CN115250908B - Tissue culture propagation method of extremely endangered plant azalea - Google Patents
Tissue culture propagation method of extremely endangered plant azalea Download PDFInfo
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- CN115250908B CN115250908B CN202210353259.9A CN202210353259A CN115250908B CN 115250908 B CN115250908 B CN 115250908B CN 202210353259 A CN202210353259 A CN 202210353259A CN 115250908 B CN115250908 B CN 115250908B
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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
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Abstract
The invention discloses a tissue culture propagation method of an extremely endangered plant, namely, a Lipoacha, which is suitable for tissue culture and rapid propagation of the Lipoacha of the Ericaceae, and belongs to the technical field of plant biology. The invention uses the current year newly-developed tender stem segments of current year branches, and effectively solves the problems of rare population of the extremely endangered plant Lipoacha, and difficult introduction and domestication through a series of steps of explant disinfection, callus induction, differentiation, seedling strengthening and rooting. The tissue culture rapid propagation method provided by the invention has the advantages that the propagation coefficient is 10-20, the rooting rate is 80% -90%, the transplanting survival rate is 88% -94% within 1 month, the propagation coefficient of the azalea is greatly improved, a large number of regression seedlings are provided in a larger amount and at a higher speed, the rescue protection is realized, and the state of extinction of the endangered species is relieved.
Description
Technical Field
The invention relates to a plant tissue culture method in plant biotechnology, in particular to a tissue culture and rapid propagation method of endangered plant azalea.
Background
Rhododendron simsii (L.) belonging to the genus Rhododendron of the family EricaceaeRhododendronL.), which was first discovered in 1997 and formally published in 2003, was a unique rhododendron species distributed in the national level natural protection area of majoram in Guizhou, and reported by Chen Zhengren and the like that the species were distributed only in the protection area to form holes, the total number of which was about 37, and the Lipozzolan was classified as a very dangerous (CR) grade in The Red Listof Rhododendrons published in the international plant protection consortium (BGCI) 2013, and as a very dangerous grade in the "high-grade plant volume (2013) in the" red directory of Chinese biodiversity ".
At present, tissue culture and rapid propagation become an important means for producing seedlings of Chinese medicinal materials, flowers and endangered species. The study reports that the adaptability of the azalea plants to the culture medium is closely related to the genotype of the azalea plants, and the adaptability of different varieties of azalea plants to the culture medium is different. For the azalea plants, basic culture media suitable for tissue culture are reported to be Read, WPM,1/2MS,1/4MS and the like, but the azalea plants are rich in variety and large in inter-species difference, and element adjustment is needed to be carried out by combining different azalea subgenera and even subgroups during application.
To date, no research and report of propagation of the related biotechnology method of the azalea is available, in order to prevent the azalea from going to the extinct risk, solve the problems of natural starvation, low seed germination rate, difficult introduction and domestication and serious endangered condition of the azalea, provide a large number of regressive seedlings in larger quantity and faster speed, implement the rescuing protection, need to study the tissue culture of the azalea, and form a set of special tissue culture propagation technical system.
Disclosure of Invention
The invention aims to provide a tissue culture method of the azalea, which can implement the rescue protection under the condition of extremely endangered, accelerate the seedling propagation and provide a great number of regressive seedlings to provide a practical and effective method.
In order to achieve the purpose of the invention, the invention provides the following technical scheme:
preparation of modified WPM medium: the modified WPM culture medium is prepared by mixing NH in original culture medium 4 NO 3 400mg/L is reduced to 330mg/L, KNO in original culture medium 3 The concentration is reduced from 990mg/L to 780mg/L, and the rest elements remain unchanged.
A tissue culture propagation method of Lipoach comprises the steps of explant selection, disinfection, callus induction, differentiation, strong seedling and rooting, taking the newly developed tender stem of Lipoach in the current year after flowering as the explant, disinfecting, washing under tap water for 30s, then soaking and washing in 1L of aqueous solution for 3-5min by using 3ml of detergent to fix volume, washing cleanly by using clear water, washing to be foam-free, transferring to an ultra-clean workbench, disinfecting for 20s by using 75% alcohol, washing 3-5 times by using sterile water, carrying out surface disinfection for 8-12min by using 0.1% mercuric chloride solution (0.1 g mercuric chloride solid is dissolved and fixed volume is fixed in 100ml of water), and culturing as follows:
1) Callus induction: the callus induction culture medium is modified WPM+1.5mg/L2-iP (2-isopentenyl adenine) +0.5 mg/L2, 4-D (2, 4-dichlorophenoxyacetic acid) +1.0mg/L IAA (indoleacetic acid), pH is 5.8, sucrose 30g/L, 1800-strength agar 7g/L, pH is 5.8, and after dark culture for 7 days, light culture is carried out for 30-40 days, wherein the light dark period is 10h/14h;
2) And (3) differentiation culture: transferring the callus in the culture medium to modified WPM+0.8 mg/L2-iP+0.1 mg/L IAA+0.1mg/L IBA (indolebutyric acid), sucrose 30g/L, 1800-strength agar 7g/L, pH5.8, and differentiating culture for 30-40 days with a light-dark period of 12h/12h;
3) Strong seedling and rooting culture: cutting the differentiated seedling, transferring to a seedling strengthening and rooting integrated culture medium, namely improving WPM+2mg/L IAA, sucrose 35g/L, 1800-strength agar 7g/L, pH5.8 and light-dark period of 14h/10h.
The culture conditions are all that the light intensity is 2500LUX-3000LUX and the temperature is 20-23 ℃.
When transplanting, the young seedling grows to 3cm in height in the bottle, the stem thickness reaches 1.5mm-2mm, and after dense fibrous roots are found, the young seedling roots are washed under water, and then transplanting is carried out. The transplanting matrix is 1 part of perlite, 1 part of humus soil, 1 part of imported peat soil and 1 part of laterite. The temperature of the greenhouse is 20-30 ℃ and the humidity is 40-60%.
The technical proposal of the invention is based on the following research basis: the azalea is the only and extremely endangered plant of the azalea family in Guizhou province, which was discovered for the first time in 1997, formally published as a new species in 2003, reported by Chen Zhengren and the like to be distributed only in protected areas to hole mountains, the total number of which is about 37, and the azalea is classified as a very dangerous (CR) grade in The Red List of Rhododendrons published in 2013 of the International plant protection Consortium (BGCI) and as a very dangerous grade in the higher plant volume (2013) of the China biodiversity red directory. The number of the azalea population is small, the seed germination rate is low, the endangered condition is serious, and the artificial introduction and domestication are difficult. In order to solve the endangered situation, achieve mass propagation, carry out rescue protection, conservation and regression planting, the invention provides a tissue culture technology of the azalea.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention establishes an effective tissue culture and rapid propagation method of the azalea, and solves the current situation that the endangered condition is serious, the seed germination rate is low, and the artificial introduction and domestication are difficult.
2. The seedlings obtained through tissue culture and rapid propagation of the invention greatly shortens the seedling time, is easy to grow seedlings, maintains the excellent woody characters, can provide a large number of regression seedling continuation species, and is further developed and utilized by the market.
The propagation coefficient of the Lipoacha in the tissue culture rapid propagation method is 10-20 within 1 month, the rooting rate is 80% -90%, the transplanting survival rate is 88% -94%, the propagation coefficient of the Lipoacha is greatly improved, and a very effective propagation method is provided for the protection and the regression planting of the species.
The technical features of the present invention will be further described with reference to specific examples, but the present invention is not limited thereto.
Detailed Description
1. Method example 1:
taking a current year tender stem segment of a flowering azalea as an explant, sterilizing the explant as a conventional tissue culture explant, cutting wounds at two ends of the explant, washing the explant under tap water for 30s, soaking and washing the explant in 1L of aqueous solution for 3min with 3ml of detergent to a constant volume, washing the explant with sterile water until no foam is generated, transferring the washed product to an ultra-clean bench, sterilizing the product with 75% alcohol for 20s, washing the product with sterile water for 4 times, sterilizing the product with 0.1% mercuric solution (0.1 g mercuric solid is dissolved in a small amount of 95% absolute ethyl alcohol and then the volume is fixed in 100ml of water) for 9min, washing the product with sterile water for 5 times, cutting the sterilized tender stem segment with at least one axillary bud on each segment. Oblique insertion of axillary bud-carrying stem segments into callus induction medium: modified WPM+1.5 mg/L2-iP+0.5 mg/L2, 4-D+1.0mg/L IAA, pH5.8, 30g/L sucrose, 1800 strength agar 7g/L, and artificial auxiliary light induction for 35 days after dark induction for 7 days, wherein the light-dark period is 10h/14h. After callus grows out, transferring the callus into differentiation culture: modified WPM+1.0mg/L2-iP+0.1mg/L IAA+0.1mg/L IBA, 30g/L sucrose, 1800 strength agar 7g/L, and light-dark period of 12h/12h for 33 days; after the seedlings are differentiated, the seedlings are separated for strengthening and rooting, and the seedlings are transferred into a seedling strengthening and rooting integrated culture medium which is modified WPM+2mg/L IAA, sucrose 35g/L and 1800-strength agar 7g/L, pH is 5.8, and the light and dark period is 14h/10h. The culture conditions are all that the light intensity is 2500LUX-3000LUX and the temperature is 20-23 ℃. When the seedlings grow to 3cm in the bottle, the stem thickness reaches 1.5mm-2mm, the seedlings are taken out of the bottle to be acclimatized after dense fibrous roots are present, the seedlings are taken out after 30 days, and the seedling root culture medium is washed under running water and then transplanted. The transplanting matrix is 1 part of perlite, 1 part of humus soil, 1 part of imported peat soil and 1 part of laterite. The temperature of the greenhouse is 20-30 ℃ and the humidity is 40-60%.
2. Method example 2:
picking up current year's twig after flowering of Rhododendron simsii, cutting off leaves, inserting into water, and taking it back to laboratory. Rinse under tap water for 30s. 3ml of detergent is taken and dissolved in 1L of tap water, the material is soaked in the detergent, the surface of the detergent is brushed for 5min by a small brush, the detergent is placed under flowing tap water for 5min, and then the detergent is washed for 3 times by sterile water. Taking the material onto an ultra-clean bench, sterilizing with 75% alcohol for 20s, washing with sterile water for 4 times, preparing 0.1% mercuric chloride solution (0.1 g mercuric chloride constant volume to 1L), sterilizing the field material for 11min, sterilizing for 7min, continuously oscillating, washing with sterile water for 3 times, sterilizing with 0.1% mercuric chloride for 4min, and washing with sterile water for 5 times. Cutting the material into a bud segment, and obliquely inserting the bud segment into a callus induction culture medium, namely, modified WPM+1.5mg/L2-iP+0.5mg/L2, 4-D+1.0mg/L IAA, sucrose 30g/L, 1800-strength agar 7g/L and pH5.8; the artificial auxiliary light induction is carried out after the dark induction is carried out for 7 days, the light intensity is 2500LUX-3000LUX, the light dark period is 10h/14h, and the temperature is 18-23 ℃. After 35 days of culture, calli were cut into 0.5cm x 0.5cm pieces on a super clean bench and inoculated into differentiation medium: modified WPM+1.0mg/L2-iP+0.1 mg/L IAA+0.1mg/L IBA, 30g/L sucrose, 7 g/L1800-strength agar, pH5.8, and performing differentiation culture with light intensity of 2500LUX-3000LUX, light-dark period of 12h/12h, and temperature of 18-23deg.C. After differentiation for 40 days, separating the seedlings, transferring the seedlings to a strong seedling and rooting integrated culture medium for improving WPM+2mg/L IAA, culturing in sucrose 35g/L, 1800-strength agar 7g/L and pH5.8, wherein the light intensity is 2500LUX-3000LUX, the light dark period is 14h/10h, and the illumination condition of the strong seedling and rooting integrated culture is artificial auxiliary light 2500LUX-3000LUX, and the temperature is 18-23 ℃. After culturing for 40 days, selecting 3cm high, and seedling with stem thickness of 1.5mm-2mm and thick fibrous root for outdoor hardening, and according to the condition in bottle seedling, generally taking 20-30 days, transplanting when the leaf turns dark green, and cleaning root culture medium with tap water before transplanting. The transplanting matrix is 1 part of perlite, 1 part of humus soil, 1 part of imported peat soil and 1 part of laterite (before transplanting, the transplanting matrix needs to be sprayed and disinfected by 800 times of carbendazim), and the pH is 5.8. After transplanting, placing the plant in a greenhouse, wherein the culture temperature is 20-30 ℃ and the humidity is 40-60%. 10-20 differentiated test-tube seedlings can be obtained from one stem within 30-40 days, the seedlings with the length of more than 2cm are about 70%, the rooting rate is 80-90%, and the transplanting survival rate is 88-94%.
Drawings
FIG. 1 is a diagram of the differentiation seedling of Rhododendron simsii.
Claims (1)
1. The tissue culture propagation method of the azalea includes the steps of explant selection, disinfection, callus induction, differentiation, seedling strengthening and rooting, and is specifically characterized by comprising the following steps: taking a new tender stem section of a flowering current year of a azalea as an explant, sterilizing, firstly washing under tap water for 30s, then soaking and cleaning in 1L of water solution for 3-5min by using 3ml of detergent, then washing cleanly by using clear water, washing with sterile water until no foam exists, transferring to an ultra-clean workbench, sterilizing for 20s by using 75% alcohol, washing 3-5 times by using sterile water, sterilizing for 8-12min by using 0.1% mercuric chloride solution (0.1 g mercuric chloride solid is dissolved and then fixed in 100ml water), and culturing as follows:
1) Medium improvement: the modified WPM culture medium is prepared by mixing NH in original culture medium 4 NO 3 400mg/L is reduced to 330mg/L, KNO in original culture medium 3 The concentration is reduced from 990mg/L to 780mg/L, and the rest elements are kept unchanged;
2) Callus induction: the callus induction culture medium is modified WPM+1.5mg/L2-iP (2-isopentenyl adenine) +0.5 mg/L2, 4-D (2, 4-dichlorophenoxyacetic acid) +1.0mg/L IAA (indoleacetic acid), pH is 5.8, sucrose 30g/L, 1800-strength agar 7g/L, pH is 5.8, and after dark culture for 7 days, light culture is carried out for 30-40 days, and the light dark period is 10h/14h;
3) And (3) differentiation culture: transferring the callus in the culture medium to modified WPM+0.8 mg/L2-iP+0.1 mg/L IAA+0.1mg/L IBA (indolebutyric acid), sucrose 30g/L, 1800-strength agar 7g/L, pH5.8, and differentiating culture for 30-40 days with a light-dark period of 12h/12h;
4) Strong seedling and rooting culture: cutting the differentiated seedlings, transferring the cut differentiated seedlings to a seedling strengthening and rooting integrated culture medium, namely improving WPM+2mg/L IAA, sucrose 35g/L, 1800-strength agar 7g/L, pH5.8 and light-dark period of 14h/10h;
the culture conditions are that the light intensity is 2500LUX-3000LUX and the temperature is 20-23 ℃;
during transplanting, the seedlings grow to 3cm in height in the bottle, the stem thickness reaches 1.5mm-2mm, and after dense fibrous roots are found, the roots of the seedlings are washed under water, and then transplanting is carried out; the transplanting matrix is 1 part of perlite, 1 part of humus soil, 1 part of imported peat soil and 1 part of laterite; the temperature of the greenhouse is 20-30 ℃ and the humidity is 40-60%.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102090337A (en) * | 2010-12-13 | 2011-06-15 | 江苏省农业科学院 | Rapid propagation method of rhododendron latoucheae |
| CN108935106A (en) * | 2018-08-31 | 2018-12-07 | 贵州思源农旅综合开发有限公司 | A kind of tissue culture propagation and in-vitro conservation method of critical endangered plant Guizhou great Hua cuckoo |
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- 2022-04-06 CN CN202210353259.9A patent/CN115250908B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102090337A (en) * | 2010-12-13 | 2011-06-15 | 江苏省农业科学院 | Rapid propagation method of rhododendron latoucheae |
| CN108935106A (en) * | 2018-08-31 | 2018-12-07 | 贵州思源农旅综合开发有限公司 | A kind of tissue culture propagation and in-vitro conservation method of critical endangered plant Guizhou great Hua cuckoo |
Non-Patent Citations (1)
| Title |
|---|
| 蒋淑磊等.照山白杜鹃愈伤组织诱导及植株再生技术.《分子植物育种》.2022,第20卷(第5期),1629-1634. * |
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