CN102422816A - Method for rapidly culturing shoot tips in vitro - Google Patents
Method for rapidly culturing shoot tips in vitro Download PDFInfo
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- CN102422816A CN102422816A CN2011103274230A CN201110327423A CN102422816A CN 102422816 A CN102422816 A CN 102422816A CN 2011103274230 A CN2011103274230 A CN 2011103274230A CN 201110327423 A CN201110327423 A CN 201110327423A CN 102422816 A CN102422816 A CN 102422816A
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Abstract
The invention provides a method for rapidly culturing shoot tips in vitro, which is characterized in that in the first step, selecting stolon of the strawberries sprouted at the same year, washing to be clean and disinfecting; in the second step, cutting 0.1-0.5 mm shoot tips of the disinfected and cleaned strawberries stolon under the anatomical lens, inoculating to a culture medium for germination culture, cutting the new sprouts after the 0.5-1.5 cm new sprouts are grown on the shoot tips, inoculating to an enrichment medium for inducing clustered shoots; in the third step, cutting the cluster buds to simple buds, inoculating to a rooting medium for rooting culture; transferring test tube plantlets to a substrate formed by mixing perlite and peat, watering and covering a plastic film for insulation and moisture retention, revealing the plastic film for gradually hardening, transferring the seedling to a plastic potted tray filled with pure peat, watering and covering the plastic film for insulation and moisture retention, and directly planting the seedling to a field. The invention has the advantages of high reproductive rate, rapid reproduction speed and high survival rate.
Description
Technical field
The present invention relates to the stem apex in-vitro rapid culture method of a kind of strawberry, utilize shoot tip meristem induced bundle after sterilizing of taking out then on the living stolon to sprout, after cultivating, obtain complete test-tube plantlet again, belong to strawberry cultural method technical field.
Background technology
Strawberry is a kind of high-quality high-grade fruit, and is nutritious, and economic worth is high.For a long time, the modes of reproduction of strawberry is main with division propagation and axillalry bud cottage propagation always, but these traditional modess of reproduction are easy to make the strawberry infective virus, and its output and quality are descended, and can't satisfy the need of producing with seedling.
Summary of the invention
The purpose of this invention is to provide the strawberry cultural method that a kind of reproduction rate is high, reproduction speed is fast, reproductive effect is excellent.
In order to achieve the above object, the invention provides the stem apex in-vitro rapid culture method of a kind of strawberry, it is characterized in that concrete steps are:
The first step: choose the stolon that strawberry sprouts then, rinse well, it is subsequent use to sterilize;
Second step: the strawberry stolon that will sterilize clean cuts the long stem apex of 0.1～0.5mm under anatomical lens; Be inoculated in the cultivation of sprouting in the medium takes place; After treating to grow on the stem apex the long sprouting of 0.5～1.5cm; Sprouting is downcut, be inoculated into and carry out inducing clumping bud in the proliferated culture medium, wherein; Said generation medium is on the basis of MS medium, to have added 6-benzylaminopurine (6-BA) and methyl (NAA), and said proliferated culture medium is on the basis of MS medium, to have added 6-benzylaminopurine (6-BA), kinetin (KT) and indolebutyric acid (IBA);
The 3rd step: the bud of will growing thickly is cut into simple bud, is inoculated in to carry out culture of rootage in the root media, and said root media is on the basis of 1/2MS medium, to have added methyl (NAA);
In the 4th step, it is in the perlite of 4～6:1 and the matrix that peat mixes that the test-tube plantlet that will take root moves into by mass ratio, preserves moisture with the plastic film covering and heat insulating after watering sufficient water; Open film after 5～9 days and progressively refine seedling; Every day, the blade face water spray was 2～3 times, and 10～20 days after-applied fertilizer liquid moved to the plastics cave dish of filling pure peat with seedling after 50～70 days; Preserve moisture with the plastic film covering and heat insulating after watering, direct transplanting is to big Tanaka after cultivating in 1.5～2.5 months.
Preferably; The concrete operations of the sterilization in the said first step are: on superclean bench, sterilize with 70～80vol% ethanolic solution earlier; Use the quick solution disinfection of clean that of 0.05～0.15vol% then; With the sterilization of 0.05～0.15vol% mercuric chloride solution, use aseptic water washing at last, use the aseptic filter paper suck dry moisture again.
Preferably, the concentration of 6-benzylaminopurine is 1～3mg/L in the said generation medium, and the concentration of methyl is 0.1～0.3mg/L, and the pH value is 5～6.
Preferably, the concentration of 6-benzylaminopurine is 0.1～1.0mg/L in the said proliferated culture medium, and the concentration of kinetin is 0.1～0.3mg/L, and the concentration of indolebutyric acid is 0.1～0.3mg/L.
Preferably, the concentration of methyl is 0.1～1.0mg/L in the said root media.
Sprouting in the said first step, to cultivate be under 20～30 ℃, intensity of illumination 2000～3000LX and 8～16 hours/day condition of light application time, to carry out.
Inducing clumping bud is under 20～30 ℃, intensity of illumination 3000～4000LX and 8～16 hours/day condition of light application time, to carry out in said second step.
Said second step carries out carrying out shoot proliferation behind the inducing clumping bud, breeds once in every 30-35 days, and the rate of increase is 1:3-4.
Culture of rootage is at 20～30 ℃, intensity of illumination 3000～4000LX in said the 3rd step, carries out under 8～16 hours/day the condition of light application time.
Contain 0.05～0.15wt% ammonium nitrate and 0.05～0.15wt% potassium dihydrogen phosphate in the fertilizer liquid of using in said the 4th step.
Compared with prior art, the present invention has following beneficial effect:
1, virus elimination rate high (adopt and strip 0.3mm size stem apex under the anatomical lens), the stem apex growth is fast, and the stem apex incidence is high, generally up to 96%.Reproduction rate is high, and reproduction speed is fast, can in short-term, batch production produce seedling.
2, test-tube plantlet growth is vigorous, can eliminate " vitrifying " phenomenon.
3, the rooting of vitro seedling rate is high, generally can reach 99%.
Embodiment
Specify the present invention below in conjunction with embodiment.
Embodiment 1
The MS medium component is following:
Annotate: the 1/2MS medium is that above-mentioned macroelement and trace element are reduced by half, and other components unchanged preparations obtain.
The stem apex in-vitro rapid culture method of strawberry, concrete steps are following:
Phase I: generating material cleaning and sterilizing.Choose the stolon that strawberry sprouts then, rinse well with washing agent, flushing is 2 hours under flowing water.On superclean bench,, use clean your quick solution disinfection 15 minutes of 0.1vol% then, sterilized 15 minutes with the 0.1vol% mercuric chloride solution at last earlier with 75vol% ethanolic solution sterilization 1 minute, with aseptic water washing three times, the aseptic filter paper suck dry moisture;
Second stage, the aseptic strain inducing culture.The strawberry stolon of sterilization is cut the long stem apex of 0.3mm under anatomical lens, be inoculated in and take place on the medium, its prescription is MS+6-BA2mg/L+NAA0.2mg/L, medium pH 5.8.The stem apex of inoculation is put culturing room and is cultivated, 25 ℃ of temperature, and intensity of illumination 2500LX, illumination 12 hours every days is with the inducing culture aseptic strain.
Phase III, the aseptic strain shoot proliferation.After treating to grow on the stem apex the long sprouting of 1cm, sprouting is downcut, be inoculated on the proliferated culture medium, its prescription is MS+6-BA0.5mg/L+KT0.2mg/L+IBA0.2mg/L, medium pH 5.8.The sprouting of inoculation places culturing room to cultivate, 23 ℃ of temperature, intensity of illumination 3500LX, and inducing clumping bud and shoot proliferation are carried out in illumination 12 hours every days, breed once in per 32 days, and the rate of increase is 1:3;
Stage, the aseptic strain culture of rootage.The bud of will growing thickly is cut into simple bud, is inoculated in the root of hair medium, and its prescription is 1/2MS+NAA0.5mg/L, medium pH 5.8.At 23 ℃, intensity of illumination 3500LX, carry out culture of rootage under 12 hours/day the condition of light application time;
Five-stage, test-tube plantlet refining seedling is cultivated.It is in the perlite of 5:1 and the matrix that peat mixes that the test-tube plantlet that will take root moves into by mass ratio; Preserve moisture with the plastic film covering and heat insulating after watering sufficient water, open film after the week and progressively refine seedling, every day, the blade face water spray was 2 times; (fertilizing amount is 1000ml/ ㎡ to the fertilizer liquid of the 15 days after-applied 0.1wt% of containing ammonium nitrate and 0.1wt% potassium dihydrogen phosphate; Every month twice), after 60 days seedling is moved to the plastics cave dish (72 hole) of filling pure peat, preserve moisture with the plastic film covering and heat insulating in the back of watering; Direct transplanting is to big Tanaka after cultivating in two months, and survival rate can reach more than 95%.
Through comparative trial, A group has been taked the inventive method, is employed in the strawberry stem tip that strips the 0.3mm size under the anatomical lens fast culture that exsomatizes.B group is control group, the strawberry stem tip that only with the naked eye strips size about the 1mm fast culture that exsomatizes.
Result of the test is following:
| ? | Virus elimination rate | " glass seedling " incidence | Rooting rate |
| The A group | Up to more than 95% | Vitality is vigorous, and no glass seedling produces | Up to 99% |
| The B group | Less than 50%, have in addition not detoxification | Have the glass seedling to produce, test-tube plantlet growth is unusual | Below 50%, even do not take root |
Through test data, prove that the present invention has more useful effect than original technology.
Claims (10)
1. the stem apex in-vitro rapid culture method of a strawberry is characterized in that, concrete steps are:
The first step: choose the stolon that strawberry sprouts then, rinse well, it is subsequent use to sterilize;
Second step: the strawberry stolon that will sterilize clean cuts the long stem apex of 0.1～0.5mm under anatomical lens; Be inoculated in the cultivation of sprouting in the medium takes place; After treating to grow on the stem apex the long sprouting of 0.5～1.5cm; Sprouting is downcut, be inoculated into and carry out inducing clumping bud in the proliferated culture medium, wherein; Said generation medium is on the basis of MS medium, to have added 6-benzylaminopurine and methyl, and said proliferated culture medium is on the basis of MS medium, to have added 6-benzylaminopurine, kinetin and indolebutyric acid;
The 3rd step: the bud of will growing thickly is cut into simple bud, is inoculated in to carry out culture of rootage in the root media, and said root media is on the basis of 1/2MS medium, to have added methyl;
In the 4th step, it is in the perlite of 4～6:1 and the matrix that peat mixes that the test-tube plantlet that will take root moves into by mass ratio, preserves moisture with the plastic film covering and heat insulating after watering sufficient water; Open film after 5～9 days and progressively refine seedling; Every day, the blade face water spray was 2～3 times, and 10～20 days after-applied fertilizer liquid moved to the plastics cave dish of filling pure peat with seedling after 50～70 days; Preserve moisture with the plastic film covering and heat insulating after watering, direct transplanting is to big Tanaka after cultivating in 1.5～2.5 months.
2. the stem apex in-vitro rapid culture method of strawberry as claimed in claim 1; It is characterized in that; The concrete operations of the sterilization in the said first step are: on superclean bench, earlier with the sterilization of 70～80vol% ethanolic solution, use the quick solution disinfection of clean that of 0.05～0.15vol% then, sterilize with 0.05～0.15vol% mercuric chloride solution at last; Use aseptic water washing, use the aseptic filter paper suck dry moisture again.
3. the stem apex in-vitro rapid culture method of strawberry as claimed in claim 1 is characterized in that, the concentration of 6-benzylaminopurine is 1～3mg/L in the said generation medium, and the concentration of methyl is 0.1～0.3mg/L, and the pH value is 5～6.
4. the stem apex in-vitro rapid culture method of strawberry as claimed in claim 1; It is characterized in that; The concentration of 6-benzylaminopurine is 0.1～1.0mg/L in the said proliferated culture medium, and the concentration of kinetin is 0.1～0.3mg/L, and the concentration of indolebutyric acid is 0.1～0.3mg/L.
5. the stem apex in-vitro rapid culture method of strawberry as claimed in claim 1 is characterized in that, the concentration of methyl is 0.1～1.0mg/L in the said root media.
6. the stem apex in-vitro rapid culture method of strawberry as claimed in claim 1 is characterized in that, sprouting in said second step, to cultivate be under 20～30 ℃, intensity of illumination 2000～3000LX and 8～16 hours/day condition of light application time, to carry out.
7. the stem apex in-vitro rapid culture method of strawberry as claimed in claim 1 is characterized in that, inducing clumping bud is under 20～30 ℃, intensity of illumination 3000～4000LX and 8～16 hours/day condition of light application time, to carry out in said second step.
8. the stem apex in-vitro rapid culture method of strawberry as claimed in claim 1 is characterized in that, culture of rootage is at 20～30 ℃, intensity of illumination 3000～4000LX in said the 3rd step, carries out under 8～16 hours/day the condition of light application time.
9. the stem apex in-vitro rapid culture method of strawberry as claimed in claim 1 is characterized in that, said second step carries out carrying out shoot proliferation behind the inducing clumping bud, breeds once in every 30-35 days, and the rate of increase is 1:3-4.
10. the stem apex in-vitro rapid culture method of strawberry as claimed in claim 1 is characterized in that, contains 0.05～0.15wt% ammonium nitrate and 0.05～0.15wt% potassium dihydrogen phosphate in the fertilizer liquid of using in said the 4th step.
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Cited By (22)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102893869A (en) * | 2012-10-22 | 2013-01-30 | 浙江省农业科学院 | Root tip detoxification and rapid propagation technology of strawberries |
| CN102919011A (en) * | 2012-10-09 | 2013-02-13 | 合肥市艳九天农业科技有限公司 | Stock plant nutrition supply method for strawberry seedling breeding |
| CN102986534A (en) * | 2012-12-14 | 2013-03-27 | 苏州和美生物科技有限公司 | Initial culture medium special for preventing brown stain of strawberries and method for producing tissue culture strawberry seedlings by using initial culture medium |
| CN103283595A (en) * | 2013-05-17 | 2013-09-11 | 高红胜 | Primary culture method of stem tip tissue of strawberry |
| CN103283594A (en) * | 2013-05-13 | 2013-09-11 | 北京林业大学 | Micro-propagation liquid culture method for strawberries |
| CN103329802A (en) * | 2013-06-21 | 2013-10-02 | 苏州市阳澄湖现代农业发展有限公司 | Pollution-free high-yield planting method for strawberries |
| CN103348918A (en) * | 2013-07-19 | 2013-10-16 | 合肥瑞谷农业科技有限公司 | Efficient detoxification tissue cultivating method of strawberries |
| CN103798135A (en) * | 2012-11-06 | 2014-05-21 | 甄子萱 | Method using continuous cropping soil leaching liquor to breed strawberry anti continuous cropping obstacle new germplasm |
| CN103858758A (en) * | 2012-12-12 | 2014-06-18 | 东港市草莓研究所 | Strawberry seedling tissue culture rapid propagation technology |
| 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 |
| CN104041411A (en) * | 2014-06-05 | 2014-09-17 | 卞佳林 | Seedling hardening and rooting culture medium for strawberry |
| CN104041410A (en) * | 2014-06-05 | 2014-09-17 | 卞佳林 | Strong strawberry seedling rooting culture method |
| CN104186351A (en) * | 2014-09-24 | 2014-12-10 | 江苏农林职业技术学院 | Tissue culture method of strawberries |
| CN104429940A (en) * | 2014-10-23 | 2015-03-25 | 绍兴文理学院 | Method for acquiring virus-free strawberry seedlings |
| CN105210868A (en) * | 2015-10-08 | 2016-01-06 | 新疆生产建设兵团第六师农业科学研究所 | The acquisition methods of strawberry detoxification breeder's stock seedling and store method |
| CN107135949A (en) * | 2017-06-16 | 2017-09-08 | 黔东南民族职业技术学院 | Strawberry adventitious bud induction culture base and preparation method and application |
| CN108464240A (en) * | 2018-04-13 | 2018-08-31 | 河北富硕农业科技发展有限公司 | The method of Snow White's strawberry detoxifying fast breeding |
| CN109076960A (en) * | 2018-09-29 | 2018-12-25 | 河南云帮农业科技有限公司 | A kind of Plantlets of Strawberry detoxication and tissue culture strong sprout method |
| CN109769694A (en) * | 2019-03-28 | 2019-05-21 | 南京富一农业科技有限公司 | A kind of strawberry stem tip in-vitro regeneration method |
| CN111955346A (en) * | 2020-09-03 | 2020-11-20 | 云南华农农业有限公司 | Novel method for inhibiting browning and improving artificial rapid propagation efficiency of Monte raspberries |
| CN112425508A (en) * | 2020-12-08 | 2021-03-02 | 黑龙江省农业科学院园艺分院 | Culture medium for inducing differentiation, proliferation and rooting of strawberry stem tips |
| CN115517171A (en) * | 2022-10-18 | 2022-12-27 | 太原师范学院 | Method for producing strawberry seedlings by rapid plant tissue culture |
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Cited By (25)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102919011A (en) * | 2012-10-09 | 2013-02-13 | 合肥市艳九天农业科技有限公司 | Stock plant nutrition supply method for strawberry seedling breeding |
| CN102893869A (en) * | 2012-10-22 | 2013-01-30 | 浙江省农业科学院 | Root tip detoxification and rapid propagation technology of strawberries |
| CN103798135A (en) * | 2012-11-06 | 2014-05-21 | 甄子萱 | Method using continuous cropping soil leaching liquor to breed strawberry anti continuous cropping obstacle new germplasm |
| CN103858758A (en) * | 2012-12-12 | 2014-06-18 | 东港市草莓研究所 | Strawberry seedling tissue culture rapid propagation technology |
| CN102986534A (en) * | 2012-12-14 | 2013-03-27 | 苏州和美生物科技有限公司 | Initial culture medium special for preventing brown stain of strawberries and method for producing tissue culture strawberry seedlings by using initial culture medium |
| CN103283594A (en) * | 2013-05-13 | 2013-09-11 | 北京林业大学 | Micro-propagation liquid culture method for strawberries |
| CN103283594B (en) * | 2013-05-13 | 2014-07-16 | 北京林业大学 | Micro-propagation liquid culture method for strawberries |
| CN103283595A (en) * | 2013-05-17 | 2013-09-11 | 高红胜 | Primary culture method of stem tip tissue of strawberry |
| CN103283595B (en) * | 2013-05-17 | 2015-04-22 | 高红胜 | Primary culture method of stem tip tissue of strawberry |
| CN103329802A (en) * | 2013-06-21 | 2013-10-02 | 苏州市阳澄湖现代农业发展有限公司 | Pollution-free high-yield planting method for strawberries |
| CN103348918A (en) * | 2013-07-19 | 2013-10-16 | 合肥瑞谷农业科技有限公司 | Efficient detoxification tissue cultivating method of strawberries |
| 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 |
| CN104041410A (en) * | 2014-06-05 | 2014-09-17 | 卞佳林 | Strong strawberry seedling rooting culture method |
| CN104041411A (en) * | 2014-06-05 | 2014-09-17 | 卞佳林 | Seedling hardening and rooting culture medium for strawberry |
| CN104186351A (en) * | 2014-09-24 | 2014-12-10 | 江苏农林职业技术学院 | Tissue culture method of strawberries |
| CN104429940A (en) * | 2014-10-23 | 2015-03-25 | 绍兴文理学院 | Method for acquiring virus-free strawberry seedlings |
| CN104429940B (en) * | 2014-10-23 | 2016-06-29 | 绍兴文理学院 | A kind of method obtaining Fructus Fragariae Ananssae detoxic seedling |
| CN105210868A (en) * | 2015-10-08 | 2016-01-06 | 新疆生产建设兵团第六师农业科学研究所 | The acquisition methods of strawberry detoxification breeder's stock seedling and store method |
| CN107135949A (en) * | 2017-06-16 | 2017-09-08 | 黔东南民族职业技术学院 | Strawberry adventitious bud induction culture base and preparation method and application |
| CN108464240A (en) * | 2018-04-13 | 2018-08-31 | 河北富硕农业科技发展有限公司 | The method of Snow White's strawberry detoxifying fast breeding |
| CN109076960A (en) * | 2018-09-29 | 2018-12-25 | 河南云帮农业科技有限公司 | A kind of Plantlets of Strawberry detoxication and tissue culture strong sprout method |
| CN109769694A (en) * | 2019-03-28 | 2019-05-21 | 南京富一农业科技有限公司 | A kind of strawberry stem tip in-vitro regeneration method |
| CN111955346A (en) * | 2020-09-03 | 2020-11-20 | 云南华农农业有限公司 | Novel method for inhibiting browning and improving artificial rapid propagation efficiency of Monte raspberries |
| CN112425508A (en) * | 2020-12-08 | 2021-03-02 | 黑龙江省农业科学院园艺分院 | Culture medium for inducing differentiation, proliferation and rooting of strawberry stem tips |
| CN115517171A (en) * | 2022-10-18 | 2022-12-27 | 太原师范学院 | Method for producing strawberry seedlings by rapid plant tissue culture |
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Application publication date: 20120425 |