CN101248758A - Tissue culture method for fine stalk double butterflies - Google Patents
Tissue culture method for fine stalk double butterflies Download PDFInfo
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- CN101248758A CN101248758A CNA2008100307599A CN200810030759A CN101248758A CN 101248758 A CN101248758 A CN 101248758A CN A2008100307599 A CNA2008100307599 A CN A2008100307599A CN 200810030759 A CN200810030759 A CN 200810030759A CN 101248758 A CN101248758 A CN 101248758A
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Abstract
The invention provides a tissue culture method of the Tripterospermum filicaule, which belongs to the plant tissue culture method of the Tripterospermum and relates to the plant tissue culture method. The method includes four steps of obtaining and culturing aseptic seedling, propagation culturing, rooting culturing and transplanting. The culture mediums of the culture condition therein are all selected from L9 (3<4>) orthogonal tests and analysis of variance. (1) germinating the culture medium: MS+ZT 1.0mg.L-<1>; (2) proliferating the culture medium: MS+6BA 1.8mg.L-<1>; (3) rooting the culture medium: 1/2MS+IBa 0.8mg.L-<1>. The culture mediums are all added by 3% sucrose and 1.1% carrageenan with the PH value of 5.8 and the culture temperature at 25 plus or minus 1 DEG C. The automatic-controlled fluorescent lamp light source has a light intensity of 2000LX and a light duration of 14h.d<-1> and a replacement every 30d when proliferating. The method can increase the inductivity, the reproduction coefficient and the transplanting survival rate of the sprout, which solves the problems of the difficulty and the complication of the medical plant seed.
Description
Technical field
The present invention is the method for tissue culture of fine stalk double butterflies, relates to the breeding effectively fast of this medicinal plant, development and use.
Technical background
To the Tripterospermum Study on plants, document seldom, domestic mainly is the article of classification, development, external and China Taiwan Province has carried out the research of plant chemical ingredient and pharmic function active component aspect to 5 kinds of this platymiscium.The fine stalk double butterflies plant, seed is tiny, resting stage is long, and germination rate is extremely low, and the applicant was through experiment in 3 years, when finding this plant seed breeding, its seedling has a brephic that does not have actual effect, germinates to can not be completed two leaf stage in back 6 months, so not only germination rate is extremely low, the back planting percent that germinates is also extremely low, so the seminal propagation difficulty is very big.
Summary of the invention
The technical problem that the present invention need solve is:, germinate back planting percent low, seminal propagation difficulty big problem low at fine stalk double butterflies plant germination rate, and set up a kind of tissue culture method of fine stalk double butterflies efficiently.
The object of the present invention is achieved like this:
The first step, aseptic seedling obtains and cultivates: get annual plant rhizome (near the terrestrial stem on 10 centimeters on ground, generally from the mixed forest of the tea woods of acid red loam or oil tea and masson pine, tung oil tree, Chinese fir down the collection fine stalk double butterflies do not have the stem of damage by disease and insect as explant.Because the rhizome near ground is younger than the physiological age of the twining stem above the rhizome, the explant that physiological age is young more, its lateral bud sprouts sprouting on the bud inductive differentiation medium time more early, bud ratio is high more, fine stalk double butterflies does not form callus substantially, all is to be sprouted by the dormancy sprouting of lateral bud.) removal blade and petiole, be cut into the long stem section of 6cm, first clean back with saturated washing powder solution rinsing 1-5 minute with running water, again with running water flowing water flushing 1-2 hour, use then after distilled water flushing 1-2 time in desinfection chamber on superclean bench, alcohol with 75% soaks 30s, and constantly shakes, so that fully sterilization.Outwell alcohol, the mercuric chloride solution of adding 0.1% and a Tween 80 soaked 8-10 minute, with further sterilization.After outwelling mercuric chloride solution, use aseptic water washing again 6 times, place on the dry aseptic filter paper of sterile petri dish, blot surface moisture.Be cut into about long 1.5cm with scraper, every section stem with bud that a joint is arranged inserts in the medium with the lower end, and joint and axillalry bud are exposed at outside the medium, are inoculated on the medium.The dark cultivation after 5 days changes illumination cultivation over to.After 6 days, explant is sprouted successively on the germination medium, grows tender shoots.
Second step: enrichment culture: inoculation back 30-35d, during the long 1-2cm of bud, select the bud of healthy cleaning-less bacteria infection to move into together with explant and carry out numerous bud cultivation on the proliferated culture medium.Inoculate after 40 days, about height of seedling 5cm, tool 3-4 joint, reproduction coefficient 5.1, later every 30-35d changes a subculture.
The 3rd step: culture of rootage: long to 2-3cm when indefinite bud, during tool 3-4 sheet leaf, with its cutting-out, switching is gone on the root media, carries out inducing of root, cultivates about 30 days, begins to grow adventive root.
The 4th step: acclimatization and transplants: when about test-tube plantlet plant height 4cm, during the long 2-3cm of root, the selection robust growth, the plant of well developed root system carries out acclimatization and transplants.Earlier seedling is shifted out group training chamber, place in the greenhouse of shade rate (with comparing under the daylight) about 70% and close a bottle hardening 3d, carry out the illumination adaptive training, (through to the research of this plant species original producton location environmental ecology limiting factor, find that this plant happiness is distributed under the camellia oleifera lam on hillside of one side backlight in afternoon.Measure through illuminometer and psychrometer, at shade rate about 70%, well-grown in the poor environment in gulf of cloud barrier mist cover, stem length can reach more than one meter, and the individual plant cauline leaf weighs about 0.25kg.) will cultivate bottle cap again and open half, half-open bottle hardening 1 day, uncork hardening again a day.Then seedling is taken out, clean the root medium, transplant in perlite is housed: red soil: river sand ratio is in the little flowerpot of plastics of 1: 2: 1 matrix, (its matrix should be 0.1% liquor potassic permanganate or 0.1% bacterium final hit was handled 24 hours), clean blade face and stem surface through spraying (every 100kg water+a small amount of MS culture fluid+1 streptomycin), after treating that blade surface does not have ponding, seal the alms bowl mouth with transparent plastic bag, from second day, sealing hole that chopsticks are thick of thorn on the plastic sack every day, remove after 15 days and seal film, enter normal growth, transplanting survival rate is more than 95%.Transplant to oil tea Right wood sylvan life like the environment facies of original producton location, twining stem reached more than the 50cm in 1 year, can yield positive results in two years, gathered in the crops its plant corpus and was used as medicine.
Wherein: above-mentioned condition of culture, medium is by L9 (3
4) orthogonal experiment, variance analysis select.
1. germination medium: MS+ZT 1.0mg.L
-1
2. proliferated culture medium: MS+6BA 1.8mg.L
-1
3. root media: 1/2MS+IBA 0.8m.L
-1
Above medium all adds 3% sucrose, 1.1% carragheen, PH5.8,25 ± 1 ℃ of cultivation temperature, automatic control fluorescent light source, light intensity 2000LX, light application time 14h.d
-1., every 30d changes a subculture during propagation.
Adopt said method, have following advantage:
1. bud lures differential medium to adopt ZTmg/L (zeatin) to bring up to 47%. as the inductivity that growth stimulant replaces 6-BA1.5mg/L induced dormancy bud to sprout into sprouting from 15%
2. clearly make explant without twining stem certainly, and with sturdy rhizome near the ground 10 centimeters as explant, explant physiological age youth not only like this, and it is nutritious, the time that makes rudiment is ahead of time more than 1 day, bud is also sturdy, and this also is the reason that reproduction coefficient improves, and the bar number of root increases by 0.4/strain.
3. in the proliferated culture medium, the 6-BA umber is increased to 1.8mg/L by 1.5mg/L, is the another reason that reproduction coefficient improves, and can breed 4.6 buds in per two months.
4. when acclimatization and transplants: 1. in the greenhouse, carry out earlier three days illumination adaptive trainings and 2. cultivate bottle cap and divided two days, two steps opened, the back plants to select for use the transparent plastic film bag to seal behind the flowerpot to preserve moisture, prevent the volatilization of self antibiotic substance, pierce through the sealed bag film gradually, make it to be subjected to the humidity adaptive training, increase antibacterial ability.3. thoroughly handle germ in the matrix with potassium permanganate and bacterium final hit, transplant and seal up transparent membrane after the back is sprayed once with the streptomycin weak solution, when making training tissue culture seedling because the cortex prosperity of stem, colloid substance is many, be easy to bacteria infection and cause the high conspicuous contradiction of lethality better to be solved, also make this plant because of originating in the high and cold mountain area sylvan life thick grass, air humidity is big, stem transportation moisture ability, the contradiction that adapts to the dry air ability is solved.4. the humus soil in the matrix formulations is changed into the red soil of original producton location 3.5-5.5, make putrefactive microorganisms minimizing in the soil, the habitat adds to add an amount of MS nutrient solution in matrix more near the original producton location condition, so make transplanting survival rate improve 6%, reaches more than 95%.
5. make explant through the group training---there is not the brephic that does not have actual effect in the plant that nourishes and generates and produce with the material of growing up.It is big to have overcome the hard to tackle degree of this plant species seed, restricts the huge contradiction that this kind medicinal plant extensive exploitation utilizes.
Embodiment
The invention will be further described below in conjunction with example:
The first step: obtain aseptic seedling: the rhizome of getting annual plant, remove blade and petiole, be cut into the long stem section of 6cm, first clean back with saturated washing powder solution rinsing 5 minutes with running water, again with running water flowing water flushing two hours, use after the distilled water flushing 2 times in desinfection chamber on superclean bench alcohol-pickled 30s then with 75%, and constantly shaking, so that fully sterilization.Outwell alcohol, the mercuric chloride solution of adding 0.1% and a Tween-80 soaked 10 minutes, with further sterilization.After outwelling mercuric chloride solution, use aseptic water washing again 6 times, place on the dry aseptic filter paper of sterile petri dish, blot surface moisture.Be cut into about long 1.5cm with scraper, every section stem with bud that a joint is arranged inserts in the medium with the lower end, and joint and axillalry bud are exposed at outside the medium, are inoculated on the medium.The dark cultivation after 5 days changes illumination cultivation over to.After 6 days, explant is sprouted successively on the germination medium, grows tender shoots.
Second step: enrichment culture: inoculation back 30d, during the long 2cm of bud, select the bud of healthy cleaning-less bacteria infection to move into together with explant and carry out numerous bud cultivation on the proliferated culture medium.Inoculate after 40 days, about height of seedling 5cm, tool 3-4 joint, reproduction coefficient 5.1, later every 35d changes a subculture.
The 3rd step: culture of rootage: long to 2cm when indefinite bud, during tool 3-4 sheet leaf, with its cutting-out, switching is gone on the root media, carries out inducing of root, cultivates 30 days, begins to grow adventive root.
The 4th step: acclimatization and transplants: when about test-tube plantlet plant height 4cm, during the long 3cm of root, the selection robust growth, the plant of well developed root system carries out acclimatization and transplants.Earlier seedling is shifted out group training chamber, place and close bottle in the greenhouse of shade rate (with comparing under the daylight) about 70% and carry out illumination adaptive training 3d, will cultivate bottle cap again and open half, half-open bottle of hardening 1 day, uncork hardening again a day.Then seedling is taken out, clean the root medium, transplant in perlite is housed: red soil: river sand ratio is in the little flowerpot of plastics of 1: 2: 1 matrix, (its matrix should be 0.1% liquor potassic permanganate or 0.1% bacterium final hit was handled 24 hours), clean blade face and stem surface through spraying (every 100kg water+a small amount of MS culture fluid+1 streptomycin), after treating that blade surface does not have ponding, seal the alms bowl mouth with transparent plastic bag, from second day, sealing hole that chopsticks are thick of thorn on the plastic sack every day, remove after 15 days and seal film, enter normal growth.Then, transplant to oil tea Right wood sylvan life like the environment facies of original producton location.
Wherein: above-mentioned condition of culture, medium is by L9 (3
4) orthogonal experiment, variance analysis select.
1. germination medium: MS+ZT 1.0mg.L
-1
2. proliferated culture medium: MS+6BA 1.8mg.L
-1
3. root media: 1/2MS+IBA 0.8m.L
-1
Above medium all adds 3% sucrose, 1.1% carragheen, PH5.8,25 ℃ of cultivation temperature, automatic control fluorescent light source, light intensity 2000LX, light application time 14h.d
-1., every 30d changes a subculture during propagation.
Claims (2)
1. the method for tissue culture of fine stalk double butterflies is characterized in that adopting following step:
The first step, aseptic seedling obtains and cultivates: the rhizome of getting annual plant, remove blade and petiole, be cut into the long stem section of 6cm, clean the back with saturated washing powder solution rinsing 1-5 minute, again with running water flowing water flushing 1-2 hour with running water earlier, use then after distilled water flushing 1-2 time in desinfection chamber on superclean bench, alcohol with 75% soaks 20-30s, and constantly shakes, so that fully sterilization; Outwell alcohol, the mercuric chloride solution of adding 0.1% and a Tween 80 soaked 8-10 minute, with further sterilization, after outwelling mercuric chloride solution, use aseptic water washing 3-6 time again, place on the dry aseptic filter paper of sterile petri dish, blot surface moisture, be cut into about long 1.5cm, every section stem with bud that a joint is arranged with scraper, insert in the germination medium with the lower end, joint and axillalry bud are exposed at outside the germination medium, are inoculated on the germination medium, secretly cultivate after 5 days and change illumination cultivation over to, after 6 days on the germination medium explant sprout successively, grow tender shoots;
Second step: enrichment culture: inoculation back 30-35d, during the long 1-2cm of bud, select the bud of healthy cleaning-less bacteria infection to move into together with explant and carry out numerous bud cultivation on the proliferated culture medium; Inoculate after 40 days, about height of seedling 5cm, tool 3-4 joint, reproduction coefficient 5.1, later every 30-35d changes a subculture;
The 3rd step: culture of rootage: long to 2-3cm when indefinite bud, during tool 3-4 sheet leaf, with its cutting-out, switching is gone on the root media, carries out inducing of root, cultivates 30 days;
The 4th step: acclimatization and transplants: when about test-tube plantlet plant height 4cm, during the long 2-3cm of root, select robust growth, the plant of well developed root system carries out acclimatization and transplants, earlier seedling is shifted out group training chamber, place with daylight under to compare shade rate be to close a bottle hardening 3d in 70% the greenhouse, carry out the illumination adaptive training, to cultivate bottle cap again and open half, half-open bottle hardening 1 day, uncork hardening again a day, then seedling is taken out, clean the root medium, transplant in perlite is housed: red soil: river sand ratio is that its matrix should be 0.1% liquor potassic permanganate or 0.1% bacterium final hit was handled 24 hours in 1: 2: 1 the little flowerpot of plastics of matrix, clean blade face and stem surface through every 100kg water+a small amount of MS culture fluid+1 streptomycin spraying, after treating that blade surface does not have ponding, seal the alms bowl mouth, from second day with transparent plastic bag, sealing hole that chopsticks are thick of thorn on the plastic sack every day, remove after 15 days and seal film, enter normal growth, transplant to oil tea Right wood sylvan life like the environment facies of original producton location.
2. the method for tissue culture of fine stalk double butterflies according to claim 1, it is characterized in that: above-mentioned medium is by L9 (3
4) orthogonal experiment, variance analysis select, wherein:
1. germination medium: MS+ZT 1.0mg.L
-1
2. proliferated culture medium: MS+6BA 1.8mg.L
-1
3. root media: 1/2MS+IBA 0.8m.L
-1
Above medium all adds 3% sucrose, 1.1% carragheen, PH5.8,25 ± 1 ℃ of cultivation temperature, automatic control fluorescent light source, light intensity 2000LX, light application time 14h.d
-1., every 30d changes a subculture during propagation.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008100307599A CN101248758B (en) | 2008-03-07 | 2008-03-07 | Tissue culture method for fine stalk double butterflies |
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| Application Number | Priority Date | Filing Date | Title |
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| CN2008100307599A CN101248758B (en) | 2008-03-07 | 2008-03-07 | Tissue culture method for fine stalk double butterflies |
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| CN101248758A true CN101248758A (en) | 2008-08-27 |
| CN101248758B CN101248758B (en) | 2011-05-11 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102908383A (en) * | 2012-11-12 | 2013-02-06 | 吉首大学 | Method for preparing antivenom oral liquid |
| CN103718964A (en) * | 2013-12-25 | 2014-04-16 | 吉首大学 | Seed breeding technology of tripterospermum cordatum |
| WO2018068281A1 (en) * | 2016-10-14 | 2018-04-19 | 泉州市泉美生物科技有限公司 | Method for cultivating plant in transparent sealed container and medium therefor |
-
2008
- 2008-03-07 CN CN2008100307599A patent/CN101248758B/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102908383A (en) * | 2012-11-12 | 2013-02-06 | 吉首大学 | Method for preparing antivenom oral liquid |
| CN102908383B (en) * | 2012-11-12 | 2014-04-09 | 吉首大学 | Method for preparing antivenom oral liquid |
| CN103718964A (en) * | 2013-12-25 | 2014-04-16 | 吉首大学 | Seed breeding technology of tripterospermum cordatum |
| CN103718964B (en) * | 2013-12-25 | 2015-05-20 | 吉首大学 | Seed breeding technology of tripterospermum cordatum |
| WO2018068281A1 (en) * | 2016-10-14 | 2018-04-19 | 泉州市泉美生物科技有限公司 | Method for cultivating plant in transparent sealed container and medium therefor |
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| CN101248758B (en) | 2011-05-11 |
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