CN107455261B - A kind of method of Acer saccharum tissue culture quick breeding - Google Patents
A kind of method of Acer saccharum tissue culture quick breeding Download PDFInfo
- Publication number
- CN107455261B CN107455261B CN201710835544.3A CN201710835544A CN107455261B CN 107455261 B CN107455261 B CN 107455261B CN 201710835544 A CN201710835544 A CN 201710835544A CN 107455261 B CN107455261 B CN 107455261B
- Authority
- CN
- China
- Prior art keywords
- explant
- acer saccharum
- culture
- bud
- tissue culture
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 244000046139 Acer saccharum Species 0.000 title claims abstract description 49
- 235000004421 Acer saccharum Nutrition 0.000 title claims abstract description 48
- 238000000034 method Methods 0.000 title claims abstract description 27
- 238000009395 breeding Methods 0.000 title claims abstract description 22
- 230000001488 breeding effect Effects 0.000 title claims abstract description 22
- 239000000463 material Substances 0.000 claims abstract description 24
- 241000196324 Embryophyta Species 0.000 claims abstract description 18
- 230000004069 differentiation Effects 0.000 claims abstract description 13
- 239000000835 fiber Substances 0.000 claims abstract description 6
- 238000004659 sterilization and disinfection Methods 0.000 claims description 35
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 27
- 239000001963 growth medium Substances 0.000 claims description 16
- 230000006698 induction Effects 0.000 claims description 16
- 239000002609 medium Substances 0.000 claims description 16
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 15
- 230000012010 growth Effects 0.000 claims description 12
- 238000005286 illumination Methods 0.000 claims description 12
- 238000005406 washing Methods 0.000 claims description 11
- 229920001817 Agar Polymers 0.000 claims description 9
- 229930006000 Sucrose Natural products 0.000 claims description 9
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 9
- 239000008272 agar Substances 0.000 claims description 9
- 235000016709 nutrition Nutrition 0.000 claims description 9
- 230000035764 nutrition Effects 0.000 claims description 9
- 239000005720 sucrose Substances 0.000 claims description 9
- 238000012258 culturing Methods 0.000 claims description 8
- 235000019362 perlite Nutrition 0.000 claims description 8
- 239000010451 perlite Substances 0.000 claims description 8
- 239000012286 potassium permanganate Substances 0.000 claims description 8
- 230000008929 regeneration Effects 0.000 claims description 8
- 238000011069 regeneration method Methods 0.000 claims description 8
- 239000004576 sand Substances 0.000 claims description 8
- 239000002689 soil Substances 0.000 claims description 8
- 238000005520 cutting process Methods 0.000 claims description 7
- 239000012879 subculture medium Substances 0.000 claims description 7
- 230000001640 apoptogenic effect Effects 0.000 claims description 6
- RCTYPNKXASFOBE-UHFFFAOYSA-M chloromercury Chemical compound [Hg]Cl RCTYPNKXASFOBE-UHFFFAOYSA-M 0.000 claims description 6
- 230000009514 concussion Effects 0.000 claims description 6
- 238000004851 dishwashing Methods 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- 238000002791 soaking Methods 0.000 claims description 6
- 230000006378 damage Effects 0.000 claims description 5
- 241000607479 Yersinia pestis Species 0.000 claims description 4
- 238000004140 cleaning Methods 0.000 claims description 4
- 201000010099 disease Diseases 0.000 claims description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 4
- 230000008569 process Effects 0.000 claims description 4
- 230000003442 weekly effect Effects 0.000 claims description 4
- 238000012545 processing Methods 0.000 claims description 3
- 239000007921 spray Substances 0.000 claims description 3
- 238000007654 immersion Methods 0.000 claims description 2
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims 3
- 241000894006 Bacteria Species 0.000 claims 1
- 238000005516 engineering process Methods 0.000 abstract description 3
- 230000007613 environmental effect Effects 0.000 abstract description 3
- 230000004083 survival effect Effects 0.000 description 8
- 230000035755 proliferation Effects 0.000 description 6
- 230000001954 sterilising effect Effects 0.000 description 6
- 238000012360 testing method Methods 0.000 description 5
- 230000000694 effects Effects 0.000 description 4
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 3
- 206010020649 Hyperkeratosis Diseases 0.000 description 3
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 description 3
- 244000046151 Acer negundo Species 0.000 description 2
- 235000004422 Acer negundo Nutrition 0.000 description 2
- 235000010328 Acer nigrum Nutrition 0.000 description 2
- 235000010157 Acer saccharum subsp saccharum Nutrition 0.000 description 2
- TWFZGCMQGLPBSX-UHFFFAOYSA-N Carbendazim Natural products C1=CC=C2NC(NC(=O)OC)=NC2=C1 TWFZGCMQGLPBSX-UHFFFAOYSA-N 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- JNPZQRQPIHJYNM-UHFFFAOYSA-N carbendazim Chemical compound C1=C[CH]C2=NC(NC(=O)OC)=NC2=C1 JNPZQRQPIHJYNM-UHFFFAOYSA-N 0.000 description 2
- 239000006013 carbendazim Substances 0.000 description 2
- 230000000249 desinfective effect Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 230000035784 germination Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 241000208140 Acer Species 0.000 description 1
- 235000010319 Acer grandidentatum Nutrition 0.000 description 1
- 235000002629 Acer saccharinum Nutrition 0.000 description 1
- 241001123297 Acer saccharum subsp. saccharum Species 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- 244000086443 Craterellus fallax Species 0.000 description 1
- 235000007926 Craterellus fallax Nutrition 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 241000409198 Packera aurea Species 0.000 description 1
- 240000000111 Saccharum officinarum Species 0.000 description 1
- 235000007201 Saccharum officinarum Nutrition 0.000 description 1
- 239000005708 Sodium hypochlorite Substances 0.000 description 1
- 238000005452 bending Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000006652 catabolic pathway Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000007598 dipping method Methods 0.000 description 1
- 238000005553 drilling Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 239000007943 implant Substances 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 235000015110 jellies Nutrition 0.000 description 1
- 239000008274 jelly Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 229960002523 mercuric chloride Drugs 0.000 description 1
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 description 1
- 229910052753 mercury Inorganic materials 0.000 description 1
- BQPIGGFYSBELGY-UHFFFAOYSA-N mercury(2+) Chemical compound [Hg+2] BQPIGGFYSBELGY-UHFFFAOYSA-N 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000004899 motility Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000008635 plant growth Effects 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000004513 sizing Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000009331 sowing Methods 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 235000012069 sugar maple Nutrition 0.000 description 1
- 238000012090 tissue culture technique Methods 0.000 description 1
- 238000009966 trimming Methods 0.000 description 1
Classifications
-
- 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
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a kind of methods of Acer saccharum tissue culture quick breeding, belong to woody plant tissure culture raising technology field.The present invention include explant selection, explant pretreatment, the foundation of sterile system, Fiber differentiation, Multiplying culture, culture of rootage, hardening tame and etc..It using the histoorgan of plant, under the conditions of sterile system, is cultivated by manual control external environmental condition, achievees the purpose that quickly to breed nursery stock, effectively keep the stable character of excellent female parent.Compared with conventional cuttage and grafting vegetative propagation technique, this technology materials amount is small, avoids the restriction of natural conditions, and breeding coefficient is up to 5-20 times, is more suitable for neophyte, solves because of its inadequate resource, caused by breeding and popularization problem.
Description
Technical field
The present invention relates to a kind of methods of Acer saccharum tissue culture quick breeding, belong to woody plant tissure culture breeding skill
Art field.
Background technique
Acer saccharum is tree-like extremely graceful, and leaf uniqueness enters after autumn, and leaf color starts slowly from the green yellow, orange of becoming
The colors such as color, colour, dark red are world-renowned color leaf ornamental trees.In addition, it is also one of three big sugar material xylophytas,
Containing the maple sugar of 3-10% or so in sap, the sugar maple tree of general diameter of a cross-section of a tree trunk 1.3 meters above the ground 15cm or more can start drilling and tap liquid glucose, continuously
Acquisition time at least 50 years, it is known as " iron sugarcane ".The one kind of its timber as Hard maple, wooden hard tough, density is big,
It has a fine grain, there is high anti-wear intensity and anti-steam bending performance, for the economic tree of ideal of " first cutting sugar, use material afterwards "
Kind.Acer saccharum starts tentatively to push away in China each province and city as novel economizer woods trees, landscape ornamental tree species and commerical tree species
Extensively, this drives forestry economic development to be of great significance for enriching China's forest resourceies.
Acer saccharum introduces a fine variety, at present domestic quick breeding to Acer saccharum and industrialization warp shorter in China's time limit
Battalion's research still belongs to blank.For its introducing and planting and application research still in its infancy.Acer saccharum growth speed
Degree is more slow, and 10 years raw general 5m of tree, 10-15 start to bloom, the above tree beginning mast-fruiting event of life in 20 years.China adds at present
Acer negundo germ plasm resource of putting on airs all relies on import, and expensive and quarantine program is complicated.Modes of reproduction is educated with traditional sowing
Based on seedling, germination accelerating method is complicated, the period is long, and bud ratio is about 70% or so, and neophyte is influenced vulnerable to natural environmental condition,
Obtaining seedling rate is only 50%.Studies have shown that seedling variation larger, color property stability of the Acer saccharum by seminal propagation
It is not high, therefore be difficult in seedling wide popularization and application in the market.
Vegetative propagation includes cutting propagation, propagation by grafiting and tissue cultures.For Acer saccharum, cuttage and grafting are numerous
It grows also in test, cuttage survival rate is only 30%, graft survival rate 5%, and it is larger by natural environment influences such as region, temperature,
It is especially difficult to adapt to the damp and hot natural environment of south China summer high temperature, not yet explores stable method at present；Breeding coefficient
It is not high and domestic not largely at the propagation material of tree offer abundance, certain limitation is provided to large area large-scaled propugation.Add and takes
It is at home and abroad still rarely reported in terms of the tissue cultures of big Acer negundo, but woody tissue culture technique have been relatively mature, materials
Convenient, growing environment is controllable, and breeding coefficient is high, and the good advantage of genetic stability is increasingly applied in modern nursery.
Summary of the invention
Technical problem to be solved by the invention is to provide a kind of methods of Acer saccharum tissue culture quick breeding, it is intended to benefit
It is cultivated under the conditions of sterile system by manual control external environmental condition with the histoorgan of plant, reaches quickly numerous
Grow the purpose of nursery stock.This technology materials are convenient, and growing environment is controllable, and breeding coefficient is high, and genetic stability is good, solve and introduce a fine variety just
The deficiency of phase Acer saccharum resource, Steady breed and utilization and extention to Acer saccharum all have very important meaning
Justice.
In order to solve the above technical problems, the technical solution used in the present invention is: a kind of Acer saccharum tissue culture rapid
Numerous method, which comprises the following steps:
(1) explant selects
Perennial healthy and strong, no disease and pests harm Acer saccharum is chosen, using the spray of its current year raw, non-lignifying as explant
Body；
(2) explant pre-processes
Explant in step (1) is removed into blade, petiole, after 75% alcohol wipe, is trimmed at least one axillary bud of band
Or the stem section of terminal bud, then dish washing liquid concussion cleaning；Then stem section being immersed in pure water and being placed in temperature is at 18-22 DEG C
It is handled in ultrasonic cleaner, uses pure water rinsing later；
(3) foundation of sterile system
Will after step (2) are handled explant in aseptic operating platform with 75% alcohol soaking disinfection, with aseptic water washing,
With 0.1% mercury chloride shake sterilize, with aseptic water washing, blot, then cut off stem section both ends disinfection apoptotic fraction after obtain it is sterile
Material；
(4) Fiber differentiation
6-BA 0.5mg/L, sucrose 30g, agar 6.5g are added on the basis of MS culture medium, configures induced medium, are adjusted
Save induced medium pH to 5.8；The differentiation that progress bud in induced medium is inserted into step (3) sterilizable material morphology lower end is lured
Culture is led, the sprouting and growth of bud are promoted；
(5) Multiplying culture
6-BA 0.5mg/L, GA are added on the basis of MS culture medium30.5mg/L, sucrose 30g, agar 6.5g, configuration
Subculture medium adjusts subculture medium pH to 5.8, the Acer saccharum axillary bud in step (4) is entirely accessed squamous subculture
Base carries out Multiplying culture；
(6) induction of root
IBA 0.5mg/L, sucrose 20g, agar 6.5g are added on the basis of MS culture medium, configures root media, are adjusted
Root media pH to 5.8 is saved, the seedling access root media of step (5) is subjected to rooting induction；
(7) the hardening domestication of aseptic seedling
The Acer saccharum of step (6) aseptic seedling of taking root from culturing room is moved into greenhouse culture, then moves into light base
Continue to cultivate in matter into complete regeneration plant；Wherein Light media is mixed with 2: 1: 1 volume ratio by Nutrition Soil, river sand and perlite
The disinfection of merging potassium permanganate is made.
A further technical solution lies in: step (1) carries out the explant time as mid-April to mid-June.
A further technical solution lies in: step (2) specific steps are as follows: the explant in step (1) is not being damaged into armpit
Blade, petiole are removed on the basis of bud and terminal bud, dip 75% alcohol wipe 1-2 after with gauze, then be trimmed to 3-5cm at least
Stem section with an axillary-bud or top-bud, after dish washing liquid concussion cleaning 2 times, flowing water is cleaned 8-10 minutes, then impregnates stem section
Equipped with pure water container in be placed in temperature be 18-22 DEG C at ultrasonic cleaner in handle 8-12 minutes, later with pure
Water rinses 1-2 times.
A further technical solution lies in: the specific steps of step (3) are as follows: by explant after step (2) processing sterile
It is impregnated 20-40 seconds in station with 75% alcohol and carries out surface sterilization, with aseptic water washing 1-2 times, shaken with 0.1% mercury chloride
It soaking disinfection 2-3 minutes, with aseptic water washing 4-6 times, blots, then obtains sterile material after cutting off stem section both ends disinfection apoptotic fraction
Material.
A further technical solution lies in: in the step (4) bud induction control cultivation temperature at 24-26 DEG C,
Light application time 12-14h/d, intensity of illumination 2000-2500lx, illumination cultivation 20-30d.
A further technical solution lies in: strong seedling culture temperature is at 24-26 DEG C in the step (5), light application time 12-
14h/d, intensity of illumination 2000-2500lx cultivate 20-30d.
A further technical solution lies in: 20-25 DEG C of cultivation temperature of root, light application time 10-16h/ in the step (6)
D, intensity of illumination 2000-2500lx cultivate 15-20d.
A further technical solution lies in: the step (7) the specific steps are the Acer saccharum of step (6) is taken root
Aseptic seedling moves to greenhouse from culturing room, unclamps bottle cap culture 3-5d, then open bottle cap culture 5-7d, then moves into Light media
In continue cultivate at complete regeneration plant；Wherein Light media is mixed by Nutrition Soil, river sand and perlite with 2: 1: 1 volume ratio
And it is made of potassium permanganate disinfection；Therebetween, it is primary that carbendazim is sprayed weekly；In outside air temperature is for 1 week be lower than 30 DEG C or less when
It moves in the seedbed of crop field, appropriate dense planting, temperature takes sunshade net when being greater than 30 DEG C, guarantee moisture supply.
The Acer saccharum of step (6) aseptic seedling of taking root from culturing room is moved into greenhouse culture, then moves into light base
Continue to cultivate in matter into complete regeneration plant；Wherein Light media is mixed with 2: 1: 1 volume ratio by Nutrition Soil, river sand and perlite
The disinfection of merging potassium permanganate is made.
A further technical solution lies in: with 1mol/LNaOH adjusting medium pH in step (4)-(6).
The beneficial effects of adopting the technical scheme are that
The present invention has found that field explant is affected by environment larger during the test, the time of exposure after shoot stripping
Longer, tissue cultures pollution rate is higher, so the disinfection of explant, building sterile system, which becomes, carries out tissue using field material
The key of culture.Through repetition test, the comparison of test of many times result just obtains specific embodiments of the present invention, i.e. mid-April
It draws materials to mid-June, which is in growth animated period, and newborn branch quantity is compared with horn of plenty, exposure duration section
And branch not yet lignifying, differentiation rate are higher.The frequency of 1-2 materials can not only guarantee that plant growth was unaffected weekly, but also
Trimming sizing can be carried out to plant.Test uses alcohol wipe-surface cleaner flushing-ultrasonic cleaning-alcohol disinfecting-liter
Mercury disinfection, increases disinfection early period means, reduces mercuric chloride disinfection residual and the too long damage to explant of disinfecting time, reaches reason
The sterilization effect thought, explant number needed for ensure that Fiber differentiation.
Early period of the invention uses degradation pathways method for tissue culture using stem section and blade, i.e., during the cultivation process from each device
Generate callus on official, culture callus using being differentiated to form aftergrowth again, callus growth it is good but regardless of
Chemical conversion bud cannot still reach good differentiation effect after Numerous formulations are attempted.It is used using the stem section with axillary bud and terminal bud
Direct way method for tissue culture isolates the organ to suit the requirements from plant, by sterile working, in manual control item
It is cultivated under part to obtain regenerated intact plant, it is fast to Acer saccharum progress numerous, cooperate optimal induction, proliferation, life
Root culture medium prescription can efficiently obtain a large amount of Acer saccharum seedling, substantially reduce growing-seedling period, solve inheritance stability
Property problem, is conducive to promote and apply.Pass through the selection to explant, induced medium, subculture medium and root media, bud
Differentiation rate be up to 95%, rooting rate 90% or so, 80% or more planting percent can efficiently obtain the high-quality money of Acer saccharum
Source.
The present invention solve Growing season materials and field explant disinfection problem after, with axillary bud, terminal bud stem section a variety of
Stronger differentiation rate is shown in culture medium, and the best induction for facilitating application, subculture, culture of rootage are obtained after data statistics
Based formulas is conducive to operate application in nursery stock production.The growth of Acer saccharum root system is more slow, and the present invention will be lured taking root
Acer saccharum is obtained after leading and is moved into Light media container and continues to cultivate, and is promoted the growth of radicula, is advantageously ensured that survival rate.Newly draw
Kind of trees influence vulnerable to amblent air temperature, especially originate in the Acer saccharum of high latitude area, non-refractory, intolerant to ponding,
Intolerant to transplanting, suitable weather is preferably selected to transplant seedlings to crop field seedbed.
Detailed description of the invention
The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.
Fig. 1-2 is the experimental result after explant Fiber differentiation 5d, 15d respectively；
Experimental result after Fig. 3-4 culture of rootage 7d；
Fig. 5 is influence diagram of the different sampling stages to explant；
Fig. 6 is the influence diagram of the different external implant body pollution rates of sterilization method.
Specific embodiment
With reference to the attached drawing in the embodiment of the present invention, technical solution in the embodiment of the present invention carries out clear, complete
Ground description, it is clear that described embodiment is only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other
Embodiment shall fall within the protection scope of the present invention.
In the following description, numerous specific details are set forth in order to facilitate a full understanding of the present invention, but the present invention can be with
Implemented using other than the one described here other way, those skilled in the art can be without prejudice to intension of the present invention
In the case of do similar popularization, therefore the present invention is not limited by the specific embodiments disclosed below.
The present invention provides a kind of methods of Acer saccharum tissue culture quick breeding, which comprises the following steps:
(1) explant selects
Perennial healthy and strong, no disease and pests harm Acer saccharum is chosen, with its current year in mid-March, 2016 to mid-August
The branch of raw, non-lignifying is as explant；
(2) explant pre-processes
Explant in step (1) is removed into blade, petiole and is trimmed to 3-5cm after dipping 75% alcohol wipe with gauze
At least stem section of one axillary-bud or top-bud of band, then dish washing liquid concussion are cleaned 10 minutes；Then stem section is immersed in pure water simultaneously
Being placed in temperature is handle 10 minutes in ultrasonic cleaner at 20 DEG C, later with pure water rinsing 2 times；
(3) foundation of sterile system
Will through step (2) handle after explant in aseptic operating platform with 75% alcohol soaking disinfection 30 seconds, use sterile water
It rinses 3 times, shakes disinfection 1 minute, 1.5 minutes, 2 points respectively with 0.1% mercury chloride (), 30% hydrogen peroxide, 20% sodium hypochlorite
Clock, 2.5 minutes, 3 minutes are obtained after filter paper blots, then after cutting off stem section both ends disinfection apoptotic fraction with aseptic water washing 5 times
Sterilizable material；
(4) Fiber differentiation
6-BA, sucrose 30g, the agar 6.5g that various concentration is added on the basis of MS culture medium, configure induced medium,
Adjust induced medium pH to 5.4,5.6,5.8,6.0；Step (3) sterilizable material morphology lower end is inserted into induced medium
The induction culture for carrying out bud, promotes the sprouting and growth of bud；
(5) Multiplying culture
6-BA, NAA, GA of various concentration are added on the basis of MS culture medium3, sucrose 30g, agar 6.5g, configuration after
For culture medium, subculture medium pH to 5.8 is adjusted, the Acer saccharum axillary bud in step (4) is entirely accessed into subculture medium
Carry out Multiplying culture
(6) induction of root
IBA, sucrose 20g, the agar 6.5g that various concentration is added on the basis of MS culture medium, configure root media,
Root media pH to 5.8 is adjusted, the proliferation seedling access root media of step (5) is subjected to rooting induction；
(7) the hardening domestication of aseptic seedling
The Acer saccharum of step (6) aseptic seedling of taking root from culturing room is moved into greenhouse culture, then moves into light base
Continue to cultivate in matter into complete regeneration plant；Wherein Light media is mixed with 2: 1: 1 volume ratio by Nutrition Soil, river sand and perlite
The disinfection of merging potassium permanganate is made.
Preferably, step (1) carries out the explant time as Acer saccharum Growing season.
Preferably, step (1) carries out the explant time as mid-April to mid-June.
Preferably, step (2) specific steps are as follows: step (2) specific steps are as follows: do not damaging the explant in step (1)
Hurt and remove blade, petiole on the basis of axillary bud and terminal bud, dips 75% alcohol wipe 1-2 after with gauze, then be trimmed to 3-5cm
At least stem section of one axillary-bud or top-bud of band, after dish washing liquid concussion cleaning 2 times, flowing water is cleaned 8-10 minutes, then by stem section
Being immersed in and being placed in temperature in the container equipped with pure water is to handle 8-12 minutes in ultrasonic cleaner at 18-22 DEG C, later
With pure water rinsing 1-2 times.
Preferably, the specific steps of step (3) are as follows: by explant after step (2) processing with 75% in aseptic operating platform
Alcohol impregnates progress surface sterilization in 20-40 seconds, with aseptic water washing 1-2 times, with soaking disinfection 2-3 points of 0.1% mercury chloride concussion
Clock is blotted with aseptic water washing 4-6 times, then obtains sterilizable material after cutting off stem section both ends disinfection apoptotic fraction.
Preferably, the induction control cultivation temperature of bud is at 24-26 DEG C in the step (4), light application time 12-14h/
D, intensity of illumination 2000-2500lx, illumination cultivation 20-30d.
Preferably, strong seedling culture temperature in 24-26 DEG C, light application time 12-14h/d, intensity of illumination is in the step (5)
2000-2500lx cultivates 20-30d.
Preferably, 20-25 DEG C of cultivation temperature, light application time 10-16h/d of root in the step (6), intensity of illumination is
2000-2500lx cultivates 15-20d.
Preferably, the step (7) the specific steps are the Acer saccharum of step (6) is taken root aseptic seedling from culturing room
Greenhouse is moved to, unclamps bottle cap culture 3-5d, then open bottle cap culture 5-7d, then moves into Light media and continues to cultivate at complete
Whole regeneration plant；Wherein Light media is mixed by Nutrition Soil, river sand and perlite with 2: 1: 1 volume ratio and is disappeared with potassium permanganate
Poison is made；Therebetween, it is primary that carbendazim is sprayed weekly；In outside air temperature is for 1 week be lower than 30 DEG C or less when move in field plant bed,
Appropriate dense planting, temperature take sunshade net when being greater than 30 DEG C, guarantee moisture supply.
The Acer saccharum of step (6) aseptic seedling of taking root from culturing room is moved into greenhouse culture, then moves into light base
Continue to cultivate in matter into complete regeneration plant；Wherein Light media is mixed with 2: 1: 1 volume ratio by Nutrition Soil, river sand and perlite
The disinfection of merging potassium permanganate is made.
A further technical solution lies in: with 1mol/LNaOH adjusting medium pH in step (4)-(6).
Embodiment
Perennial healthy and strong, no disease and pests harm Acer saccharum is chosen, with its current year in mid-March, 2017 to mid-August
The branch of raw, non-lignifying obtains 4 by comparing the death rate between different months, pollution rate, survival rate as explant
The middle of the month to mid-June is the best period of materials.March, pollution rate was although relatively low but material is less, mid-April to June
The explant of the middle ten days materials, the death rate and pollution rate are relatively low, and preferably, this may be in growth animated period phase with plant for growth
It closes.After mid-June, the pollution rate and the death rate of explant obviously rise, and mainly arrive with plum rain season, and high temperature and humidity causes
Microbial activities is frequent, explant sterilizing relative difficulty, and branch starts lignifying, and differentiation capability weakens.
From the point of view of disinfection way and disinfecting time, 0.1% mercury chloride (HgCl2) compared with 30% hydrogen peroxide (H2O2), 20% chlorine
Sour sodium (NaClO) shakes the disinfection that disinfection is more suitable for Acer saccharum respectively.Work as 0.1%HgCl2,When sterilizing 3 minutes, pollution
Lower rate is 6.67%, and survival rate is up to 93.3%；When between when sterilized more than 3 minutes, survival rate starts sharp fall, makes
It may be that a small amount of mercury ion has encroached on explant at the reason of this result, so as to cause death.So being pre-processed by early period
Afterwards, then with ethanol postincubation 30s add 0.1%HgCl2Disinfection Effect it is preferable.It is specifically shown in Fig. 5-6.
Influence of the different pH value of table 1 to explant survival rate and culture medium
Medium pH is adjusted by adjusting 1mol/LNaoh, compares different pH value to the growth effect of explant, pH 5.8
When, explant survival rate is up to 83.3%, and transparent jelly state is presented in culture medium, and hardness is moderate.Stem section is deposited when pH is 5.4
Motility rate is very low, it may be possible to which, because culture medium is semiliquid, explant immersion is not easy to survive wherein.When pH is 6.0, culture medium is
Faint yellow solid, explant can not absorb nutrition.
2 hormon of table is with the influence for comparing bud induction
3 hormon of table is with the influence for comparing proliferation
The influence that 4 hormon of table is taken root with comparison
The hormone ratio being added in the medium is different, and formation time and the inductivity for forming bud are also different, in 6-BA
Under the formula of 0.5mg/L, the first sprout time of bud is 5d, germination rate 93.3%.For the Multiplying culture 6-BA and GA of bud3's
The proliferation rate of combined treatment ratio 6-BA and NAA combination is high, and optimum formula is 6-BA 0.5mg/L, GA3 0.5mg/L, proliferation rate
For 90%, GA3Cell proliferation differentiation is accelerated, the elongation of stem, the growth of bud and leaf are promoted.In rooting induction, IBA is added
The root induction rate of 0.5mg/L is up to 86.7%.
Claims (9)
1. a kind of method of Acer saccharum tissue culture quick breeding, which comprises the following steps:
(1) explant selects
Perennial healthy and strong, no disease and pests harm Acer saccharum is chosen, using the spray of its current year raw, non-lignifying as explant；
(2) explant pre-processes
Explant in step (1) is removed into blade, petiole, after 75% alcohol wipe, is trimmed at least one axillary bud of band or top
Then the stem section of bud is shaken with dish washing liquid and is cleaned；Then it is super at 18-22 DEG C for stem section being immersed in and being placed in temperature in pure water
It is handled in sound wave washer, uses pure water rinsing later；
(3) foundation of sterile system
It explant will be used with 75% alcohol soaking disinfection with aseptic water washing in aseptic operating platform after step (2) are handled
The concussion disinfection of 0.1% mercury chloride, with aseptic water washing, is blotted, then obtains sterile material after cutting off stem section both ends disinfection apoptotic fraction
Material；
(4) Fiber differentiation
6-BA0.5mg/L, sucrose 30g, agar 6.5g are added on the basis of MS culture medium, configures induced medium, and adjusting lures
Medium pH is led to 5.8；Step (3) sterilizable material morphology lower end is inserted into induced medium to the induction training for carrying out bud
It supports, promotes the sprouting and growth of bud；
(5) Multiplying culture
6-BA0.5mg/L, GA are added on the basis of MS culture medium30.5mg/L, sucrose 30g, agar 6.5g, configuration is after being commissioned to train
Base is supported, subculture medium pH to 5.8 is adjusted, the Acer saccharum axillary bud in step (4) is entirely accessed into subculture medium and is carried out
Multiplying culture；
(6) induction of root
IBA0.5mg/L, sucrose 20g, agar 6.5g are added on the basis of MS culture medium, configures root media, and adjusting is taken root
Medium pH carries out rooting induction to 5.8, by the seedling access root media of step (5)；
(7) the hardening domestication of aseptic seedling
The Acer saccharum of step (6) aseptic seedling of taking root from culturing room is moved into greenhouse culture, is then moved into Light media
Continue to cultivate into complete regeneration plant；Wherein Light media is mixed simultaneously by Nutrition Soil, river sand and perlite with 2: 1: 1 volume ratio
It is made of potassium permanganate disinfection.
2. a kind of method of Acer saccharum tissue culture quick breeding according to claim 1, it is characterised in that: step (1)
The progress explant time is 4 the middle ten days to mid-June.
3. a kind of method of Acer saccharum tissue culture quick breeding according to claim 1, it is characterised in that: step (2)
Specific steps are as follows: the explant in step (1) is removed into blade, petiole on the basis of not damaging axillary bud and terminal bud, uses gauze
75% alcohol wipe 1-2 is dipped after, then is trimmed to the stem section of 3-5cm at least one axillary-bud or top-bud of band, is shaken with dish washing liquid
After cleaning 2 times, flowing water is cleaned 8-10 minutes, and it is 18-22 that then stem section, which is immersed in the container equipped with pure water, and is placed in temperature
It handles in ultrasonic cleaner at DEG C 8-12 minutes, uses pure water rinsing 1-2 times later.
4. a kind of method of Acer saccharum tissue culture quick breeding according to claim 1, it is characterised in that: step (3)
Specific steps are as follows: explant after step (2) processing is subjected to surface in 20-40 seconds with the immersion of 75% alcohol in aseptic operating platform
Disinfection is shaken soaking disinfection 2-3 minutes with aseptic water washing 1-2 times with 0.1% mercury chloride, with aseptic water washing 4-6 times, is inhaled
It is dry, then sterilizable material is obtained after cutting off stem section both ends disinfection apoptotic fraction.
5. a kind of method of Acer saccharum tissue culture quick breeding according to claim 1, it is characterised in that: the step
(4) the induction control cultivation temperature of bud is at 24-26 DEG C, light application time 12-14h/d, intensity of illumination 2000- in
2500lx, illumination cultivation 20-30d.
6. a kind of method of Acer saccharum tissue culture quick breeding according to claim 1, it is characterised in that: the step
(5) strong seedling culture temperature cultivates 20-30d at 24-26 DEG C, light application time 12-14h/d, intensity of illumination 2000-2500lx in.
7. a kind of method of Acer saccharum tissue culture quick breeding according to claim 1, it is characterised in that: the step
(6) 20-25 DEG C of the cultivation temperature of root in, light application time 10-16h/d, intensity of illumination 2000-2500lx cultivate 15-20d.
8. a kind of method of Acer saccharum tissue culture quick breeding according to claim 1, it is characterised in that: the step
(7) the specific steps are the Acer saccharum of step (6) aseptic seedling of taking root from culturing room is moved to greenhouse, unclamp bottle cap training
3-5d is supported, then opens bottle cap culture 5-7d, then moves into Light media and continues to cultivate into complete regeneration plant；Wherein Light media
It is made with 2: 1: 1 volume ratio mixing and of Nutrition Soil, river sand and perlite of potassium permanganate disinfection；Therebetween, it sprays weekly more
Bacterium spirit is primary；In outside air temperature is for 1 week be lower than 30 DEG C or less when move in field plant bed, appropriate dense planting, temperature be greater than 30 DEG C
When take sunshade net, guarantee moisture supply.
9. a kind of method of Acer saccharum tissue culture quick breeding according to claim 1, it is characterised in that: in step
(4) Medium's PH Value is adjusted with 1mol/L NaOH in-(6).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710835544.3A CN107455261B (en) | 2017-09-15 | 2017-09-15 | A kind of method of Acer saccharum tissue culture quick breeding |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710835544.3A CN107455261B (en) | 2017-09-15 | 2017-09-15 | A kind of method of Acer saccharum tissue culture quick breeding |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107455261A CN107455261A (en) | 2017-12-12 |
| CN107455261B true CN107455261B (en) | 2019-08-13 |
Family
ID=60552529
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710835544.3A Active CN107455261B (en) | 2017-09-15 | 2017-09-15 | A kind of method of Acer saccharum tissue culture quick breeding |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107455261B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108990800B (en) * | 2018-07-27 | 2022-02-11 | 江苏农林职业技术学院 | Tissue culture method of Acer buergerianum |
| CN109247238B (en) * | 2018-11-30 | 2021-09-24 | 四川七彩林科股份有限公司 | Acer negundo L tissue culture seedling ex-vitro rooting method |
| CN110301352B (en) * | 2019-07-02 | 2020-12-08 | 浙江理工大学 | Tissue culture method of sasanqua |
| CN110810032A (en) * | 2019-10-24 | 2020-02-21 | 浙江人文园林股份有限公司 | Dream micro-cuttage technical method for Japanese red maple variant orange |
| CN112314438A (en) * | 2020-11-18 | 2021-02-05 | 杭州云树园艺有限公司 | Culture method for acer yankee callus induction |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH11318209A (en) * | 1998-05-12 | 1999-11-24 | Ryoji Mita | Production of nursery stock of acer nikoense maximowicz |
| CN103477983B (en) * | 2013-09-06 | 2015-08-12 | 巴中七彩林业科技有限公司 | Acer palmatum red maple tissue culture propagation technique |
| CN104285818A (en) * | 2014-10-30 | 2015-01-21 | 江苏省农业科学院 | Tissue culture rapid propagation method of acer palmatum |
| CN104488703B (en) * | 2014-11-25 | 2016-02-24 | 巴中七彩林业科技有限公司 | A kind of tissue culture and rapid propagation method of gold leaf Acer negundo. L |
| CN104920225B (en) * | 2015-07-03 | 2017-10-24 | 西北农林科技大学 | A kind of tissue culture and rapid propagation method of gold leaf Acer negundo. L |
| CN106922538B (en) * | 2017-04-26 | 2018-11-23 | 中国科学院合肥物质科学研究院 | A kind of method of Acer saccharum Regeneration in Vitro |
-
2017
- 2017-09-15 CN CN201710835544.3A patent/CN107455261B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN107455261A (en) | 2017-12-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107455261B (en) | A kind of method of Acer saccharum tissue culture quick breeding | |
| CN101473747B (en) | Micro-graft and fast propagation method of tree | |
| CN107996166B (en) | Rapid cuttage breeding method for catalpa bungei | |
| CN111616052A (en) | Rapid propagation and sugar-free rooting culture method and application of apple rootstock catalpa bungei | |
| CN106538392B (en) | A kind of white oriental cherry tissue culture and rapid proliferation method | |
| CN108293878A (en) | A kind of tissue culture method of snakegourd tender leaf | |
| CN108739370B (en) | Method for rapid propagation by utilizing mature lotus embryos | |
| CN107197746B (en) | Breeding method of cunninghamia lanceolata field excellent resources | |
| CN103430845A (en) | Strawberry tissue culturing method | |
| CN107711513B (en) | Tissue culture and rapid propagation method for spartina alterniflora | |
| CN105210870A (en) | The tissue culture propagation technology of No. 1, the anti-anvil of peach in Peach rootstock | |
| CN104396759A (en) | Method for tissue culture and rapid breeding of Fraxinus chinensis | |
| CN109042330A (en) | A kind of method for tissue culture of spindle tree | |
| CN108782247A (en) | A kind of method for tissue culture of late cherry " Yu Yihuang " kind of Japan | |
| CN106489737A (en) | A kind of culture medium of Hybrid Tea tissue cultures and method | |
| CN106900307A (en) | A kind of division propagation method of jade hairpin | |
| CN111034613A (en) | Tissue culture rapid propagation method for superior paulownia catalpa trees | |
| CN110402818A (en) | A kind of method of excellent kind of Chinese chestnut Mature Embryo Tissue culture rapid seedling cultivation | |
| CN107006367B (en) | 'sunshine' cherry tissue culture rapid propagation method | |
| CN109041847A (en) | A kind of method of Common zenia bearing tree childrenization rejuvenation and asexual multiplication seedling | |
| CN101611698B (en) | Method for culturing cephalotaxus excised embryos and regenerating plants | |
| CN103858768A (en) | Tissue culture method of plumeria rubra L.cv.Acutifolia | |
| CN107517854A (en) | A kind of tissue culture and rapid propagation method of roundleaf eucalyptus | |
| CN110024676B (en) | Rapid rooting and seedling raising method for water culture cutting of betula alnoides | |
| CN105660398B (en) | A kind of tissue cultivation rapid breeding method of the big wood paint in mao of dam |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |