CN107810850B

CN107810850B - Cultivation method of regenerated seedlings of hybrid seeds of populus microphylla and populus diversifolia

Info

Publication number: CN107810850B
Application number: CN201710721972.3A
Authority: CN
Inventors: 王方琳; 尉秋实; 柴成武; 王飞
Original assignee: Gansu Desert Control Research Institute
Current assignee: Gansu Desert Control Research Institute
Priority date: 2017-08-22
Filing date: 2017-08-22
Publication date: 2020-06-16
Anticipated expiration: 2037-08-22
Also published as: CN107810850A

Abstract

The invention discloses a cultivation method of regenerated seedlings of hybrid seeds of populus microphylla and populus diversifolia, which comprises the following steps of establishing an explant sterilization and sterile system, inducing and proliferating adventitious buds, carrying out rooting culture and hardening seedling transplantation; the invention adopts a tissue culture method, realizes the rapid propagation of the high-quality test-tube plantlet of the small and large populus diversifolia in a short time, only needs about 60 days from the induction and propagation of the cluster buds to the rooting of the test-tube plantlet in the whole propagation process, greatly improves the seedling forming speed of the small and large populus diversifolia, has important effects on the large-scale, industrialized production and propagation of the plant, and can continuously provide high-quality nursery stocks for the improvement of salinized land in northwest arid desert regions and the forestation and forestation in the desert regions in China.

Description

Cultivation method of regenerated seedlings of hybrid seeds of populus microphylla and populus diversifolia

The technical field is as follows:

the invention belongs to the technical field of plant regeneration seedling cultivation, and particularly relates to a cultivation method of regenerated seedlings of hybrid seeds of populus microphylla and populus diversifolia.

Background art:

the improvement and utilization of large-area desertification and salinization land distribution exist in northwest China, the improvement and utilization of the land are important tasks for ecological environment construction and sustainable development of forestry in China, the shortage of afforestation tree species is one of the main difficulties for improving the desertification salinization land, and in order to quickly and effectively solve the problem, the breeding of salt-tolerant and drought-tolerant varieties must be vigorously developed simultaneously besides the introduction and domestication of the salt-tolerant tree species.

Populus simonii (Populus simonii x P. eupahratica) is a new hybrid poplar variety which is drought-resistant, saline-alkali resistant and capable of growing under wider ecological conditions and successfully cultivated in 1980 by virtue of the Retianci of scholars in China, and is a good variety completely with the double-parent fusion character and obtained after repeated hybridization experiments with Populus simonii (Populus simonii Carr.) as female parent and Populus huttitica (P.eupahratica Oliv.) as male parent. In general, the populus euphratica has the advantages of both the populus tremuloides and the populus euphratica, namely, the populus euphratica is more saline-alkali resistant, is more drought resistant and grows rapidly than the populus euphratica, so the populus euphratica becomes a promising tree species in saline land improvement and tree planting and forestation in arid areas.

At present, the research reports about populus euphratica abroad are blank, domestic scholars mainly develop some researches on the aspects of crossbreeding technology, hardwood cutting propagation, introduction and domestication, drought resistance of seedlings and the like, and the research reports about tissue culture and rapid propagation of populus euphratica are not reported so far.

Tissue culture is an asexual propagation method developed on the basis of plant physiology and cytology. The method has the advantages that the method can enable plant planting to get rid of a long period, can be controlled in a centralized manner, can be produced in a large scale and automatically, is not influenced by external natural conditions, can well keep the excellent properties of a parent plant of the propagated nursery stock, and the like, is widely applied to various fields of molecular biotechnology, quick propagation of nursery stock, production of detoxified seedlings, breeding, production of secondary metabolites, preservation of germplasm resources and the like, and generates huge economic benefits and ecological benefits. Therefore, by using modern biological technology, the tissue culture of the small and populus euphratica is developed on the premise of not changing the excellent characters of the parent plant, high-quality seedlings can be successfully cultured in a short time, and reference is provided for improvement of salinized land in northwest arid desert regions and selection of nursery stocks for tree planting in the desert regions in China.

The invention content is as follows:

the technical problem to be solved by the invention is as follows: provides a cultivation method which realizes the rapid propagation of high-quality test-tube plantlets of populus euphratica in a short time and has high survival rate.

In order to solve the technical problems, the invention is realized by the following technical scheme: a method for cultivating regenerated seedlings of hybrid seeds of populus microphylla and populus diversifolia comprises the following steps,

s1, establishing an explant sterilization and sterile system: in 4-5 months, cutting current-year branches with the length of 20-30 cm from small and populus diversifolia plants growing outdoors, dipping tap water added with a cleaning agent by using a soft brush to clean stains and dust on the surfaces of the branches of the explants, dipping a glass rod by using a drop of Tween 80 to deeply clean the branches, washing the branches of the explants under running water for 4-6 hours, and then placing the washed branches of the explants on an ultra-clean workbench to perform sterilization treatment;

s2, adventitious bud induction and proliferation:

A) placing the explant material subjected to sterilization treatment in a culture dish which is subjected to high-pressure sterilization treatment and paved with double-layer filter paper, cutting off cuts at the front end and the rear end, reserving a stem section and a leaf section, cutting the stem section into a small section with the length of 0.5cm, cutting the leaf section into small pieces with the length of 0.5cm multiplied by 0.5cm, cutting off peripheral edge parts, then spreading and inoculating the cut sections on the surface of an adventitious bud induction culture medium, slightly pressing the cut sections of the leaf section and the stem section with tweezers to fully contact with the culture medium respectively, and finally sealing and culturing;

B) and culture conditions are as follows: after inoculation, firstly culturing in a dark environment for 3 days, and then culturing in an environment with the temperature of 24 +/-2 ℃, the illumination intensity of 1500-2000 lx and the illumination time of 14 h/d;

s3, rooting culture:

(1) selecting the small and adventitious buds of the populus euphratica which are cultured for 30-40 days in the step S2 and have strong growth and 2-3 cm high as test materials, shearing the adventitious buds, inoculating the adventitious buds into a bottle containing a rooting culture medium, and sealing to culture test-tube plantlets;

(2) the culture conditions are as follows: setting the temperature of the culture room to 25 ℃, firstly culturing in the dark for 4 days, gradually enhancing the illumination intensity after 4 days, and controlling the illumination intensity to be 1800-2200 lx and the illumination time to be 12h/d after culturing for 40-50 days;

s4, hardening and transplanting: selecting test-tube plantlets which are cultured for 40-50 days and have developed roots and strong growth, firstly opening 1/2 bottle caps, then injecting 100ml of distilled water, placing the test-tube plantlets in room under natural light for 3 days, then completely opening the bottle caps, adding 50ml of distilled water, continuing placing the test-tube plantlets in room under natural light for 2 days, then transferring the test-tube plantlets to a greenhouse, completely opening the bottle caps, placing the test-tube plantlets in the greenhouse for 2 days, cleaning the culture medium at the root of the bottle seedling with clear water, transplanting the cleaned culture medium into a mixed matrix of vermiculite, sawdust and peat soil, after the transplanting is finished, the seedling is placed on a seedbed, the upper part of the seedbed is covered with a double-layer shading net with the specification of 50-70 percent, the direct irradiation of sunlight is avoided, the sprinkling irrigation and watering are carried out every evening, the soil humidity in a nutrition pot is ensured to be 60-80 percent, after the test-tube seedlings are transplanted and fixedly planted for 15 days, and (4) properly increasing and decreasing the watering amount according to the dry and wet conditions of the soil, and carrying out open field transplanting and field planting after growing for 30 days under the greenhouse condition.

Preferably, the specific operation steps of the sterilization treatment in the step S1 are as follows, firstly, 75% ethanol is adopted to sterilize the explant branches for 10S to 30S, and then sterile water is used for washing for 3 to 5 times; the second step uses HgC1 with a concentration of 0.1%₂Sterilizing for 3-5 min, washing with sterile water for 5-7 times, and autoclavingThe filter paper of the bacteria is inoculated after absorbing the residual water on the surface of the explant.

Preferably, the formula of the induction medium in the step S2 is modified WPM +6-BA0.5mg/L + NAA0.3mg/L + KT0.1mg/L + activated carbon 3.0g/L + agar 4.5g/L + sucrose 30g/L, and the induction medium Ph6.0.

Preferably, the formula of the rooting medium in the step S3 is 1/2WPM + peptone 1g/L + IBA0.4mg/L + banana 150g/L + activated carbon 3.0g/L agar 4.5g/L + sucrose 30 g/L.

Preferably, in step S4, the volume ratio of the vermiculite to the sawdust to the peat soil is 2:1: 1.

compared with the prior art, the invention has the advantages that:

1. the invention adopts a tissue culture method, realizes the rapid propagation of the high-quality test-tube plantlet of the small and large populus diversifolia in a short time, only needs about 60 days from the induction and propagation of the cluster buds to the rooting of the test-tube plantlet in the whole propagation process, greatly improves the seedling forming speed of the small and large populus diversifolia, has important effects on the large-scale, industrialized production and propagation of the plant, and can continuously provide high-quality nursery stocks for the improvement of salinized land in northwest arid desert regions and the forestation and forestation in the desert regions in China;

2. the invention adopts a tissue culture method to carry out experimental research on the rapid propagation of high-quality seedlings of populus euphratica to indicate that the WPM culture medium is the most suitable basic culture medium formula for the rapid induced propagation of populus euphratica, wherein the improved WPM, 6-BA0.5mg/L, NAA0.3mg/L, KT0.1mg/L, 3.0g/L of active carbon, 4.5g/L of agar and 30g/L of cane sugar are the most suitable culture medium formula for the adventitious bud induction and propagation, and the propagation rate reaches 89.3 percent;

3. according to the invention, the rapid propagation of high-quality seedlings of small and old poplars is tested and researched by adopting a tissue culture method, wherein 1/2WPM + peptone 1g/L + IBA0.4mg/L + banana 150g/L + active carbon 3.0g/L agar 4.5g/L + sucrose 30g/L is a formula which is most suitable for rooting culture of small and old poplars, and the rooting rate reaches 96.7%;

4. in the seedling hardening process, a method of opening a 1/2 bottle cap in the first 3 days and then gradually and completely opening the bottle cap is adopted, and finally the test-tube seedlings are successfully transplanted to three substrates of vermiculite, sawdust and peat soil according to the volume ratio of 2:1: in the mixed matrix with the proportion of 1, the survival rate after transplanting reaches 94.85 percent.

Description of the drawings:

the invention is further described below with reference to the accompanying drawings.

FIG. 1 is a schematic diagram of the cultivation method of the present invention.

FIG. 2 is a graph comparing the survival rate of stem segments and leaves after different agents are sterilized.

FIG. 3 is a graph of the growth induction of poplar stem segments under different treatment conditions.

FIG. 4 is a graph showing rooting of populus euphratica under different treatment conditions.

FIG. 5 is a graph comparing the effect of different substrate ratios on the survival rate of hardened seedlings and populus euphratica transplantation.

The specific implementation mode is as follows:

the invention is described in detail below with reference to the following figures and embodiments:

the cultivation method of the regenerated seedlings of the hybrid of the populus tremuloides and the populus diversifolia as shown in figure 1 comprises the following steps,

1. establishment of sterile System

1.1 treatment of explant Material

Cutting small and current-year populus diversifolia branches growing outdoors and having the length of 20-30 cm, taking the branches back to a laboratory, dipping tap water added with a cleaning agent into the branches by using a soft brush to clean stains and dust on the surfaces of the branches of the explants, dipping a glass rod into a drop of Tween 80 to carry out deep cleaning, washing the explants under the tap water for 4-6 hours after cleaning, and then placing the explants on an ultra-clean workbench to carry out sterilization treatment. The specific sterilization time settings and the tests used for sterilization were as follows: arranging experimental contents by adopting an orthogonal test method, sterilizing explants for 10S, 20S and 30S respectively by using 75% ethanol in the first step, and then washing the explants for 3-5 times by using sterile water; in the second step, HgCL with a concentration of 0.1%₂Sterilizing (mercuric chloride) for 3min, 4min and 5min respectively, washing with sterile water for 5-7 times, and finally, absorbing residual water on the surface of the explant by using autoclaved filter paper and then inoculating.

1.2 analysis of results

As shown in FIG. 2, 75% ethanol and 0.1% HgCL were used₂Sterilizing leaves and stem segments of populus euphratica in different treatment time, wherein the results show that the survival rate of the stem segments and the leaves is higher when the leaves and the stems are treated with ethanol for 10s, the survival rate of the stem segments reaches the maximum value of 10s of ethanol and 4min of mercury chloride, the survival rate is 76.95%, and then the survival rate is gradually reduced along with the prolonging of the treatment time of the ethanol and the mercury chloride; the maximum value of the leaves is ethanol for 10s and mercuric chloride for 3min, the survival rate is 53.31%, the survival rate is gradually reduced along with the prolonging of the treatment time of the ethanol and the mercuric chloride, and the leaves are completely withered and die when the ethanol is treated for 30s, which shows that the longer treatment time of the ethanol is not beneficial to sterilizing the small populus euphratica leaves.

2. Adventitious bud induction and proliferation

2.1 explant inoculation

Placing the stem section and leaf material of the explant subjected to the sterilization treatment in a culture dish which is subjected to the high-pressure sterilization treatment and is paved with double-layer filter paper, cutting off the cuts at the front end and the rear end of the stem section, and cutting the stem section to a small section with the length of about 0.5 cm; sucking water on the surface of the leaf blade by using filter paper, cutting the leaf blade into small pieces of 0.5 multiplied by 0.5cm, cutting the peripheral edge parts of the leaf blade and the stem blade, respectively spreading and inoculating the two materials to the surface of an adventitious bud induction culture medium, slightly pressing by using tweezers to enable the cut parts of the leaf blade and the stem blade to respectively and fully contact with the culture medium, and finally sealing and culturing.

2.2 culture conditions

The pH of the adventitious bud induction culture medium is 6.0, the adventitious bud induction culture medium is firstly cultured in a dark environment for 3 days after inoculation, and then the culture medium is cultured in an environment with the temperature of 24 +/-2 ℃, the illumination intensity of 1500-2000 lx and the illumination time of 14 h/d.

2.3 results and analysis

As shown in figure 3, the culture medium for inducing and proliferating adventitious buds of populus euphratica is WPM basic culture medium, 6-BA (0.3mg/L, 0.5mg/L, 1.0mg/L), NAA (0.1mg/L, 0.2mg/L, 0.3mg/L), KT (0.1mg/L, 0.3mg/L, 0.5mg/L) and active carbon (1g/L, 3g/L, 5g/L) are respectively added into the culture medium, the adventitious bud induction and proliferation effects of the stem segment of the explant are shown in figure 3, the culture medium adopts the improved WPM basic culture medium in comparison, no additive is added, and the explant dies in yellow and does not induce 15 days after the stem segment is inoculated; when the explant is treated by 1-9 shown in the figure 3, adventitious buds can grow on stem segments of the explant through induction, but the difference between the proliferation rate and the growth condition of the adventitious buds is larger when different treatments are adopted, when the concentration of 6-BA in a culture medium is 0.3mg/L, the induction rate of the adventitious buds is gradually increased along with the increase of the concentrations of NAA, KT and active carbon, when the concentration of 6-BA is 0.5mg/L, the induction rate of the adventitious buds is also gradually increased along with the increase of the concentrations of other additives, and when the concentration of NAA is 0.3mg/L, KT 0.1.1 mg/L and the concentration of active carbon is 3g/L, the induction rate of the adventitious buds of the stem segments reaches the maximum value of 89.30%, at the moment, the induction rate of the adventitious buds is high, the proliferation rate is high, and the bud seedlings grow well; when the concentration of 6-BA in the culture medium is increased to 1.0mg/L, the induced proliferation rate is gradually reduced, and more induced adventitious buds generate deformity; therefore, the formula of the culture medium which is most suitable for adventitious induction and multiplication culture of populus euphratica is improved WPM +6-BA0.5mg/L + NAA0.3mg/L + KT0.1mg/L + active carbon 3.0g/L + agar 4.5g/L + sucrose 30 g/L.

In addition, studies have shown that when leaves of California populus diversifolia explants are inoculated into the culture medium shown in FIG. 3, the leaves do not undergo any change in the outer tube except for slight expansion of the peripheral incisions, which indicates that the use of leaves as explants is not favorable for adventitious bud induction and proliferation culture of California populus diversifolia.

3. Rooting culture

3.1 inoculation of rooting Material

Selecting the small and adventitious buds of populus euphratica which are cultured for 30-40 days in the step 2, grow robustly and are 2-3 cm high as test materials, and vertically inoculating the test materials into a rooting culture medium for culturing after shearing; the control was 1/2WPM minimal medium without any hormones added.

3.2 culture conditions

The temperature of the culture room is set to be 25 ℃, the culture room is firstly cultured in the dark for 4 days, the illumination intensity is gradually enhanced after 4 days, the illumination time is 12h/d, and the illumination intensity is controlled to be 1800-2200 lx after 40-50 days of culture and the illumination time is 12 h/d.

3.3 results and analysis

As shown in FIG. 4, after adventitious buds of populus euphratica were inoculated, callus was generated at the cut site at the earliest day of 9, adventitious roots were grown at 15 days, and the adventitious roots were generated in all the treatments except the control treatment, but the number of roots, the height of the plants, the growth conditions of the plants and the like were greatly different in the different treatments. When the IBA concentration is 0.2mg/L, the rooting rate is gradually increased along with the increase of other additives; in the treatment stage with IBA of 0.4mg/L, the rooting rate of populus euphratica is respectively 94.85%, 84.57% and 82.00%, and then the rooting rate is gradually reduced along with the increase of IBA in the culture medium. Therefore, the formula of the culture medium which is most suitable for rooting culture of the small populus euphratica is 1/2WPM, IBA0.4mg/L, peptone 1g/L, banana 150g/L, activated carbon 3.0g/L, agar 4.5g/L and sucrose 30 g/L.

4. Hardening off and transplanting

The seedling hardening is a transition stage of indoor culture and outdoor seedling culture of test-tube seedlings, and mainly aims to enable the seedlings to have a process adaptive to the external environment before being transplanted to a field, and the stage needs to pay attention to the management of light and temperature.

4.1 exercise of bottle seedlings

After rooting culture is carried out for 40-50 days, hardening and transplanting test-tube seedlings, selecting test-tube seedlings with developed root systems and strong growth after rooting culture, firstly opening 1/2 bottle caps, injecting 100ml of distilled water, placing the test-tube seedlings in room natural light for 3 days, then completely opening the bottle caps, adding 50ml of distilled water, continuously placing the test-tube seedlings in room natural light for 2 days, then transferring the test-tube seedlings to a greenhouse, completely opening the bottle caps, placing the greenhouse for 2 days, washing the culture medium at the roots of the test-tube seedlings by tap water, washing the culture medium, transplanting the test-tube seedlings into three substrates of vermiculite, sawdust and peat soil according to different mixing ratios, placing the test-tube seedlings on a seedbed after transplanting, covering a double-layer shading net with the specification of 50-70% on the upper part of the seedbed, avoiding direct irradiation of sunlight, watering at the evening every day, ensuring that the humidity of soil in a nutrition pot is 60-80%, properly increasing and reducing the watering amount according to the dry, and after growing for 30 days under the greenhouse condition, open field transplanting and field planting can be carried out.

4.2 preparation before transplantation

4.2.1 preparation of the substrate

The soilless seedling raising substrate is required to have good water and fertilizer retention capacity, good air permeability, pH of 5.5-6.5, stable physical and chemical properties and best sterilization. Generally, 2-3 substrates are mixed for use, such as turf and vermiculite according to a volume ratio of 2:1, as shown in experimental data in fig. 5, the survival rate of plants is highest, the plants grow robustly, leaves are dark green, the growth speed is high, the well-mixed and wetted substrates are placed into a plug tray 1 day before seedling transplantation, and light pressure is performed; pouring the mixture thoroughly with 1000 times of pulex to kill pathogenic bacteria remained in the matrix. The water content of the substrate is about 80 percent when seedlings are transplanted the next day, and the substrate is suitable for transplanting the seedlings.

4.2.2 preparation of the seedbed

The environment in which the seedlings are placed should be easily controlled with respect to humidity, temperature and illumination. Making high ridges in the north-south direction in a sunlight greenhouse, wherein the height is 10-15 cm, and the width is 120 cm; 2 plug trays (54cm × 2 ═ 108cm) can be transversely placed; a small arched shed is made on the high ridge, and a sunshade net can be arranged besides the function of heat preservation and moisture preservation.

4.2.3 preparation of the tool

If some proper tools are made in the processes of lifting and transplanting seedlings, the efficiency can be improved, and the survival rate can also be improved, such as tools used when the seedlings are taken out from culture bottles, tools used when the seedlings are transplanted, and the like. When the seedling transplanting amount is large, the arrangement of labor conditions, personnel and work flows and the like are also considered.

4.3 transplantation of seedlings

4.3.1 lifting, grading, washing seedlings

Pouring out the seedlings in the culture bottle, removing the culture medium adhered to the roots of the seedlings, putting the seedlings into clear water for light washing, and cleaning the culture medium adhered to the roots without damaging roots, tender shoots and leaves as much as possible. Then classifying the seedlings according to the sizes, and separating the seedlings without roots.

4.3.2 transplantation

Inserting a hole in the substrate of the plug tray by using a prepared tool, and slightly putting the root of the seedling into the hole to prevent the root from being indented as much as possible; lightly cover and slightly compact. After the seedling leaves are moved, the sprayer is used for spraying water lightly, so that the substrate on the seedling leaves can be washed, the root system is in closer contact with the substrate, and the growth of the root system is facilitated. For non-rooted seedlings, the rooting powder (agent) with proper concentration can be firstly stained and then transplanted.

4.4 post-transplant management

After seedling transplanting, the environment where seedlings grow needs to be kept at high humidity, the humidity is 85-95%, and the higher humidity is beneficial to reducing the transpiration rate of leaves and reducing the burden of water absorption of root systems. A small arched shed can be built on the seedbed to effectively preserve moisture. But diseases are easy to breed while the humidity is high, in order to reduce the occurrence of the diseases, ventilation is carried out for 5min every 4-5 h, the ventilation time is gradually increased day by day, and a small shed for moisturizing can be scattered after about 7 days for normal management. The environmental temperature after transplantation is slightly higher than the environmental temperature of seedling growth before transplantation, and can be controlled at 23-26 ℃ in the daytime and not lower than 13-18 ℃ at night. In order to reduce the transpiration rate of the leaves, shading management needs to be carried out after transplantation, the shading needs to be carried out for the first 2 days after transplantation, light is gradually exposed until the light is not shaded, and the process needs about 10-14 days. Because the environment humidity is large and the temperature is moderate, the breeding of various germs is facilitated. In order to prevent diseases, the ventilation and the humidity reduction are enhanced, and the prevention, the treatment and the treatment of the diseases are combined with medicaments. 750 folds of pulex can be sprayed on the 4 th day after seedling transplantation and every 4 days thereafter. The spraying and water supplementing can be combined, and the ventilation time is prolonged after the spraying and water supplementing. After 1 week, fertilizing by combining water supplement; the high-phosphorus compound fertilizer can be sprayed, and the concentration is 0.2 percent, so as to promote the comprehensive growth of roots and plants. The survival rate of transplantation can be determined after 10 days.

It is to be emphasized that: it will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.

Claims

1. A cultivation method of regenerated seedlings of hybrid seeds of populus microphylla and populus diversifolia is characterized by comprising the following steps: comprises the following steps of (a) carrying out,

s2, adventitious bud induction and proliferation:

s3, rooting culture:

A) selecting the small and adventitious buds of the populus euphratica which are cultured for 30-40 days in the step S2 and have strong growth and 2-3 cm high as test materials, shearing the adventitious buds, inoculating the adventitious buds into a bottle containing a rooting culture medium, and sealing to culture test-tube plantlets;

B) and culture conditions are as follows: setting the temperature of the culture room to 25 ℃, firstly culturing in the dark for 4 days, gradually enhancing the illumination intensity after 4 days, and controlling the illumination intensity to be 1800-2200 lx and the illumination time to be 12h/d after culturing for 40-50 days;

The formula of the induction culture medium in the step S2 is improved WPM +6-BA0.5mg/L + NAA0.3mg/L + KT0.1mg/L + activated carbon 3.0g/L + agar 4.5g/L + sucrose 30g/L, and the induction culture medium Ph6.0.

2. The method for breeding regenerated seedlings of hybrid populus tremuloides and populus diversifolia seedlings according to claim 1, wherein the method comprises the following steps: the specific operation steps of the sterilization treatment in the step S1 are as follows, firstly, 75% ethanol is adopted to sterilize the explant branches for 10S-30S, and then sterile water is used to wash the explant branches for 3-5 times; the second step uses HgC1 with a concentration of 0.1%₂And (3) sterilizing for 3-5 min, washing with sterile water for 5-7 times, and finally, absorbing residual water on the surface of the explant by using autoclaved filter paper and then inoculating.

3. The cultivation method of the hybrid regenerated seedling is characterized by comprising the following steps: the method for breeding regenerated seedlings of hybrid populus tremuloides and populus diversifolia seedlings according to claim 1, wherein the method comprises the following steps: the formula of the rooting medium in the step S3 is 1/2WPM + peptone 1g/L + IBA0.4mg/L + banana 150g/L + activated carbon 3.0g/L agar 4.5g/L + sucrose 30 g/L.

4. The method for breeding regenerated seedlings of hybrid populus tremuloides and populus diversifolia seedlings according to claim 1, wherein the method comprises the following steps: in the step S4, the volume ratio of the vermiculite to the sawdust to the peat soil is 2:1: 1.