CN107223566B - A kind of Wulian poplar method for tissue culture - Google Patents
A kind of Wulian poplar method for tissue culture Download PDFInfo
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- CN107223566B CN107223566B CN201710449631.5A CN201710449631A CN107223566B CN 107223566 B CN107223566 B CN 107223566B CN 201710449631 A CN201710449631 A CN 201710449631A CN 107223566 B CN107223566 B CN 107223566B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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Abstract
The present invention relates to field of plant tissue culture, especially a kind of Wulian poplar method for tissue culture.The present invention provides a kind of Wulian poplar method for tissue culture, comprises the steps of：Select leaf stem section as explant, carry out disinfection disinfecting action, it is cultivated in implantation MS blank cultures, incubation time is 25d, it carries out Primary culture and obtains seedling primary, it is inoculated in vitrifying culture medium from non-vitrifying stem section is chosen in seedling primary, carry out vitrifying culture 35d, obtain vitrifying seedling, vitrifying seedling will be gone to be inoculated in progress squamous subculture 35d in proliferated culture medium, obtain Regenerated plant, culture of rootage is carried out to Regenerated plant and obtains rooted seedling, acclimatization and transplants operation is carried out to rooted seedling, obtains healthy and strong Wulian poplar seedling.Method for tissue culture provided by the invention is easy to operate, reduces vitrified incidence during the poplar tissue culture of Wulian, reduces Wulian poplar and cultivates cost, pushes the commercialization culture of Wulian poplar.
Description
Technical field
The present invention relates to field of plant tissue culture, especially a kind of Wulian poplar method for tissue culture.
Background technique
Wulian poplar (Populus wulianensis) belongs to Salicaceae (Salicaceae), Populus (Populus) white poplar
Group is tree species difficult to take root.Itself also known as Kunyu Mountain poplar are the peculiar rare tree species in Shandong, be distributed mainly on Shandong Rizhao (Wulian mountain) and
The ground such as Yantai (Kunyu Shan Mountain recruits Tiger Mountain), are born in the shaw of the gully 300~500m of height above sea level, scattered or formation piece woods.It is situated between in form
It is good between aspen (Polulus davidiana Dode.) and Populus adenopoda (Polulus adenopuda Maxim.)
Commerical tree species, there is excellent resistance to water logging ability, have a very strong resilience to big flood and stormy waves, adapt to the powerful life of wind-force
Long environment.
Poplar Breeds resources are bred using method for tissue culture, are not limited by season and growth time, Ke Yi
A large amount of nursery stocks are obtained with faster speed in short time.In conventional poplar tissue culture procedures, especially Properties of Populus Clones is planted
In the tissue cultures of object, easily there is vitrification phenomenon.Vitrification phenomenon is distinctive a kind of physiology mistake in Plant Tissue Breeding
Adjust or physiology lesion, show as test tube seedling be translucent shape, chlorosis, leaf-shrinkage curling, it is frangible.Wulian poplar is in Initial culture mistake
Vitrifying incidence is 50%~80% in journey.The vitrifying incidence during tissue culture is reduced, the training of poplar tissue is become
Feeding primary study problem.
Summary of the invention
The present invention is intended to provide a kind of Wulian poplar method for tissue culture, solves vitrifying incidence during Wulian poplar tissue culture
Height, survival rate is low, can not promote the problem of Wulian poplar scale breeding.
The technical proposal of the invention is realized in this way, and steps are as follows：
(1) Initial culture：Explant is inoculated into initial culture base and carries out Primary culture, obtains seedling primary；
(2) vitrifying culture is gone：By seedling primary obtained in the previous step, it is inoculated in vitrifying culture medium and carries out glass
Change culture, obtains vitrifying seedling；
(3) Multiplying culture：Vitrifying seedling will be gone to be inoculated in proliferated culture medium and carry out Multiplying culture, obtain Regenerated plant；
(4) culture of rootage：Regenerated plant is transferred in root media and carries out culture of rootage, obtains rooted seedling；
The initial culture base is, adds the MS basal medium of 25g/L sucrose+5.5g/L agar powder, adjust pH value to
5.8；
It is described to go the vitrifying culture medium to be, add 0.05mg/LNAA+0.4mg/L 6-BA+30g/L sucrose+6.5g/L fine jade
1/2 (N) MS culture medium of cosmetics adjusts pH value to 5.8；
The proliferated culture medium is to add 0.03mg/LNAA+0.4~0.5mg/L 6-BA+30g/L sucrose+6.5g/L fine jade
The MS culture medium of cosmetics adjusts pH value to 5.8；
The root media is to add the 1/2MS culture medium of 0.05mg/LNAA+15g/L sucrose+6.5g/L agar powder,
PH value is adjusted to 5.8.
Embodiment as one preferred, the explant select the band stem eye section of 1.5~2.0cm.
Embodiment as one preferred, the band stem eye section are the new pumping top tape buds of acquisition mid-March in spring
Stem section.
Embodiment as one preferred, the Initial culture further include before explant disinfecting process, the explant
Body disinfecting process：Explant surface is cleaned with washing powder, rinses explant overnight with thread water, the explant that will be rinsed well,
It is placed on superclean bench and sterilizes, using 75% ethanol postincubation 35s, carried out disinfection place with 5% sodium hypochlorite processing 9min
Reason, band stem eye section is cut into 0.8~1.5cm leaf stem section after disinfection, is placed in initial culture base and cultivates.
The condition of culture of embodiment as one preferred, the tissue cultivation rapid breeding method is：Day temperature is 25
± 2 DEG C, 18 ± 2 DEG C of night, light application time 16h, intensity of illumination is 3000~5000LX.
Embodiment as one preferred, the time of the Initial culture are 25d, the time for going vitrifying culture
It is 35d, the time of the Multiplying culture is 35d.
Embodiment as one preferred, the method for tissue culture further include hardening, the hardening：Rooted seedling is moved
It plants and seals hardening in greenhouse, after 5d, remove sterility cover, continue 3~4d of hardening.
Embodiment as one preferred, the method for tissue culture further include transplanting, the transplanting：Clear water is flushed into
Seedling living is transplanted to the Light media sterilized by 0.5% potassium permanganate, and the Light media includes turf and vermiculite, institute
Stating the ratio of turf and vermiculite in Light media is 1:1.
Beneficial effects of the present invention：Method for tissue culture provided by the invention increases consolidation step, will be by just
The aseptic seedling obtained after supporting of being commissioned to train is inoculated on vitrifying culture medium, by going consolidation step to reduce Wulian poplar tissue culture glass
Change incidence, goes vitrifying Medium Proportion simple, while in order to more preferably solve technical problem, the explant that the present invention selects is excellent
Stem with bud at the top of the new pumping of acquisition mid-March in spring is first selected, 1.5~2.0cm length is cut into, carries out tissue cultures, is led to
The proliferation for crossing bud is quickly bred, and ensure that the stability of heredity, materials are easy；Meanwhile tissue cultures provided by the invention
Method reduces tissue breeding cost, improves Wulian poplar tissue culture efficiency.
Detailed description of the invention
Fig. 1 is the photo figure for the explant that sterilizing survives；
Fig. 2 is the photo figure of Wulian Yang Wei vitrifying seedling；
Fig. 3 is the photo figure of vitrifying tissue-cultured seedling；
Fig. 4 is the photo figure of proliferation and subculture culture；
Fig. 5 is the photo figure of cultivation stage of taking root；
Fig. 6 is that acclimatization and transplants survive rear plant photo figure.
Specific embodiment
Technical solution of the present invention is clearly and completely retouched below in conjunction with specific embodiments of the present invention and attached drawing
It states, it is clear that described embodiment is only a part of the embodiment of the present invention, instead of all the embodiments.Based in the present invention
Embodiment, every other embodiment obtained by those of ordinary skill in the art without making creative efforts,
It shall fall within the protection scope of the present invention.
Embodiment one
A kind of Wulian poplar method for tissue culture, steps are as follows：
(1) explant disinfection culture
The band stem eye section that 1.5~2.0cm is quickly cut with secateurs, uses thread with washing powder clean the surface as explant
Water rinses overnight, and the stem with bud rinsed well is placed on superclean bench, using 75% ethanol postincubation 35s, with 5% chlorine
Sour sodium processing 9min sterilizes, and band stem eye section after detoxification is cut into 0.8~1.5cm with scalpel, is placed in MS blank cultures
Middle culture, incubation time are 25d, and cultivation results are referring to Fig.1.
(2) vitrifying culture is gone：From aseptic seedling obtained in the previous step, non-vitrifying, the high 1.5~2.0cm of stem are chosen
Tufted seedling stem section is inoculated in vitrifying culture medium and carries out vitrifying culture 35d, obtains vitrifying seedling.Non- vitrifying
Seedling is referring to Fig. 2.Spent vitrifying culture medium is tested, is to choose MS or 1/2 (N) MS as mother liquor, selectivity adds NAA, 6-
Two kinds of archusias of BA and active carbon (AC), culture medium pH value are adjusted to 5.8, and addition the results are shown in Table 1.
Table 1 goes the selection of vitrifying culture medium
Test method：The stem section of the non-vitrifying of selection, the high 1.5~2.0cm of stem is divided into 11 groups, every group of 48 stem sections,
Subjects are inoculated in different culture medium listed by table 1 and are cultivated, unified illumination cultivation condition：Day temperature is 25
± 2 DEG C, 18 ± 2 DEG C of night, light application time 16h, intensity of illumination is 3000~5000LX, and cultivated days are 35d, obtain each group
Vitrifying incidence data, test result are shown in Table 2.
The vitrifying incidence data of 2 different culture medium of table
As can be seen from Table 2, N content halves and can extenuate vitrifying in MS mother liquor, growth coefficient is improved.After AC is added, vitrifying
Degree is not eased, and can obtain herein, and AC is added and does not play beneficial effect to Wulian poplar vitrifying.According to integrated data
Analysis, 1/2 (N) MS+0.05mg/LNAA+0.4mg/L 6-BA culture medium vitrifying incidence are 10.1%, and value-added coefficient reaches
7.37, the average a height of 3.33cm of stem；1/2 (N) MS+0.05mg/LNAA+0.5mg/L 6-BA culture medium vitrifying incidence is reduced to
14%, for value-added coefficient up to 7.35, the average a height of 3.51cm of stem, above two culture medium, which all has, significantly goes vitrifying effect.
Second has been carried out on the basis of the above to test：The stem section for choosing non-vitrifying, the high 1.5~2.0cm of stem, is divided into 8
Group, every group 48, in 1/2 (N) MS+0.05mg/LNAA+0.4mg/L 6-BA culture medium and 1/2 (N) MS+0.05mg/LNAA+
The sucrose and agar powder of various concentration are added on 0.5mg/L 6-BA medium base respectively, culture medium pH value is adjusted to 5.8, into one
Step carries out vitrifying and tests.
What table 3 added sucrose and agar removes vitrifying culture medium
| Processing | Mother liquor | NAA(mg/L) | 6-BA(mg/L) | Sucrose (g/L) | Agar powder (g/L) |
| 1 | 1/2(N)MS | 0.05 | 0.4 | 25 | 6 |
| 2 | 1/2(N)MS | 0.05 | 0.4 | 25 | 6.5 |
| 3 | 1/2(N)MS | 0.05 | 0.4 | 30 | 6 |
| 4 | 1/2(N)MS | 0.05 | 0.4 | 30 | 6.5 |
| 5 | 1/2(N)MS | 0.05 | 0.5 | 25 | 6 |
| 6 | 1/2(N)MS | 0.05 | 0.5 | 25 | 6.5 |
| 7 | 1/2(N)MS | 0.05 | 0.5 | 30 | 6 |
| 8 | 1/2(N)MS | 0.05 | 0.5 | 30 | 6.5 |
After test tube seedling stem section is inoculated into culture medium, illumination cultivation condition is：Day temperature is 25 ± 2 DEG C, night 18 ± 2
DEG C, light application time 16h, intensity of illumination is 3000~5000LX.35d is cultivated, cultivation results are shown in Table 4.
What table 4 added sucrose and agar goes vitrifying test result
By table 4 as it can be seen that increasing the addition of sucrose and agar powder, vitrifying incidence is advantageously reduced, adds 30g/L sucrose
When+6.5g/L agar powder, there is vitrifying incidence minimum；From comprehensive data analysis it is found that 1/2 (N) MS+0.05mg/
The culture medium vitrifying incidence minimum of LNAA+0.4mg/L 6-BA+30g/L sucrose+6.5g/L agar powder is 0, proliferation system
Number is 7.65, and average stem height is 3.75cm.Obtained from the average glass degree horizontal data result of table 4, high concentration sucrose and
The vitrifying that the addition of agar is conducive to Wulian poplar test tube seedling removes.Therefore the vitrifying culture medium that goes selected is to add 0.05mg/
1/2 (N) MS culture medium of LNAA+0.4mg/L 6-BA+30g/L sucrose+6.5g/L agar powder, cultivation results are referring to Fig. 3.
(4) Multiplying culture：Vitrifying seedling will be gone to be inoculated in progress Multiplying culture 35d in proliferated culture medium, obtain Regenerated plant,
Test proliferated culture medium used adds the NAA and 6-BA of various concentration using MS culture medium as mother liquor, and addition situation is shown in Table 5.
The test of 5 Wulian poplar proliferated culture medium of table
| Processing | NAA(mg/L) | 6-BA(mg/L) |
| 1 | 0.03 | 0.4 |
| 2 | 0.04 | 0.4 |
| 3 | 0.05 | 0.4 |
| 4 | 0.03 | 0.5 |
| 5 | 0.04 | 0.5 |
| 6 | 0.05 | 0.5 |
| 7 | 0.03 | 0.6 |
| 8 | 0.04 | 0.6 |
| 9 | 0.05 | 0.6 |
Test method：Healthy and strong test tube tufted seedling stem section is divided into 9 groups, every group of 48 stem sections, respectively MS+30g/L sucrose+
It is added in 6.5g/L agar powder in the proliferated culture medium of the NAA and 6-BA of various concentration and carries out orthogonal test, culture medium pH value tune
It is 5.8.Condition of culture is：Day temperature be 25 ± 2 DEG C, 18 ± 2 DEG C of night, light application time 16h, intensity of illumination be 3000~
5000LX.35d is cultivated, each group cultivation effect is shown in Table 6.
6 Wulian poplar Multiplying culture result of table
As shown in Table 6, the growth coefficient of processing 1 and processing 4 is more prominent, and effective seedling and stem height have high value, handles 1 glass
It is 0 that glass incidence, which has minimum value,；Handling 4 vitrifying incidences is 1.4%.Compared with table 5, first carries out vitrifying and train
It supports, fine adjustment hormone concentration is conducive to the amplification of tissue-cultured seedling growth coefficient and the elongation of stem, and promotes the increase of effective seedling.
From vitrifying incidence minimum, addition 0.03mg/LNAA+0.4mg/L 6-BA is selected conducive to the culture medium of Wulian poplar proliferation
The MS culture medium of+30g/L sucrose+6.5g/L agar powder, the cultivation results obtained on this culture medium, referring to Fig. 4.
(5) culture of rootage：Regenerated plant is transferred to progress culture of rootage 35d in root media, obtains rooted seedling.Used
Root media selects in 1/2MS+6.5g/L agar powder the root media for adding various concentration sucrose, NAA or IBA, culture
Base selection the results are shown in Table 7.
The test of 7 root media of table
| Processing | NAA(mg/L) | IBA(mg/L) | Sucrose (g/L) |
| 1 | 0.01 | 30 | |
| 2 | 0.02 | 30 | |
| 3 | 0.03 | 30 | |
| 4 | 0.04 | 30 | |
| 5 | 0.05 | 30 | |
| 6 | 0.05 | 15 | |
| 7 | 0.04 | 30 | |
| 8 | 0.08 | 30 | |
| 9 | 0.12 | 30 | |
| 10 | 0.16 | 30 | |
| 11 | 0.2 | 30 | |
| 12 | 0.2 | 15 |
Test method：Healthy and strong test tube tufted seedling stem section is divided into 12 groups, every group of 48 stem sections, respectively in 1/2MS+6.5g/L
Concentration gradient test, culture medium pH value tune are carried out in the root media of agar powder and additional various concentration sucrose, NAA or IBA
It is 5.8.Unifying condition of culture is：Day temperature is 25 ± 2 DEG C, and 18 ± 2 DEG C of night, light application time 16h, intensity of illumination is
3000~5000LX, cultivated days are 35d.Each culture medium cultivation results are shown in Table 8.
The different root media test results of table 8
As shown in Table 8, integrally reach peak-peak and be both present in processing 6, for rooting rate up to 91.7%, average stem is a height of
4.58cm, vitrifying incidence are 0.From addition single factors analysis, hormone NAA function and effect are better than IBA；After sucrose halves,
Conducive to Wulian poplar tissue culture seedling rooting.
Therefore it is conducive to Wulian poplar root media, selects addition 0.05mg/LNAA+15g/L sucrose+6.5g/L agar powder 1/
2MS culture medium, the cultivation results on this root media are referring to Fig. 5.
(6) hardening is sealed in hardening, the transplantation of seedlings that will take root in greenhouse, after 5d, is removed sterility cover, is continued hardening 3d, with clear
The culture medium that washing goes to root system surface to adhere to.
(7) it transplants, selects the seedling survived, the culture medium of seedling root is rinsed out with clear water, is transplanted to through 0.5%
The Light media of potassium permanganate disinfection, the Light media includes turf and vermiculite, and the ratio of turf and vermiculite is in the Light media
1:1, transplant survival situation is referring to Fig. 6.
Embodiment two
Difference with embodiment one is：The proliferated culture medium of selection is to add 0.03mg/LNAA+0.5mg/L 6-BA+
The MS culture medium of 30g/L sucrose+6.5g/L agar powder.
Vitrifying seedling will be gone to be inoculated in progress Multiplying culture 35d in proliferated culture medium, obtain Regenerated plant, test increasing used
Culture medium is grown using MS culture medium as mother liquor, adds the NAA and 6-BA of various concentration, and addition situation is shown in Table 9.
The test of 9 Wulian poplar proliferated culture medium of table
Test method：Healthy and strong test tube tufted seedling stem section is divided into 9 groups, every group of 48 stem sections, respectively MS+30g/L sucrose+
It is added in 6.5g/L agar powder in the proliferated culture medium of the NAA and 6-BA of various concentration and carries out orthogonal test, culture medium pH value tune
It is 5.8.Condition of culture is：Day temperature be 25 ± 2 DEG C, 18 ± 2 DEG C of night, light application time 16h, intensity of illumination be 3000~
5000LX.35d is cultivated, each group cultivation effect is shown in Table 10.
10 Wulian poplar Multiplying culture result of table
As shown in Table 10, the growth coefficient of processing 1 and processing 4 is more prominent, and effective seedling and stem height have high value, processing 1
It is 0 that vitrifying incidence, which has minimum value, and effective seedling is 6.67 plants, and growth coefficient is 10.5, the average high 5.88cm of stem；Handle 4 glass
Glass incidence is 1.4%, and effective seedling is 6.67 plants, growth coefficient 11.58, the average a height of 5.96cm of stem, handle 4 increasing
It grows coefficient and is higher than processing 1, average stem height is higher than processing 1, and under comprehensive comparison, the culture medium conducive to Wulian poplar proliferation selects addition
The MS culture medium of 0.03mg/LNAA+0.5mg/L6-BA+30g/L sucrose+6.5g/L agar powder, the training obtained on this culture medium
Support result.
Embodiment three
Difference with embodiment one is：Wulian poplar tissue culture procedures are followed successively by this implementation：Explant sterilizes, is primary
It cultivates, go vitrifying culture, Multiplying culture, culture of rootage, hardening, transplanting, go before vitrifying culture, i.e., explant sterilizes it
After increase Initial culture step.Steps are as follows for Initial culture：
Primary culture is carried out by initial culture base is inoculated by the explant of disinfection, culture obtains aseptic seedling primary, just
The MS basal medium of addition 25g/L sucrose+5.5g/L agar powder, pH value adjustment to 5.8, condition of culture are selected for culture medium
For：Day temperature is 25 ± 2 DEG C, and 18 ± 2 DEG C of night, light application time 16h, intensity of illumination is 3000~5000LX, when culture
Between be 25d, the seedling primary after culture is seeded in vitrifying culture culture and carries out vitrifying culture.
Common initial culture base is for Induced cultures axillary bud sprouting, to obtain the sterile spire of callus induction,
It usually selects and adds growth hormone, mitogen etc. on the basis of MS basal medium.8 groups are selected, every group 48 sterile stem sections are made
For experimental subjects, relatively common initial culture base, the i.e. basis the MS culture of addition 0.5mg/L6-BA+0.05mg/LNAA are selected
Base as a control group, applies in the present embodiment, compares with the MS basal medium of 25g/L sucrose+5.5g/L agar powder is selected
Cultivation results, control group are followed successively by with experimental group incubation step：Explant disinfection, Initial culture go vitrifying culture, proliferation training
It supports, culture of rootage, hardening, transplanting, Initial culture selects 8 groups, and every group of 48 sterile stem sections, glass rate the results are shown in Table 11.
11 initial culture base test result of table
By in table 11, it can be seen that carry out Initial culture, glass to poplar stem section with the initial culture base of the present embodiment
Rate is low, and tissue cultures are high-efficient.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Within mind and principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (7)
1. a kind of Wulian poplar method for tissue culture, steps are as follows：
（1）Initial culture：Explant is inoculated into initial culture base and carries out Primary culture, obtains seedling primary, the explant choosing
With the stem with bud of 1.5~2.0 cm；
（2）Go vitrifying culture：By seedling primary obtained in the previous step, it is inoculated in vitrifying culture medium and carries out vitrifying and train
It supports, obtains vitrifying seedling；
（3）Multiplying culture：Vitrifying seedling will be gone to be inoculated in proliferated culture medium and carry out Multiplying culture, obtain Regenerated plant；
（4）Culture of rootage：Regenerated plant is transferred in root media and carries out culture of rootage, obtains rooted seedling；
The initial culture base is to add the MS basal medium of+5.5 g/L agar powder of 25 g/L sucrose, adjusts pH value
To 5.8；
It is described to go the vitrifying culture medium to be, add+30 g/L sucrose+6.5 of+0.4 mg/L 6-BA of 0.05 mg/L NAA
The N of g/L agar powder halves MS culture medium, adjusts pH value to 5.8；
The proliferated culture medium is to add+30 g/L sucrose+6.5 of 0.03 mg/L NAA+, 0.4~0.5 mg/L 6-BA
The MS culture medium of g/L agar powder adjusts pH value to 5.8；
The root media is the 1/2MS training of addition+6.5 g/L agar powder of+15 g/L sucrose of 0.05 mg/L NAA
Base is supported, adjusts pH value to 5.8.
2. a kind of Wulian poplar method for tissue culture according to claim 1, which is characterized in that the stem with bud is spring
Acquire the top stem with bud of new pumping mid-March.
3. a kind of Wulian poplar kind method for tissue culture according to claim 1, which is characterized in that before the Initial culture
It further include explant disinfecting process, the explant disinfecting process：Explant surface is cleaned with washing powder, is rushed overnight with thread water
Explant is washed, is placed it on superclean bench, using 75% ethanol postincubation, 35 s, 9 min is handled with 5 % sodium hypochlorite and carries out
Detoxic, sterilizing, stem with bud is cut into 0.8~1.5 cm leaf stem section after detoxification, is placed in initial culture base and cultivates, and incubation time is
25 d。
4. according to claim 1 to a kind of Wulian poplar method for tissue culture described in 3 any one, which is characterized in that described first
Feeding time of being commissioned to train is 25 d, and the time for going vitrifying culture is 35 d, and the time of the Multiplying culture is 35 d.
5. a kind of Wulian poplar method for tissue culture according to claim 4, which is characterized in that the method for tissue culture
Condition of culture is：Day temperature be 25 ± 2 DEG C, 18 ± 2 DEG C of night, light application time be 16 h, intensity of illumination be 3000~
5000 LX。
6. a kind of Wulian poplar kind method for tissue culture according to claim 5, which is characterized in that the method for tissue culture
It further include hardening, the hardening：Hardening is sealed in the transplantation of seedlings that will take root in greenhouse, after 5 d, removes sterility cover, continue hardening 3~
4 d。
7. a kind of Wulian poplar method for tissue culture according to claim 6, which is characterized in that the method for tissue culture is also
Including transplanting, the transplanting：Clear water rinses the seedling survived, is transplanted to the Light media sterilized by 0.5 % potassium permanganate,
The Light media includes turf and vermiculite, and the ratio of turf and vermiculite is 1 in the Light media:1.
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