CN103430845A - Strawberry tissue culturing method - Google Patents
Strawberry tissue culturing method Download PDFInfo
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Abstract
The invention discloses a strawberry tissue culturing method. The method comprises the steps of sterilization, primary culture, multiplication propagation culture, rooting culture and habituated culture. To be specific, the method comprises the following steps: using strawberry stolon points as explant, inoculating the explant in a sterile way to a primary culture medium to obtain cluster seedlings, transfering the seedlings into a multiplication propagation culture medium, performing rooting culture on the multiplication propagation cultured single-plant seedlings to obtain the rooting seedlings, and then performing hardening and habituation on the rooting seedlings, so as to obtain regeneration plants. According to the strawberry tissue culturing method provided by the invention, through the adoption of the tissue culturing method, a lot of sterile seedlings can be obtained during a short time, so that the strawberry is easier to propagate; the propagation period of strawberry is greatly shortened; the propagation speed of strawberry is accelerated; furthermore, the planting area can be quickly expanded. In addition, the method can break through limitation of climate and soil to strawberry propagation. The tissue culturing method effectively solves the problems that the seeding and propagation are slow and the cutting survival rate is low.
Description
Technical field
The present invention relates to a kind of method for plant tissue culture, relate in particular to a kind of strawberry method for tissue culture.
Background technology
Strawberry is rose family Fragaria perennial evergreen draft, the tool stolon, and plant height generally is no more than 30 centimetres, and its branches and tendrils are emerald green, and fruit is blackish red, spends in vain bunch bunch, both ornamentals, fruit is edible again, also can be processed into strawberry jam, strawberry wine etc.
Strawberry originates in Chile, extensively cultivation all over the world now, and kind reaches more than 2000.Strawberry is liked light, and happiness is moist, and water funk stain is not drought-enduring, more cold-resistant, likes fertile ventilative good sandy loam.Spring temperature rises to 5 degree when above, and plant starts to sprout, and optimum growth temperature is the 20-26 degree.Jiangsu Province, late Febuary sprouts, April, early and middle ten days was bloomed, mid or late May fruit maturation.Autumn has set in, and temperature is lower than 17 degree, and flower bud differentiation under the short-day condition of 10-12 hour, and the short-term dormancy is arranged winter.
Summary of the invention
Goal of the invention: technical problem to be solved by this invention is to provide a kind of strawberry method for tissue culture, and the method can shorten the breeding cycle of strawberry, improves its germination rate and survival rate.
Summary of the invention: for solving the problems of the technologies described above, the technical solution adopted in the present invention is:
A kind of strawberry method for tissue culture, comprise the steps:
Step 1, sterilization:
Choose the then newborn strawberry stem tip sterilization treatment that carries out disinfection;
Step 2, first culture: the strawberry stem tip 0.3～0.4mm after the cancellation poison is inoculated on MS+6-BA0.5mg/L+NAA0.2mg/L+ sucrose 30g/L+6.5g/L agar powder medium, the pH value of medium is 5.5, by stem apex at 25 ± 2 ℃ of temperature, intensity of illumination 1500～2000lux, under illumination every day 12-14 hour, cultivate 40-45 days;
Step 3, expanding propagation is cultivated: the every 5--7 strain of the seedling of growing thickly after first culture is cut into to one, is inoculated on MS medium+6-BA0.5mg/L+NAA0.01mg/L medium, the pH value of medium is 5.8,23～27 ℃ of temperature, intensity of illumination 2000lux, illumination every day 12-14 hour, cultivate seedling after 30 days and cut apart formation individual plant seedling, in turning the seedling process, remove primary blade and be conducive to the new talent differentiation, remove at any time callus in expanding propagation, be conducive to new seedling proliferation and growth;
Step 4, culture of rootage: the bud seedling after expanding propagation is cultivated is transferred on root media and is cultivated, root media is 1/2MS+IBA0.5mg/L+NAA0.1mg/L+0.3% active carbon+sucrose 20g/L, in temperature, it is 23～27 ℃, intensity of illumination 2000lux, illumination every day 12 hours, cultivate after 20-25 days, the bud seedling that forms short of white is tamed to cultivation, from being inoculated into the seedling of taking root, grow up to, the cultivation temperature during this generally should be stabilized in 25 ± 2 ℃, and illumination every day is about 12 hours, intensity of illumination is 2000Lx, and culturing room's temperature is 18～26 ℃;
Step 5, domestication is cultivated: the bud seedling after culture of rootage is moved in perlite, quartz sand and the vermiculite domestication matrix that 1:1:2 mixes by volume and cultivated, cultivation temperature is 15～20 ℃, and humidity is 80%, and shading rate is 50%, cultivate 5～7 days, after one week, can strengthen gradually solar radiation, adopt spray form to water, the water yield is unsuitable excessive, after falling to doing, sprays again.
Wherein, in step 1, described sterilization processing refers to first immerses strawberry stem tip the alcohol that the quality percentage composition is 75%, after moistening 1～2min, then strawberry stem tip is immersed to the aqueous sodium hypochlorite solution that the quality percentage composition is 10%, soaks 10～15min.
Wherein, in step 2, before inoculation, medium adopts 1～1.1kg/cm
2atmospheric pressure hot pressing sterilization 15～20 minutes.
Wherein, in step 5, before domestication, the blake bottle that the bud seedling of taking root is housed under the dislocation natural conditions, is opened to bottle cap ventilative, after 24 hours from culturing room, take out seedling from bottle, remove the medium at clean root and collar place, then plant in nursery or cave dish and tame cultivation.
Wherein, in step 5, domestication matrix is become thoroughly decomposed wood sawdust and leaf mould mixture or garden mould and the cinder mixture that 2:1 mixes by volume in any proportion through sterilization treatment, domestication matrix is rinsed well with clear water, or mix sterilizing with carbendazim or thiophanate methyl, while with cinder, making matrix, then with 0.2% glacial acetic acid neutralization, alkalescence is reduced.
Beneficial effect: strawberry method for tissue culture of the present invention is by adopting method for tissue culture, can obtain at short notice a large amount of aseptic seedling, make the breeding of strawberry more easy, greatly shortened the breeding cycle of strawberry, accelerated the reproduction speed of strawberry, thereby can enlarge fast cultivated area, method of the present invention has been broken the restriction to the strawberry breeding of weather and soil in addition, by adopting method for tissue culture, effectively solved seed propagation slowly and the low problem of cuttage survival rate.
Embodiment
According to following embodiment, the present invention may be better understood.Yet, those skilled in the art will readily understand, the described content of embodiment is only for the present invention is described, and should also can not limit the present invention described in detail in claims.
Embodiment 1
A kind of strawberry method for tissue culture, comprise the steps:
Step 1, sterilization:
Choose the stem apex of crawling of just having sprouted then, after running water rinses, the alcohol that the quality percentage composition of take on superclean bench is 75%, after moistening 1～2min, proceed to again the aqueous sodium hypochlorite solution that the quality percentage composition is 10%, after soaking 10～15min, then use preprepared aseptic water washing 4 times, after draining the water, the stem apex after sterilization is inoculated on first culture base;
Step 2, first culture: before inoculation, medium is adopted to 1～1.1kg/cm
2atmospheric pressure hot pressing sterilization 15～20 minutes, after the medium sterilization, strawberry stem tip 0.3～0.4mm after the cancellation poison is inoculated on MS+6-BA0.5mg/L+NAA0.2mg/L+ sucrose 30g/L+6.5g/L agar powder medium, the pH value of medium is 5.5, by stem apex at 25 ± 2 ℃ of temperature, intensity of illumination 1500～2000lux, under illumination every day 12-14 hour, cultivate 40-45 days;
Step 3, expanding propagation is cultivated: the every 5--7 strain of the seedling of growing thickly after first culture is cut into to one, is inoculated on MS medium+6-BA0.5mg/L+NAA0.01mg/L medium, the pH value of medium is 5.8,23～27 ℃ of temperature, intensity of illumination 2000lux, illumination every day 12-14 hour, cultivate seedling after 30 days and cut apart formation individual plant seedling, in turning the seedling process, remove primary blade and be conducive to the new talent differentiation, remove at any time callus in expanding propagation, be conducive to new seedling proliferation and growth;
Step 4, culture of rootage: the bud seedling after expanding propagation is cultivated is transferred on root media and is cultivated, root media is 1/2MS+IBA0.5mg/L+NAA0.1mg/L+0.3% active carbon+sucrose 20g/L, in temperature, it is 23～27 ℃, intensity of illumination 2000lux, illumination every day 12 hours, cultivate after 20-25 days, the bud seedling that forms short of white is tamed to cultivation, from being inoculated into the seedling of taking root, grow up to, the cultivation temperature during this generally should be stabilized in 25 ± 2 ℃, and illumination every day is about 12 hours, intensity of illumination is 2000Lx, and culturing room's temperature is 18～26 ℃;
Step 5, domestication is cultivated: before domestication, by blake bottle that the bud seedling of taking root is housed from culturing room under the dislocation natural conditions, open bottle cap ventilative, after 24 hours, take out seedling from bottle, remove the medium at clean root and collar place, plant again in nursery or cave dish and tame cultivation, domestication matrix is perlite, quartz sand and vermiculite 1:1:2 by volume mix, cultivation temperature is 15～20 ℃, humidity is 80%, shading rate is 50%, cultivate 5～7 days, after one week, can strengthen gradually solar radiation, the employing spray form waters, the water yield is unsuitable excessive, after falling to doing, spray again.Domestication matrix is rinsed well with clear water, or mixes sterilizing with carbendazim or thiophanate methyl.
Embodiment 2
A kind of strawberry method for tissue culture, comprise the steps:
Step 1, sterilization:
Choose the stem apex of crawling of just having sprouted then, after running water rinses, the alcohol that the quality percentage composition of take on superclean bench is 75%, after moistening 1～2min, proceed to again the aqueous sodium hypochlorite solution that the quality percentage composition is 10%, after soaking 10～15min, then use preprepared aseptic water washing 4 times, after draining the water, the stem apex after sterilization is inoculated on first culture base;
Step 2, first culture: before inoculation, medium is adopted to 1～1.1kg/cm
2atmospheric pressure hot pressing sterilization 15～20 minutes, after the medium sterilization, strawberry stem tip 0.3～0.4mm after the cancellation poison is inoculated on MS+6-BA0.5mg/L+NAA0.2mg/L+ sucrose 30g/L+6.5g/L agar powder medium, the pH value of medium is 5.5, by stem apex at 25 ± 2 ℃ of temperature, intensity of illumination 1500～2000lux, under illumination every day 12-14 hour, cultivate 40-45 days;
Step 3, expanding propagation is cultivated: the every 5--7 strain of the seedling of growing thickly after first culture is cut into to one, is inoculated on MS medium+6-BA0.5mg/L+NAA0.01mg/L medium, the pH value of medium is 5.8,23～27 ℃ of temperature, intensity of illumination 2000lux, illumination every day 12-14 hour, cultivate seedling after 30 days and cut apart formation individual plant seedling, in turning the seedling process, remove primary blade and be conducive to the new talent differentiation, remove at any time callus in expanding propagation, be conducive to new seedling proliferation and growth;
Step 4, culture of rootage: the bud seedling after expanding propagation is cultivated is transferred on root media and is cultivated, root media is 1/2MS+IBA0.5mg/L+NAA0.1mg/L+0.3% active carbon+sucrose 20g/L, in temperature, it is 23～27 ℃, intensity of illumination 2000lux, illumination every day 12 hours, cultivate after 20-25 days, the bud seedling that forms short of white is tamed to cultivation, from being inoculated into the seedling of taking root, grow up to, the cultivation temperature during this generally should be stabilized in 25 ± 2 ℃, and illumination every day is about 12 hours, intensity of illumination is 2000Lx, and culturing room's temperature is 18～26 ℃;
Step 5, domestication is cultivated: before domestication, by blake bottle that the bud seedling of taking root is housed from culturing room under the dislocation natural conditions, open bottle cap ventilative, after 24 hours, take out seedling from bottle, remove the medium at clean root and collar place, plant again in nursery or cave dish and tame cultivation, domestication matrix is become thoroughly decomposed wood sawdust and leaf mould mixture in any proportion through sterilization treatment, cultivation temperature is 15～20 ℃, humidity is 80%, shading rate is 50%, cultivate 5～7 days, after one week, can strengthen gradually solar radiation, the employing spray form waters, the water yield is unsuitable excessive, after falling to doing, spray again.Domestication matrix is rinsed well with clear water, or mixes sterilizing with carbendazim or thiophanate methyl.
Embodiment 3
A kind of strawberry method for tissue culture, comprise the steps:
Step 1, sterilization:
Choose the stem apex of crawling of just having sprouted then, after running water rinses, the alcohol that the quality percentage composition of take on superclean bench is 75%, after moistening 1～2min, proceed to again the aqueous sodium hypochlorite solution that the quality percentage composition is 10%, after soaking 10～15min, then use preprepared aseptic water washing 4 times, after draining the water, the stem apex after sterilization is inoculated on first culture base;
Step 2, first culture: before inoculation, medium is adopted to 1～1.1kg/cm
2atmospheric pressure hot pressing sterilization 15～20 minutes, after the medium sterilization, strawberry stem tip 0.3～0.4mm after the cancellation poison is inoculated on MS+6-BA0.5mg/L+NAA0.2mg/L+ sucrose 30g/L+6.5g/L agar powder medium, the pH value of medium is 5.5, by stem apex at 25 ± 2 ℃ of temperature, intensity of illumination 1500～2000lux, under illumination every day 12-14 hour, cultivate 40-45 days;
Step 3, expanding propagation is cultivated: the every 5--7 strain of the seedling of growing thickly after first culture is cut into to one, is inoculated on MS medium+6-BA0.5mg/L+NAA0.01mg/L medium, the pH value of medium is 5.8,23～27 ℃ of temperature, intensity of illumination 2000lux, illumination every day 12-14 hour, cultivate seedling after 30 days and cut apart formation individual plant seedling, in turning the seedling process, remove primary blade and be conducive to the new talent differentiation, remove at any time callus in expanding propagation, be conducive to new seedling proliferation and growth;
Step 4, culture of rootage: the bud seedling after expanding propagation is cultivated is transferred on root media and is cultivated, root media is 1/2MS+IBA0.5mg/L+NAA0.1mg/L+0.3% active carbon+sucrose 20g/L, in temperature, it is 23～27 ℃, intensity of illumination 2000lux, illumination every day 12 hours, cultivate after 20-25 days, the bud seedling that forms short of white is tamed to cultivation, from being inoculated into the seedling of taking root, grow up to, the cultivation temperature during this generally should be stabilized in 25 ± 2 ℃, and illumination every day is about 12 hours, intensity of illumination is 2000Lx, and culturing room's temperature is 18～26 ℃;
Step 5, domestication is cultivated: before domestication, by blake bottle that the bud seedling of taking root is housed from culturing room under the dislocation natural conditions, open bottle cap ventilative, after 24 hours, take out seedling from bottle, remove the medium at clean root and collar place, plant again in nursery or cave dish and tame cultivation, domestication matrix is garden mould and the cinder mixture that 2:1 mixes by volume, adding the quality percentage composition is 0.2% glacial acetic acid again, the temperature that domestication is cultivated is 15～20 ℃, humidity is 80%, shading rate is 50%, cultivate 5～7 days, after one week, can strengthen gradually solar radiation, the employing spray form waters, the water yield is unsuitable excessive, after falling to doing, spray again.Domestication matrix is rinsed well with clear water, or mixes sterilizing with carbendazim or thiophanate methyl.
Claims (5)
1. a strawberry method for tissue culture, is characterized in that: comprise the steps:
Step 1, sterilization:
Choose the then newborn strawberry stem tip sterilization treatment that carries out disinfection;
Step 2, first culture: the strawberry stem tip 0.3～0.4mm after the cancellation poison is inoculated on MS+6-BA0.5mg/L+NAA0.2mg/L+ sucrose 30g/L+6.5g/L agar powder medium, the pH value of medium is 5.5,25 ± 2 ℃ of temperature, intensity of illumination 1500～2000lux, illumination every day 12-14 hour, cultivate 40-45 days;
Step 3, expanding propagation is cultivated: the every 5--7 strain of the seedling of growing thickly after first culture is cut into to one, be inoculated on MS medium+6-BA0.5mg/L+NAA0.01mg/L medium, the pH value of medium is 5.8,23～27 ℃ of temperature, intensity of illumination 2000lux, illumination every day 12-14 hour, cultivate seedling after 30 days and cut apart formation individual plant seedling;
Step 4, culture of rootage: the bud seedling after expanding propagation is cultivated is transferred on root media and is cultivated, root media is 1/2MS+IBA0.5mg/L+NAA0.1mg/L+0.3% active carbon+sucrose 20g/L, in temperature, it is 23～27 ℃, intensity of illumination 2000lux, illumination every day 12 hours, cultivated after 20-25 days, and the bud seedling that forms short of white is tamed to cultivation;
Step 5, domestication is cultivated: the bud seedling after culture of rootage is moved in perlite, quartz sand and the vermiculite domestication matrix that 1:1:2 mixes by volume and cultivated, and cultivation temperature is 15～20 ℃, and humidity is 80%, and shading rate is 50%, cultivates 5～7 days.
2. strawberry method for tissue culture according to claim 1, it is characterized in that: in step 1, described sterilization processing refers to first immerses strawberry stem tip the alcohol that the quality percentage composition is 75%, after moistening 1～2min, again strawberry stem tip is immersed to the aqueous sodium hypochlorite solution that the quality percentage composition is 10%, soak 10～15min.
3. strawberry method for tissue culture according to claim 1 is characterized in that: in step 2, before inoculation, medium adopts 1～1.1kg/cm
2atmospheric pressure hot pressing sterilization 15～20 minutes.
4. strawberry method for tissue culture according to claim 1, it is characterized in that: in step 5, before domestication, by blake bottle that the bud seedling of taking root is housed from culturing room under the dislocation natural conditions, open bottle cap ventilative, after 24 hours, take out seedling from bottle, remove the medium at clean root and collar place, then plant in nursery or cave dish and tame cultivation.
5. strawberry method for tissue culture according to claim 1, is characterized in that: in step 5, tame matrix for become thoroughly decomposed wood sawdust and leaf mould mixture or garden mould and the cinder mixture that 2:1 mixes by volume in any proportion through sterilization treatment.
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Cited By (7)
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| CN103975854A (en) * | 2014-04-29 | 2014-08-13 | 卞佳林 | Culture medium for culturing strawberry stem tips and inducing plant differentiation and culture method thereof |
| CN104186351A (en) * | 2014-09-24 | 2014-12-10 | 江苏农林职业技术学院 | Tissue culture method of strawberries |
| CN105010123A (en) * | 2014-04-28 | 2015-11-04 | 上海市农业科学院 | Method and culture medium for obtaining strawberry distant hybrid through in-vitro rescue culture |
| CN107155700A (en) * | 2017-05-18 | 2017-09-15 | 贵州务川科华生物科技有限公司 | The preparation method of broussonetia papyrifera seedlings culture medium |
| CN107333659A (en) * | 2017-09-15 | 2017-11-10 | 江苏省农业科学院 | A kind of day neutrality Strawberry Plantlets fast breeding culture medium and tissue culture and rapid propagation method |
| CN107517847A (en) * | 2017-06-22 | 2017-12-29 | 合肥华典生物科技股份有限公司 | A kind of strawberry mitogeneticization callus fast culture process |
| CN116019012A (en) * | 2022-12-29 | 2023-04-28 | 宿迁市设施园艺研究院 | Detoxification seedling method for strawberries of snow rabbits |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105010123A (en) * | 2014-04-28 | 2015-11-04 | 上海市农业科学院 | Method and culture medium for obtaining strawberry distant hybrid through in-vitro rescue culture |
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| CN103975854A (en) * | 2014-04-29 | 2014-08-13 | 卞佳林 | Culture medium for culturing strawberry stem tips and inducing plant differentiation and culture method thereof |
| CN104186351A (en) * | 2014-09-24 | 2014-12-10 | 江苏农林职业技术学院 | Tissue culture method of strawberries |
| CN107155700A (en) * | 2017-05-18 | 2017-09-15 | 贵州务川科华生物科技有限公司 | The preparation method of broussonetia papyrifera seedlings culture medium |
| CN107155700B (en) * | 2017-05-18 | 2019-12-10 | 贵州务川科华生物科技有限公司 | Preparation method of broussonetia papyrifera seedling culture medium |
| CN107517847A (en) * | 2017-06-22 | 2017-12-29 | 合肥华典生物科技股份有限公司 | A kind of strawberry mitogeneticization callus fast culture process |
| CN107333659A (en) * | 2017-09-15 | 2017-11-10 | 江苏省农业科学院 | A kind of day neutrality Strawberry Plantlets fast breeding culture medium and tissue culture and rapid propagation method |
| CN107333659B (en) * | 2017-09-15 | 2019-01-22 | 江苏省农业科学院 | A kind of day neutral Strawberry Plantlets fast breeding culture medium and tissue culture and rapid propagation method |
| CN116019012A (en) * | 2022-12-29 | 2023-04-28 | 宿迁市设施园艺研究院 | Detoxification seedling method for strawberries of snow rabbits |
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Application publication date: 20131211 |