Method for improving in-vitro seedling efficiency of creeping stems of yellow-hair strawberries and application

Abstract

The invention belongs to the technical field of plant in-vitro culture, and discloses a method and application for improving in-vitro seedling efficiency of yellow-hair strawberry stolons, and the method comprises the following steps: taking the stolons of healthy yellow-hair strawberry plants which are pure in variety and free from plant diseases and insect pests, stripping one leaf primordium and two bud primordium under a microscope, inoculating into an initial culture medium, and culturing in a culture medium; after the leaf primordium and the bud primordium grow up, inoculating into an adventitious bud culture medium to induce adventitious buds, and then cutting the adventitious buds into single buds for rooting culture. The method comprises the following steps: cleaning a culture medium between root systems of rooting tissue culture seedlings, planting the seedlings in a plug tray, culturing the seedlings in a domestication shed, planting the seedlings in a strawberry production shed in March of the next year, carrying out botany, fruit and other character variation confirmation, and carrying out research and production application on related mechanisms of excellent character utilization of the yellow-hair strawberries after the confirmation passes. According to the method, the in-vitro culture production efficiency of the yellow-hair strawberries can be remarkably improved and is three times that of a traditional stem tip stripping method, and the method is efficient, safe and stable.

Description

Technical Field

[0001] The invention belongs to the technical field of plant in vitro culture and discloses a method for improving the in vitro seedling efficiency of yellow-haired strawberry runners and an application thereof. Background Art

[0002] Strawberry (Fragaria ananassa) is a perennial herbaceous plant of the genus Fragaria in the Rosaceae family. It is widely planted around the world due to its high economic value and huge market potential. However, since strawberry breeding is almost entirely based on intraspecific hybridization between octoploid cultivars, the genetic background is narrow and there are obvious deficiencies in resistance and flavor, which seriously restricts the improvement of strawberry flavor and the selection of breakthrough new varieties.

[0003] Wild yellow-haired strawberry (Fragaria nilgrrensis), commonly known as Baipao, Baimeng, Mengmeng, etc., is a wild diploid strawberry species unique to eastern and southeastern Asia, and is mainly distributed in the southwest of my country. Yellow-haired strawberry is distantly related to cultivated strawberry species. The fruit contains a strong peach aroma, and has strong resistance to adversity and pests and diseases. It is an important genetic resource for improving the quality of cultivated strawberries. In addition, yellow-haired strawberry also has the effects of clearing heat and detoxifying, and repairing tendons and bones. It is often used for wind-heat cough, whooping cough, oral ulcers, hematuria, urinary tract infections, etc. In recent years, yellow-haired strawberries have also been transplanted into fields and planted in patches to help the people increase their income and become rich.

[0004] In vitro seedlings are an important basis for the development and utilization of yellow-haired strawberry germplasm resources. They can provide experimental materials for research on ploidy breeding, genetic transformation, gene editing, interaction mechanisms, and on this basis, breed new varieties and carry out production applications. However, there is a certain degree of variation in the characteristics of flowers and fruits and growth potential of the offspring of in vitro seedlings, so it is necessary to confirm the variation. The explants used for in vitro seedlings of yellow-haired strawberry have been reported to include stem tips, anthers, and leaves. Among them, stem tip culture is the main method, and the technology is relatively mature. However, in addition to the stem tip growth point, a strawberry runner also has a leaf primordium and two bud primordia. The traditional method often only peels off the stem tip for culture, while discarding one leaf primordium and two bud primordia, which not only causes a waste of resources, but also has a low efficiency of peeling. Summary of the invention

[0005] The purpose of the present invention is to solve the defects of the above-mentioned existing in vitro seedling technology of yellow-haired strawberry, and to innovate a method for in vitro seedling formation from leaf primordia and bud primordia of yellow-haired strawberry. The present invention provides a method and application for improving the efficiency of in vitro seedling formation of yellow-haired strawberry runners, comprising the following steps:

[0006] 1) In April of each year, take the runners of healthy yellow-haired strawberry plants that have been treated with high temperature (40°C) at noon for one month and are of pure variety and free of diseases and insect pests.

[0007] 2) The runner segments were washed with sterile water and placed on a clean bench. The surface was first disinfected with 75% ethanol for 30 seconds, then with 2% sodium hypochlorite for 8 minutes, and finally rinsed with sterile water for 5 times with constant shaking. The strawberry leaf primordium and bud primordium were peeled off under a microscope, inoculated into the initial culture medium and cultured for 25-35 days. The conditions of the light room were: 14 hours of light per day, light intensity of 5000-10000 lux, and a constant temperature of 25±1°C day and night.

[0008] 3) After the leaf primordium and bud primordium of the yellow-haired strawberry in step 2) grow up, inoculate them in an adventitious bud culture medium and culture them for 25-35 days in the same lighting room conditions as in step 2.

[0009] 4) The adventitious buds induced from the leaf primordium and bud primordium of the yellow-haired strawberry in step 3) are cut into single buds, inoculated into a rooting medium and cultured for 25-35 days in the same light-growing room conditions as in step 3.

[0010] 5) The rooted tissue culture seedlings obtained in step 4) are cleaned of the root culture medium, planted in 24-50 cell trays, and cultured in an acclimatization shed for 1 week before entering daily management.

[0011] 6) In March of the following year, a certain number of yellow-haired strawberry seedlings from step 5) are planted in strawberry production sheds to confirm the variation of botanical and fruit traits.

[0012] 7) After the mutation is confirmed, the "Method for improving the efficiency of in vitro seedling formation of yellow-haired strawberry runners" will be used for research on the relevant mechanisms and production applications of the excellent traits of yellow-haired strawberry.

[0013] Preferably, it is characterized in that the runners described in step 1) are treated at a high temperature of 40 degrees at noon in a greenhouse for 1 month, and the diameter of the runner stem segments is 2-2.5 mm and the length is 3-5 cm.

[0014] Preferably, it is characterized in that the strawberry leaf primordium and bud primordium described in step 2) need to be peeled off by 0.1-0.2 mm using a dissecting needle under a 50-100 times microscope;

[0015] Preferably, it is characterized in that the initial culture medium in step 2) comprises the following components per 1 L: MS 4.74 g, sucrose 30 g, agar 6 g and the balance water; pH 5.5-5.8.

[0016] Preferably, it is characterized in that the strawberry adventitious bud culture medium in step 3) comprises the following components per 1 L: MS 4.74 g, sucrose 30 g, agar 6 g, 6-BA 0.5 mg and the balance water; pH 5.5-5.8.

[0017] Preferably, it is characterized in that the strawberry rooting medium in step 4) comprises the following components per 1 L: MS 2.37 g, sucrose 30 g, agar 8 g, IBA 0.2 mg and the balance water; pH 5.5-5.8.

[0018] Preferably, the plug tray described in step 5) has a matrix formula of 7:2:1 of peat soil:perlite:vermiculite, an EC value of less than 4.0, and a pH value of 5.5-6.5.

[0019] Preferably, it is characterized in that the conditions of the acclimation shed in step 5) are: 20-25°C, humidity not less than 95%, light intensity 5000-10000 lux, the acclimation shed must be thoroughly disinfected before use, and an insect-proof net of 120 mesh or more is used to avoid invasion by virus-transmitting media such as aphids.

[0020] Preferably, the daily management described in step 5) is characterized in that water and fertilizer irrigation, disease and insect pest prevention are implemented, old leaves are removed in time, and 4-5 functional leaves are maintained.

[0021] Preferably, it is characterized in that the botanical and fruit traits mentioned in step 6) variation confirmation should be carried out in the middle of the fruit harvest duration, and the comprehensive evaluation includes but is not limited to botanical characteristics and fruit traits. Specific indicators are shown in Tables 2 and 3 of the specification.

[0022] Preferably, it is characterized in that step 7) the in vitro seedlings provided by the present invention are required for the research on the related mechanisms of the excellent traits of yellow-haired strawberry, including ploidy breeding, genetic transformation, gene editing, interaction mechanism, etc., and the production applications include yellow-haired strawberry seedling cultivation and planting.

[0023] The present invention also provides application of the in vitro seedling method described in the above technical solution in yellow-haired strawberry runners.

[0024] The present invention provides a method and application for improving the efficiency of in vitro seedling formation of yellow-haired strawberry runners, and proposes for the first time that leaf primordia and bud primordia of yellow-haired strawberry can be in vitro seedling formation, breaking the original theory that only the stem tip can be in vitro seedling formation. Secondly, one runner can be stripped out of one leaf primordium and two bud primordia, increasing the in vitro seedling production efficiency of yellow-haired strawberry to three times the original. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The present invention provides pictures related to the in vitro seedlings of yellow-haired strawberry, wherein A′ yellow-haired′ strawberry runner (A-1 appearance; A-2 bud primordium; A-3 bud primordium; A-4 leaf primordium); B peeled leaf primordium and bud primordium; X leaf primordium and bud primordium culture. DETAILED DESCRIPTION

[0026] The present invention provides a method for improving the efficiency of in vitro seedling formation of yellow-haired strawberry runners, comprising the following steps:

[0027] 1) In April of each year, take the runners of healthy yellow-haired strawberry plants that have been treated with high temperature (40°C) at noon for one month and are of pure variety and free of diseases and insect pests.

[0028] 2) The runner segments were washed with sterile water and placed on a clean bench. The surface was first disinfected with 75% ethanol for 30 seconds, then with 2% sodium hypochlorite for 8 minutes, and finally rinsed with sterile water for 5 times with constant shaking. The strawberry leaf primordium and bud primordium were peeled off under a microscope, inoculated into the initial culture medium and cultured for 25-35 days. The conditions of the light room were: 14 hours of light per day, light intensity of 5000-10000 lux, and a constant temperature of 25±1°C day and night.

[0029] 3) After the leaf primordium and bud primordium of the yellow-haired strawberry in step 2) grow up, they are inoculated in an adventitious bud culture medium and cultured for 25-35 days in the same light room conditions as in step 2.

[0030] 4) The adventitious buds induced from the leaf primordium and bud primordium of the yellow-haired strawberry in step 3) are cut into single buds, inoculated into a rooting medium and cultured for 25-35 days in the same light room conditions as in step 3.

[0031] 5) The rooted tissue culture seedlings obtained in step 4) are cleaned of the root culture medium, planted in 24-50 cell trays, and cultured in an acclimatization shed for 1 week before entering daily management.

[0032] 6) In March of the following year, a certain number of yellow-haired strawberry seedlings from step 5) are planted in strawberry production sheds to confirm the variation of botanical and fruit traits.

[0033] 7) After the mutation is confirmed, the "Method for improving the efficiency of in vitro seedling formation of yellow-haired strawberry runners" will be used for research on the relevant mechanisms and production applications of the excellent traits of yellow-haired strawberry.

[0034] The present invention takes the runners of healthy plants of yellow-haired strawberry that have been treated with high temperature (40°C) at noon for one month in April every year, and are of pure variety and free of pests and diseases. In the present invention, the runners are derived from yellow-haired strawberry, preferably treated with high temperature of 40 degrees at noon in a greenhouse for one month, and the runner stem segments have a diameter of 2-2.5 mm and a length of 3-5 cm. The present invention has no special limitation on the source of the wild yellow-haired strawberry, and the conventional source of wild yellow-haired strawberry well known to those skilled in the art can be used.

[0035] The stolon segment is washed with sterile water and placed on a clean bench. The surface is first disinfected with 75% ethanol for 30 seconds, then disinfected with 2% sodium hypochlorite for 8 minutes, and finally rinsed with sterile water for 5 times. During the period, the strawberry leaf primordium and bud primordium are peeled off under a microscope, and inoculated in the initial culture medium for 25-35 days. The conditions of the light room are: 14 hours of light per day, light intensity of 5000-10000 lux, and constant temperature of 25±1°C day and night. In the present invention, the strawberry leaf primordium and bud primordium need to be peeled off 0.1-0.2mm by a dissecting needle under a 50-100 times microscope. In the present invention, the initial culture medium includes the following components per 1L: MS4.74g, sucrose 30g, agar 6g and the balance of water; pH 5.5-5.8.

[0036] After the leaf primordium and bud primordium of yellow-haired strawberry grow up, they are inoculated in an adventitious bud culture medium and cultured for 25-35 days in a light room. In the present invention, the strawberry adventitious bud culture medium comprises the following components per 1L: MS 4.74g, sucrose 30g, agar 6g, 6-BA 0.5mg and the balance water; pH 5.5-5.8.

[0037] Adventitious buds induced by the leaf primordium and bud primordium of yellow-haired strawberry are cut into single buds, inoculated into a rooting medium and cultured for 25-35 days in a light room. In the present invention, the strawberry rooting medium comprises the following components per 1L: MS 2.37g, sucrose 30g, agar 8g, IBA 0.2mg and the balance water; pH 5.5-5.8.

[0038] The rooted tissue culture seedlings obtained are cleaned of the root inter-culture medium, planted in a 24-50 hole tray, and cultured in the acclimation shed for 1 week before entering daily management. In the present invention, the matrix formula in the hole tray is: peat soil: perlite: vermiculite is 7:2:1, the EC value is less than 4.0, and the pH value is between 5.5-6.5. In the present invention, the conditions of the acclimation shed are: 20-25°C, humidity is not less than 95%, and the light intensity is 5000-10000lux. The acclimation shed must be thoroughly disinfected before use, and an insect-proof net of 120 mesh or more is used to avoid invasion by virus transmission media such as aphids. In the present invention, the daily management is characterized by implementing water and fertilizer irrigation, disease and insect pest prevention, timely removal of old leaves, and maintaining 4-5 functional leaves.

[0039] In March of the following year, a certain number of yellow-haired strawberry seedlings were selected and planted in the strawberry production shed to confirm the variation of botanical and fruit traits. In the present invention, the variation confirmation should be comprehensively evaluated in the middle of the fruit harvest duration. In the present invention, the indicators of the comprehensive evaluation preferably include: botanical characteristics and fruit traits. In the present invention, the botanical characteristics include one or more of leaf color, stolon color, petiole color, number of flowers, fruit initial maturity and yield. The present invention preferably screens the four indicators of leaf color, stolon color, petiole color and number of flowers. In the present invention, the fruit traits include one or more of fruit shape, fruit shape consistency, average single fruit weight, fruit surface color, flesh color, flesh texture and flavor. The present invention preferentially screens the five indicators of fruit shape, fruit shape consistency, average single fruit weight, fruit surface color and flesh color.

[0040] After the mutation is confirmed, the "Method for Improving the Efficiency of In Vitro Seedlings of Yellow-haired Strawberry Runners" is used for research on the mechanism of utilization of excellent traits of yellow-haired strawberry and production application. In the present invention, the research on the mechanism of utilization of excellent traits of yellow-haired strawberry includes the in vitro seedlings obtained by the method of the present invention required for research on ploidy breeding, genetic transformation, gene editing, interaction mechanism, etc., and the production application includes yellow-haired strawberry seedling cultivation and planting.

[0041] The present invention also provides the application of the in vitro seedling method described in the above technical solution in yellow-haired strawberry runners. The in vitro seedling method described in the present invention is also applicable to other wild strawberry species.

[0042] The method for improving the in vitro seedling efficiency of yellow-haired strawberry runners and its application according to the present invention are further introduced in detail below in conjunction with specific embodiments. The technical solutions of the present invention include but are not limited to the following embodiments.

[0043] Example

[0044] 1) In April of each year, take the runners of healthy plants of pure yellow-haired strawberry without diseases and insect pests that have been treated with high temperature (40℃) at noon for one month (the runners are shown in the Figure 1 A-1 in ).

[0045] 2) The runner segments were washed with sterile water and placed on a clean bench. The surface was first disinfected with 75% ethanol for 30 seconds, then with 2% sodium hypochlorite for 8 minutes, and finally rinsed with sterile water for 5 times. During the period, the strawberry leaf primordium and bud primordium were peeled off under a microscope (see the micrograph of the bud primordium). Figure 1 A-2 and A-3 in Figure 2, leaf primordium micrographs are shown in Figure 1 A-4 in the figure, peeling diagram is shown in Figure 1 B), inoculated into the initial culture medium and cultured for 25-35 days, the conditions of the light room are: 14 hours of light per day, light intensity of 5000-10000 lux, and constant temperature of 25±1℃ day and night.

[0046] 3) After the leaf primordium and bud primordium of the yellow-haired strawberry in step 2 grow up (the leaf primordium and bud primordium grown up after cultivation see Figure 1 X) in the sample was inoculated into adventitious bud medium and cultured for 25-35 days in the same light room conditions as in step 2.

[0047] 4) The adventitious buds induced from the leaf primordium and bud primordium of the yellow-haired strawberry in step 3) are cut into single buds, inoculated into a rooting medium and cultured for 25-35 days in the same light room conditions as in step 3.

[0048] 5) The rooted tissue culture seedlings obtained in step 4) are cleaned of the root culture medium, planted in 24-50 cell trays, and cultured in an acclimatization shed for 1 week before entering daily management.

[0049] 6) In March of the following year, a certain number of yellow-haired strawberry seedlings from step 5) are planted in strawberry production sheds to confirm the variation of botanical and fruit traits.

[0050] 7) After the mutation is confirmed, the "Method for improving the efficiency of in vitro seedling formation of yellow-haired strawberry runners" will be used for research on the relevant mechanisms and production applications of the excellent traits of yellow-haired strawberry.

[0051] To make the objects and advantages of the present invention clearer, the method and application of improving the efficiency of in vitro seedling formation of yellow-haired strawberry runners described in the present invention are further introduced in detail below in combination with specific comparative examples. The technical solutions of the present invention include but are not limited to the following comparative examples.

[0052] Comparative Example

[0053] 1.Production efficiency

[0054] The traditional yellow-haired strawberry stem tip peeling was used as a control, and the production efficiency of the in vitro seedling formation from leaf primordium and bud primordium of the present invention was demonstrated (see Table 1), and the survival rate (survival number / original inoculation number), adventitious bud generation rate (adventitious bud number / original inoculation number), rooting situation, and production efficiency (seedling number / stolon number) were statistically analyzed. The experiment was repeated 3 times, and all data were statistically analyzed using DPS16.05 and Excel 2007 software.

[0055] Table 1 Comparison of production efficiency of three in vitro culture methods

[0056]

[0057] Note: The same letters in the same column indicate that the differences were not significant at the 0.05 level after Duncan's multiple range test.

[0058] As can be seen from Table 1, taking the shoot apex culture as the control, the survival rate and adventitious bud formation rate of the leaf primordium and bud primordium culture were not significantly different from those of the shoot apex culture, and the rooting condition was good, and no variation was detected. However, the production efficiency was increased to 3 times that of the shoot apex culture, that is, the production efficiency of a single runner was increased to 3 times the original.

[0059] 2. Mutation Confirmation

[0060] With bare-root seedlings (seedlings propagated by runners in the natural growth area of ​​yellow-haired strawberry) as a control, the advantages of the in vitro seedlings of the leaf primordium and bud primordium of the yellow-haired strawberry of the present invention are demonstrated (see Table 2 and Table 3). The yellow-haired strawberry seedlings and bare-root seedlings were planted in the experimental greenhouse in late August 2023, with a row spacing of 20cm×25cm. According to the experimental design, a total of 3 test seedlings were planted, 10 plants per plot of each seedling, 3 replicates, and a total of 90 plants. The botanical characteristics and fruit traits were compared with the strawberry germplasm resource description specifications and data standards and industry standard NY / T2346-2013 compiled by Zhao Mizhen et al. The experiment was repeated 3 times, and all data were statistically analyzed using DPS16.05 and Excel 2007 software.

[0061] Table 2 Comparison of botanical characteristics of three strawberry seedlings

[0062]

[0063] Note: The same letters in the same column indicate that the differences were not significant at the 0.05 level after Duncan's multiple range test.

[0064] The growth potential of in vitro seedlings of yellow-haired strawberry is slightly better than that of bare-root seedlings (Table 2). The leaves are dark green, the runners are green, the petioles are yellow-green, the inflorescences grow continuously, and the number of flowers in a single inflorescence is 9 to 18. In the central Guizhou region, the initial maturity period is in late April, with no variation, and the yield is higher than that of bare-root seedlings.

[0065] Table 3 Comparison of fruit traits of three strawberry seedlings

[0066]

[0067] Note: The same letters in the same column indicate that the differences were not significant at the 0.05 level after Duncan's multiple range test.

[0068] The fruit shape of the in vitro yellow-haired strawberry seedlings is conical and consistent. The average single fruit weight is slightly higher than that of the bare-root seedlings. The first-grade fruit is about 5.73g. The fruit surface is white, the flesh is white, the flesh is soft, and the flavor is moderately sweet and sour (Table 3).

[0069] In summary, compared with bare-root seedlings, the seedlings cultured in vitro from the leaf primordium and bud primordium of yellow-haired strawberry in this patent have slightly stronger growth, slightly higher average single fruit weight and yield, no botanical variation, and the production efficiency of a runner is increased to 3 times that of stem tip culture. Therefore, a method for in vitro seedling cultivation from the leaf primordium and bud primordium of yellow-haired strawberry is safer, more stable, and more efficient, and can provide experimental materials for the research on the mechanism of utilization of the excellent traits of yellow-haired strawberry, including ploidy breeding, genetic transformation, gene editing, and interaction mechanism research, and can also be used for research on yellow-haired strawberry seedling cultivation and planting.

[0070] Finally, it should be noted that the above embodiments and comparative examples are only used to illustrate the technical solutions of the present invention, rather than to limit them. It should be pointed out that a person skilled in the art can still modify the technical solutions described in the comparative examples and embodiments, or make equivalent substitutions for some of the technical features therein, and these modifications or substitutions should also be regarded as within the protection scope of the present invention.

Claims

1. A method for improving the efficiency of in vitro seedling formation of yellow-haired strawberry runners and its application, comprising the steps of: 1) In April of each year, take the runners of healthy yellow-haired strawberry plants that have been treated with high temperature (40°C) at noon for one month and are of pure variety and free of diseases and insect pests. 2) The runner segments were washed with sterile water and placed on a clean bench. The surface was first disinfected with 75% ethanol for 30 seconds, then with 2% sodium hypochlorite for 8 minutes, and finally rinsed with sterile water for 5 times with constant shaking. The strawberry leaf primordium and bud primordium were peeled off under a microscope, inoculated into the initial culture medium and cultured for 25-35 days. The conditions of the light room were: 14 hours of light per day, light intensity of 5000-10000 lux, and a constant temperature of 25±1°C day and night. 3) After the leaf primordium and bud primordium of the yellow-haired strawberry in step 2) grow up, inoculate them in an adventitious bud culture medium and culture them for 25-35 days in the same lighting room conditions as in step 2. 4) The adventitious buds induced from the leaf primordium and bud primordium of the yellow-haired strawberry in step 3) are cut into single buds, inoculated into a rooting medium and cultured for 25-35 days in the same light-growing room conditions as in step 3. 5) The rooted tissue culture seedlings obtained in step 4) are cleaned of the root culture medium, planted in 24-50 cell trays, and cultured in an acclimatization shed for 1 week before entering daily management. 6) In March of the following year, a certain number of yellow-haired strawberry seedlings from step 5) are planted in strawberry production sheds to confirm the variation of botanical and fruit traits. 7) After the mutation is confirmed, the "Method for improving the efficiency of in vitro seedling formation of yellow-haired strawberry runners" will be used for research on the relevant mechanisms and production applications of the excellent traits of yellow-haired strawberry.

2. The in vitro seedling method according to claim 1, characterized in that: The runners described in step 1) are treated at a high temperature of 40 degrees at noon in a greenhouse for one month, and the diameter of the runner stem segments is 2-2.5 mm and the length is 3-5 cm.

3. The in vitro seedling method according to claim 1, characterized in that: The strawberry leaf primordium and bud primordium described in step 2) need to be peeled off by 0.1-0.2 mm using a dissecting needle under a 50-100 times microscope.

4. The in vitro seedling method according to claim 1, characterized in that: The initial culture medium in step 2) comprises the following components per 1 L: 4.74 g MS, 30 g sucrose, 6 g agar and the balance water; pH 5.5-5.

8.

5. The in vitro seedling method according to claim 1, characterized in that: The strawberry adventitious bud culture medium described in step 3) comprises the following components per 1 L: MS 4.74 g, sucrose 30 g, agar 6 g, 6-BA 0.5 mg and the balance water; pH 5.5~5.

8.

6. The in vitro seedling method according to claim 1, characterized in that: The strawberry rooting medium described in step 4) comprises the following components per 1 L: MS 2.37 g, sucrose 30 g, agar 8 g, IBA 0.2 mg and the balance water; pH 5.5~5.

8.

7. The in vitro seedling method according to claim 1, characterized in that: The plug tray described in step 5) has a matrix formula of 7:2:1 of peat soil:perlite:vermiculite, an EC value of less than 4.0, and a pH value between 5.5-6.

5.

8. The in vitro seedling method according to claim 1, characterized in that: The conditions of the acclimation shed in step 5) are: 20-25°C, humidity not less than 95%, and light intensity 5000-10000 lux. The acclimation shed should be thoroughly disinfected before use, and an insect-proof net with a mesh size of 120 or more should be used to prevent the invasion of virus-transmitting media such as aphids.

9. The in vitro seedling method according to claim 1, characterized in that: The daily management described in step 5) is characterized in that water and fertilizer irrigation, disease and insect pest prevention are implemented, old leaves are removed in time, and 4-5 functional leaves are maintained.

10. The in vitro seedling method according to claim 1, characterized in that: Step 6) Confirmation of variation: The botanical and fruit traits should be carried out in the middle of the fruit harvest period. The comprehensive evaluation includes but is not limited to botanical characteristics and fruit traits. Specific indicators are shown in Tables 2 and 3 of the specification.

11. The in vitro seedling method according to claim 1, characterized in that: Step 7) The excellent traits of yellow-haired strawberry are utilized for related mechanism research, including ploidy breeding, genetic transformation, gene editing, interaction mechanism research, etc., using the experimental materials provided by the present invention, and production applications include yellow-haired strawberry seedling cultivation and planting.

12. Use of the in vitro seedling method according to any one of claims 1 to 9 in plant runners.

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