Hongyan strawberry stem tip tissue culture seedling method

Abstract

The invention discloses a culture seedling method of strawberries, and particularly relates to a stem tip tissue culture seedling method of Hongyan strawberries. The method comprises the following steps: selecting an explant, namely selecting a strawberry stock plant, taking a first stolon grown at the base of the strawberry stock plant when a first leaf is unfolded and less than 1 / 2, and a second leaf is not unfolded; sterilization of explants: cleaning stolons, and sterilizing the stolons with alcohol and a sodium hypochlorite aqueous solution; stem tip stripping: stripping the sterilized stolons of the explants by using a scalpel tip, and cutting a growing point with 1-2 leaf primordia; inducing cluster buds; carrying out rooting culture on single cluster buds; and transplanting rooted seedlings. According to the method, stem tip stripping is matched with the optimal stripping selection state of stolons, the optimal disinfection time of sodium hypochlorite, culture medium screening suitable for propagation and rooting, and the optimal matrix ratio during virus-free seedling transplanting, so that the method has the characteristics of good virus-free effect, high seedling rate, easiness in implementation, reduction of production cost and high transplanting survival rate.

Description

Technical Field

[0001] The invention relates to a strawberry seedling cultivation method, in particular to a red-face strawberry stem tip tissue culture seedling cultivation method, and belongs to the technical field of fruit and vegetable cultivation. Background Art

[0002] 'Hongyan' strawberry (Fragaria×ananassa Duch.CV.Benihoppe) is the main strawberry variety grown in Liaoning Province. There is a large demand for strawberry seedlings, but the quality of seedlings on the market is uneven. The traditional breeding method of strawberry seedlings is to use runner breeding, which is more likely to produce virus accumulation in actual production, leading to problems such as leaf wrinkling, quality degradation, and low yield, thus failing to maintain the excellent traits of this variety. There are four main strawberry viruses: strawberry mild yellow edge virus, strawberry vein banding virus, strawberry mottle virus, and strawberry crinkle virus. The widespread infection of seedlings with viruses is the main problem encountered in strawberry production. The yield of virus-infected strawberries is significantly lower than that of non-infected strawberries, and the fruit quality is also poor. The use of virus-free strawberry seedlings in actual strawberry production has been proven to be one of the most effective ways. The virus-free mother seedlings are mainly obtained by strawberry virus-free tissue culture technology, that is, by removing the virus pathogens through anther tissue culture, micro-stem tip tissue culture and other technologies to obtain virus-free strawberry regeneration plants, so that the strawberry seedlings can restore the excellent species of the original species of the variety to achieve the purpose of high quality and high yield. At present, the traditional strawberry virus-free seedling cultivation is not easy, and the condition control during the cultivation process is insufficient, which will cause the problem of low survival rate of seedlings. Therefore, the development of a strawberry seedling cultivation method with good virus-free effect and high seedling rate has become a technical problem that those skilled in the art need to solve urgently. Summary of the invention

[0003] The technical problem to be solved by the present invention is to provide a method for tissue culture seedlings of red strawberry stem tips, which has the characteristics of low mutation rate, good detoxification effect, seedling rate as high as 98%, easy implementation and reduced production cost.

[0004] In order to solve the above technical problems, the present invention is implemented by adopting the following technical solutions:

[0005] A method for growing strawberry stem tips into seedlings through tissue culture, the method comprising the following steps:

[0006] (1) Explant selection: Select a mother strawberry plant and take the first runner from its base, when the first leaf is unfolded and less than 1 / 2, and the second leaf is not unfolded;

[0007] (2) Explant sterilization: After cleaning, the runners are sterilized with alcohol and sodium hypochlorite aqueous solution to obtain sterilized explants;

[0008] (3) Shoot tip peeling: peel the sterilized explant runner with the tip of a scalpel and cut the growth point with 1-2 leaf primordia;

[0009] (4) Induction of clustered shoots: Place the growth point with leaf primordia in a subculture medium and culture at 22-26°C. Change the medium during the culture period and subculture for 7 times to obtain clustered shoots.

[0010] (5) Rooting culture of individual buds: Take out individual buds from the clump, place them in a rooting medium, and culture them for 40-45 days using conventional methods to obtain rooted seedlings;

[0011] (6) Transplantation of rooted seedlings: Transplant the rooted seedlings into the substrate and culture them for 35-45 days to obtain mature seedlings.

[0012] Preferably, the strawberry mother plant described in step (1) above is a 'Hongyan' strawberry that is healthy and strong, has good fruit quality, and has no deformed fruits.

[0013] The cleaning described in the above step (2) is performed by using running water for 20-30 minutes, and the alcohol sterilization is performed by using 75% alcohol for 30 seconds.

[0014] The sodium hypochlorite aqueous solution sterilization described in the above step (2) is carried out by treating with a 2.5% sodium hypochlorite aqueous solution for 10 minutes.

[0015] The size of the growth point in the above step (3) is 0.2-0.3 mm.

[0016] The composition of the subculture medium described in step (4) above is: MS basic medium supplemented with 0.5 mg / l BA, 30 g / l sucrose, and 6 g / l agar.

[0017] The composition of the rooting medium described in step (5) above is: 1 / 2MS basic medium supplemented with 0.5 mg / l IBA, 30 g / l sucrose, and 6 g / l agar.

[0018] The matrix described in the above step (6) is composed of the following raw materials in the above mass ratio: weathered sand: peat soil: garden soil = 1:1:1.

[0019] Due to the adoption of the above technical solution, the present invention has the following beneficial effects:

[0020] The strawberry stem apex meristem is the tissue where its cells grow and divide most vigorously. There is no vascular bundle in this part, and the virus can only be transmitted through the intercellular filaments, which is slower than the cell division and growth rate, so that the number of growth points in the meristem area carrying viruses is extremely small. The present invention adopts stem apex peeling, the state of optimal peeling selection of runners, the optimal time of sodium hypochlorite disinfection, the screening of suitable culture medium for propagation and rooting, and the optimal matrix ratio when transplanting virus-free seedlings, so that it has the characteristics of good virus-free effect, high seedling rate, easy implementation, reduced production cost, and high transplant survival rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a diagram of the state of the stolon.

[0022] Figure 2 It is a growth point diagram with one leaf primordium.

[0023] Figure 3 This is a map of the planting situation.

[0024] Figure 4 This is a picture of flowering conditions.

[0025] Figure 5 This is a result diagram. DETAILED DESCRIPTION

[0026] The following will be combined with the drawings in the specific embodiments of the present invention to clearly and completely describe the technical solutions in the specific embodiments of the present invention. Obviously, the specific embodiments described are only part of the specific embodiments of the present invention, not all of the specific embodiments. Based on the specific embodiments of the present invention, all other specific embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] The preparation methods in the following embodiments and test examples are conventional methods unless otherwise specified, and the percentage contents are by mass unless otherwise specified.

[0028] Example 1

[0029] A method for growing strawberry stem tips into seedlings through tissue culture, comprising the following steps:

[0030] (1) Explant selection: Select strawberry mother plants, which are 'Hongyan' strawberries that are healthy, have good fruit quality, and have no deformed fruits. Take the first runner from the base of the strawberry mother plant, when the first leaf is unfolded and less than 1 / 2, and the second leaf is not unfolded;

[0031] (2) Explant sterilization: The runners were washed with running water (tap water) for 20 minutes, sterilized with 75% alcohol for 30 seconds, and then sterilized with a 2.5% sodium hypochlorite aqueous solution for 10 minutes to obtain sterilized explants;

[0032] (3) Shoot tip peeling: peel the sterilized explant runner with the tip of a scalpel and cut out the growth point with one leaf primordium. The size of the growth point is 0.2-0.3 mm.

[0033] (4) Induction of clustered shoots: The growth point with leaf primordia was placed in a subculture medium and cultured at 22°C. The medium was changed during the culture period and subcultured 7 times to obtain clustered shoots. The composition of the subculture medium was: MS basic medium supplemented with 0.5 mg / l BA, 30 g / l sucrose, and 6 g / l agar.

[0034] (5) Rooting culture of individual clustered shoots: Take out individual shoots from the clustered shoots, place them in a rooting medium, and culture them for 40 days using conventional methods to obtain rooted shoots; the composition of the rooting medium is: 1 / 2MS basic medium supplemented with 0.5 mg / l IBA, 30 g / l sucrose, and 6 g / l agar;

[0035] (6) Transplantation of rooted seedlings: The rooted seedlings are transplanted into a substrate and cultured for 35 days to obtain mature seedlings. The substrate is composed of the following raw materials in the mass ratio: weathered sand: peat soil: garden soil = 1:1:1.

[0036] Example 2

[0037] A method for growing strawberry stem tips into seedlings through tissue culture, comprising the following steps:

[0038] (1) Explant selection: Select strawberry mother plants, which are 'Hongyan' strawberries that are healthy, have good fruit quality, and have no deformed fruits. Take the first runner from the base of the strawberry mother plant, when the first leaf is unfolded and less than 1 / 2, and the second leaf is not unfolded;

[0039] (2) Explant sterilization: The runners were washed with running water (tap water) for 25 minutes, sterilized with 75% alcohol for 30 seconds, and then sterilized with a 2.5% sodium hypochlorite aqueous solution for 10 minutes to obtain sterilized explants;

[0040] (3) Shoot tip peeling: peel the sterilized explant runner with the tip of a scalpel and cut out the growth point with two leaf primordia. The size of the growth point is 0.2-0.3 mm.

[0041] (4) Induction of clustered shoots: The growth points with leaf primordia were placed in a subculture medium and cultured at 24°C. The medium was changed during the culture period and subcultured 7 times to obtain clustered shoots. The composition of the subculture medium was: MS basic medium supplemented with 0.5 mg / l BA, 30 g / l sucrose, and 6 g / l agar.

[0042] (5) Rooting culture of individual clustered shoots: Individual shoots were taken out from the clustered shoots and placed in a rooting medium. The conventional method was used to culture the shoots for 42 days to obtain rooted shoots. The rooting medium consisted of 1 / 2 MS basic medium supplemented with 0.5 mg / l IBA, 30 g / l sucrose, and 6 g / l agar.

[0043] (6) Transplantation of rooted seedlings: Transplant the rooted seedlings into a substrate and cultivate them for 40 days to obtain mature seedlings. The substrate is composed of the following raw materials in the mass ratio: weathered sand: peat soil: garden soil = 1:1:1.

[0044] Example 3

[0045] A method for growing strawberry stem tips into seedlings through tissue culture, comprising the following steps:

[0046] (1) Explant selection: Select strawberry mother plants, which are 'Hongyan' strawberries that are healthy, have good fruit quality, and have no deformed fruits. Take the first runner from the base of the strawberry mother plant, when the first leaf is unfolded and less than 1 / 2, and the second leaf is not unfolded;

[0047] (2) Explant sterilization: The runners were washed with running water (tap water) for 30 minutes, sterilized with 75% alcohol for 30 seconds, and then sterilized with a 2.5% sodium hypochlorite aqueous solution for 10 minutes to obtain sterilized explants;

[0048] (3) Shoot tip peeling: peel the sterilized explant runner with the tip of a scalpel and cut out the growth point with 1-2 leaf primordia. The size of the growth point is 0.2-0.3 mm.

[0049] (4) Induction of clustered shoots: The growth points with leaf primordia were placed in a subculture medium and cultured at 26°C. The culture medium was changed during the culture period and subcultured 7 times to obtain clustered shoots. The composition of the subculture medium was: MS basic medium supplemented with 0.5 mg / l BA, 30 g / l sucrose, and 6 g / l agar.

[0050] (5) Rooting culture of individual clustered shoots: Take out individual shoots from the clustered shoots, place them in a rooting medium, and culture them for 45 days using conventional methods to obtain rooted shoots; the composition of the rooting medium is: 1 / 2MS basic medium supplemented with 0.5 mg / l IBA, 30 g / l sucrose, and 6 g / l agar;

[0051] (6) Transplantation of rooted seedlings: Transplant the rooted seedlings into a substrate and culture them for 45 days to obtain mature seedlings. The substrate is composed of the following raw materials in the mass ratio: weathered sand: peat soil: garden soil = 1:1:1.

[0052] The present invention is further described below through test data.

[0053] Test example

[0054] 1 Materials and methods

[0055] 1.1 Materials

[0056] The test material is the 'Hongyan' strawberry. The mother plants with strong growth, good fruit quality and no deformed fruits are selected, and a runner growing from its base is selected. The entire test period is from March 2023 to August 2024.

[0057] 1.2 Methods

[0058] 1.2.1 Sodium hypochlorite treatment

[0059] Place the 3-4 cm long runners in running water, wash for 20-30 min, treat with 75% alcohol for 30 s, treat with sodium hypochlorite (2.5%) for 0 min, 5 min, 10 min, 15 min, 20 min, and then peel off. Peel off the runners with the tip of a scalpel, cut off the growth point (protruding sphere) with 1-2 leaf primordia, and the peeled state of the stem tip is as follows: Figure 2 shown.

[0060] The culture medium formula is MS+6-BA (0.5 mg / l)+sucrose (30 g / l)+agar (6 g / l), the pH is between 5.8-6.0, and the contamination rate and survival rate are observed and counted after 7-15 days.

[0061] 1.2.2 Optimal stolons for stem tip stripping after disinfection

[0062] Randomly select the first runner that emerges, select strawberry runners that are healthy and have no obvious diseases and insect pests, select 50 in each state, and use the same stem tip stripping method as in 1.2.1. Count the number of successful stem tip stripping in each state and calculate the success rate of stem tip stripping.

[0063] The success rate of stem tip stripping = the number of successful stem tip stripping / the number of stem tips

[0064] 1.2.3 Screening of subculture and rooting medium

[0065] 1.2.3.1 Screening of subculture medium

[0066] Buds with consistent growth were cut and placed on subculture proliferation medium. Ten bottles were inoculated for each treatment, with three buds in each bottle.

[0067] The culture medium is designed separately:

[0068] Formulation 1: MS + 6-BA (0.25 mg / l) + sucrose (30 g / l) + agar (6 g / l);

[0069] Formulation 2: MS + 6-BA (0.5 mg / l) + sucrose (30 g / l) + agar (6 g / l);

[0070] Formulation 3: MS + 6-BA (0.75 mg / l) + sucrose (30 g / l) + agar (6 g / l);

[0071] Formula 4: MS + 6-BA (1 mg / l) + sucrose (30 g / l) + agar (6 g / l)

[0072] After inoculation, place it in a tissue culture room with a temperature of 22-26°C. Observe the growth of the buds once every 35 days or so, replace the culture medium during this period, and subculture 7 times to obtain seedlings.

[0073] 1.2.3.2 Screening of rooting medium

[0074] Select individual strawberry seedlings with consistent growth from seedlings and inoculate them on rooting medium, which are designed as follows:

[0075] Formula 1: 1 / 2MS + IBA (0.2mg / l) + sucrose (30g / l) + agar (6g / l)

[0076] Formula 2: 1 / 2MS + IBA (0.3 mg / l) + sucrose (30 g / l) + agar (6 g / l)

[0077] Formula 3: 1 / 2MS + IBA (0.4 mg / l) + sucrose (30 g / l) + agar (6 g / l)

[0078] Formula 4: 1 / 2MS + IBA (0.5 mg / l) + sucrose (30 g / l) + agar (6 g / l)

[0079] 1.2.4 Selection of different substrates and observation of flowering and fruiting after transplanting

[0080] 1.2.4.1 Screening of different matrices

[0081] On January 8, 2024, the plug tray transplantation was carried out. The rooted seedlings were hardened for 3-5 days and then transplanted into substrates mixed in different proportions. The substrate mixing ratios were weathered sand (100%), garden soil (100%), peat soil (100%), weathered sand: peat soil: garden soil = 1:1:1, weathered sand: peat soil: garden soil = 2:1:1. 100 tissue culture seedlings were planted in each substrate. After 35 days, the survival rate, number of newly grown roots, root length, stem thickness and plant height were counted.

[0082] 1.2.4.2 Observation of flowering and fruiting after planting

[0083] At the end of April, transplant the seedlings from the tray to a protected area for planting, manage water and fertilizer routinely, and observe flowering and fruiting in the field from June to August.

[0084] 2 Results Analysis

[0085] 2.1 Effect of sodium hypochlorite treatment time on explant survival rate

[0086] As can be seen from Table 1, after 75% alcohol treatment for 30s, the contamination rate and survival rate of 2.5% sodium hypochlorite gradually decreased as the treatment time of the shoot tip became longer; when the treatment time was 10min, 15min, and 20min, the shoot tip contamination rate was 0, and the survival rate was 96.7%, 93.3%, and 83.3%, respectively. However, when the disinfection time was 10min and 15min, the survival rate was not much different. The disinfection time with the highest survival rate of the shoot tip was 10min. Combined with the contamination rate and shoot tip survival rate, the best time for explant disinfection was 75% alcohol treatment for 30s and 2.5% sodium hypochlorite treatment for 10min.

[0087] Table 1 Effects of different disinfection time treatments on explants

[0088] 2.5% sodium hypochlorite (min) Number of pollution Number of survival Contamination rate (%) Survival rate (%) 1 0 30±0.00a 30±0.00a 100％±0.00a 100％±0.00a 2 5 21±1.00b 30±0.00a 69.7％±0.04b 100％±0.00a 3 10 0±0.00c 29±1.00a 0±0.00c 96.5%±0.04ab 4 15 0±0.00c 28±1.00a 0±0.00c 93.3％±0.03b 5 20 0±0.00c 25±2.31b 0±0.00c 83.3％±0.06c

[0089] Note: Different lowercase letters in the same column indicate significant differences at the 0.05 level, the same below

[0090] 2.2 Effect of the developmental state of runners on the success rate of stem tip stripping

[0091] According to the field observation of strawberry runners (the first runner to grow), the first leaf to sprout is the first leaf of the runner. According to the time sequence and state of leaf germination, the runners are divided into the following 6 periods, as shown in Table 2 and Figure 1 shown.

[0092] Table 2 Division of the creeping period of strawberries

[0093] period Condition of stolon leaves 1 The stolons have no leaves 2 Creeping first leaf <1 / 2 expanded and second leaf not expanded 3 1 / 2≤The first leaf of the runner is expanded<1 and the second leaf is not expanded 4 First leaf of stolon fully expanded and second leaf <1 / 2 expanded 5 The first leaf of the stolon is fully expanded and 2 / 1≤The second leaf is expanded<1 6 Two leaves of the stolon fully expanded

[0094] The stem tip was peeled off under a 50x dissecting microscope. As shown in Table 3, the success rate of stem tip peeling of the explant after disinfection decreased with the degree of expansion of the stolon leaves. The optimal state for stem tip peeling of the red strawberry stolon is state 2, that is, the expansion rate of the first leaf of the stolon is <1 / 2, and the second leaf is not expanded ( Figure 2 In 2), the success rate of stem tip peeling was 42%; in the lowest state of stem tip peeling 6, that is, the two leaves of the stolon were fully unfolded, the success rate of stem tip peeling was 18%.

[0095] Table 3 Peeling of the stem tips of “Hongyan” strawberry runners in different runner states

[0096] period Number of peeled stem tips Number of successful stem tip removal Success rate (%) 1 50 19 38％ 2 50 21 42％ 3 50 18 36％ 4 50 15 30％ 5 50 14 28％ 6 50 9 18％

[0097] 2.3 Subculture and rooting medium

[0098] 2.3.1 Subculture

[0099] According to observation, when the 6-BA concentration was 0.5mg / l and 0.75mg / l, the leaves were normal and the clumping ability was strong. When the 6-BA concentration was 1mg / l, the leaves were small but accompanied by callus. When the 6-BA concentration was 0.25mg / l, the leaves were normal and dark green, but the clumping ability was average.

[0100] Table 4. Growth of buds under different concentrations of 6-BA

[0101]

[0102]

[0103] 2.3.2 Rooting culture

[0104] As can be seen from Table 5, different concentrations of IBA have a significant effect on the rooting of Red Strawberry tissue culture. When the culture medium is 1 / 2MS+IBA (0.5mg / l), the number of roots is the largest and the root length is the longest, which are 15 and 4.1cm respectively. When it is 1 / 2MS+IBA (0.4mg / l), the number of roots is the same, both are 15, but the root length is significantly different. When the culture medium is 1 / 2MS+IBA (0.2mg / l), the root length is the shortest and the number of roots is the least, which are 2.92cm and 9.67 respectively.

[0105] Table 5 Effects of different IBA contents on rooting

[0106]

[0107] Note: Different lowercase letters in the same column indicate significant differences at the 0.05 level, the same below

[0108] 2.4 Screening of different substrates and observation of flowering and fruiting after transplanting

[0109] 2.4.1 Screening of different matrices

[0110] As shown in Table 6, after the same period of time, different substrates have a significant effect on the tissue culture seedlings of Red Strawberry. In terms of survival rate, weathered sand: peat soil: garden soil = 1:1:1, peat soil (100%) has the highest survival rate, and the difference is not obvious. On the newly grown roots, the number of newly grown roots in weathered sand: peat soil: garden soil = 1:1:1 and weathered sand: peat soil: garden soil = 1:1:1 is 11.4, and the number of newly grown roots in weathered sand (100%) is 8.89, and the difference between the two is not obvious. The number of new roots in garden soil (100%) and peat soil (100%) is the least; when weathered sand: peat soil: garden soil = 1:1:1, the plant height is the highest, the stem is the thickest, and the root length is the longest. The plant height is 22.8cm, the root length is 15.29cm, and the stem thickness is 0.62cm.

[0111] Table 6 Effects of different substrates on the growth of strawberry tissue culture seedlings

[0112]

[0113] Note: Different lowercase letters in the same column indicate significant differences at the 0.05 level, the same below

[0114] 2.4.2 Observation of flowering and fruiting after planting

[0115] After the tissue culture seedlings were planted in the field, the flowering and fruiting were observed, and they were normal. Figure 3 For the colonization situation, Figure 4 For flowering conditions, Figure 5 For the result situation.

[0116] The present invention improves the accuracy and speed of cutting the growth point by combining the state of the stolon development state with the state of the stem tip growth point with 1 to 2 leaf primordia as the benchmark. Sodium hypochlorite is used instead of mercuric chloride to disinfect the strawberry stem tip, and combined with the development state of the stolon, it is verified that the leaf expansion rate of the stolon leaf of the 'Hongyan' strawberry is <1 / 2; when the second leaf is not expanded, 75% alcohol disinfection for 30 seconds and 2.5% sodium hypochlorite treatment for 10 minutes are the best.

[0117] The present invention uses only one hormone for propagation and rooting, and selects a low concentration while ensuring the ability to grow in clusters. It is believed that the optimal propagation medium for 'Hongyan' is MS+0.5mg / l6-BA+30g / l sucrose+6g / l agar; the optimal rooting medium is 1 / 2MS+0.5mg / lIBA++30g / l sucrose+6g / l agar. The present invention selects different ratios of peat soil, garden soil, and weathered sand. The test results show that when weathered sand: peat soil: garden soil = 1:1:1, the survival rate is the highest, and related indicators such as plant height and stem thickness are better.

Claims

1. A method for cultivating red strawberry stem tip seedlings, characterized in that The method comprises the following steps: (1) Explant selection: Select a mother strawberry plant and take the first runner from its base, when the first leaf is unfolded and less than 1 / 2, and the second leaf is not unfolded; (2) Explant sterilization: After cleaning, the runners are sterilized with alcohol and sodium hypochlorite aqueous solution to obtain sterilized explants; (3) Shoot tip peeling: peel the sterilized explant runner with the tip of a scalpel and cut out the growth point with 1-2 leaf primordia; (4) Induction of clustered shoots: Place the growth point with leaf primordia in a subculture medium and culture at 22-26°C. Change the medium during the culture period and subculture for 7 times to obtain clustered shoots. (5) Rooting culture of clustered buds: Take out a single plant from the cluster, place it in a rooting medium, and culture it for 40-45 days using conventional methods to obtain rooted seedlings; (6) Transplantation of rooted seedlings: Transplant the rooted seedlings into the substrate and culture them for 35-45 days to obtain mature seedlings.

2. A method for cultivating the stem tip of red-faced strawberry seedlings according to claim 1, characterized in that The strawberry mother plant described in step (1) is a 'Hongyan' strawberry that is healthy and strong, has good fruit quality, and has no deformed fruits.

3. A method for cultivating the stem tip of red-faced strawberry seedlings according to claim 1, characterized in that The cleaning in step (2) is performed by using running water for 20-30 minutes, and the alcohol sterilization is performed by using 75% alcohol for 30 seconds.

4. A method for cultivating the stem tip of red-faced strawberry seedlings according to claim 1, characterized in that The sodium hypochlorite aqueous solution sterilization described in step (2) is carried out by treating with a 2.5% sodium hypochlorite aqueous solution for 10 minutes.

5. A method for cultivating the stem tip of red-faced strawberry into seedlings according to claim 1, characterized in that The size of the growth point in step (3) is 0.2-0.3 mm.

6. A method for cultivating the stem tip of red-faced strawberry into seedlings according to claim 1, characterized in that The composition of the subculture medium described in step (4) is: MS basic medium supplemented with 0.5 mg / l BA, 30 g / l sucrose, and 6 g / l agar.

7. A method for cultivating the stem tip of red-faced strawberry into seedlings according to claim 1, characterized in that The rooting medium described in step (5) is composed of: 1 / 2MS basic medium supplemented with 0.5 mg / l IBA, 30 g / l sucrose, and 6 g / l agar.

8. A method for cultivating the stem tip of red-faced strawberry into seedlings according to claim 1, characterized in that The matrix in step (6) is composed of the following raw materials in the mass ratio: weathered sand: peat soil: garden soil = 1:1:1.

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