CN107371880A - A kind of apple rootstock tissue culturing fast seedling-cultivating method - Google Patents
A kind of apple rootstock tissue culturing fast seedling-cultivating method Download PDFInfo
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- CN107371880A CN107371880A CN201710628657.6A CN201710628657A CN107371880A CN 107371880 A CN107371880 A CN 107371880A CN 201710628657 A CN201710628657 A CN 201710628657A CN 107371880 A CN107371880 A CN 107371880A
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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
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G31/00—Soilless cultivation, e.g. hydroponics
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05D—INORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C; FERTILISERS PRODUCING CARBON DIOXIDE
- C05D9/00—Other inorganic fertilisers
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F11/00—Other organic fertilisers
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
- C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
Abstract
The present invention provides a kind of apple rootstock tissue culturing fast seedling-cultivating method, using stem section as explant, adventitious bud inducing differential medium, subculture multiplication medium, root media etc. are optimized, the growth coefficient of adventitious bud is 4.2, final rooting rate is up to 100%, and number of averagely taking root is up to 7.3.By controlling tissue-cultured seedling greenhouse to tame the design parameter in each stage, transplanting survival rate is up to 90% after hardening 30d.
Description
Technical field
The present invention relates to a kind of apple rootstock tissue culturing fast seedling-cultivating method, belong to field of plant tissue culture technique.
Background technology
" blue or green anvil No.1 " be Qingdao Institute of Agricultural Sciences and Shandong Agricultural University using hupehensis Rehd as it is maternal, with column type
Apple strain CO is the apple rootstock new lines that paternal hybrid is bred as, also excellent in addition to the merit for possessing hupehensis Rehd
Dwarfing performance." blue or green anvil No.1 " well developed root system, tree body stationarity is strong after Grafted Variety, and Dwarf Effect on Dwarf is obvious, early into spending easily
Phase high yield strong stress resistance, the promotion effect in Shaanxi, Shandong and Xinjiang is fine, is China's independent research for having very much development potentiality
Stock variety.
At present, " blue or green anvil No.1 " mainly by seed propagation, and conventional seed planting breeding need to take a large amount of soils, and the cycle compared with
It is long.Therefore, bred with the method for tissue culture expanding propagation.And also only blade is efficient in vitro in experimental stage Primary Study by forefathers
The foundation of regenerating system.Such as Wu Ruigang《The foundation of the in vitro high-efficiency regeneration system of apple rootstock " blue or green anvil No.1 "》(plant physiology
Journal, 2013,49 (10):In 1053-1056), using " blue or green anvil No.1 " blade as explant, from the adventitious bud inducing of excised leaf
Differentiation, shoot proliferation, test tube seedling root induction and acclimatization and transplantses, establish efficient vitro Regeneration System, are " blue or green anvil one
Number " numerous, genetic conversion system the foundation of expansion of stock and its character improvement provide technical support.
Existing " blue or green anvil No.1 " vitro Regeneration System also rests on the small-scale conceptual phase in laboratory, is moved after taming hardening
Plant survival rate only 60% or so.Needs of production can not be met.Therefore, it is necessary to which the tissue cultures for developing more high-survival rate are quick
Method for culturing seedlings.
The content of the invention
The present invention is in order to overcome the above-mentioned deficiencies of the prior art, there is provided a kind of apple rootstock tissue culturing fast seedling-cultivating side
Method, adventitious bud inducing differential medium, subculture multiplication medium, life as explant, are optimized using " blue or green anvil No.1 " stem section
Root culture etc., final rooting rate is up to 100%, and number of averagely taking root is up to 7.3；Transplanting survival rate is up to 90% after hardening 30d.
For achieving the above object, the technical solution adopted by the present invention is that a kind of apple rootstock tissue cultures are quickly educated
Seedling-growing method, including indoor tissue cultures stage and greenhouse domestication stage, the indoor tissue cultures stage, using stem section as explant
Body, after disinfecting, carry out inoculated and cultured.Each stage culture medium prescription is as follows：
Differential medium：MS+2.0mg·L-16-BA+0.2mg·L-1NAA+25g·L-1Sucrose+6gL-1Agar powder；
Subculture medium：MS+1.0mg·L-16-BA+0.1mg·L-1NAA+25g·L-1Sucrose+6gL-1Agar powder；
Root media：1/2MS+0.5mg·L-1IBA+1.5mg·L-1IAA+20g·L-1Sucrose+6gL-1Agar powder+
0.2g·L-1Activated carbon；
The pH value of each culture medium is 5.6~5.8.
Specifically, a kind of apple rootstock tissue culturing fast seedling-cultivating method of the present invention, step is as follows,
First, the indoor tissue cultures stage
(1) selection, sterilization and the inoculated and cultured of explant
Adopt and cut annual healthy and strong branch of the rest period with tender shoots, soak 20min with bromogeramine, clean；Tiltedly it is cut into 6-
Disinfected after 8cm stem section：1min is rinsed in sterilized water concussion；75% alcohol vibration rinses 12s；Sterilized water concussion punching
Wash 1min；0.1% mercuric chloride solution, instills few drops of Tween 80s, and 8min is rinsed in concussion；1min is rinsed in sterilized water concussion；Repeat
Aseptic water washing 7 times.
Then the stem section after sterilization is cut off into wound with operating scissors, stem section is cut into two sections with 1-2 bud, is inoculated in
In blank MS culture mediums.
The explant being inoculated with is positioned between culture, is cultivated, and condition of culture is as follows：25 DEG C ± 3 DEG C of temperature, humidity
25%-50%, intensity of illumination 3000lx-6000lx, light application time 10h-12h；15-20d rolling bottles continue to cultivate.
It is preferred that first segment is as explant.
(2) differentiation culture
The stem section that young shoot is carried after cultivating is taken, the wound of upper and lower ends is cut off with operating scissors, is then inserted into differentiation culture
In base, differential medium formula is as follows：MS+2.0mg·L-16-BA+0.2mg·L-1NAA+25g·L-1Sucrose+6gL-1Agar
(agar powder gel strength is 1300g/cm to powder2)；Seedling after inoculation is positioned between culture and cultivated.Condition of culture is same as above, training
Support cycle 20-25d.
(3) subculture expands numerous culture
After differentiation culture terminates, seedling, which is transferred in subculture medium, carries out the numerous culture of subculture expansion；Subculture expands breeding culture medium
For：MS+1.0mg·L-16-BA+0.1mg·L-1NAA+25g·L-1Sucrose+6gL-1Agar powder；Condition of culture is same as above, culture
Cycle 20-25d.Every Seedling Differentiation goes out 4-6 new talent.
(4) culture of rootage
The healthy and strong seedling of more than 4cm is divided into single, is connected in root media；Root media is as follows：1/2MS+
0.5mg·L-1IBA+1.5mg·L-1IAA+20g·L-1Sucrose+6gL-1(agar powder gel strength is 1300g/ to agar powder
cm2)+0.2g·L-1Activated carbon (creates the dark surrounds favorably taken root)；Condition of culture is as follows：25 DEG C ± 3 DEG C of temperature, humidity
25%-50%, intensity of illumination 2500-4000lx, light application time 10-12h.15d is cultivated, there can be root to bear.
Further, in order to improve the utilization rate of tissue-cultured seedling and success rate of taking root, step (3) subculture expands numerous culture
Afterwards, the relatively weak seedling of growing way is gone in strong seedling culture base, carries out strong seedling culture；
Strong seedling culture base is as follows：MS+0.25mg·L-16-BA+0.05mg·L-1NAA+25g·L-1Sucrose+6gL-1Fine jade
Cosmetics.Treat that seedling grows 20 days or so in strong seedling culture base, the weaker tissue-cultured seedling of growing way becomes strong, can carry out training of taking root
Support.
2nd, the greenhouse domestication stage
When 80% seedling root long degree is more than 3cm, acclimation is carried out：
(1) greenhouse experiment controls
Temperature control daytime, night was not less than 18 DEG C, relative humidity 60%-80% at 23-28 DEG C or so.The 1-2 days, light
Humidity nearly 100% in 4000lx or so, bottle is controlled by force, and bottle seedling of taking root for the 3rd day pine lid, intensity control is in 4000-6000lx, bottle
Interior humidity is 90% or so, the 4-5 days increase light intensity to 6000-8000lx, humidity 80%-90% in bottle, the 6th day bottle seedling of taking root
Uncapping, intensity control humidity 80% or so in 8000lx or so, bottle, the 7-9 days, intensity control was in 8000-12000lx, bottle
Interior humidity is in 70%-80%.
(2) it is colonized
Seedling is washed after 10th day, is colonized.Management after field planting：The temperature control of overall situation is relatively wet at 23-28 DEG C in greenhouse
Degree is in 60%-70%.The 1st day composite fertilizer for spraying 0.1% after field planting, sprayed with 0.5 ‰ carbendazim within the 2nd day, make phase
90% is not less than to humidity, intensity control is in 4000lx or so；The 3-5 days, intensity control was in 4000-6000lx, relative humidity
Between 85%-90%；6th day, one jiao of Small plastic shed film, increase light intensity to 6000-8000lx are opened, relative humidity control exists
80% or so；The 7-9 days, intensity control was in 8000lx or so, relative humidity 75%-80%, the 9th day, opening Small plastic shed film
1/3, for humid control 75% or so, light intensity brings up to 8000-10000lx；The 10-11 days, relative humidity was controlled in 70%-
75%, intensity control is in 10000lx or so；12nd day, the 2/3 of Small plastic shed film is opened, relative humidity is 70% or so, light intensity
10000-12000lx, the overall situation close in greenhouse；The 13-14 days, two day time is buffered, can fully open canopy at the 15th day
It is thin, greenhouse overall situation is adapted to, soil humidity should not be excessive, controls 70% or so, in order to avoid occur macerating root.Tamed successfully after 30d
Hole tray shoot survival percent be 90%.
Substrate composition is perlite：Vermiculite：Turf=1：1：3, put the 10-15d that is exposed to the sun under the sun with the sterilization of 2 ‰ carbendazim.
The present invention has the advantages that：
(1) from stem section as explant.The present invention does explant from the stem section with bud first, from stem after young shoot sprouting
Section is peeled off, and can be grown to serve as seedling, needs to carry out dedifferentiation and again break up that seedling could be formed compared to using blade as explant,
Substantially reduce the time and reduce production difficulty.
(2) for the present invention using the stem section with bud as explant, the growth coefficient of adventitious bud is 4.2.Existing blue or green anvil is in vitro again
Raw body system, using blade as explant, evoking adventive bud, the growth coefficient of adventitious bud is 2.7.
(3) number is averagely taken root in the present invention up to 7.3, and existing regenerating system averagely takes root number up to 5.7.
(4) domestication survival rate of the invention is up to 90%, and transplanting survival rate is up to 60% after existing acclimatization technology hardening.
Existing blue or green anvil vitro Regeneration System also rests on the small-scale conceptual phase in laboratory, and the correlation technique of the present invention all have passed through
The checking of large-scale production, and indices are all higher than the index that prior art is reached.
Brief description of the drawings
Fig. 1 is differentiation seedling of the blue or green anvil No.1 stem section explant after differential medium culture 25d.
Fig. 2 is that blue or green anvil No.1 breaks up rooted seedling of the seedling after root media culture 20d.
Fig. 3 is after blue or green anvil No.1 rooted seedling tames 30d in greenhouse.
Embodiment
Technical scheme and its caused technique effect are carried out with reference to specific test method and accompanying drawing
Further elucidated above, the description below is merely to explain the present invention, but the present invention is not any limitation as in any way, based on this
Any conversion or replacement that invention training centre is made, belong to protection scope of the present invention.
Method used in the present invention is this area conventional method unless otherwise specified.It is used in following embodiments
Test material, reagent etc., unless otherwise specified, commercially obtain.
The green grass or young crops anvil No.1 tissue culturing fast seedling-cultivating methods of embodiment one
First, in vitro band bud branch, which carries out disinfection, obtains aseptic seedling.Way is：1st, cut rest period with tender shoots 1 year is adopted
Raw healthy and strong branch, soak 20min with bromogeramine, branch surface is scrubbed clean into (particularly near bud), Ran Houyong with hairbrush
Flowing water is rinsed well.2nd, 6-8cm stem section is tiltedly cut into water with fruit shear, it is ensured that every section above carries 2-3 bud, is placed on dry
In net tissue culture bottle.3rd, load 3/4ths pure water with the tissue culture bottle of 240ml specifications, covered tightly with the lid without ventilated membrane,
Sterilized water is made with 125 DEG C of sterilizing 30min of high-pressure sterilizing pot, cooling is standby.4th, the inoculator that will be placed in superclean bench
Sterilizer is opened, the instrument insertion inoculator sterilizer sterilizing such as operating scissors, scalpel, gun-shaped forceps, by 75% alcohol, sterilized water
It is put into superclean bench, the uviol lamp between inoculation with superclean bench is opened, closed sterilizing 30min Deng instrument.5th, dress
Good sterile clothes, wear before sterile emgloves is just seated at superclean bench, the stem section rinsed is put into superclean bench.6th, will
The blue or green anvil stem section rinsed is poured into 1 bottle of sterilized water, and 1min is rinsed in concussion, outwells sterilized water.7th, 75% alcohol is poured into four
At/tri- bottles, bottle cap is covered, concussion rinses 12s, outwells alcohol.8th, sterilized water to be poured into, covers bottle cap, 1min is rinsed in concussion,
Outwell sterilized water.9th, 0.1% mercuric chloride solution is poured into, instills few drops of Tween 80s, covers bottle cap, concussion is rinsed 8min, outwelled
Solution.10th, the blue or green anvil stem section after sterilization is poured into the tissue culture bottle equipped with sterilized water, 1min is rinsed in concussion, outwells sterilized water, weight
Multiple this operates 7 times.
Second, carry out inoculation operation.Comprise the concrete steps that：1st, the blue or green anvil stem section for bacterium of having gone out is placed in bottle, covers lid, it is standby
With.2nd, stainless steel inoculation disk alcolhol burner barbecue sterilizing is gripped with the gun-shaped forceps for being inserted in bacterium of having been gone out in inoculator sterilizer, each
Position will be roasted fully, to reach sterilization effect.3rd, 75% alcohol is saturated with the clean small handkerchief for being saturated with 75% alcohol, is wiped
Wipe the blake bottle equipped with blank MS culture mediums, the neat edge for being emitted on superclean bench.4th, the stem section for bacterium of having gone out is gripped, is put
Put in disk is inoculated with, wound is cut off with operating scissors, stem section is cut into two sections with 1-2 bud.5th, blake bottle is turned on, by bottle cap
Mouth is placed on inside superclean bench upward, and trying one's best, it is inner to place, and is placed on eminence.Bottleneck is roasted on alcolhol burner, when roasting
Rotating with Uniform bottle.6th, the right hand holds the stem section that gun-shaped forceps gripping shears, and left hand holds blake bottle, makes bottleneck inside, by stem section gently
Culture medium to be inserted, keeps stem section to stand in the medium, it is ensured that down, every bottle can be inoculated with 1-2 for the biology lower end of stem section,
Bottle cap is covered, the culture medium after inoculation is placed on one side.7 according to the method described above, and the stem section after all disinfect all is inoculated with
In blank MS culture mediums, title material, disinfecting time and date are indicated on tissue culture bottle.
3rd, the explant being inoculated with is positioned between culture, is cultivated, and condition of culture is as follows：Temperature should be between 1 culture
25℃±3℃.Humidity should be between 25%-50% between 2 cultures.3rd, intensity of illumination should be between 3000lx-6000lx, during illumination
Between between 10h-12h.4th, bottle seedling should discharge neatly, bottle spacing 3cm or so.
4th, the stem section browning being inoculated between culture and pollution condition should be observed daily.Carried out when in strict accordance with above-mentioned steps
During operation, contamination rate can be typically controlled below 20%.When there is browning, upgrade in time culture medium；When occur bacterium or
During person's fungal contamination, it should remove in time, and do sterilization treatment.According to above-mentioned condition, typically in 15-20d or so, the bud meeting of stem section
Sprout, treat that young shoot is gradually grown up, can continue to cultivate to carry out rolling bottle.
5th, differential period.Required inoculation condition is identical with explant sterilization, comprises the following steps that:1st, with bacterium of having gone out
Gun-shaped forceps gripping with young shoot stem section, the wound of upper and lower ends is cut off with operating scissors, is then inserted into differential medium,
Differential medium formula is as follows：MS+2.0mg·L-16-BA+0.2mg·L-1NAA+25g·L-1Sucrose+6gL-1Agar powder (fine jade
Cosmetics gel strength is 1300g/cm2) 2, by the seedling after inoculation be positioned over culture between cultivate, cycle about 20-25d or so.
3rd, seedling growth is observed daily, and pollution seedling is removed and sterilized in time.4th, after 20-25d or so, seedling gradually grows up,
It can be transferred in new culture medium, carry out squamous subculture.
6th, subculture expands numerous stage.
Subculture medium is：MS+1.0mg·L-16-BA+0.1mg·L-1NAA+25g·L-1Sucrose+6g·L-1Agar powder.
Now every seedling can differentiate 4-5 new talent, can carry out vast propagation production.Now, every seedling can at most be divided into
5-6 new talent.The tissue-cultured seedling obtained is as shown in Figure 1.
After blue or green anvil children seedling and propagating is to certain amount, the differentiation seedling of robust growth directly can be transferred in root media
Row culture of rootage, and the relatively weak seedling of growing way need to be turning initially to carrying out strong seedling culture in strong seedling culture base.Strong seedling culture base is such as
Under：MS+0.25mg·L-16-BA+0.05mg·L-1NAA+25g·L-1Sucrose+6gL-1Agar powder.Treat seedling in strong seedling culture
Grown 20 days or so in base, the weaker tissue-cultured seedling of growing way becomes strong, can carry out culture of rootage.
7th, culture of rootage.Step is as follows：1st, with gun-shaped forceps and operating scissors by differential medium and strong seedling culture base
Blue or green anvil seedling healthy and strong more than 4cm is divided into single, is connected in root media.Root media is as follows：1/2MS+0.5mg·L- 1IBA+1.5mg·L-1IAA+20g·L-1Sucrose+6gL-1(agar powder gel strength is 1300g/cm to agar powder2)+0.2g·
L-1Activated carbon (creates the dark surrounds favorably taken root).3rd, the seedling after inoculation is positioned between culture and cultivated, culture 15d is left
The right side, there can be root to bear.4th, observation seedling takes root situation daily, and the seedling rooting rate being typically inoculated in basal culture medium can reach
100%, average every seedling takes root bar number more than 7.Tamed and dociled when 80% seedling root long degree is more than 3cm it is necessary to enter greenhouse
Change is handled.Rooted seedling is as shown in Figure 2.
8th, the greenhouse domestication of tissue-cultured seedling：1st, the control of overall situation is in greenhouse：Temperature control daytime is on a 23-28 DEG C of left side
The right side, night are not less than 18 DEG C, and relative humidity is typically in 60%-80%.Bottle seedling of being taken root in greenhouse the 1-2 days, intensity control exists
4000lx or so, humidity nearly 100% in bottle, bottle seedling of taking root for the 3rd day pine lid, intensity control humidity in 4000-6000lx, bottle exist
90% or so, the 4-5 days increase light intensity are to 6000-8000lx, humidity 80%-90% in bottle, the 6th day bottle seedling uncapping of taking root, light
Humidity 80% or so in 8000lx or so, bottle are controlled by force, and the 7-9 days, intensity control humidity in 8000-12000lx, bottle existed
70%-80%, seedling can be washed within the 10th day.2nd, matrix prepares：Substrate composition is perlite：Vermiculite：Turf=1：1：3, more than 2 ‰
The 10-15d that is exposed to the sun under the sun is put in bacterium spirit sterilization.The matrix disinfected is fitted into 50 hole disks, poured before planting stock it is permeable after, place
It is stand-by to reach semi-wet to matrix.3rd, seedling is washed：It is careful not to hurt root system when washing seedling, 1000 times of carbendazim of washed seedling
5min is sterilized, is placed on standby in water.4th, it is colonized：Nest root is careful not to during field planting and is colonized too deep in order to avoid burying the heart.Side is colonized back
Water spray, will irrigate root water in time, then cover very thin plastic sheeting and seal after field planting, upper covering shading screen.5th, it is colonized
Management afterwards：The temperature control of overall situation is at 23-28 DEG C in greenhouse, and relative humidity is in 60%-70%.Spray within the 1st day after field planting
0.1% composite fertilizer, sprayed with 0.5 ‰ carbendazim within the 2nd day, relative humidity is not less than 90%, intensity control exists
4000lx or so；The 3-5 days, intensity control was in 4000-6000lx, and relative humidity is between 85%-90%；6th day, open small
One jiao of shed film, increase light intensity to 6000-8000lx, relative humidity are controlled 80% or so；The 7-9 days, intensity control existed
8000lx or so, relative humidity 75%-80%, the 9th day, open Small plastic shed film 1/3, humid control is 75% or so, light intensity
Bring up to 8000-10000lx；The 10-11 days, relative humidity was controlled in 70%-75%, and intensity control is in 10000lx or so；The
12 days, the 2/3 of Small plastic shed film is opened, relative humidity is big close in greenhouse in 70% or so, light intensity 10000-12000lx
Environment；The 13-14 days, buffer two day time, the 15th day can fully open canopy it is thin, adapt to greenhouse overall situation, soil humidity is not
It is excessive, control 70% or so, in order to avoid occur macerating root.6th, rich water quality management and the prevention and control of plant diseases, pest control：Must when using fertilizer
Suitable concentration is grasped, prevents the fertilizer of high concentration from causing fertilizer damage to nursery stock.The preventing and treating of pest and disease damage, relies mainly on prevention in domestication.30d
After tame successful hole tray shoot survival percent as 90%, as shown in Figure 3.
Embodiment two, the inventive method and conventional method contrast
1. test material and method
1.1 material
Blue or green anvil No.1
1.2 test method
Cellar culture and cultural method of the present invention is respectively adopted；The inventive method, i.e., using the method for embodiment one.It is conventional
Method is as follows：
Explant chooses blade, and after being cleaned with liquid detergent, 30min or so is rinsed under flowing water；Then in superclean bench
It is upper respectively with 75% alcohol and 1gL-1Mercuric chloride solution sterilization 30s and 8min, then with aseptic water washing 3-5 times.Blade is put
On aseptic filter paper, leaf margin and blade tip are cut off with scalpel, is cut into leaf bulk.
Each stage used medium and incubation time are as follows,
Minimal medium is MS.
Differential medium：MS+5.0mg·L-16-BA+0.3mg·L-1NAA+25g·L-1Sucrose+6gL-1Agar powder (fine jade
Cosmetics gel strength is 1300g/cm2)。
Proliferated culture medium：MS+1.0mg·L-16-BA+0.1mg·L-1IAA+25g·L-1Sucrose+6gL-1Agar powder (fine jade
Cosmetics gel strength is 1300g/cm2)。
Root media：1/2MS+0.5mg·L-1IBA+1.5mg·L-1IAA+20g·L-1Sucrose+6gL-1Agar powder
(agar powder gel strength is 1300g/cm2)+0.2g·L-1Activated carbon.
The pH value 5.6~5.8 of each culture medium used.The conditions such as each stage cultivation temperature, humidity and illumination and side of the present invention
Method is identical.
2. result
Using each phase contrast's situation of two kinds of compound formulation cultures, 1 is shown in Table
1 tissue culture method of the present invention of table contrasts with conventional tissue culture method tissue culture situation
| The inventive method | Conventional method | |
| Explant | Stem section | Blade |
| Differential period | Coefficient of differentiation 4 or so, cycle 20-25d, | Coefficient of differentiation 2.5 or so, cycle 25-30d |
| Expand numerous stage | Coefficient of differentiation 5 or so, cycle 20-25d | Coefficient of differentiation 3 or so, cycle 25-30d |
| Take root the stage | Take root bar number 7.3,15-20d | Take root bar number 5.7,20-30d |
| Transplant domestication rate | 90% or so | 60% or so |
Claims (7)
1. a kind of apple rootstock tissue culturing fast seedling-cultivating method, including indoor tissue cultures stage and greenhouse domestication stage, its
It is characterized in, the indoor tissue cultures stage, using stem section as explant, after disinfecting, carries out inoculated and cultured；Each stage
Culture medium prescription is as follows：
Differential medium：MS+2.0mg·L-16-BA+0.2mg·L-1NAA+25g·L-1Sucrose+6gL-1Agar powder；
Subculture medium：MS+1.0mg·L-16-BA+0.1mg·L-1NAA+25g·L-1Sucrose+6gL-1Agar powder；
Root media：1/2MS+0.5mg·L-1IBA+1.5mg·L-1IAA+20g·L-1Sucrose+6gL-1Agar powder+
0.2g·L-1Activated carbon；
The pH value of each culture medium is 5.6~5.8.
2. apple rootstock tissue culturing fast seedling-cultivating method as claimed in claim 1, it is characterized in that, the indoor tissue cultures
In the stage, it is as follows to specifically include step：
(1) selection, sterilization and the inoculated and cultured of explant
Adopt and cut annual healthy and strong branch of the rest period with tender shoots, soak 20min with bromogeramine, clean；Tiltedly it is cut into 6-8cm's
Disinfected after stem section：1min is rinsed in sterilized water concussion；75% alcohol vibration rinses 12s；Sterilized water concussion is rinsed
1min；0.1% mercuric chloride solution, instills few drops of Tween 80s, and 8min is rinsed in concussion；1min is rinsed in sterilized water concussion；Repeat nothing
Bacterium water rinses 7 times；
Then the stem section after sterilization is cut off into wound with operating scissors, stem section is cut into two sections with 1-2 bud, is inoculated in blank
In MS culture mediums；
The explant being inoculated with is positioned between culture, is cultivated；Condition of culture is as follows：25 DEG C ± 3 DEG C of temperature, humidity 25%-
50%, intensity of illumination 3000lx-6000lx, light application time 10h-12h；15-20d rolling bottles continue to cultivate；
(2) differentiation culture
The stem section that young shoot is carried after cultivating is taken, the wound of upper and lower ends is cut off with operating scissors, is then inserted into differential medium,
Cultivate 20-25d；
(3) subculture expands numerous culture
After differentiation culture terminates, seedling, which is transferred in subculture medium, carries out the numerous culture 20-25d of subculture expansion；
(4) culture of rootage
The healthy and strong seedling of more than 4cm is divided into single, is connected in root media；Condition of culture is as follows：25 DEG C ± 3 DEG C of temperature,
Humidity 25%-50%, intensity of illumination 2500-4000lx, light application time 10-12h；Cultivate 15d-20d.
3. apple rootstock tissue culturing fast seedling-cultivating method as claimed in claim 2, it is characterized in that,
After step (3) subculture expands numerous culture, the relatively weak seedling of growing way is gone in strong seedling culture base, carries out strong sprout training
Support；
Strong seedling culture base is as follows：MS+0.25mg·L-16-BA+0.05mg·L-1NAA+25g·L-1Sucrose+6gL-1Agar powder；
Culture 20 days, treat that the weaker tissue-cultured seedling of growing way becomes strong, culture of rootage can be carried out.
4. apple rootstock tissue culturing fast seedling-cultivating method as claimed in claim 1 or 2, it is characterized in that, the stem section is the
One stem segment.
5. apple rootstock tissue culturing fast seedling-cultivating method as claimed in claim 1, it is characterized in that, rank is tamed in the greenhouse
Section, is comprised the following steps that,
(1) greenhouse experiment controls
Temperature control, 23-28 DEG C of daytime, night are not less than 18 DEG C, relative humidity 60%-80%；The 1-2 days, intensity control existed
4000lx, humidity nearly 100% in bottle；Bottle seedling of taking root for 3rd day pine lid, intensity control humidity 90% in 4000-6000lx, bottle；
The 4-5 days increase light intensity are to 6000-8000lx, humidity 80%-90% in bottle；6th day bottle seedling uncapping of taking root, intensity control exist
8000lx, humidity 80% in bottle；The 7-9 days, intensity control in 8000-12000lx, bottle humidity in 70%-80%；
(2) it is colonized
Seedling is washed after 10th day, is colonized；Management after field planting：At 23-28 DEG C, relative humidity exists the temperature control of overall situation in greenhouse
60%-70%；The 1st day composite fertilizer for spraying 0.1% after field planting, sprayed within the 2nd day, made relatively wet with 0.5 ‰ carbendazim
Degree is not less than 90%, and intensity control is in 4000lx；The 3-5 days, intensity control was in 4000-6000lx, and relative humidity is in 85%-
Between 90%；6th day, one jiao of Small plastic shed film, increase light intensity to 6000-8000lx are opened, relative humidity is controlled 80%；The
7-9 days, intensity control was in 8000lx, relative humidity 75%-80%；9th day, Small plastic shed film 1/3 is opened, humid control exists
75%, light intensity brings up to 8000-10000lx；The 10-11 days, relative humidity control existed in 70%-75%, intensity control
10000lx；12nd day, the 2/3 of Small plastic shed film is opened, relative humidity is in 70%, light intensity 10000-12000lx, close to temperature
Indoor overall situation；The 13-14 days, buffer two day time, the 15th day can fully open canopy it is thin, adapt to greenhouse overall situation, matrix
Humid control is 70%.
6. apple rootstock tissue culturing fast seedling-cultivating method as claimed in claim 5, it is characterized in that, the substrate composition is,
Perlite：Vermiculite：Turf=1：1：3, put the 10-15d that is exposed to the sun under the sun after being sterilized with 2 ‰ carbendazim.
7. apple rootstock tissue culturing fast seedling-cultivating method as claimed in claim 5, it is characterized in that, the stage is tamed in the greenhouse
It can be carried out when being more than 3cm from 80% seedling root long degree.
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