CN112931226B - Tissue culture rapid propagation method for alnus ferox - Google Patents

Tissue culture rapid propagation method for alnus ferox Download PDF

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CN112931226B
CN112931226B CN202110453518.0A CN202110453518A CN112931226B CN 112931226 B CN112931226 B CN 112931226B CN 202110453518 A CN202110453518 A CN 202110453518A CN 112931226 B CN112931226 B CN 112931226B
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陈陆琴
任保青
曹建庭
马俊
赵玉琳
李文龙
韵敏
南光耀
陈珍
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Taiyuan Botanical Garden
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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
    • A01H4/008—Methods for regeneration to complete plants
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
    • Y02P60/40—Afforestation or reforestation

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Abstract

The invention relates to the technical field of plant tissue culture, in particular to a tissue culture and rapid propagation method for alnus orientalis. The method has the advantages that the highest induction rate of adventitious buds is 71.6%, the rooting rate is 75%, the step of washing a culture medium is omitted in test-tube seedling transplantation by using vermiculite as a medium, root damage is reduced, the survival rate reaches more than 95%, and the method has practical significance in large-scale seedling culture of the alnus japonica.

Description

Tissue culture and rapid propagation method for alnus orientalis
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to a tissue culture and rapid propagation method of alnus orientalis.
Background
Alder of alder family of Oriental alder, arbor, up to 10-15m, soft, can be used for general building, making furniture and utensil material; the infructescence, the leaves and the bark are mixed, can extract tannin extract or be used as dye, and can diminish inflammation and stop bleeding when used as a medicine because of the tannin; most importantly, the fertilizer forms root nodules with actinomycetes, plays a role in nitrogen fixation, can be used as pioneer tree species for improving soil fertility, and is used for afforestation of barren mountains. The Oriental alder is distributed in the southwest of Sireiya, Turkey, northwest of Syria, etc., and its seeds and seedlings are difficult to obtain, and there is a lack of applicable seedlings. The plant tissue culture propagation technology can obtain a large number of seedlings in a short time and meet the market demand, but the research on tissue culture of the alder orientalis is not reported yet at present.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides a tissue culture and rapid propagation method of alnus japonica, which can obtain a large amount of high-quality test-tube seedlings in a short time by applying a plant tissue culture technology, is easy to realize large-scale industrial production and solves the problem of few alnus japonica seedlings.
In order to solve the technical problems, the invention adopts the technical scheme that:
a tissue culture and rapid propagation method of alnus orientalis comprises the following steps:
s1, explant preparation: selecting current-year stem segments or stem tips of alder alnus japonica planted in greenhouse as explants, cleaning the surfaces of the explant, sterilizing the explant, and using HgCl2Soaking, washing with sterile water for later use;
s2, primary culture: inoculating the explant into a culture medium to culture and induce adventitious buds, wherein the culture conditions are as follows: at 22-25 ℃, the illumination intensity is 1800-;
s3, subculture: cutting the bud cluster meeting the subculture requirement into small clusters, and inoculating the small clusters in a subculture medium under the culture conditions of: culturing at 22-25 deg.C with illumination intensity of 1800 and 2500Lux and photoperiod of 14h/d for 20-25d to obtain bud seedling;
s4, rooting culture: and (3) placing a single seedling of the bud seedling cultured in the S3 into a rooting culture medium for culturing, wherein the culture conditions are as follows: culturing for 20d to root to obtain test-tube plantlets, wherein the temperature is 22-25 ℃, the illumination intensity is 1800 and 2500Lux, the photoperiod is 14 h/d;
s5, hardening seedlings: when the roots of the test-tube plantlets cultured in the S4 grow to 1-2cm, placing the tissue culture boxes in a seedling hardening room for 3d for transplanting;
s6, transplanting: and (4) taking out the seedlings cultured in the S5, transplanting the seedlings into a transplanting matrix, putting the seedlings into a small arched shed in a greenhouse, keeping the humidity of the arched shed at 75-85% and the temperature of the arched shed at 20-25 ℃, gradually opening the small arched shed after transplanting for one week, and performing conventional management after 20 days.
Further, the explant cleaning and disinfection in step S1 specifically comprises: cutting off leaf of explant, soaking in washing powder for 15min, washing with tap water for 1 hr, sterilizing with 75% alcohol for 1min, and washing with sterile water for 3 times.
Further, the HgCl in step S12With 0.1% HgCl2Soaking for 3min, and washing with sterile water for 5 times.
Furthermore, in the step S2, the culture medium uses WPM culture medium as basic culture medium, and 0.3-1.0mg/L of 6-benzylaminopurine, 0.01mg/L of indolebutyric acid, 30g/L of sucrose, and 6g/L of agar are added, and the pH is 5.8-6.5.
Further, in the subculture medium in step S3, the WPM medium is used as a minimal medium, and 0.5mg/L of 6-benzylaminopurine, 0.01mg/L of indolebutyric acid, 30g/L of sucrose and 6g/L of agar are added, and the pH is 5.8-6.5.
Further, the rooting medium in step S4 contains 412.5mgNH per liter4NO3、475mg KNO3、92.5mg MgSO4·7H2O、42.5mg KH2PO4、16.96mg MnSO4·H2O、8.6mg ZnSO4·7H2O、6.2mg H3BO3、0.84mg KI、0.25mg Na2MoO4·2H2O、0.0025mg CuSO4·5H2O、0.0025mg CoCl2·6H2O、110mg CaCl2·2H2O、37.4mgNa2EDTA、27.8mg FeSO4·7H2O, IBA and the balance of water, the content of IBA is 0.1mg, 0.3mg or 0.5 mg.
Further, in the step S4, after the rooting medium is contained in the tissue culture box of vermiculite, the seedling is subjected to rooting culture.
Further, in step S6, the transplanting medium is peatmoss, perlite and vermiculite in a ratio of 3:1: 1.
Further, the matrix is disinfected by 1000 times of solution of 50% carbendazim wettable powder before transplantation.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides an alnus japonica tissue culture rapid propagation method, which solves the problem of large-scale culture of alnus japonica by applying a plant tissue culture technology and adopting an axillary bud induction culture medium, a subculture medium and a rooting culture medium of stem segments. The method has the advantages that the highest inductivity of adventitious buds is 71.6%, the rooting rate is 75%, the step of washing a culture medium is omitted in test-tube seedling transplanting with vermiculite as a medium, root damage is reduced, the survival rate is over 95%, and the method has practical significance in large-scale seedling culture of the alder orientalis.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
A tissue culture and rapid propagation method of alnus orientalis comprises the following steps:
s1, explant preparation: selecting current-year stem segments or stem tips of alder alnus japonica planted in greenhouse as explants, cleaning the surfaces of the explant, sterilizing the explant, and using HgCl2Soaking, washing with sterile water for later use;
s2, primary culture: inoculating the explant into a culture medium to culture and induce adventitious buds, wherein the culture conditions are as follows: at 22-25 ℃, the illumination intensity is 1800-;
s3, subculture: cutting the bud cluster meeting the subculture requirement into small clusters, and inoculating the small clusters in a subculture medium under the culture conditions of: culturing at 22-25 deg.C and illumination intensity of 1800 Lux and photoperiod of 14h/d for 20-25d to obtain bud seedling;
s4, rooting culture: and (3) placing a single seedling of the bud seedling cultured in the S3 into a rooting culture medium for culturing, wherein the culture conditions are as follows: culturing for 20d to root to obtain test-tube plantlets at the temperature of 22-25 ℃, the illumination intensity of 1800 and 2500Lux and the photoperiod of 14 h/d;
s5, hardening seedlings: when the roots of the test-tube plantlets cultured in the S4 grow to 1-2cm, placing the tissue culture boxes in a seedling hardening room for 3d for transplanting;
s6, transplanting: and (4) taking out the seedlings cultured by the S5, transplanting the seedlings into a transplanting matrix, putting the seedlings into a small arched shed in a greenhouse, keeping the humidity of the arched shed at 75-85% and the temperature of the arched shed at 20-25 ℃, gradually opening the small arched shed after transplanting for one week, and performing conventional management after 20 days.
Further, the explant cleaning and disinfection in step S1 specifically comprises: cutting off leaf of explant, soaking in washing powder for 15min, washing with tap water for 1 hr, sterilizing with 75% alcohol for 1min, and washing with sterile water for 3 times.
Further, the HgCl in step S12With 0.1% HgCl2Soaking for 3min, and washing with sterile water for 5 times.
Furthermore, in the step S2, the culture medium uses WPM culture medium as basic culture medium, and 0.3-1.0mg/L of 6-benzylaminopurine, 0.01mg/L of indolebutyric acid, 30g/L of sucrose, and 6g/L of agar are added, and the pH is 5.8-6.5.
Further, in the subculture medium in step S3, the WPM medium is used as a minimal medium, and 0.5mg/L of 6-benzylaminopurine, 0.01mg/L of indolebutyric acid, 30g/L of sucrose and 6g/L of agar are added, and the pH is 5.8-6.5.
Further, the rooting medium in step S4 contains 412.5mgNH per liter4NO3、475mg KNO3、92.5mg MgSO4·7H2O、42.5mg KH2PO4、16.96mg MnSO4·H2O、8.6mg ZnSO4·7H2O、6.2mg H3BO3、0.84mg KI、0.25mg Na2MoO4·2H2O、0.0025mg CuSO4·5H2O、0.0025mg CoCl2·6H2O、110mg CaCl2·2H2O、37.4mgNa2EDTA、27.8mg FeSO4·7H2O, IBA and the balance of water, the content of IBA being 0.1mg, 0.3mg or 0.5 mg.
Further, in the step S4, after the rooting medium is contained in the tissue culture box of vermiculite, the seedling is subjected to rooting culture.
Further, in step S6, the transplanting medium is peatmoss, perlite and vermiculite in a ratio of 3:1: 1.
Further, the matrix is disinfected by 1000 times of solution of 50% carbendazim wettable powder before transplantation.
Example 1
The tissue culture and rapid propagation method of alnus orientalis comprises the following steps:
(1) preparing an explant: selecting current-year stem segment or stem tip of alder planted in greenhouse and having axillary bud as explant, cutting off leaf and cutting into 2-3cm segments, soaking in washing powder water for 15min, washing with tap water for 1h, placing in ultra-clean bench, sterilizing with 75% alcohol surface for 1min, washing with sterile water for 3 times, and then washing with 0.1% HgCl2Soaking for 3min, and washing with sterile water for 5 times.
(2) Primary culture: inoculating the explant to WPM, 1.0 mg/L6-benzylamino adenine (6-BA), 0.01mg/L indolebutyric acid (IBA), 30g/L sucrose and 6g/L agar, and culturing in a culture medium with pH of 5.8-6.5 under the following conditions: at 22-25 ℃, the illumination intensity is 1800 and 2500Lux, the light period is 14h/d, and subculture is carried out after the adventitious bud germinates.
(3) Subculturing: cutting buds meeting the subculture requirement into small clusters, inoculating the small clusters into a subculture medium, taking a WPM medium as a basic medium, adding 0.5mg/L of 6-benzylaminopurine (6-BA), 0.01mg/L of indolebutyric acid (IBA), 30g/L of sucrose and 6g/L of agar to prepare the subculture medium with the pH of 5.8-6.5, and culturing for 20-25d under the same culture condition (2).
(4) Rooting culture: the rooting medium contained 412.5mg NH per liter4NO3、475mg KNO3、92.5mg MgSO4·7H2O、42.5mg KH2PO4、16.96mg MnSO4·H2O、8.6mg ZnSO4·7H2O、6.2mg H3BO3、0.84mg KI、0.25mg Na2MoO4·2H2O、0.0025mg CuSO4·5H2O、0.0025mg CoCl2·6H2O、110mg CaCl2·2H2O、37.4mgNa2EDTA、27.8mg FeSO4·7H2O, IBA and the balance of water, wherein the content of the IBA is 0.1mg, a rooting culture medium is poured into a tissue culture box containing vermiculite, a single seedling of the bud seedling cultured in the step (3) is placed in the rooting culture medium for culturing under the same culture condition (2), and the bud seedling can grow after 20 daysAnd (4) root.
(5) Hardening seedlings: and (4) when the roots of the test-tube seedlings cultured in the step (4) grow to 1-2cm, placing the tissue culture box in a seedling hardening room for 3d, and preparing for transplanting.
(6) Transplanting: and (3) sterilizing the test-tube seedling transplanting matrix by 1000 times of liquid of 50% carbendazim wettable powder before transplanting, taking out the seedlings cultured in the step (5), transplanting the seedlings into the matrix, putting the matrix into a small arched shed in a greenhouse, keeping the humidity of the arched shed at 75-85% and the temperature of the arched shed at 20-25 ℃, gradually opening the small arched shed after transplanting for one week, and performing conventional management after 20 days.
Example 2
The tissue culture and rapid propagation method of alnus orientalis comprises the following steps:
(1) preparing an explant: selecting current-year stem segment or stem tip of alder planted in greenhouse and having axillary bud as explant, cutting off leaf and cutting into 2-3cm segments, soaking in washing powder water for 15min, washing with tap water for 1h, placing in ultra-clean bench, sterilizing with 75% alcohol surface for 1min, washing with sterile water for 3 times, and then washing with 0.1% HgCl2Soaking for 3min, and washing with sterile water for 5 times.
(2) Primary culture: inoculating the explant to WPM, 0.5 mg/L6-benzylamino adenine, 0.01mg/L indolebutyric acid (IBA), 30g/L sucrose, 6g/L agar and a culture medium with the pH of 5.8-6.5, and culturing to induce adventitious buds under the following culture conditions: at 22-25 ℃, the illumination intensity is 1800 and 2500Lux, the light period is 14h/d, and subculture is carried out after the adventitious buds germinate.
(3) Subculturing: cutting buds meeting the subculture requirement into small clusters, inoculating the small clusters into a subculture medium, taking a WPM medium as a basic medium, adding 0.5mg/L of 6-benzylaminopurine (6-BA), 0.01mg/L of indolebutyric acid (IBA), 30g/L of sucrose and 6g/L of agar to prepare the subculture medium with the pH of 5.8-6.5, and culturing for 20-25d under the same culture condition (2).
(4) Rooting culture: the rooting medium contained 412.5mg NH per liter4NO3、475mg KNO3、92.5mg MgSO4·7H2O、42.5mg KH2PO4、16.96mg MnSO4·H2O、8.6mg ZnSO4·7H2O、6.2mg H3BO3、0.84mg KI、0.25mg Na2MoO4·2H2O、0.0025mg CuSO4·5H2O、0.0025mg CoCl2·6H2O、110mg CaCl2·2H2O、37.4mg Na2EDTA、27.8mg FeSO4·7H2O, IBA and the balance of water, wherein the content of the IBA is 0.3mg, the rooting culture medium is poured into a tissue culture box containing vermiculite, the bud seedling cultured in the step (3) is placed into the rooting culture medium for culturing, the culturing conditions are the same as those in the step (2), and the bud seedling can take roots after 20 days.
(5) Hardening seedlings: and (4) when the roots of the test-tube seedlings cultured in the step (4) grow to 1-2cm, placing the tissue culture box in a seedling hardening room for 3d, and preparing for transplanting.
(6) Transplanting: and (3) sterilizing the test-tube seedling transplanting matrix by 1000 times of liquid of 50% carbendazim wettable powder before transplanting, taking out the seedlings cultured in the step (5), transplanting the seedlings into the matrix, putting the matrix into a small arched shed in a greenhouse, keeping the humidity of the arched shed at 75-85% and the temperature of the arched shed at 20-25 ℃, gradually opening the small arched shed after transplanting for one week, and performing conventional management after 20 days.
Example 3
The tissue culture rapid propagation method of alnus japonica comprises the following steps:
(1) preparing an explant: selecting current-year stem segment or stem tip of alder planted in greenhouse and having axillary bud as explant, cutting off leaf and cutting into 2-3cm segments, soaking in washing powder water for 15min, washing with tap water for 1h, placing in ultra-clean bench, sterilizing with 75% alcohol surface for 1min, washing with sterile water for 3 times, and then washing with 0.1% HgCl2Soaking for 3min, and washing with sterile water for 5 times.
(2) Primary culture: inoculating the explant to WPM, 0.3 mg/L6-benzylamino adenine, 0.01mg/L indolebutyric acid (IBA), 30g/L sucrose, 6g/L agar and a culture medium with the pH of 5.8-6.5, and culturing to induce adventitious buds under the following culture conditions: at 22-25 ℃, the illumination intensity is 1800 and 2500Lux, the light period is 14h/d, and subculture is carried out after the adventitious bud germinates.
(3) Subculturing: cutting buds meeting the subculture requirement into small clusters, inoculating the small clusters into a subculture medium, taking a WPM medium as a basic medium, adding 0.5mg/L of 6-benzylaminopurine (6-BA), 0.01mg/L of indolebutyric acid (IBA), 30g/L of sucrose and 6g/L of agar to prepare the subculture medium with the pH of 5.8-6.5, and culturing for 20-25d under the same culture condition (2).
(4) Rooting culture: the rooting medium contained 412.5mg NH per liter4NO3、475mg KNO3、92.5mg MgSO4·7H2O、42.5mg KH2PO4、16.96mg MnSO4·H2O、8.6mg ZnSO4·7H2O、6.2mg H3BO3、0.84mg KI、0.25mg Na2MoO4·2H2O、0.0025mg CuSO4·5H2O、0.0025mg CoCl2·6H2O、110mg CaCl2·2H2O、37.4mgNa2EDTA、27.8mg FeSO4·7H2O, IBA and the balance of water, wherein the content of the IBA is 0.5mg, the rooting culture medium is poured into a tissue culture box containing vermiculite, the bud seedling cultured in the step (3) is placed into the rooting culture medium for culturing, the culturing conditions are the same as those in the step (2), and the bud seedling can take roots after 20 days.
(5) Hardening seedlings: and (4) when the roots of the test-tube seedlings cultured in the step (4) grow to 1-2cm, placing the tissue culture box in a seedling hardening room for 3d, and preparing for transplanting.
(6) Transplanting: and (3) sterilizing the test-tube seedling transplanting matrix by 1000 times of liquid of 50% carbendazim wettable powder before transplanting, taking out the seedlings cultured in the step (5), transplanting the seedlings into the matrix, putting the matrix into a small arched shed in a greenhouse, keeping the humidity of the arched shed at 75-85% and the temperature of the arched shed at 20-25 ℃, gradually opening the small arched shed after transplanting for one week, and performing conventional management after 20 days.
In step (2) of each example described above, the explant was inoculated on an induction medium and cultured for 25 days to obtain the adventitious bud induction rates shown in Table 1.
TABLE 1 adventitious bud Induction Rate
Figure BDA0003039658760000051
Figure BDA0003039658760000061
As can be seen from Table 1, the explants cultured on the induction medium for 25 days can induce adventitious buds, wherein the induction rate of the adventitious buds is the highest with the induction medium of example 2, and the seedlings have good growth potential.
In the above-mentioned step (4) of each example, the multiple shoots were excised from the base, and the single shoots were inoculated into a rooting medium and cultured for 20 days to obtain the rooting rates shown in Table 2.
TABLE 2 adventitious bud rooting percentage
Culture medium Rooting rate
Example 1 58.3％
Example 2 75％
Example 3 62.4％
As can be seen from Table 2, the clumped sprouts were excised from the base and the single shoots were inoculated into the rooting medium and cultured for 20 days, and the adventitious sprout rooting rate varied with the IBA concentration, with the highest rooting rate on the rooting medium of example 2.
In summary, according to the tissue culture and rapid propagation method of alnus orientalis, the optimal formula of the adventitious bud induction culture medium is as follows: WPM +0.5 mg/L6-BA +0.01mg/L IBA; the rooting culture medium comprises: 412.5mg NH4NO3、475mg KNO3、92.5mg MgSO4·7H2O、42.5mg KH2PO4、16.96mg MnSO4·H2O、8.6mg ZnSO4·7H2O、6.2mg H3BO3、0.84mg KI、0.25mg Na2MoO4·2H2O、0.0025mg CuSO4·5H2O、0.0025mg CoCl2·6H2O、110mg CaCl2·2H2O、37.4mgNa2EDTA、27.8mg FeSO4·7H2O, IBA and the balance of water, wherein the content of IBA is 0.3mg, and the medium is vermiculite.
Although only the preferred embodiments of the present invention have been described in detail, the present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the knowledge of those skilled in the art, and all changes are encompassed in the scope of the present invention.

Claims (6)

1. A tissue culture and rapid propagation method of alnus orientalis is characterized by comprising the following steps:
s1, explant preparation: selecting annual stem segment or stem tip with axillary bud of Alnus cremastogyne potted in greenhouse as explant, cleaning surface, sterilizing, and adding HgCl2Soaking, washing with sterile water for later use;
s2, primary culture: inoculating the explant into a culture medium to culture and induce adventitious buds, wherein the culture medium takes a WPM culture medium as a basic culture medium, 0.3-1.0mg/L of 6-benzylaminopurine, 0.01mg/L of indolebutyric acid, 30g/L of sucrose and 6g/L of agar are added, the pH value is 5.8-6.5, and the culture conditions are as follows: at 22-25 ℃, the illumination intensity is 1800-;
s3, subculture: cutting buds meeting the subculture requirement into small clusters, inoculating the small clusters into a subculture medium, taking a WPM culture medium as a basic culture medium, adding 0.5mg/L of 6-benzylaminopurine, 0.01mg/L of indolebutyric acid, 30g/L of sucrose and 6g/L of agar, controlling the pH to be 5.8-6.5, and culturing under the conditions that: culturing at 22-25 deg.C with illumination intensity of 1800 and 2500Lux and photoperiod of 14h/d for 20-25d to obtain bud seedling;
s4, rooting culture: placing single sprout cultured in S3 in rooting culture medium containing 412.5mg NH per liter for culturing4NO3、475mg KNO3、92.5mg MgSO4·7H2O、42.5mg KH2PO4、16.96mg MnSO4·H2O、8.6mg ZnSO4·7H2O、6.2mg H3BO3、0.84mg KI、0.25mg Na2MoO4·2H2O、0.0025mg CuSO4·5H2O、0.0025mg CoCl2·6H2O、110mg CaCl2·2H2O、37.4mg Na2EDTA、27.8mg FeSO4·7H2O, IBA and the balance of water, the content of IBA is 0.1mg, 0.3mg or 0.5mg, and the culture conditions are as follows: culturing for 20d to root to obtain test-tube plantlets at the temperature of 22-25 ℃, the illumination intensity of 1800 and 2500Lux and the photoperiod of 14 h/d;
s5, hardening seedlings: when the roots of the test-tube plantlets cultured in the S4 grow to 1-2cm, placing the tissue culture boxes in a seedling hardening room for 3d for transplanting;
s6, transplanting: and (4) taking out the seedlings cultured in the S5, transplanting the seedlings into a transplanting matrix, putting the seedlings into a small arched shed in a greenhouse, keeping the humidity of the arched shed at 75-85% and the temperature of the arched shed at 20-25 ℃, gradually opening the small arched shed after transplanting for one week, and performing conventional management after 20 days.
2. The tissue culture rapid propagation method of alder orientalis according to claim 1, wherein: the explant cleaning and disinfection in the step S1 specifically comprises the following steps: cutting off leaf of explant, soaking in washing powder for 15min, washing with tap water for 1 hr, sterilizing with 75% alcohol for 1min, and washing with sterile water for 3 times.
3. The tissue culture rapid propagation method of alder orientalis according to claim 1, wherein: HgCl as described in step S12With 0.1% HgCl2Soaking for 3min, and washing with sterile water for 5 times.
4. The tissue culture rapid propagation method of alder orientalis according to claim 1, wherein: and in the step S4, after the rooting medium is contained in the tissue culture box of vermiculite, carrying out rooting culture on the bud seedlings.
5. The tissue culture rapid propagation method of alder orientalis according to claim 1, wherein: in the step S6, the transplanting matrix is turfy soil, perlite and vermiculite =3:1: 1.
6. The tissue culture rapid propagation method of alnus orientalis as claimed in claim 1 or 5, wherein: and (3) disinfecting the substrate with 1000 times of solution of 50% carbendazim wettable powder before transplanting.
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