CN115005096A - Ethnic medicine alangium platanifolium seedling breeding method - Google Patents
Ethnic medicine alangium platanifolium seedling breeding method Download PDFInfo
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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
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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
- A01H4/002—Culture media for tissue culture
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- 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
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- 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
Abstract
The invention relates to a ethnic medicine seedling breeding method, in particular to a ethnic medicine alangium chinense seedling breeding method, which comprises the following steps in sequence: collecting alangium chinense explants, carrying out callus induction culture on the explants, carrying out bud differentiation culture, carrying out subculture, carrying out rooting culture, and transplanting in a field; the induction culture medium adopted by the induction culture of the explant callus comprises: MS culture medium, 1.5-3.0 mg/L6-BA, 1.0-2.0 mg/L KT, 1.0-2.0 mg/L NAA, 20-30 g/L sucrose, 3.5-4.0 g/L agar, 0.1-0.5 g/L active carbon, and pH of the induction culture medium is 5.4-5.8.
Description
Technical Field
The invention relates to a method for plant tissue culture, in particular to a method for breeding national medicament alangium platanifolium seedlings.
Background
The national medicine Alangium chinense Harms (Alangium chinense Harms) belongs to the branch root and fibrous root of the plant of the genus Alangium of the family Alangicaceae, also called as the Alangium chinense, the platinum bar, the illicium verum, the Huaguamu and the like, is mostly a national medicine, is used in the ethnic groups of Miao nationality, Bai nationality, Zhuang nationality, Tujia nationality and the like, has the effects of dispelling wind and dredging collaterals, dissipating blood stasis and relieving pain, anaesthetizing and relaxing muscles and the like, can be used for treating rheumatic arthritis, rheumatoid arthritis, numbness and paralysis, heart failure, strain and lumbago, traumatic injury, bone tuberculosis, tuberculosis and the like, is used as the main medicine of the preparations of 'rheumatism-fixing tablet', 'Feng He Li rheumatism-dispelling wine', 'Xiao Bi Ling mixture' and the like, and has good development prospect. The Chinese alangium is mainly used in the provinces of the Yangtze river basin and the Yangtze river basin, such as Guangxi, Hubei, Hunan, Guizhou, Yunnan, Sichuan and the like, and originates in mountainous regions or forests with the elevation below 1500 m. At present, the alangium platanifolium is mainly used for seedling culture in a seeding and breeding mode, but the phenomena of unstable seedling heredity and degeneration exist, so that the yield is low, the leaves are small, the branches and the stems are fine, and the medicinal value is not high.
At present, the tissue culture technology of the Alangium platanifolium and the application of the tissue culture and rapid propagation patent of the Alangium platanifolium are not reported at home and abroad.
Disclosure of Invention
The invention aims to provide a ethnic drug alangium platanifolium seedling breeding method, which selects the current-year non-lignified segmented stem section of alangium platanifolium as an explant, and performs the steps of callus induction, bud differentiation, proliferation, rooting, seedling hardening, transplanting and the like, so that the callus induction rate reaches over 78.6 percent, the seedling transplanting survival rate reaches over 95 percent, and the fast breeding of ethnic drug alangium platanifolium seedlings is realized, thereby realizing the purpose of the invention.
The technical scheme provided by the invention is as follows: a ethnic medicine alangium platanifolium seedling propagation method comprises the following steps in sequence: collecting an explant of the alangium chinense, inducing callus, differentiating buds, proliferating, rooting and transplanting;
the induction culture medium adopted by the induction culture of the explant callus comprises: MS culture medium, 1.5-3.0 mg/L6-BA, 1.0-2.0 mg/L KT, 1.0-2.0 mg/L NAA, 20-30 g/L sucrose, 3.5-4.0 g/L agar and 0.1-0.5 g/L active carbon, wherein the pH of the induction culture medium is 5.4-5.8.
The method for the explant callus induction culture operation comprises the following steps: sterilizing the current-year non-lignified segmented stems of the alangium platanifolium, shearing the sterilized segments into 2.5-3.0 cm segmented stems, horizontally placing the segmented stems into an induction culture medium, and carrying out full-black dark culture at 25-28 ℃ for 60-90 days to obtain callus.
The bud differentiation culture medium used for inducing bud differentiation operation comprises: MS culture medium, 1.5-3.0 mg/L6-BA, 1.0-2.0 mg/L NAA, 20-30 g/L sucrose, 3.5-4.0 g/L agar, 0.1-0.5 g/L active carbon, and the pH value of bud differentiation culture medium is 5.4-5.8.
The bud differentiation culture operation specifically comprises the following steps: transferring the callus obtained by the explant induced culture operation into a bud differentiation culture medium for culture for 25-30 days to differentiate adventitious buds.
The proliferation culture medium used for the proliferation culture operation comprises: MS culture medium, 1.0-2.0 mg/L6-BA, 0.5-1.0 mg/L NAA, 20-30 g/L sucrose, 3.5-4.0 g/L agar, 0.1-0.5 g/L activated carbon, and pH of the multiplication culture medium is 5.4-5.8.
The proliferation culture operation specifically comprises the following steps: removing the top end of the adventitious bud obtained by bud differentiation culture operation, cutting the adventitious bud into a stem section with the length of 1.0-2.0 cm, inoculating the stem section into a proliferation culture medium, and culturing for 20-25 days to realize proliferation of the adventitious bud.
The rooting culture medium used in the rooting culture operation comprises: 1/2MS culture medium, 2.0-3.0 mg/L ABT-6, 1.0-2.0 mg/L NAA, 15-20 g/L sucrose, 3.0-4.0 g/L agar, and the pH value of the rooting culture medium is 5.4-5.8.
The rooting culture operation method comprises the following steps: and (3) inoculating the rootless adventitious buds with the length of 1.5-2.0 cm obtained by the multiplication culture operation into a rooting culture medium for culturing for 25-30 days, and then realizing the rooting of the test-tube plantlet.
The specific method of the transplanting operation comprises the following steps: and (3) hardening the test-tube plantlets which are obtained by rooting culture and have the height of about 4-5 cm, thick root systems, strong growth and dark green leaf colors for 3-7 days under natural light of a greenhouse, and transplanting the culture medium with the roots cleaned to the sterilized culture medium to be cultured into seedlings, so that the seedlings are obtained. The proportion of the culture medium is 8:1:1 of carbon soil, farmyard manure and river sand.
The culture conditions in the bud differentiation culture, the proliferation culture operation and the rooting culture operation are as follows: the culture temperature is 25-28 ℃, the illumination intensity is 2000-3000 lx, and the illumination time is 10-14 hours/day.
Has the advantages that:
the invention selects the current-year non-lignified stem with nodes of the alangium platanifolium as an explant, and establishes the tissue culture technology of the national medicament alangium platanifolium through the steps of callus induction, bud differentiation, proliferation, rooting, seedling hardening, transplanting and the like. The method has the characteristics of strong technical performance, low cost, simple process and the like, can be directly used for the industrial production of the alangium platanifolium seedlings, and has important practical significance for promoting the industrial development and utilization of national medicine alangium platanifolium.
Detailed Description
The technical solution of the present invention will be further described in detail with reference to the following embodiments, but the present invention is not limited thereto.
Example one
(1) Explant collection: selecting the annual non-lignified segmented stem section of the alangium platanifolium plant which grows robustly and has no obvious diseases as an explant in the field, immediately carrying out water retention and moisture retention treatment after collection, and bringing the explant back to a laboratory in time.
(2) Explant callus induction culture: when the explants collected in the step (1) and returned to the laboratory are washed under tap water for 5 hours, the explants are placed in an ultraclean workbench to be disinfected in 75% ethanol solution for 15 seconds, the explants are washed with sterile water for 3 times and then are placed in 0.1% mercuric chloride solution to be disinfected for 15 minutes, the explants are washed with sterile water for 5 times and then are cut into segmented stem sections with the length of about 2.5-3.0 cm after being dried with sterile filter paper and inoculated into an induction culture medium, and the callus can be induced to form after being placed at 25 ℃ for full-black dark culture for 60 days, wherein the induction rate is 84.3%, and the pollution rate is lower than 10%. The induction culture medium comprises: MS culture medium +3.0 mg/L6-BA +2.0mg/L KT +2.0mg/L NAA +20g/L sucrose +3.5g/L agar +0.1g/L activated carbon, pH 5.4.
(3) Adventitious bud differentiation: transferring the callus obtained by induction in the step (2) to a bud differentiation culture medium for culturing for 25 days to obtain the adventitious bud, wherein the bud differentiation rate reaches 85.6 percent. The bud differentiation culture medium comprises: MS culture medium +3.0 mg/L6-BA +2.0mg/L NAA +20g/L sucrose +3.5g/L agar +0.1g/L active carbon, pH 5.4.
(4) Adventitious bud proliferation: and (4) cutting the rootless adventitious buds obtained by induction in the step (3) into stem sections with the length of about 1.0-2.0 cm, removing the top ends of the stem sections, inoculating the stem sections into a proliferation culture medium, and culturing for 20 days to realize the proliferation of the adventitious buds, wherein the proliferation coefficient is 6.2 times. The proliferation culture medium comprises: MS culture medium +2.0 mg/L6-BA +1.0mg/L NAA +20g/L sucrose +3.5g/L agar +0.1g/L active carbon, pH 5.4.
(5) Rooting culture: and (3) cutting the rootless adventitious buds with the length of about 1.5-2.0 cm obtained by proliferation in the step (4) from the base part, inoculating the rootless adventitious buds into a rooting culture medium, and culturing for 25 days to realize rooting of the test-tube plantlet, wherein the rooting rate is 96.2%. The rooting culture medium comprises: 1/2MS culture medium +3.0mg/L ABT-6 (rooting powder) +2.0mg/L NAA +20g/L sucrose +4.0g/L agar, pH 5.4.
(6) Transplanting: and (3) hardening the test-tube seedlings which are 4-5 cm high, thick in root system, strong in growth and dark green in leaf color in the step (5) under natural light of a greenhouse for 3 days, transplanting the culture medium with cleaned roots into a sterilized culture medium (the proportion of the culture medium is carbon soil: farmyard manure: river sand: 8:1:1) to be cultivated into seedlings, and obtaining the seedlings, wherein the survival rate is 95.3% after 30 days of transplanting.
The culture conditions in the above steps (3) to (5) are: the culture temperature was 25 ℃, the light intensity was 2000lx, and the light time was 10 hours/day.
Example two
(1) Explant collection: selecting the annual non-lignified segmented stem section of the alangium platanifolium plant which grows robustly and has no obvious diseases as an explant in the field, immediately carrying out water retention and moisture retention treatment after collection, and bringing the explant back to a laboratory in time.
(2) Explant callus induction culture: when the explants collected in the step (1) and returned to the laboratory are washed under tap water for 7 hours, the explants are placed in an ultraclean workbench to be disinfected in 75% ethanol solution for 20 seconds, the explants are washed with sterile water for 4 times and then are placed in 0.1% mercuric chloride solution to be disinfected for 20 minutes, the explants are washed with sterile water for 5 times and then are cut into segmented stem sections with the length of about 2.5-3.0 cm after being dried with sterile filter paper and inoculated into an induction culture medium, and the callus can be induced to form after being placed at 26 ℃ for full-black dark culture for 75 days, wherein the induction rate is 81.7%, and the pollution rate is lower than 8%. The induction culture medium comprises: MS culture medium +2.0 mg/L6-BA +1.5mg/L KT +1.5mg/L NAA +25g/L sucrose +3.7g/L agar +0.3g/L activated carbon, pH 5.6.
(3) Adventitious bud differentiation: transferring the callus obtained by induction in the step (2) to a bud differentiation culture medium for culturing for 27 days to obtain the adventitious bud, wherein the bud differentiation rate reaches 82.1%. The bud differentiation culture medium comprises: MS culture medium +2.0 mg/L6-BA +1.5mg/L NAA +25g/L sucrose +3.7g/L agar +0.3g/L active carbon, pH 5.6.
(4) Adventitious bud proliferation: and (4) cutting the rootless adventitious buds obtained by induction in the step (3) into stem sections with the length of about 1.0-2.0 cm, removing the top ends of the stem sections, inoculating the stem sections into a proliferation culture medium, and culturing for 23 days to realize the proliferation of the adventitious buds, wherein the proliferation coefficient is 5.1 times. The proliferation culture medium comprises: MS culture medium +1.5 mg/L6-BA +0.7mg/L NAA +25g/L sucrose +3.7g/L agar +0.3g/L active carbon, pH 5.6.
(5) Rooting culture: and (3) cutting the rootless adventitious buds with the length of about 1.5-2.0 cm obtained by proliferation in the step (4) from the base part, inoculating the rootless adventitious buds into a rooting culture medium, and culturing for 25 days to realize rooting of the test-tube plantlet, wherein the rooting rate is 96.2%. The rooting culture medium comprises: 1/2MS culture medium +2.5mg/L ABT-6 (rooting powder) +1.5mg/L NAA +17g/L sucrose +3.5g/L agar, pH 5.6.
(6) Transplanting: and (3) hardening the test-tube seedlings which are 4-5 cm high, thick in root system, strong in growth and dark green in leaf color in the step (5) under natural light in a greenhouse for 5 days, transplanting the culture medium with cleaned roots into a sterilized culture medium (the proportion of the culture medium is carbon soil: farmyard manure: river sand: 8:1:1) to be cultivated into seedlings, and obtaining the seedlings, wherein the survival rate is 95% after 30 days of transplanting.
The culture conditions in the above steps (3) to (5) are: the culture temperature was 26 ℃, the light intensity was 2500lx, and the light time was 12 hours/day.
EXAMPLE III
(1) Explant collection: selecting annual non-lignified segmented stem section of the alangium platanifolium plant which grows robustly and has no obvious diseases as an explant in the field, immediately carrying out water retention and moisture preservation treatment after collection, and bringing back to a laboratory in time.
(2) Explant callus induction culture: when the explants collected in the step (1) and returned to the laboratory are washed under tap water for 10 hours, the explants are placed in an ultraclean workbench to be disinfected in 75% ethanol solution for 25 seconds, washed with sterile water for 5 times and then surface water drops are sucked dry by sterile filter paper, the explants are placed in 0.1% mercuric chloride solution to be disinfected for 25 minutes, washed with sterile water for 6 times and then surface water drops are sucked dry by sterile filter paper, the explants are cut into segmented stem sections with the length of about 2.5-3.0 cm and inoculated into an induction culture medium, and the culture medium is placed at 28 ℃ for complete dark culture for 90 days to induce and form callus, wherein the induction rate is 78.6%, and the pollution rate is lower than 5%. The induction culture medium comprises: MS culture medium +1.5 mg/L6-BA +1.0mg/L KT +1.0mg/L NAA +30g/L sucrose +4.0g/L agar +0.5g/L activated carbon, pH 5.8.
(3) Adventitious bud differentiation: transferring the callus obtained by induction in the step (2) to a bud differentiation culture medium for culturing for 30 days to obtain the adventitious bud, wherein the bud differentiation rate reaches 76.8%. The bud differentiation culture medium comprises: MS culture medium +1.5 mg/L6-BA +1.0mg/L NAA +30g/L sucrose +4.0g/L agar +0.5g/L active carbon, pH 5.8.
(4) Adventitious bud proliferation: and (4) cutting the rootless adventitious buds obtained by induction in the step (3) into stem sections with the length of about 1.0-2.0 cm, removing the top ends of the stem sections, inoculating the stem sections into a proliferation culture medium, and culturing for 25 days to realize the proliferation of the adventitious buds, wherein the proliferation coefficient is 4.7 times. The proliferation culture medium comprises: MS culture medium +1.0 mg/L6-BA +0.5mg/L NAA +30g/L sucrose +4.0g/L agar +0.5g/L active carbon, pH 5.8.
(5) Rooting culture: and (3) cutting the rootless adventitious buds with the length of about 1.5-2.0 cm obtained by proliferation in the step (4) from the base part, inoculating the rootless adventitious buds into a rooting culture medium, and culturing for 30 days to realize rooting of the test-tube plantlet, wherein the rooting rate is 90.8%. The rooting culture medium comprises: 1/2MS culture medium +2.0mg/L ABT-6 (rooting powder) +1.0mg/L NAA +20g/L sucrose +4.0g/L agar, pH 5.8.
(6) Transplanting: and (3) hardening the test-tube seedlings which are 4-5 cm high, thick in root system, strong in growth and dark green in leaf color in the step (5) for 7 days under natural light in a greenhouse, transplanting the culture medium with cleaned roots into a sterilized culture medium (the proportion of the culture medium is carbon soil: farmyard manure: river sand: 8:1:1) to be cultivated into seedlings, and obtaining the seedlings, wherein the survival rate is 98.2% after 30 days of transplanting.
The culture conditions in the above steps (3) to (5) are: the culture temperature is 28 ℃, the illumination intensity is 3000lx, and the illumination time is 14 hours/day.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such modifications are intended to be included in the scope of the present invention.
Claims (10)
1. A breeding method of national drug Alangium platanifolium seedling is characterized in that the method comprises the following steps of Alangium platanifolium explant collection, explant callus induction culture, bud differentiation culture, proliferation culture, rooting culture and field transplantation in sequence;
the induction culture medium adopted by the induction culture of the explant callus comprises: MS culture medium, 1.5-3.0 mg/L6-BA, 1.0-2.0 mg/L KT, 1.0-2.0 mg/L NAA, 20-30 g/L sucrose, 3.5-4.0 g/L agar and 0.1-0.5 g/L active carbon, wherein the pH of the induction culture medium is 5.4-5.8.
2. The tissue culture and rapid propagation method of ethnic drug Alangium platanifolium as claimed in claim 1, wherein the method of explant callus induction culture operation is: sterilizing the current-year non-lignified segmented stems of the alangium platanifolium, shearing the sterilized segments into 2.5-3.0 cm segmented stems, horizontally placing the segmented stems into an induction culture medium, and carrying out full-black dark culture at 25-28 ℃ for 60-90 days to obtain callus.
3. The tissue culture and rapid propagation method of the ethnic drug Alangium platanifolium according to claim 1, wherein the bud differentiation medium used for inducing bud differentiation operation comprises: MS culture medium, 1.5-3.0 mg/L6-BA, 1.0-2.0 mg/L NAA, 20-30 g/L sucrose, 3.5-4.0 g/L agar, 0.1-0.5 g/L active carbon, and the pH value of bud differentiation culture medium is 5.4-5.8.
4. The tissue culture and rapid propagation method of ethnic drug Alangium platanifolium as claimed in claim 3, wherein the bud differentiation culture operation is specifically: transferring the callus obtained by the explant induced culture operation into a bud differentiation culture medium for culture for 25-30 days to differentiate adventitious buds.
5. The tissue culture and rapid propagation method of the ethnic drug alangium platanifolium according to claim 1, wherein the propagation culture medium used in the propagation culture operation comprises: MS culture medium, 1.0-2.0 mg/L6-BA, 0.5-1.0 mg/L NAA, 20-30 g/L sucrose, 3.5-4.0 g/L agar, 0.1-0.5 g/L activated carbon, and pH of the multiplication culture medium is 5.4-5.8.
6. The tissue culture and rapid propagation method of ethnic drug Alangium platanifolium as claimed in claim 5, wherein the proliferation culture operation is specifically: the adventitious bud obtained by bud differentiation culture operation is cut into stem sections with the length of 1.0-2.0 cm after the top end of the adventitious bud is removed, and the stem sections are inoculated into a proliferation culture medium for culturing for 20-25 days, so that the proliferation of the adventitious bud can be realized.
7. The tissue culture and rapid propagation method of the ethnic drug Alangium platanifolium according to claim 1, wherein the rooting medium used in the rooting culture operation comprises: 1/2MS culture medium, 2.0-3.0 mg/L ABT-6, 1.0-2.0 mg/L NAA, 15-20 g/L sucrose, 3.0-4.0 g/L agar, and the pH value of the rooting culture medium is 5.4-5.8.
8. The tissue culture and rapid propagation method of the ethnic drug Alangium platanifolium according to claim 7, characterized in that the rooting culture operation method is: and (3) inoculating the rootless adventitious buds with the length of 1.5-2.0 cm obtained by the multiplication culture operation into a rooting culture medium for culturing for 25-30 days, and then realizing the rooting of the test-tube plantlet.
9. The tissue culture and rapid propagation method of ethnic drug Alangium platanifolium according to claim 1, characterized in that the specific method of the transplanting operation is: and (3) hardening the test-tube plantlets which are obtained by rooting culture and have the height of about 4-5 cm, thick root systems, strong growth and dark green leaf colors for 3-7 days under natural light of a greenhouse, and transplanting the culture medium with the roots cleaned to the sterilized culture medium to be cultured into seedlings, so that the seedlings are obtained.
10. The tissue culture and rapid propagation method of the ethnic drug Alangium platanifolium according to any one of claims 1 to 8, wherein the culture conditions in the bud differentiation culture, proliferation culture operation and rooting culture operation are as follows: the culture temperature is 25-28 ℃, the illumination intensity is 2000-3000 lx, and the illumination time is 10-14 hours/day.
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