CN110073978A - It is a kind of using bud as the quick-breeding method of the U.S. Liriodendron chinense in Asia of explant - Google Patents
It is a kind of using bud 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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Abstract
The invention discloses a kind of using bud as the quick-breeding method of the U.S. Liriodendron chinense in the Asia of explant, pretreatment and disinfection, initial culture base, subculture medium, strong seedling culture base, root media and acclimatization and transplants including the sub- U.S. mandarin jacket wood bud of tri- kinds of genotype of XY1, XY2 and T8 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.For T8 genotype under optimal culture condition, the starting rate average value of highest Initial culture reaches 72% it has been confirmed by experiments that；The growth coefficient average value of the maximum squamous subculture of XY1 genotype reaches 3.70；The average single-strain blade number average value of most strong seedling cultures of XY2 genotype reaches 8.37, and the rooting rate average value of highest culture of rootage reaches 51.67%, and final acclimatization and transplants survival rate is up to 85%.
Description
Technical field
The present invention relates to a kind of woody plant tissure culture propagation methods, more specifically to one kind using bud 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.Direct organ is to develop formation directly from explant without the callus stage
Root or bud, generally can from apical meristem (stem apex), axillary bud separate living tissue, bud former base bud separate living tissue induce it is indefinite
Bud, or adventitious bud is differentiated by explant surface layer.Tissue cultures can be divided into direct organ and occur to occur two kinds with indirect organ
Approach, direct organ are a kind of rapid propagation methods for being more generally also easy to grasp, and Apparatuses formation is directly from original
Explant, incubation is relatively easy, and growth rate is fast, and Variations of Regenerated Plants is small, heritability stablize, can protect well
Hold the merit (Tang Wei, Ou Yangfan, 1997) of parent.In conclusion the fast breeding technique hand based on tissue culture technique
It is low with seed production efficiency that section can effectively solve the problems, such as that the breeding of sub- U.S. Liriodendron chinense is promoted to a certain extent.
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 and influence factor of tissue culture regeneration, including sampling time, disinfecting time, explant
Selection and processing, hormone combination and acclimatization and transplants, the Regeneration in Vitro body of sub- U.S. Liriodendron chinense is established with direct adventitious organogenesis
System, it is intended to provide excellent tissue culture technology and scheme for the quick breeding and popularization of sub- U.S. Liriodendron chinense superior genotypes.
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.
Technical solution: to solve the above-mentioned problems, the technical solution adopted in the present invention is as follows:
It is a kind of using bud as the quick-breeding method of the U.S. Liriodendron chinense in Asia of explant, comprising the following steps:
(1) terminal bud or lateral bud are acquired as explant, pretreatment and disinfection；
(2) explant of the acquisition after step (1) being pre-processed and sterilized, which is inoculated into initial culture base, to be cultivated, wherein
The initial culture base is added with 1.0~3.0mg/L 6-BA, 0.3~1.0mg/L NAA and 0.1 based on MS culture medium
~0.5mg/L IBA；
(3) the callus tender shoots that Initial culture obtains is transferred in subculture medium and is cultivated, wherein the subculture medium with
Based on 3/4MS culture medium, it is added with 0.5~2.0mg/L 6-BA, 0.1~0.5mg/L IBA and 0.1~0.5mg/L KT；
(4) Multiple Buds for obtaining squamous subculture are transferred in strong seedling culture base and cultivate, 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, initial culture base is added with 2.0mg/L 6-BA, 0.5mg/L based on MS culture medium in step (2)
NAA, 0.1mg/L IBA and 5.0mg/L VC.
Preferably, in step (3) subculture medium based on 3/4MS culture medium, added with 1.0mg/L6-BA,
0.5mg/L IBA, 0.3mg/L KT and 5.0mg/L VC.
Preferably, strong seedling culture base is added with 0.5mg/L 6-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 refrigerators of explant of acquisition place 1-2d, dish washing liquid
Cleaning explant 2 times, peels off the outermost one layer of bract of bud and is placed in conical flask with diluted 84 medicining liquid dipping 5-10min, uses
One layer of gauze ties, and places tap water down-flow water and rinses 2-4h, is transferred to superclean bench.The flushed explant of flowing water is put
It in the conical flask for entering bacterium of having gone out in advance, is first cleaned 2 times with the sterile shaking water of autoclaved cooling, with the alcohol of 75% concentration
30s is impregnated, has impregnated again with sterile water wash 1 time, has been sterilized with the mercuric chloride of 0.1% concentration, disinfecting time is set as 5~20min；
Mercuric chloride keeps conical flask oscillating and shaking during sterilizing, and makes mercuric chloride sufficiently and material；Finally with sterile water wash 5-6 times, often
Secondary 5min dispels remaining mercuric chloride completely as far as possible；By 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 bud as the quick-breeding method of the U.S. Liriodendron chinense in the Asia of explant, comprising the following steps:
(1) the sub- U.S. Liriodendron chinense terminal bud of acquisition in April 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 initial culture base, to be cultivated, wherein
The initial culture base based on MS culture medium, added with 2.0mg/L 6-BA, 0.5mg/L NAA, 0.1mg/L IBA and
5.0mg/L VC；
(3) the callus tender shoots of Initial culture is transferred in subculture medium and is cultivated, wherein the subculture medium is with 3/
Based on 4MS culture medium, it is added with 1.0mg/L 6-BA, 0.5mg/L IBA, 0.3mg/L KT and 5.0mg/L VC；
(4) Multiple Buds for obtaining squamous subculture are transferred in strong seedling culture base and cultivate, 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 2000
1x, indoor relative humidity control are 75%.
Preferably, using bud as the quick-breeding method of the U.S. Liriodendron chinense in the Asia of explant, comprising the following steps:
(1) the sub- U.S. Liriodendron chinense lateral bud of acquisition in April 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 initial culture base, to be cultivated, wherein
The initial culture base based on MS culture medium, added with 2.0mg/L 6-BA, 0.5mg/L NAA, 0.1mg/L IBA and
5.0mg/L VC；
(3) the callus tender shoots of Initial culture is transferred in subculture medium and is cultivated, wherein the subculture medium is with 3/
Based on 4MS culture medium, it is added with 1.0mg/L 6-BA, 0.5mg/L IBA, 0.3mg/L KT and 5.0mg/L VC；
(4) Multiple Buds for obtaining squamous subculture are transferred in strong seedling culture base and cultivate, 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 2000
1x, indoor relative humidity control are 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 starting rate average value of highest Initial culture reaches 72%, is T8 genotype；The growth coefficient of maximum squamous subculture is flat
Mean value reaches 3.70, is XY1 genotype；The average single-strain blade number average value of most strong seedling cultures reaches 8.37, is XY2 gene
Type；The rooting rate average value of highest culture of rootage reaches 51.67%, is XY2 genotype；Final acclimatization and transplants survival rate is up to
85%.
Detailed description of the invention
Fig. 1 is that the sub- U.S. mandarin jacket wood bud of three kinds of genotype is proliferated Initial culture figure: A-C:T8 is inoculated into starting expansion blade mistake
Journey；D-F:XYI is inoculated into starting expansion blade process figure: G-I:XY2 is inoculated into starting expansion blade process；
Fig. 2 is the sub- U.S. mandarin jacket wood bud shoot proliferation figure of three kinds of genotype: A:XY1 bud proliferation and subculture；B:XY2 bud proliferation after
Generation；C:T8 bud proliferation and subculture；
Fig. 3 is that the sub- U.S. mandarin jacket wood bud of three kinds of genotype is proliferated adventitious bud strong sprout figure: A:XY1 bud is proliferated adventitious bud strong sprout；B:
XY2 bud is proliferated adventitious bud strong sprout；C:T8 bud is proliferated adventitious bud strong sprout；
Fig. 4 is the sub- U.S. Liriodendron chinense culture of rootage figure of three kinds of genotype: A:XY1 bud proliferation is taken root；B:XY2 bud proliferation is taken root；
C:T8 bud proliferation is taken root；
Fig. 5 is the sub- U.S. mandarin jacket wood bud acclimatization and transplants figure of three kinds of genotype: A:XY1 transplanted seedling；B:XY2 transplanted seedling；C:T8 is moved
Plant seedling；
Fig. 6 is influence diagram of the different hardening number of days to sub- U.S. Liriodendron chinense 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 3 plants of sub- U.S. Liriodendron chinense superior genotypes T8, XY1, XY2, and wherein T8 picks up from Jiangsu
Practice forest farm in another name for Sichuan Province under the Nanjing Forestry University of Jurong, XY1 and XY2 are picked up from Nanjing Forestry University campus.
Condition of culture: condition of culture is illumination constant temperature incubation, and cultivation temperature is 25 ± 2 DEG C, lighting delay number 14h/d, light
According to about 2000 1x of intensity, indoor relative humidity control is 75%.
Data statistical analysis method:
After explant cultivates 7d on culture medium, statistics different sampling stages, the pollution rate of different disinfecting times, browning
Rate, the death rate；After cultivating 20d, statistics hormon handles the starting rate of lower explant, starting time；After cultivating 40d, statistics
The lower bud appreciation rate of hormon processing；After cultivating 90d, statistics hormon handles lower single plant average leaf number and record growth
Situation culture；After 120d, statistics hormon handles the rooting rate of lower aseptic seedling and number of taking root；After transplanting, different refinings are counted
The survival rate of seedling time 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+eij；In formula: YijFor i-th of processing the
The character value of j sample, u indicate population mean, TiFor i-th of Treatment Effects, 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: explant acquisition, the selection of disinfecting time
Explant acquisition time and method: acquisition time be respectively in September, 2016, in December, 2016,2 months 2017,
In April, 2017, in June, 2017.Fine day is selected, full terminal bud and lateral bud is acquired at 2 points or so in the afternoon, is packed into valve bag, does
Good label, is put into ice chest and takes back laboratory.
The pretreatment of explant and sterilization method: 4 DEG C of refrigerators of explant of acquisition place 1-2d, and dish washing liquid cleans explant
It 2 times, peels off the outermost one layer of bract of bud and is placed in conical flask, is pricked with one layer of gauze with diluted 84 medicining liquid dipping 5-10min
It is good, it places tap water down-flow water and rinses 2-4h, be transferred to superclean bench.The flushed explant of flowing water is put into and is gone out in advance
It in the conical flask of bacterium, is first cleaned 2 times with the sterile shaking water of autoclaved cooling, impregnates 30s, leaching with the alcohol of 75% concentration
Finish again with sterile water wash 1 time, sterilized with the mercuric chloride of 0.1% concentration, disinfecting time be set as 5min, 10min, 15min,
20min；Mercuric chloride keeps conical flask oscillating and shaking during sterilizing, and makes mercuric chloride sufficiently and material；Finally use sterile water wash 5-6
Secondary, each 5min dispels remaining mercuric chloride completely as far as possible；The explant being disposed is blotted into water with aseptic filter paper
Point, carry out subsequent experimental.
(1) selection of disinfecting time
It can analyze and obtain according to table 1-1 experimental result: working as 0.1%HgCl2When disinfecting time is 5min, genotype XY1
Pollution rate highest, be 53%；When disinfecting time is 20min, pollution rate is minimum, is 21%.Pollution rate substantially with disinfection when
Between increase and downward trend is presented.But with the increase of disinfecting time, the melting brown rate and the death rate of explant also increase therewith
Add, in 5min, melting brown rate and the death rate are only 5.5% and 0% respectively, and reach 41.2% and 30.6% in 30min.
Although the injury to explant is also apparent so the increase of disinfecting time can reduce pollution rate.It is between when sterilized
When 10min and 15min, close to 19.4% minimum pollution rate, melting brown rate and the death rate are also maintained at lower pollution rate
Level, melting brown rate are 6.2% and 7.2%, and the death rate is 3.7% and 6.0%.So for genotype XY1,0.1%HgCl2
Reasonable disinfecting time is 15min.
Table 1-1 0.1%HgCl2Influence of the disinfecting time for genotype XY1 explant
It can analyze and obtain according to table 1-2 experimental result: for genotype XY2, working as 0.1%HgCl2Disinfecting time is
When 5min, pollution rate is up to 61%, when disinfecting time is 10min, pollution rate minimum 21%；It is between when sterilized
When 30min, melting brown rate reaches highest 49.5%, and disinfecting time is minimum when being 5min, is 1.4%；It is 30min between when sterilized
When, death rate highest is 32.7%, and when disinfecting time is 10min, minimum is only 0.9%.Pollution rate is with disinfecting time totality
Downward trend is presented, minimum in 10min, as disinfecting time is at ascendant trend, the death rate is fallen before to be reached melting brown rate in 10min
After minimum, and ascendant trend is presented.So disinfecting time reasonable for genotype XY2 is 0.1%HgCl2Sterilize 10min.
Table 1-2 0.1%HgCl2Influence of the disinfecting time for genotype XY2 explant
| 0.1%HgCl2Disinfecting time | 5min | 10min | 15min | 20min | 30min |
| Inoculation number (a) | 132 | 132 | 132 | 132 | 132 |
| It pollutes number (a) | 61 | 21 | 28 | 23 | 31 |
| Pollution rate (%) | 46.2 | 15.9 | 21.2 | 17.4 | 23.5 |
| Browning number (a) | 1 | 3 | 4 | 34 | 50 |
| Melting brown rate (%) | 1.4 | 2.7 | 3.8 | 31.2 | 49.5 |
| Death toll (a) | 4 | 1 | 4 | 30 | 33 |
| The death rate (%) | 5.6 | 0.9 | 3.8 | 27.5 | 32.7 |
Can analyze and obtain according to table 1-3 experimental result: for genotype T8, when sterilized between be 5min when, explant
Pollution rate highest 45.2%；It is minimum when 15min, only 14.0%；10min takes second place, and is 16.1%；It is 15min between when sterilized
When, melting brown rate and the death rate are minimum, and respectively 2.5% and 2.5%；10min still takes second place, and respectively 5.1% and 3.8%.
So optimum disinfecting time is 0.1%HgCl for genotype T8 explant2Sterilize 15min.
Table 1-3 0.1%HgCl2Influence of the disinfecting time for genotype T8 explant
In conclusion with 0.1%HgCl2Comparatively ideal level can be reached when sterilizing 15min, pollution can either be controlled again
Damage of the mercuric chloride for explant can be reduced to a certain extent, provide preferable disinfection way to cultivate aseptic seedling.
(2) selection of explant acquisition time
Can analyze and obtain according to table 1-4 experimental result: pollution rate of three genotype in December reaches minimum water
It is flat, XY1 15.5%, XY2 17.8%, T8 12.2%；For three genotype at April, it is low that pollution rate maintains second
Level, XY1 17.0%, XY2 18.1%, T8 14.3%, with December without too big gap.XY1 is in death in April
Rate minimum 4.6%, starting rate is up to 77.3%；XY2 death rate minimum 6.8% in September part, starting rate when April
Up to 86.5%；The T8 death rate minimum 6.3% at June, taking second place April is 6.7%, and starting rate is in highest in June
It is 92.1%, taking second place April is 87.8%.
Influence of the table 1-4 sampling time to explant starting rate
In conclusion comprehensively consider pollution rate, the death rate, starting rate, when April ideal nutrition.In this way
It can either achieve the purpose that sterilize explant, and the death rate can be reduced to the greatest extent, improve the starting rate of explant.
Embodiment 2: sub- U.S. Liriodendron chinense Initial culture
Initial culture: the present embodiment using MS culture medium as minimal medium, and be added to various concentration 6-BA, IBA,
Tri- kinds of hormones of NAA, wherein sucrose 30g/L, agar 7g/L, Medium's PH Value are adjusted to 5.72.In addition, different 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 uses after solidifying.Experimental design 3 because
The horizontal orthogonal test L9 (3 of element 33), each genotype is inoculated with 20 bottles under each processing, is repeated 3 times.
The main process of Initial culture: firstly, filter paper, scissors, tweezers culture dish are put 121 DEG C of 20min of high-pressure sterilizing pot
Sterilizing, bacterium of having gone out are placed on superclean bench, open ultraviolet lamp sterilizing 30min, open ventilation after the completion of super-clean bench sterilizing.Then
On the clean filter paper of the sterilized bud culture dish holding of mercuric chloride, with the cooling scissors tweezers of infrared inoculation sterilizing instrument high temperature sintering
The extra xylem part of terminal bud, lateral bud base portion is cut off and is inoculated on the culture medium of each processing, in vitro culture is carried out.Inoculation
Complete one bottle, Yao Genghuan mono- opens filter paper, and by high temperature sintering and the cooling again of scissors tweezers.It will be in strict accordance with sterile behaviour in inoculation
It is required, preventing from bringing Browning object into culture medium causes to pollute.
Culture medium inoculated 10d that the explant of three different genotypes is handled in various concentration or so largely has been able to
Starting, outermost two layers of bract open, and grow 1-2 piece tender leaf；25d or so is cultivated, explant base portion expands callus, forms children
Tender callus tender shoots, blade grow 4-5 piece (Fig. 1).
(1) influence of the hormone combination to XY1 Initial culture
It can be seen that in 9 processing from table 2-1, processing 5 reaches highest for the starting rate of XY1 Initial culture, puts down
Mean value reaches 68.3%, wherein reaching as high as 77%；Followed by processing 4, average value reaches 61.7%, reaches as high as 72%.It is right
Genotype XY1 Initial culture result carries out variance analysis, and it is horizontal (table 2-2) to have reached significant difference between 9 processing.
Influence of the table 2-1 hormone combination to XY1 Initial culture
Table 2-2 genotype XY1 Initial culture variance analysis
By further Duncan multiple alignment, from table 2-3 can be seen that processing 4, processing 5 is reached with other 7 processing
Significant difference and extremely significant difference have been arrived, but has handled and significant difference and extremely significant difference is not present between 4 and processing 5.It is logical
It crosses and the mean value of starting rate between the two is compared, 5 average values of processing are 68.3%, and the average value for handling 4 is 61.7%, processing 5
Better than processing 4, illustrate processing 5 better than other processing.
The Duncan multiple alignment that table 2-3 9 processing carry out
| Processing | 5% conspicuousness | 1% conspicuousness |
| 1 | ab | ABC |
| 2 | ab | ABC |
| 3 | bc | BC |
| 4 | a | A |
| 5 | a | A |
| 6 | ab | ABC |
| 7 | bc | BC |
| 8 | bc | BC |
| 9 | c | C |
To sum up, XY1 Initial culture optimization process is processing 5, i.e., 6-BA concentration is 2.0mg/L, and NAA concentration is 0.5mg/L,
IBA concentration is 0.1mg/L, to show that genotype XY1 optimal medium primary is MS+2.0mg/L 6-BA+0.5mg/L NAA
+0.1mg/L IBA+5.0mg/L VC。
(2) influence of the hormone combination to XY2 Initial culture
It can be seen that in 9 processing from table 2-4, processing 5 reaches highest for the starting rate of XY2 Initial culture, puts down
Mean value reaches 68.3%, wherein reaching as high as 75%；Followed by processing 4, average value reaches 59.0%, up to 73%.To base
Because type XY2 Initial culture result carries out variance analysis, it is horizontal (table 2-5) that 9 processing have reached significant difference.
Influence of the table 2-4 hormone combination to XY2 Initial culture
Table 2-5 genotype XY2 Initial culture variance analysis
It by further Duncan multiple alignment, can be seen that from table 2-6, processing 5 reaches with other 8 processing
Significant difference and extremely significant difference, and handle 5 starting rate average value 68.3%, hence it is evident that is handled greater than other 8 is flat
Mean value illustrates better than other 8 processing of processing 5.
The Duncan multiple alignment that table 2-6 9 processing carry out
So the optimization process of XY2 Initial culture is processing 5, i.e., 6-BA concentration is 2.0mg/L, and NAA concentration is 0.5mg/
L, IBA concentration are 0.1mg/L, to show that genotype XY2 optimal medium primary is MS+2.0mg/L 6-BA+0.5mg/L
NAA+0.1mg/L IBA+5.0mg/L VC。
(3) influence of the hormone combination to T8 Initial culture
It can be seen that in 9 processing from table 2-7, processing 5 reaches highest for the starting rate of T8 Initial culture, puts down
Mean value reaches 72%, wherein reaching as high as 75.0%；Followed by processing 6, average value reaches 61.3%, up to 64%.To base
Because type T8 Initial culture result carries out variance analysis, it is horizontal (table 2-8) that 9 processing have reached significant difference.
Influence of the table 2-7 hormone combination to T8 Initial culture
Table 2-8 genotype T8 Initial culture variance analysis
By further Duncan multiple alignment, it can be seen that processing 5 from table 2-9 and exist significantly with other 8 processing
Sex differernce and extremely significant sex differernce, and significant difference is not present between 8 processing；Moreover, the starting rate average value of processing 5
72.0%, hence it is evident that the average value handled greater than other 8 illustrates better than other 8 processing of processing 5.
The Duncan multiple alignment that table 2-9 carries out 9 processing
| Processing | 5% conspicuousness | 1% conspicuousness |
| 1 | b | B |
| 2 | b | B |
| 3 | b | B |
| 4 | b | B |
| 5 | a | A |
| 6 | b | B |
| 7 | b | B |
| 8 | b | B |
| 9 | b | B |
So the optimization process of T8 Initial culture is processing 5, i.e., 6-BA concentration is 2.0mg/L, and NAA concentration is 0.5mg/
L, IBA concentration are 0.1mg/L, to show that genotype T8 optimal medium primary is MS+2.0mg/L 6-BA+0.5mg/L
NAA+0.1mg/L IBA+5.0mg/L VC。
Embodiment 3: sub- U.S. Liriodendron chinense squamous subculture
Squamous subculture: in squamous subculture, using 3/4MS as minimal medium, and 6-BA, IBA, KT tri- of various concentration is added
Kind hormone, 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/
The VC of L.The high-pressure sterilizing pot sterilizing that all culture mediums pass through 121 DEG C of 20min uses after solidifying.The horizontal orthogonal examination of 3 factor 3
Test L9 (33), each genotype is inoculated with 20 bottles under each processing, is repeated 3 times.
Squamous subculture key step: it goes out firstly, filter paper, scissors, tweezers culture dish are put 121 DEG C of 20min of high-pressure sterilizing pot
Bacterium；Bacterium of having gone out is placed on superclean bench, opens ultraviolet lamp sterilizing 30min, opens ventilation after the completion of super-clean bench sterilizing.Then will
The sterilizable material of Initial culture is pressed from both sides with tweezers to filter paper in super-clean bench before this, and with scissors by its dead leaf and the portion of browning
Cutting removes in order to avoid influencing the follow-up cultivation of aseptic seedling.If it is more than the indefinite of 1cm that some startings, which grow comparatively fast and form bud height,
When bud, adventitious bud can be scaled off from root, place and continue to cultivate in culture medium.It does not exist together finally, carrying out different explants
The label of reason prevents from obscuring, and is placed culturing room and continues to cultivate.
As shown in Fig. 2, the tender shoots formed further growth on subculture medium, blade start to increase, stem is also opened
Beginning elongation growth.Bastem portion callusization is more obvious simultaneously, and under the regulation of hormone, it is indefinite to emerge since base portion callus
Bud.Squamous subculture 35d or so, adventitious buds differentiation is more obvious, and entire bud forms Multiple Buds shape, and growth coefficient increases.
(1) influence of the hormone combination to XY1 squamous subculture
It can be seen that in 9 processing from table 3-1, processing 6 reaches highest for the growth coefficient of XY1 squamous subculture,
Average value reaches 3.70, wherein up to 3.1；Followed by processing 5, average value 2.80, up to 3.1.To genotype XY1 subculture
Cultivation results carry out variance analysis, and it is horizontal (table 3-2) that 9 processing have reached significant difference.
Influence of the table 3-1 hormone combination to XY1 squamous subculture
Table 3-2 genotype XY1 squamous subculture variance analysis
By further Duncan multiple alignment, it can be seen that processing 6 from table 3-3 and exist significantly with other 8 processing
Sex differernce and extremely significant sex differernce；Processing 4, processing 5, processing 9 and other 5 processing in addition to processing 6 have reached conspicuousness
With extremely significant property level difference, but between three and there was no significant difference.From the point of view of the average value of growth coefficient, processing 6 is
3.70, hence it is evident that higher than processing 4,2.57,2.80, the 2.57 of 5, processing 9 is handled, so better than other 8 processing of processing 6.
The Duncan multiple alignment that table 3-3 carries out 9 processing
| Processing | 5% conspicuousness | 1% conspicuousness |
| 1 | d | D |
| 2 | bc | BC |
| 3 | bc | BC |
| 4 | b | B |
| 5 | b | B |
| 6 | a | A |
| 7 | bc | BC |
| 8 | bc | BC |
| 9 | b | B |
Therefore, the optimization process of XY1 squamous subculture is processing 6, i.e., 6-BA concentration is 1.0mg/L, and IBA concentration is 0.5mg/
L, KT concentration are 0.3mg/L, to show that genotype XY1 squamous subculture optimal medium is 3/4MS+1.0mg/L 6-BA+
0.5mg/L IBA+0.3mg/L KT+5.0mg/L VC。
(2) influence of the hormone combination to XY2 squamous subculture
It can be seen that in 9 processing from table 3-4, processing 6 reaches highest for the growth coefficient of XY2 squamous subculture,
Average value reaches 3.33, up to 3.6；Followed by processing 5, average value 2.70, up to 2.9.To genotype XY2 squamous subculture
As a result variance analysis is carried out, there are significant difference (table 3-5) for 9 processing.
Influence of the table 3-4 hormone combination for XY2 squamous subculture
Table 3-5 genotype XY2 squamous subculture variance analysis
By further Duncan multiple alignment, processing 5, processing 6 have reached significant difference with other 7 processing
It is horizontal with extremely significant sex differernce, significant difference has also been reached between processing 5 and processing 6 and extremely significant sex differernce is horizontal；But locate
The average value for managing 6 growth coefficients is 3.33, and the average value for handling 5 is 2.70, and processing 6 illustrates processing 6 better than it better than processing 5
His 8 processing (table 3-6).
3-69 processing Duncan multiple alignment of table
| Processing | 5% conspicuousness | 1% conspicuousness |
| 1 | f | E |
| 2 | e | BCDE |
| 3 | de | BCD |
| 4 | bc | BC |
| 5 | b | B |
| 6 | a | A |
| 7 | cde | BCD |
| 8 | cd | BCD |
| 9 | bcd | BC |
Therefore, the optimization process of XY2 squamous subculture is processing 6, i.e., 6-BA concentration is 1.0mg/L, and IBA concentration is 0.5mg/
L, KT concentration are 0.3mg/L, to show that genotype XY2 squamous subculture optimal medium is 3/4MS+1.0mg/L 6-BA+
0.5mg/L IBA+0.3mg/L KT+5.0mg/L VC。
(3) influence of the hormone combination to T8 squamous subculture
It can be seen that in 9 processing from table 3-7, processing 6 reaches highest for the growth coefficient of T8 squamous subculture,
Average value reaches 3.57, up to 3.8；Followed by processing 5 and processing 9, average value has reached 2.40.To genotype T8 subculture
Cultivation results carry out variance analysis, and there are significant difference (table 3-8) for 9 processing.
Influence of the table 3-7 hormone combination to T8 squamous subculture
Table 3-8 genotype T8 squamous subculture variance analysis
By further Duncan multiple alignment, processing 6 can be seen that with processing 5, processing 9 in the presence of significant from table 3-9
Sex differernce, there are significant differences for processing 5, processing 9 and 6 processing in addition to processing 6；Processing 6 is also deposited with other 8 processing
Extremely significant sex differernce is not present in extremely significant sex differernce, but between other 8 processing.Moreover, the mean value of 6 growth coefficients of processing is
3.57, hence it is evident that be greater than other 8 processing, illustrate processing 6 better than other processing.
The Duncan multiple alignment of 3-9, table processing
Therefore, the optimization process of T8 squamous subculture is processing 6, i.e., 6-BA concentration is 1.0mg/L, and IBA concentration is 0.5mg/
L, KT concentration are 0.3mg/L, to show that genotype T8 squamous subculture optimal medium is 3/4MS+1.0mg/L 6-BA+
0.5mg/L IBA+0.3mg/L KT+5.0mg/L VC。
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 main process of strong seedling culture: firstly, filter paper, scissors, tweezers culture dish are put 121 DEG C of 20min of high-pressure sterilizing pot
Sterilizing, bacterium of having gone out are placed on superclean bench, open ultraviolet lamp sterilizing 30min, open ventilation after the completion of super-clean bench sterilizing.Then
Explant is quickly transferred on strong seedling culture base with heat sterilization cooling tweezers, seals sealed membrane, different disposal carries out mark
Note, is put in culturing room and continues to cultivate.
When stem length is to about 1cm, proliferation Multiple Buds are cut from base portion, are inoculated in strong seedling culture base.It is left to be inoculated with 30d
The right side can obviously observe the stem elongation growth of adventitious bud, average stem length 2-3cm, the blade of adventitious bud also by initial 4-5 piece,
Increase to 5-8 piece (Fig. 3).
(1) influence of the hormone combination to XY1 strong seedling culture
It can be seen that in 9 processing from table 4-1, average single-strain blade of the processing 5 for XY1 strong seedling culture culture
Number reaches at most, and average value reaches 8.33, up to 9.1；Followed by processing 4, average value is up to 7.93, highest 8.3.To genotype
XY1 strong seedling culture result carries out variance analysis, and there are significant difference (table 4-2) for 9 processing.
Influence of the table 4-1 hormone combination to XY1 strong seedling culture
Table 4-2 genotype XY1 strong seedling culture variance analysis
By further Duncan multiple alignment, from table 4-3 can be seen that processing 4, processing 5 is reached with other processing
It has arrived conspicuousness and extremely significant sex differernce is horizontal, also reached conspicuousness and extremely significant sex differernce water between processing 4 and processing 5
It is flat；But from the point of view of single plant average leaf number average value, 5 average values 8.33 of processing are greater than processing average value 47.93, illustrate processing 5
Better than other processing.
The Duncan multiple alignment that table 4-3 carries out 9 processing
Therefore, XY1 strong seedling culture optimization process is processing 5, i.e., 6-BA concentration is 0.5mg/L, and IBA concentration is 0.3mg/L,
KT concentration is 0.1mg/L, to show that genotype XY1 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 strong seedling culture
It can be seen that in 9 processing from table 4-4, processing 5 reaches the average single-strain blade number of XY2 strong seedling culture
To most, average value reaches 8.37, up to 9.4；Followed by processing 4, average value is up to 7.57, highest 8.5.To genotype XY2
Strong seedling culture result carries out variance analysis, and there are significant difference (table 4-5) for 9 processing.
Influence of the table 4-4 hormone combination to XY2 strong seedling culture
Table 4-5 genotype XY2 strong seedling culture variance analysis
By further Duncan multiple alignment, it can be seen that processing 4, processing 5 and other processing from table 4-6 and exist
Significant difference, there is also significant differences between processing 4 and processing 5；There is also extremely significant property is poor with other processing for processing 5
It is different, but handle 4 and be not present；In terms of average single-strain blade number average value, 5 average values of processing are 8.37, hence it is evident that are greater than its elsewhere
Reason illustrates processing 5 better than other processing.
The Duncan multiple alignment of 4-6, table processing
| Processing | 5% conspicuousness | 1% conspicuousness |
| 1 | bc | AB |
| 2 | bc | AB |
| 3 | bc | AB |
| 4 | ab | AB |
| 5 | a | A |
| 6 | bc | AB |
| 7 | c | B |
| 8 | c | B |
| 9 | c | B |
Therefore, the optimization process of XY2 strong seedling culture 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 strong seedling culture optimal medium is MS+0.5mg/L 6-BA+0.3mg/
L IBA+0.1mg/L KT+5.0mg/L VC。
(3) influence of the hormone combination to T8 strong seedling culture
It can be with from table 4-7, it will be seen that in 9 processing, processing 5 reaches the average single-strain blade number of T8 strong seedling culture
At most, average value reaches 8.53, up to 9.3；Followed by processing 4, average value is up to 7.50, up to 8.3.It is strong to genotype T8
Seedling cultivation results carry out variance analysis, and there are significant difference (table 4-8) for 9 processing.
Influence of the table 4-7 hormone combination to T8 strong seedling culture
Table 4-8 genotype T8 strong seedling culture variance analysis
By further Duncan multiple alignment, from table 4-9 can be seen that processing 4, processing 5 is deposited with other processing
There is also significant differences and extremely significant sex differernce between significant difference and extremely significant sex differernce, processing 4 and processing 5；?
In terms of average single-strain blade number average value, 5 average values of processing are 8.53, and 4 average values of processing are 7.50, and processing 5, which is significantly greater than, to be located
Reason 4 illustrates processing 5 better than other processing.
The Duncan multiple alignment of 4-9, table processing
| Processing | 5% conspicuousness | 1% conspicuousness |
| 1 | bc | B |
| 2 | bc | B |
| 3 | bc | B |
| 4 | ab | AB |
| 5 | a | A |
| 6 | bc | B |
| 7 | c | B |
| 8 | c | B |
| 9 | c | B |
Therefore, the optimization process of T8 strong seedling culture 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 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 tissue culture 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.
Root media, which can be seeded to, when stem length to 2cm or so, blade are 9 or so carries out culture of rootage.Ya Mei
Liriodendron chinense tissue-cultured seedling belongs to callus type and takes root, and re-forms root after forming callus.It is seeded to root media 15d or so, bastem portion
Callus increases, and milky protrusion callus occurs in surface；After cultivating about 25d, gradually there is root to be formed in milky callus, and constantly stretch
Long growth；After 35d, root is basically formed, and surface gradually fades to nattierblue by tender white.Although the root that bud proliferation seedling is formed compares
Stalwartness, but quantity is few (Fig. 4).
(1) influence of various concentration NAA, the IBA for XY1 culture of rootage
It can be seen that in 9 processing from table 5-1, processing 2 reaches highest for the rooting rate of XY1 culture of rootage, puts down
Mean value reaches 48.3%, and highest rooting rate reaches 51%；Followed by processing 8, average value is up to 35.0%, highest 40%.To base
Because type XY1 culture of rootage result carries out variance analysis, there are significant difference (table 5-2) for 9 processing.
Influence of table 5-1 various concentration NAA, the IBA for XY1 culture of rootage
Table 5-2 genotype XY1 culture of rootage variance analysis
By further Duncan multiple alignment, from table 5-3 can be seen that processing 2, processing 8 is reached with other processing
It has arrived significant difference and extremely significant sex differernce is horizontal, also reached significant difference and extremely significant property between processing 2 and processing 8
Level of difference；But the average value 48.3% for handling 2 rooting rates is apparently higher than the average value 35.0% of processing 8, illustrates that processing 2 is better than
Other processing.
The Duncan multiple alignment that table 5-3 carries out 9 processing
| Processing | 5% conspicuousness | 1% conspicuousness |
| 1 | d | C |
| 2 | a | A |
| 3 | d | C |
| 4 | bcd | BC |
| 5 | bc | BC |
| 6 | bc | BC |
| 7 | d | C |
| 8 | b | B |
| 9 | cd | BC |
So the optimization process of XY1 culture of rootage 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 culture of rootage is 1/2MS+0.2mg/L IBA+0.1mg/L NAA+5.0mg/L VC.
(2) influence of various concentration NAA, the IBA for XY2 culture of rootage
It can be seen that in 9 processing from table 5-4, processing 2 reaches highest for the rooting rate of XY2 culture of rootage, puts down
Mean value reaches 51.67%, and highest rooting rate reaches 55%；Followed by processing 8, average value is up to 34.33%, highest 37%.It is right
Genotype XY2 culture of rootage result carries out variance analysis, and there are significant difference (table 5-5) for 9 processing.
Influence of table 5-4 various concentration NAA, the IBA for XY2 culture of rootage
Table 5-5 genotype XY2 culture of rootage variance analysis
By further Duncan multiple alignment, from table 5-6 can be seen that processing 2, processing 8 reaches with other processing
Significant difference is horizontal, and significant difference level has also been reached between processing 2 and processing 8；Processing 2 is gone back with other 8 processing
Reach extremely significant level of difference, and extremely significant sex differernce is not present between other 8 processing, and handles the average value of 2 rooting rates
51.67%, hence it is evident that be higher than other processing, illustrate processing 2 better than other processing.
The Duncan multiple alignment that table 5-6 carries out 9 processing
| Processing | 5% conspicuousness | 1% conspicuousness |
| 1 | bc | B |
| 2 | a | A |
| 3 | bc | B |
| 4 | bc | B |
| 5 | bc | B |
| 6 | bc | B |
| 7 | c | B |
| 8 | b | B |
| 9 | bc | B |
So the optimization process of XY2 culture of rootage 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 culture of rootage is 1/2MS+0.2mg/L IBA+0.1mg/L NAA+5.0mg/L VC.
(3) various concentration NAA, IBA handles the influence to T8 culture of rootage
It can be seen that in 9 processing from table 5-7, processing 2 reaches highest for the rooting rate of T8 culture of rootage, puts down
Mean value reaches 46.33%, and highest rooting rate reaches 49%；Processing 8 is inferior to processing 2 for the rooting rate of T8 culture of rootage, and
There is 12% gaps for rooting rate between the two.Variance analysis is carried out to genotype T8 culture of rootage result, 9 processing exist
Significant difference (table 5-8).
Influence of table 5-7 various concentration NAA, the IBA for T8 culture of rootage
Table 5-8 genotype T8 culture of rootage variance analysis
By further Duncan multiple alignment, it can be seen that processing 2 from table 5-9 and reached aobvious with other processing
It writes sex differernce and extremely significant sex differernce is horizontal；Processing 6, processing 8 have also reached significant difference and extremely significant property with other processing
Level of difference, there was no significant difference between processing 6 and processing 8；Processing 2, processing 6 and handle 8 rooting rate average value be respectively
46.33%, 31.33% and 34.33%, 2 > of processing handle 8 > processing 6, illustrate processing 2 better than other processing.
The Duncan multiple alignment that table 5-9 carries out 9 processing
So the optimization process of T8 culture of rootage 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 culture of rootage.
Embodiment 6: the training tissue culture seedling culture 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 (Fig. 5).After two weeks, blade is gradually become dark green by yellow green, and leaf area increases, and stem is not yet for culture
Disconnected elongation growth, original children's tender stem start lignifying occur, and surface becomes light brown.
From fig. 6, it can be seen that compared with the control group, transplanting survival rate can be significantly improved for the hardening of tissue-cultured seedling, most
Improve 26% more.In hardening number of days, preceding 5d, as time goes by, transplanting survival rate is in rising trend, and 5d reaches highest
Value 85%, and in 7d, transplanting survival rate is again on a declining curve, and only 75%.It follows that when hardening 5d, tissue-cultured seedling pair
It has been adapted to substantially in external environment, has become seedling, more suitable period when transplanting at this time.
Claims (10)
1. a kind of using bud as the quick-breeding method of the U.S. Liriodendron chinense in the Asia of explant, which comprises the following steps:
(1) terminal bud or lateral bud are acquired as explant, pretreatment and disinfection；
(2) explant of the acquisition after step (1) being pre-processed and sterilized, which is inoculated into initial culture base, to be cultivated, wherein described
Initial culture base based on MS culture medium, added with 1.0~3.0mg/L 6-BA, 0.3~1.0mg/L NAA and 0.1~
0.5mg/L IBA；
(3) the callus tender shoots that Initial culture obtains is transferred in subculture medium and is cultivated, wherein the subculture medium is with 3/
Based on 4MS culture medium, it is added with 0.5~2.0mg/L 6-BA, 0.1~0.5mg/L IBA and 0.1~0.5mg/L KT；
(4) Multiple Buds for obtaining squamous subculture are transferred in strong seedling culture base and cultivate, 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 bud as the U.S. Liriodendron chinense quick-breeding method in the Asia of explant described in claim 1, which is characterized in that primary in step (2)
Culture medium is added with 2.0mg/L 6-BA, 0.5mg/L NAA, 0.1mg/L IBA and 5.0mg/L based on MS culture medium
VC。
3. using bud as the U.S. Liriodendron chinense quick-breeding method in the Asia of explant described in claim 1, which is characterized in that subculture in step (3)
Culture medium is added with 1.0mg/L 6-BA, 0.5mg/L IBA, 0.3mg/L KT and 5.0mg/L based on 3/4MS culture medium
VC。
4. using bud 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 bud 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 bud 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 bud as the U.S. Liriodendron chinense quick-breeding method in the Asia of explant
Method are as follows: 4 DEG C of refrigerators of explant of acquisition place 1-2d, and dish washing liquid cleans explant 2 times, peels off the outermost one layer of bract of bud, use
Diluted 84 medicining liquid dipping 5-10min, is placed in conical flask, is tied with one layer of gauze, places tap water down-flow water and rinses 2-
4h is transferred to superclean bench.The flushed explant of flowing water is put into the conical flask for bacterium of having gone out in advance, high pressure sterilization is first used
The sterile shaking water of cooling clean 2 times, impregnate 30s with the alcohol of 75% concentration, impregnated again with sterile water wash 1 time, use
The mercuric chloride of 0.1% concentration sterilizes, and disinfecting time is set as 5~20min；Mercuric chloride keeps conical flask oscillating and shaking during sterilizing, and makes
Mercuric chloride is sufficiently and material；It finally uses sterile water wash 5-6 times, each 5min, remaining mercuric chloride is dispelled as far as possible dry
Only；By the explant being disposed aseptic filter paper suck dry moisture.
8. a kind of using bud 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 bud 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) the sub- U.S. Liriodendron chinense terminal bud of acquisition in April 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 initial culture base, to be cultivated, wherein described
Initial culture base is added with 2.0mg/L 6-BA, 0.5mg/L NAA, 0.1mg/L IBA and 5.0mg/ based on MS culture medium
L VC；
(3) the callus tender shoots of Initial culture is transferred in subculture medium and is cultivated, wherein the subculture medium is trained with 3/4MS
Based on supporting base, it is added with 1.0mg/L 6-BA, 0.5mg/L IBA, 0.3mg/L KT and 5.0mg/L VC；
(4) Multiple Buds for obtaining squamous subculture are transferred in strong seedling culture base and cultivate, 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 bud 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) the sub- U.S. Liriodendron chinense lateral bud of acquisition in April 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 initial culture base, to be cultivated, wherein described
Initial culture base is added with 2.0mg/L 6-BA, 0.5mg/L NAA, 0.1mg/L IBA and 5.0mg/ based on MS culture medium
L VC；
(3) the callus tender shoots of Initial culture is transferred in subculture medium and is cultivated, wherein the subculture medium is trained with 3/4MS
Based on supporting base, it is added with 1.0mg/L 6-BA, 0.5mg/L IBA, 0.3mg/L KT and 5.0mg/L VC；
(4) Multiple Buds for obtaining squamous subculture are transferred in strong seedling culture base and cultivate, 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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| CN117378503A (en) * | 2023-11-23 | 2024-01-12 | 南京林业大学 | Tissue culture and rapid propagation method of hybrid tulip tree |
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| CN117378503A (en) * | 2023-11-23 | 2024-01-12 | 南京林业大学 | Tissue culture and rapid propagation method of hybrid tulip tree |
| CN117378503B (en) * | 2023-11-23 | 2024-07-05 | 南京林业大学 | Tissue culture and rapid propagation method of hybrid tulip tree |
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