CN110122331A - It is a kind of using leaf as the quick-breeding method of the U.S. Liriodendron chinense in Asia of explant - Google Patents
It is a kind of using leaf as the quick-breeding method of the U.S. Liriodendron chinense in Asia of explant Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
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- A—HUMAN NECESSITIES
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- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
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Abstract
The invention discloses a kind of using leaf as the quick-breeding method of the U.S. Liriodendron chinense in Asia of explant, the pretreatment of the leaf including the sub- U.S. Liriodendron chinense of tri- kinds of genotype of XY1, XY2 and T8 and disinfection, calli induction media, differential medium, strong seedling culture base, root media and acclimatization and transplants and etc..Method of the invention has that reproduction speed is fast, growth coefficient is big suitable for the fast numerous of sub- U.S. Liriodendron chinense, the high advantage of transplanting survival rate.It has been confirmed by experiments that under optimal culture condition, the inductivity average value of the highest callus induction of XY1 genotype is 93.33%, and the growth coefficient average value of maximum callus differentiation is 7.10；The average single-strain blade number average value of most strong seedling cultures of XY2 genotype is up to 9.4；The rooting rate average value of the highest culture of rootage of T8 genotype is up to 56.00%, and final acclimatization and transplants survival rate is up to 89%.
Description
Technical field
The present invention relates to a kind of woody plant tissure culture propagation methods, more specifically to one kind using leaf as explant
The quick-breeding method of the U.S. Liriodendron chinense in the Asia of body, belongs to woody plant tissure culture seedling-raising technique field.
Background technique
Sub- U.S. Liriodendron chinense [Liriodendron chinense (Hemsl.) Sarg. × Liriodendron
Tulipifera L.] it is 1963 Nanjing Forestry University's Ye Peizhong professor's first passage artificial hybridization yellow poplars
The interspecific hybrid that (L.tulipifera Linn.) and Liriodendron (L.chinense sarg.) obtain, growth is fast, and form is logical
Directly, pattern is gorgeous, and rare pest and disease damage, wide adaptability, hybrid vigour is obvious, and ornamental value is high, is suitable for use as garden landscape and row
Road tree species.Since sub- U.S. Liriodendron chinense self-reproduction ability is poor, Natural seed setting rate is only 1% or so (Fan Ruwen, 1992), in addition
For artificialpollination by weather conditions, florescence nonsynchronous influence, cuttage, propagation by grafiting are relatively difficult, constrain Ya Meima
The breeding popularization of gown wood and seed production efficiency.
Plant Tissue Breeding is the plant device of culture of isolated on the culture medium for being suitble to plant growth by sterile working
Official, tissue, cell, embryo and protoplast and illumination appropriate and temperature have developed into their callus, adventitious bud etc.
The process of whole plant.Mature plant cell under certain condition can dedifferentiation and differentiation, form complete plant, be the complete of cell
Energy property is the basis for realizing tissue cultures.Therefore the fast breeding technique means based on tissue culture technique can be in certain journey
It is low with seed production efficiency effectively to solve the problems, such as that the breeding of sub- U.S. Liriodendron chinense is promoted on degree.
Although Plant Tissue Breeding regeneration individual succeeds on several hundred kinds of plants, the plant not belonged to, even
There is also biggish differences between kindred plant different genotype.In general, angiosperm is easier to train compared with gymnosperm
It supports, most of successful cultivations for reporting Solanaceae, begonia, red-spotted stonecrop, Gesneriaceae and brassicaceae in angiosperm.Same plant
There are larger differences in culture effect for the different genotype of object.Even if thinking the plant most easily cultivated, some genotype are still
It is difficult to cultivate successfully, such as " the bright formula tobacco " in tobacco is a typical example.The mechanism for generating this phenomenon is current
It is still unclear, thus it is speculated that the possible reason is reacting related mechanisms of gene regulation with culture there are certain in plant.It is different
The sensibility that genotype starts these regulatory mechanisms is different, to cause the difference to culture reaction.
The tissue culture technology about sub- U.S. Liriodendron is rarely reported at present.The application is with the sub- U.S. Liriodendron chinense of 3 superior genotypes
Outstanding single plant is material, from each link of tissue culture regeneration and Study on influencing factors sampling time, disinfecting time, explant
Selection is influenced with processing, hormone combination and acclimatization and transplants, with indirect adventitious organogenesis (blade) establish sub- U.S. Liriodendron chinense from
Body regenerating system, it is intended to provide excellent tissue culture technology and side for the quick breeding and popularization of sub- U.S. Liriodendron chinense superior genotypes
Case.
Summary of the invention
Goal of the invention: in view of the above-mentioned problems existing in the prior art, the purpose of the present invention is to provide a kind of sub- U.S. mandarin jackets
The quick-breeding method of wood, solves that sub- U.S. Liriodendron chinense self-reproduction is difficult, cuttage, propagation by grafiting also difficult problem.
It is a kind of using leaf as the quick-breeding method of the U.S. Liriodendron chinense in Asia of explant, comprising the following steps:
(1) it acquires blade or petiole is explant, pretreatment and disinfection；
(2) explant of the acquisition after step (1) being pre-processed and sterilized, which is inoculated into calli induction media, to be cultivated,
In, the calli induction media is added with 1.0~3.0mg/L 2,4-D, 0.1~0.5mg/L based on MS culture medium
6-BA and 0.1~0.5mg/L NAA；
(3) the callus structure that callus Fiber differentiation obtains is transferred in differential medium and is cultivated, wherein the differentiation culture
Base is added with 1.0~3.0mg/L 6-BA, 0.1~0.5mg/L IBA and 0.3~1.0mg/L KT based on MS culture medium；
(4) Multiple Buds that differentiation culture obtains are transferred in strong seedling culture base and are cultivated, wherein the strong seedling culture is trained with MS
Based on supporting base, it is added with 0.3~1.0mg/L 6-BA, 0.1~0.5mg/L IBA and 0.1~0.3mg/LKT；
(5) adventitious bud for obtaining strong seedling culture, which is transferred in root media, cultivates, wherein the root media is with 1/
Based on 2MS culture medium, it is added with 0.1~0.3mg/L IBA and 0.1~0.3mg/L NAA；
(6) tissue-cultured seedling for obtaining culture of rootage cleans root culture medium, and transplanting hardening into the matrix of hardening obtains small
Seedling.
Preferably, in step (2) calli induction media based on MS culture medium, be added with 2.0mg/L2,4-D,
0.5mg/L 6-BA, 0.3mg/L NAA and 500mg/L CH.
Preferably, differential medium is added with 2.0mg/L6-BA, 0.3mg/L based on MS culture medium in step (3)
IBA, 0.3mg/L KT and 500mg/L CH.
Preferably, strong seedling culture base is added with 0.5mg/L6-BA, 0.3mg/L based on MS culture medium in step (4)
IBA, 0.1mg/L KT and 5.0mg/L VC.
Preferably, in step (5) root media based on 1/2MS culture medium, added with 0.2mg/L IBA,
0.1mg/L NAA and 5.0mg/L VC.
Preferably, the U.S. Liriodendron chinense in the Asia includes tri- genotype of XY1, XY2 and T8.
Preferably, processing and the method sterilized in step (1) are as follows: 4 DEG C of refrigerator overnights will be placed with petiolate blade, and used
Dish washing liquid impregnates 3~5min, and banister brush is gently brushed along a direction, and the beaker that rim of a cup envelope has gauze is put into after rinsing well
In, 30min is rinsed in tap water down-flow water.The flushed explant of flowing water is put into the conical flask for bacterium of having gone out in advance, high pressure
The sterile shaking water of the cooling of sterilizing cleans 2 times, then impregnates 30s with the alcohol of 75% concentration, has impregnated with sterile water wash 1
It is secondary, then use 0.1%HgCl2Disinfection, disinfecting time are set as 3~15min；Mercuric chloride keeps conical flask concussion to shake during sterilizing
It is dynamic, make mercuric chloride sufficiently and material；Last sterile water wash 5-6 times, each 5min dispels remaining mercuric chloride as far as possible
Completely；The explant being disposed aseptic filter paper suck dry moisture.
Preferably, acclimatization and transplants matrix is the plant ash, perlite, vermiculite of sterilizing completion according to 7: 2: 1 in step (6)
Ratio mixing is added nutrient solution and is mixed evenly.
Preferably, using leaf as the quick-breeding method of the U.S. Liriodendron chinense in the Asia of explant, comprising the following steps:
(1) blade of the sub- U.S. Liriodendron chinense of acquisition XY1 genotype is as explant, pretreatment and disinfection；
(2) explant of the acquisition after step (1) being pre-processed and sterilized, which is inoculated into calli induction media, to be cultivated simultaneously
Dark treatment 15d, wherein the calli induction media is added with 2.0mg/L2,4-D, 0.5mg/L based on MS culture medium
6-BA, 0.3mg/L NAA and 500mg/L CH；
(3) the callus structure that callus Fiber differentiation obtains is transferred in differential medium and is cultivated, wherein the differentiation culture
Base is added with 2.0mg/L 6-BA, 0.3mg/L IBA, 0.3mg/L KT and 500mg/L CH based on MS culture medium；
(4) Multiple Buds that differentiation culture obtains are transferred in strong seedling culture base and are cultivated, wherein the strong seedling culture is trained with MS
Based on supporting base, it is added with 0.5mg/L 6-BA, 0.3mg/L IBA, 0.1mg/L KT and 5.0mg/LVC；
(5) adventitious bud for obtaining strong seedling culture, which is transferred in root media, cultivates, wherein the root media is with 1/
Based on 2MS culture medium, it is added with 0.2mg/L IBA, 0.1mg/L NAA and 5.0mg/L VC；
(6) tissue-cultured seedling for obtaining culture of rootage cleans root culture medium, and transplanting hardening 5d into the matrix of hardening is obtained
Seedling.Step (2)~(6) condition of culture are as follows: cultivation temperature is 25 ± 2 DEG C, lighting delay number 14h/d, intensity of illumination 2000lx,
Indoor relative humidity control is 75%.
Preferably, using leaf as the quick-breeding method of the U.S. Liriodendron chinense in the Asia of explant, comprising the following steps:
(1) blade of the sub- U.S. Liriodendron chinense of acquisition T8 genotype is as explant, pretreatment and disinfection；
(2) explant of the acquisition after step (1) being pre-processed and sterilized, which is inoculated into calli induction media, to be cultivated simultaneously
Dark treatment 15d, wherein the calli induction media is added with 2.0mg/L2,4-D, 0.5mg/L based on MS culture medium
6-BA, 0.3mg/L NAA and 500mg/L CH；
(3) the callus structure that callus Fiber differentiation obtains is transferred in differential medium and is cultivated, wherein the differentiation culture
Base is added with 2.0mg/L 6-BA, 0.3mg/L IBA, 0.3mg/L KT and 500mg/L CH based on MS culture medium；
(4) Multiple Buds that differentiation culture obtains are transferred in strong seedling culture base and are cultivated, wherein the strong seedling culture is trained with MS
Based on supporting base, it is added with 0.5mg/L 6-BA, 0.3mg/L IBA, 0.1mg/L KT and 5.0mg/L VC；
(5) adventitious bud for obtaining strong seedling culture, which is transferred in root media, cultivates, wherein the root media is with 1/
Based on 2MS culture medium, it is added with 0.2mg/L IBA, 0.1mg/L NAA and 5.0mg/L VC；
(6) tissue-cultured seedling for obtaining culture of rootage cleans root culture medium, and transplanting hardening 5d into the matrix of hardening is obtained
Seedling.Step (2)~(6) condition of culture are as follows: cultivation temperature is 25 ± 2 DEG C, lighting delay number 14h/d, intensity of illumination 2000lx,
Indoor relative humidity control is 75%.
The utility model has the advantages that compared with the prior art, the quick-breeding method of sub- U.S. Liriodendron chinense provided by the invention is suitable for Ya Meima
Gown wood it is fast numerous, have that reproduction speed is fast, growth coefficient is big, the high advantage of transplanting survival rate.It is most preferably cultivating it has been confirmed by experiments that
Under the conditions of, the inductivity average value of highest callus induction is 93.33%, is XY1 genotype；The growth coefficient of maximum callus differentiation
Average value is 7.10, is XY1 genotype；The average single-strain blade number average value of most strong seedling cultures is up to 9.4, is XY2 gene
Type；The rooting rate average value of highest culture of rootage is T8 genotype up to 56.00%；Final acclimatization and transplants survival rate is up to 89%.
Detailed description of the invention
Fig. 1 is the callus induced map of blade, petiole: A-D: the process of petiole formation callus；E-H: leaf dish forms callus
Process；
Fig. 2 is the state diagram of different dark treatment callus induction: A: no dark culture processing；B: dark treatment 5d；C: dark treatment
10d；D: dark treatment 15d；E: dark culture always；
Fig. 3 is three kinds of genotype blade callus induction figures: A-C: leaf dish forms callus process；The callus that D:XY1 is induced；
The callus that E:XY2 is induced；The callus that F:T8 is induced；
Fig. 4 is three kinds of genotype callus differentiation figures: A-C: callus breaks up adventitious bud process；D:XY1 callus adventitious bud；E:
XY2 callus adventitious bud；F:T8 callus adventitious bud；
Fig. 5 is three kinds of genotype callus adventitious bud strong seedling culture figures；
Fig. 6 is three genotype callus adventitious bud rooting culture figures: A:XY1 callus adventitious bud rooting；B:XY2 callus is indefinite
Bud is taken root；C:T8 callus adventitious bud rooting；
Fig. 7 is three genotype acclimatization and transplants figures；
Fig. 8 is influence diagram of the different hardening number of days to transplanting survival rate.
Specific embodiment
The present invention is further described below combined with specific embodiments below.Material as used in the following examples, reagent
Deng being commercially available unless otherwise specified.
Following embodiment material source is in plantation in the leaf of Nanjing Forestry University campus Nei Yamei Liriodendron chinense single plant XY1, XY2
Piece, petiole, and pick up from the practice of another name for Sichuan Province under Nanjing Forestry University forest farm Liriodendron hybrid progeny test forost fine individual plant T8 blade,
Petiole；And blade, the petiole of aseptic seedling XY1, XY2, the T8 cultivated from direct organ.
Condition of culture: be broadly divided into after no light culture, 5d dark culture illumination cultivation after illumination cultivation, 10d dark culture,
Illumination cultivation and full exposure culture after 15d dark culture, cultivation temperature are 25 ± 2 DEG C, and later period lighting delay number is 14h/d, illumination
Intensity about 1500lx, indoor relative humidity control are 75%.
Callus abductive approach: the application adds the 2 of various concentration using MS as minimal medium, 4-D, NAA, 6-BA tri-
The CH, sucrose 30g/L, agar 8.0g/L of kind hormone and 500mg/L, Medium's PH Value are adjusted to 5.72 and are used as callus Fiber differentiation
Base.In addition, different processing is added to the VC of 5.0g/L.The high-pressure sterilizing pot that all culture mediums pass through 121 DEG C of 20min goes out
It is used after bacterium solidification.Blade, petiole are cut into the leaf dish of 1cm × 1cm and the dissection of 1cm respectively in super-clean bench.3 factor 3 is horizontal
Orthogonal test L9 (33), each genotype is inoculated with 20 wares under each processing, is repeated 3 times.
Data statistical analysis method:
After explant cultivates 7d on culture medium, pollution rate, the death rate of blade difference disinfecting time are counted；Cultivate 30d
Afterwards, melting brown rate, Callus induction rate and the growth shape for recording callus under statistics blade, petiole are handled under different lighting processes
Condition；After cultivating 70d, callus differentiation and proliferation coefficient is counted；After cultivating 90d, statistics hormon handles lower single plant average leaf number
And record upgrowth situation culture；After cultivating 120d, the rooting rate of aseptic seedling is counted and number of taking root；After transplanting, statistics is different
The survival rate of quantity of taking root transplanting.Calculation method is as shown in the table:
The data obtained uses SPSS (Statistical Product and Service Solution) in embodiment
20.0 versions are for statistical analysis, using generalized linear model (GLM, General Linear Model), single argument variance point
It analyses (Univariate).The analysis of variance model of hormon processing are as follows: Yij=u+Ti+eii；
In formula: YijThe character value of j-th of sample is handled for i-th, u indicates population mean, TiFor i-th of processing effect
It answers, eijFor random error；
The analysis of variance model of different genotype are as follows: Yij=u+Gi+eij；In formula: YijFor i-th of genotype, j-th of sample
Character value, u indicate population mean, GiFor i-th of genotype effects, eijFor random error.If there is significant difference,
The Multiple range test for carrying out average is then examined using Duncan.
Embodiment 1: disinfecting time, explant, the selection of dark treatment time
Explant acquisition: selection fine day, takes the tender leaf of green sprouting at 2 points or so in the afternoon, cuts from branch together with petiole
Under, it marks, is put into after ice chest takes back laboratory, 4 DEG C of refrigerators are stood overnight.
(1) selection of disinfecting time
The pretreatment of explant and sterilization method: will stand overnight with petiolate blade, 3 are impregnated with dish washing liquid~
5min, banister brush are gently brushed along a direction, and rim of a cup envelope is put into after rinsing well to be had in the beaker of gauze, are flowed down in tap water
Water rinses 30min.The flushed explant of flowing water is put into the conical flask for bacterium of having gone out in advance, autoclaved cooling it is sterile
Shaking water cleans 2 times, then impregnates 30s with the alcohol of 75% concentration, has impregnated with sterile water wash 1 time, then with 0.1%
HgCl2Disinfection, disinfecting time are set as 3min, 5min, 10min, 15min；Mercuric chloride keeps conical flask oscillating and shaking during sterilizing,
Make mercuric chloride sufficiently and material；Last sterile water wash 5-6 times, each 5min, remaining mercuric chloride is dispelled as far as possible dry
Only；The explant being disposed aseptic filter paper suck dry moisture carries out subsequent experimental.
Can be seen that from table 1-1 for genotype XY1 pollution rate is successively 5min, 15min < 10min < from low to high
3min, minimum pollution rate are 10.0%；Melting brown rate is successively 5min < 3min < 10min < 15min from low to high, minimum brown
Rate is 3.7%；Death rate rate is successively 5min < 3min < 10min < 15min from low to high；Genotype XY2 is polluted
Rate is successively 15min < 5min, 10min < 3min from low to high, and minimum pollution rate is 6.7%；Melting brown rate from low to high according to
Secondary is 3min < 5min < 10min < 15min, and minimum melting brown rate is 8.7%；Death rate rate is successively 5min < from low to high
3min < 10min < 15min；It is successively 15min < 5min < 3min, 10min from low to high for genotype T8 pollution rate, most
Low pollution rate is 10.0%；Melting brown rate is successively 5min < 3min < 15min < 10min from low to high, and minimum melting brown rate is
4.2%；Death rate rate is successively 3min < 5min < 10min < 15min from low to high；
Table 1-1 0.1%HgCl2Influence of the different disinfecting times for three genotype
XY1 pollution rate is with the trend that disinfecting time is in relative drop, and melting brown rate and the death rate are in opposite ascendant trend,
But in this node of 5min, pollution rate, melting brown rate, the death rate are all minimum；XY2 pollution rate is in that decline becomes with disinfecting time
Gesture, melting brown rate and the death rate are in rising trend, and 3min and 5min are the relatively low and poor between the two of melting brown rate and the death rate
Away from not being very big；T8 pollution rate fall before after rise again downward trend, be in 5min and 15min it is relatively low, between the two
Gap is also unobvious, and melting brown rate is that rising is minimum obvious with 10min highest gap in downward trend, 5min after falling before,
The HgCl of blade 0.1%2The properly pasteurized time is 5min.
(2) selection of explant
In super-clean bench, it is 1cm that blade, which is cut into area,2The leaf dish of size, the dissection that petiole is cut into 1cm length connect respectively
Kind is in calli induction media.7d or so is cultivated, blade edge starts to curl up；10d or so is cultivated, petiole both ends start to expand；
Illustrate that dedifferentiation enters induction period to cell；Cultivate 20d or so, blade edge incision have begun to be formed it is a small amount of transparent
Callus；25d or so is cultivated, transparent quality is integrally presented in petiole fragments, wraps up one layer of callus above；35d or so is cultivated, leaf dish is whole
Body has formed callus structural volume and has also significantly increased；42d or so is cultivated, petiole is whole also to form callus structure, is wrapped in whole
A dissection (Fig. 1).
It can be seen that for pollution rate from table 1-2, either blade or petiole, the pollution rate of aseptic seedling is obvious
Blade, petiole lower than disinfection；For blade, petiole, either sterile or disinfection, the pollution rate of blade is low
In the pollution rate of petiole.Melting brown rate is also the blade of germ-free condition, petiole lower than disinfection, but the blade of germ-free condition, petiole
Without browning, and the melting brown rate of the petiole sterilized is significantly lower than blade.It is high that inductivity remains the blade of germ-free condition, petiole
In disinfection, sterile blade is up to 90.0%；The explant of two states, the inductivity of blade are above petiole, but sterile
The inductivity of petiole is higher than the blade of disinfection, but gap is not very big.
The influence that the different explants of table 1-2 different conditions induce callus
In conclusion the sequence of the explant for selecting proper callus induction is: aseptic blade > sterile petiole
> disinfection blade > disinfection petiole.
(3) influence that different dark treatment cultures induce callus
It is found by comparative experiments, is integrally in dark brown by the callus that the explant of 0d dark treatment induces, redgreen,
Browning is serious, and quality is hard, and the volume of callus is also smaller, and the later period loses vigor without continued growth substantially；By the dark place 5d
Managing the callus induced is integrally in light green, and surface is relatively more wet, and quality is partially soft, and volume growth is more apparent, and late growth is very fast,
It but is in water stain shape；The callus surface visible particle sense induced by 10d dark treatment, quality is neither too hard, nor too soft, whole that green is presented,
Volume increases obviously, has the potentiality for developing into embryo callus subculture；The callus surface particles sense induced by 15d dark treatment is very
By force, quality densification is solid, and volume increases obvious, the characteristic with typical embryo callus subculture；It is chronically under dark surrounds and induces
Callus out, whole in filbert, there is the callus of milky and transparent quality on surface, and quality is softer, callus volume increase also compared with
Obviously, green callus (Fig. 2, table 1-3) can also be gradually formed by being gone under light environment.
The influence that table 1-3 dark treatment culture induces callus
Note :+: it is small in size, ++: small volume, +++: volume is medium, ++++: volume is big
I: browning degree is low, II: browning degree is heavier, III: browning degree weight
It can be seen that the dark treatment time reasonable for explant induced synthesis embryo callus subculture is 15d.
Embodiment 2: the callus induction of sub- U.S. Liriodendron chinense
It is 1cm that blade, which is cut into area,2The leaf dish of size is inoculated in calli induction media, culture 7d or so, blade edge
Edge starts to curl up；20d or so is cultivated, blade edge incision has begun to form a small amount of transparent callus；Cultivate 35d or so, leaf
Consolidation body has formed callus structural volume and has also significantly increased；Hereafter callus is constantly proliferated, and volume also constantly increases, and cultivates 50d
Left and right, callus structure are formed completely, and the form (Fig. 3) that there is granular sensation on the fine and close solid and surface of green is presented.
(1) influence that hormone combination induces XY1 callus
It can be seen that in 9 processing from table 2-1, processing 6 reaches highest for the inductivity of XY1 callus induction, puts down
Mean value reaches 93.33%, wherein reaching as high as 96%；Followed by processing 5, average value is up to 78.67%, highest 80%.To gene
Type XY1 callus induces result to carry out variance analysis, and there are significant difference (table 2-2) for 9 processing.
The influence that table 2-1 hormone combination induces XY1 callus
Table 2-2 genotype XY1 callus induces variance analysis
By further Duncan multiple alignment, from table 2-3 can be seen that processing 5, processing 6 is deposited with other processing
In conspicuousness and extremely significant sex differernce, and between processing 5 and processing 6, there is also conspicuousnesses and extremely significant sex differernce；Handle 5 callus
The average value of induction is 78.67%, and the average value for handling 6 is 93.33%, and processing 6 is significantly higher than processing 5, illustrates that processing 5 is better than
Other processing.
The Duncan multiple alignment of table 2-3 9 processing
| Processing | 5% conspicuousness | 1% conspicuousness |
| 1 | e | C |
| 2 | de | C |
| 3 | bc | BC |
| 4 | bc | BC |
| 5 | b | B |
| 6 | a | A |
| 7 | c | BC |
| 8 | d | C |
| 9 | bc | BC |
To sum up, the optimization process of XY1 callus induction is processing 6, i.e. 2,4-D concentration is 2.0mg/L, and 6-BA concentration is
0.5mg/L, NAA concentration are 0.3mg/L, to show that genotype XY1 callus induction optimal medium is MS+2.0mg/L2,4-D
+0.5mg/L 6-BA+0.3mg/L NAA+500mg/L CH。
(2) influence of hormone combination XY2 callus induction
It can be seen that in 9 processing from table 2-4, processing 6 reaches highest for the inductivity of XY2 callus induction, puts down
Mean value reaches 86.33%, wherein reaching as high as 91%；Followed by processing 5 and processing 9, has reached 82.33%.To genotype
XY2 callus induces result to carry out variance analysis, and there are significant difference (table 2-5) for 9 processing.
The influence that table 2-4 hormone combination induces XY2 callus
Table 2-5 genotype XY2 callus induces variance analysis
By further Duncan multiple alignment, it can be seen that processing 6 from table 2-6 and reached aobvious with other 8 processing
It is horizontal to write sex differernce, processing 5, processing 9 and processing 1, there are significant differences for processing 2；Processing 6 extremely aobvious with 1 presence of processing
Sex differernce is write, handles electrodeless significant difference with other.The callus induction average value of processing 6 is 86.33%, is higher than other 8
Processing illustrates processing 6 better than other processing.
The Duncan multiple alignment of table 2-6 9 processing
| Processing | 5% conspicuousness | 1% conspicuousness |
| 1 | c | B |
| 2 | b | A |
| 3 | ab | A |
| 4 | ab | A |
| 5 | ab | A |
| 6 | a | A |
| 7 | ab | A |
| 8 | ab | A |
| 9 | ab | A |
To sum up, the optimization process of XY2 callus induction is processing 6, i.e. 2,4-D concentration is 2.0mg/L, and 6-BA concentration is
0.5mg/L, NAA concentration are 0.3mg/L, to show that genotype XY2 callus induction optimal medium is MS+2.0mg/L2,4-D
+0.5mg/L6-BA+0.3mg/L NAA+500mg/L CH。
(3) influence that hormone combination induces T8 callus
It can be seen that in 9 processing from table 2-7, processing 6 reaches highest for the inductivity of T8 callus induction, puts down
Mean value reaches 87.67%, wherein reaching as high as 90%；Followed by processing 7, mean value is up to 87%, up to 92%.To genotype T8
Callus induces result to carry out variance analysis, and there are significant difference (table 2-8) for 9 processing.
The influence that table 2-7 hormone combination induces T8 callus
Table 2-8 genotype T8 callus induces variance analysis
By further Duncan multiple alignment, it can be seen that processing 6 from table 2-9 and reached aobvious with other 8 processing
It is horizontal to write sex differernce, only there are extremely significant sex differernces with processing 1, and extremely significant sex differernce is not present with other；Handle 7 and processing
1 has reached significance.It handles 6 Callus induction rate average values and is above other 8 processing, illustrate processing 6 better than its elsewhere
Reason.
The Duncan multiple alignment of table 2-9 9 processing
To sum up, the optimization process of T8 callus induction is processing 6, i.e. 2,4-D concentration is 2.0mg/L, and 6-BA concentration is
0.5mg/L, NAA concentration are 0.3mg/L, to show that genotype T8 callus induction optimal medium is MS+2.0mg/L2,4-D+
0.5mg/L 6-BA+0.3mg/L NAA+500mg/L CH。
Embodiment 3: sub- U.S. Liriodendron chinense callus differentiation
Differentiation culture: the present embodiment adds tri- kinds of hormones of 6-BA, IBA, KT of various concentration using MS as minimal medium
And the CH of 500mg/L, sucrose 30g/L, agar 7.0g/L are adjusted to 5.72 as differential medium, Medium's PH Value.In addition, not
Same processing is added to the VC of 5.0g/L.The high-pressure sterilizing pot sterilizing that all culture mediums pass through 121 DEG C of 20min makes after solidifying
With.The horizontal orthogonal test L9 (3 of 3 factor 33), each genotype is inoculated with 20 bottles under each processing, is repeated 3 times.
Embryo callus subculture is forwarded to differential medium, inoculation 25d or so, it is convex that callus surface bulge particle gradually becomes bud,
Tender white is become by green；It is inoculated with 35d or so, the convex light green bud of gradually emerging of the bud of tender white；It is inoculated with 50d or so, callus shape
At Multiple Buds shape, and very capable (Fig. 4) continued to multiply.
(1) influence that hormone combination breaks up XY1 callus
It can be seen that in 9 processing from table 3-1, processing 5 reaches highest proliferation system for the proliferation of XY1 callus differentiation
Number, average coefficient of proliferation 7.10, wherein reaching as high as 7.5；Followed by processing 9, average value is up to 5.33, highest 6.8.For
The result of XY1 callus differentiation carries out variance analysis, and there are significant difference (table 3-2) for 9 processing.
The influence that table 3-1 hormone combination breaks up XY1 callus
Table 3-2 genotype XY1 callus breaks up variance analysis
It by further Duncan multiple alignment, can be seen that from table 3-3, processing 5 has reached significant with other 8 processing
Sex differernce and extremely significant sex differernce, that there are conspicuousnesses is poor between processing 1, processing 2, processing 3, processing 4 and processing 7 for processing 9
It is different, significant difference is not present with processing 6, processing 8.The callus differentiation and proliferation coefficient of processing 5 is all larger than other 8 processing, says
Better than other 8 processing of daylight reason 5.
The Duncan multiple alignment of table 3-3 9 processing
| Processing | 5% conspicuousness | 1% conspicuousness |
| 1 | c | B |
| 2 | c | B |
| 3 | c | B |
| 4 | b | AB |
| 5 | a | A |
| 6 | ab | AB |
| 7 | c | B |
| 8 | ab | AB |
| 9 | ab | AB |
To sum up, XY1 callus differentiation optimization process is processing 5, i.e., 6-BA concentration is 2.0mg/L, and IBA concentration is 0.3mg/L,
KT concentration is 0.3mg/L, to show that genotype XY1 callus differentiation optimal medium is MS+2.0mg/L 6-BA+0.3mg/L
IBA+0.3mg/L KT+500mg/L CH。
(2) influence that hormone combination breaks up XY2 callus
It can be seen that in 9 processing from table 3-4, processing 5 reaches highest proliferation system for the proliferation of XY2 callus differentiation
Number, average coefficient of proliferation 6.4, wherein reaching as high as 7.7；Followed by processing 6, average value is up to 5.73, highest 7.3.For XY2
The result of callus differentiation carries out variance analysis, and there are significant difference (table 3-5) for 9 processing.
The influence that table 3-4 hormone combination breaks up XY2 callus
Table 3-5 genotype XY2 callus breaks up variance analysis
By further Duncan multiple alignment, processing 5 can be seen that with other 8 processing in the presence of aobvious from table 3-6
Write sex differernce and extremely significant sex differernce；Significant difference is not present with processing 8 in processing 6, but that there are conspicuousnesses is poor with other processing
Different and extremely significant sex differernce.The growth coefficient average out to 6.4 of the callus differentiation of processing 5, hence it is evident that be greater than other 8 processing, explanation
Processing 5 is better than other processing.
The Duncan multiple alignment of table 3-6 9 processing
To sum up, the optimization process of XY2 callus differentiation is processing 5, i.e., 6-BA concentration is 2.0mg/L, and IBA concentration is 0.3mg/
L, KT concentration are 0.3mg/L, to show that genotype XY2 callus differentiation optimal medium is MS+2.0mg/L 6-BA+0.3mg/
L IBA+0.3mg/L KT+500mg/L CH。
(3) influence that hormone combination breaks up T8 callus
It can be seen that in 9 processing from table 3-7, processing 5 reaches highest proliferation system for the proliferation of T8 callus differentiation
Number, average coefficient of proliferation 6.63, wherein reaching as high as 8.3；Followed by processing 6, average value is up to 6.00, highest 7.7.For T8
The result of callus differentiation carries out variance analysis, and there are significant difference (table 3-8) for 9 processing.
The influence that table 3-7 hormone combination breaks up T8 callus
Table 3-8 genotype T8 callus breaks up variance analysis
By further Duncan multiple alignment, it can be seen that processing 5 from table 3-9 and reach with other 8 processing
Conspicuousness and extremely significant sex differernce are horizontal；Processing 6 has also reached conspicuousness with other processing and extremely significant sex differernce is horizontal；More
In terms of hurting differentiation and proliferation coefficient, the average value of 5 growth coefficients of processing is 6.63, and the average value for handling 6 is 6.00, and processing 5 is greater than
Processing 6, while also greater than other processing, illustrate processing 5 better than other processing.
The Duncan multiple alignment of table 3-9 9 processing
| Processing | 5% conspicuousness | 1% conspicuousness |
| 1 | c | C |
| 2 | c | BC |
| 3 | c | ABC |
| 4 | abc | ABC |
| 5 | a | A |
| 6 | ab | AB |
| 7 | bc | ABC |
| 8 | abc | ABC |
| 9 | abc | ABC |
To sum up, the optimization process of T8 callus differentiation is processing 5, i.e., 6-BA concentration is 2.0mg/L, and IBA concentration is 0.3mg/
L, KT concentration are 0.3mg/L, to show that genotype T8 callus differentiation optimal medium is MS+2.0mg/L6-BA+0.3mg/L
IBA+0.3mg/L KT+500mg/L CH。
Embodiment 4: sub- U.S. Liriodendron chinense strong seedling culture
Strong seedling culture: the present embodiment is using MS as minimal medium, and tri- kinds of 6-BA, IBA, KT for adding various concentration swash
Element, wherein sucrose 30g/L, agar 7g/L, Medium's PH Value are adjusted to 5.72.In addition, different processing is added to 5.0g/L's
VC.The high-pressure sterilizing pot sterilizing that all culture mediums pass through 121 DEG C of 20min uses after solidifying.The horizontal orthogonal test of 3 factor 3
L9(33), each genotype is inoculated with 20 bottles under each processing, is repeated 3 times.The key step of strong seedling culture: firstly, by filter paper,
Scissors, tweezers culture dish put 121 DEG C of 20min sterilizings of high-pressure sterilizing pot, and bacterium of having gone out is placed on superclean bench, opens ultraviolet lamp and go out
Bacterium 30min opens ventilation after the completion of super-clean bench sterilizing.Then explant is quickly transferred to strengthen with heat sterilization cooling tweezers
On seedling culture medium, sealed membrane is sealed, different disposal marks, and is put in culturing room and continues to cultivate.
After callus is differentiated to form Multiple Buds, bud is cut together with a little callus of base portion and is connect to about 1cm by the stem length to bud
Kind arrives strong seedling culture base.20d or so is cultivated, the obvious elongation growth of stem of bud can be observed, 2- is risen to by original 1cm or so
3cm；Blade also grows bottle-green roomy blade by original smaller and hard light green, and quantity increases to 6-9 by 5-6 piece
Piece (Fig. 5).
(1) influence of the hormone combination to XY1 callus adventitious bud strong seedling culture
It can be seen that in 9 processing from table 4-1, processing 5 is for the flat of XY1 callus adventitious bud strong seedling culture culture
Equal single-strain blade number reaches at most, and average value reaches 8.93, up to 9.1；Followed by processing 4, average value is up to 7.37, highest
7.9.Variance analysis is carried out to genotype XY1 strong seedling culture result, there are significant difference (table 4-2) for 9 processing.
Influence of the table 4-1 hormone combination for XY1 callus adventitious bud strong seedling culture
Table 4-2 genotype XY1 callus adventitious bud strong seedling culture variance analysis
By further Duncan multiple alignment, it can be seen that processing 5 from table 4-3 and reach with other 8 processing
Conspicuousness and extremely significant sex differernce are horizontal；Processing 4 has reached significant difference level with other processing, but extremely significant property is not present
Difference.In terms of average single-strain blade number, the average value for handling 5 is 8.93, greater than the average value 7.37 of processing 4, also greater than it
He is handled, and illustrates processing 5 better than other processing.
The Duncan multiple alignment of table 4-3 9 processing
Therefore, the optimization process of XY1 callus adventitious bud strong sprout is processing 5, i.e. 6-BA concentration is 0.5mg/L, and IBA concentration is
0.3mg/L, KT concentration are 0.1mg/L, to show that genotype XY1 callus adventitious bud strong seedling culture optimal medium is MS+
0.5mg/L 6-BA+0.3mg/L IBA+0.1mg/L KT+5.0mg/L VC。
(2) influence of the hormone combination to XY2 callus adventitious bud strong seedling culture
It can be seen that in 9 processing from table 4-4, processing 5 is for the flat of XY2 callus adventitious bud strong seedling culture culture
Equal single-strain blade number reaches at most, and average value reaches 8.90, up to 9.4；Followed by processing 4, average value is up to 8.2, highest
8.5.Variance analysis is carried out to genotype XY2 strong seedling culture result, there are significant difference (table 4-5) for 9 processing.
Influence of the table 4-4 hormone combination for XY2 callus adventitious bud strong seedling culture
Table 4-5 genotype XY2 callus adventitious bud strong seedling culture variance analysis
By further Duncan multiple alignment, it is equal to can be seen that processing 4, processing 5 and other 7 processing from table 4-6
Significant difference is reached and extremely significant sex differernce is horizontal, between processing 4 and processing 5 and there was no significant difference；But in average list
Strain the number of blade in terms of, handle 5 average value 8.9 be greater than processing 4 average value 8.2, processing 5 better than processing 4, also superior to its elsewhere
Reason.
The Duncan multiple alignment of table 4-6 9 processing
| Processing | 5% conspicuousness | 1% conspicuousness |
| 1 | bc | BC |
| 2 | b | B |
| 3 | c | C |
| 4 | a | A |
| 5 | a | A |
| 6 | b | B |
| 7 | c | C |
| 8 | bc | BC |
| 9 | bc | BC |
To sum up, the optimization process of XY2 callus adventitious bud strong sprout is processing 5, i.e. 6-BA concentration is 0.5mg/L, and IBA concentration is
0.3mg/L, KT concentration are 0.1mg/L, to show that genotype XY2 callus adventitious bud strong seedling culture optimal medium is MS+
0.5mg/L 6-BA+0.3mg/L IBA+0.1mg/L KT+5.0mg/LVC。
(3) influence of the hormone combination to T8 callus adventitious bud strong seedling culture
It can be seen that in 9 processing from table 4-7, average single-strain blade number of the processing 5 for T8 strong seedling culture culture
Reach at most, average value reaches 8.33, up to 8.9；Followed by processing 4, average value is up to 7.9, highest 8.1.To genotype T8
Strong seedling culture result carries out variance analysis, and there are significant difference (table 4-8) for 9 processing.
Influence of the table 4-7 hormone combination for T8 callus adventitious bud strong seedling culture
Table 4-8 genotype T8 callus adventitious bud strong seedling culture variance analysis
Duncan Multiple range test (table 4-9) is the results show that processing 4, processing 5 and processing 6 reach with other 6 processing
Conspicuousness and extremely significant sex differernce are horizontal, but simultaneously there was no significant difference between three.
The Duncan multiple alignment of table 4-9 9 processing
| Processing | 5% conspicuousness | 1% conspicuousness |
| 1 | bc | B |
| 2 | b | B |
| 3 | c | B |
| 4 | a | A |
| 5 | a | A |
| 6 | a | A |
| 7 | bc | B |
| 8 | b | B |
| 9 | b | B |
To sum up, the optimization process of T8 callus adventitious bud strong sprout is processing 5, i.e. 6-BA concentration is 0.5mg/L, and IBA concentration is
0.3mg/L, KT concentration are 0.1mg/L, to show that genotype T8 callus adventitious bud strong seedling culture optimal medium is MS+
0.5mg/L 6-BA+0.3mg/L IBA+0.1mg/L KT+5.0mg/L VC。
Embodiment 5: sub- U.S. Liriodendron chinense culture of rootage
Culture of rootage: the present embodiment adds two kinds of hormones of IBA, NAA of various concentration using 1/2MS as minimal medium,
Wherein sucrose 30g/L, agar 6.5g/L, Medium's PH Value are adjusted to 5.72.In addition, different processing is added to 5.0g/L's
VC.The high-pressure sterilizing pot sterilizing that all culture mediums pass through 121 DEG C of 20min uses after solidifying.The horizontal orthogonal test of 2 factor 3
L6(32), each genotype is inoculated with 8 bottles under each processing, is repeated 3 times.
Adventitious bud Jing Guo strong seedling culture is inoculated into root media, inoculation 13d or so, bastem portion together with callus
Callus starts milky protrusion occur；About 20d, milky protrusion are gradually emerged the tender white tip of a root；Hereafter the continuous elongation growth of root,
And it is formed from taking root；Culture 28d or so root basically forms, and the quantity of average root reaches 2-3 item, and the growth potential of adventitious bud is also better
Good (Fig. 6).
(1) influence of various concentration NAA, the IBA to XY1 callus adventitious bud rooting culture
It can be seen that in 9 processing from table 5-1, rooting rate of the processing 2 for XY1 callus adventitious bud rooting culture
Reach highest, average value reaches 52.33%, and highest rooting rate reaches 57%；Followed by processing 5, average value up to 38.33%,
Highest 40%.Variance analysis is carried out to genotype XY1 culture of rootage result, there are significant difference (table 5-2) for 9 processing.
Influence of table 5-1 various concentration NAA, the IBA for XY1 callus adventitious bud rooting culture
Table 5-2 genotype XY1 callus adventitious bud rooting culture variance analysis
By further Duncan multiple alignment, it can be seen that processing 2 from table 5-3 and reached aobvious with other 8 processing
It writes sex differernce and extremely significant sex differernce is horizontal；Processing 5 with other processing there are significant difference, but electrodeless significant difference.?
In terms of rooting rate, the average value for handling 2 is 52.33%, is higher than other processing, illustrates processing 2 better than other processing.
The Duncan multiple alignment that table 5-3 carries out 9 processing
| Processing | 5% conspicuousness | 1% conspicuousness |
| 1 | bc | B |
| 2 | a | A |
| 3 | bc | B |
| 4 | bc | B |
| 5 | b | B |
| 6 | bc | B |
| 7 | c | B |
| 8 | bc | B |
| 9 | bc | B |
So XY1 callus adventitious bud rooting optimization process is processing 2, i.e. IBA concentration is 0.2mg/L, and NAA concentration is
0.1mg/L, so that the optimal medium for obtaining XY1 callus adventitious bud rooting culture is 1/2MS+0.2mg/L IBA+0.1mg/L
NAA+5.0mg/L VC。
(2) influence of various concentration NAA, the IBA to XY2 callus adventitious bud rooting culture
It can be seen that in 9 processing from table 5-4, rooting rate of the processing 2 for XY2 callus adventitious bud rooting culture
Reach highest, average value reaches 51.33%, and highest rooting rate reaches 55%；Followed by processing 5, average value 39.67%, most
It is high by 42%.Variance analysis is carried out to genotype XY2 culture of rootage result, there are significant difference (table 5-5) for 9 processing.
Influence of table 5-4 various concentration NAA, the IBA for XY2 callus adventitious bud rooting culture
Table 5-5 genotype XY2 callus adventitious bud rooting culture variance analysis
By further Duncan multiple alignment, it can be seen that processing 2 from table 5-6 and reached aobvious with other 8 processing
It writes sex differernce and extremely significant sex differernce is horizontal；Processing 5 has reached significant difference level with other processing, but electrodeless conspicuousness is poor
It is different.In terms of rooting rate, 2 rooting rate average value 51.33% is handled higher than other processing, illustrates processing 2 better than other processing.
The Duncan multiple alignment that table 5-6 carries out 9 processing
To sum up, XY2 callus adventitious bud rooting optimization process is processing 2, i.e. IBA concentration is 0.2mg/L, and NAA concentration is
0.1mg/L, so that the optimal medium for obtaining XY2 callus adventitious bud rooting culture is 1/2MS+0.2mg/L IBA+0.1mg/L
NAA+5.0mg/L VC。
(3) influence of various concentration NAA, the IBA to T8 callus adventitious bud rooting culture
It can be seen that in 9 processing from table 5-7, processing 2 reaches the rooting rate of T8 callus adventitious bud rooting culture
To highest, average value reaches 56.00%, and highest rooting rate reaches 63%；Followed by processing 3, average value is up to 40.33%, most
It is high by 44%.Variance analysis is carried out to genotype T8 culture of rootage result, there are significant difference (table 5-8) for 9 processing.
Influence of table 5-7 various concentration NAA, the IBA for T8 callus adventitious bud rooting culture
Table 5-8 genotype T8 callus adventitious bud rooting culture variance analysis
By further Duncan multiple alignment, it can be seen that processing 2 from table 5-9 and there is limitation with other 8 processing
Sex differernce and extremely significant sex differernce, and there are no significant difference between 8 processing.In terms of rooting rate, the rooting rate for handling 2 is put down
Mean value is 56%, is higher than other 8 processing, illustrates processing 2 better than other processing.
The Duncan multiple alignment that table 5-9 carries out 9 processing
| Processing | 5% conspicuousness | 1% conspicuousness |
| 1 | b | B |
| 2 | a | A |
| 3 | b | B |
| 4 | b | B |
| 5 | b | B |
| 6 | b | B |
| 7 | b | B |
| 8 | b | B |
| 9 | b | B |
To sum up, the optimization process that T8 callus is taken root is processing 2, i.e., IBA concentration is 0.2mg/L, and NAA concentration is 0.1mg/L,
It is 1/2MS+0.2mg/L IBA+0.1mg/L NAA+ to obtain the optimal medium of T8 callus adventitious bud rooting culture
5.0mg/L VC。
Embodiment 6: the training tissue culture seedling transplanting of sub- U.S. Liriodendron chinense
Acclimatization and transplants: firstly, plant ash, perlite, vermiculite are fitted into woven bag, the high pressure for being put into 121 DEG C of 20min is gone out
Bacterium pot sterilizing.The tissue-cultured seedling taken root is removed from culturing room simultaneously, is placed at outdoor shading and carries out strong light hardening, time 1d,
3d,5d,7d；Then, plant ash, perlite, the vermiculite sterilizing completed are mixed according to 7: 2: 1 ratio, and it is mixed that nutrient solution is added
Conjunction stirs evenly, and is fitted into culturing pot.Finally, gently being pressed from both sides out tissue-cultured seedling with tweezers, pay attention to protecting tender root in the process,
The culture medium of root is washed completely under flowing water, bad root phenomenon occurs after preventing transplanting.By tissue culture transplantation of seedlings to being equipped with matrix
Culturing pot in, nest root of trying not gently is compacted matrix, and pours quantitative root water, comes into full contact with root with matrix.With
Preservative film seals tissue-cultured seedling, prevents humidity too low, make tissue-cultured seedling dehydration wilt, be put into incubator and control humidity, it is latter
It is all to be watered daily using spraying, so that humidity is maintained 85% or so.Preservative film can be removed after a week, to tissue-cultured seedling robust growth
After can transplant.
The preceding 3d just transplanted, some wiltings of transplanting seedling leaf, or even there is the withered phenomenon of blade, it to keep at this time wetter
The growing environment of profit prevents the excessive loss of transplanted seedling moisture.After 3d, transplanted seedling has adapted to external environment, is not in substantially
The phenomenon that excessive water is lost.After two weeks, blade is gradually become dark green by yellow green, and leaf area increases, and stem also constantly stretches for culture
Long growth, original children's tender stem start lignifying occur, and surface becomes light brown (Fig. 7).
From figure 8, it is seen that compared with the control group, transplanting survival rate can be significantly improved for the hardening of tissue-cultured seedling, most
Improve 23% more.In hardening number of days, preceding 5d, as time goes by, transplanting survival rate is in rising trend, and 5d reaches highest
Value 89%, and in 7d, transplanting survival rate is again on a declining curve.It follows that tissue-cultured seedling is for external ring when hardening 5d
Border adapts to substantially, becomes seedling, more suitable period when transplanting at this time.
Claims (10)
1. a kind of using leaf as the quick-breeding method of the U.S. Liriodendron chinense in the Asia of explant, which comprises the following steps:
(1) it acquires blade or petiole is explant, pretreatment and disinfection；
(2) explant of the acquisition after step (1) being pre-processed and sterilized, which is inoculated into calli induction media, to be cultivated, wherein
The calli induction media is added with 1.0~3.0mg/L 2,4-D, 0.1~0.5mg/L 6-BA based on MS culture medium
With 0.1~0.5mg/L NAA；
(3) the callus structure that callus Fiber differentiation obtains is transferred in differential medium and is cultivated, wherein the differential medium with
Based on MS culture medium, it is added with 1.0~3.0mg/L 6-BA, 0.1~0.5mg/L IBA and 0.3~1.0mg/L KT；
(4) Multiple Buds that differentiation culture obtains are transferred in strong seedling culture base and are cultivated, wherein the strong seedling culture is with MS culture medium
Based on, it is added with 0.3~1.0mg/L 6-BA, 0.1~0.5mg/L IBA and 0.1~0.3mg/L KT；
(5) adventitious bud for obtaining strong seedling culture, which is transferred in root media, cultivates, wherein the root media is with 1/2MS
Based on culture medium, it is added with 0.1~0.3mg/L IBA and 0.1~0.3mg/L NAA；
(6) tissue-cultured seedling for obtaining culture of rootage cleans root culture medium, and transplanting hardening into the matrix of hardening obtains seedling.
2. using leaf as the U.S. Liriodendron chinense quick-breeding method in the Asia of explant described in claim 1, which is characterized in that callus in step (2)
Induced medium based on MS culture medium, be added with 2.0mg/L2,4-D, 0.5mg/L 6-BA, 0.3mg/L NAA and
500mg/L CH。
3. using leaf as the U.S. Liriodendron chinense quick-breeding method in the Asia of explant described in claim 1, which is characterized in that differentiation in step (3)
Culture medium is added with 2.0mg/L 6-BA, 0.3mg/L IBA, 0.3mg/L KT and 500mg/L CH based on MS culture medium.
4. using leaf as the U.S. Liriodendron chinense quick-breeding method in the Asia of explant described in claim 1, which is characterized in that strong sprout in step (4)
Culture medium is added with 0.5mg/L 6-BA, 0.3mg/L IBA, 0.1mg/L KT and 5.0mg/L VC based on MS culture medium.
5. using leaf as the U.S. Liriodendron chinense quick-breeding method in the Asia of explant described in claim 1, which is characterized in that step is taken root in (5)
Culture medium is added with 0.2mg/L IBA, 0.1mg/L NAA and 5.0mg/L VC based on 1/2MS culture medium.
6. according to claim 1 using leaf as the U.S. Liriodendron chinense quick-breeding method in the Asia of explant, the U.S. Liriodendron chinense packet in the Asia
Include tri- genotype of XY1, XY2 and T8.
7. handling and sterilizing in step (1) according to claim 1 using leaf as the U.S. Liriodendron chinense quick-breeding method in the Asia of explant
Method are as follows: 4 DEG C of refrigerator overnights will be placed with petiolate blade, and impregnate 3~5min with dish washing liquid, banister brush is along a direction
It gently brushes, rim of a cup envelope is put into after rinsing well to be had in the beaker of gauze, rinses 30min in tap water down-flow water.Flowing water is rinsed
The explant crossed is put into the conical flask for bacterium of having gone out in advance, and the sterile shaking water of autoclaved cooling cleans 2 times, then with 75%
The alcohol of concentration impregnates 30s, has impregnated with sterile water wash 1 time, has then used 0.1%HgCl2Disinfection, disinfecting time are set as 3
~15min；Mercuric chloride keeps conical flask oscillating and shaking during sterilizing, and makes mercuric chloride sufficiently and material；Last sterile water wash 5-6
Secondary, each 5min dispels remaining mercuric chloride completely as far as possible；The explant being disposed aseptic filter paper suck dry moisture.
8. a kind of using leaf as the quick-breeding method of the U.S. Liriodendron chinense in the Asia of explant according to claim 1, which is characterized in that step
(6) acclimatization and transplants matrix is that plant ash, perlite, vermiculite that sterilizing is completed are mixed according to 7: 2: 1 ratio in, and nutrient solution is added
It is mixed evenly.
9. according to claim 1 using leaf as the quick-breeding method of the U.S. Liriodendron chinense in the Asia of explant, which is characterized in that including following
Step:
(1) blade of the sub- U.S. Liriodendron chinense of acquisition XY1 genotype is as explant, pretreatment and disinfection；
(2) explant of the acquisition after step (1) being pre-processed and sterilized is inoculated into culture and dark place in calli induction media
Manage 15d, wherein the calli induction media is added with 2.0mg/L2,4-D, 0.5mg/L 6- based on MS culture medium
BA, 0.3mg/L NAA and 500mg/L CH；
(3) the callus structure that callus Fiber differentiation obtains is transferred in differential medium and is cultivated, wherein the differential medium with
Based on MS culture medium, it is added with 2.0mg/L 6-BA, 0.3mg/L IBA, 0.3mg/L KT and 500mg/L CH；
(4) Multiple Buds that differentiation culture obtains are transferred in strong seedling culture base and are cultivated, wherein the strong seedling culture is with MS culture medium
Based on, it is added with 0.5mg/L 6-BA, 0.3mg/L IBA, 0.1mg/L KT and 5.0mg/L VC；
(5) adventitious bud for obtaining strong seedling culture, which is transferred in root media, cultivates, wherein the root media is with 1/2MS
Based on culture medium, it is added with 0.2mg/L IBA, 0.1mg/L NAA and 5.0mg/L VC；
(6) tissue-cultured seedling for obtaining culture of rootage cleans root culture medium, and transplanting hardening 5d into the matrix of hardening is obtained small
Seedling.
Step (2)~(6) condition of culture are as follows: cultivation temperature is 25 ± 2 DEG C, lighting delay number 14h/d, intensity of illumination 2000lx,
Indoor relative humidity control is 75%.
10. according to claim 1 using leaf as the quick-breeding method of the U.S. Liriodendron chinense in Asia of explant, which is characterized in that including with
Lower step:
(1) blade of the sub- U.S. Liriodendron chinense of acquisition T8 genotype is as explant, pretreatment and disinfection；
(2) explant of the acquisition after step (1) being pre-processed and sterilized is inoculated into culture and dark place in calli induction media
Manage 15d, wherein the calli induction media is added with 2.0mg/L2,4-D, 0.5mg/L 6- based on MS culture medium
BA, 0.3mg/L NAA and 500mg/L CH；
(3) the callus structure that callus Fiber differentiation obtains is transferred in differential medium and is cultivated, wherein the differential medium with
Based on MS culture medium, it is added with 2.0mg/L 6-BA, 0.3mg/L IBA, 0.3mg/L KT and 500mg/L CH；
(4) Multiple Buds that differentiation culture obtains are transferred in strong seedling culture base and are cultivated, wherein the strong seedling culture is with MS culture medium
Based on, it is added with 0.5mg/L 6-BA, 0.3mg/L IBA, 0.1mg/L KT and 5.0mg/L VC；
(5) adventitious bud for obtaining strong seedling culture, which is transferred in root media, cultivates, wherein the root media is with 1/2MS
Based on culture medium, it is added with 0.2mg/L IBA, 0.1mg/L NAA and 5.0mg/L VC；
(6) tissue-cultured seedling for obtaining culture of rootage cleans root culture medium, and transplanting hardening 5d into the matrix of hardening is obtained small
Seedling.
Step (2)~(6) condition of culture are as follows: cultivation temperature is 25 ± 2 DEG C, lighting delay number 14h/d, intensity of illumination 2000lx,
Indoor relative humidity control is 75%.
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