CN100420369C - Method for obtaining hormone-free tissue culture detoxified seedling of chrysanthemum for tea use - Google Patents
Method for obtaining hormone-free tissue culture detoxified seedling of chrysanthemum for tea use Download PDFInfo
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- CN100420369C CN100420369C CNB2006100189875A CN200610018987A CN100420369C CN 100420369 C CN100420369 C CN 100420369C CN B2006100189875 A CNB2006100189875 A CN B2006100189875A CN 200610018987 A CN200610018987 A CN 200610018987A CN 100420369 C CN100420369 C CN 100420369C
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Abstract
The present invention discloses a method of a virus-free seedling obtained from tissue culture with no hormone of chrysanthemum for tea. The present invention comprises the working procedures of the procuration of stem apex explant of the chrysanthemum for tea; the culture of the stem apex explant of the chrysanthemum for tea, and the formation of multiple shoots; the virus detection of the multiple shoots of the chrysanthemum for tea; the proliferation of the multiple shoots of the chrysanthemum for tea; the radication and the transplantation of proliferation seedlings of the chrysanthemum for tea. The method of the virus-free tissue culture seedling with no hormone of the chrysanthemum for tea of the present invention, has the advantages of stable system, easy operation, high proliferation rate and virus removing rate of the tissue culture seedling, easy radication of the obtained seedling, haleness, adversity resistance, strong disease resistance ability and insect resistance ability, regular anthesis, high quality and easy cultivation management, and the cost is about 50% less than the conventional cost.
Description
Technical field
The invention belongs to the cultural method of plant, be specifically related to a kind of method of chrysanthemum for tea use obtaining hormone-free tissue culture detoxified seedling.
Background technology
The traditional propagation method of chrysanthemum for tea use mainly is to utilize propagation methods such as cuttage, plant division, but these propagation methods need a large amount of maternal plants on the one hand, the aspect also need spend a large amount of human and material resources, financial resources, time etc. in addition, and reproduction speed is slow, for large-scale chrysanthemum for tea use production, traditional propagation method can not satisfy the needs in the production far away.Simultaneously because the long-term asexual reproduction methods such as plant division, cuttage that adopt, make plant infective virus serious (Cai Zhunan etc., 1992; Song Ruilin etc., 1997).The present virus of invasion and attack chrysanthemum kind surplus in the of 10 down is as the chrysanthemum viroids CSC that stunts, chrysanthemum tomato aspermy virus TAV, tobacco mosaic virus TMV, cucumber mosaic virus CMV, Potyvirus PVX, marmor upsilon etc.And the state of an illness runs down with the prolongation of implantation time, and a little less than the plant strain growth gesture, output reduces, quality variation, even therefore the improved seeds that have are lost.For a long time, still there is not effective method about the control of chrysanthemum virus disease.Development along with modern biotechnology, the apical meristem that it is found that plant is not infected by the virus, thereby adopt Shoot Tip Culture to be expected to obtain virus-free material, cultivate at the potato stem apex, sweet potato stem tip is cultivated, strawberry stem tip obtains virus-free seedling (high rainbow and Li Mei, 1994 of losing of succeeing in cultivating; Shang Youfen etc., 1996).The advantage of detoxic seedling (virus-free seedling) has: hold up strong kind, recover kind of a property, improve the yield and quality; Robust growth, well developed root system; Improve output.Resistance strengthens, and extensive management reduces production costs.Need not or reduce chemical pesticide control, reduced pesticide pollution and residue of pesticide, can obtain the nuisanceless plant of green Chinese herbal medicine, have good economic and social benefit.About the stem apex detoxify of chrysanthemum cultivate focus mostly in the past view and admire and cut-flower chrysanthemum kind aspect (Cai Zhunan etc., 1992; Qiu Wenda etc., 1983; Xu Lijuan etc., 1997; Wang Kang ability etc., 2000; Jiang is carefully prosperous etc., and 2003).Said method is not suitable for the edible demand of chrysanthemum for tea use.
Summary of the invention
The object of the present invention is to provide a kind of method that is applicable to the chrysanthemum for tea use obtaining hormone-free tissue culture detoxified seedling, to address the above problem.
Technical scheme of the present invention is: the method for chrysanthemum for tea use obtaining hormone-free tissue culture detoxified seedling, step is: the acquisition of (1) chrysanthemum for tea use stem apex explant: after will sterilizing with the chrysanthemum for tea use stem section of terminal bud, peel the spire of terminal bud outside off, expose stem apex, cut stem apex; (2) formation of the cultivation of chrysanthemum for tea use stem apex explant and the bud of growing thickly: stem apex is cultivated 4～6 all backs form the bud of growing thickly in inducing culture; Inducing culture is a MS+ sucrose 3%+ agar 0.75%, pH6.0; (3) the grow thickly virus of bud of chrysanthemum for tea use detects: when the length of the bud of growing thickly is 2.5～3.5cm, each individual plant that is produced by stem apex is carried out virus detection; (4) the chrysanthemum for tea use propagation of bud of growing thickly: will detect the back through virus and confirm not have the individual plant of virus and be transferred on the root media and cultivate, when plant grows to 6～8cm, plant is cut into the stem section of 1～2 joint breeds, described root media is a 1/2MS+ sucrose 3%+ agar 0.75%, pH6.0; (proliferation conditions is: fluorescent light source, and continuous illumination every day 12h, intensity of illumination is 2000～3000lx, cultivation temperature (25 ± 1) ℃.); (5) the taking root and transplant of chrysanthemum for tea use propagation seedling: will breed after seedling is cut into stem section with 1～2 joint, after taking root on the root media, hardening, take out test-tube plantlet, after cleaning the root system of test-tube plantlet with clear water again, be transplanted into the detoxic seedling that is promptly obtained not having by chrysanthemum for tea use the acquisition of hormone tissue culture by cultivation in sterilization peat and the sterilization perlite matrix of mixing, root media is a 1/2MS+ sucrose 3%+ agar 0.75%, pH6.0.
Sterilization is that chrysanthemum for tea use stem section was immersed in 70% the alcohol after 20～50 seconds in the described step (1), use aseptic water washing, again with the sterilization of 0.2% mercuric chloride, aseptic water washing, after described mercuric chloride is sterilized with aseptic water washing at least once.
The described cultivation of described step (2) is: illumination, and continuous illumination every day 10-14h, intensity of illumination is 2000～3000lx, cultivation temperature 24-26 ℃, 4～6 weeks of incubation time.
The described cultivation of described step (4) is: illumination, and continuous illumination every day 10-14h, intensity of illumination is 2000～3000lx, cultivation temperature 24-26 ℃, 4～6 weeks of incubation time.
After the described culture of rootage of described step (5), after seedling has 4～5 leaves and 5～6 roots, carry out hardening again.
The described hardening of described step (5): blake bottle is taken out culturing room, remove and seal film, place under the normal temperature and cultivated seedling 2-5 days.
It is stable that chrysanthemum for tea use of the present invention does not have the method system of hormone detoxification tissue culturing seedling, operate easier, the rate of increase and tissue cultivating seedling virus removal rate height; The seedling that obtains takes root easily, and healthy and strong degeneration-resistant, resistant to diseases and insects is strong, and cost is lower by about 50% than conventional, and it is neat to bloom, fresh flower quality height, and cultivation management is easier to.
Embodiment
1, the acquisition of chrysanthemum for tea use stem apex explant:
Get the chrysanthemum for tea use stem section of band terminal bud, be about 2cm, in flowing water, wash 4～6h, on superclean bench, material is immersed 70% alcohol 30s then, use aseptic water washing 3 times, again with 0.2% mercuric chloride sterilization 3min, aseptic water washing 5～6 times, the mercuric chloride sterilization repeats 2 times.The material that disinfects is put into aseptic culture dish, carefully peel the spire of terminal bud outside off under anatomical lens, till seeing smooth surface clearly be conical stem apex under anatomical lens, cut stem apex, length is 0.3～0.5.
2, the formation of the cultivation of chrysanthemum for tea use stem apex explant and the bud of growing thickly:
Stem apex is cultivated in inducing culture, and the stem apex inducing culture is a MS+ sucrose 3%+ agar 0.75%, and pH6.0 promptly adds the sucrose of 30 grams and the agar of 7.5 grams in 1000ml medium MS.Fluorescent light source, continuous illumination every day 12h, intensity of illumination is 2000～3000lx, cultivation temperature (25 ± 1) ℃.Cultivate 4～6 weeks back formation clump bud, stem apex induces differentiation rate to reach more than 80%.
3, the grow thickly virus of bud of chrysanthemum for tea use detects:
When the length of the bud of growing thickly is 2.5～3.5cm, during 2～3 leaves of tool, can be cut into individual plant, each individual plant that is produced by stem apex is carried out virus detect, after confirming not have virus, just can carry out large-scale breeding fast, the production virus-elimination seedlings.
4, the chrysanthemum for tea use propagation of bud of growing thickly:
To detect confirm not have individual plant after the virus through virus and be transferred on the root media and breed, root media is: 1/2MS+ sucrose 3%+ agar 0.75%, pH6.0 promptly add the sucrose of 30 grams and the agar of 7.5 grams in 500ml medium MS.Condition of culture is the same.When following the bottom root to form after general 1 week, the growth of top robust plant, stem is thick, leaf is big, look green.When plant grows to 6～8cm, plant is cut into the stem section of 1～2 joint breeds (blade on the stem section removes or only stays 1/3～1/4).After 25～30 days, the bud growth coefficient reaches 4～7 times.
5, chrysanthemum for tea use is bred taking root of seedling and is transplanted:
After breeding the seedling that obtains some, the propagation seedling can be cut into stem section with 1～2 joint, on above-mentioned root media, take root.Rooting rate reaches 100% after 2 weeks, and the quantity of taking root is many, and root system is sturdy.Work as seedling after 3 weeks and grow to about 5, have 4～5 leaves, during 5～6 roots, test-tube plantlet can be carried out suitable hardening, be about to blake bottle and take out culturing room, remove and seal film, place normal temperature lower refining seedling 3d.Take out test-tube plantlet again from blake bottle, clean the agar that sticks on the root system with clear water again, can be transplanted into the matrix of being mixed (1: 1) by sterilization peat with perlite, sterilization peat and perlitic part by weight can be but be not limited to 1: 1.Matrix elder generation water is drenched, cover 1 week of preserving moisture with plastic film then after, open film, 3～5 time guarantee matrix humidity and high humidity of air with atomizer spray every day.2 all left and right sides transplant survivals, transplanting survival rate is more than 95%.
Utilize chrysanthemum for tea use not have the hormone method for tissue culture and obtain detoxic seedling, strong to the hygrothermal environment adaptive capacity, its stem is stood upright, and pattern is pure, and quality improves 1～2 grade, and being worth increases by 20%～40%, and this research does not appear in the newspapers both at home and abroad as yet.Compare with similar products at home and abroad, our product is with strong points, utilize detoxification chrysanthemum for tea use tissue culturing seedling to be planting material, with adopt asexual reproduction methods such as plant division, cuttage to compare in the traditional chrysanthemum for tea use production, the detoxic seedling that obtains, neither contain hormone, also have advantages such as disease resistance is strong, growth potential is prosperous, purification and rejuvenation, high yield, help implant mass and obtain green non-pollution health care chrysanthemum for tea use.Therefore, in the production of novel non-pollution chrysanthemum for tea use, this method and the detoxic seedling that is obtained by this method have improved the scientific and technological content that chrysanthemum for tea use is produced, has stronger competitiveness, to realizing the standardized planting of chrysanthemum for tea use, the novel non-pollution health care chrysanthemum for tea use product that obtains high yield, quality product matter for the modern production of chrysanthemum for tea use plays critical effect, has better market application.
Claims (6)
1. the method for a chrysanthemum for tea use obtaining hormone-free tissue culture detoxified seedling is characterized in that: the acquisition of (1) chrysanthemum for tea use stem apex explant: after will sterilizing with the chrysanthemum for tea use stem section of terminal bud, peel the spire of terminal bud outside off, expose stem apex, cut stem apex; (2) formation of the cultivation of chrysanthemum for tea use stem apex explant and the bud of growing thickly: stem apex is cultivated 4～6 all backs form the bud of growing thickly in inducing culture; Inducing culture is a MS+ sucrose 3%+ agar 0.75%, and pH 6.0; (3) the grow thickly virus of bud of chrysanthemum for tea use detects: when the length of the bud of growing thickly is 2.5～3.5cm, each individual plant that is produced by stem apex is carried out virus detection; (4) the chrysanthemum for tea use propagation of bud of growing thickly: will detect the back through virus and confirm not have the individual plant of virus and be transferred on the root media and cultivate, when plant grows to 6～8cm, plant is cut into the stem section of 1～2 joint breeds, described root media is 1/2 MS+ sucrose 3%+ agar 0.75%, and pH 6.0; (5) the taking root and transplant of chrysanthemum for tea use propagation seedling: will breed after seedling is cut into stem section with 1～2 joint, after taking root on the root media, hardening, take out test-tube plantlet, after cleaning the root system of test-tube plantlet with clear water again, be transplanted into the detoxic seedling that is promptly obtained not having by chrysanthemum for tea use the acquisition of hormone tissue culture by cultivation in sterilization peat and the sterilization perlite matrix of mixing, root media is 1/2 MS+ sucrose 3%+ agar 0.75%, and pH 6.0.
2. the method for chrysanthemum for tea use obtaining hormone-free tissue culture detoxified seedling according to claim 1, it is characterized in that: sterilization is that chrysanthemum for tea use stem section was immersed in 70% the alcohol after 20～50 seconds in the step (1), use aseptic water washing, sterilize with 0.2% mercuric chloride again, aseptic water washing, described mercuric chloride sterilization back with aseptic water washing at least once.
3. the method for chrysanthemum for tea use obtaining hormone-free tissue culture detoxified seedling according to claim 1, it is characterized in that: the described cultivation of step (2) is: illumination, and continuous illumination every day 10-14h, intensity of illumination is 2000～30001x, cultivation temperature 24-26 ℃, 4～6 weeks of incubation time.
4. the method for chrysanthemum for tea use obtaining hormone-free tissue culture detoxified seedling according to claim 1, it is characterized in that: the described cultivation of step (4) is: illumination, and continuous illumination every day 10-14h, intensity of illumination is 2000～30001x, cultivation temperature 24-26 ℃, 4～6 weeks of incubation time.
5. the method for chrysanthemum for tea use obtaining hormone-free tissue culture detoxified seedling according to claim 1 is characterized in that: after the described culture of rootage of step (5), after seedling has 4～5 leaves and 5～6 roots, carry out hardening again.
6. the method for chrysanthemum for tea use obtaining hormone-free tissue culture detoxified seedling according to claim 1 is characterized in that: the described hardening of step (5): blake bottle is taken out culturing room, remove and seal film, place under the normal temperature and cultivated seedling 2-5 days.
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| CN105309304A (en) * | 2014-07-14 | 2016-02-10 | 东北林业大学 | Rapid induction method of ground-grow chrysanthemum "flame" indirect somatic embryo |
| CN114788495B (en) * | 2022-04-07 | 2023-04-14 | 南充市农业科学院 | Hormone-free strawberry stem tip culture method |
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| CN1286905A (en) * | 1999-09-07 | 2001-03-14 | 北京锦绣大地农业股份有限公司 | Industrial fast test tube reproduction method for African chrysanthemum |
| CN1344489A (en) * | 2001-10-29 | 2002-04-17 | 华南师范大学 | Fast excised African chrysanthemum propagation method |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1178067A (en) * | 1997-10-10 | 1998-04-08 | 中国科学院昆明植物研究所 | Technology for germ plasma preservation and quick breeding by group culture of pyrethrum |
| CN1286905A (en) * | 1999-09-07 | 2001-03-14 | 北京锦绣大地农业股份有限公司 | Industrial fast test tube reproduction method for African chrysanthemum |
| CN1344489A (en) * | 2001-10-29 | 2002-04-17 | 华南师范大学 | Fast excised African chrysanthemum propagation method |
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| 茶(饮) 用菊花的引种初步研究. 蒋细旺，陈卫红，杨涛.湖北林业科技，第2期. 2005 |
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