A method for rapid breeding seedlings of drought-resistant kiwi rootstock seedlings

Abstract

This invention provides a method for rapid propagation of seedlings from drought-resistant kiwifruit rootstock seeds, comprising the following steps: S1, selecting individual plants that still exhibit excellent performance after 20-30 days without watering at 32-37℃, extracting the seeds from the fruit, and drying them for later use; S2, soaking the seeds in a 0.15mg / L gibberellin solution for 24-48 hours, then rinsing and disinfecting them; S3, culturing the disinfected seeds in a culture medium until seedlings emerge, followed by acclimatization and transplanting. Kiwifruit rootstocks cultivated using this method maintain well-developed root systems, vigorous growth, and do not wilt or die even after 20-30 days without watering during the hot summer months. This significantly promotes the kiwifruit industry in kiwifruit-growing areas with poor soil and irrigation conditions, thereby improving economic benefits.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture technology, specifically relating to a method for rapid propagation of seedlings from drought-resistant kiwifruit rootstock seeds. Background Technology

[0002] Ankang City, located in the heart of the Qinling-Bashan Mountains, has a large amount of hillside and abandoned land, making it very difficult to grow grain crops. This has made it a new area for the development of the kiwi fruit industry.

[0003] In Ankang City, land suitable for kiwifruit cultivation is generally characterized by scattered, undeveloped plots, shallow and infertile soil, and hard, poorly aerated soil, severely impacting the profitability of kiwifruit cultivation. Through years of research on kiwifruit cultivation techniques in mountainous areas, as well as field surveys and experiments, the Ankang Academy of Agricultural Sciences has discovered that kiwifruit trees have fleshy root systems with weak extension capabilities. In hard soils, they struggle to penetrate deeply and expand, are prone to water loss during droughts, and are susceptible to rotting in waterlogged conditions. Therefore, they require highly loose and well-aerated soil. Thus, the selection and rapid propagation of kiwifruit rootstocks with high drought and flood resistance are crucial for the cultivation and promotion of kiwifruit on the slopes of the Qinling-Bashan Mountains. Summary of the Invention

[0004] In view of this, in order to screen and breed kiwifruit rootstocks with high drought and waterlogging resistance, this invention provides a method for rapid propagation of seedlings from drought-resistant kiwifruit rootstock seeds.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A method for rapid propagation of seedlings from drought-resistant kiwifruit rootstock seeds includes the following steps:

[0007] S1. Select individual plants that still perform well after being kept dry for 20 to 30 consecutive days at a high temperature of 32 to 37℃ without watering, remove the seeds from the fruits and dry them for later use.

[0008] S2. Soak the above seeds in a 0.15 mg / L gibberellin solution for 24–48 hours, then rinse and disinfect them.

[0009] S3. Place the disinfected seeds in a culture medium for cultivation. After the seedlings have grown, they can be acclimatized and transplanted.

[0010] Further, the disinfection step in step S2 is as follows: First, add the seeds to a 75% ethanol solution for disinfection for 1-2 minutes, rinse with sterile water, then add a 4% sodium hypochlorite solution for disinfection for 7-9 minutes, rinse with sterile water, then soak in a 3% hydrogen peroxide solution for 5 minutes, and finally rinse with sterile water and pour onto sterile filter paper to absorb the moisture, thus completing the disinfection process.

[0011] Furthermore, the seeds in step S1 include young seeds and mature seeds. The young seeds are taken from the seeds of kiwifruit fruits grown in September-October, and the mature seeds are taken from the seeds of kiwifruit fruits grown in November-December.

[0012] Furthermore, when the seeds are young seeds, the specific cultivation process in step S3 is as follows:

[0013] Remove the apical embryo 0.1–0.3 mm from the seed tip, retaining the endosperm; inoculate the endosperm into callus medium to cultivate callus tissue; inoculate the callus tissue into shoot cluster medium to cultivate shoot clusters; when the shoot clusters have grown to have multiple tender leaves, longitudinally cut the shoots to ensure that each shoot base has callus tissue, and inoculate them into proliferation medium to proliferate and differentiate into a large number of tender shoots; when the tender shoots grow to 4–5 cm, cut them from the internodes into stem segments of about 2 cm with buds and inoculate them into rooting medium; when each seedling grows 4–6 roots with a root length of 2–3 cm, seedlings are obtained.

[0014] When the seed is a mature seed, the specific cultivation process in step S3 is as follows:

[0015] Make a longitudinal cut of 0.05-0.1 mm about 0.1-0.2 mm from the top of the seed, and inoculate it into culture medium I. When the germination rate reaches more than 90%, transfer it to culture medium II. Seedlings are obtained when each seedling grows 4-6 roots with a root length of 2-3 cm.

[0016] Furthermore, the callus culture medium is formulated as follows: MS + TDZ (0.25-1 mg / L) + 6-BA (0.5-2.0 mg / L) + NAA (0.05-1.0 mg / L) + sucrose (20 g / L) + agar (5 g / L);

[0017] The budding culture medium formula is as follows: MS + ZT (0.1-1 mg / L) + 6-BA (1.0-3.0 mg / L) + IBA (0.2-1.0 mg / L) + sucrose (20 g / L) + agar (5 g / L);

[0018] The proliferation medium formulation is as follows: MS + GA3 (0.1-1.5 mg / L) + 6-BA (1.0-2.0 mg / L) + IBA (0.1-0.5 mg / L) + sucrose (20 g / L) + agar (5 g / L);

[0019] The rooting medium formula is as follows: 1 / 2 MS + IBA (0.2-1.0 mg / L) + NAA (0.1-0.5 mg / L) + IAA (0.01-0.15 mg / L) + sucrose (20 g / L) + agar (5 g / L);

[0020] The culture medium I is formulated as follows: MS + TDZ (0.25-1 mg / L) + 6-BA (0.5-2.0 mg / L) + NAA (0.05-1.0 mg / L) + sucrose (20 g / L) + agar (5 g / L);

[0021] The culture medium II formulation is as follows: 1 / 2 MS + IBA (0.2-1.0 mg / L) + NAA (0.1-0.5 mg / L) + IAA (0.01-0.15 mg / L) + sucrose (20 g / L) + agar (5 g / L).

[0022] Furthermore, the young seeds were cultured in callus medium, shoot bud medium, and proliferation medium under the following conditions: light intensity 2000–2500 lx, air humidity 60%, constant temperature 25°C, and light duration 14 h / d.

[0023] The culture conditions in the rooting medium were as follows: first, low light culture was carried out, with a light intensity of 400 lx, a light duration of 12 h / d, and a temperature of 20–24 ℃. After 13–16 days of culture, the light intensity was increased to 3000–3200 lx, and culture continued.

[0024] The mature seeds were cultured in culture medium I under the following conditions: light intensity 2000–2500 lx, air humidity 60%, constant temperature 25°C, and light duration 14 h.

[0025] The culture conditions in medium II were as follows: first, low light culture was carried out with a light intensity of 400 lx, a light duration of 12 h / d, and a temperature of 20–24 °C. After 13–16 days of culture, the light intensity was increased to 3000–3200 lx, and culture continued.

[0026] Furthermore, the acclimatization process is as follows: the seedlings, together with the culture medium, are placed under conditions of 20-24°C and 50%-70% shading for 5-7 days. Then, the culture medium is washed off and the seedlings are transferred to a substrate for cultivation. The water content is controlled at 80%, and the seedlings are cultivated under normal light conditions for 15-25 days to complete the acclimatization.

[0027] Furthermore, the matrix is ​​prepared by mixing sand, humus and perlite in a mass ratio of 2:5:1.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0029] (1) In traditional kiwifruit tissue culture, most researchers choose to use kiwifruit stem segments, leaves, and mature seeds for tissue culture. However, kiwifruit stem segments and leaves are prone to carrying viruses that cause kiwifruit canker, and mature seeds have weak differentiation capabilities. This invention collects kiwifruit seeds at two stages and, through processing, obtains young and mature seeds. Young seeds have the most vigorous differentiation and proliferation capabilities and are used for tissue induction culture, which can quickly produce a large number of drought-resistant kiwifruit rootstock seedlings. Mature seeds can be cultured into seedlings in one step using an optimized culture medium, shortening the propagation time.

[0030] (2) Through the induction method of the present invention, the callus induction rate of young seeds can reach 98.36%, the bud differentiation rate can reach 98.10%, the proliferation coefficient can reach 9.32, and the rooting rate can reach 99.12%; the germination rate of mature seeds can reach 99.33% and the rooting rate can reach 97.72%.

[0031] (3) The seedlings bred by this invention, after being domesticated and transplanted, still have well-developed root systems, vigorous growth, and high survival rate even if they are not watered for 20 to 30 consecutive days during the hot summer season. Attached Figure Description

[0032] Figure 1 This is a diagram of the shoot clusters formed when young kiwifruit seeds are inoculated into the No. 5 shoot cluster induction medium formula in this invention.

[0033] Figure 2 This image shows the young shoots of kiwifruit seed cultured in this invention, inoculated into the No. 8 proliferation medium formula.

[0034] Figure 3 This is a diagram showing the rooting of seedlings from the proliferation culture of young kiwifruit seeds in this invention, after being inoculated into the No. 4 rooting medium formula.

[0035] Figure 4 This is a germination diagram of mature kiwifruit seeds inoculated with the No. 3 germination medium formula in this invention.

[0036] Figure 5 This is a diagram showing the rooting of seedlings cultivated from mature kiwifruit seeds in this invention, after being inoculated into rooting medium formula No. 6.

[0037] Figure 6 Comparison of root systems of rootstocks for drought resistance test (left: seedlings and roots cultivated in Comparative Example 1, middle: seedlings and roots cultivated from young seeds in this application, right: seedlings and roots cultivated from mature seeds in this application). Detailed Implementation

[0038] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention.

[0039] Example 1

[0040] This embodiment provides a method for selecting seedlings from highly drought-resistant kiwifruit rootstocks, as detailed below:

[0041] During the summer months of July and August, when outdoor temperatures ranged from 32°C to 37°C, field trials were conducted to assess the drought tolerance of kiwifruit rootstocks. Individual plants that performed well even after 20-30 consecutive days without watering under high temperatures were tagged. Fruits at the stage of cessation of growth were harvested in September and October, and the seeds were washed out with water to collect the young seeds. Fruits at the stage of maturity were harvested in October and November, and the seeds were washed out with water to collect the mature seeds. The young and mature seeds obtained were then selected based on their intact shape and plumpness, dried in a well-ventilated area, and stored at room temperature.

[0042] Example 2

[0043] This embodiment provides a method for disinfecting seedlings from highly drought-resistant kiwifruit rootstock, as detailed below:

[0044] Place the obtained young and mature seeds on filter paper, spreading them evenly. Pour 0.15 mg / L gibberellin solution into a petri dish, ensuring the solution covers the seeds. Let the seeds soak for 24–48 hours. Remove the seeds and rinse them under running water for 1–2 hours. After blotting the surface moisture with filter paper, place them in a clean bench. Using sterile forceps, place the seeds into a 25 mL sterile centrifuge tube (no more than 20 seeds). Add 75% ethanol solution, tighten the cap, and manually shake the centrifuge tube to ensure full contact between the seeds and ethanol for 1–2 minutes. Rinse 3–5 times with sterile water. Add 4% sodium hypochlorite solution and disinfect for 7–9 minutes. Rinse 3–5 times with sterile water, then soak in 3% hydrogen peroxide solution for 5 minutes. Finally, rinse 3–5 times with sterile water and pour the seeds onto sterile filter paper to blot off excess moisture.

[0045] The sterilized seeds were inoculated onto MS basal medium, and the seed contamination rate and germination rate were observed. The details are shown in Table 1 below:

[0046] Table 1. Effects of different disinfection methods on seed germination rate

[0047]

[0048] As shown in Table 1 above, the best disinfection effect is achieved when 75% ethanol, 4% sodium hypochlorite, and 3% hydrogen peroxide are used in combination. Among them, when the treatment time of 4% sodium hypochlorite is 7 to 9 minutes, the contamination rate is 5.23% to 8.11%, and the germination rate is as high as 85.63% to 95.54%.

[0049] Example 3

[0050] This embodiment provides a seed culture program for young seeds, as detailed below:

[0051] Remove the apical embryo 0.1–0.3 mm from the seed tip, retaining the endosperm; inoculate the endosperm into callus medium to cultivate callus tissue; inoculate the callus tissue into shoot cluster medium to cultivate shoot clusters; when the shoot clusters have grown to have multiple tender leaves, longitudinally cut the shoots to ensure that each shoot base has callus tissue, and inoculate them into proliferation medium to proliferate and differentiate into a large number of tender shoots; when the tender shoots grow to 4–5 cm, cut them into stem segments of about 2 cm with buds from the internodes and inoculate them into rooting medium; when each seedling grows 4–6 roots with a root length of 2–3 cm, the seedlings are obtained.

[0052] The specific formulations for each culture medium are as follows:

[0053] Callus culture medium formula: MS + TDZ (0.25~1mg / L) + 6-BA (0.5~2.0mg / L) + NAA (0.05~1.0mg / L) + sucrose (20g / L) + agar (5g / L);

[0054] The formula for the budding medium is: MS + ZT (0.1-1 mg / L) + 6-BA (1.0-3.0 mg / L) + IBA (0.2-1.0 mg / L) + sucrose (20 g / L) + agar (5 g / L);

[0055] The culture medium formula is: MS + GA3 (0.1-1.5 mg / L) + 6-BA (1.0-2.0 mg / L) + IBA (0.1-0.5 mg / L) + sucrose (20 g / L) + agar (5 g / L);

[0056] Rooting medium formula: 1 / 2 MS + IBA (0.2-1.0 mg / L) + NAA (0.1-0.5 mg / L) + IAA (0.01-0.15 mg / L) + sucrose (20 g / L) + agar (5 g / L).

[0057] Under specific conditions at each stage of cultivation:

[0058] The conditions for culturing young seeds in callus medium, shoot bud medium, and proliferation medium were: light intensity 2000–2500 lx, air humidity 60%, constant temperature 25℃, and light duration 14 h / d. The conditions for culturing in rooting medium were: first, low light culture with light intensity 400 lx, light duration 12 h / d, and temperature 20–24℃. After 13–16 days of culture, the light intensity was increased to 3000–3200 lx, and culture continued.

[0059] Furthermore, the effects of different hormone concentrations in different culture media on the culture effect were investigated, and the results are shown in Table 2-5:

[0060] Table 2 Effects of different hormone treatments on callus induction

[0061]

[0062] Table 3 Effects of different hormone treatments on shoot induction

[0063]

[0064] Table 4. Effects of different hormone treatments on proliferation coefficient

[0065]

[0066] Table 5. Effects of different hormone treatments on rooting efficacy

[0067]

[0068] Table 2-5 shows that the culture media provided in this application for different stages of young seeds all showed good culture effects. Among them, when the callus culture medium formula was MS + TDZ (0.6 mg / L) + 6-BA (1.5 mg / L) + NAA (0.5 mg / L) + sucrose (20 g / L) + agar (5 g / L), the callus induction rate reached as high as 98.36%; when the shoot culture medium formula was MS + ZT (0.4 mg / L) + 6-BA (2.0 mg / L) + IBA (0.6 mg / L) + sucrose (20 g / L) + agar, the callus induction rate reached as high as 98.36%. The shoot induction rate reached 98.10% when the medium was MS + GA3 (1.5 mg / L) + 6-BA (1.5 mg / L) + IBA (0.5 mg / L) + sucrose (20 g / L) + agar (5 g / L); the proliferation coefficient reached 9.32 when the medium was MS + IBA (0.4 mg / L) + NAA (0.1 mg / L) + IAA (0.1 mg / L) + sucrose (20 g / L) + agar (5 g / L); and the rooting rate reached 99.12% when the medium was 1 / 2 MS + IBA (0.4 mg / L) + NAA (0.1 mg / L) + IAA (0.1 mg / L) + sucrose (20 g / L) + agar (5 g / L).

[0069] Example 4

[0070] This embodiment provides a mature seed cultivation method, as detailed below:

[0071] Make a longitudinal cut of 0.05-0.1 mm about 0.1-0.2 mm from the top of the seed, and inoculate it into culture medium I. When the germination rate reaches more than 90%, transfer it to culture medium II. Seedlings are obtained when each seedling grows 4-6 roots with a root length of 2-3 cm.

[0072] The specific formulations for each culture medium are as follows:

[0073] The culture medium I formula is as follows: MS + TDZ (0.25~1mg / L) + 6-BA (0.5~2.0mg / L) + NAA (0.05~1.0mg / L) + sucrose (20g / L) + agar (5g / L);

[0074] The culture medium II formula is as follows: 1 / 2 MS + IBA (0.2-1.0 mg / L) + NAA (0.1-0.5 mg / L) + IAA (0.01-0.15 mg / L) + sucrose (20 g / L) + agar (5 g / L).

[0075] Under specific conditions at each stage of cultivation:

[0076] The mature seeds were cultured in culture medium I under the following conditions: light intensity 2000–2500 lx, air humidity 60%, constant temperature 25°C, and light duration 14 h.

[0077] The culture conditions in medium II were as follows: first, low light culture was carried out with a light intensity of 400 lx, a light duration of 12 h / d, and a temperature of 20–24 °C. After 13–16 days of culture, the light intensity was increased to 3000–3200 lx, and culture continued.

[0078] Furthermore, the effects of different hormone concentrations in different culture media on the culture effect were investigated, and the results are shown in Tables 6 and 7:

[0079] Table 6. Effects of different hormone treatments on germination rate

[0080]

[0081] Table 7 Effects of different hormone treatments on rooting efficacy

[0082]

[0083]

[0084] As shown in Tables 6-7, the culture media provided in this application for different stages of mature seeds all showed good results. Among them, the highest germination rate of medium I (MS + TDZ (0.3 mg / L) + 6-BA (2.0 mg / L) + NAA (1.0 mg / L) + sucrose (20 g / L) + agar (5 g / L)) reached 99.33%. The highest rooting rate of medium II (1 / 2 MS + IBA (0.4 mg / L) + NAA (0.5 mg / L) + IAA (0.04 mg / L) + sucrose (20 g / L) + agar (5 g / L) reached 97.72%.

[0085] Comparative Example 1

[0086] This comparative example provides a conventional kiwifruit rootstock seedling cultivation process, as follows: Collect seeds of common kiwifruit rootstock, select seeds with intact shape and plump grains, and sow them in seedling trays. During this period, water appropriately to keep the soil moist and strengthen daily management. Once the seeds germinate and grow to 5-10cm, seedlings are obtained.

[0087] drought resistance test

[0088] First, the seedlings obtained by the preparation method of this application are subjected to acclimatization treatment. The specific steps are as follows: the seedlings and culture medium are placed together under the conditions of 20-24℃ and 50%-70% shading rate for 5-7 days. Then, the culture medium is washed off and the seedlings are transferred to a substrate (prepared by mixing sand, humus and perlite in a mass ratio of 2:5:1) for cultivation. The water content is controlled at 80%, and the seedlings are cultivated under normal light conditions for 15-25 days to complete the acclimatization.

[0089] One hundred seedlings from the acclimatized seedlings and 100 seedlings from Comparative Example 1 were planted in the experimental orchard. Initially, the temperature in the experimental field was maintained at 18–28℃, humidity at 40%–60%, and soil moisture content at 70%–80% to ensure rapid seedling growth. A small amount of organic fertilizer could be applied during this period to ensure healthy tree growth. Subsequently, the ambient temperature was controlled between 30℃ and 38℃, and watering was withheld for 20–30 consecutive days. The growth of the kiwifruit rootstock was then investigated. Specific results are shown in Table 8.

[0090] Table 8. Effects of different growing environments on the survival rate of kiwifruit rootstock seedlings

[0091]

[0092] As shown in Table 8, compared with kiwifruit rootstock seedlings cultivated by conventional methods, the seedlings cultivated in this application have higher drought resistance. They maintain a high survival rate even after 20 to 30 consecutive days without watering when the ambient temperature is between 30℃ and 38℃. The survival rate reaches 100% when the temperature is between 30℃ and 32℃. However, at the higher temperature of 38℃, the survival rate of seedlings cultivated by conventional methods is only 15%, while the survival rate of seedlings cultivated in this application reaches 90%.

[0093] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for rapid propagation of seedlings from drought-resistant kiwifruit rootstock seeds, characterized in that, Includes the following steps: S1. Select individual plants that still perform well after being kept dry for 20 to 30 consecutive days at a high temperature of 32 to 37℃ without watering, remove the seeds from the fruits and dry them for later use. S2. Soak the above seeds in a 0.15 mg / L gibberellin solution for 24-48 hours, then rinse and disinfect them. S3. Place the disinfected seeds in a culture medium for cultivation. After the seedlings have grown, they can be domesticated and transplanted. The disinfection steps described in step S2 are as follows: First, add the seeds to a 75% ethanol solution for 1-2 minutes for disinfection, rinse with sterile water, then add a 4% sodium hypochlorite solution for 7 minutes for disinfection, rinse with sterile water, then soak in a 3% hydrogen peroxide solution for 5 minutes, and finally rinse with sterile water and pour onto sterile filter paper to absorb the moisture, thus completing the disinfection process. The seeds in step S1 include young seeds and mature seeds. The young seeds are taken from the seeds of kiwifruit fruits grown from September to October, and the mature seeds are taken from the seeds of kiwifruit fruits grown from November to December. When the seeds are young seeds, the specific cultivation process in step S3 is as follows: Remove the apical embryo 0.1-0.3 mm from the seed tip, retaining the endosperm; inoculate the endosperm into callus medium to cultivate callus tissue; inoculate the callus tissue into shoot cluster medium to cultivate shoot clusters; when the shoot clusters have grown to have multiple tender leaves, longitudinally cut the shoots to ensure that each shoot base has callus tissue, and inoculate them into proliferation medium to proliferate and differentiate into a large number of tender shoots; when the tender shoots grow to 4-5 cm, cut them from the internodes into stem segments of about 2 cm with buds and inoculate them into rooting medium; when each seedling grows 4-6 roots with a root length of 2-3 cm, the seedlings are obtained. When the seed is a mature seed, the specific cultivation process in step S3 is as follows: Make a longitudinal cut of 0.05-0.1 mm about 0.1-0.2 mm from the top of the seed, and inoculate it into culture medium I. When the germination rate reaches more than 90%, transfer it to culture medium II. Seedlings are obtained when each seedling grows 4-6 roots with a root length of 2-3 cm. The callus culture medium formula is as follows: MS + TDZ 0.6 mg / L + 6-BA 1.5 mg / L + NAA 0.5 mg / L + sucrose 20 g / L + agar 5 g / L; The budding culture medium formula is as follows: MS + ZT 0.4 mg / L + 6-BA 2.0 mg / L + IBA 0.6 mg / L + sucrose 20 g / L + agar 5 g / L; The proliferation medium formulation is as follows: MS + GA 3 1.5 mg / L + 6-BA 1.5 mg / L + IBA 0.5 mg / L + sucrose 20 g / L + agar 5 g / L; The rooting medium formula is as follows: 1 / 2 MS + IBA 0.4 mg / L + NAA 0.1 mg / L + IAA 0.1 mg / L + sucrose 20 g / L + agar 5 g / L; The culture medium I has the following formulation: MS + TDZ 0.3 mg / L + 6-BA 2.0 mg / L + NAA 1.0 mg / L + sucrose 20 g / L + agar 5 g / L; The culture medium II formulation is as follows: 1 / 2 MS + IBA 0.4 mg / L + NAA 0.5 mg / L + IAA 0.04 mg / L + sucrose 20 g / L + agar 5 g / L.

2. The method according to claim 1, characterized in that, The young seeds were cultured in callus medium, shoot bud medium and proliferation medium under the following conditions: light intensity 2000~2500 lx, air humidity 60%, constant temperature 25℃, and light duration 14h / d. The culture conditions in the rooting medium were as follows: first, low light culture was carried out, with a light intensity of 400 lx, a light duration of 12 h / d, and a temperature of 20–24 ℃. After 13–16 days of culture, the light intensity was increased to 3000–3200 lx, and culture continued. The mature seeds were cultured in culture medium I under the following conditions: light intensity of 2000-2500 lx, air humidity of 60%, constant temperature of 25℃, and light duration of 14h. The culture conditions in medium II were as follows: first, low light culture was carried out, with a light intensity of 400 lx, a light duration of 12 h / d, and a temperature of 20~24℃. After 13~16 days of culture, the light intensity was increased to 3000~3200 lx, and the culture continued.

3. The method of claim 1, wherein, The acclimatization process is as follows: The seedlings, together with the culture medium, are placed in a temperature of 20-24℃ and a shading rate of 50%-70% for 5-7 days. Then, the culture medium is washed off and the seedlings are transferred to a substrate for cultivation. The water content is controlled at 80%, and the seedlings are cultivated under normal light conditions for 15-25 days to complete the acclimatization.

4. The method of claim 3, wherein, The matrix is ​​prepared by mixing sand, humus and perlite in a mass ratio of 2:5:1.

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