CN106234224A - The tissue culture of great Ye Lignum cinnamomi camphorae and method for quickly breeding - Google Patents
The tissue culture of great Ye Lignum cinnamomi camphorae and method for quickly breeding Download PDFInfo
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- CN106234224A CN106234224A CN201610670422.9A CN201610670422A CN106234224A CN 106234224 A CN106234224 A CN 106234224A CN 201610670422 A CN201610670422 A CN 201610670422A CN 106234224 A CN106234224 A CN 106234224A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
Abstract
The tissue culture of a kind of great Ye Lignum cinnamomi camphorae and method for quickly breeding, step includes: take the shoot that great Ye Lignum cinnamomi camphorae maternal plant sprouts then, being cut into the segment of 0.5 1.5cm long band axillalry bud, cold preservation, sterile water wash, the ethanol infiltration of 70%, the mercuric chloride of 0.1% 0.2% are sterilized 6 12min, aseptic water washing；It is seeded to after blotting water in MS culture medium obtain axillalry bud, it is seeded to light culture 18 25 days in the culture medium of MS+6 BA2.0mg/L+NAA0.2mg/L, form callus, access in the culture medium of MS+6 BA0.3mg/L+NAA0.1mg/L, the little sorite of plexi occurred after 12 18 days；After 25 35 days little for plexi sorite according to 35 buds/standard cut, then proceed in the proliferated culture medium of MS+6 BA0.2 0.5mg/L+NAA0.1mg/L, after continuous 35 generations of generation, the sturdy simple bud of H ＞ 2.5cm in sorite is chosen down, in the root media of access 1/2MS+IAA0.3mg/L, after 15 20 days, root starts to sprout, and the root having the Seedling of 95% to bear 23 long 0.4 0.6cm after 40 45d i.e. completes the breeding of Seedling.Have and ensure that kind can provide again the most neat great Ye Lignum cinnamomi camphorae Seedling advantage in the short time in the case of pure.
Description
Technical field
The present invention relates to group culturation rapid propagating technology field, be specifically related to tissue culture and the Fast-propagation side of a kind of great Ye Lignum cinnamomi camphorae
Method.
Background technology
Great Ye Lignum cinnamomi camphorae (Cinnamomum camphora), Lauraceae Cinnamomum, is a specific physiologic type of Cinnamomum porrectum (Roxb.) Kosterm., 1990
Age, Institute of Zoology Zhu Liangfeng researcher in south China found first in Guangdong Province, and what its leaf was rubbed has strong Lignum cinnamomi camphorae sweet-smelling
Gas.For the representative seeds of Subtropical Evergreen Broad-leaf Forest, the main place of production be TaiWan, China, Fujian, Jiangxi, Guangdong, Guangxi, Hunan,
Hubei, Yunnan and zhejiang and other places, be southern china preciousness material and spy's economic tree.Because of its life-span length, tree performance grandness, the four seasons
Evergreen, sight preferably, has fabulous afforestation to be worth, and is just deeply liked by numerous people since ancient times；Great Ye Lignum cinnamomi camphorae is survived
Phase is long, can be grown to the towering old trees of hundreds and thousands of years, has the strongest smoking to lay the dust, water conservation, the sand prevention of solid soil and beautify
The ability of environment.Its grain of wood is careful, proportion 0.53-0.64, indeformable, resistance to damages by worms, and has the peppery aromatic odor of light perfume (or spice), easily
Processing, is a kind of well building and furniture woods, becomes one of various famous and precious timber；Timber, branch, root, leaf all can obtain through refining Camphor tree
Brain, camphor oil, for medicine, chemical industry, spice, anticorrosion, parasite killing etc..Seed contains oils and fats, can extract oil, for soapmaking very good material.
Lignum cinnamomi camphorae is one the highest containing linalool in five Natural Types of Radix Cinnamomi porrecti, and linalool and corresponding derivant thereof are fragrant
The primary raw material of chemical industry, very big in whole world demand, but natural Radix Cinnamomi porrecti alcohol only accounts for few part, and the linalool synthesized is at gas
Taste, note, sweet main physical and chemical performance such as grade differs greatly than natural Radix Cinnamomi porrecti alcohol, therefore, market is to excellent great Ye Lignum cinnamomi camphorae nursery stock
Demand is very big.And how to ensure that its kind can provide the most neat excellent great Ye Lignum cinnamomi camphorae Seedling in the short time in the case of pure
It is referred to as the technical problem that industry is urgently to be resolved hurrily.
Summary of the invention
The present invention is directed to the above-mentioned deficiency of prior art, it is provided that again can be in the short time in the case of a kind of guarantee kind is pure
Tissue culture and the method for quickly breeding of the great Ye Lignum cinnamomi camphorae of the most neat great Ye Lignum cinnamomi camphorae Seedling are provided.
In order to solve above-mentioned technical problem, the technical solution used in the present invention is: the tissue culture of a kind of great Ye Lignum cinnamomi camphorae and
Method for quickly breeding, step includes:
(1) take the shoot that perennial excellent great Ye Lignum cinnamomi camphorae maternal plant sprouts then, be cut into the little of 0.5-1.5cm long band axillalry bud
Section, puts into cold compartment of refrigerator 10-15h with hermetic container after installing, taking-up is clean by sterile water wash, then with sterilized water in vibration
Oscillation cleaning 30-50min on device；0.3-0.8min, aseptic water washing 23 times is infiltrated again with the ethanol of 70%；Use 0.1%-
The mercuric chloride sterilization 6-12min of 0.2%, aseptic water washing 3--5 time；
(2) being seeded to after blotting water with filter paper in MS culture medium, after 36-42 days, axillalry bud length is to 0.5-1cm；Axillalry bud is chosen
Under be seeded to light culture 18-in the culture medium of MS+6-BA (6-benzyl aminoadenine) 2.0mg/L+NAA (naphthalene acetic acid) 0.2mg/L
After 25 days, its base portion is formed yellowish partially green, and the measured callus of matter that density degree is moderate accesses MS+6-callus
In the culture medium of BA0.3mg/L+NAA0.1mg/L, plexi little sorite occurred after 12-18 days；
(3) after 25-35 days little for plexi sorite according to 35 buds/standard cut, then proceed to MS+6-
In the proliferated culture medium of BA0.2-0.5mg/L+NAA0.1mg/L, being that a cycle is continuous for once with 30 days, in continuous generation, once produces
The individual sub-bud of 5--8/；
(5) after proliferated culture medium is continuous for 3-5 generation, the sturdy simple bud of H in sorite (highly) ＞ 2.5cm is chosen down, access 1/
In the root media of 2MS+IAA0.3mg/L, after 15 20 days, root starts to sprout, and has the Seedling of 95% to bear 2-3 bar after 40 45d
The root of long 0.4-0.6cm i.e. completes the breeding of Seedling.
Said method also includes transplanting step: the seedling taken root is put into cool canopy seedling exercising about 10 days (d) so that it is fully
Lignifying, leaf complexion changed is green, after seedling is taken out from culture vessel, wash away culture medium and be transplanted into 1/3 coconut palm bran+2/3 peat soil
In substrate, note heat and moisture preserving, seedling quickly normal growth.
Above culture medium is all with pure water, containing sucrose 3%, carrageenan 0.75%, pH value 5.8, cultivation temperature 24--26 DEG C, light
According to 10 12h/d, light intensity 1500 2000lx.
Advantages of the present invention and beneficial effect:
1. the present invention is by specific culture medium and composition, and the control of cultivation light application time, pH etc., at short notice
The great Ye Lignum cinnamomi camphorae Seedling that kind is pure, neat can be obtained；Therefore it is the excellent process preparing Lignum cinnamomi camphorae.In addition method is simple, pass through
Tissue culture stability is strong, and the requirement to equipment is relatively low, and the Seedling heritability produced is good.The present invention uses shoot as induction
Object, it is easy to obtain, it is ensured that the cycle is short, rooting rate is high, easily survives；The result of the present invention shows, at condition of culture: culture medium
All with pure water, containing sucrose 3%, carrageenan 0.75%, pH value 5.8, cultivation temperature 24--26 DEG C, illumination 10 12h/d, light intensity
Under the conditions of 1500 2000lx, the Seedling of 95% can bear the root of the long 0.4-0.6cm of 2-3 bar.
2. the seedling taken root being put into cool canopy seedling exercising about 10 days (d) so that it is fully lignifying, leaf complexion changed is green, after will
Seedling takes out from culture vessel, washes away in the substrate that culture medium is transplanted into 1/3 coconut palm bran+2/3 peat soil, notes heat and moisture preserving,
Seedling quickly normal growth, survival rate more than 85%
Accompanying drawing explanation
Axillalry bud during accompanying drawing 1 bud inducement of the present invention.
Wound healing during accompanying drawing 2 bud inducement of the present invention and bud.
The propagation sorite (I) of the birth process of accompanying drawing 3 bud of the present invention.
The propagation sorite (II) of the birth process of accompanying drawing 4 bud of the present invention.
The propagation sorite (III) of the birth process of accompanying drawing 5 bud of the present invention.
The sections of the birth process of accompanying drawing 6 bud of the present invention is agglomerating.
Root to be generated in the Induction Process of accompanying drawing 7 root of the present invention.‘
Seedling of taking root in the Induction Process of accompanying drawing 8 root of the present invention.
Seedling of taking root (I) in the Induction Process of accompanying drawing 9 root of the present invention.
Seedling of taking root (II) in the Induction Process of accompanying drawing 10 root of the present invention.
Detailed description of the invention
Describe the present invention in detail below by embodiment, but the present invention is not limited solely to following example.
Embodiment
1. test material and method
Take the shoot that perennial excellent great Ye Lignum cinnamomi camphorae maternal plant sprouts then, be cut into the segment of about 1cm long band axillalry bud.Note
The time that the unavailable scalpel got rusty wound to be reduced expose in atmosphere, otherwise stem section can be due to polyphenol oxidase etc.
Oxidation and otch brownization, increase production of hybrid seeds difficulty.The stem section hermetic container adopted back puts into cold compartment of refrigerator about 12h after installing
Clean by sterile water wash afterwards, then with sterilized water more than oscillation cleaning 30min on the oscillator.Infiltrate about with the ethanol of 70% afterwards
0.5min, aseptic water washing 23 times.Again regard material size, old tender degree respectively with 0.1% and 0.2% mercuric chloride sterilization 6--
10min and 8--12min, aseptic water washing 3--5 time, it is seeded to after blotting water with filter paper in MS minimal medium, disappears after 40d
Poison successfully axillalry bud length is to about 0.5-1cm.Axillalry bud is chosen down in the culture medium being seeded to MS+6-BA2.0mg/L+NAA0.2mg/L
After light culture about 20d, its base portion is formed yellowish partially green, and the measured callus of matter that density degree is moderate connects callus
Enter in the culture medium of MS+6-BA0.5mg/L+NAA0.1mg/L, plexi budlet occurs after about 15d, after 30d, sorite is cut into
35 bud/successive transfer culture.
Above culture medium is all with pure water, containing sucrose 3%, carrageenan 0.75%, pH value 5.8.Cultivation temperature 24--26 DEG C, light
According to 10 12h/d, light intensity 1500 2000lx.
2 calluss and the induction of bud
Culture medium prescription the most suitably directly affects differentiation and the growth of outer implant, and the proportioning of various growth regulators seems
Particularly critical.The axillalry bud being about 0.5-1cm is that 1 propagation unit is inoculated in callus induction culture medium by this experiment.Test knot
Fruit is shown in Table 1 (minimal medium MS)
Table 1 hormon concentrations on callus induction experiment result
As known from Table 1: axillalry bud is more sensitive to 6-BA comparison KT and 2ip, though KT with 2ip institute concentration is the same with 6-BA
Height, but it is much lower to form wound healing rate.6-BA is best with No. 4 culture medium again, and formed wound healing is of high quality.No. 5, No. 6 trainings
Though supporting wound healing number ratio more than No. 4 that base is formed, but the white puffy of part wound healing, be invalid wound healing, especially No. 6 formation wound healing in
Reveal water soaking mode vitrification, of low quality.Add NAA higher than the ratio that IAA forms callus, concrete outcome is shown in accompanying drawing
1-2。
The induction of 3 buds
The callus induced is cut into the little agglomerate of about 0.2-0.3cm, is inoculated in the culture medium of induced bud.
Result of the test is shown in Table 2 (minimal medium MS)
Table 2 different hormone combinations is to bud inducement result of the test
Comprehensive data above analysis, basic element of cell division 2ip, 6-KT, 6-BA all can elicit bud from callus, and 6-BA is again
Being preferred with 0.3mg/L, low concentration then bud is on the low side, and higher concentration callus is deteriorated again, under the influence of for the rate of increase.Auxin side
Face NAA outline from data is better than IAA.Being to sum up best with d culture medium, bud is most, though callus increases substantially and
Its quality is not deteriorated, concrete outcome is shown in accompanying drawing 3-6.
4. the propagation of bud
By the budlet clump induced band a little wound healing be divided into 2--3 bud/, proceed in the proliferated culture medium preferably gone out, respectively
Process refers to table 3, is continuous generation in a cycle once can to produce 5--8 sub-bud with 30d, and the concentration of 6-BA is in certain scope
Concentration is the highest, and sub-bud is the most, but concentration its rate of increase the highest can decline on the contrary.And along with the rising of 6-BA concentration, blade
Can come to a point and the base portion of Seedling there will be more invalid wound healing, affect its rate of increase on the contrary.Therefore, closely to see during continuous generation
Examine Seedling condition, adjust the concentration of 6-BA at any time, to guarantee to improve its expanding propagation system as far as possible on the premise of ensureing propagation Seedling quality
Number.By experiment, Suo Changjiang etc. then think that MS+6-BA5.0mg/L+NAA0.5mg/L cultivation effect is preferable.Peng Donghui then thinks
MS+6-BA3.0mg/L+IBA0.2mg/L cultivation effect is preferable；The culture medium that the embodiment of the present invention uses is shown in Table 3, therefrom screens
Most preferably proportioning.
Another: after continuing for 3--5 generation with the proliferated culture medium chosen, the sturdy individual plant choosing H ＞ 2.5CM from sorite will
The 6-BA culture medium of the sections higher concentration that its stem section is divided into about 0.3cm/ section makes its agglomerating expanding propagation, with 50d as cycle.Everywhere
Reason refers to table 4, and because of every strain individual plant 4-7 sections of stem Duan Kefen, therefore its rate of increase can be multiplied and the quality of Seedling is through many generations
Observation can ensure that.Thus reach the purpose producing in a large number and emerging continually and steadily.
Table 3 sorite growth perfonnance in proliferated culture medium respectively processes
Comprehensive data above analysis, A, B, C process is used equally to sorite propagation expanding propagation, concrete select visual Seedling condition and
Fixed, also can cross-reference.It is bad that D, E process Seedling condition, should not use.
Table 4 sturdy individual plant segmentation growth perfonnance in proliferated culture medium respectively processes
Comprehensive data above is found out, No. 3 process culture medium for sections agglomerating propagation expanding propagation preferably, concrete visible accompanying drawing 7-
8。
The induction of 5
The sturdy simple bud of H ＞ 2.5cm in sorite is chosen down, accesses in root media, 15 20d in preferred culture medium
Rear starts to sprout, and has the Seedling of 95% to bear 2-3 bar and be about the root of 0.5cm after 40 45d.Concrete different culture media is taken root situation
It is shown in Table 5:
Table 5 different culture media is taken root situation
Comprehensive data above analysis, No. c process culture medium is best for seedling rooting up to standard.Root media should not add life
Long element NAA, otherwise Seedling base portion or rhizome junction are easily generated callus, make rooting rate reduce and affect quality of rooting.And Wu
By experiment, Jin Shou etc. then think that 1/2MS+IBA0.8mg/L+NAA0.2mg/L rooting efficiency is preferable, concrete visible accompanying drawing 7-8.
6. transplanting: the seedling taken root is put into cool canopy seedling exercising about 10d so that it is fully lignifying, leaf complexion changed is green, after will
Seedling takes out from culture vessel, washes away in the substrate that culture medium is transplanted into 1/3 coconut palm bran+2/3 peat soil, notes heat and moisture preserving,
Seedling quickly normal growth, survival rate more than 85%；After transplanting, growth conditions is shown in accompanying drawing 9-10.
Claims (3)
1. the tissue culture of a great Ye Lignum cinnamomi camphorae and method for quickly breeding, it is characterised in that: step includes:
(1) take the shoot that perennial excellent great Ye Lignum cinnamomi camphorae maternal plant sprouts then, be cut into the segment of 0.5-1.5cm long band axillalry bud,
Putting into cold compartment of refrigerator 10-15h with hermetic container after installing, taking-up is clean by sterile water wash, then with sterilized water at agitator
Upper oscillation cleaning 30-50min；0.3-0.8min, aseptic water washing 23 times is infiltrated again with the ethanol of 70%；Use 0.1%-
The mercuric chloride sterilization 6-12min of 0.2%, aseptic water washing 3--5 time；
(2) being seeded to after blotting water with filter paper in MS culture medium, after 36-42 days, axillalry bud length is to 0.5-1cm；Axillalry bud is chosen down and connects
Kind form callus, healing to its base portion after light culture in the culture medium of MS+6-BA2.0mg/L+NAA0.2mg/L 18-25 days
Injured tissue accesses in the culture medium of MS+6-BA0.3mg/L+NAA0.1mg/L, occurs the little sorite of plexi after 12-18 days；
(3) after 25-35 days little for plexi sorite according to 35 buds/standard cut, then proceed to MS+6-BA0.2-
In the proliferated culture medium of 0.5mg/L+NAA0.1mg/L, it was that a cycle is continuous for once with 30 days；
(4) after proliferated culture medium is continuous for 3-5 generation, the sturdy simple bud of H ＞ 2.5cm in sorite is chosen down, access 1/2MS+
In the root media of IAA0.3mg/L, after 15 20 days, root starts to sprout, and has the Seedling of 95% to bear 2-3 bar long after 40 45d
The root of 0.4-0.6cm i.e. completes the breeding of Seedling.
The tissue culture of great Ye Lignum cinnamomi camphorae the most according to claim 1 and method for quickly breeding, it is characterised in that: step is also wrapped
Include transplanting step: the Seedling taken root is put into cool canopy seedling exercising 8-12 days, after leaf complexion changed is green, seedling taken out from culture vessel,
Wash away culture medium and be transplanted in the substrate of 1/3 coconut palm bran+2/3 peat soil growth.
The tissue culture of great Ye Lignum cinnamomi camphorae the most according to claim 1 and method for quickly breeding, it is characterised in that: described training
Foster base is all solvent with pure water, and containing sucrose 3%, carrageenan 0.75%, pH value 5.8；Cultivation temperature 24--26 DEG C, illumination 10
12h/d, light intensity 1500 2000lx.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
| CN113080059A (en) * | 2021-03-27 | 2021-07-09 | 吉安市林业科学研究所 | Tissue culture propagation method of linalool type sassafras |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113080059A (en) * | 2021-03-27 | 2021-07-09 | 吉安市林业科学研究所 | Tissue culture propagation method of linalool type sassafras |
| 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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