methods 0.000 claims description 14
- 210000002257 embryonic structure Anatomy 0.000 claims description 11
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 10
- 229960002523 mercuric chloride Drugs 0.000 claims description 10
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 claims description 10
- 239000003415 peat Substances 0.000 claims description 6
- 239000011159 matrix material Substances 0.000 claims description 3
- 230000004083 survival effect Effects 0.000 abstract description 7
- 239000006259 organic additive Substances 0.000 abstract description 3
- 239000005648 plant growth regulator Substances 0.000 abstract description 3
- 238000003976 plant breeding Methods 0.000 abstract description 2
- 239000000758 substrate Substances 0.000 description 18
- 238000002474 experimental method Methods 0.000 description 8
- 239000000463 material Substances 0.000 description 7
- 239000002609 medium Substances 0.000 description 6
- 241000196324 Embryophyta Species 0.000 description 5
- 238000010586 diagram Methods 0.000 description 4
- 230000003287 optical effect Effects 0.000 description 4
- 238000012360 testing method Methods 0.000 description 3
- PRPINYUDVPFIRX-UHFFFAOYSA-N 1-naphthaleneacetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CC=CC2=C1 PRPINYUDVPFIRX-UHFFFAOYSA-N 0.000 description 2
- NWBJYWHLCVSVIJ-UHFFFAOYSA-N N-benzyladenine Chemical compound N=1C=NC=2NC=NC=2C=1NCC1=CC=CC=C1 NWBJYWHLCVSVIJ-UHFFFAOYSA-N 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 230000035784 germination Effects 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 238000002054 transplantation Methods 0.000 description 2
- 208000004998 Abdominal Pain Diseases 0.000 description 1
- 241000722826 Ardisia Species 0.000 description 1
- 240000007817 Olea europaea Species 0.000 description 1
- 208000002193 Pain Diseases 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 238000000540 analysis of variance Methods 0.000 description 1
- 239000007640 basal medium Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229910052956 cinnabar Inorganic materials 0.000 description 1
- 238000013401 experimental design Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000012869 germination medium Substances 0.000 description 1
- 241000411851 herbal medicine Species 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000009331 sowing Methods 0.000 description 1
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention belongs to the technical field of plant breeding, and relates to a method for rapid propagation of ardisia crenata tissues, which comprises the following steps: sterilizing Ardisia crenata fruit, and collecting embryo; placing the seed embryo in a growth culture medium for culturing to obtain an aseptic seedling; the growth culture medium comprises a basal culture medium, mashed potato, agar, sucrose, 2,4-dichlorophenoxyacetic acid and kinetin; and (4) hardening the aseptic seedlings, and transplanting the aseptic seedlings into a mixture of a seedling culture medium and humus soil for cultivation. The growth quality of aseptic seedlings and the survival rate of aseptic seedlings of the ardisia crenata can be obviously improved by adding the organic additives and the plant growth regulators into the growth culture medium of the embryo.
Description
Technical Field
The invention belongs to the technical field of plant breeding, and particularly relates to a method for rapid propagation of ardisia crenata tissues.
Background
Ardisia crenata (scientific name: ardisia crenata Sims) is a plant of genus Ardisia of family Ardisiaceae. The ardisia crenata has strong shade resistance, the root system of the ardisia crenata is more deeply and widely distributed than that of herbaceous plants, and the higher ornamental value can be reflected by arranging ground cover objects under arbor and shrub forests. In addition, the ardisia crenata belongs to common folk Chinese herbal medicines and has higher medicinal value, the root of the ardisia crenata can be used for treating abdominal pain when being decocted and taken, and the root and the leaf can be used for dispelling wind and eliminating dampness, eliminating stasis and relieving pain, and clearing and activating the channels and collaterals.
At present, the propagation method of the ardisia crenata comprises the methods of sowing, cutting, layering, tissue culture and the like. However, the existing ardisia crenata tissue culture propagation method has the problems of low survival rate and poor seedling quality.
Disclosure of Invention
The embodiment of the invention aims to provide a method for rapid propagation of ardisia crenata tissue, aiming at solving the problems in the background art.
The embodiment of the invention is realized in such a way that the method for the rapid propagation of the ardisia crenata tissue comprises the following steps:
sterilizing Ardisia crenata fruit, and collecting embryo;
placing the seed embryo in a growth culture medium for culturing to obtain an aseptic seedling; the growth culture medium comprises a basal culture medium, mashed potato, agar, sucrose, 2,4-dichlorophenoxyacetic acid and kinetin, wherein the concentration of the mashed potato is 30-50 g/L, the concentration of the 2,4-dichlorophenoxyacetic acid is 0.5-1.5 mg/L, and the concentration of the kinetin is 0.005-0.015 mg/L;
and (4) hardening the aseptic seedlings, and transplanting the aseptic seedlings into a mixture of a seedling culture medium and humus soil for cultivation.
In a preferable embodiment of the present invention, the step of sterilizing is performed by treating with 70-80% by volume of alcohol and 0.05-0.15% by mass of mercuric chloride solution.
In another preferable embodiment of the present invention, in the step, the alcohol treatment time is 1 to 3min, and the mercuric chloride solution treatment time is 10 to 12min.
As another preferable scheme of the embodiment of the invention, the ardisia crenata fruit is ardisia crenata fruit with fruit bearing of 35-50 days.
In another preferred embodiment of the present invention, in the step, the temperature of the cultivation is 25. + -. 2 ℃, the light cycle is 10-14 h/d, and the light intensity is 2200-2800 lx.
In another preferred embodiment of the present invention, the agar is present in the growth medium at a concentration of 5 to 7g/L.
In another preferred embodiment of the present invention, the concentration of sucrose in the growth medium is 25 to 35g/L.
In another preferred embodiment of the present invention, the pH of the growth medium is 5.5 to 6.
As another preferable mode of the embodiment of the present invention, the basal medium is 1/2MS medium.
As another preferable scheme of the embodiment of the invention, the mass ratio of the seedling substrate to the humus soil is 1 (0.8-1.2).
Has the beneficial effects that:
1. the method for the rapid propagation of the ardisia crenata tissue provided by the embodiment of the invention comprises the steps of disinfection and sterilization, seed embryo taking, culture, seedling hardening, transplanting and the like, and the growth quality of aseptic seedlings can be obviously improved by adding an organic additive and a plant growth regulator into a growth culture medium of the seed embryos; in addition, the seedling culture substrate and the humus soil are used for cultivating the aseptic seedling of the ardisia crenata, so that the survival rate of the aseptic seedling of the ardisia crenata can be obviously improved.
2. The invention determines the best disinfection and sterilization process through the determination experiment of the disinfection and sterilization process as follows: 1min for 75% alcohol treatment and 10min for 0.1% mercuric chloride treatment.
3. Through the determination experiment of the sampling period of the ardisia crenata fruit, the optimal scheme is preferably as follows: the fruits bearing 35-50 days are soft, the seed embryos are easy to take out, and the fruits bearing 35-50 days are preliminarily determined in the adoption period.
4. The optimal growth medium for aseptic seedlings is determined by the formula research experiment of the growth medium, and comprises the following components: 1/2M S + 1.0mg/L2,4-D +0.01mg/L KT +40g/L mashed potato +6g/L agar +30g/L sucrose (pH = 5.8).
Drawings
FIG. 1 is a graph comparing survival rates of aseptic seedlings of Ardisia crenata transplanted to different types of substrates.
FIG. 2 is a diagram of a breeding process of the method provided in embodiment 5 of the present invention.
Detailed Description
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, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.
Example 1
The embodiment provides a method for rapid propagation of ardisia crenata tissues, which comprises the following steps:
s1, taking 35d of radix Ardisiae Crenatae as a material, carrying out disinfection and sterilization treatment on the radix Ardisiae Crenatae with alcohol with the practical volume fraction of 70% for 1min, then carrying out disinfection and sterilization treatment on the radix Ardisiae Crenatae with 0.05% mercuric chloride solution for 10-12min, and taking the embryo of the radix Ardisiae Crenatae for later use.
S2, placing the embryo in a growth culture medium for culturing for 45d to obtain a sterile seedling; wherein the culture temperature is 23 ℃, the illumination period is 10h/d, and the illumination intensity is 2200lx; in addition, the growth medium had a pH of 5.5 and comprised 1/2MS medium, 30g/L mashed potatoes, 5g/L agar, 25g/L sucrose, 0.5 mg/L2,4-dichlorophenoxyacetic acid (2,4-D) and 0.005mg/L Kinetin (KT).
And S3, hardening the aseptic seedlings for 3d, transplanting the aseptic seedlings into a mixture of a seedling culture substrate and humus soil according to a mass ratio of 1. The seedling substrate may be a commercially available substrate containing peat or the like.
Example 2
The embodiment provides a method for rapid propagation of ardisia crenata tissues, which comprises the following steps:
s1, taking 50d of bearing fruit of Ardisia crenata Kane as a material, carrying out disinfection and sterilization treatment on Ardisia crenata Kane with alcohol with the practical volume fraction of 80% for 3min, then carrying out disinfection and sterilization treatment on Ardisia crenata Kane with 0.15% mercuric chloride solution for 12min, and taking the embryo of the Ardisia crenata Kane for later use.
S2, placing the embryo in a growth culture medium for culturing for 60d to obtain a sterile seedling; wherein the culture temperature is 27 ℃, the illumination period is 14h/d, and the illumination intensity is 2800lx; in addition, the growth medium had a pH of 6 and comprised 1/2MS medium, 50g/L mashed potato, 7g/L agar, 35g/L sucrose, 1.5 mg/L2,4-dichlorophenoxyacetic acid (2,4-D) and 0.015mg/L Kinetin (KT).
And S3, hardening the aseptic seedlings for 4d, transplanting the aseptic seedlings into a mixture of a seedling culture substrate and humus soil according to a mass ratio of 1.2. The seedling substrate may be a commercially available substrate containing peat or the like.
Example 3
The embodiment provides a method for rapid propagation of ardisia crenata tissues, which comprises the following steps:
s1, taking 40 d-bearing Ardisia crenata Kao as a material, carrying out disinfection and sterilization treatment on Ardisia crenata Kao for 2min by using alcohol with the practical volume fraction of 75%, then carrying out disinfection and sterilization treatment on Ardisia crenata Kao for 11min by using 0.1% mercuric chloride solution, and taking the embryo of the Ardisia crenata Kao for later use.
S2, placing the embryo in a growth culture medium for culturing for 50d to obtain an aseptic seedling; wherein the culture temperature is 25 ℃, the illumination period is 12h/d, and the illumination intensity is 2500lx; in addition, the growth medium had a pH of 5.8 and comprised 1/2MS medium, 35g/L mashed potatoes, 5.5g/L agar, 28g/L sucrose, 0.8 mg/L2,4-dichlorophenoxyacetic acid (2,4-D) and 0.008mg/L Kinetin (KT).
S3, hardening the aseptic seedlings for 3d, and transplanting the aseptic seedlings into a mixture of a seedling culture substrate and humus soil according to the mass ratio of 1:1 for cultivation. The seedling substrate may be a commercially available substrate containing peat or the like.
Example 4
The embodiment provides a method for rapid propagation of ardisia crenata tissues, which comprises the following steps:
s1, taking Ardisia crenata Kao fruits with fruit hanging for 45d as a material, carrying out disinfection and sterilization treatment on the Ardisia crenata Kao fruits for 2min by using alcohol with the practical volume fraction of 75%, then carrying out disinfection and sterilization treatment on the Ardisia crenata Kao fruits for 10min by using 0.1% mercuric chloride solution, and then taking embryo of the Ardisia crenata Kao fruits for standby.
S2, placing the embryo in a growth culture medium for culturing for 55d to obtain a sterile seedling; wherein the culture temperature is 25 ℃, the illumination period is 12h/d, and the illumination intensity is 2500lx; in addition, the pH of the growth medium was 5.8, which included 1/2MS medium, 45g/L mashed potatoes, 6.5g/L agar, 32g/L sucrose, 1.2 mg/L2,4-dichlorophenoxyacetic acid (2,4-D) and 0.012mg/L Kinetin (KT).
S3, hardening off the aseptic seedlings for 5d, and transplanting the aseptic seedlings into a mixture of a seedling raising matrix and humus soil according to the mass ratio of 1:1 for cultivation. The seedling substrate may be a commercially available substrate containing peat or the like.
Example 5
The embodiment provides a method for rapid propagation of ardisia crenata tissue, which comprises the following steps:
s1, taking 50d of bearing fruit of Ardisia crenata Kane as a material, carrying out disinfection and sterilization treatment on Ardisia crenata Kane with alcohol with the practical volume fraction of 75% for 1min, then carrying out disinfection and sterilization treatment on Ardisia crenata Kane with 0.1% mercuric chloride solution for 10min, and taking the embryo of the Ardisia crenata Kane for later use.
S2, placing the embryo in a growth culture medium for culturing for 60d to obtain a sterile seedling; wherein the culture temperature is 25 ℃, the illumination period is 12h/d, and the illumination intensity is 2500lx; in addition, the growth medium had a pH of 5.8 and comprised 1/2MS medium, 40g/L mashed potatoes, 6g/L agar, 30g/L sucrose, 1 mg/L2,4-dichlorophenoxyacetic acid (2,4-D) and 0.01mg/L Kinetin (KT).
S3, hardening the aseptic seedlings for 3d, and transplanting the aseptic seedlings into a mixture of a seedling culture substrate and humus soil according to the mass ratio of 1:1 for cultivation. The seedling substrate may be a commercially available substrate containing peat or the like.
Experimental example:
1. determination experiment of the sterilization process:
taking the fruits in the olive period of the ardisia crenata as materials, taking MS as a seed embryo germination culture medium, treating 15 fruits each, sterilizing, taking seed embryos, inoculating 3 seed embryos to each bottle, inoculating 5 bottles in total, culturing in the dark at 25 ℃ for 3 days, observing the pollution condition, and counting, wherein the statistical result is shown in Table 1.
TABLE 1
As can be seen from table 1, the optimal sterilization process is: 1min for 75% alcohol treatment and 10min for 0.1% mercuric chloride treatment.
2. Determination experiment of Ardisia crenata fruit sampling period:
the influence of different use periods on the germination of the seed embryo is studied by taking MS + 6-benzylaminopurine (6-BA) 8.0mg/L + naphthylacetic acid (NAA) 0.5mg/L + agar 5g/L + sucrose 30g/L (pH = 5.8) as a germination medium. Each treatment was repeated 3 times with 5 flasks and 3 embryos per flask, and the results are shown in Table 2.
TABLE 2
As can be seen from Table 2, the embryos in the shade-dried mature fruits were used as controls; the fruit peel is in a light yellow stage, and the characteristics of the stage are as follows: the fruit is hard and embryo extraction is difficult. The fruits bearing 35-50 days are soft, the seed embryos are easy to take out, and the fruits bearing 35-50 days are preliminarily determined in the adoption period.
3. Growth medium formulation study experiments:
growth media was prepared according to the orthogonal experimental design. And (3) inoculating 5 bottles to each test, inoculating 3 embryos to each bottle, repeating for 3 times, and counting the plant height, the leaf number and the root number for 60d, wherein the counting results are shown in tables 3-5. Wherein, the culture conditions are as follows: the temperature is set to be (25 +/-2) ° C, the illumination period is 12h/d, and the illumination intensity is 2500lx (the culture conditions are the same in the whole experiment).
TABLE 3 orthogonal test factor horizon
TABLE 4
TABLE 5 analysis of variance
| Test No. | Plant height/cm | Number of blades | Number of root |
| 1 | 1.19±0.24 cd | 4.50±1.91 ab | 1.50±1.00 b |
| 2 | 1.19±0.52 cd | 3.50±1.91 ab | 2.00±0.82 Ab |
| 3 | 2.13±0.63 bd | 3.25±1.50 b | 3.00±1.83 Ab |
| 4 | 2.02±0.90 bd | 3.75±1.50 ab | 1.75±0.96 b |
| 5 | 3.14±1.23 Aa | 6.25±1.26 a | 6.25±0.96 Aa |
| 6 | 2.41±0.78 Aa | 4.00±1.50 ab | 2.00±0.82 b |
| 7 | 2.70±0.89 Aab | 3.25±1.89 ab | 2.75±1.71 Ab |
| 8 | 2.45±0.72 Aab | 4.00±1.50 ab | 1.75±0.96 b |
| 9 | 1.83±0.50 c | 5.00±1.91 ab | 2.75±0.96 Ab |
As can be seen from tables 3-5, the optimal growth medium for aseptic seedlings is: 1/2MS + 1.0mg/L2,4-D +0.01mg/L KT +40g/L mashed potato +6g/L agar +30g/L sucrose (pH = 5.8).
4. Hardening and transplanting experiments:
taking the aseptic seedlings cultured by the optimal aseptic seedling growth medium as materials, hardening the seedlings for 3d, and respectively transplanting the seedlings into a mixture (JZ 1) formed by mixing a seedling culture medium and humus according to a 1:1 mass ratio, a mixture (JZ 2) formed by mixing a seedling culture medium and yellow loam according to a 1:1 mass ratio and a pure seedling culture medium (JZ 3) for cultivation. Wherein 30 plants of each matrix are transplanted, 3 times of repetition are carried out, the survival rate is counted after 30 days, and the result is shown in figure 1. In fig. 1, P represents a significant difference from JZ1, P <0.05. As can be seen from FIG. 1, the hardening-seedling transplantation performed by the method provided by the embodiment of the invention has higher survival rate.
In conclusion, the embodiment of the invention can obviously improve the growth quality of aseptic seedlings by adding the organic additives and the plant growth regulator into the growth culture medium of the seed embryos; in addition, the seedling culture substrate and the humus soil are used for cultivating the aseptic seedling of the ardisia crenata, so that the survival rate of the aseptic seedling of the ardisia crenata can be obviously improved.
5. The method provided in the above example 5 is used to breed the ardisia crenata embryo, and the breeding process is shown in fig. 2. In FIG. 2, A is an optical image of Ardisia crenata seed embryos inoculated onto a growth medium; b is an optical diagram of the germinated seedling of the seed embryo after 15d of inoculation; c is an optical diagram of the ardisia crenata tissue culture seedling after 45 days of inoculation; d is the cinnabar root embryo (50 times) under the stereoscope; and E is an optical diagram of the transplanted seedling of the ardisia crenata after 30d of transplantation. Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Claims (2)
1. A method for rapid propagation of Ardisia crenata tissue is characterized by comprising the following steps:
sterilizing the ardisia crenata fruits of 35 to 50d in the fruiting period, respectively treating the ardisia crenata fruits with 75% of alcohol by volume and 0.1% of mercuric chloride solution by mass for 1min, and taking out the embryos after sterilization;
culturing the seed embryo in a growth culture medium at the temperature of 25 +/-2 ℃, the illumination period of 10-14h/d and the illumination intensity of 2200-2800lx; obtaining aseptic seedlings; the growth culture medium is 1/2MS culture medium, mashed potato with the concentration of 40g/L, agar of 6g/L, sucrose of 30g/L, 2,4-dichlorophenoxyacetic acid of 1.0mg/L and kinetin of 0.01 mg/L;
and (3) hardening the aseptic seedlings, and transplanting the aseptic seedlings into a mixture of peat-containing matrix and humus soil in a mass ratio of 1:1 for cultivation.
2. The method according to claim 1, wherein the pH of the growth medium is 5.5 to 6.
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