CN103875529A - Blueberry tissue culture propagation and ex-vitro rooting method - Google Patents
Blueberry tissue culture propagation and ex-vitro rooting method Download PDFInfo
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- CN103875529A CN103875529A CN201410054055.0A CN201410054055A CN103875529A CN 103875529 A CN103875529 A CN 103875529A CN 201410054055 A CN201410054055 A CN 201410054055A CN 103875529 A CN103875529 A CN 103875529A
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Abstract
The invention relates to the technical field of industrialized seedling production and in particular relates to a blueberry tissue culture propagation and ex-vitro rooting method. The method mainly aims to solve the technical problems that rapid industrialized blueberry industrialized seedling production is restrained due to low rooting rate, long rooting period, complex rooting program, high rooting cost and the like during in-vitro blueberry rooting. The method comprises the following steps: selecting and sterilizing an explant, inducing lateral bud germination, performing multiplication culture, and finally performing ex-vitro rooting, wherein in the culture medium for inducing lateral bud germination, the components of the culture medium comprises an improved WPM basic culture medium, 0.8-2mg/L of ZT, 25-35mg/L of white sugar and 5-10g/L of agar; the pH value is regulated to be 5.4; according to the improved WPM basic culture medium, the original potassium sulfate and calcium chloride are replaced by calcium nitrate hydrate and potassium nitrate.
Description
Technical field
The present invention relates to a kind of industrial breeding technique field, a kind of method that especially relates to blueberry tissue culture and expand numerous and outside sprout-cultivating-bottle.
Background technology
Blueberry (
vaccinium spp.), Ericaceae Vaccinium.Fruit bluish violet, unique flavor, nutritious.Blueberry nutrient composition is not limited only to fructose, fiber, vitamin A and C, the more important thing is compounds such as being rich in antioxidant, bacterial growth inhibitor, folic acid, anthocyanin, removing, inhibition and reverse human senility for people's interior free yl have substantial connection, and therefore the international food and agricultural organization of Ye Bei classifies one of the mankind's five large healthy food as.
Blueberry not only has the good world, domestic market prospect, and is changing agriculture forestry planting industry structure, can take the course of its own in the aspect such as improve the ecological environment, and is the ideal that can take into account social benefit, economic benefit and the ecological benefits kind that cultivates apple trees.China starts introducing and planting in phase early 1980s, and current cultivation place is developed rapidly from north to south.
The method of blueberry sapling multiplication is more, as cuttage, green wood cutting, division propagation, seminal propagation, grafting, tissue cultivation etc.At present, the blueberry of China cultivation on producing normally taking cottage propagation as main.In recent years, by scientific research personnel's continuous effort, and further investigation to tissue culture technique, for having explored a kind of blueberry propagation method that can be comparatively simple and quick in various places, tissue is cultivated factorial seedling-culturing method and has been widely used in blueberry seedling breeding.The development of this technology and application, produce the seedling that a large amount of nontoxic high-qualitys are provided to blueberry, become gradually the direction that blueberry high quality seedling is produced from now on.But in blueberry tissue incubation, adopt the technology of taking root in bottle to have many problems, as: rooting rate is low, the cycle of taking root is long, the program of taking root is complicated and the cost of taking root is more high, and the quick factorial seedling growth of blueberry cannot be realized.
Summary of the invention
A kind of method that the present invention is to provide blueberry tissue culture and expands numerous and outside sprout-cultivating-bottle, it is mainly to solve that while taking root in blueberry bottle, rooting rate is low, the cycle of taking root is long, the program of taking root is complicated and the technical problem of the quick factorial seedling growth of the more high restriction blueberry of the cost of taking root.
Above-mentioned technical problem of the present invention is mainly solved by following technical proposals:
A kind of blueberry tissue culture of the present invention expands the method for numerous and outside sprout-cultivating-bottle, it is characterized in that described method comprises:
A. the choosing and sterilizing of explant: select robust growth without damage by disease and insect plant, clip semi-lignified branch, remove blade, under flowing water, rinse, on superclean bench, carry out disinfection with alcohol, then use aseptic water washing 2-3 time, proceed in mercuric chloride solution and carry out disinfection, then use aseptic water washing 3-4 time;
B. induce sprouting of lateral bud: in superclean bench, sterilizable material obtained in the previous step is inoculated in the medium of induction sprouting of lateral bud, nutrient media components is: WPM minimal medium+ZT0.8-2mg/L+ white sugar 25-35g/L+ agar 5-10g/L of improvement, pH is adjusted to 5.4, and it is to replace original potassium sulfate and calcium chloride with nitric hydrate calcium and potassium nitrate that the WPM wherein improveing cultivates substantially;
C. propagation is cultivated: the stem section that aseptic lateral bud obtained in the previous step is cut into 2-3cm proceeds to subculture in proliferated culture medium and expands numerous, proliferation culture medium formula component is: WPM minimal medium+ZT0.8-2mg/L+ white sugar 25-35g/L+ agar 5-10g/L of improvement, and pH is adjusted to 5.4;
D. outside sprout-cultivating-bottle: by healthy and strong 8-10cm obtained in the previous step aseptic seedling, open bottle cap, be covered with wet towel at ventilation hardening 2-3 days, note during this time keeping towel in moisture state, before transplanting domestication, seedbed and matrix are processed and sterilized, matrix adopts domestic one-level sphagna, sterilization after washing, drying, pulverizing, the thickness 4-6cm in seedbed; To be cut into 3-4cm segment through the healthy and strong blueberry tissue culture seedling of ventilation hardening, cuttage is in above-mentioned seedbed, average every strain can be cut into 2-3 section, every section of need of cuttage process dip root-growing agent, seedbed overlay film after cuttage completes, and in canopy, relative moisture is controlled at 60-80%, temperature is controlled at 18-25 DEG C, every a water of spray in 2-3 days, after this sprayed once the more foliage fertilizer of flower every 10 days, finally obtain the blueberry finished product of taking root.
The present invention adopts the WPM minimal medium medium of improvement, replace original potassium sulfate and calcium chloride with nitric hydrate calcium and potassium nitrate, thereby make the propagation multiple of blueberry in propagation incubation improve 2-3 doubly, adopt outside sprout-cultivating-bottle technology, reduce the rooting process in tissue culture bottle, thereby shorten cycle of factorial seedling growth, reduce group training cost, improved the survival rate of transplanting hardening simultaneously, in outside sprout-cultivating-bottle process, every strain group training seedling is adopted to the mode of cutting segment cuttage, its multiplication rate exceeds 2-3 doubly compared with the blueberry seedling of taking root in conventional bottle, thereby reduce the cost of transplanting domestication.
As preferably, in described step a, the concentration of mercuric chloride solution is 0.1%.
As preferably, the sterilization of described steps d mesostroma need be the carbendazim sterilization 24 hours of 1000 times by concentration.
As preferably, described root-growing agent is 1.0mg/L NAA.
Embodiment
Below by embodiment, technical scheme of the present invention is described in further detail.
Embodiment 1: a kind of blueberry tissue culture of this example expands the method for numerous and outside sprout-cultivating-bottle, the steps include:
A. the choosing and sterilizing of explant: select robust growth without damage by disease and insect plant, clip 8-10cm semi-lignified branch, remove blade, under flowing water, rinse 15-20 minute, 75% alcohol disinfecting 30s in superclean bench, then aseptic water washing 2-3 time, proceeds in 0.1% mercuric chloride and sterilizes 8 minutes, aseptic water washing 3-4 time;
B. induce sprouting of lateral bud: in superclean bench, sterilizable material obtained in the previous step is inoculated in the medium of induction sprouting of lateral bud, nutrient media components is: WPM minimal medium+ZT 1.0mg/L+ white sugar 25g/L+ agar 7g/L of improvement, pH is adjusted to 5.4;
C. propagation is cultivated: the stem section that aseptic lateral bud obtained in the previous step is cut into 2-3cm proceeds in proliferated culture medium subculture and expands numerously, and proliferation culture medium formula component is: WPM minimal medium+ZT 0.5mg/L+ white sugar 25g/L+ agar 7g/L of improvement, and pH is adjusted to 5.4;
D. outside sprout-cultivating-bottle: by healthy and strong 8-10cm obtained in the previous step aseptic seedling, open bottle cap, be covered with wet towel at ventilation hardening 2-3 days, note during this time keeping towel in moisture state.Before transplanting domestication, seedbed and matrix are processed and sterilized, matrix adopts domestic one-level sphagna, needs, with 1000 times of carbendazim sterilizations 24 hours, then to wash, dry, pulverize, the thickness 5cm in seedbed; To be cut into 3-4cm segment through the healthy and strong blueberry tissue culture seedling of ventilation hardening, cuttage is in above-mentioned seedbed, average every strain becomes 2-3 section as seen, every section of need of cuttage process dip the root-growing agent of 0.5mg/L NAA, seedbed overlay film after cuttage completes, in canopy, relative moisture is controlled at 60-80%, temperature is controlled at 18-25 DEG C, every a water of spray in 2-3 days, within 7-10 days, observe and have new root to generate, the rooting rate of 20 days, more than 80%, after this sprayed once the more foliage fertilizer of flower every 10 days, the survival rate of transplanted seedling is the highest obtains 90%.
Its condition of culture of setting up of above-mentioned blueberry sterile system is: 25 ± 2 DEG C of temperature, intensity of illumination 2500-3000lx, light application time 14-16 hour.
Embodiment 2: a kind of blueberry tissue culture of this example expands the method for numerous and outside sprout-cultivating-bottle, the steps include:
A. the choosing and sterilizing of explant: select robust growth without damage by disease and insect plant, clip 8-10cm semi-lignified branch, remove blade, under flowing water, rinse 15-20 minute, 75% alcohol disinfecting 30s in superclean bench, then aseptic water washing 2-3 time, proceeds in 0.1% mercuric chloride and sterilizes 10 minutes, aseptic water washing 3-4 time;
B. induce sprouting of lateral bud: in superclean bench, sterilizable material obtained in the previous step is inoculated in the medium of induction sprouting of lateral bud, nutrient media components is: WPM minimal medium+ZT 2.0mg/L+ white sugar 30g/L+ agar 8g/L of improvement, pH is adjusted to 5.4;
C. propagation is cultivated: the stem section that aseptic lateral bud obtained in the previous step is cut into 2-3cm proceeds in proliferated culture medium subculture and expands numerously, and proliferation culture medium formula component is: WPM minimal medium+ZT 1.0mg/L+ white sugar 30g/L+ agar 8g/L of improvement, and pH is adjusted to 5.4;
D. outside sprout-cultivating-bottle: by healthy and strong 8-10cm obtained in the previous step aseptic seedling, open bottle cap, be covered with wet towel at ventilation hardening 2-3 days, note during this time keeping towel in moisture state.Before transplanting domestication, seedbed and matrix are processed and sterilized, matrix adopts domestic one-level sphagna, needs, with 800 times of carbendazim sterilizations of concentration 24 hours, then to wash, dry, pulverize, the thickness 5cm in seedbed.To be cut into 3-4cm segment through the healthy and strong blueberry tissue culture seedling of ventilation hardening, cuttage is in above-mentioned seedbed, average every strain can be cut into 2-3 section, every section of need of cuttage process dip the root-growing agent of 1.0mg/L NAA, seedbed overlay film after cuttage completes, in canopy, relative moisture is controlled at 60-80%, temperature is controlled at 18-25 DEG C, every a water of spray in 2-3 days, within 7-10 days, observe and have new root to generate, the rooting rate of 20 days, more than 85%, after this sprayed once the more foliage fertilizer of flower every 10 days, the survival rate of transplanted seedling is the highest obtains 95%.
Its condition of culture of setting up of above-mentioned blueberry sterile system is: 25 ± 2 DEG C of temperature, intensity of illumination 2500-3000lx, light application time 14-16 hour.
The foregoing is only specific embodiments of the invention, but architectural feature of the present invention is not limited to this, any those skilled in the art is in the field of the invention, and the variation of doing or modification are all encompassed among the scope of the claims of the present invention.
Claims (4)
1. blueberry tissue culture expands a method for numerous and outside sprout-cultivating-bottle, it is characterized in that described method comprises:
A. the choosing and sterilizing of explant: select robust growth without damage by disease and insect plant, clip semi-lignified branch, remove blade, under flowing water, rinse, on superclean bench, carry out disinfection with alcohol, then use aseptic water washing 2-3 time, proceed in mercuric chloride solution and carry out disinfection, then use aseptic water washing 3-4 time;
B. induce sprouting of lateral bud: in superclean bench, sterilizable material obtained in the previous step is inoculated in the medium of induction sprouting of lateral bud, nutrient media components is: WPM minimal medium+ZT0.8-2mg/L+ white sugar 25-35mg/L+ agar 5-10g/L of improvement, and pH is adjusted to 5.4;
C. propagation is cultivated: the stem section that aseptic lateral bud obtained in the previous step is cut into 2-3cm proceeds to subculture in proliferated culture medium and expands numerous, proliferation culture medium formula component is: WPM minimal medium+ZT0.8-2mg/L+ white sugar 25-35mg/L+ agar 5-10g/L of improvement, and pH is adjusted to 5.4;
D. outside sprout-cultivating-bottle: by healthy and strong 8-10cm obtained in the previous step aseptic seedling, open bottle cap, be covered with wet towel at ventilation hardening 2-3 days, note during this time keeping towel in moisture state, before transplanting domestication, seedbed and matrix are processed and sterilized, matrix adopts domestic one-level sphagna, sterilization after washing, drying, pulverizing, the thickness 4-6cm in seedbed; To be cut into 3-4cm segment through the healthy and strong blueberry tissue culture seedling of ventilation hardening, cuttage is in above-mentioned seedbed, average every strain can be cut into 2-3 section, every section of need of cuttage process dip root-growing agent, seedbed overlay film after cuttage completes, and in canopy, relative moisture is controlled at 60-80%, temperature is controlled at 18-25 DEG C, every a water of spray in 2-3 days, after this sprayed once the more foliage fertilizer of flower every 10 days, finally obtain the blueberry finished product seedling of taking root.
2. the method that a kind of blueberry tissue culture according to claim 1 expands numerous and outside sprout-cultivating-bottle, is characterized in that the concentration of mercuric chloride solution in described step a is 0.1%.
3. the method that a kind of blueberry tissue culture according to claim 1 expands numerous and outside sprout-cultivating-bottle, is characterized in that the sterilization of described steps d mesostroma need be sterilized 24 hours with the carbendazim that concentration is 1000 times.
4. the method that a kind of blueberry tissue culture according to claim 1 expands numerous and outside sprout-cultivating-bottle, is characterized in that described root-growing agent is 1.0mg/L NAA.
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Cited By (13)
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| CN104585039A (en) * | 2015-02-09 | 2015-05-06 | 上海青浦现代农业园区发展有限公司 | Tissue culture and rapid propagation method of blueberry |
| CN104823857A (en) * | 2015-05-18 | 2015-08-12 | 江苏农林职业技术学院 | Seedling raising method for promoting ex-vitro rooting of blueberry tissue culture seedlings |
| CN104885773A (en) * | 2015-06-29 | 2015-09-09 | 云南省农业科学院花卉研究所 | Method for rapidly cultivating early shaping tissue culture commodity seedlings of blueberries |
| CN105009908A (en) * | 2015-07-29 | 2015-11-04 | 广西壮族自治区林业科学研究院 | Out-bottle one-step plantlet developing method for rootless test-tube cunninghamia lanceolata seedlings |
| CN105028209A (en) * | 2015-08-26 | 2015-11-11 | 江苏省中国科学院植物研究所 | Method for improving rooting of vaccinium ashei tissue culture seedlings |
| CN105210874A (en) * | 2015-10-15 | 2016-01-06 | 铜仁市金农绿色农业科技有限公司 | A kind of blueberry seedling-cultivating method |
| CN106106182A (en) * | 2016-08-05 | 2016-11-16 | 四川省农业科学院经济作物育种栽培研究所 | A kind of root media cultivated for blueberry tissue culture Seedling and cultural method thereof |
| CN108307823A (en) * | 2018-02-05 | 2018-07-24 | 黑龙江省农业科学院园艺分院 | A method of carrying out cutting propagation using blueberry tissue culture seedling |
| CN108668659A (en) * | 2018-07-18 | 2018-10-19 | 苏州枫彩生态科技集团有限公司 | A kind of method of the outer cuttage root-taking of blueberry bottle |
| CN108848977A (en) * | 2017-12-20 | 2018-11-23 | 江苏省林业科学研究院 | A kind of promotion blueberry tissue-cultured seedling rooting method |
| CN115005105A (en) * | 2022-07-26 | 2022-09-06 | 辽宁省果树科学研究所 | Blueberry tissue culture method |
| CN115517167A (en) * | 2022-09-28 | 2022-12-27 | 上海市农业科学院 | Culture method for inducing blueberry chromosome doubling |
| CN116686716A (en) * | 2023-07-25 | 2023-09-05 | 广州智源农业科技发展有限公司 | Tissue culture seedling raising method for blueberries |
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Cited By (16)
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| CN104585039A (en) * | 2015-02-09 | 2015-05-06 | 上海青浦现代农业园区发展有限公司 | Tissue culture and rapid propagation method of blueberry |
| CN104823857A (en) * | 2015-05-18 | 2015-08-12 | 江苏农林职业技术学院 | Seedling raising method for promoting ex-vitro rooting of blueberry tissue culture seedlings |
| CN104885773B (en) * | 2015-06-29 | 2017-03-15 | 云南省农业科学院花卉研究所 | A kind of method of quickly breeding blue berry early stage sizing tissue culture commercial seedling |
| CN104885773A (en) * | 2015-06-29 | 2015-09-09 | 云南省农业科学院花卉研究所 | Method for rapidly cultivating early shaping tissue culture commodity seedlings of blueberries |
| CN105009908A (en) * | 2015-07-29 | 2015-11-04 | 广西壮族自治区林业科学研究院 | Out-bottle one-step plantlet developing method for rootless test-tube cunninghamia lanceolata seedlings |
| CN105009908B (en) * | 2015-07-29 | 2017-11-24 | 广西壮族自治区林业科学研究院 | The method of the outer forming seedling through one step culture of China fir unrooted test tube seedling bottle |
| CN105028209A (en) * | 2015-08-26 | 2015-11-11 | 江苏省中国科学院植物研究所 | Method for improving rooting of vaccinium ashei tissue culture seedlings |
| CN105210874A (en) * | 2015-10-15 | 2016-01-06 | 铜仁市金农绿色农业科技有限公司 | A kind of blueberry seedling-cultivating method |
| CN106106182A (en) * | 2016-08-05 | 2016-11-16 | 四川省农业科学院经济作物育种栽培研究所 | A kind of root media cultivated for blueberry tissue culture Seedling and cultural method thereof |
| CN108848977A (en) * | 2017-12-20 | 2018-11-23 | 江苏省林业科学研究院 | A kind of promotion blueberry tissue-cultured seedling rooting method |
| CN108307823A (en) * | 2018-02-05 | 2018-07-24 | 黑龙江省农业科学院园艺分院 | A method of carrying out cutting propagation using blueberry tissue culture seedling |
| CN108668659A (en) * | 2018-07-18 | 2018-10-19 | 苏州枫彩生态科技集团有限公司 | A kind of method of the outer cuttage root-taking of blueberry bottle |
| CN115005105A (en) * | 2022-07-26 | 2022-09-06 | 辽宁省果树科学研究所 | Blueberry tissue culture method |
| CN115517167A (en) * | 2022-09-28 | 2022-12-27 | 上海市农业科学院 | Culture method for inducing blueberry chromosome doubling |
| CN116686716A (en) * | 2023-07-25 | 2023-09-05 | 广州智源农业科技发展有限公司 | Tissue culture seedling raising method for blueberries |
| CN116686716B (en) * | 2023-07-25 | 2024-03-22 | 广州智源农业科技发展有限公司 | Tissue culture seedling raising method for blueberries |
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