CN102511399B - Acer mono axillary bud path tissue culture method - Google Patents
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- 241000219240 Acer pictum subsp. mono Species 0.000 title abstract description 7
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- 238000000034 method Methods 0.000 claims abstract description 10
- 230000006698 induction Effects 0.000 claims abstract description 5
- 239000012879 subculture medium Substances 0.000 claims abstract description 4
- 241000208140 Acer Species 0.000 claims description 36
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- 229910052739 hydrogen Inorganic materials 0.000 claims description 2
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- 231100000614 poison Toxicity 0.000 claims description 2
- RCTYPNKXASFOBE-UHFFFAOYSA-M chloromercury Chemical compound [Hg]Cl RCTYPNKXASFOBE-UHFFFAOYSA-M 0.000 claims 1
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Abstract
The invention provides an acer mono maxim axillary bud path tissue culture and propagation method, which comprises the following steps that: firstly, explant disinfection is carried out, the disinfected explants are inoculated into an induction culture medium for bud differentiation induction, the normal culture is carried out for 20 days under the condition of controlling the culture temperatureto be 25 DEG C, the light illumination time to be 10 to 16 hours/day and the light illumination intensity to be 1600 to 2,000Ix, then, the obtained acer mono maxim tender tips are cut and are then inoculated into a subculture medium for subculture for 30 days, finally, the acer mono maxim aseptic seedlings are inoculated into a rooting culture medium for rooting culture for 15 to 20 days, the culture temperature is 20 to 26 DEG C, the light illumination time is 10 to 16 hours/day, and the light illumination degree is 1,600 to 2,000Ix. After the rooting aseptic seedlings are obtained, the rooting aseptic seedlings are transplanted into a seedling bed for domestication culture. The problem of fast acer mono maxim popularization can be effectively solved by using the method.
Description
Technical field
The present invention relates to look wood maple (Acer mono Maxim) tissue culture propagation, belong to the technical field of woody plant seedling breeding.
Background technology
Look wood maple (Acer mono Maxim) belongs to Aceraceae (Aceraceae) maple and belongs to (Acer) plant.Have another name called five jiaos of maples, deciduous tree, high 15-20m; Bark is coarse, normal lobe, taupe; Sprig is carefully thin, without hair, gives birth to then spray green or purple green, perennial branch grey or light gray, the circular hole skin of tool; Hibernaculum is bordering on sphere, and scale is avette.The long 4-6cm of leaf opposite leaf handle, carefully thin, without hair; Blade papery, appearance are closely oval, long 6-8cm, and wide 9-11cm, 5 split, and 3 split and 7 split with life one tree sometimes; Avette or the wide triangle of sliver, long gradually point, full edge without hair, only has tuft between the master pulse armpit, 5 of master pulses are showing in the above, the lateral vein two sides is not all showing, above light green, below light green.Spend majority, polygamy, male flower and hermaphrodite flower homophyletic, most normal one-tenth are coniform, and give birth on the corymb top, without hair; The lace green-yellow, total long 1-2cm of bennet; Sepal 5, Long Circle, yellow green; Petal 5, ellipse, pale; Stamen 8, shorter than petal without hair, flower pesticide is yellow; Ovary is without hair, agensis in male flower, and style is without hair, and column cap 2 splits, warp.Purple green when samara is tender, faint yellow when ripe, nutlet is flattened shape, long 1-1.3cm, wide 5-8mm; The wing Long Circle, wide 5-10mm, together with the long 2-2.5cm of nutlet, flare up acute angle or be bordering on the obtuse angle, May at florescence, fruit September phase.Major Natural is distributed in northeast, North China, Central China, East China, southwestern various places.Because look wood maple gesture is graceful, is thick with leaves, and is leaf beautiful, tender leaf is red, autumn has set in become orange or red, very attractive in appearance.Therefore, where no matter these seeds planted in, invariably feels fascinating, both can be used as the ground such as good colored tree is planted in the lawn, mound, small stream limit, Chi Pan in gardens, can be used as again street tree and utilize.In addition, the look wood maple grain of wood is clear, and structure is thin and even, wooden heavy, hard, corrosion resistant, and easily processing, tangent plane is smooth, and good springiness is the upper good material of making top-grade furniture, musical instrument, farm implements and plywood.
The at present research and development of the domestic wooden maple of checking colors are used less.Research for its Fast-propagation and cultivation management still is in blank.Look wood maple is at present take seed growing as main, and correlative study shows that look wood maple is larger by the seedling variance ratio of seminal propagation, and shape is unstable, so extremely shortage of seedling resource on the market.Also among test, the full rate of Aceraceae seed is extremely low for cuttage and grafting, only ten thousand/and several.So not only reproduction coefficient is little, and the seedling time greatly prolong, be difficult to the formation scale,, do not satisfy the demand in market.
The woody plant tissure culture studies is started in early 1950s, until the later stage seventies just obtains larger development.Tissue cultivate to obtain regeneration plant and mainly is divided into two kinds of approach, and one is the cultivation of broad sense tissue, namely isolates the organ that suits the requirements etc. from plant corpus, by sterile working, cultivates the whole plant that obtains to regenerate under the manual control condition; It two is cultivated for the narrow sense tissue, namely produces callus from each organ in incubation, cultivates callus again through being differentiated to form aftergrowth again.
At present, incomplete statistics has more than 300 kind of woody plant of genus more than 90 to cultivate the acquisition regeneration plant through tissue.And about the research that the Aceraceae tissue is cultivated, report seldom both at home and abroad.Nearly 5 years, rarely seen have the report tender stem of wooden maple of checking colors (to see Plant Physiology Communications, the 2nd phase in 2008, the tissue of look wood maple is cultivated and fast breeding technique), acer fabri (is seen northern gardening with tender stem, the 9th phase in 2010, the tissue of acer fabri is cultivated and fast breeding technique) and tender leaf (see Sino-South African Forestry University of Science and Technology's journal, the 4th phase in 2010, view seeds acer fabri induction of callus) for explant carries out the cultivation of inducing of callus, and there is not yet report about axillalry bud (organ) the approach rapid propagation in vitro research of look wood maple.
In sum, taking tissue culture propagating look wood maple is one of important channel that solves look wood maple shortage of resources, and the exploitation of the wooden maple of checking colors and utilization and extention all have very important meaning.
Summary of the invention
The technical problem that the present invention mainly solves is the axillalry bud approach tissue culture propagation of open look wood maple.
Adopt following technical scheme for solving this technology:
A) explant sterilization: get that stems with bud is explant on the annual cuttage seeding current-year branch of look wood maple early or mid September, rinse well with clear water, putting into mass concentration after the cleaning and be 3% H2O2 soaked 15～20 minutes, take out to change over to immediately to contain in the 0.1%HgCI2 solution that mass concentration is 1～5% Tween-80 and soaked 10～20 minutes, then take out with aseptic water washing 5～6 times and with sterilization blotting paper water is blotted.
B) axillalry bud is sprouted: the explant behind the cancellation poison is cut into the long stem section of 1.0-1.5cm, and wherein each stem section is with an axillalry bud, and the stem section is inoculated in inducing culture WPM+6-BA1.0mg/L+IBA0.01mg/L+PVP500mg/L+GA
3Induce 1.0mg/L carry out the sprouting of axillalry bud among the PH6.0, the control cultivation temperature is carried out illumination cultivation 20d at 25 ℃, and described illumination cultivation is: light application time 10-16h/d, and intensity of illumination is 1600-2000lx;
C) subculture cultivation: with step B) the rear access of the tender tip cutting of middle institute Dry Sack wood maple carried out subculture cultivation 30d among the subculture medium WPM+6-BA0.3mg/L+IBA0.05mg/L+PVP500mg/L PH6.0,25 ℃ of cultivation temperature, light application time 10-16h/d, intensity of illumination is 1600-2000lx;
D) inducing of root: with step C) carry out culture of rootage 15-20d among institute's Dry Sack wood maple test-tube plantlet access root media 1/2MS+IBA3.0mg/L PH6.0, cultivation temperature 20-26 ℃, light application time 10-16h/d, intensity of illumination is 1600-2000Ix.
E) aseptic seedling domestication and continue to cultivate:: with step D) the institute Dry Sack wood maple aseptic seedling of taking root cultivates 3-5d opening under the environment of bottle cap, then move into and continue in the light matrix container that is fit to this seeds growing environment to cultivate, light matrix is to be mixed by soil, river sand and the fertilizer ratio with 2: 1: 2, and with the sterilization of 0.4% potassium permanganate, cultivate and after 10-15 days nursery stock is transplanted with matrix and dense planting continues cultivation in the soil that adapts to.
The beneficial effect of the relative prior art of the present invention is:
1, the present invention finds in process of the test, and explant is killed and wounded after the operation sterilization routinely, can not obtain effective explant number of inducing cultivation required.Through repetition test, many experiments result's comparison just obtains specific embodiments of the present invention, and namely flowing water flushing-hydrogen peroxide-mercuric chloride has not only obtained desirable sterilization effect, has also guaranteed the explant number of inducing cultivation required.This result and invention institute for special object look wooden maple ingredient much relations are arranged.It is on the basis of theory research that the present invention adopts look wood maple explant sterilization method, obtains the effective sterilizing method that many experimental results is supported.
2, owing to different plants, root growth mode and length are different, and the ability of taking root is different, and therefore different hypothalluses has a great impact the rooting rate of plant.The test-tube plantlet of the present invention after with a secondary root moves into and continues in the light matrix that is made of soil, river sand, fertilizer to cultivate, and carries out two secondary roots, greatly improved rooting rate, further improved the survival rate of look wood maple.Two secondary roots are different from a secondary root, because the fringe bar root system after the secondary root is relatively more flourishing, sturdy, absorbing capacity is high, so the present invention is according to these characteristics, for taking root of maple of look wood makes the higher light matrix of nutrient component, with potassium permanganate light matrix is carried out disinfection simultaneously, can avoid harmful components wherein that seedling growth is impacted.
3, the method that adopts axillalry bud approach tissue the to cultivate wooden maple of checking colors breeds, cooperate best induce, subculture, prescription of rooting medium, can effectively obtain a large amount of look wood maple aseptic seedling, solve the problem of look wooden maple shortage of resources; By to the choosing of explant, inducing culture, subculture medium and root media, can be so that the rate of increase reach 70 times, the domestication survival rate reaches 98-100%, obtains efficiently look wood maple seedling.
Embodiment
Embodiment one
1, chooses the healthy and strong annual cuttage seeding of look wood maple of 10 strains.Put into hot-house culture in resting stage in January, 2010.On September 1st, 2010, clip is given birth to stem segment with axillary bud then, removes blade, keeps the 1cm petiole, and take axillalry bud as explant, every stem section is with an axillalry bud, is about 2cm.Explant is rinsed well with clear water, putting into mass concentration after the cleaning and be 3% H2O2 soaked 15～20 minutes, take out to change over to immediately to contain in the 0.1%HgCI2 solution that mass concentration is 1～5% Tween-80 and soaked 10～20 minutes, then take out with aseptic water washing 5～6 times and with sterilization blotting paper water is blotted.0.5cm is excised respectively at the explant two ends that sterilization is crossed, to remove injured part.Access inducing culture WPM+6-BA1.0mg/L+IBA0.01mg/L+PVP500mg/L+GA
31.0mg/L among the PH6.0.Every bottle graft 3 strains access 30 bottles altogether.25 ℃ of temperature, light application time 10h/d under the condition of light intensity 2000lx, cultivates.
2, behind the explant access medium 4d, explant begins to have axillalry bud to sprout.During to 20d, germination rate reaches 95%, obtains altogether not first for aseptic seedling with root of 86 strains.The about 4cm of aseptic seedling plant height, on every strain stem with 2 to 4 of the axillalry buds of having sprouted.Blade is removed, and trunk is cut to stem with bud, is with an axillalry bud for every section, gets altogether 170 sections.Among the access shoot proliferation medium WPM+6-BA0.3mg/L+IBA0.05mg/L+PVP500mg/L PH6.0, condition of culture is the same.After cultivating 10d, axillalry bud begins to go out successively the tip, and branch begins sclerosis behind the 15d, and axillalry bud begins full, and behind the 30d, the germination rate of 170 axillalry buds reaches 98%, and the rate of increase reaches 3.8 times, namely on average contains 3.8 axillalry buds on every strain subculture seedling, obtains altogether 633 of axillalry buds.
3, the aseptic seedling of subculture is cut into segment with 2-3 axillalry bud, access Rooting and hardening-off culture base 1/2MS+IBA3.0mg/LPH6.0 accesses 600 strains altogether, and condition of culture is the same.After cultivating 15d, begin to take root, rooting rate reaches 100% behind the 20d, so far obtains 600 strains of band root aseptic seedling.So far the aseptic seedling rate of increase reaches 60 times.
4, the blake bottle bottle cap of 600 strain band root aseptic seedling is opened, then move into and continue in the light matrix container that is fit to this seeds growing environment to cultivate, light matrix is to be mixed by soil, river sand and the fertilizer ratio with 2: 1: 2, and with the sterilization of 0.4% potassium permanganate, cultivate and after 10-15 days nursery stock is transplanted with matrix and dense planting continues cultivation in the soil that adapts to.Seedling percent reaches 99%, obtains seedling 594 strains.
Embodiment two
1, chooses the healthy and strong annual cuttage seeding of look wood maple of 20 strains.Put into hot-house culture in resting stage in January, 2010.On September 16th, 2010, clip is given birth to stem segment with axillary bud then, removes blade, keeps the 1cm petiole, and take axillalry bud as explant, every stem section is with an axillalry bud, is about 2cm.Explant is rinsed well with clear water, putting into mass concentration after the cleaning and be 3% H2O2 soaked 15～20 minutes, take out to change over to immediately to contain in the 0.1%HgCI2 solution that mass concentration is 1～5% Tween-80 and soaked 10～20 minutes, then take out with aseptic water washing 5～6 times and with sterilization blotting paper water is blotted.0.5cm is excised respectively at the explant two ends that sterilization is crossed, to remove injured part.Access inducing culture WPM+6-BA1.0mg/L+IBA0.01mg/L+PVP500mg/L+GA
31.0mg/L among the PH6.0.Every bottle graft 3 strains access 60 bottles altogether.24 ℃ of temperature, light application time 16h/d under the condition of light intensity 20001x, cultivates.
2, behind the explant access medium 6d, explant begins to have axillalry bud to sprout.During to 20d, germination rate reaches 97%, obtains altogether not first for aseptic seedling with root of 175 strains.The about 4cm of aseptic seedling plant height, on every strain stem with 2 to 4 of the axillalry buds of having sprouted.Blade is removed, and trunk is cut to stem segment with axillary bud, is with an axillalry bud for every section, gets altogether 400 sections.Among the access shoot proliferation medium WPM+6-BA0.3mg/L+IBA0.05mg/L+PVP500mg/L PH6.0, condition of culture is the same.After cultivating 10d, axillalry bud begins to go out successively the tip, and branch begins sclerosis behind the 15d, and axillalry bud begins full, and behind the 30d, the germination rate of 400 axillalry buds reaches 99%, and the rate of increase reaches 3.6 times, namely on average contains 3.6 axillalry buds on every strain subculture seedling, obtains altogether 1425 of axillalry buds.
3 will be cut into through the aseptic seedling of subculture the segment with 2-3 axillalry bud, and access Rooting and hardening-off culture base 1/2MS+IBA3.0mg/LPH6.0 accesses 1410 strains altogether, and condition of culture is the same.After cultivating 15d, begin to take root, rooting rate reaches 100% behind the 20d, so far obtains 1410 strains of band root aseptic seedling.Through above-mentioned cultivation, the aseptic seedling rate of increase reaches 70.5 times.
5, the blake bottle bottle cap of 1410 strain band root aseptic seedling is opened, be positioned under the good environment of room temperature, ventilation condition and cultivate 5d, then move into and continue in the light matrix container that is fit to this seeds growing environment to cultivate, light matrix is to be mixed by soil, river sand and the fertilizer ratio with 2: 1: 2, and with the sterilization of 0.4% potassium permanganate, cultivate and after 10-15 days nursery stock is transplanted with matrix and dense planting continues cultivation in the soil that adapts to.Seedling percent reaches 98%, obtains seedling 1382 strains.
Claims (1)
1. a look wood maple axillalry bud approach tissue culture propagation is characterized in that, may further comprise the steps:
A) explant sterilization: early or mid September is chosen the then living stem segment with axillary bud of the annual cuttage seeding of look wood maple as explant, disinfection;
B) induction of bud: the explant behind the cancellation poison is cut into the long stem section of 1.0-1.5cm, and wherein each stem section is with an axillalry bud, and the stem section is inoculated in inducing culture WPM+6-BA1.0mg/L+IBA0.01mg/L+PVP500mg/L+GA
31.0mg/L carry out the induction of bud among the pH6.0, control cultivation temperature at 25 ℃, illumination cultivation 20d, described illumination cultivation is: light application time 10-16h/d, intensity of illumination is 1600-2000lx;
C) subculture is cultivated and propagation: with step B) the rear access of the tender tip cutting of middle institute Dry Sack wood maple subculture medium WPM+6-BA0.3mg/L+IBA0.05mg/L+PVP500mg/L, carry out subculture among the pH6.0 and cultivate 30d, 25 ℃ of cultivation temperature, light application time 10-16h/d, intensity of illumination is 1600-2000lx;
D) inducing of root: with step C) institute's Dry Sack wood maple test-tube plantlet access root media 1/2MS+IBA3.0mg/L carries out culture of rootage 15-20d among the pH6.0, and cultivation temperature 20-26 ℃, light application time 10-16h/d, intensity of illumination is 1600-2000lx;
E) aseptic seedling domestication and continue to cultivate: with step D) the institute Dry Sack wood maple aseptic seedling of taking root is cultivated 3-5d opening under the environment of bottle cap, then move into and continue in the light matrix container that is fit to this seeds growing environment to cultivate, light matrix is to be mixed by soil, river sand and the fertilizer ratio with 2: 1: 2, and with the sterilization of 0.4% potassium permanganate, cultivate and after 10-15 days nursery stock is transplanted with matrix and dense planting continues cultivation in the soil that adapts to;
Described steps A) the explant disinfecting process in is: put into mass concentration after the cleaning and be 3% H
2O
2Middle immersion 15～20 minutes, taking-up changes over to immediately and contains the 0.1%HgCl that mass concentration is 1～5% Tween-80
2Soaked in the solution 10～20 minutes, and then took out with aseptic water washing 5～6 times and with sterilization blotting paper water is blotted.
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| CN102715087A (en) * | 2012-06-30 | 2012-10-10 | 吴昌海 | Tissue culture reproduction method for liquidambar styraciflua |
| CN103404438B (en) * | 2013-08-06 | 2015-03-25 | 巴中七彩林业科技有限公司 | Acer palmatum seed tissue culture method |
| CN103392601B (en) * | 2013-08-09 | 2014-10-22 | 南京金埔园林股份有限公司 | Michelia compressa tissue culture propagation method |
| 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 |
| CN109662032A (en) * | 2019-03-05 | 2019-04-23 | 延边大学 | A kind of culture medium and method of acer pseudo-sieboldianum tissue cultures |
| CN116724889B (en) * | 2023-05-31 | 2024-06-21 | 东北林业大学 | Method for in-vitro rapid propagation by using axillary buds of Acer mono |
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