CN115812599B - Efficient regeneration method using Japanese larch cotyledon as explant - Google Patents

Abstract

The invention discloses a method for inducing adventitious buds and obtaining regenerated plants using cotyledons of Japanese larch as explants, which includes the steps of sowing, disinfection, induction culture, proliferation culture, rooting culture, seedling hardening and transplanting. The invention utilizes induction medium, proliferation medium and rooting medium containing different hormone ratios, so that Japanese larch has high differentiation efficiency, high proliferation coefficient and high rooting rate, short plant growth time requirement, strong adaptability after cultivation, strong and upright seedlings, good growth, uniformity, greatly shortening the seedling cultivation process and saving a lot of costs. The invention establishes an adventitious bud induction system starting from cotyledons of Japanese larch, and successfully obtains regenerated plants, which provides a technical means for the rapid propagation of Japanese larch seedlings, and also provides a platform for the establishment of an efficient Japanese larch transgenic system in the future.

Description

A highly efficient regeneration method using Japanese larch cotyledons as explants

Technical Field

The invention belongs to the technical field of plant cultivation and reproduction, and in particular relates to a high-efficiency regeneration method using Japanese larch cotyledons as explants.

Background technique

Japanese larch is a deciduous tree belonging to the Pinaceae family and the genus Larix. There are 25 species of Japanese larch in the world, which are naturally distributed in temperate mountains, cold temperate plains and alpine climate zones. In China, Japanese larch is distributed in the Greater and Lesser Khingan Mountains at an altitude of 300-1200 meters, with a large stock volume and a large forest area. Japanese larch has the characteristics of strong adaptability, rapid growth in the early stage, fast forestation, few diseases and pests, and excellent material quality. It is an important pulpwood and construction material species in the subalpine regions of Northeast, Northwest, North China and South China, and is also the main afforestation species for returning farmland to forest and shelterbelt projects.

Conventional breeding of Japanese larch is greatly affected by natural conditions, has a long growth cycle, is difficult to genetically manipulate, and consumes a lot of manpower and material resources, which cannot meet the needs of modern forest genetic breeding. In recent years, with the rapid development of genetic engineering breeding, more and more studies have been conducted on genetic transformation of coniferous plants. At present, there are few reports on transgenic Japanese larch in China. The lack of a good Japanese larch regeneration system is an important reason. Therefore, establishing an efficient and stable coniferous plant tissue culture system is the basic prerequisite for genetic transformation. There have been many reports on the tissue culture system of Japanese larch. Qi Liwang et al. used the tender stem segments of the current year to induce adventitious buds; Wu Kexian et al. used the apical buds of the lateral branches of Changbai Japanese larch as explants for in vitro culture to induce clustered buds, but the efficiency of inducing adventitious buds was low; Wang Weida et al. used immature zygotic embryos as explants to induce adventitious buds, and the low rooting rate restricted plant regeneration and genetic improvement.

The invention uses the cotyledons of Japanese larch seedlings as explants, induces adventitious buds, elongates the adventitious buds, successfully roots them to a certain extent, and transplants them to a greenhouse, thereby establishing a complete Japanese larch regeneration system, which lays a good foundation for the tissue culture, rapid propagation and genetic improvement of Japanese larch in the later stage. The adventitious buds formed by the Japanese larch cotyledon regeneration system are large in number and have good genetic stability, and the cotyledons have a large particle receiving area, which is suitable for both gene gun-mediated genetic transformation and application research, and Agrobacterium-mediated genetic transformation system.

Summary of the invention

The purpose of the present invention is to provide an efficient regeneration method using Japanese larch cotyledons as explants, which has the advantages of convenient material acquisition, low cost, simple operation, fast propagation speed, high reproduction coefficient, etc., and can provide an efficient regeneration system for the Japanese larch genetic transformation system.

To achieve the above object, the present invention adopts the following technical solution:

An efficient regeneration method using Japanese larch cotyledons as explants comprises the following steps:

1) Sowing: Select mature and plump seeds, soak them in tap water for 24 hours, and then sow them in the substrate. After the seeds germinate for 15 to 20 days, select the healthy seedlings as the materials for the next experiment;

2) Disinfection: Wash the seedlings obtained in step 1) with tap water, remove the roots, and soak them in 75 vol% alcohol for 30-60 s, 0.1 vol% mercuric chloride solution for 5-7 min, and rinse with sterile water 5 times;

3) Induction culture: taking cotyledon segments of the seedlings disinfected in step 2) as explants, inoculating them on an induction medium and culturing them for 30 days to obtain adventitious buds; the formula of the induction medium is: DCR medium + 1.5 mg/L 6-BA + 0.001 mg/L TDZ;

4) Proliferation culture: the adventitious buds obtained in step 3) are cut and transferred to a proliferation medium for 60 days to obtain adventitious bud clusters; the formula of the proliferation medium is: DCR medium + 1.5 mg/L 6BA + 0.001 mg/L TDZ + 0.2 g/L activated carbon;

5) Rooting culture: inoculate the adventitious bud clusters obtained by the culture in step 4) onto a rooting medium and culture for 30 to 60 days to obtain rooted seedlings; the formula of the rooting medium is: DCR medium + 0.05 mg/L NAA + 0.2 g/L activated carbon;

6) Hardening the seedlings: The rooted seedlings obtained in step 5) are transferred to a hardening room for hardening;

7) Transplanting: After 10 days of hardening, transplant the rooted seedlings into the substrate and culture them in the greenhouse for 1-2 weeks, followed by shade and diffuse light culture.

The matrix is prepared by mixing nutrient soil, vermiculite and perlite in a volume ratio of 2:2:1.

The conditions for the induction culture, proliferation culture and rooting culture are: light rate of 80 μmol·m ^-2 ·s ^-1 , light time of 16 h/d, and room temperature of 25±2°C.

The step 6) is specifically as follows: putting the rooted seedlings cultured in step 5) into tap water in a half-open bottle for hardening, the temperature in the hardening room is 25°C, the light duration is 16 h/d, the light intensity is 80 μmol·m ^-2 ·s ^-1 , and the humidity is 65%.

The step 7) is specifically as follows: after hardening the seedlings for 10 days, when the root system changes from white to grayish white, the rooted seedlings are transplanted. When transplanting, the rooted seedlings are first rinsed in clean water to remove the culture medium of the roots, and then the surface moisture of the roots is dried, and then the rooted seedlings are transplanted into a container containing a substrate, and cultured in a greenhouse for 1-2 weeks. The greenhouse culture conditions are: light time 16h/d, light intensity 2000 lux, temperature 21-26°C, followed by shade and diffuse light culture, spraying water every day, and maintaining the humidity at 40%-60%.

The above-mentioned high-efficiency regeneration method is applied to the in vitro propagation of Japanese larch.

The above-mentioned high-efficiency regeneration method is used in establishing a Japanese larch cultivation system.

The above-mentioned high-efficiency regeneration method is applied in the germplasm preservation of superior Japanese larch strains.

Due to the adoption of the above technical solution, the beneficial effects of the present invention are:

(1) The present invention uses cotyledons of Japanese larch seedlings germinated in a greenhouse as the source of explants, which not only has the characteristics of simple material collection and is not limited by time and quantity, but also has the advantages of saving time and labor.

(2) The present invention utilizes a culture medium containing different hormone ratios to provide factors required by the explant during the rooting and seedling growth period, thereby providing good rooting conditions, promoting healthy and strong seedlings, and obtaining regenerated seedlings of Japanese larch. The culture method is convenient for obtaining materials, low in cost, simple to operate, and has the characteristics of fast propagation speed and high reproduction coefficient, and can provide a good regeneration system for Japanese larch after genetic transformation.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Effects of different concentrations of naphthaleneacetic acid (NAA) on adventitious bud induction of Japanese larch.

Figure 2: Effects of different concentrations of activated carbon on the elongation of adventitious buds of Japanese larch.

Figure 3: Regeneration process of Japanese larch cotyledons as explants. A is the explant after disinfection; B is the adventitious bud generated after the explant was inoculated with the induction medium for 15 days; C is the adventitious bud generated after the explant was inoculated with the induction medium for 30 days; D is a single adventitious bud about 50 days after the adventitious bud was inoculated with the proliferation medium; E is the proliferation of the adventitious bud in the culture dish; F is the bud cluster cultured in the seedling medium; G is the rooted regenerated seedling; H is the regenerated seedling after transplanting. The scale in the figure is 1 cm.

Figure 4: Schematic diagram of the histochemistry of the formation process of adventitious buds in Japanese larch cotyledons. A is the longitudinal section of the cotyledon just inoculated; B is the longitudinal section of the cotyledon 10 days after inoculation; C is the meristem protrusion; D-E is the longitudinal section of the bud primordium protrusion; F is the longitudinal section of the formed adventitious bud. The scale bar in the figure is 0.1 mm.

Detailed ways

In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

Example 1

The effects of different concentrations of thidiazuron (TDZ) on adventitious bud induction of Japanese larch were investigated. The steps were as follows:

1) Sowing: Select mature and plump Japanese larch seeds, soak them in warm water for 24 hours, and sow them on a cultivation substrate containing vermiculite. After the seeds germinate for 15 to 20 days, select healthy seedlings as materials for the next step of the experiment.

2) Disinfection: Wash the seedlings obtained in step 1) with tap water, remove the roots, soak them in 75 vol% alcohol for 30-60 seconds, soak them in 0.1 vol% mercuric chloride solution for 5-7 minutes, and rinse them with sterile water 5 times, each time for 1 minute;

3) Induction culture: The cotyledon segments of the sterilized seedlings are cut as explants and inoculated onto an induction medium. The culture is carried out for 30 days under the conditions of a light rate of 80 μmol·m ^-2 ·s ^-1 , a light duration of 16 h/d, and a culture temperature of 25±2°C. The induction of adventitious buds is observed and recorded. The formula of the induction medium is selected from any one of the following:

①DCR medium + 0.75 mg/L 6-BA + 0.001 mg/L TDZ;

②DCR medium + 0.75 mg/L 6-BA + 0.01 mg/L TDZ;

③DCR culture medium + 0.75mg/L 6-BA + 0.05mg/L TDZ.

The analysis showed that the TDZ concentration significantly affected the frequency of bud induction. When TDZ was 0.001 mg/L, the induction rate of cotyledon adventitious buds was the highest, reaching 19.35%. As the concentration of TDZ increased, the induction rate showed a decreasing trend. When TDZ was 0.05 mg/L, the induction rate of adventitious buds was only 10.14%. It can be seen that adding 0.001 mg/L TDZ has a good effect on the induction of adventitious buds of Japanese larch (Table 1).

Table 1 Effects of different concentrations of TDZ on adventitious bud induction of Japanese larch

Note: Data are the mean ± error of three replicates. Different letters in the same column indicate significant differences at the 0.05 level, and the same letters indicate insignificant differences.

Example 2

The effects of different concentrations of naphthaleneacetic acid (NAA) on adventitious bud induction of Japanese larch were investigated. The steps are as follows:

1) Sowing: Select mature and plump Japanese larch seeds, soak them in warm water for 24 hours, and sow them on a cultivation substrate containing vermiculite. After the seeds germinate for 15 to 20 days, select healthy seedlings as materials for the next step of the experiment.

2) Disinfection: Wash the seedlings obtained in step 1) with tap water, remove the roots, soak them in 75 vol% alcohol for 30-60 s, soak them in 0.1 vol% mercuric chloride solution for 5-7 min, and rinse them with sterile water 5 times, each time for 1 min.

3) Induction culture: The cotyledon segments of the sterilized seedlings are cut as explants and inoculated onto an induction medium. The culture is carried out for 30 days under the conditions of a light rate of 80 μmol·m ^-2 ·s ^-1 , a light duration of 16 h/d, and a culture temperature of 25±2°C. The induction of adventitious buds is observed and recorded. The formula of the induction medium is selected from any one of the following:

①DCR medium + 0.75 mg/L 6-BA + 0.001 mg/L TDZ;

②DCR medium + 0.75mg/L 6-BA + 0.001mg/L TDZ + 0.1mg/L NAA;

③DCR medium + 0.75mg/L 6-BA + 0.001mg/L TDZ + 0.3mg/L NAA;

④DCR medium + 0.75mg/L 6-BA + 0.001mg/L TDZ + 0.5mg/L NAA;

⑤DCR medium + 0.75mg/L 6-BA + 0.001mg/L TDZ + 1mg/L NAA.

The observation results show (Figure 1) that when NAA is not added, the average adventitious bud induction rate reaches the maximum; as the concentration of added NAA increases, the induction efficiency of larch adventitious buds gradually decreases; when the concentration increases to 0.3 mg/L, the adventitious bud induction efficiency decreases to 6.5%; and when the NAA concentration reaches 1 mg/L, the induction of adventitious buds begins to be inhibited, and the state of the adventitious buds begins to deteriorate. This shows that low concentrations or no addition of NAA combined with TDZ can effectively promote the induction of adventitious buds, while high concentrations of NAA will inhibit the formation of adventitious buds. In view of the fact that the induction effect of NAA on the cotyledon adventitious buds of Japanese larch seedlings is not obvious, NAA will no longer be added in this experiment.

Example 3

The effects of different concentrations of 6-benzyladenine (6-BA) and thidiazuron (TDZ) on adventitious bud induction of Japanese larch were investigated. The steps were as follows:

1) Sowing: Select mature and plump Japanese larch seeds, soak them in warm water for 24 hours, and sow them on a cultivation substrate containing vermiculite. After the seeds germinate for 15 to 20 days, select healthy seedlings as materials for the next step of the experiment.

2) Disinfection: Wash the seedlings obtained in step 1) with tap water, remove the roots, soak them in 75 vol% alcohol for 30-60 s, soak them in 0.1 vol% mercuric chloride solution for 5-7 min, and rinse them with sterile water 5 times, each time for 1 min.

3) Induction culture: The cotyledon segments of the sterilized seedlings are cut as explants and inoculated onto an induction medium. The culture is carried out for 30 days under the conditions of a light rate of 80 μmol·m ^-2 ·s ^-1 , a light duration of 16 h/d, and a culture temperature of 25±2°C. The induction of adventitious buds is observed and recorded. The formula of the induction medium is selected from any one of the following:

①DCR medium + 0.75 mg/L 6-BA + 0.001 mg/L TDZ;

②DCR medium + 0.75 mg/L 6-BA + 0.01 mg/L TDZ;

③DCR medium + 0.75 mg/L 6-BA + 0.05 mg/L TDZ;

④DCR medium + 1.5 mg/L 6-BA + 0.001 mg/L TDZ;

⑤DCR medium + 1.5 mg/L 6-BA + 0.01 mg/L TDZ;

⑥DCR medium + 1.5 mg/L 6-BA + 0.05 mg/L TDZ;

⑦DCR medium + 3mg/L 6-BA + 0.001mg/L TDZ;

⑧DCR medium + 3 mg/L 6-BA + 0.01 mg/L TDZ;

⑨DCR medium + 3 mg/L 6-BA + 0.05 mg/L TDZ.

The results showed (Table 2) that when the 6-BA concentration was 0.75 mg/L and 3 mg/L, the induction rate of adventitious buds was low; when the 6-BA concentration was 1.5 mg/L, it showed a relatively high efficiency, indicating that Japanese larch cotyledons have a certain tolerance to 6-BA, and too high or too low is not conducive to the production of adventitious buds; therefore, the DCR medium with added 0.001 mg/L TDZ and 1.5 mg/L 6-BA has the highest efficiency in inducing larch explants.

Table 2 Effects of different concentrations of 6-BA combined with TDZ on adventitious bud induction of Japanese larch

Example 4

The effects of different concentrations of activated carbon on the elongation of adventitious buds of Japanese larch were investigated as follows:

1) Sowing: Select mature and plump Japanese larch seeds, soak them in warm water for 24 hours, and sow them on a cultivation substrate containing vermiculite. After the seeds germinate for 15 to 20 days, select healthy seedlings as materials for the next step of the experiment.

2) Disinfection: Wash the seedlings obtained in step 1) with tap water, remove the roots, soak them in 75 vol% alcohol for 30-60 s, soak them in 0.1 vol% mercuric chloride solution for 5-7 min, and rinse them with sterile water 5 times, each time for 1 min.

3) Induction culture: The cotyledon segments of the sterilized seedlings were cut into pieces as explants and inoculated into the induction medium. The culture was carried out for 30 days under the conditions of a light rate of 80 μmol·m ^-2 ·s ^-1 , a light duration of 16 h/d, and a culture temperature of 25±2°C. The induction of adventitious buds was observed and recorded. The formula of the induction medium was: DCR medium + 1.5 mg/L 6-BA + 0.001 mg/LTDZ.

4) Proliferation culture: the adventitious buds obtained by culture are cut and transferred to a proliferation medium, and cultured under the conditions of a light rate of 80 μmol·m ^-2 ·s ^-1 , a light duration of 16 h/d, and a culture temperature of 25±2°C, and the growth of the adventitious buds is observed and recorded, and the elongation rate is calculated after 60 days; the formula of the proliferation medium is selected from any one of the following:

①DCR medium + 1.5 mg/L 6-BA + 0.001 mg/L TDZ;

②DCR culture medium + 1.5mg/L 6-BA + 0.001mg/L TDZ + 0.2g/L activated carbon;

③DCR culture medium + 1.5mg/L 6-BA + 0.001mg/L TDZ + 0.5g/L activated carbon;

④DCR culture medium + 1.5 mg/L 6-BA + 0.001 mg/L TDZ + 1 g/L activated carbon.

The results showed (Figure 2) that with the increase of activated carbon dosage, the elongation of adventitious buds first increased and then decreased, and the elongation rate reached the maximum value when the activated carbon dosage was 0.2 g/L. Therefore, 0.2 g/L of activated carbon was added to the culture medium in subsequent experiments.

Example 5

An efficient regeneration method using Japanese larch cotyledons as explants, the steps are as follows:

1) Sowing: Select mature and plump Japanese larch seeds, soak them in warm water for 24 hours, and sow them on a substrate made of nutrient soil, vermiculite and perlite in a ratio of 2:2:1 (v/v). After the seeds germinate for 15 to 20 days, select healthy seedlings as materials for the next experiment.

2) Disinfection: Wash the seedlings obtained in step 1) with tap water, remove the roots, soak them in 75 vol% alcohol for 30-60 s, soak them in 0.1 vol% mercuric chloride solution for 5-7 min, and rinse them with sterile water 5 times, each time for 1 min.

3) Induction culture: The cotyledon segments of the sterilized seedlings were cut into pieces as explants, inoculated into an induction medium, and cultured for 30 days under the conditions of a light rate of 80 μmol·m ^-2 ·s ^-1 , a light duration of 16 h/d, and a culture temperature of 25±2°C to obtain adventitious buds; the formula of the induction medium was: DCR medium + 1.5 mg/L 6-BA + 0.001 mg/L TDZ.

4) Proliferation culture: the adventitious buds obtained by culture are cut and transferred to a proliferation culture medium, and cultured for 60 days under the conditions of a light rate of 80 μmol·m ^-2 ·s ^-1 , a light duration of 16 h/d, and a culture temperature of 25±2°C to obtain adventitious bud clusters; the formula of the proliferation culture medium is: DCR culture medium + 1.5 mg/L 6-BA + 0.001 mg/L TDZ + 0.2 g/L activated carbon.

5) Rooting culture: The adventitious bud clusters obtained by culture are inoculated into the rooting medium, and cultured for 30-60 days under the conditions of a light rate of 80 μmol·m ^-2 ·s ^-1 , a light duration of 16 h/d, and a culture temperature of 25±2°C to obtain rooted seedlings; the formula of the rooting medium is: DCR medium + 0.05 mg/L NAA + 0.2 g/L activated carbon. Under this condition, the rooting rate is 50%.

6) Hardening of seedlings: Place the rooted seedlings obtained by culture in a half-open bottle of tap water for hardening in the seedling hardening room with a temperature of 25°C, a light duration of 16 h/d, a light intensity of 80 μmol·m ^-2 ·s ^-1 , and a humidity of 65%.

7) Transplantation of rooted seedlings: After 10 days of hardening, when the roots turn from white to grayish white, the rooted seedlings are transplanted. When transplanting, first rinse the rooted seedlings in clean water to remove the culture medium of the roots, then dry the surface moisture of the roots, and then transplant the rooted seedlings into a container filled with a substrate made of nutrient soil, vermiculite and perlite mixed in a ratio of 2:2:1 (v/v). Cultivate in the greenhouse for 1-2 weeks. The greenhouse cultivation conditions are: light time 16 h/d, light intensity 2000 lux, temperature 21-26℃, followed by shade and diffuse light cultivation, spray water every day, and keep the humidity at 40%-60%.

The regeneration process is shown in Figure 3. The cotyledons of Japanese larch (Figure 3A) were inoculated on the adventitious bud induction medium for 15-20 days of induction culture. The cotyledons began to swell and the cells began to be tightly arranged around the cell wall. At about 21 days of culture, small protrusions began to gradually appear on the surface of the cotyledons, which was a signal for the occurrence of adventitious buds. At 30 days of culture, adventitious buds gradually formed (Figures 3B~3C); at about 60 days of proliferation culture, the adventitious buds elongated to a certain length and obtained adventitious bud clusters (Figures 3D~3E); the obtained adventitious bud clusters were moved to the rooting medium to promote rooting and continued elongation (Figures 3F~3G); at 30-60 days of rooting culture, the seedlings were taken out of the bottle for hardening, transplanted to the soil, and continued to grow (Figure 3H).

According to the results of paraffin sections (Figure 4), the occurrence of adventitious buds in Japanese larch cotyledons did not go through the callus stage, and the adventitious buds maintained vascular connection with the mother plant, which belongs to the direct organogenesis pathway (Figure 4B~4D). Under the stimulation of hormones, the cells on the surface and below the surface of the cotyledons rapidly divided to form meristems and began to protrude (Figure 4C). The cells on both sides of the meristems also began to divide rapidly under the stimulation of hormones to form bud primordia (Figure 4D), and further formed adventitious buds visible to the naked eye (Figure 4E). The adventitious buds that reached a certain height were transferred to the rooting medium, and the root primordium could be observed 30 days after inoculation.

The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims (4)

1. A highly efficient regeneration method using Japanese larch cotyledons as explants, characterized in that it comprises the following steps: 1) Sowing: Select mature and plump seeds, soak them in tap water for 24 hours, and then sow them in the substrate. After the seeds germinate for 15 to 20 days, select the healthy seedlings as the materials for the next experiment; 2) Disinfection: Wash the seedlings obtained in step 1) with tap water, remove the roots, and soak them in 75 vol% alcohol for 30-60 seconds, 0.1 vol% mercuric chloride solution for 5-7 minutes, and rinse with sterile water 5 times; 3) Induction culture: taking cotyledon segments of the seedlings disinfected in step 2) as explants, inoculating them on an induction medium and culturing them for 30 days to obtain adventitious buds; the formula of the induction medium is: DCR medium + 1.5 mg/L 6-BA + 0.001 mg/L TDZ; 4) Proliferation culture: the adventitious buds obtained in step 3) are cut and transferred to a proliferation medium for 60 days to obtain adventitious bud clusters; the formula of the proliferation medium is: DCR medium + 1.5 mg/L 6-BA + 0.001 mg/L TDZ + 0.2 g/L activated carbon; 5) Rooting culture: inoculate the adventitious bud clusters obtained by the culture in step 4) onto a rooting medium and culture for 30 to 60 days to obtain rooted seedlings; the formula of the rooting medium is: DCR medium + 0.05 mg/L NAA + 0.2 g/L activated carbon; 6) Hardening the seedlings: The rooted seedlings obtained in step 5) are transferred to a hardening room for hardening; 7) Transplanting: After 10 days of hardening, transplant the rooted seedlings into the substrate and culture them in the greenhouse for 1 to 2 weeks, followed by shade and diffuse light cultivation. keep; The matrix is prepared by mixing nutrient soil, vermiculite and perlite in a volume ratio of 2:2:1; The conditions for the induction culture, proliferation culture and rooting culture are: light rate 80 μmol·m ^-2 ·s ^-1 , light time 16 h/d, room temperature 25±2°C; The step 6) is specifically as follows: placing the rooted seedlings cultured in step 5) into tap water in a half-open bottle for hardening, wherein the temperature in the hardening room is 25°C, the light duration is 16h/d, the light intensity is 80μmol·m ^-2 ·s ^-1 , and the humidity is 65%; The step 7) is specifically as follows: after hardening the seedlings for 10 days, when the root system changes from white to grayish white, the rooted seedlings are transplanted. When transplanting, the rooted seedlings are first rinsed in clean water to remove the culture medium of the roots, and then the surface moisture of the roots is dried, and then the rooted seedlings are transplanted into a container containing a substrate, and cultured in a greenhouse for 1 to 2 weeks. The greenhouse culture conditions are: light time 16h/d, light intensity 2000lux, temperature 21 to 26°C, followed by shade and diffuse light culture, spraying water every day, and maintaining the humidity at 40% to 60%. 2. Application of the high-efficiency regeneration method according to claim 1 in in vitro propagation of Japanese larch. 3. Use of the high-efficiency regeneration method according to claim 1 in establishing a Japanese larch cultivation system. 4. Application of the high-efficiency regeneration method according to claim 1 in the germplasm preservation of superior strains of Japanese larch.