CN103348917B - Rapid propagation method of ginger virus-free seedlings by one step - Google Patents
Rapid propagation method of ginger virus-free seedlings by one step Download PDFInfo
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- CN103348917B CN103348917B CN201310301668.5A CN201310301668A CN103348917B CN 103348917 B CN103348917 B CN 103348917B CN 201310301668 A CN201310301668 A CN 201310301668A CN 103348917 B CN103348917 B CN 103348917B
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Abstract
The invention discloses a rapid propagation method of ginger virus-free seedlings by one step. The rapid propagation method comprises the following steps: A, selecting tender shoots germinating on ginger rhizomes as explants; B, carrying out heat treatment to the explants, so as to detoxify the explants; C, disinfecting the detoxified explants with ethyl alcohol and mercury bichloride; D, peeling stem tips by a dissecting microscope; E, inducing the stem tips to grow micro ginger cluster buds under light condition and constant temperature condition; F, carrying out virus detection on the micro ginger cluster buds by an enzyme-linked immunosorbent assay (ELISA); G, carrying out subculture multiplication culture on the virus-free micro ginger cluster buds; H, transplanting virus-free micro ginger cluster buds with roots after plant division. By the utilization of the rapid propagation method, various viruses and germs in ginger bodies are removed effectively, germ-free and virus-free ginger seedlings are cultured, virus-free healthy cultivation of the ginger is realized, space occupation of propagation is small, propagation speed is high, one virus-free bud can propagate more than hundred thousand virus-free ginger seedlings for one year, a problem of virus-free ginger seedling supplying in plantation of ginger is solved effectively, good quality of the ginger is recovered, the disease occurrence is reduced, and the output and quality of the ginger are improved.
Description
Technical field
The invention belongs to field of plant tissue culture technique, be specifically related to a kind of ginger one step and become detoxic seedling method for quickly breeding, the method utilizes minimal medium and additional hormon composition to reach the Fast-propagation of detoxification ginger seedling.
Background technology
Ginger (Zingiber officinale Rosc.) belongs to Zingiber ginger and belongs to perennial root herbaceous plant, and young ginger can be cooked vegetables, and old ginger can be used as spices and medicine, is a kind of important both for medicine and food economic crops.The weather of ginger happiness warm and moist, cold-resistant, be afraid of humidity, be afraid of high light direct projection, be the good assistant that hill farmer is shaken off poverty and set out on the road to prosperity.Because ginger does not bloom or seldom blooms, so the long-term vegetative propagation of ginger causes the multiple germ virus of ginger cylinder accumulation, wherein bacterium is the most serious with the withered false pseudomonas bacillus of green grass or young crops, and this bacterium is very easily propagated by kind of a ginger accumulation, brings out ginger plague.Oneself becomes the restraining factors of ginger production development ginger plague, the morbidity general underproduction 20～30% in plot, and severe one reaches 50～70%, even without receiving; Plant the accumulation of ginger virus and conventionally cause Ginger Yield to decline 5～45%, make its quality reduce, resistance decline simultaneously.Meanwhile, in conventional cultivation, get the kind ginger stripping and slicing plantation of rudiment, per hectare need be planted ginger 4500～6000kg, not only consumes a large amount of commodity ginger, and needs a large amount of soils to reserve seed for planting, and production cost is higher.Ginger is very large in China's cultivated area, by group, is trained with thermal treatment a large amount of virus-elimination seedlingses can be provided, and reduces infecting of bacterial wilt of ginger and virus disease, increases substantially output and quality.
Summary of the invention
The object of the invention is to be to provide a kind of ginger one step to become detoxic seedling method for quickly breeding, easy to implement the method, easy to operate, reproduction speed is fast, can accelerate detoxification ginger Fast-propagation.
In order to achieve the above object, the present invention adopts following technical measures:
Ginger one step becomes detoxic seedling method for quickly breeding, the steps include:
A, choose cadmium yellow, glossy, bud is many and without the ginger piece of scar, full stalwartness, after running water is rinsed well, is embedded in clean husky bed vernalization under 20 ℃～25 ℃ conditions.When tender shoots grows to 1～2cm length, with blade, cut, with running water, rinse and remove sandy soil;
B, tender shoots are placed in insulating box, process 5min at 50 ℃ to complete detoxification;
C, on superclean bench, with the alcoholic solution of 75% volume ratio, soak 10～30s, aseptic water washing, then with 0.1% weight ratio mercuric chloride solution sterilization 10～12min, sterile water rushes 3～5 times;
D, drain away the water after, under aseptic condition, treated material is dissected under anatomical lens, cut meristematic tissue 0.1～0.3mm growing point, be inoculated in inducing culture rapidly.The formula of inducing culture is: MS+KT1.0～1.5mg/L+NAA0.5～0.6mg/L+ agar 4000～6000mg/L+ white sugar 20000～30000mg/L+pH5.8;
E, illumination cultivation, temperature is 25～28 ℃, light intensity 1500～3000Lx, illumination every day 10～16h, cultivates 30～60d, grows miniature Jiang Cong bud;
F, from each miniature Jiang Cong bud, select 1 strain, the above vegetable material of clip bud basal part of stem 0.5cm.By enzyme linked immunosorbent assay (ELISA kit) (the biological Co., Ltd of Shanghai Yan Ji), carry out cucumber mosaic virus (CMT) and tobacco mosaic virus (TMV) detection, the miniature Jiang Cong bud that detoxification is unclean is rejected, nontoxic miniature Jiang Cong bud subculture is cultivated;
G, the miniature Jiang Cong bud of detoxification is cut into the miniature Jiang Cong bud of 0.4cm～0.6cm size, each bud stem is cut from base portion 0.5～1cm, and removes all, proceeds to the medium in D step, carry out the shoot proliferation of bud and cultivate, later every 25～35d subculture 1 time;
H, according to producing season, subculture is cultivated to miniature Jiang Cong bud uncork hardening 5～7d of 25～35d, the band root seedling that 3～7cm is high is divided into single, with 0.003%～0.005% weight ratio NAA solution, soak shoot root base portion 10～15min, to organize training transplantation of seedlings to the matrix (peat composed of rotten mosses/perlite/vermiculite=2/1/1, volume ratio) in, water sufficient normal root water, cover little shed film heat and moisture preserving, early stage, temperature remained on 15～25 ℃, humidity 80～90%, and after 7～15d, take off film 1h or 2h noon, after extend gradually and take off the film time, until overlay film not.Every 2～3 weeks, water and execute 0.1%～0.3% weight ratio urea and 0.1%～0.3% weight ratio KH
2pO
4or thin liquid dung, note carrying out the extermination of disease and insect pest, group training shoot survival percent reaches more than 95%, will survive transplantation of seedlings.
The composition of described MS medium (unit: mg/L):
The present invention compares with general planting technology, has the following advantages and effect:
The foundation of detoxification ginger forming seedling through one step culture rapid propagation system, can effectively solve the detoxification ginger seedling supply problem in ginger cultivation.The production system set up is stable, and that breeding takes up room is little, the cycle is short, reproduction rate is high, with low cost, can meet the large-scale production of ginger seedling.The method, in medium layoutprocedure, replaces sucrose with white sugar, with running water, replaces distilled water, effectively reduces production costs.Detoxification Jiang Miao from explant induction, breed, take root completes a kind of medium, reduces different culture media configuration step and culture of rootage switch over operation step.Utilize miniature Jiang Cong bud shoot proliferation, planting percent is high, and plant is sturdy, well developed root system, and the production cycle shortens.The propagation multiple of 30～50d reaches 7～10 times, and within 1 year, a detoxification bud can be bred 100,000 above detoxification ginger seedlings.With a bud 40d subculture once, each breeding rate is 7, and front twice subculture simple bud do not breed calculating.
| Number of days (d) | 30 | 70 | 110 | 150 | 190 | 230 | 270 | 310 | 350 |
| Seedling number () | 1 | 1 | 1 | 7 | 49 | 343 | 2401 | 16807 | 117649 |
Embodiment
Embodiment 1:
Ginger one step becomes detoxic seedling method for quickly breeding, the steps include:
1. choose cadmium yellow, glossy, bud is many and without the ginger piece of scar, full stalwartness, after running water is rinsed well, be embedded in clean husky bed, vernalization under 20 ℃ or 21 ℃ or 22 ℃ or 24 ℃ or 24 ℃ or 25 ℃ of conditions.When tender shoots grows to 1cm or 1.5cm or 2cm length, with blade, cut, through running water, rinse and remove sandy soil;
2. tender shoots is placed in insulating box, processes 5min at 50 ℃ to complete detoxification;
3. on superclean bench, the alcoholic solution by 75% volume ratio soaks 10s or 15s or 20s or 25s or 30s, aseptic water washing 3 times, then use 0.1%(weight ratio) and mercuric chloride solution sterilization 10min or 11min or 12min, sterile water punching 3 times or 4 times or 5 times;
4. after draining away the water, under aseptic condition, treated material is dissected under anatomical lens, cut the most advanced and sophisticated 0.1mm of meristematic tissue or 0.2mm or 0.3mm growing point, be inoculated in inducing culture rapidly.The effective ingredient of inducing culture is: MS+KT1.0mg/L+NAA0.5mg/L+ agar 5000mg/L+ white sugar 30000mg/L, pH5.8;
5. illumination cultivation, temperature is 25 ℃ or 26 ℃ or 27 ℃ or 28 ℃, light intensity 1500Lx or 2000Lx or 2500Lx or 3000Lx, illumination every day 10h or 12h or 14h or 16h, cultivate 30d or 40d or 50d or 60d, grows miniature Jiang Cong bud;
6. from each miniature Jiang Cong bud, select 1 strain, the above vegetable material of clip bud basal part of stem 0.5cm.By enzyme linked immunosorbent assay (ELISA kit) (the biological Co., Ltd of Shanghai Yan Ji), carry out cucumber mosaic virus (CMT) and tobacco mosaic virus (TMV) detection, the miniature Jiang Cong bud that detoxification is unclean is rejected, nontoxic miniature Jiang Cong bud subculture is cultivated;
7. the miniature Jiang Cong bud of detoxification is cut into the miniature Jiang Cong bud of 0.4cm or 0.5cm or 0.6cm size, each bud stem is cut from base portion 0.5cm or 0.6cm or 0.7cm or 0.8cm or 0.9cm or 1cm, and remove all, proceed to the shoot proliferation that carries out bud in the medium in step 4 and cultivate, later every 25d or 30d or 35d subculture 1 time;
8. according to producing season, subculture is cultivated to miniature Jiang Cong bud uncork hardening 5d or 6d or the 7d of 25d or 30d or 35d, the band root seedling of height of seedling 3cm or 4cm or 5cm or 6cm or 7cm is divided into single seedling, with 0.003%～0.005% weight ratio NAA solution, soak shoot root base portion 12min, to organize training transplantation of seedlings to the matrix (peat composed of rotten mosses/perlite/vermiculite=2/1/1, volume ratio) in, water sufficient normal root water, consumption take wet all over medium of seedling bed be degree, cover little shed film heat and moisture preserving, early stage, temperature remained on 15 ℃ or 17 ℃ or 19 ℃ or 21 ℃ or 23 ℃ or 25 ℃, more than humidity 80% or 85% or 90%, after 7d or 9d or 11d or 13d or 15d, progressively take off film, take off film 1h or 2h noon, extend and take off the film time gradually, during taking off film, keep mean temperature at 15 ℃～25 ℃, humidity is 80%～90%.Every 2～3 weeks, water and execute the urea of 0.2% weight ratio and the KH of 0.1% weight ratio
2pO4 or thin liquid dung, guarantee that canopy rich water quality management condition in interior vegetative period is consistent, notes carrying out the extermination of disease and insect pest, and group training shoot survival percent reaches more than 95%.
Embodiment 2:
Ginger one step becomes detoxic seedling method for quickly breeding, the steps include:
1. choose cadmium yellow, glossy, bud is many and without the ginger piece of scar, full stalwartness, after running water is rinsed well, is embedded in clean husky bed vernalization under 25 ℃ of conditions.When tender shoots grows to 1.5cm length, with blade, cut, through running water, rinse and remove sandy soil;
2. tender shoots is placed in insulating box, processes 5min at 50 ℃ to complete detoxification;
3. on superclean bench, the alcoholic solution by 75% volume ratio soaks 20s, aseptic water washing 3 times, then use 0.1%(weight ratio) and mercuric chloride solution sterilization 12min, sterile water punching 5 times;
4. after draining away the water, under aseptic condition, to dissect treated material under anatomical lens, cut meristematic tissue 0.2mm growing point, be inoculated in inducing culture rapidly.The effective ingredient of inducing culture is: MS+KT1.0mg/L+NAA0.5mg/L+ agar 5000mg/L+ white sugar 30000mg/L, pH5.8;
5. illumination cultivation, temperature is 25 ℃, light intensity 2000Lx, illumination every day 14h, cultivates 30d, and stem apex expands and induces miniature Jiang Cong bud;
6. from each miniature Jiang Cong bud, select 1 strain, the above vegetable material of clip bud basal part of stem 0.5cm.By enzyme linked immunosorbent assay (ELISA kit) (the biological Co., Ltd of Shanghai Yan Ji), carry out cucumber mosaic virus (CMT) and tobacco mosaic virus (TMV) detection, the miniature Jiang Cong bud that detoxification is unclean is rejected, nontoxic miniature Jiang Cong bud subculture is cultivated;
7. the miniature Jiang Cong bud of detoxification is cut into the miniature Jiang Cong bud of 0.5cm size, each bud stem is from cutting apart from base portion 0.5cm, and removes all, proceeds to the shoot proliferation that carries out bud in the medium in step 4 and cultivates, later every 30d subculture 1 time;
8. according to producing season, subculture is cultivated to the miniature Jiang Cong bud uncork hardening 5d of 30d, the band root seedling of height of seedling 6cm is divided into single seedling, with 0.004% weight ratio NAA solution, soak shoot root base portion 12min, to organize training transplantation of seedlings to the matrix (peat composed of rotten mosses/perlite/vermiculite=2/1/1, volume ratio) in, water sufficient normal root water, consumption take wet all over medium of seedling bed be degree, cover little shed film heat and moisture preserving, early stage, temperature remained on 21 ℃, humidity is 85%, after 7d, take off film 1h noon, progressively extend and take off the film time, until overlay film not, during taking off film, maintain the temperature at 18～20 ℃, humidity is 85%.Every 2 weeks, water and execute the urea of 0.2% weight ratio and the KH of 0.1% weight ratio
2pO
4, guarantee that canopy rich water quality management condition in interior vegetative period is consistent, note carrying out the extermination of disease and insect pest, group training shoot survival percent reaches 98%.
Claims (2)
1. ginger one step becomes detoxic seedling method for quickly breeding, the steps include:
A, choose cadmium yellow, glossy, bud is many and without the ginger piece of scar, full stalwartness, after running water is rinsed well, be embedded in clean husky bed vernalization under 20 ℃～25 ℃ conditions, when tender shoots grows to 1～2cm length, with blade, cut, through running water, rinse and remove sandy soil;
B, tender shoots are placed in insulating box, process 5min at 50 ℃ to complete detoxification;
C, on superclean bench, with the alcoholic solution of 75% volume ratio, soak 10～30s, aseptic water washing, then with 0.1% weight ratio mercuric chloride solution sterilization 10～12min, sterile water rushes 3～5 times;
D, drain away the water after, under aseptic condition, treated material is dissected under anatomical lens, cut meristematic tissue tip 0.1～0.3mm growing point, be inoculated in inducing culture, the formula of inducing culture is: MS+KT1.0～1.5mg/L+NAA0.5～0.6mg/L+ agar 4000～6000mg/L+ white sugar 20000～30000mg/L, pH5.8;
E, illumination cultivation, temperature is 25～28 ℃, light intensity 1500～3000Lx, illumination every day 10～16h, cultivates 30～60d, grows miniature Jiang Cong bud;
F, from each miniature Jiang Cong bud, select 1 strain, the above vegetable material of clip bud basal part of stem 0.5cm, carries out cucumber mosaic virus and tobacco mosaic virus detects with ELISA kit, and the miniature Jiang Cong bud that detoxification is unclean is rejected, and nontoxic miniature Jiang Cong bud subculture is cultivated;
G, the miniature Jiang Cong bud of detoxification is cut into the miniature Jiang Cong bud of 0.4cm～0.6cm size, each bud stem cuts away from base portion 0.5～1cm, and removes all, proceeds to the medium in D step, carry out the shoot proliferation of miniature Jiang Cong bud and cultivate, later every 25～35d subculture 1 time;
H, according to producing season, subculture is cultivated to miniature Jiang Cong bud uncork hardening 5～7d of 25～35d, the band root seedling that 3～7cm is high is divided into single, with 0.003%～0.005% weight ratio NAA solution, soak shoot root base portion 10～15min, to organize training transplantation of seedlings in matrix, water sufficient normal root water, cover little shed film heat and moisture preserving, early stage, temperature remained on 15～25 ℃, humidity 80～90%, takes off film after 7～15d, extend and take off the film time gradually, until overlay film not watered every 2～3 weeks and executes 0.1%～0.3% weight ratio urea and 0.1%～0.3% weight ratio KH
2pO
4or thin liquid dung, will survive transplantation of seedlings;
Described matrix is: the peat composed of rotten mosses/perlite/vermiculite=2/1/1, volume ratio.
2. propagation method according to claim 1, is characterized in that, during taking off film, keeps mean temperature at 15 ℃～25 ℃, and humidity is 80%～90%.
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| CN105475135A (en) * | 2015-12-04 | 2016-04-13 | 昆明学院 | Method for tissue culturing and rapid propagation of Dichorisandra thyrsiflora |
| CN105766646B (en) * | 2016-03-31 | 2018-04-10 | 内蒙古自治区农牧业科学院 | A kind of method for tissue culture of vesicle grass |
| CN106922528B (en) * | 2017-03-13 | 2019-02-22 | 仲恺农业工程学院 | Method for promoting rapid in-vitro propagation of mioga ginger |
| CN107372104A (en) * | 2017-06-15 | 2017-11-24 | 绍兴益康生物科技有限公司 | A kind of detoxicated ginger takes off the rapid propagation method of bacterium |
| CN111771725A (en) * | 2020-07-31 | 2020-10-16 | 潍坊兴旺生物种业有限公司 | Optimized ginger virus-free seedling regeneration propagation method and development of industrialization process thereof |
| CN111837959A (en) * | 2020-08-05 | 2020-10-30 | 四川农业大学 | Micro ginger block induction method based on ginger test-tube plantlet and application |
| CN113598000B (en) * | 2021-08-27 | 2022-08-30 | 湖北省农业科学院经济作物研究所 | Method for one-step formation of ginger rootless tissue culture seedlings into field seedlings |
| CN115281081B (en) * | 2021-11-22 | 2023-05-12 | 湘西土家族苗族自治州农业科学研究院 | Breeding method of miniature test tube detoxified ginger seeds |
| CN115589945A (en) * | 2022-10-10 | 2023-01-13 | 长江大学（Cn） | Method for openly culturing ginger test-tube plantlets |
| CN115735767A (en) * | 2022-11-10 | 2023-03-07 | 潍坊峡兴农业科技有限公司 | Rapid propagation method of sugar-free virus-free ginger seedlings |
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| KR0160086B1 (en) * | 1995-06-21 | 1998-11-16 | 김광희 | The method of cultivating ginger seedlings |
| JP2007259749A (en) * | 2006-03-28 | 2007-10-11 | Kirin Agribio Co Ltd | Method for proliferating plant, characterized by ethylene treatment |
| CN1930951B (en) * | 2006-09-29 | 2010-12-01 | 徐坤 | Method of cultivating detoxicated ginger seedling |
| CN102144565A (en) * | 2011-03-03 | 2011-08-10 | 中国药科大学 | Method for cultivating ginger-free viruses and cultivating virus-free seedlings by means of propagating and rooting synchronously |
| CN102939903A (en) * | 2012-11-23 | 2013-02-27 | 绍兴文理学院 | Ginger detoxification, sterilization and rapid propagation method |
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