Strawberry two-stage seedling raising method

Abstract

The invention relates to plant cultivation, in particular to a strawberry two-stage seedling raising method for reducing somatic clone variation in the production process, which comprises the following steps: inoculating strawberry explants into a culture medium containing 6-BA with hormone of 0.35 mg / L for tissue culture to obtain original seedlings, blooming and fruiting once, screening out variant plants with malformed fruits, and carrying out secondary seedling raising. Breeding of original seedlings is conducted through stolon production, and the original seedlings are directly used as production seedlings. According to the method, the culture medium with low hormone concentration is adopted, the multiplication coefficient can be guaranteed, the mutation rate can be reduced, the subculture frequency is controlled to be 3-4 times, the mutation rate can also be effectively reduced, the obtained breeder seed female parent seedlings are subjected to a round of flowering and fruit bearing, variant plants with malformed fruits are screened out, then the original seedlings are bred, and the yield of the breeder seeds is improved. The obtained original seedlings are directly used as production seedlings, the seedling raising period is shortened while strawberry variation is controlled, the risk of disease and pest infection is reduced, and the seedling raising efficiency is improved.

Description

Technical Field

[0001] The invention relates to a secondary strawberry seedling raising method in plant cultivation. Background Art

[0002] strawberry( Fragaria × ananassa Fragaria duch. is a perennial herbaceous plant in the genus Fragaria in the Rosaceae family, widely beloved for its vibrant fruit color, pleasant aroma, and rich flavor. Demand for strawberries is high, and the quality of strawberry seedlings directly impacts both yield and quality. Therefore, obtaining strawberry seedlings with superior traits is crucial for farmers. Farmers often use runner progeny as seedlings for seedling propagation. However, long-term continuous cropping and self-propagation can increase the incidence and severity of strawberry virus diseases, which in turn affect strawberry quality and yield. Strawberry virus diseases can cause weak growth, low fruit set, reduced yield, and poor fruit quality. Producing virus-free seedlings using tissue culture can fundamentally address this issue. Cultivating virus-free seedlings is an effective means of maintaining quality and increasing strawberry yield. In recent years, through virus detection and testing, strawberry virus-free technology has been continuously optimized and widely applied. Virus-free strawberry seedlings exhibit vigorous growth, strong disease resistance, and high yield, demonstrating promising cultivation prospects.

[0003] There are many strawberry virus-free technologies, including anther detoxification, stem tip detoxification, ultra-low temperature detoxification, and heat treatment detoxification. The most commonly used is stem tip tissue culture rapid propagation. Strawberry stem tip detoxification involves using the micro-stem tips of strawberry runners as explants. After disinfection, they are inoculated onto MS culture medium and subjected to multiple in vitro culture, inoculation, and seedling cultivation to ultimately produce sterile seedlings.

[0004] With the development of strawberry tissue culture technology, tissue culture seedlings have begun to be used in large quantities in production, but mutations will occur. Strawberry is a berry plant that is prone to genetic mutations. When strawberries reproduce rapidly, tissue culture somatic cell asexual line mutations will occur. Somatic cell asexual line mutation refers to the mutation of cultured cells or plants during long-term continuous culture, which manifests as genetic material mutations or epigenetic mutations. It is common in tissue culture processes. Mutations lead to genetic instability in regenerated plants. Studies have found that the pollen sterility rate of tissue culture variants can reach up to 21.63%, the flowering time varies greatly, and the fruit yield and quality of the variants are reduced.

[0005] Because tissue culture seedlings have high genotypic and phenotypic instability, and the incorrect use of high concentrations of cytokinins during the rapid propagation stage of tissue culture increases the risk of strawberry mutation. Therefore, strawberry nursery breeding stipulates that tissue culture seedlings must undergo at least three nursery production cycles in the field, that is, the three-level strawberry seedling system: through virus-free tissue culture, the stem tip cells are cultured in a sterile environment to produce virus-free seedlings F0, which are bred into virus-free seedlings F1, which are bred into virus-free seedlings F2, and which are bred into production seedlings E. The E that pass the test will be sold to growers for strawberry production.

[0006] The original stock seedlings F0 are obtained through the rapid propagation technology of shoot apex tissue, starting from the meristem, terminal bud or axillary bud. In this method, the size of the meristem should be controlled between 0.2-0.4mm to ensure that it is virus-free. In order to avoid the problem of somatic asexual variation, the number of subcultures of tissue culture seedlings should be less than 10 times. The acclimated F0 is planted in a cultivation box filled with sterile substrate in the spring of the second year, and the original stock seedlings F1 are propagated by runners and harvested in winter. This process requires a full growing season, about 8-9 months, with a reproduction coefficient of 60-100. The F1 is dormant and is preserved until the spring of the third year to be cultivated and propagated F2, with a reproduction coefficient of about 80. The F2 is planted in the spring of the following year to reproduce the production seedlings E, and the production seedlings that pass the inspection will eventually be sold.

[0007] This seedling production system has a long production cycle, high costs, and cannot effectively eliminate mutant plants. After repeated propagation in greenhouses or in the field, the seedlings still face a high risk of disease and insect infestation, making the safety of the produced seedlings impossible to ensure. If the seedlings mutate and produce deformed fruit, it will seriously affect farmers' profits. Summary of the Invention

[0008] The technical problem to be solved by the present invention is how to improve the strawberry tissue culture seedling preparation and cultivation system and reduce somatic cell asexual line variation in the production process.

[0009] To solve the above technical problems, the present invention first provides a secondary strawberry seedling cultivation method, which comprises the steps of inoculating strawberry explants into a culture medium containing 0.35 mg / L 6-BA as a hormone to obtain original seedlings through tissue culture, flowering and fruiting once, screening out mutants that produce deformed fruits, and then breeding the original seedlings through runner production, and directly using the original seedlings as production seedlings.

[0010] In the above method, the culture medium containing 0.35 mg / L 6-BA as a plant growth regulator is a solid culture medium obtained by adding sucrose, a coagulant, activated carbon and the plant growth regulator 6-BA to an MS liquid culture medium.

[0011] In the above method, the coagulant in the culture medium containing 0.35 mg / L 6-BA as a plant growth regulator may be agar.

[0012] In the above method, the culture medium containing 0.35 mg / L 6-BA as a plant growth regulator has a sucrose content of 30 g / L, an agar content of 7 g / L, and an activated carbon content of 2 g / L.

[0013] In the above method, the pH value of the culture medium containing 0.35 mg / L 6-BA as a plant growth regulator is 5.85.

[0014] In the above method, the original seedlings are obtained by tissue culture, which is subcultured after the primary culture, and the number of subcultures is 0-4 times.

[0015] In the above method, the original seedlings are obtained by tissue culture, which is subcultured after the primary culture, and the number of subcultures is 3-4 times.

[0016] In the above method, the primary culture is cultured for 30 days.

[0017] In the above method, the subculture is carried out for 30 days per generation.

[0018] In the above method, the explant is the stolon tip.

[0019] In the above method, the culture conditions of the tissue culture are light intensity 2000-3000 Lx, light duration 14 h, temperature 25±2°C, and humidity 45%-50%.

[0020] In the above method, obtaining the original seedlings also includes the steps of hardening and transplanting.

[0021] In the above method, the flowering and fruiting need to be promoted by low temperature and short day treatment.

[0022] The present invention uses a culture medium with a relatively low hormone concentration for strawberry seedling cultivation, which is the optimal choice for both ensuring the proliferation coefficient and reducing the mutation rate. Controlling the number of subcultures to 3-4 times can also effectively reduce the mutation rate. After a round of flowering and fruiting testing, the obtained original parent seedlings are screened out for mutated original seedlings that produce deformed fruits. The original seedlings are then bred and the original seedlings are directly used as production seedlings. This can shorten the seedling cultivation cycle while controlling strawberry mutation, reduce the risk of disease and insect pest infection, and improve seedling cultivation efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a deformed fruit produced by excessive number of subcultures in Example 1 of the present invention.

[0024] Figure 2These are fruits that produce normal fruit after 3-4 subcultures in Example 1 of the present invention.

[0025] Figure 3 The electrophoresis results of some samples amplified by primer D3 in Example 1 of the present invention are shown. Wherein: M is a DNA marker, and lanes 44-66 are the PCR results of the fourth generation tissue culture seedlings.

[0026] Figure 4 The electrophoresis results of some samples amplified by primer D1 in Example 1 of the present invention are shown in FIG. Wherein: M is a DNA marker, and lanes 44-66 are the PCR electrophoresis results of the fourth generation tissue culture seedlings.

[0027] Figure 5 The electrophoresis results of some samples amplified by primer D2 in Example 1 of the present invention are shown. Wherein: M is DNA Marker, lanes 21-28 are the PCR electrophoresis results of the third generation tissue culture seedlings; lanes 29-43 are the PCR electrophoresis results of the fourth generation tissue culture seedlings.

[0028] Figure 6 The electrophoresis results of some samples amplified by primer D2 in Example 1 of the present invention are shown. Wherein: M is a DNA marker, and lanes 44-66 are the PCR electrophoresis results of the fourth generation tissue culture seedlings.

[0029] Figure 7 The electrophoresis results of some samples amplified by primer D4 in Example 1 of the present invention are shown. Wherein: M is a DNA marker, and lanes 44-66 are the PCR electrophoresis results of the fourth generation tissue culture seedlings. DETAILED DESCRIPTION

[0030] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0031] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0032] The mother strawberry seedlings of the commercial strawberry variety 'Hongyan' used in this example were purchased from Beijing Larsen Agricultural Development Co., Ltd.

[0033] Example 1 Numerous studies have shown that during in vitro strawberry culture, the type and content of growth regulators, the number of subcultures, and the duration of tissue culture can all lead to variations in chromosome number or traits. Factors influencing somatic asexual variation include subculture duration and number, culture medium composition, and hormone concentration. Hormones can directly induce somatic asexual variation, and the probability of variation increases with longer culture times, more subcultures, higher concentrations of plant growth regulators, and a greater variety of plant growth regulators.

[0034] The specific steps of this embodiment are as follows: 1. Explant disinfection and stem tip removal During the early stages of runner formation in the strawberry cultivar 'Hongyan', pinch off the stem tip approximately 3-4 cm from the front with scissors. Rinse the runner under running water and gently rub the stem tip with your fingers. Degrease the runner using dish soap on a magnetic stirrer. Stir for 15 minutes. Rinse the stem tip with water and rinse under running water for a final 2 hours. Drain with filter paper and place in a Petri dish. Seal the dish with Parafilm and set aside.

[0035] In a clean bench, the stems were sterilized with 75% alcohol (purchased from Shandong Anjie High-Tech Disinfection Technology Co., Ltd.) by shaking for 10 seconds and rinsed three times with sterile distilled water for 1 minute each. The stems were then washed with 2% sodium hypochlorite solution (purchased from Sangon Biotech (Shanghai) Co., Ltd.) by shaking for 8 minutes and rinsed five times with sterile distilled water for 1 minute, 30 seconds, 1 minute, 30 seconds, and 1 minute, respectively. The surface moisture of the runners was blotted dry with filter paper. Under a stereomicroscope in a sterile room, the leaf primordium at the top of the runner was peeled off layer by layer. A 0.5 mm long shoot tip was cut with a sterile blade and inoculated as an explant on primary culture medium.

[0036] 2. Low cytokinin controls variation 2.1 Primary culture medium screening In this example, six different hormone concentrations were set to explore the optimal hormone concentration of the primary culture medium, as follows: The primary culture medium is a solid medium obtained by adding hormones to MS solid medium as a basal medium. The specific preparation method (taking 1 L as an example) is as follows: 4.43 g of MS dry powder (purchased from PhytoTech), 7 g of agar (purchased from Beijing Bio-Toda Technology Co., Ltd.), 30 g of sucrose (purchased from Tianjin Damao Chemical Reagent Factory), 2 g of activated carbon, and hormones (purchased from Beijing Bio-Toda Technology Co., Ltd.) are dissolved in water and the volume is adjusted to 1 L. The pH is adjusted to 5.85 using standard 1 mol / L HCl and 1 mol / L NaOH. Sterilize under autoclave at 121°C for 20 min and condense for use.

[0037] The hormone type and dose were used as variables to set up different concentration gradients, with a total of six groups. The treatment groups are as follows: A1: MS+sucrose+agar+6-BA (0.35 mL·L -1 ) + activated carbon (2 g / L) A2: MS+sucrose+agar+6-BA (0.35 mL·L -1 ) + NAA (0.1 mL·L -1 ) + activated carbon (2 g / L) A3: MS+sucrose+agar+6-BA (0.45 mL·L -1 ) + activated carbon (2 g / L) A4: MS + sucrose + agar + 6-BA (0.45 mL·L -1 ) + NAA (0.1 mL·L -1 ) + activated carbon (2 g / L) A5: MS+sucrose+agar+6-BA (0.5 mL·L -1 ) + activated carbon (2 g / L) A6: MS+sucrose+agar+6-BA (0.5 mL·L -1 ) + NAA (0.1 mL·L -1 ) + activated carbon (2 g / L) Six bottles were inoculated per treatment, with one shoot tip in each bottle, replicated three times. The growth dynamics of the meristem were observed and recorded weekly after inoculation. Cultures were maintained in the tissue culture room at a temperature of 25 ± 2°C, a humidity of 45%-50%, and a light intensity of 2,000-3,000 lx for 14 hours per day. After 30 days of primary culture, the bud induction rate and callus induction rate were calculated to select the optimal culture medium for inducing clustered buds.

[0038] Bud induction rate (%) = number of explants with bud clusters / total number of inoculated explants × 100% Induction callus rate (%) = number of explants forming callus / total number of inoculated explants × 100% Table 1 Effects of different hormone ratios on shoot bud induction

[0039] Note: The data in the table are mean ± standard deviation; different lowercase letters in the same column indicate significant differences among treatments (ANOVA, p <0.05, Duncan), n=6.

[0040] Experimental results showed that after 10-15 days of inoculation, new, tender green buds formed at the growing point of the plant, and new shoots emerged from the buds after 15-30 days. During shoot tip culture, new shoots emerged first when cytokinin concentrations were high. Low cytokinin concentrations slowed bud formation, but no callus formed during shoot tip culture, resulting in a high seedling emergence rate. In the group treated with NAA, callus formed at the growing point of the shoot tip and gradually proliferated, resulting in slow bud growth and affecting seedling development. Statistical data analysis showed that the bud induction rates of the A1, A3, and A5 treatments were significantly higher than those of the A2, A4, and A6 treatments, with A1 and A3 achieving rates of 84.12% and 81.67% respectively. The callus induction rates of the A2, A4, and A6 treatments supplemented with NAA were significantly higher than those of the A1, A3, and A5 treatments treated with 6-BA alone, with A2 exhibiting the highest callus induction rate. The A1 treatment group, which only added 6-BA at a concentration of 0.35 mg / L, grew well, with young leaves stretching and very few calluses, achieving the best induction effect (Table 1).

[0041] 2.2 Screening of secondary culture medium After 1-2 months of culture, the shoot tips grow into small buds with leaf primordia. The buds are transferred to subculture medium for culture. The tissue culture room is maintained at a temperature of 25±2°C, a humidity of 45%-50%, a light intensity of 2,000-3,000 lx, and a light duration of 14 h / d.

[0042] The subculture medium is a solid medium obtained by adding hormones to the MS solid medium as a basal medium. The specific preparation method is as follows (taking 1 L as an example): 4.43 g of MS dry powder (purchased from PhytoTech), 7 g of agar (purchased from Beijing Bio-Toda Technology Co., Ltd.), 30 g of sucrose (purchased from Tianjin Damao Chemical Reagent Factory), 2 g of activated carbon, and hormones (purchased from Beijing Bio-Toda Technology Co., Ltd.) are dissolved in water and the volume is adjusted to 1 L. The pH is adjusted to 5.85 using standard 1 mol / L HCl and 1 mol / L NaOH. Sterilize under autoclave at 121°C for 20 min and condense for use.

[0043] The hormone type and dose were used as variables to set up different concentration gradients, with a total of six groups. The treatment groups are as follows: A1: MS+sucrose+agar+6-BA (0.35 mL·L -1 ) + activated carbon (2 g / L) A2: MS+sucrose+agar+6-BA (0.35 mL·L -1 ) + NAA (0.1 mL·L -1 ) + activated carbon (2 g / L) A3: MS+sucrose+agar+6-BA (0.45 mL·L -1) + activated carbon (2 g / L) A4: MS + sucrose + agar + 6-BA (0.45 mL·L -1 ) + NAA (0.1 mL·L -1 ) + activated carbon (2 g / L) A5: MS+sucrose+agar+6-BA (0.5 mL·L -1 ) + activated carbon (2 g / L) A6: MS+sucrose+agar+6-BA (0.5 mL·L -1 ) + NAA (0.1 mL·L -1 ) + activated carbon (2 g / L) Each treatment was inoculated with 6 bottles, repeated 3 times. Thirty days after inoculation, the proliferation of shoots at different hormone concentrations was counted, and the treatment with lower hormone concentration and better seedling growth was selected.

[0044] Proliferation coefficient = total number of buds after 30 days of inoculation / total number of buds at the initial stage of inoculation Table 2 Effects of different hormone ratios on strawberry proliferation coefficient

[0045] Note: The data in the table are mean ± standard deviation; different lowercase letters in the same column indicate significant differences among treatments (ANOVA, p <0.05, Duncan), n=6.

[0046] Statistical analysis of the effects of different hormone-treated culture media on the proliferation of 'Hongyan' strawberry revealed that, at the same 6-BA concentration, the proliferation coefficient of the group treated with NAA was lower than that of the group without NAA. The proliferation coefficient gradually increased with increasing 6-BA concentration. Treatments A1, A3, and A5, treated only with 6-BA, exhibited superior proliferation coefficients compared to the other treatments. A5, treated with 0.5 mg / L 6-BA, exhibited the highest proliferation coefficient, reaching 3.0. A1, treated with 0.35 mg / L 6-BA, exhibited a low hormone concentration and a proliferation coefficient of 2.8, resulting in excellent seedling growth, making it the optimal choice for the experiment (Table 2).

[0047] 3. Limited subculture reduces variation Two bottles of virus-free strawberry seedlings from the primary culture treated with 0.35 mg / L 6-BA (A1) were randomly selected for subculture. Each generation was cultured for 30 days. Leaves from each generation were collected and used as material for mutation detection. The sample types tested included 2 original seedlings, 4 first-generation seedlings, 7 second-generation seedlings, 15 third-generation seedlings, and 38 fourth-generation seedlings, for a total of 66 tissue culture progeny.

[0048] The IRAP molecular marker method was used to detect genetic variation in the virus-free tissue culture seedlings of 'Hongyan' strawberry during the tissue culture subculture process, proving that the number of subcultures is an influencing factor in somatic cell asexual variation and that the IRAP molecular marker method is suitable for detecting genetic variation in 'Hongyan' strawberry. Four IRAP primers (Table 3) were designed based on the long terminal repeat sequence (LTRs) structural region sequence of strawberry plant retrotransposons retrieved from NCBI. These primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. and purified by PAGE. Sixty-six tissue culture progeny were tested using the four IRAP primers (primer sequences are shown in Table 3). Primer D3 showed no variation in the first to fourth generation plants (partial results are shown in Figure 3 ), three primers (primers D1, D2, D4) detected three plants with mutations and all of them were in the fourth generation (partial results see Figure 4 、 Figure 5 、 Figure 6 and Figure 7 Electrophoresis revealed that these three plants had bands missing or added compared to the DNA bands of the original plant. There were three band types, three mutation sites, and a mutation rate of 7.90%. Compared to the DNA bands of the original plant, plant No. 31 had one more band at 1500-2000 bp ( Figure 5 Plant No. 64 lost a band at about 2000 bp ( Figure 4 ); Plant No. 66 lacked a band at 740 bp ( Figure 7 ). Table 3 Primer sequences

[0049] 4. Acclimation and transplanting The culture medium for primary culture was A1 with a lower hormone concentration, and the virus-free strawberry seedlings with good seedling growth were randomly selected for subculture. The culture medium for subculture also used A1 with a lower hormone concentration, and the number of subcultures was set to 0, 1, 2, 3, 4, and 5, respectively.

[0050] Inoculate 6 bottles for each subculture number, repeat 3 times, and culture in the culture room.

[0051] After subculture, when the shoot tip grows to about 3 cm in height, rooting culture is carried out on rooting medium as follows: cut off the callus tissue and the lower leaves with a scalpel, and insert the explants vertically into the rooting medium (MS dry powder 4.43 g / L, agar 7 g / L, sucrose 30 g / L, pH = 5.85) with a depth of about 5 mm.

[0052] After four weeks of rooting, healthy seedlings, reaching a height of more than 4 cm, with a well-developed root system and 3-5 leaves, are transferred to a greenhouse for acclimatization and transplanting. The greenhouse temperature should be maintained at 22-25°C during the day and 12-15°C at night. Shade nets should be used during the day to provide shade and protect against the sun. Leave the bottle cap open for the first day, unscrew it on the second day, and remove it on the third day. Mist the seedlings with water once daily, morning, noon, and evening. After a week of uncapping, remove the seedlings with tweezers, clean the culture medium to which the roots are attached, and transplant them into nutrient pots (prepared with a 1:1:1 ratio of peat, vermiculite, and perlite). Maintain moisture and provide shade after transplanting.

[0053] 30 days after transplanting, the strawberry seedlings will be treated with low temperature and short day (temperature 15℃, light time 10h / d). Low temperature and short day are necessary and sufficient conditions for flower bud differentiation. Flowering test will be carried out to observe the results.

[0054] By acclimating and transplanting tissue culture seedlings with different subculture times, and subjecting them to low temperature and short day treatment to promote flowering and fruiting, the strawberry fruits were observed. Comparative observations showed that excessive subculture times (more than 8 times) at a concentration of 0.5 mg / L 6-BA caused the strawberries to mutate and produce deformed fruits ( Figure 1 This may be because the relatively high cytokinin levels interfere with the balance of endogenous hormones, causing the cells to enter an abnormal reproductive state. As the number of subcultures increases, the risk of chromosomal aberrations increases significantly, leading to the production of deformed fruits.

[0055] By screening the hormones of the primary and secondary culture media, culturing in a low cytokinin proliferation medium, and controlling the limited number of subcultures for 3-4 times, the plants did not mutate, the fruits were normal, and no deformed fruits were produced ( Figure 2 ).

[0056] 5. The secondary seedling raising system improves seedling raising efficiency The traditional three-stage seedling system: Using virus-free tissue culture, stem tip cells are cultured in a sterile environment to produce virus-free seedling stock F0. This F0 is then bred into virus-free seedling stock F1, which is then bred into virus-free seedling F2, which is then bred into production seedling E. Only after testing can these plants be sold to growers for strawberry production. This seedling system undergoes three field production cycles, with a seedling production cycle of 4-5 years.

[0057] Compared to the three-stage seedling raising system, the two-stage seedling raising system of the present invention involves using tissue culture (cultivated in a low-cytokinin proliferation medium, with 3-4 subcultures after the primary culture) to test for flowering and fruiting. Variant seedlings producing deformed fruit are then screened out. The seedlings are then propagated from runners to produce daughter seedlings, which are then used directly as production seedlings. This seedling raising system eliminates the need to propagate seedlings from seedlings, reduces the number of field production runs, lowers the risk of pest and disease infection, controls strawberry variation, shortens the seedling raising cycle, and improves seedling raising efficiency.

[0058] In summary, A1 is the optimal medium for inducing bud growth from the stem tips of 'Hongyan' strawberry, with a survival rate of 87.30% and a bud induction rate of 84.12%. The hormone NAA is not suitable for primary culture. The combination of 6-BA and NAA easily produces callus, which is not conducive to inducing budding from the stem tip. A5 has the best effect on stem tip proliferation, followed by A1 and A3. The addition of 6-BA has no significant effect on proliferation. Because hormone concentration affects the probability of somatic asexual variation, medium with lower hormone concentration has a lower variation rate. Therefore, A1 is the optimal choice for both maintaining the proliferation coefficient and reducing the variation rate. Limiting the number of subcultures to 3-4 can also effectively reduce the variation rate. After a round of flowering and fruiting tests, the obtained original seed mother seedlings are screened out and the mutated original seed seedlings that produce deformed fruits are then bred. The obtained original seed seedlings are directly used as production seedlings. This can control strawberry mutations while shortening the seedling cycle, reducing the risk of disease and pest infection, and improving seedling efficiency.

[0059] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. A strawberry seedling raising method, characterized in that: The method comprises the steps of inoculating strawberry explants into a culture medium containing 0.35 mg / L 6-BA as a plant growth regulator to obtain original seedlings through tissue culture, flowering and fruiting once, screening out mutants that produce deformed fruits, breeding the original seedlings through runner production, and directly using the original seedlings as production seedlings.

2. The method according to claim 1, wherein: The culture medium containing 0.35 mg / L 6-BA as a plant growth regulator is a solid culture medium obtained by adding sucrose, a coagulant, activated carbon and the plant growth regulator 6-BA to an MS liquid culture medium.

3. The method according to claim 1, wherein: The original seedlings obtained by tissue culture are subcultured after primary culture, and the number of subcultures is 0-4 times.

4. The method according to claim 3, wherein: The original seedlings obtained by tissue culture are subcultured after primary culture, and the number of subcultures is 3-4 times.

5. The method according to claim 1, wherein: The primary culture was cultured for 30 days.

6. The method according to claim 1, wherein: The subculture is carried out for 30 days per generation.

7. The method according to claim 1, wherein: The explant is the stolon tip.

8. The method according to claim 1, wherein: The culture conditions of the tissue culture are: light intensity 2000-3000 Lx, light duration 14 h, temperature 25±2°C, and humidity 45%-50%.

9. The method according to claim 1, wherein: The method of obtaining the original seedlings also includes the steps of hardening the seedlings and transplanting.

10. The method according to any one of claims 1 to 8, characterized in that: The flowering and fruiting need to be promoted by low temperature and short day treatment.

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