CN112715365A

CN112715365A - Tissue culture method of populus euphratica and culture medium composition thereof

Info

Publication number: CN112715365A
Application number: CN202110106226.XA
Authority: CN
Inventors: 周扬颜; 陈文秀; 莫翱玮
Original assignee: Shandong Dafengyuan Agricultural Co ltd
Current assignee: Shandong Dafengyuan Agricultural Co ltd; Yunnan Fengwo Agriculture Co.,Ltd.
Priority date: 2021-01-26
Filing date: 2021-01-26
Publication date: 2021-04-30
Anticipated expiration: 2041-01-26
Also published as: CN112715365B

Abstract

The invention discloses a populus euphratica tissue propagation culture medium combination, wherein an induced callus culture medium is a WPM culture medium comprising 5-8g/L agar, 25-35g/L sucrose, 0.4-1.0 mg/L6-BA, 0.1-0.8mg/L KT and 0.3-1.0mg/L NAA; the germination culture medium is WPM culture medium comprising 5-9g/L agar, 25-35g/L sucrose, 0.3-1.0mg/L6-BA, 0.1-0.7mg/L KT, 0.1-0.4mg/L NAA; the rooting culture medium is WPM culture medium comprising agar 5-9g/L, sucrose 25-35g/L, active carbon 0.2-0.8g/L and IBA 0.9-1.6 mg/L. The invention discloses a method for carrying out tissue propagation culture on populus euphratica by using the culture medium. By using the culture medium combination and the method, the induction rate of the populus euphratica callus in the adventitious bud induction culture medium can reach 88.2 percent, the callus germination rate can reach 90.3 percent, the rooting rate can reach 89.2 percent, and the seedling survival rate can reach 90.5 percent.

Description

Tissue culture method of populus euphratica and culture medium composition thereof

Technical Field

The invention belongs to the technical field of plant tissue culture, and relates to a tissue culture method of populus euphratica and a culture medium composition thereof.

Background

Populus tremuloides (with a scientific name: Populus lasiocarpa Oliv.) is a perennial woody plant of Populus of Salicaceae, is one of the oldest and most primitive species in Populus, is a special local tree species in China, is native to China, has wild species distribution only in Hubei, Shaanxi, Sichuan, Guizhou and Yunnan provinces, and has no wild species distribution abroad. The populus euphratica is suitable for mountainous regions and has high ecological value. However, the existing research shows that the cuttage survival rate of populus tremuloides is extremely low, the survival rate of branch cutting treated by different rooting agents is zero, and the survival rate of root cutting is very low. Although the grafting survival rate is relatively high, the cost is high, and only the rootstocks of the populus tremuloides can be grafted and survived, which all lead to the germplasm resource preservation and utilization research work of the populus tremuloides to be urgently solved, so that the rapid propagation and germplasm preservation of the populus tremuloides through the tissue culture technology of the populus tremuloides is of great significance.

Disclosure of Invention

In order to solve the defects in the prior art, the invention aims to provide a tissue culture method of populus tremuloides, establish a tissue culture system of the populus tremuloides with high efficiency, convenience and low cost, and provide technical support for solving the problem of seedlings of the populus tremuloides in production. The invention provides a tissue culture method and a culture medium of populus euphratica, belonging to the technical field of plant tissue culture. In the invention, the in-vitro populus tremuloides branches are subjected to axillary bud regeneration and sterile culture to obtain tender branches and leaves; then sterilizing and cutting the tender leaves, inoculating the tender leaves to a callus induction culture medium for callus induction to obtain callus; transferring the callus onto an adventitious bud induction culture medium for bud formation culture to obtain adventitious buds; then inoculating the adventitious bud on a rooting culture medium for culturing to obtain a rooted seedling; and finally hardening the tissue culture seedlings, and transplanting the tissue culture seedlings to the field to obtain the tissue culture populus euphratica seedlings. The tissue culture method of the populus tremuloides provided by the invention is simple, low in cost, short in period and high in efficiency, and is suitable for large-scale tissue culture and propagation expansion of the populus tremuloides.

The invention provides a culture medium combination for expanding propagation of populus euphratica tissues in a first aspect, wherein the culture medium combination comprises an induced callus culture medium, a germination culture medium and a rooting culture medium;

the callus induction medium comprises 5-9g/L (such as 6g/L, 7g/L, 8g/L or any range between two values) of agar, 25-35g/L of sucrose (such as 26g/L, 27g/L, 28g/L, 29g/L, 30g/L, 31g/L, 32g/L, 33g/L, 34g/L or any range between two values), 0.4-1.0mg/L (such as 0.45mg/L, 0.5mg/L, 0.55mg/L, 0.6mg/L, 0.65mg/L, 0.7mg/L, 0.75mg/L, 0.8mg/L, 0.85mg/L, 0.9mg/L, 0.95mg/L or any range between two values), and 6-9 mg/L of BA, 0.1-0.8mg/L (e.g., 0.15mg/L, 0.2mg/L, 0.25mg/L, 0.3mg/L, 0.35mg/L, 0.4mg/L, 0.45mg/L, 0.5mg/L, 0.55mg/L, 0.6mg/L, 0.65mg/L, 0.7mg/L, 0.75mg/L, or any range therebetween) of NAM, 0.3-1.0mg/L (e.g., any range of NAM between 0.35mg/L, 0.4mg/L, 0.45mg/L, 0.5mg/L, 0.55mg/L, 0.6mg/L, 0.65mg/L, 0.7mg/L, 0.75mg/L, 0.8mg/L, 0.85mg/L, 0.9mg/L, 0.95mg/L, or any range of NAM in the culture medium);

the germination medium is a BA comprising 5-9g/L (e.g., any one of 6g/L, 7g/L, 8g/L or a range between any two of them), 25-35g/L (e.g., any one of 26g/L, 27g/L, 28g/L, 29g/L, 30g/L, 31g/L, 32g/L, 33g/L, 34g/L or a range between any two of them), 0.3-1.0mg/L (e.g., 0.35mg/L, 0.4mg/L, 0.45mg/L, 0.5mg/L, 0.55mg/L, 0.6mg/L, 0.65mg/L, 0.7mg/L, 0.75mg/L, 0.8mg/L, 0.85mg/L, 0.9mg/L, 0.95mg/L or a range between any one of them) or a BA, 0.1-0.7mg/L (e.g., 0.15mg/L, 0.2mg/L, 0.25mg/L, 0.3mg/L, 0.35mg/L, 0.4mg/L, 0.45mg/L, 0.5mg/L, 0.55mg/L, 0.6mg/L, 0.65mg/L, or a range between any two values) KT, 0.1-0.4mg/L (e.g., 0.15mg/L, 0.2mg/L, 0.25mg/L, 0.3mg/L, 0.35mg/L, or a range between any two values) NAA;

the rooting medium comprises 5-9g/L (such as any one or a range between any two of 6g/L, 7g/L and 8 g/L) agar, 25-35g/L sucrose (such as any one or a range between any two of 26g/L, 27g/L, 28g/L, 29g/L, 30g/L, 31g/L, 32g/L, 33g/L and 34 g/L), 0.2-0.8g/L activated carbon, 0.9-1.6mg/L (such as 0.95mg/L, 1.0mg/L, 1.05mg/L, 1.1mg/L, 1.15mg/L, 1.2mg/L, 1.25mg/L, 1.3mg/L, 1.35mg/L, 1.4mg/L, 1.45 mg/L), Any one of 1.5mg/L, 1.55mg/L, or a range between any two values) WPM medium of IBA.

In some embodiments, the induction callus medium is a WPM medium comprising 5-8g/L agar, 25-35g/L sucrose, 0.5-0.8 mg/L6-BA, 0.2-0.4mg/L KT, 0.4-0.6mg/L NAA.

In some embodiments, the induction callus medium is a WPM medium comprising 6.5g/L agar, 30g/L sucrose, 0.7mg/L6-BA, 0.3mg/L KT, 0.5mg/L NAA.

In some embodiments, the inducing callus medium has a PH of 5.8 ± 0.5.

In some embodiments, the germination medium is a WPM medium comprising 5-9g/L agar, 25-35g/L sucrose, 0.6-0.8mg/L6-BA, 0.4-0.6mg/L KT, 0.1-0.2mg/L NAA.

In some embodiments, the germination medium is a WPM medium comprising 6.5g/L agar, 30g/L sucrose, 0.7mg/L6-BA, 0.5mg/L KT, 0.15mg/L NAA.

In some embodiments, the germination medium has a PH of 5.8 ± 0.5.

In some embodiments, the rooting medium is a WPM medium comprising 5-9g/L agar, 25-35g/L (e.g., 26g/L, 27g/L, 28g/L, 29g/L, 30g/L, 31g/L, 32g/L, 33g/L, 34g/L, or a range between any two of these values) sucrose, 0.4-0.6g/L activated charcoal, 1.1-1.3mg/L IBA.

In some embodiments, the rooting medium is a WPM medium comprising 7g/L agar, 30g/L sucrose, 0.5g/L activated charcoal, 1.2mg/L IBA.

In some embodiments, the rooting medium has a PH of 5.8 ± 0.5.

In some embodiments, the combination of media further comprises callus subculture media; the callus subculture medium is a WPM medium comprising 5-8g/L (e.g., any one of 6g/L and 7g/L or a range between any two of the values) agar, 20-30g/L (e.g., any one of 21g/L, 22g/L, 23g/L, 24g/L, 25g/L, 26g/L, 27g/L, 28g/L and 29g/L or a range between any two of the values) sucrose, 0.05-0.5mg/L6-BA, 0.05-0.5mg/L KT and 0.05-0.4mg/L NAA.

In some embodiments, the callus subculture medium is a WPM medium comprising 5-8g/L agar, 20-30g/L sucrose, 0.1-0.3mg/L6-BA, 0.1-0.25mg/L KT, 0.05-0.2mg/L NAA.

In some embodiments, the callus subculture medium is a WPM medium comprising 6g/L agar, 25g/L sucrose, 0.3mg/L6-BA, 0.2mg/L KT, 0.1mg/L NAA.

In some embodiments, the callus subculture medium has a PH of 5.8 ± 0.5.

In some embodiments, the WPM medium further comprises: NH (NH)₄NO₃ 400mg/L、Ca(NO₃)₂386mg/L、K₂SO₄ 990mg/L、CaCl₂72.5 mg/L、KH₂PO₄ 170mg/L、Na₂MoO₄·2H₂O 0.25mg/L、MgSO₄180.7 mg/L、MnSO₄ 22.3mg/L、ZnSO₄·7H₂O 8.6mg/L、Fe₂(SO₄)₃·7H₂O 27.85mg/L、CuSO₄·5H₂O 0.25mg/L、Na₂37.3mg/L of EDTA, 100mg/L of inositol, 10.1mg/L of vitamin B, 0.5mg/L of nicotinic acid, 60.5mg/L of vitamin B and 2.0mg/L of glycine.

The second aspect of the invention provides a method for cultivating populus euphratica tissue in a propagation manner, which comprises the following steps:

(a) callus induction

Inoculating the populus euphratica explant into an induced callus culture medium for induced culture to form a populus euphratica callus;

the induced callus culture medium is the induced callus culture medium in the populus euphratica tissue propagation culture medium combination of the first aspect of the invention;

(b) culturing for bud

Cutting the populus tremuloides callus into callus clusters, inoculating the callus clusters into a germination culture medium for germination culture to form a populus tremuloides plant with adventitious buds;

the germination medium is the germination medium in the populus euphratica tissue propagation medium combination of the first aspect of the invention;

(c) rooting culture

Inoculating the populus tremuloides plants with adventitious buds to a rooting culture medium for rooting culture to form the populus tremuloides plants with adventitious buds and roots;

the rooting medium is the rooting medium in the populus euphratica tissue propagation medium composition of the first aspect of the invention.

In some embodiments, the populus tremuloides explant is a populus tremuloides leaf.

In some embodiments, the populus tremuloides leaf is obtained by the following method: washing, soaking and water culture of the branches of the populus euphratica, wherein tender leaves growing on the branches of the populus euphratica are the leaves of the populus euphratica.

In some embodiments, the flush time is 18-30 h.

In some embodiments, the temperature of the soaking is 20-28 ℃ and the time of the soaking is 36-60 h.

In some embodiments, the callus induction is performed after the populus euphratica leaves are sterilized.

In some embodiments, the sterilization process sequentially comprises a first sterile water rinse, an alcohol solution soak, a second sterile water rinse, a sodium hypochlorite solution soak, and a third sterile water rinse.

In some embodiments, the first sterile water flush is 3-5 times, the second sterile water flush is 3-5 times, and the third sterile water flush is 3-5 times.

In some embodiments, the alcohol solution has a concentration of 70-80% by volume, and the alcohol solution is soaked for 25-35 s.

In some embodiments, the sodium hypochlorite solution has a mass concentration of 0.8-1.5%, and the sodium hypochlorite solution is soaked for 4-6 min.

In some embodiments, in step (a), the populus tremuloides leaves are divided into 0.3-0.4cm²The pieces were inoculated.

In some embodiments, in step (a), the induction culture is a dark culture at a temperature of 18-25 ℃ for 2-3 weeks.

In some embodiments, in step (a), the inducing callus medium is replaced every 2-3 weeks.

In some embodiments, in step (b), the cultivation temperature of the sprout cultivation is 20-25 ℃, the light-dark period is (14-18)/(6-10) h/d (e.g., any one of 15/9h/d, 16/8h/d, 17/7h/d or a range between any two values), the illumination intensity is 1500-.

In some embodiments, in step (b), the germination medium is replaced every 15 to 20 days.

In some embodiments, in step (c), the rooting culture is performed at a temperature of 18-25 ℃, with a light-dark cycle of (14-18)/(6-10) h/d (e.g., any one or a range between 15/9h/d, 16/8h/d, 17/7 h/d), with an illumination intensity of 1500-.

In some embodiments, the culturing method further comprises the steps of:

(d) subculture of callus

Inoculating the populus euphratica callus to a callus subculture medium for callus subculture, and subculturing the populus euphratica callus;

the callus subculture medium is the callus subculture medium in the populus euphratica tissue propagation medium composition according to the first aspect of the invention.

In some embodiments, the callus subculture is carried out at a temperature of 23. + -. 4 ℃ with a light-dark cycle of (14-18)/(6-10) h/d (e.g., any one of 15/9h/d, 16/8h/d, 17/7h/d or a range between any two values) for a period of 3-4 weeks.

In some embodiments, the culturing method further comprises the steps of:

(e) hardening off seedlings

And (3) carrying out hardening-seedling culture on the populus tremuloides plants with adventitious buds and roots.

In some embodiments, the conditions of the acclimatization culture are: 1500-3500lx, a light-dark period (14-18)/(6-10) h/d (e.g., any one or a range of 15/9h/d, 16/8h/d, 17/7 h/d), and a culture temperature of 25 + -3 ℃.

In some embodiments, the culturing method further comprises the steps of:

(f) transplanting

Taking out the hardened poplar seedlings from the bottle and transplanting the seedlings into soil.

In some embodiments, the soil is a mixture of peatmoss and vermiculite.

In some embodiments, the growing environment of the populus tremuloides seedling within 10-20 days after transplanting is: the temperature is 20-25 deg.C, and the humidity is 80 + -5%.

Compared with the prior art, the invention has the beneficial effects that:

the invention provides a method for tissue culture of populus tremuloides, which takes populus tremuloides leaves as explants. The method has low cost, short propagation time and high propagation coefficient. The method provided by the invention effectively solves the limitation that the breeding of the populus tremuloides depends on seed breeding for a long time in production, provides technical support for the production, breeding and large-scale planting of the populus tremuloides, and solves the problems of large seed consumption and low breeding coefficient in seed breeding and cultivation.

According to the tissue culture method of the populus tremuloides, fungi in branches can be greatly reduced by washing, and disinfection in the subsequent steps can be facilitated; the washed branches of the populus euphratica are soaked at room temperature and then are subjected to aseptic culture, so that the condition of fungal pollution in the tissue culture process can be effectively reduced, the disinfection strength of leaves is reduced, and necessary conditions are provided for inducing callus from the leaves of the populus euphratica. When the callus culture medium provided by the invention is used for induction, the disinfected small leaf blocks firstly expand to form callus, and adventitious buds grow from the callus. The invention can induce adventitious buds from the sterile-treated leaves only by 6-8 weeks, thus obviously shortening the tissue culture time, and the callus obtained by leaf induction can be cultured to the adventitious buds for rooting; further, the purpose of obtaining a plurality of seedlings from one leaf is realized. The tissue culture propagation method of the populus tremuloides greatly reduces the tissue culture time of the populus tremuloides, and the callus obtained by leaf induction can be cultured to root again to adventitious buds, so that the tissue culture time is effectively shortened, and the operation steps are simplified; the method provided by the invention can propagate one leaf into a plurality of seedlings, breaks through the limitation that only one seedling can be propagated by seeds, and has the advantages of less material required by the system, low cost and high rooting rate.

Drawings

FIG. 1 is a photograph showing callus growth conditions in example 1 of the present invention.

FIG. 2 is one of photographs showing the case of callus subculture in example 1 of the present invention.

FIG. 3 is a photograph showing the conditions of shoot culture in example 1 of the present invention.

FIG. 4 is a photograph showing the root growth in example 1 of the present invention.

FIG. 5 is one of photographs showing the callus culture of comparative example 2 according to the present invention.

FIG. 6 is one of photographs showing the conditions of the sprouting culture in comparative example 2 of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be described in detail with reference to the accompanying drawings.

The steps not described in detail in the present invention are all the operations conventional in the art, and the materials not described in detail are all the materials conventional in the art.

Example 1

Preparation of culture Medium

A group of formulas of populus euphratica tissue culture system comprises a culture medium with the following formula

(1) Culture medium for callus induction of populus euphratica

The basic culture medium is a WPM culture medium, and specifically comprises inorganic salts: NH (NH)₄NO₃ 400mg/L、Ca(NO₃)₂386mg/L、K₂SO₄ 990mg/L、CaCl₂72.5 mg/L、KH₂PO₄ 170mg/L、Na₂MoO₄·2H₂O 0.25mg/L、MgSO₄180.7 mg/L、MnSO₄ 22.3mg/L、ZnSO₄·7H₂O 8.6mg/L、Fe₂(SO₄)₃·7H₂O 27.85mg/L、CuSO₄·5H₂O 0.25mg/L、Na₂EDTA 37.3mg/L, organics: 100mg/L inositol, 10.1mg/L vitamin B, 0.5mg/L nicotinic acid, 60.5mg/L vitamin B, and 2.0mg/L glycine. The formula also comprises 6.5g/L agar and 30g/L sucrose. The hormones in the culture medium are 6-BA, KT and NAA, and the contents of the hormones are respectively shown in Table 1. The pH was 5.8.

(2) Populus megalobium callus subculture medium

The basic culture medium is a WPM culture medium, and specifically comprises inorganic salts: NH (NH)₄NO₃ 400mg/L、Ca(NO₃)₂386mg/L、K₂SO₄ 990mg/L、CaCl₂72.5 mg/L、KH₂PO₄ 170mg/L、Na₂MoO₄·2H₂O 0.25mg/L、MgSO₄180.7 mg/L、MnSO₄ 22.3mg/L、ZnSO₄·7H₂O 8.6mg/L、Fe₂(SO₄)₃·7H₂O 27.85mg/L、CuSO₄·5H₂O 0.25mg/L、Na₂EDTA 37.3mg/L, organics: 100mg/L inositol, 10.1mg/L vitamin B, 0.5mg/L nicotinic acid, 60.5mg/L vitamin B, and 2.0mg/L glycine. The formula also comprises 6g/L agar and 25g/L sucrose. The hormones in the culture medium are 6-BA, KT and NAA, and the contents of the hormones are respectively shown in Table 2. The pH was 5.8.

(3) Buxus sinica germination culture medium

The basic culture medium is a WPM culture medium, and specifically comprises inorganic salts: NH (NH)₄NO₃ 400mg/L、Ca(NO₃)₂386mg/L、K₂SO₄ 990mg/L、CaCl₂72.5 mg/L、KH₂PO₄ 170mg/L、Na₂MoO₄·2H₂O 0.25mg/L、MgSO₄180.7 mg/L、MnSO₄ 22.3mg/L、ZnSO₄·7H₂O 8.6mg/L、Fe₂(SO₄)₃·7H₂O 27.85mg/L、CuSO₄·5H₂O 0.25mg/L、Na₂EDTA 37.3mg/L, organics: 100mg/L inositol, 10.1mg/L vitamin B, 0.5mg/L nicotinic acid, 60.5mg/L vitamin B, and 2.0mg/L glycine. The formula also comprises 6.5g/L agar and 30g/L sucrose. The hormones in the culture medium are 6-BA, KT and NAA, and the contents of the hormones are respectively shown in Table 3. The pH was 5.8.

(4) Buxus sinica rooting culture medium

The basic culture medium is a WPM culture medium, and specifically comprises inorganic salts: NH (NH)₄NO₃ 400mg/L、Ca(NO₃)₂386mg/L、K₂SO₄ 990mg/L、CaCl₂72.5 mg/L、KH₂PO₄ 170mg/L、Na₂MoO₄·2H₂O 0.25mg/L、MgSO₄180.7 mg/L、MnSO₄ 22.3mg/L、ZnSO₄·7H₂O 8.6mg/L、Fe₂(SO₄)₃·7H₂O 27.85mg/L、CuSO₄·5H₂O 0.25mg/L、Na₂EDTA 37.3mg/L, organics: 100mg/L inositol, 10.1mg/L vitamin B, 0.5mg/L nicotinic acid, 60.5mg/L vitamin B, and 2.0mg/L glycine. The formula also comprises 6.5g/L agar and 30g/L sucrose. The rooting medium 1-6 components only contain different hormones IBA and active carbon, and the specific components and contents are shown in Table 4. The pH was 5.8.

(II) callus induction culture

Taking populus tremuloides branches collected in 3 months, removing old leaves, and washing with tap water for 24 hours.

Soaking the washed branches of the populus tremuloides in tap water for 2 days at room temperature, wherein the soaking temperature is 20-25 ℃, then carrying out water culture, changing water every day, and growing new twigs and tender leaves after the axillary buds break dormancy and germinate.

Sterilizing in a clean bench. Placing the young twigs of the primarily cleaned populus tremuloides in a beaker, washing the young twigs with sterile water for 5 times, then disinfecting the twigs with alcohol with the volume fraction of 75% for 30s, washing the twigs with sterile water for 5 times, soaking the twigs in sodium hypochlorite aqueous solution with the mass fraction of 1.0% for 5min, continuously stirring the twigs during the washing process, washing the twigs with the sterile water for 5 times, cutting the leaves (including petioles) on the twigs into small blocks of about (0.4 multiplied by 0.4) cm, and respectively inoculating the small blocks into 8 callus induction culture media shown in the table 1, wherein the number of bottles of each culture medium is 10. The callus is differentiated from the edge of the leaf after 2-3 weeks of culture at the culture temperature of 22 +/-2 ℃ in a dark room, wherein a picture of one bottle is shown in figure 1, and the callus induction rate is shown in table 1.

Therefore, the method can effectively induce the populus euphratica explants to generate callus.

(III) subculture of callus

Selection of callus: and (3) selecting the callus with good growth vigor, strong growth power and fluffy tissue in the step (II), placing the callus on an ultra-clean workbench of a sterile room, and performing sterile operation on the ultra-clean workbench in the later operation.

The selected callus was cut into callus clusters having a length and width of (0.5X 0.5) cm using a sterile scalpel, and the callus clusters were respectively implanted into 8 types of populus euphratica callus subculture media shown in Table 2 for subculture, and the number of bottles in each medium was 10.

Subculturing the above callus, culturing in dark room for 3-4 weeks at 23 + -2 deg.C, and observing callus proliferation state, wherein a bottle of photograph is shown in FIG. 2. See table 2 for proliferation coefficients.

It can be seen that the method of the present invention enables efficient subculture of callus.

(IV) adventitious bud Induction

And (3) placing the callus with good growth vigor in the step (II) in an aseptic super clean workbench, properly cutting the callus into callus groups with the length and width of (0.5 multiplied by 0.5) cm by using an aseptic scalpel, respectively transferring the callus groups into 8 adventitious bud induction culture media shown in the table 3, wherein 5 to 15 callus groups are placed in each bottle, the callus groups are cultured for about 25 days under the conditions that the culture temperature is 22 +/-2 ℃, the light-dark period is 16/8h/d, and the illumination intensity is 1800 and 3000lx, adventitious buds are differentiated, the germination rate and the seedling growth vigor after germination are recorded, the number of each culture medium bottle is 10, the picture of one bottle is shown in the table 3, and the germination rate is shown in the table 3.

Therefore, the method can effectively induce the callus of the populus euphratica to sprout.

(V) root induction

Cutting the bud-growing plantlets which grow vigorously in the step (IV), and respectively inoculating the bud-growing plantlets on 8 rooting culture media shown in the table 4 for culture to obtain rooted plantlets; the culture conditions were: culturing for 3-4 weeks under the conditions of 22 + -2 deg.C, 16/8h/d light dark period, 1800-3000lx light intensity. One of the bottles is shown in FIG. 4, and the rooting percentage is shown in Table 4.

Therefore, the method can effectively induce the adventitious bud plant of the populus euphratica to root.

(VI) hardening off the seedlings

Performing acclimatization culture on the populus tremuloides plant with adventitious buds and roots, opening a culture bottle, filling clear water with the thickness of 0.5cm, changing every day, gradually opening a bottle cap, and acclimatizing the seedling for 7-10 days under the illumination of 2000-3000lx with the light-dark period of 16/8h/d (namely 16h of light each day and 8h of dark culture), wherein the culture temperature is 25 +/-1 DEG C

(VII) transplantation

Taking out the populus tremuloides seedlings after 7-10 days of seedling hardening from the bottle, washing the culture medium with clear water, transplanting the seedlings into the mixed soil of turfy soil and vermiculite, removing leaves at the bottom end, keeping top buds, and covering a transparent plastic cover for moisturizing. Keeping the temperature in the cover at 21-23 deg.C and humidity of 80 + -5% within 15d after transplanting, and removing the cover after transplanting for 15d to obtain Populus deltoides seedling.

In the experiment, the induction rate of the populus euphratica callus in the adventitious bud induction culture medium can reach 88.2%, the callus germination rate can reach 90.3%, the rooting rate can reach 89.2%, the seedling survival rate can reach 90.5%, and about 90 days is needed from the inoculation of the leaves to the callus induction culture medium to the survival of the seedlings after the seedlings are transplanted to obtain the populus euphratica seedlings.

The annual multiplication number of the leaves of the populus euphratica can reach 1000 tissue culture seedlings by calculating according to a formula shown below.

∑＝a·b^η，

Wherein, a: the number of blades; eta: the number of adventitious buds per cultivation period; b: number of fertile cycles throughout the year. The number of times of the annual multiplication cycle refers to the adventitious bud obtained from the first inductionThe propagation culture is carried out on the basis, and the times of the propagation culture can be continuously increased. In this experiment, when a is 1, the number η of adventitious buds per culture period is 10, and the number of cycles b in which the buds can be proliferated throughout the year is 3. The number of seedlings sigma-1 x 10 obtained from a whole year of populus euphratica³1000 strains.

TABLE 1. leaf induction callus culture medium and culture results of populus euphratica

Numbering	6-BA(mg/L)	KT(mg/L)	NAA(mg/L)	Callus induction rate (%)	Callus growth state
						1	0.5	0.5	0.5	65.5％	+++
2	0.5	0.3	0.5	71.7％	+++
						3	0.5	0.5	1.0	30.5％	+
4	0.5	0.3	1.0	35％	+
						5	0.7	0.5	0.5	74.1％	+++
6	0.7	0.3	0.5	88.2％	++++
						7	0.7	0.5	1.0	28.5％	+
8	0.7	0.3	1.0	30.9％	+

Note: "+" indicates callus growth status.

The best growth state is shown as ++++, the healing time is short, the growth is fast, the callus mass is large, granular, loose in structure and light yellow;

"+ + + +" indicates better callus growth status, short callus growth time, faster callus growth, granular, light yellow;

"+ +" indicates that the callus is in a normal growth state, slow in growth, and in a block shape, yellow green;

"+" indicates that the callus has poor growth state, long healing time, slow growth, block shape, compact structure, hard texture and yellow green.

TABLE 2. Populus tremuloides callus subculture medium and culture results

Numbering	6-BA(mg/L)	KT(mg/L)	NAA(mg/L)	Coefficient of proliferation
					1	0.1	0.1	0.1	1.8
2	0.1	0.2	0.1	2.5
					3	0.3	0.1	0.1	3.4
4	0.3	0.2	0.1	4.2
					5	0.1	0.1	0.3	2.0
6	0.1	0.2	0.3	2.2
					7	0.3	0.1	0.3	2.5
8	0.3	0.2	0.3	3.0

TABLE 3 adventitious bud culture Medium and culture results of Populus tremuloides

Numbering	6-BA(mg/L)	KT(mg/L)	NAA(mg/L)	Percentage of sprouting (%)	Growth state
						1	0.5	0.3	0.3	55.64	Weak growth and yellow-green leaves
2	0.5	0.5	0.3	66.2	General growth and green leaves
						3	0.7	0.3	0.3	42.7	Weak growth and green leaves
4	0.7	0.5	0.3	43.2	Slow germination, weak growth and yellow leaves
						5	0.5	0.3	0.15	65.2	General growth and green leaves
6	0.5	0.5	0.15	70.5	General growth and green leaves
						7	0.7	0.3	0.15	85.2	Multiple shoots, strong growth and green leaves
8	0.7	0.5	0.15	90.3	Fast germination, more sprouts, strong growth vigor and green leaves

TABLE 4 rooting medium and cultivation results for populus euphratica

Numbering	IBA(mg/L)	Activated carbon (g/L)	Rooting percentage (%)	Growth state
					1	1.0	0.3	65％	More adventitious roots
2	1.0	0.5	68.2％	More adventitious roots
					3	1.2	0.3	73.1％	More adventitious roots
4	1.2	0.5	89.2％	Short rooting time and more adventitious roots
					5	1.5	0.3	53.2％	Slow and less adventitious roots
6	1.5	0.5	57.5％	Slow and less adventitious roots

Comparative example 1 callus culture

The difference from the second step of example 1 is only in the culture medium: the formula of the callus induction culture medium of the comparative example is as follows:

formulation a1:

the callus induction medium is WPM medium comprising 6.5g/L agar, 30g/L sucrose, 1.0mg/L LNAA, and 2.0 mg/L6-BA.

Formulation a2:

the callus induction medium is WPM medium comprising 6.5g/L agar, 30g/L sucrose, 1.0mg/L LNAA, and 1.5 mg/L6-BA.

Formulation a3:

the callus induction culture medium is WPM culture medium comprising 6.5g/L agar, 30g/L sucrose, 1.0mg/L LNAA, 1.5mg/L6-BA and 0.5 mg/LKT.

The results of the culture are shown in Table 5.

TABLE 5 callus induction culture results of Populus diversifolia

Numbering	Callus induction rate (%)
		a1	2.7
a2	5.1
		a3	0

Comparative example 2

Compared with example 1, the leaf induction callus culture medium 6, the shoot growth culture medium 8 and the rooting culture medium 4 of the populus tremuloides in example 1 are adopted, except that: the callus induction culture flask is placed in a light-dark period of 16/8h/d and under the condition of the light intensity of 1800lx for culture.

TABLE 6 callus induction, subculture and culture results under light of populus euphratica

Numbering	Callus induction rate (%)	Callus proliferation coefficient	Germination percentage (%)	Growth state
					b1	72.1	1.9	73.9	The healing time is long, and the callus clusters are small; slow germination, general growth and green leaves.

The callus of the populus euphratica is induced under light, the callus grows for a longer time in the dark, the subculture differentiation capability is weak, the callus induced under the light sprouts slowly, and the growth vigor is weaker than the callus induced under the dark. One of the bottles of the light-induced callus is shown in FIG. 5, and one of the bottles of the light-induced callus is shown in FIG. 6.

Comparative example 3

Compared with example 1, the leaf induction callus culture medium 6, the shoot growth culture medium 8 and the rooting culture medium 4 of the populus tremuloides in example 1 are adopted, except that: the explants were selected for the stem segments with axillary buds.

Comparative example no	Callus induction rate (%)	Percentage of sprouting (%)	Rooting percentage (%)	Coefficient of proliferation
					B1	60.2	80.1	81.1	2.0
B2	63.8	78.3	80.6	2.6

The stem section of the populus tremuloides with axillary buds is used for tissue culture of the populus tremuloides, the callus induction rate is lower than that of the leaves of the populus tremuloides, and the multiplication capacity is weaker than that of the leaves of the populus tremuloides for tissue culture.

It will be appreciated by those skilled in the art that the invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The embodiments disclosed above are therefore to be considered in all respects as illustrative and not restrictive. All changes which come within the scope of or equivalence to the invention are intended to be embraced therein.

Claims

1. A culture medium composition for expanding propagation of populus tremuloides tissue comprises an induction callus culture medium, a germination culture medium and a rooting culture medium;

the induction callus culture medium is a WPM culture medium comprising 5-9g/L agar, 25-35g/L sucrose, 0.4-1.0 mg/L6-BA, 0.1-0.8mg/L KT and 0.3-1.0mg/L NAA;

the germination culture medium is a WPM culture medium comprising 5-9g/L agar, 25-35g/L sucrose, 0.3-1.0mg/L6-BA, 0.1-0.7mg/L KT and 0.1-0.4mg/L NAA;

the rooting culture medium is a WPM culture medium comprising 5-9g/L agar, 25-35g/L sucrose, 0.2-0.8g/L active carbon and 0.9-1.6mg/L IBA.

2. The culture medium combination of claim 1, wherein the callus induction medium is WPM medium comprising 5-8g/L agar, 25-35g/L sucrose, 0.5-0.8 mg/L6-BA, 0.2-0.4mg/L KT, 0.4-0.6mg/L NAA;

preferably, the callus induction culture medium is WPM culture medium comprising 6.5g/L agar, 30g/L sucrose, 0.7mg/L6-BA, 0.3mg/L KT, 0.5mg/L NAA;

preferably, the pH of the callus induction medium is 5.8 +/-0.5;

preferably, the germination medium is a WPM medium comprising 5-9g/L agar, 25-35g/L sucrose, 0.6-0.8mg/L6-BA, 0.4-0.6mg/L KT, 0.1-0.2mg/L NAA;

preferably, the germination medium is a WPM medium comprising 6.5g/L agar, 30g/L sucrose, 0.7mg/L6-BA, 0.5mg/L KT, 0.15mg/L NAA;

preferably, the pH of the germination medium is 5.8 +/-0.5;

preferably, the rooting medium is a WPM medium comprising 5-9g/L agar, 25-35g/L sucrose, 0.4-0.6g/L activated carbon and 1.1-1.3mg/L IBA;

preferably, the rooting medium is a WPM medium comprising 7g/L agar, 30g/L sucrose, 0.5g/L activated carbon and 1.2mg/L IBA;

preferably, the rooting medium has a pH of 5.8. + -. 0.5.

3. The culture medium combination of claim 1, wherein the culture medium combination further comprises callus subculture medium; the callus subculture medium is a WPM medium comprising 5-8g/L agar, 20-30g/L sucrose, 0.05-0.5mg/L6-BA, 0.05-0.5mg/L KT and 0.05-0.4mg/L NAA;

preferably, the callus subculture medium is a WPM medium comprising 5-8g/L agar, 20-30g/L sucrose, 0.1-0.3mg/L6-BA, 0.1-0.25mg/L KT and 0.05-0.2mg/L NAA;

preferably, the callus subculture medium is a WPM medium comprising 6g/L agar, 25g/L sucrose, 0.3mg/L6-BA, 0.2mg/L KT and 0.1mg/L NAA;

preferably, the callus subculture medium has a pH of 5.8. + -. 0.5.

4. The culture medium combination of claim 1, wherein the WPM medium further comprises: NH (NH)₄NO₃ 400mg/L、Ca(NO₃)₂ 386mg/L、K₂SO₄ 990mg/L、CaCl₂72.5mg/L、KH₂PO₄ 170mg/L、Na₂MoO₄·2H₂O 0.25mg/L、MgSO₄180.7mg/L、MnSO₄ 22.3mg/L、ZnSO₄·7H₂O 8.6mg/L、Fe₂(SO₄)₃·7H₂O 27.85mg/L、CuSO₄·5H₂O 0.25mg/L、Na₂37.3mg/L of EDTA, 100mg/L of inositol, 10.1mg/L of vitamin B, 0.5mg/L of nicotinic acid, 60.5mg/L of vitamin B and 2.0mg/L of glycine.

5. A method for cultivating populus euphratica tissue in a propagation manner comprises the following steps:

(a) callus induction

the induced callus medium is the induced callus medium in the populus euphratica tissue propagation medium combination of any one of claims 1 to 4;

(b) culturing for bud

the germination medium is the germination medium in the populus euphratica tissue propagation medium combination of any one of claims 1 to 4;

(c) rooting culture

the rooting medium is the rooting medium in the populus euphratica tissue propagation medium combination of any one of claims 1-4.

6. The culture method according to claim 5, wherein the Populus tremula explant is a leaf of Populus tremula;

preferably, the populus euphratica leaf is obtained according to the following method: washing, soaking and water culture are carried out on the branches of the populus euphratica, and tender leaves growing on the branches of the populus euphratica are the leaves of the populus euphratica;

preferably, the washing time is 18-30 h;

preferably, the soaking temperature is 20-28 ℃, and the soaking time is 36-60 h;

preferably, the leaf of populus euphratica is subjected to the callus induction after being sterilized;

preferably, the sterilization treatment sequentially comprises first sterile water washing, alcohol solution soaking, second sterile water washing, sodium hypochlorite solution soaking and third sterile water washing;

preferably, the first sterile water washing is performed for 3-5 times, the second sterile water washing is performed for 3-5 times, and the third sterile water washing is performed for 3-5 times;

preferably, the volume concentration of the alcohol solution is 70-80%, and the soaking time of the alcohol solution is 25-35 s;

preferably, the mass concentration of the sodium hypochlorite solution is 0.8-1.5%, and the soaking time of the sodium hypochlorite solution is 4-6 min;

preferably, in the step (a), the leaf of Populus tremula is divided into 0.3-0.4cm²Inoculating the small blocks;

preferably, in step (a), the induction culture is dark culture, the culture temperature is 18-25 ℃, and the culture time is 2-3 weeks;

preferably, in step (a), the induction callus medium is changed every 2-3 weeks;

preferably, in the step (b), the culture temperature of the bud-growing culture is 20-25 ℃, the light-dark period is (14-18)/(6-10) h/d, the illumination intensity is 1500-;

preferably, in step (b), the germination medium is replaced every 15 to 20 days;

preferably, in step (c), the culture temperature of the rooting culture is 18-25 ℃, the light-dark period is (14-18)/(6-10) h/d, the illumination intensity is 1500-.

7. The culture method according to claim 5, further comprising the steps of:

(d) subculture of callus

the callus subculture medium is the callus subculture medium in the populus euphratica tissue propagation medium combination according to claim 3;

preferably, in the callus subculture, the culture temperature is 23 +/-4 ℃, the light-dark period is (14-18)/(6-10) h/d, and the culture time is 3-4 weeks.

8. The culture method according to claim 5 or 7, further comprising the steps of:

(e) hardening off seedlings

Carrying out hardening culture on the populus tremuloides plants with adventitious buds and roots;

preferably, the conditions of the seedling exercising culture are as follows: 1500-3500lx, the seedling is trained for 5-12 days under the light, the light-dark period (14-18)/(6-10) h/d and the culture temperature is 25 +/-3 ℃.

9. The culture method according to claim 8, further comprising the steps of:

(f) transplanting

Taking out the hardened poplar seedlings from the bottle, and transplanting the poplar seedlings into soil;

preferably, the soil is a mixed soil of turfy soil and vermiculite;

preferably, the growing environment of the populus euphratica seedlings within 10-20 days after transplanting is as follows: the temperature is 20-25 deg.C, and the humidity is 80 + -5%.