CN104737914A - Cinnamomum camphora tissue culture method - Google Patents
Cinnamomum camphora tissue culture method Download PDFInfo
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- CN104737914A CN104737914A CN201510180211.2A CN201510180211A CN104737914A CN 104737914 A CN104737914 A CN 104737914A CN 201510180211 A CN201510180211 A CN 201510180211A CN 104737914 A CN104737914 A CN 104737914A
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Abstract
The invention discloses a cinnamomum camphora tissue culture method. The method comprises the following steps: picking new sprouted semi-lignified branches of cinnamomum camphora in the same year in April to May each year, scissoring branches with 2-3 leaves, removing the leaves, oscillating and washing in sterile water for 3-5 minutes, treating by using 70% alcohol for 1-2 minutes, placing in 0.1% mercury bichloride for 6-10 minutes, and cleaning by using sterile water for 3 times; inoculating the treated semi-lignified branches into an adventitious bud induction medium, thereby obtaining an aseptic explant, culturing under illumination conditions, and thus forming callus and adventitious buds for expanding propagation; culturing in a mulitiplication expanding propagation culture medium and a root induction medium, and cultivating to obtain rooting seedlings; and transplanting the cultured cinnamomum camphora rooting seedlings, wherein the survival rate is over 95 percent.
Description
Technical field
The present invention relates to a kind of method for tissue culture, specifically a kind of borneol camphor tree method for tissue culture.
Background technology
Camphor tree (Cinnamomum comphora) is Lauraceae Cinnamomum aiphyllium seeds, is Ji'an City indigenous species.Institute of Ji'an City woods section scientific research personnel is through studying for many years, and chemical composition contained by camphor tree, is divided into the types such as borneol camphor tree, fragrant camphor tree, brain camphor tree, oily camphor tree, different camphor tree by camphor tree.Borneol camphor tree is that in above several chemical type, natural distributed is minimum, the seeds that economic worth is the highest.The natural d-borneol camphor oil extracted by borneol camphor tree coppice method plantation, is widely used in the industries such as medicine, essence and flavoring agent and cosmetics, wide market.
Breeding of borneol camphor tree has seminal propagation and cottage propagation usually, and because the borneol camphor tree elite stand of natural distributed is few, the nursery stock after seed propagation exists variation phenomenon; Cottage propagation also causes superior type seedling-wood breeding quantity very limited because superior type resource is few.Therefore, if the method for tissue cultures can be adopted to carry out borneol camphor tree superior type seedling propagation, the difficult problems such as borneol camphor tree excellent seedling propagation speed is slow, transplanting survival rate is low can be solved preferably.
Summary of the invention
The object of the present invention is to provide the borneol camphor tree method for tissue culture that a kind of reproduction speed is fast, transplanting survival rate is high, to solve the problem proposed in above-mentioned background technology.
For achieving the above object, the invention provides following technical scheme:
A kind of borneol camphor tree method for tissue culture, concrete incubation step is as follows:
A. get borneol camphor tree annual 4 ~ May and newly take out semi-lignified branch then, clip has the semi-lignified branch of 2-3 blade, after removing blade, be placed in sterile water vibration washing 3-5 minute, the ethanol postincubation 1-2 minute of 70% is used again after taking-up, then be placed in 0.1% mercuric chloride 6-10 minute, then use sterile water wash 3 times, the semi-lignified branch of obtained process;
B. the semi-lignified branch of process is inoculated in adventitious bud induction culture base, obtain aseptic explant, temperature 26-28 DEG C, after light intensity 1500-2000lx, every day 12 cultivate 40-50 days under h light condition of culture, formed for expanding numerous callus and indefinite bud, wherein adventitious bud induction culture base: MS minimal medium+6-BA0.5 ~ 1.0mg/L+NAA0.05 ~ 0.2 mg/L, pH=5.5;
C. callus and indefinite bud are put into shoot proliferation and expand breeding culture medium, obtained propagation sorite; Breeding condition: temperature 26-28 DEG C, light intensity 1500-2000lx, 12h/d; Shoot proliferation expands breeding culture medium: improvement MS+6-BA0.3 ~ 2.0mg/L+NAA0.05 ~ 0.2mg/L+Na
2s
2o
3100 ~ 300mg/L, pH=5.5;
D. propagation sorite is put into root induction medium, cultivate and obtain seedling of taking root; Breeding condition: temperature 26-28 DEG C, light intensity 1500-2000lx, 12h/d; Root induction medium: improvement MS+IBA1.0mg/L+NAA0.01 ~ 0.2mg/L, pH=5.5;
E. by the transplantation of seedlings of taking root through above cultivation;
Described improvement MS is in MS minimal medium, and potassium nitrate and ammonium nitrate component reduce by half, and calcium chloride composition increases by 20%, and sulphate composition doubles, other components unchanged.
As the further scheme of the present invention: in step b, adventitious bud induction culture base: MS minimal medium+6-BA0.8mg/L+NAA0.1mg/L, pH=5.5.
As the further scheme of the present invention: in step c, shoot proliferation expands breeding culture medium: improvement MS+6-BA 1.0mg/L+NAA 0.05mg/L+Na
2s
2o
3150mg/L, pH=5.5.
As the further scheme of the present invention: in steps d, root induction medium: improvement MS+IBA 1.0mg/L+NAA 0.05mg/L.
As the further scheme of the present invention: in step e, the seedling of taking root of having taken root to be taken away experienced seedling, practice seedling and become bottle green to stem in 20-30 days, washing away medium is again transplanted in the matrix of 1/3 coconut palm chaff+2/3 peat soil, control of temperature and humidity wherein in 25 days: temperature 26-30 DEG C, humidity 80 ~ 100%.
As the further scheme of the present invention: in described step in b, c, d, MS minimal medium or improvement MS in all containing sucrose 3%, carragheen 0.8%, pH=5.5.
Compared with prior art, the invention has the beneficial effects as follows: borneol camphor tree tissue cultures adventitious bud induction culture base of the present invention: MS minimal medium+6-BA0.8mg/L+NAA0.1mg/L, pH=5.5; Shoot proliferation expands breeding culture medium: improvement MS+6-BA 1.0mg/L+NAA 0.05mg/L+Na
2s
2o
3150mg/L, pH=5.5, root induction medium: improvement MS+IBA 1.0mg/L+NAA 0.05mg/L, by transplantation of seedlings of taking root through the above borneol camphor tree cultivated, survival rate more than 95%.
Embodiment
Below in conjunction with the embodiment of the present invention, be clearly and completely described the technical scheme in the embodiment of the present invention, obviously, described embodiment is only the present invention's part embodiment, instead of whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art, not making the every other embodiment obtained under creative work prerequisite, belong to the scope of protection of the invention.
Embodiment 1
In the embodiment of the present invention, material is taken from the raw then of the borneol camphor tree superior type of Ji'an City screening and newly takes out semi-lignified branch, and the time of drawing materials is April 30.Test method:
(1) condition of culture
All containing sucrose 3% in MS minimal medium or modified MS medium, carragheen 0.8%, pH5.5, is placed in temperature 26-28 DEG C, light intensity 1500-2000lx, and every day 12, h light was cultivated.
(2) Multiplying culture research
In following development test, test 1,2,3,4 each process 200 repetitions, each repetition 1 propagation unit (the stem section of 1 2-3 joint).35 days each proliferating cycles.
1. different pH value medium is on the impact of propagation
The medium of two kinds of process, all uses improvement MS+6-BA(6-benzyl aminoadenine) 0.8mg/L+NAA(methyl α-naphthyl acetate) 0.05 mg/L medium.But during preparation medium, respectively pH value being formulated to 6.5,5.5,5.2,5.0, take pH=6.0 as control treatment.Cultivate one proliferating cycle " Invest, Then Investigate " statistics newly take out indefinite bud quantity and growing way situation.
2. in medium the 6-BA of variable concentrations on the impact of multiplication rate
Modified MS medium+NAA0.05mg/L is put in each propagation unit, and coordinates 6-BA concentration to be respectively in 0mg/L, 0.5mg/L, 0.8mg/L, 1.0mg/L, 1.5mg/L, 2.0mg/L, 3.0mg/L, with 6-BA 0mg/L for control treatment.Cultivate one proliferating cycle " Invest, Then Investigate " statistics newly take out lateral bud quantity.
3. in medium different basic salt density on the impact of multiplication rate and growing way
Main employing MS minimal medium, 1/2MS, improvement MS and MS(3/5 1#, 2 times of 2#, 6/5 3#), coordinate 6-BA 1.0mg/L+NAA 0.05mg/L(pH=5.5).With MS minimal medium for control treatment.Cultivate one proliferating cycle " Invest, Then Investigate " statistics newly take out indefinite bud quantity and growing way situation.
Illustrate: in 1/2MS:MS minimal medium composition, the content of all the components reduces by half.
Improvement MS is in MS minimal medium, and potassium nitrate and ammonium nitrate component reduce by half, and calcium chloride composition increases by 20%, and sulphate composition doubles, other components unchanged.
MS(3/5 1#, 2 times of 2#, 6/5 3#) be: the content of each composition in MS minimal medium, the ammonium nitrate of 3/5 and potassium nitrate, the potassium iodide of 2 times, cobalt chloride and calcium chloride, the magnesium sulfate of 6/5, manganese sulphate, zinc sulphate and copper sulphate, other components unchanged.
4. how to solve the withered problem pushing away leaf
Find in shoot proliferation incubation, the sprout top tip or leaf of the stem Duan Xin cultivating propagation has withered top or fall leaf phenomenon in various degree, and growing way is poor.Adopt medium improvement MS+6-BA 1.0mg/L+NAA 0.05 mg/L, coordinate and add Na
2s
2o
3150mg/L, 300 mg/L; MgSO
4800mg/L, 1600mg/L tetra-process.Not add Na
2s
2o
3or MgSO
4for control treatment.The withered top of investigation statistics or fall leaf ratio respectively after cultivating a proliferating cycle.
(3) culture of rootage research
Explore different growth hormone and borneol camphor tree group is trained to the impact of taking root, minimal medium is improvement MS, coordinate with IBA(indolebutyric acid), NNA(4-(N-methyl-N-nitroso amine)-4-(3-pyridine radicals) butyraldehyde), IAA(indole-3-acetic acid) variable concentrations and proportioning thereof, investigation statistics 15 days, 30 days rooting rates.
(4) results and analysis
1. different pH value medium is on the impact of multiplication rate
Different pH value medium on propagation to affect result as shown in table 1.As can be seen from Table 1, proliferated culture medium pH value is advisable with 5.5.In the medium: improvement MS+6-BA 1.0mg/L+NAA 0.05mg/L(pH=5.5), the good vigor foot of sorite growing way, indefinite bud increasing number, growth coefficient is the highest, reaches 2.7, and it is also maximum that simultaneous altitude is greater than 2.0 centimetres of indefinite bud quantity, can gather in the crops seedling quantity of taking root maximum, utilization percent reaches 2.1.PH value raises or reduces, and sorite growing way is general or poor, and growth coefficient and utilization percent are all at mean level; PH value is elevated to 6.5, newly take out indefinite bud growing way generally, but vigor is not enough; PH value is reduced to 5.0, and it is poor newly to take out indefinite bud growing way, also there will be the here soft phenomenon of new leaf development.
Table 1
2. in medium the 6-BA of variable concentrations on the impact of multiplication rate
The effect of basic element of cell division 6-BA to borneol camphor tree group training propagation is embodied in: in modified MS medium, within the scope of 0 ~ 1.0 mg/L, along with the rising of 6-BA concentration, growth coefficient is higher; Tend towards stability within the scope of 1.0 ~ 3.0 mg/L, growth coefficient is about 2.5.Comprehensive growth coefficient, can utilization percent and the several index of growing way situation to consider, basic element of cell division 6-BA on multiplication rate to affect result as shown in table 2.As can be seen from Table 2,6-BA concentration is best with 0.8-1.0mg/L.
Table 2
3. in medium different basic salt density on the impact of multiplication rate and growing way
At 6-BA1.0mg/L+NAA0.05 mg/L(pH=5.5), by different salt density in adjustment medium, in medium, the different basic impact of salt density on multiplication rate and growing way is as shown in table 3.Can find: minimal medium is advisable to improve MS, its growth coefficient and utilization percent the highest, and to grow fine.As adopted full dose MS minimal medium, also there will be that bud is few, vigor is not enough and indivedual blade withered fall phenomenon.
Table 3
| Process | Process total sorite number | Indefinite bud quantity (growth coefficient) | H > 2cm indefinite bud number (utilization percent) | Growing way situation |
| CK：MS | 200 | 462（2.3） | 250（125%） | Bud is few, and vigor is not enough, indivedual leaf withered fall |
| 1/2MS | 200 | 482（2.4） | 360（180%） | Grow fine, blade pale green |
| Improvement MS | 200 | 536（2.68） | 416（208%） | Grow fine, the green vigor foot of leaf |
| MS (3/5 1#, 2 times of 2#, 6/5 3#) | 200 | 502（2.5） | 372（186%） | Growing way is general, and leaf is greener |
4. how to solve the withered problem pushing away leaf
At improvement MS+6-BA1.0mg/L+NAA0.05mg/L(pH=5.5) in medium, still there is withered top in various degree and fall leaf phenomenon in borneol camphor tree.Add in subculture medium certain density sodium thiosulfate energy efficient solution never normal bud withered top or fall leaf problem, Na
2s
2o
3on the withered top of indefinite bud and fall leaf to affect result as shown in table 4.As shown in Table 4, sorite is bred at medium improvement MS+6-BA 1.0mg/L+NAA 0.05mg/L+Na
2s
2o
3150mg/L(pH=5.5), the withered leaf rate that pushes away of indefinite bud drops to 0.5%.
Table 4
| Process medium | Process total sorite number | The withered useful amount of indefinite bud number (withered push away leaf rate) | Growth coefficient |
| CK: improvement MS+6-BA1.0mg/L+NAA0.05mg/L | 200 | 14（7.0%） | 2.6 |
| CK+Na 2S 2O 3150mg/L | 200 | 1（0.5%） | 2.67 |
| CK+Na 2S 2O 3300mg/L | 200 | 5（2.5%） | 2.5 |
| CK+MgSO 4800mg/L | 200 | 12（6.0%） | 2.6 |
| CK+MgSO 41600mg/L | 200 | 11（5.5%） | 2.5 |
5. growth hormone IAA, IBA and NAA is on the impact of taking root
Root induction test result is as shown in table 5.Growth hormone IAA is not obvious to borneol camphor tree group training rooting effect, and its rooting rate is the highest by only 30%; IBA and NAA all works to taking root, but IBA, NAA are used alone, and rooting rate is all lower, the highest by only 75%.Test shows, best to improve MS+IBA1.0mg/L+NAA0.05mg/L medium, rooting rate reaches 98%, and its root system is many and elongated, also the highest with the transplantation of seedlings survival rate of taking root that this processes, and reaches more than 95%; And improve MS+IBA1.0 mg/L+NAA0.1 (or 0.2) mg/L, though its root system is many, tubbiness is easily broken, and after transplanting, survival rate is low, only between 20-35%.
Table 5
| Process medium | Process sum | 15 born offspring numbers (rooting rate) | 30 born offspring numbers (rooting rate) | The growing way situation of root |
| CK: improvement MS | 200 | 0（0%） | 5（2.5%） | Take root slow, few root, root is elongated |
| Improvement MS+IBA0.5 | 200 | 2（1%） | 60（30%） | Radical amount is few, elongated |
| Improvement MS+IBA1.0 | 200 | 12（6%） | 142（71%） | Radical is slightly many, elongated |
| Improvement MS+IAA0.5 | 200 | 0（0%） | 40（20%） | Take root slow, few root, root is elongated |
| Improvement MS+IAA1.0 | 200 | 2（1%） | 60（30%） | Take root slow, few root, root is elongated |
| Improvement MS+NAA0.05 | 200 | 8（4.0%） | 120（60%） | Radical amount is few, and root is slightly thick |
| Improvement MS+NAA0.1 | 200 | 9（4.5%） | 75（75%） | Radical is slightly many, and root is short and thick |
| Improvement MS+IBA1.0+NAA0.05 | 200 | 12（6%） | 196（98%） | Radical is many, and root is elongated |
| Improvement MS+IBA1.0+NAA0.1 | 200 | 12（6%） | 198（99%） | Radical is many, and root is short and thick |
| Improvement MS+IBA1.0+NAA0.2 | 200 | 12（6%） | 198（99%） | Radical is many, and root is short and thick, root is crisp easily broken |
6. transplant
The plantlet in vitro of having taken root to be taken away experienced seedling, practice seedling and become bottle green to stem in 20-30 days, then wash away medium and be transplanted in the matrix of 1/3 coconut palm chaff+2/3 peat soil, notice that epidemic disaster controls in 25 days, survival rate more than 95%.
(5) conclusion
Borneol camphor tree tissue cultures adventitious bud induction culture base can adopt MS+6-BA0.5mg/L+NAA0.2mg/L(pH=5.5); Shoot proliferation expands breeding culture medium can adopt improvement MS+6-BA1.0mg/L+NAA0.05 mg/L+ Na
2s
2o
3150mg/L(pH=5.5); Root induction medium is to improve MS+IBA1.0 mg/L+NAA0.05 mg/L(pH=5.5) be advisable.
To those skilled in the art, obviously the invention is not restricted to the details of above-mentioned one exemplary embodiment, and when not deviating from spirit of the present invention or essential characteristic, the present invention can be realized in other specific forms.Therefore, no matter from which point, all should embodiment be regarded as exemplary, and be nonrestrictive, scope of the present invention is limited by claims instead of above-mentioned explanation, and all changes be therefore intended in the implication of the equivalency by dropping on claim and scope are included in the present invention.
In addition, be to be understood that, although this specification is described according to embodiment, but not each embodiment only comprises an independently technical scheme, this narrating mode of specification is only for clarity sake, those skilled in the art should by specification integrally, and the technical scheme in each embodiment also through appropriately combined, can form other embodiments that it will be appreciated by those skilled in the art that.
Claims (6)
1. a borneol camphor tree method for tissue culture, is characterized in that, concrete incubation step is as follows:
A. get borneol camphor tree annual 4 ~ May and newly take out semi-lignified branch then, clip has the semi-lignified branch of 2-3 blade, after removing blade, be placed in sterile water vibration washing 3-5 minute, the ethanol postincubation 1-2 minute of 70% is used again after taking-up, then be placed in 0.1% mercuric chloride 6-10 minute, then use sterile water wash 3 times, the semi-lignified branch of obtained process;
B. the semi-lignified branch of process is inoculated in adventitious bud induction culture base, obtain aseptic explant, temperature 26-28 DEG C, after light intensity 1500-2000lx, every day 12 cultivate 40-50 days under h light condition of culture, formed for expanding numerous callus and indefinite bud, wherein adventitious bud induction culture base: MS minimal medium+6-BA0.5 ~ 1.0mg/L+NAA0.05 ~ 0.2 mg/L, pH=5.5;
C. callus and indefinite bud are put into shoot proliferation and expand breeding culture medium, obtained propagation sorite; Breeding condition: temperature 26-28 DEG C, light intensity 1500-2000lx, 12h/d; Shoot proliferation expands breeding culture medium: improvement MS+6-BA0.3 ~ 2.0mg/L+NAA0.05 ~ 0.2mg/L+Na
2s
2o
3100 ~ 300mg/L, pH=5.5;
D. propagation sorite is put into root induction medium, cultivate and obtain seedling of taking root; Breeding condition: temperature 26-28 DEG C, light intensity 1500-2000lx, 12h/d; Root induction medium: improvement MS+IBA1.0mg/L+NAA0.01 ~ 0.2mg/L, pH=5.5;
E. by the transplantation of seedlings of taking root through above cultivation;
Described improvement MS is in MS minimal medium, and potassium nitrate and ammonium nitrate component reduce by half, and calcium chloride composition increases by 20%, and sulphate composition doubles, other components unchanged.
2. a kind of borneol camphor tree method for tissue culture according to claim 1, is characterized in that: in step b, adventitious bud induction culture base: MS minimal medium+6-BA0.8mg/L+NAA0.1mg/L, pH=5.5.
3. a kind of borneol camphor tree method for tissue culture according to claim 1, is characterized in that: in step c, and shoot proliferation expands breeding culture medium: improvement MS+6-BA 1.0mg/L+NAA 0.05mg/L+Na
2s
2o
3150mg/L, pH=5.5.
4. a kind of borneol camphor tree method for tissue culture according to claim 1, is characterized in that: in steps d, root induction medium: improvement MS+IBA 1.0mg/L+NAA 0.05mg/L.
5. a kind of borneol camphor tree method for tissue culture according to claim 1, it is characterized in that: in step e, the seedling of taking root of having taken root to be taken away experienced seedling, practice seedling and become bottle green to stem in 20-30 days, washing away medium is again transplanted in the matrix of 1/3 coconut palm chaff+2/3 peat soil, control of temperature and humidity wherein in 25 days: temperature 26-30 DEG C, humidity 80 ~ 100%.
6., according to the arbitrary described a kind of borneol camphor tree method for tissue culture of claim 1-4, it is characterized in that: in described step in b, c, d, MS minimal medium or improvement MS in all containing sucrose 3%, carragheen 0.8%, pH=5.5.
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| CN105532475A (en) * | 2016-01-29 | 2016-05-04 | 文沁 | Cultivation method of borneol cinnamomum comphora with high borneol content |
| CN105900834A (en) * | 2016-04-12 | 2016-08-31 | 华南农业大学 | Cinnamomum porrectum (Roxb.) Kosterm. in vitro plant regeneration method |
| CN106234224A (en) * | 2016-08-16 | 2016-12-21 | 吉安市林业科学研究所 | The tissue culture of great Ye Lignum cinnamomi camphorae and method for quickly breeding |
| CN106718928A (en) * | 2017-01-19 | 2017-05-31 | 三明学院 | The method for fast establishing of the borneol camphor tree regenerating system based on one three-step approach |
| CN106942060A (en) * | 2017-03-30 | 2017-07-14 | 湖南省林业科学院 | A kind of borneol camphor tree test tube seedling proliferation and rejuvenation method |
| CN106942059A (en) * | 2017-03-27 | 2017-07-14 | 河池乐康生态农业科技有限公司 | A kind of method for tissue culture of cinnamomum camphora |
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| CN105532475A (en) * | 2016-01-29 | 2016-05-04 | 文沁 | Cultivation method of borneol cinnamomum comphora with high borneol content |
| CN105900834B (en) * | 2016-04-12 | 2017-12-08 | 华南农业大学 | A kind of method of the in vitro plant regeneration of yellow camphor tree |
| CN105900834A (en) * | 2016-04-12 | 2016-08-31 | 华南农业大学 | Cinnamomum porrectum (Roxb.) Kosterm. in vitro plant regeneration method |
| CN106234224A (en) * | 2016-08-16 | 2016-12-21 | 吉安市林业科学研究所 | The tissue culture of great Ye Lignum cinnamomi camphorae and method for quickly breeding |
| CN106718928A (en) * | 2017-01-19 | 2017-05-31 | 三明学院 | The method for fast establishing of the borneol camphor tree regenerating system based on one three-step approach |
| CN106942059A (en) * | 2017-03-27 | 2017-07-14 | 河池乐康生态农业科技有限公司 | A kind of method for tissue culture of cinnamomum camphora |
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| CN106942060B (en) * | 2017-03-30 | 2018-12-07 | 湖南省林业科学院 | A kind of borneol camphor tree test tube seedling proliferation and rejuvenation method |
| CN108834902A (en) * | 2018-08-03 | 2018-11-20 | 国家林业局桉树研究开发中心 | A kind of fruitlet print adds the tissue cultivation rapid breeding method of fruit |
| CN109804884A (en) * | 2018-09-19 | 2019-05-28 | 漳州市农业科学研究所 | A kind of method of the beautiful fan brocade of test tube micro cuttage breeding |
| CN109804884B (en) * | 2018-09-19 | 2021-10-26 | 漳州市农业科学研究所 | Method for breeding Euphorbia lathyris by test tube micro-cuttage |
| CN110892862A (en) * | 2019-12-12 | 2020-03-20 | 南昌工程学院 | Water culture cutting rapid propagation method for sassafras and application thereof |
| CN110892862B (en) * | 2019-12-12 | 2021-06-11 | 南昌工程学院 | Water culture cutting rapid propagation method for sassafras and application thereof |
| CN112970588A (en) * | 2021-04-30 | 2021-06-18 | 江西省林业科学院 | Breeding method for efficiently inducing adventitious bud differentiation of sassafras callus |
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Application publication date: 20150701 |