CN110896860B - Efficient brocade rhododendron direct somatic embryogenesis method - Google Patents

Efficient brocade rhododendron direct somatic embryogenesis method Download PDF

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CN110896860B
CN110896860B CN201911216208.6A CN201911216208A CN110896860B CN 110896860 B CN110896860 B CN 110896860B CN 201911216208 A CN201911216208 A CN 201911216208A CN 110896860 B CN110896860 B CN 110896860B
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rhododendron
somatic embryogenesis
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brocade
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CN110896860A (en
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魏翔莺
张春英
郑秀霞
穆景利
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Minjiang University
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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

Abstract

The invention discloses a high-efficiency rhododendron brocade direct somatic embryogenesis method, and belongs to the technical field of rapid plant propagation and regeneration. The invention establishes a high-efficiency somatic embryogenesis system of rhododendron micranthum for the first time, takes the leaves of the seedling of the rhododendron micranthum as explants for somatic cell direct embryogenesis to regenerate plants, and is an asexual propagation mode for efficiently obtaining a large amount of tissue culture seedlings of the rhododendron micranthum. The invention perfects the tissue culture and rapid propagation system of the rhododendron evergreen subgenus plant, and solves the problems of long time consumption, easy variation generation and low cuttage propagation coefficient in the traditional sowing. The method can rapidly propagate a large number of plants with stable characters, rapidly establish the clone of a good single plant, and have important application values in the aspects of brocade rhododendron seedling production, germplasm innovation and new variety breeding.

Description

Efficient brocade rhododendron direct somatic embryogenesis method
Technical Field
The invention belongs to the technical field of rapid propagation and regeneration of plants, and particularly relates to a high-efficiency brocade rhododendron direct somatic embryogenesis method.
Background
The rhododendron micranthum serves as an important parent of the plant of the Ericaceae, has high ornamental value, is extremely cold-resistant and has fragrance. The traditional propagation mode is seed sowing or stem cutting, but the breeding period is long and the efficiency is low, so the regeneration of plants through isolated culture is a method for efficiently and quickly propagating a large number of plants. The rhododendron micranthum is a woody plant, and the plant body contains more polyphenols which can block cell division, so that the establishment of a regeneration system is difficult, and a scheme for regeneration through somatic embryogenesis is not established.
The embryonic cells have strong capacity of receiving exogenous DNA, are ideal genetic transformation competent cells, most somatic embryos developed from the embryonic cells with the characteristics of egg cells are of single cell origin, and transgenic plants obtained by transformation have few chimeras, and are the most ideal genetic transformation receptor system. Therefore, the establishment of the somatic embryogenesis system has important application value in the aspects of brocade rhododendron seedling production, germplasm innovation and new variety breeding.
Disclosure of Invention
The invention aims to provide a high-efficiency rhododendron brocade direct somatic embryogenesis method aiming at the problems that the conventional seed propagation and cutting propagation time of rhododendron brocade is slow and variation is easy to generate.
In order to achieve the purpose, the invention adopts the following technical scheme:
a high-efficiency brocade rhododendron direct somatic embryogenesis method comprises the following steps:
(1) preparing sterile seeding seedlings of rhododendron micranthum: the seeds of the rhododendron yunnanensis are sown in a culture medium after the surfaces of the seeds are disinfected, the temperature is 25 ℃, and the illumination intensity is 80mol.m-2.s-1Culturing in the tissue culture room for 2-3 months to obtain sterile seeding seedlings of the rhododendron micranthum for later use;
(2) induction of leaf somatic embryos: placing 2-4 young leaves at the top end of the rhododendron brocade seedling prepared in the step (1) on wet sterile filter paper in a super-clean workbench, cutting two ends of each leaf with a blade, enabling the front side to face upwards, flatly placing the leaves on the surface of a prepared somatic embryogenesis induction culture medium, placing four leaves on each flat plate, sealing with a sealing film, and culturing in the dark for 2 weeks at the temperature of 25 ℃; then placing the mixture in a culture room for culture at the temperature of 25 ℃ for 16h in light and for 8h in dark, wherein the light intensity is 80 lmol m-2s-1Culturing for 80-100 days; during which the process of leaf somatic embryogenesis was observed, for four typical stages of somatic embryogenesis: shooting and recording spherical embryos, heart-shaped embryos, torpedo-shaped embryos and cotyledon-shaped embryos, and counting the somatic embryo incidence rate, the transformation rate and the average seedling number of each explant;
(3) transplanting and light water management: peeling off the plantlets obtained in the step (2) from the explants, transplanting the plantlets into a plug tray, performing spray culture, stopping spraying after 20 days with the humidity of 80-90%, and keeping good ventilation and shading treatment; the water and fertilizer management is to pour water 2-3 times a week and pour 20-20-20 water-soluble fertilizer (with the concentration of 200 ppm) once a week.
The culture medium in the step (1) comprises the following components: 1/2 ER culture medium, 15g/L sucrose and 8g/L agar, and has pH of 5.0-5.2.
In the step (2), the preparation method of the somatic embryogenesis induction medium comprises the following steps: woody plant culture medium WPM +30g/L sucrose +8g/L agar, pH value is adjusted to 5.0-5.2, high pressure is carried out for 30min at 121 ℃, and after cooling to 50-60 ℃, plant growth regulator which is filtered and sterilized is added: TDZ (thidiazuron) 0.5 mg/L and NAA (naphthylacetic acid) 0.2 mg/L, the medium was dispensed into petri dishes and solidified for use (25 ml per petri dish).
The formula of the ER culture medium is as follows: NH (NH)4NO3 400 mg/L； (NH4)2SO4 132 mg/L，H3BO3 6.2 mg/L，CaCl2 332.2 mg/L，CoCl2 . 6H2O 0.025mg/L，CuSO4 . 5H2O 0.025mg/L，DTPA 39.34 mg/L，FeSO4.7H2O 27.8 mg/L，MgSO4 180.8 mg/L，MnSO4 . H2O 16.9 mg/L，Na2MoO4 . 2H2O 0.25 mg/L，C6H12O6 100 mg/L，KNO3 202 mg/L，KH2PO4 408 mg/L， C12H17ClN4OS . HCl 0.4 mg/L，ZnSO4 . 7H2O8.6 mg/L, agar 8g/L, sucrose 30g/L, and pH 5.0-5.2.
The woody plant culture medium WPM comprises the following components in percentage by weight: NH (NH)4NO3 400 mg/L，H3BO3 6.2 mg/L，CaCl272.47 mg/L，Ca(NO3)2 . 4H2O 556 mg/L，CuSO4 0.16 mg/L，C10H14N2Na2O8.2H2O 37.3 mg/L， FeSO4.7H2O 27.85 mg/L，MgSO4 180.7 mg/L，MnSO4 . H2O 22.3 mg/L，Na2MoO4 . 2H2O 0.25 mg/L，KH2PO4 170 mg/L，K2SO4 990 mg/L，ZnSO4.7H28.6mg/L of agar 8g/L, 30g/L of cane sugar and 5.0-5.2 of PH.
Filling a culture medium in the hole tray in the step (3), and transplanting after completely pouring the culture medium with a nutrient solution; the preparation method of the culture medium comprises the following steps: sand is used firstWashing several times with the running water, then soaking three days with the running water, changing 3~5 times water in the middle, air-dry for later use at last, press 2 with grass peat and prepared sand: 1, mixing and subpackaging in a standard plug tray with 96 holes; the formula of the nutrient solution is as follows: CaCl2·2H2O 0.05g/L, NaCl 0.025g/L, MgSO4·7H2O 0.15g/L,（NH4）2HPO4 0.25g/L, KH2PO40.5g/L, 0.2g/L of citric acid, 10.1mg/L of vitamin B, 15g/L of glucose and 20g/L of agar, and adjusting the pH value to 5.0-5.2.
The invention has the advantages that:
1. establishes a high-efficiency brocade rhododendron somatic embryogenesis system, and has high propagation coefficient and stable character. Solves the problems of separation of seed propagation characters and difficult cuttage rooting.
2. The invention perfects the rhododendron tissue culture rapid propagation system, the survival rate of the tissue culture seedlings after transplantation reaches more than 90%, and the seedlings do not need to undergo the rooting stage in the tissue culture bottle, thereby greatly shortening the tissue culture period, saving time and space, greatly reducing cost and having important significance for improving the tissue culture industrialization level.
Drawings
FIG. 1 shows four stages of somatic embryogenesis of Rhododendron micranthum. A: a spherical tire stage; b: a heart-shaped embryo stage; c: torpedo shaped embryo stage; d: cotyledonary embryo stage.
FIG. 2 somatic embryos are transplanted to the greenhouse after seedling establishment. A: single leaf edge production of somatic embryos; b: somatic embryos generated by a single leaf form seedlings to generate plantlets; c.: single plantlet stripped off after somatic embryo seedling formation; d: transplanting the plantlets after the somatic embryos become seedlings to the plug tray.
Detailed Description
In order to make the present invention more comprehensible, the technical solutions of the present invention are further described below with reference to specific embodiments, but the following examples are only examples of the present invention and do not represent the scope of the present invention defined by the claims.
Culture medium:
the formula of the ER culture medium is as follows: NH (NH)4NO3 400 mg/L； (NH4)2SO4 132 mg/L，H3BO3 6.2 mg/L，CaCl2 332.2 mg/L，CoCl2 . 6H2O 0.025mg/L，CuSO4 . 5H2O 0.025mg/L，DTPA 39.34 mg/L，FeSO4.7H2O 27.8 mg/L，MgSO4 180.8 mg/L，MnSO4 . H2O 16.9 mg/L，Na2MoO4 . 2H2O 0.25 mg/L，C6H12O6 100 mg/L，KNO3 202 mg/L，KH2PO4 408 mg/L， C12H17ClN4OS . HCl 0.4 mg/L，ZnSO4 . 7H2O8.6 mg/L, agar 8g/L, sucrose 30g/L, and pH 5.0-5.2.
The WPM culture medium comprises the following components: NH (NH)4NO3 400 mg/L，H3BO3 6.2 mg/L，CaCl2 72.47 mg/L，Ca(NO3)2 . 4H2O 556 mg/L，CuSO4 0.16 mg/L，C10H14N2Na2O8.2H2O 37.3 mg/L， FeSO4.7H2O 27.85 mg/L，MgSO4 180.7 mg/L，MnSO4 . H2O 22.3 mg/L，Na2MoO4 . 2H2O 0.25 mg/L，KH2PO4 170 mg/L，K2SO4 990 mg/L，ZnSO4.7H28.6mg/L of agar 8g/L, 30g/L of cane sugar and 5.0-5.2 of PH.
Example 1
A high-efficiency brocade rhododendron direct somatic embryogenesis method comprises the following steps:
(1) preparing sterile seeding seedlings of rhododendron micranthum: the seeds of the rhododendron yunnanensis are sown in a culture medium after the surfaces of the seeds are disinfected, the temperature is 25 ℃, and the illumination intensity is 80mol.m-2.s-1Culturing in the tissue culture room for 3 months to obtain sterile seeding seedlings of the rhododendron micranthum for later use;
(2) induction of leaf somatic embryos: in a clean bench, taking the step (1)) Placing 2 young leaves at the top end of the prepared brocade rhododendron seedling on wet sterile filter paper, cutting two ends of each leaf by a blade, enabling the front side to face upwards, horizontally placing the leaves on the surface of a prepared somatic embryogenesis induction culture medium, placing four leaves on each flat plate, sealing the flat plate by a sealing film, and culturing in the dark for 2 weeks at the temperature of 25 ℃; then placing the mixture in a culture room for culture at the temperature of 25 ℃ for 16h in light and for 8h in dark, wherein the light intensity is 80 lmol m-2s-1Culturing for 80 d; during which the process of leaf somatic embryogenesis was observed, for four typical stages of somatic embryogenesis: photographing and recording spherical embryos, heart-shaped embryos, torpedo-shaped embryos and cotyledon-shaped embryos (figure 1), and counting the somatic embryogenesis rate, the transformation rate and the average seedling number of each explant;
(3) transplanting and light water management: stripping the plantlet (figure 2B) of the somatic embryo seedling in the step (2) from the explant (figure 2C), transplanting the plantlet to a plug (figure 2D), performing spraying treatment, culturing for 20D, stopping spraying, and keeping good ventilation and shading treatment; the water and fertilizer management comprises watering 3 times a week, and watering once a week with 20-20-20 water soluble fertilizer (concentration of 200 ppm).
The culture medium in the step (1) comprises the following components: 1/2 ER medium +15g/L sucrose +8g/L agar, pH 5.0.
In the step (2), the preparation method of the somatic embryogenesis induction medium comprises the following steps: woody plant culture medium WPM, +30g/L sucrose +8g/L agar, pH value is adjusted to 5.1, 121 ℃ high pressure 30min, after cooling to 50 ℃, different combinations of filter sterilized plant growth regulators are added: the medium was dispensed into petri dishes and allowed to solidify for use (25 ml per dish). The growth regulator combination is as follows: the concentration of cytokinin TDZ is 0.5 mg/L, 5 gradients of auxin NAA are added, and the NAA concentrations are respectively as follows: 0 mg/L, 0.01 mg/L, 0.05 mg/L, 0.1mg/L and 0.2 mg/L. The total number of 5 somatic embryogenesis induction mediums (namely 5 treatments), 6 repeats (6 plates) are processed for each treatment, 4 leaf explants are processed for each plate, the average value of the seedling forming numbers of 4 explants in each plate is calculated when the seedling forming number of a single explant is calculated, and the average value of the last 6 repeats is calculated, namely the average seedling forming number of the single explant processed at last.
Filling a culture medium in the hole tray in the step (3), and transplanting after completely pouring the culture medium with a nutrient solution; the preparation method of the culture medium comprises the following steps: the sand is washed several times with the running water earlier, then the running water soaks three days, and the middle water that trades is 5, and it is reserve to air-dry at last, presses 2 with grass peat and sand that is prepared for: 1, mixing and subpackaging in a standard plug tray with 96 holes; the formula of the nutrient solution is as follows: CaCl2·2H2O 0.05g/L, NaCl 0.025g/L, MgSO4·7H2O 0.15g/L, （NH4）2HPO4 0.25g/L, KH2PO40.5g/L, 0.2g/L of citric acid, 10.1mg/L of vitamin B, 15g/L of glucose and 20g/L of agar, and adjusting the pH value to 5.0-5.2.
The effect of different combinations of TDZ and NAA on somatic embryogenesis of rhododendron leaves is shown in table 1.
TABLE 1 Effect of NAA and TDZ concentrations on embryogenesis of Rhododendron micranthum
Figure DEST_PATH_IMAGE002
Note: the different lower case letters after the numbers in the figure indicate that the LSD test is significantly different (P < 0.05)
The results in Table 1 show that the combination with the TDZ content of 0.5 mg/L and the NAA content of 0.2 mg/L in the induction medium for somatic embryogenesis has the most obvious induction effect on the somatic embryogenesis of rhododendron micranthum. The somatic embryogenesis rate is 96%; the transformation rate of somatic embryos is 67%, and a single explant can generate 34.83 plants on average.
The survival rate of the tissue culture seedlings after the transplantation of the embodiment reaches up to 90 percent.
Example 2
A high-efficiency brocade rhododendron direct somatic embryogenesis method comprises the following steps:
(1) preparing sterile seeding seedlings of rhododendron micranthum: the seeds of the rhododendron yunnanensis are sown in a culture medium after the surfaces of the seeds are disinfected, the temperature is 25 ℃, and the illumination intensity is 80mol.m-2.s-1Culturing in the tissue culture room for 2.5 months to obtain sterile seeding seedlings of the rhododendron micranthum for later use;
(2) induction of leaf somatic embryos: placing 4 young leaves at the top end of the rhododendron brocade seedling prepared in the step (1) on wet sterile filter paper in a super-clean workbench, cutting two ends of each leaf by using a blade, enabling the front side to face upwards, horizontally placing the leaves on the surface of a prepared somatic embryogenesis induction culture medium, placing four leaves on each flat plate, sealing the flat plates by using a sealing film, and culturing the flat plates in the dark for 2 weeks at the temperature of 25 ℃; then placing the mixture in a culture room for culture at the temperature of 25 ℃ for 16h in light and for 8h in dark, wherein the light intensity is 80 lmol m-2s-1The culture time is 100 d; during which the process of leaf somatic embryogenesis was observed, for four typical stages of somatic embryogenesis: photographing and recording the spherical embryo, the heart-shaped embryo, the torpedo-shaped embryo and the cotyledon-shaped embryo, and counting to obtain that the somatic embryo incidence rate is 97 percent and the transformation rate is 70 percent; the average number of seedlings per explant is 32.23;
(3) transplanting and light water management: peeling the plantlets obtained in the step (2) from the explants, transplanting the plantlets into a plug, performing sun-shading and sealing treatment for a week, performing spray culture, stopping spraying after the humidity reaches 90% and the time reaches 25 days, and keeping good ventilation and shading treatment; the water and fertilizer management comprises watering 2 times a week, and watering once a week with 20-20-20 water-soluble fertilizer (with concentration of 200 ppm).
The culture medium in the step (1) comprises the following components: 1/2 ER medium +15g/L sucrose +8g/L agar, pH 5.12.
In the step (2), the preparation method of the somatic embryogenesis induction medium comprises the following steps: WPM, adding 30g/L sucrose and 8g/L agar, adjusting pH to 5.2, pressurizing at 121 deg.C for 30min, cooling to room temperature, adding filter sterilized plant growth regulator: TDZ0.5 mg/L and NAA0.2 mg/L, and the medium was dispensed into petri dishes and solidified for use (25 ml per petri dish).
Filling a culture medium in the hole tray in the step (3), and transplanting the plantlets after the culture medium in the hole tray is thoroughly poured by nutrient solution; the preparation method of the culture medium comprises the following steps: the sand is washed several times with the running water earlier, then the running water soaks three days, and the middle water that trades is five times, and it is reserve to air-dry at last, presses 2 with grass peat and the sand that is prepared: 1 volume ratio of the mixture is mixed and subpackaged at 96 holes in a standard plug tray; the formula of the nutrient solution is as follows: CaCl2·2H2O 0.05g/L, NaCl 0.025g/L, MgSO4·7H2O 0.15g/L, （NH4）2HPO4 0.25g/L, KH2PO40.5g/L, 0.2g/L of citric acid, 10.1mg/L of vitamin B, 15g/L of glucose and 20g/L of agar, and the pH is adjusted to 5.2.
The survival rate of the tissue culture seedlings after the transplantation of the embodiment is 93 percent.
Example 3
A high-efficiency brocade rhododendron direct somatic embryogenesis method comprises the following steps:
(1) preparing sterile seeding seedlings of rhododendron micranthum: the seeds of the rhododendron yunnanensis are sown in a culture medium after the surfaces of the seeds are disinfected, the temperature is 25 ℃, and the illumination intensity is 80mol.m-2.s-1Culturing in the tissue culture room for 3 months to obtain sterile seeding seedlings of the rhododendron micranthum for later use;
(2) induction of leaf somatic embryos: placing 3 young leaves at the top end of the rhododendron brocade seedling prepared in the step (1) on wet sterile filter paper in a super-clean workbench, cutting two ends of each leaf by using a blade, enabling the front side to face upwards, horizontally placing the leaves on the surface of a prepared somatic embryogenesis induction culture medium, placing four leaves on each flat plate, sealing the flat plates by using a sealing film, and culturing the flat plates in the dark for 2 weeks at the temperature of 25 ℃; then placing the mixture in a culture room for culture at the temperature of 25 ℃ for 16h in light and for 8h in dark, wherein the light intensity is 80 lmol m-2s-1Culturing for 90 d; during which the process of leaf somatic embryogenesis was observed, for four typical stages of somatic embryogenesis: shooting and recording spherical embryos, heart-shaped embryos, torpedo-shaped embryos and cotyledon-shaped embryos, and counting to obtain that the somatic embryo incidence rate is 96.5 percent and the transformation rate is 70 percent; the average number of seedlings per explant was 33.53;
(3) transplanting and light water management: peeling the plantlets obtained in the step (2) from the explants, transplanting the plantlets into a plug, performing sun-shading and sealing treatment for a week, spraying, culturing at 85% humidity, stopping spraying after 22d, and keeping good ventilation and shading treatment; the water and fertilizer management is to pour water 2-3 times a week and pour 20-20-20 water-soluble fertilizer (with the concentration of 200 ppm) once a week.
The culture medium in the step (1) comprises the following components: 1/2 ER medium +15g/L sucrose +8g/L agar, pH between 5.15.
In the step (2), the preparation method of the somatic embryogenesis induction medium comprises the following steps: WPM, adding sucrose 30g/L and agar 8g/L, adjusting pH to 5.1 with 1.0M NaOH, pressurizing at 121 deg.C for 30min, cooling to room temperature, adding filter-sterilized plant growth regulator: TDZ0.5 mg/L and NAA0.2 mg/L, and the medium was dispensed into petri dishes and solidified for use (25 ml per petri dish).
Filling a culture medium in the hole tray in the step (3), and pouring a nutrient solution through the culture medium in the hole tray for transplanting; the preparation method of the culture medium comprises the following steps: the sand is washed several times with the running water earlier, then the running water soaks three days, and the middle water that trades is five times, and it is reserve to air-dry at last, presses 2 with grass peat and the sand that is prepared: 1, mixing and subpackaging in a standard plug tray with 96 holes; the formula of the nutrient solution is as follows: CaCl2·2H2O 0.05g/L, NaCl 0.025g/L, MgSO4·7H2O 0.15g/L, （NH4）2HPO4 0.25g/L, KH2PO40.5g/L, 0.2g/L of citric acid, 10.1mg/L of vitamin B, 15g/L of glucose and 20g/L of agar, and the pH is adjusted to 5.2.
The survival rate of the tissue culture seedlings after the transplanting of the embodiment reaches 95 percent.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.

Claims (1)

1. A high-efficiency brocade rhododendron direct somatic embryogenesis method is characterized by comprising the following steps:
(1) preparing sterile seeding seedlings of rhododendron micranthum: the seed surface of the rhododendron pulchrum is disinfected and then sowed in a culture medium at the temperature of 25 ℃ and the illumination intensity of 80 mu mol.m-2·s-1Culturing in the tissue culture room for 2-3 months to obtain sterile seeding seedlings of the rhododendron micranthum for later use;
(2) induction of leaf somatic embryos: in clean benchTaking 2-4 young leaves at the top end of the rhododendron brocade seedling prepared in the step (1), cutting two ends of each leaf by using a blade, enabling the front side of each leaf to face upwards, horizontally placing the leaves on the surface of a prepared somatic embryogenesis induction culture medium, placing four leaves on each flat plate, sealing the flat plate by using a sealing film, and culturing the flat plate in the dark for 2 weeks at the temperature of 25 ℃; then placing the mixture in a culture room for culture at the temperature of 25 ℃ for 16h in light and for 8h in darkness, wherein the light intensity is 80 mu mol.m-2·s-1Culturing for 80-100 days; during which the process of leaf somatic embryogenesis was observed, for four typical stages of somatic embryogenesis: photographing and recording spherical embryos, heart-shaped embryos, torpedo-shaped embryos and cotyledon-shaped embryos, and counting the somatic embryo incidence rate, the transformation rate and the average seedling number of each explant;
(3) transplanting and light water management: stripping the plantlets in the step (2) from the explants, transplanting the plantlets into a plug tray, spraying, culturing for 20 days, stopping spraying, and keeping good ventilation and shading treatment, wherein the humidity is 80% -90%; the water and fertilizer management is that water is poured for 2-3 times per week, and 20-20-20 water-soluble fertilizer with the concentration of 200ppm is poured once per week;
the culture medium in the step (1) comprises the following components: 1/2 ER culture medium, 15g/L sucrose and 8g/L agar, and the pH value is 5.0-5.2;
in the step (2), the preparation method of the somatic embryogenesis induction medium comprises the following steps: WPM +30g/L sucrose +8g/L agar, adjusting pH to 5.0-5.2, pressurizing at 121 deg.C for 30min, cooling to 50-60 deg.C, adding filter sterilized plant growth regulator: TDZ0.5 mg/L and NAA0.2 mg/L, subpackaging the culture medium into culture dishes for solidification, and 25ml of each culture dish;
filling a culture medium in the hole tray in the step (3), and before transplanting the plantlets, pouring a nutrient solution into the substrate in the hole tray for transplanting; the preparation method of the culture medium comprises the following steps: the sand is washed several times with the running water earlier, then the running water soaks three days, and the middle water that trades 3~5 times, and it is reserve to air-dry at last, presses 2 with grass carbon and the sand that is prepared for: 1, mixing and subpackaging in a standard plug tray with 96 holes; the formula of the nutrient solution is as follows: CaCl2·2H2O 0.05g/L, NaCl 0.025g/L, MgSO4·7H2O 0.15g/L,（NH4）2HPO4 0.25g/L, KH2PO40.5g/L, 0.2g/L of citric acid, 10.1mg/L of vitamin B and 15g/L of glucose, and adjusting the pH value to 5.0-5.2.
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