Living body preservation culture solution for pteridophyte spores as well as preparation method and application of living body preservation culture solution

Abstract

The invention discloses a living body preservation culture solution for pteridophyte spores. The living body preservation culture solution is prepared from ammonium nitrate, potassium nitrate, monopotassium phosphate, magnesium sulfate, calcium chloride, potassium iodide, boric acid, manganese sulfate, zinc sulfate, sodium molybdate, copper sulfate, cobalt chloride, ferrous sulfate, disodium ethylene diamine tetraacetate, glycine, thiamine hydrochloride, pyridoxine hydrochloride, nicotinic acid, vitamin B1, vitamin B6 and nicotinic acid. The living body preservation culture solution can be used for living body preservation and rapid cultivation of pteridophyte spores in a bacterium-carrying environment, the preservation time is long, the cultivation time is shortened, the method is simple, convenient and easy to operate, and the living body preservation culture solution is suitable for living body preservation and rapid seedling breeding of drynaria fortunei and cibotium barometz spore culture and is suitable for factory-like large-scale batch market production.

Description

Technical Field

[0001] This invention relates to a culture medium for the in vivo preservation of fern spores, its preparation method and application, belonging to the field of storage and propagation of true fern spores, and is applicable to the in vivo preservation and propagation of spores of species such as *Ceratophyllum demersum* and *Cibotium barometz*. Background Technology

[0002] Ferns (Pteridophyta) are a phylum within the plant kingdom. They possess true vascular tissue in their roots, stems, and leaves, and reproduce by spores. The vast majority of their leaves have sporangia on the lower surface, which aggregate into various spotted or linear clusters of sporangia, initially green, turning rusty yellow with age; some are bare, while others have caps of various shapes. The oak fern (Drynaria roosii Nakaike) belongs to the genus Drynaria in the family Polypodiaceae. It typically grows epiphytically on rocks, creeping, or epiphytically on tree trunks, climbing in a spiral pattern. *Cibotium barometz* (L.) J. Sm. is a tree-like fern belonging to the genus *Cibotium* in the family Cibotaceae. It has a prostrate, thick rhizome bearing a large leaf at its apex, with a petiole up to 120 cm long. The leaf is brownish-red, with a glossy, cushion-like base of golden-yellow hairs, while the upper part is smooth. The leaf blade is large, broadly ovate-triangular, and tripinnately divided. The leaf blade is leathery or thickly papery. The sori are borne at the tips of the lower veinlets, with a hard, brownish-red indusia. The spores are triangular-tetrahedral and transparent.

[0003] There is a lot of research on the propagation of fern spores, but the preservation and cultivation are all carried out in a sterile environment and the methods are cumbersome. The present invention aims to design a culture medium that can preserve and cultivate fern spores in a sterile environment, with a long preservation time, which is convenient for industrial application by taking live plants for industrial cultivation, shortening the cultivation time, and making the method simple, easy to operate, and suitable for large-scale market production. Summary of the Invention

[0004] The purpose of this invention is to provide a live culture medium for preserving fern spores, its preparation method, and its applications. The live culture medium for preserving fern spores prepared by this invention can preserve and cultivate fern spores in a microbial environment, facilitating industrial-scale cultivation of live spores for production applications. It also offers long preservation times, shorter cultivation times, a simple and easy-to-operate method, and is suitable for large-scale market production.

[0005] The technical solution of the present invention: A viable culture medium for preserving fern spores, comprising, by weight, 412.5–1650 parts ammonium nitrate, 475–1900 parts potassium nitrate, 42.5–170 parts potassium dihydrogen phosphate, 92.5–370 parts magnesium sulfate, 110–440 parts calcium chloride, 0.8–0.9 parts potassium iodide, 5.5–6.5 parts boric acid, 21–23 parts manganese sulfate, 8–9 parts zinc sulfate, 0.2–0.3 parts sodium molybdate, 0.02–0.03 parts copper sulfate, 0.02–0.03 parts cobalt chloride, 27–28 parts ferrous sulfate, 37–38 parts disodium EDTA, 1.5–2.5 parts glycine, 0.05–0.15 parts thiamine hydrochloride, 0.4–0.6 parts pyridoxine hydrochloride, 0.4–0.6 parts nicotinic acid, and vitamin B1. It is made from 0.05~0.15 parts of vitamin B6, 0.4~0.6 parts of vitamin B6, and 0.4~0.6 parts of niacin.

[0006] The aforementioned culture medium for the preservation of fern spores contains, by weight, 825-1650 parts ammonium nitrate, 950-1900 parts potassium nitrate, 85-170 parts potassium dihydrogen phosphate, 185-370 parts magnesium sulfate, 220-440 parts calcium chloride, 0.83 parts potassium iodide, 6.2 parts boric acid, 22.30 parts manganese sulfate, 8.6 parts zinc sulfate, 0.25 parts sodium molybdate, 0.025 parts copper sulfate, 0.025 parts cobalt chloride, 27.8 parts ferrous sulfate, 37.3 parts disodium EDTA, 2 parts glycine, 0.1 parts thiamine hydrochloride, 0.5 parts pyridoxine hydrochloride, 0.5 parts nicotinic acid, 0.1 parts vitamin B1, 0.5 parts vitamin B6, and 0.5 parts nicotinic acid.

[0007] Specifically, the aforementioned culture medium for the preservation of fern spores contains the following effective components by weight: 1650 parts ammonium nitrate, 1900 parts potassium nitrate, 170 parts potassium dihydrogen phosphate, 370 parts magnesium sulfate, 440 parts calcium chloride, 0.83 parts potassium iodide, 6.2 parts boric acid, 22.30 parts manganese sulfate, 8.6 parts zinc sulfate, 0.25 parts sodium molybdate, 0.025 parts copper sulfate, 0.025 parts cobalt chloride, 27.8 parts ferrous sulfate, 37.3 parts disodium EDTA, 2 parts glycine, 0.1 parts thiamine hydrochloride, 0.5 parts pyridoxine hydrochloride, 0.5 parts nicotinic acid, 0.1 parts vitamin B1, 0.5 parts vitamin B6, and 0.5 parts nicotinic acid.

[0008] The aforementioned method for preparing the live cell preservation culture medium is carried out according to the following steps: weigh the effective components of the live cell preservation culture medium according to the weight ratio, dissolve them in distilled water or deionized water, and make up the volume so that the concentration of ammonium nitrate is 412.5~1650mg / L. Adjust the pH value to 5.6~6.0 with 1N sodium hydroxide solution and 1N hydrochloric acid solution.

[0009] Specifically, the aforementioned method for preparing the live cell preservation culture medium is carried out according to the following steps: weigh the effective components of the live cell preservation culture medium according to the weight ratio, dissolve them in distilled water or deionized water, and then make up the volume so that the concentration of ammonium nitrate is 825~1650mg / L, and adjust the pH value to 5.6~6.0.

[0010] More specifically, the aforementioned method for preparing the live cell preservation culture medium is carried out according to the following steps: weigh the effective components of the live cell preservation culture medium according to the weight ratio, dissolve them in distilled water or deionized water, and then make up the volume so that the concentration of ammonium nitrate is 1650 mg / L, and adjust the pH value to 5.6~6.0.

[0011] The aforementioned live preservation culture medium is used for the live preservation and rapid seedling cultivation of gametophytes of *Acer buergerianum*.

[0012] The aforementioned live preservation involves directly obtaining sporangia of *Adiantum capillus-veneris* spores using a dissecting microscope during the spore maturation period and placing them in the live preservation culture medium for live preservation. The live preservation temperature is 10°C, and the light conditions for live preservation are weak light of 50-100 Lx.

[0013] The aforementioned rapid seedling cultivation involves directly obtaining sporangia of *Adiantum capillus-veneris* spores using a dissecting microscope during the spore maturation period and placing them into the aforementioned live culture medium for rapid seedling cultivation. The rapid seedling cultivation temperature is 20-25℃, and the rapid seedling cultivation light conditions are moderate light of 500-1000 Lx.

[0014] The aforementioned rapid seedling temperature is 25℃, and the aforementioned rapid seedling light conditions are medium light of 1000Lx.

[0015] The beneficial effects of this invention are: Compared to traditional aseptic operations, the advantage of this invention is that it can be operated directly in a sterile environment, which greatly reduces costs and manpower issues.

[0016] The live cell preservation culture medium prepared by this invention can preserve fern gametophytes for 24 months under weak light conditions (50-100 Lux) at a temperature controlled at 10℃, ensuring a survival rate of 80% for up to 24 months. When the live culture medium prepared by this invention is used for the rapid seedling production of ferns, the culture temperature of fern spores is controlled at 25℃, and medium-intensity light of 500~1000Lu is used. The germination rate is up to 75%, and the growth rate is faster (the growth can reach its peak in 2 months, reaching 90~95%). The culture cycle is shortened, and more importantly, the spores are controlled to prevent spore contamination of other fern spores and pests. Attached Figure Description

[0017] Figure 1: Survival of fern spores after 24 months under different temperatures, light conditions and culture media; Figure 2 : Live survival of Cibotium barometz spores after 24 months under different temperature, light, and culture conditions; Figure 3 Diagrams showing the cultivation of fern spores under different temperature, light, and culture medium conditions; Figure 4 : Images of the cultivation of Cibotium barometz spores under different temperatures, light conditions, and culture media. Detailed Implementation

[0018] The present invention will be further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

[0019] Example 1: Preparation of live cell preservation culture medium: Dissolve 1650 mg ammonium nitrate, 1900 mg potassium nitrate, 170 mg potassium dihydrogen phosphate, 370 mg magnesium sulfate, 440 mg calcium chloride, 0.83 mg potassium iodide, 6.2 mg boric acid, 22.30 mg manganese sulfate, 8.6 mg zinc sulfate, 0.25 mg sodium molybdate, 0.025 mg copper sulfate, 0.025 mg cobalt chloride, 27.8 mg ferrous sulfate, 37.3 mg disodium EDTA, 2 mg glycine, 0.1 mg thiamine hydrochloride, 0.5 mg pyridoxine hydrochloride, 0.5 mg nicotinic acid, 0.1 mg vitamin B1, 0.5 mg vitamin B6, and 0.5 mg nicotinic acid in 800 mL of distilled water, bring the volume to 1 L, and adjust the pH to 5.6-6.0 with 1 N sodium hydroxide solution and 1 N hydrochloric acid solution to obtain the live cell preservation culture medium.

[0020] Example 2: Preparation of live cell preservation culture medium: Take 825 mg ammonium nitrate, 950 mg potassium nitrate, 85 mg potassium dihydrogen phosphate, 185 mg magnesium sulfate, 220 mg calcium chloride, 0.83 mg potassium iodide, 6.2 mg boric acid, 22.30 mg manganese sulfate, 8.6 mg zinc sulfate, 0.25 mg sodium molybdate, 0.025 mg copper sulfate, 0.025 mg cobalt chloride, 27.8 mg ferrous sulfate, 37.3 mg disodium EDTA, 2 mg glycine, 0.1 mg thiamine hydrochloride, 0.5 mg pyridoxine hydrochloride, 0.5 mg nicotinic acid, 0.1 mg vitamin B1, 0.5 mg vitamin B6, and 0.5 mg nicotinic acid, dissolve in 800 mL distilled water, and bring the volume to 1 L. Adjust the pH to 5.6-6.0 with 1 N sodium hydroxide solution and 1 N hydrochloric acid solution to obtain the live cell preservation culture medium.

[0021] Example 3: Preparation of live cell preservation culture medium: Dissolve 412.5 mg ammonium nitrate, 475 mg potassium nitrate, 42.5 mg potassium dihydrogen phosphate, 92.5 mg magnesium sulfate, 110 mg calcium chloride, 0.83 mg potassium iodide, 6.2 mg boric acid, 22.30 mg manganese sulfate, 8.6 mg zinc sulfate, 0.25 mg sodium molybdate, 0.025 mg copper sulfate, 0.025 mg cobalt chloride, 27.8 mg ferrous sulfate, 37.3 mg disodium EDTA, 2 mg glycine, 0.1 mg thiamine hydrochloride, 0.5 mg pyridoxine hydrochloride, 0.5 mg nicotinic acid, 0.1 mg vitamin B1, 0.5 mg vitamin B6, and 0.5 mg nicotinic acid in 800 mL of distilled water, bring the volume to 1 L, and adjust the pH to 5.6-6.0 with 1 N sodium hydroxide solution and 1 N hydrochloric acid solution to obtain the live cell preservation culture medium.

[0022] Example 4: Preparation of live cell preservation culture medium: Dissolve 1250 mg ammonium nitrate, 1700 mg potassium nitrate, 150 mg potassium dihydrogen phosphate, 300 mg magnesium sulfate, 300 mg calcium chloride, 0.8 mg potassium iodide, 5.5 mg boric acid, 21 mg manganese sulfate, 8 mg zinc sulfate, 0.3 mg sodium molybdate, 0.03 mg copper sulfate, 0.03 mg cobalt chloride, 27 mg ferrous sulfate, 37 mg disodium EDTA, 1.5 mg glycine, 0.05 mg thiamine hydrochloride, 0.6 mg pyridoxine hydrochloride, 0.4 mg nicotinic acid, 0.15 mg vitamin B1, 0.6 mg vitamin B6, and 0.4 mg nicotinic acid in 800 mL of deionized water, bring the volume to 1 L, and adjust the pH to 5.6-6.0 with 1 N sodium hydroxide solution and 1 N hydrochloric acid solution to obtain the live cell preservation culture medium.

[0023] Example 5: Preparation of live cell preservation culture medium: Dissolve 1250 mg ammonium nitrate, 1700 mg potassium nitrate, 150 mg potassium dihydrogen phosphate, 300 mg magnesium sulfate, 300 mg calcium chloride, 0.9 mg potassium iodide, 6.5 mg boric acid, 23 mg manganese sulfate, 9 mg zinc sulfate, 0.2 mg sodium molybdate, 0.02 mg copper sulfate, 0.02 mg cobalt chloride, 28 mg ferrous sulfate, 38 mg disodium EDTA, 2.5 mg glycine, 0.15 mg thiamine hydrochloride, 0.4 mg pyridoxine hydrochloride, 0.6 mg nicotinic acid, 0.05 mg vitamin B1, 0.4 mg vitamin B6, and 0.6 mg nicotinic acid in 800 mL of deionized water, bring the volume to 1 L, and adjust the pH to 5.6-6.0 with 1 N sodium hydroxide solution and 1 N hydrochloric acid solution to obtain the live cell preservation culture medium.

[0024] Extensive experimental research was conducted to demonstrate the effectiveness of this invention. The results of this experimental research are as follows. 1. Spore pretreatment: Mature spores of Drynaria roosii Nakaike or Cibotium barometz (L.) J.Sm. were collected and stored in a 4°C refrigerator. A suitable amount of spores can be taken directly during culture.

[0025] 2. Experiments with sugar-free and inositol-free culture media 2.1 Vital preservation culture medium without sugar removal and inositol removal: Dissolve 1650 mg ammonium nitrate, 1900 mg potassium nitrate, 170 mg potassium dihydrogen phosphate, 370 mg magnesium sulfate, 440 mg calcium chloride, 0.83 mg potassium iodide, 6.2 mg boric acid, 22.30 mg manganese sulfate, 8.6 mg zinc sulfate, 0.25 mg sodium molybdate, 0.025 mg copper sulfate, 0.025 mg cobalt chloride, 27.8 mg ferrous sulfate, 37.3 mg disodium EDTA, 2 mg glycine, 0.1 mg thiamine hydrochloride, 0.5 mg pyridoxine hydrochloride, 0.5 mg nicotinic acid, 0.1 mg vitamin B1, 0.5 mg vitamin B6, and 0.5 mg nicotinic acid, along with 300 g sucrose and 20 g inositol, in 800 mL of distilled water. Make up to 1 L. Adjust the pH to 5.6-6.0 using 1 N sodium hydroxide solution and 1 N hydrochloric acid solution to obtain the live culture medium.

[0026] 2.2 Glucose-free and inositol-free in vivo preservation culture medium: Dissolve 1650 mg ammonium nitrate, 1900 mg potassium nitrate, 170 mg potassium dihydrogen phosphate, 370 mg magnesium sulfate, 440 mg calcium chloride, 0.83 mg potassium iodide, 6.2 mg boric acid, 22.30 mg manganese sulfate, 8.6 mg zinc sulfate, 0.25 mg sodium molybdate, 0.025 mg copper sulfate, 0.025 mg cobalt chloride, 27.8 mg ferrous sulfate, 37.3 mg disodium EDTA, 2 mg glycine, 0.1 mg thiamine hydrochloride, 0.5 mg pyridoxine hydrochloride, 0.5 mg nicotinic acid, 0.1 mg vitamin B1, 0.5 mg vitamin B6, and 0.5 mg nicotinic acid in 800 mL of distilled water, bring the volume to 1 L, and adjust the pH to 5.6-6.0 with 1 N sodium hydroxide solution and 1 N hydrochloric acid solution to obtain the live culture medium.

[0027] The live culture medium without sugar and inositol and the live culture medium with sugar and inositol were placed for 2 days and the results were observed. It was found that the live culture medium without sugar and inositol showed mold growth after 2 days, while the live culture medium with sugar and inositol was placed for a longer period of time.

[0028] 3. Preparation of culture medium for the Knop group and the control group 3.1 Knop culture medium: Take 800 mg of calcium nitrate, 200 mg of magnesium sulfate, 200 mg of potassium nitrate and 200 mg of potassium dihydrogen phosphate, dissolve them in 800 mL of distilled water, and make up to 1 L. Adjust the pH value to 5.6~6.0 with 1N sodium hydroxide solution and 1N hydrochloric acid solution to obtain Knop culture medium.

[0029] 3.2 Control group culture medium: ultrapure water.

[0030] 4. Effects of temperature, light, and culture medium on the preservation of live fern spores. Five to ten milligrams of *Pteris vittata* spores were added to the viable culture medium, Knop culture medium, and control group culture medium prepared in Examples 1-3, respectively. The preservation of spores in different culture media under different temperatures and light intensities was recorded, as detailed in Tables 1 and 2 below. Figure 1 and Figure 2 : Table 1 Effects of temperature, light, and culture medium on the viable preservation of *Dryopteris crassirhizoma* spores. Table 2 Effects of temperature, light, and culture medium on the viable preservation of *Cibotium barometz* spores As a result, the Knop group achieved the best survival rate of fern spores, reaching 85% after 24 months of storage at 10°C and low light conditions. In Example 1, the survival rate of fern spores of *Dryopteris cusia* and *Cibotium barometz* also reached 80% under the same conditions, second only to the Knop group.

[0031] Analysis of the experimental data shows that the preservation time of gametophytes is closely related to light, temperature, and nutrition. These results provide a method for the live preservation of spores from *Adiantum capillus-veneris* and *Cibotium barometz*.

[0032] 5. Experiment on the effects of temperature, light, and culture medium on the cultivation of fern spores 100-200 sporangia were added to the viable culture medium, Knop group culture medium, and control group culture medium prepared in Examples 1-3, respectively. The spore seedling status in different culture media under different temperatures and light intensities was recorded. The spore germination rate, growth rate, and yield were recorded. See Tables 3 and 4 below for details. Figure 3 and Figure 4 : Table 3: Effects of temperature, light, and culture medium on the cultivation of *Adiantum capillus-veneris* spores Table 4: Effects of temperature, light, and culture medium on the cultivation of Cibotium barometz spores Results: As shown in Example 1, when the temperature is controlled at 25℃ and medium-intensity light of 500~1000Lu is used, the germination rate is the highest at 75%, the growth rate is fast (the growth is most vigorous at 2 months, reaching 90%), and the quality and activity of the germinated seedlings are good, with no deformities.

[0033] Analysis of the experimental data shows that the preservation time of gametophytes is closely related to light, temperature, and nutrition. These results provide a method for rapid propagation of seedlings from *Adiantum capillus-veneris* and *Cibotium barometz* spores.

Claims

1. A culture medium for the in vivo preservation of fern spores, characterized in that: The effective components of the in vivo preservation culture medium, calculated by weight, consist of: ammonium nitrate 412.5-1650 parts, potassium nitrate 475-1900 parts, potassium dihydrogen phosphate 42.5-170 parts, magnesium sulfate 92.5-370 parts, calcium chloride 110-440 parts, potassium iodide 0.8-0.9 parts, boric acid 5.5-6.5 parts, manganese sulfate 21-23 parts, zinc sulfate 8-9 parts, sodium molybdate 0.2-0.3 parts, copper sulfate 0.02-0.03 parts, cobalt chloride 0.02-0.03 parts, ferrous sulfate 27-28 parts, disodium EDTA 37-38 parts, glycine 1.5-2.5 parts, thiamine hydrochloride 0.05-0.15 parts, pyridoxine hydrochloride 0.4-0.6 parts, nicotinic acid 0.4-0.6 parts, and vitamin B1. It is made from 0.05~0.15 parts of vitamin B6, 0.4~0.6 parts of vitamin B6, and 0.4~0.6 parts of niacin.

2. The culture medium for the in vivo preservation of fern spores according to claim 1, characterized in that: The effective components of the live culture medium, calculated by weight, are composed of 825-1650 parts ammonium nitrate, 950-1900 parts potassium nitrate, 85-170 parts potassium dihydrogen phosphate, 185-370 parts magnesium sulfate, 220-440 parts calcium chloride, 0.83 parts potassium iodide, 6.2 parts boric acid, 22.30 parts manganese sulfate, 8.6 parts zinc sulfate, 0.25 parts sodium molybdate, 0.025 parts copper sulfate, 0.025 parts cobalt chloride, 27.8 parts ferrous sulfate, 37.3 parts disodium EDTA, 2 parts glycine, 0.1 parts thiamine hydrochloride, 0.5 parts pyridoxine hydrochloride, 0.5 parts nicotinic acid, 0.1 parts vitamin B1, 0.5 parts vitamin B6, and 0.5 parts nicotinic acid.

3. The culture medium for the in vivo preservation of fern spores according to claim 1 or 2, characterized in that: The effective components of the live culture medium, calculated by weight, are composed of 1650 parts ammonium nitrate, 1900 parts potassium nitrate, 170 parts potassium dihydrogen phosphate, 370 parts magnesium sulfate, 440 parts calcium chloride, 0.83 parts potassium iodide, 6.2 parts boric acid, 22.30 parts manganese sulfate, 8.6 parts zinc sulfate, 0.25 parts sodium molybdate, 0.025 parts copper sulfate, 0.025 parts cobalt chloride, 27.8 parts ferrous sulfate, 37.3 parts disodium EDTA, 2 parts glycine, 0.1 parts thiamine hydrochloride, 0.5 parts pyridoxine hydrochloride, 0.5 parts nicotinic acid, 0.1 parts vitamin B1, 0.5 parts vitamin B6, and 0.5 parts nicotinic acid.

4. The method for preparing the live cell preservation culture medium according to any one of claims 1 to 3, characterized in that: The preparation method is carried out according to the following steps: Weigh the effective components of the live cell preservation culture medium according to the weight ratio, dissolve them in distilled water or deionized water, and make up the volume so that the concentration of ammonium nitrate is 412.5~1650mg / L. Adjust the pH value to 5.6~6.0 with 1N sodium hydroxide solution and 1N hydrochloric acid solution.

5. The method for preparing the live cell preservation culture medium according to claim 4, characterized in that: The preparation method is carried out according to the following steps: Weigh the effective components of the live cell preservation culture medium according to the weight ratio, dissolve them in distilled water or deionized water, and make up the volume so that the concentration of ammonium nitrate is 825~1650mg / L, and adjust the pH value to 5.6~6.

0.

6. The method for preparing the live cell preservation culture medium according to claim 5, characterized in that: The preparation method is carried out according to the following steps: Weigh the effective components of the live cell preservation culture medium according to the weight ratio, dissolve them in distilled water or deionized water, and make up the volume so that the concentration of ammonium nitrate is 1650 mg / L. Adjust the pH value to 5.6~6.

0.

7. The application of the live culture medium according to any one of claims 1 to 3 in the live preservation and rapid seedling cultivation of *Adiantum capillus-veneris* gametophytes, characterized in that: The live culture medium is used for the live preservation and rapid seedling cultivation of Quercus gametophytes.

8. The application according to claim 7, characterized in that: The live preservation process involves directly obtaining sporangia of fern spores using a dissecting microscope during the spore maturation period and placing them in the live preservation culture medium for live preservation. The live preservation temperature is 10℃, and the light conditions for live preservation are weak light of 50~100Lx.

9. The application according to claim 7, characterized in that: The rapid seedling cultivation involves directly obtaining sporangia of *Adiantum capillus-veneris* spores using a dissecting microscope during the spore maturation period and placing them into the in vivo preservation culture medium for rapid seedling cultivation. The rapid seedling cultivation temperature is 20-25℃, and the rapid seedling cultivation light conditions are moderate light of 500-1000 Lx.

10. The application according to claim 9, characterized in that: The rapid seedling raising temperature is 25℃, and the rapid seedling raising light conditions are medium light of 1000Lx.

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