Whole-plant cryopreservation method of strawberry seedlings and application

Abstract

The application discloses a whole-plant living body cryopreservation method of strawberry seedlings and application thereof, pretreats the strawberry seedlings and retains all functional leaves; sprays a composite protective agent on the pretreated strawberry seedlings after precooling treatment; after spraying the composite protective agent, program cooling treatment is carried out, and the strawberry seedlings are induced into a whole dormancy state and then cryopreserved. The whole-plant living body cryopreservation method can be applied to strawberry substrate seed raising and strawberry planting. The strawberry seedlings cryopreserved by using the whole-plant living body cryopreservation method are planted in a substrate or soil after resuscitation. The strawberry seedlings cryopreserved by using the method of the application are planted in a substrate or soil after resuscitation, the strawberry seedlings in a dormancy state are resuscitated, the plant growth is vigorous in an early stage, the disease resistance and stress resistance are strong, the yield and fruit quantity are large, the plant is not easy to grow vigorously in a later stage, and the good fruit production state can be continuously maintained.

Description

Technical Field

[0001] This invention relates to the field of agricultural technology, specifically to a method for long-term cryopreservation of strawberry seedlings in a deep dormant state and its applications. Background Technology

[0002] Strawberry (Fragaria annassa Duch.) is a herbaceous plant belonging to the genus Fragaria in the family Rosaceae. Horticulturally, strawberries are classified as berry-type fruit trees, with delicious flavor and rich nutrition, making them popular with consumers. Due to their short growing cycle and high economic benefits, strawberries are widely cultivated both domestically and internationally, consistently ranking first in the world in terms of cultivation area and yield among small berry producers. my country ranks first in the world in both strawberry cultivation area and total yield, with a yield of 26.24 t / hm². 2 Ranked 8th (2020 data). Strawberry industry clusters have formed in places like Donggang in Liaoning, Changfeng in Anhui, Yancheng in Jiangsu, and Jiande in Zhejiang, and are distributed throughout all provinces and cities in China, mainly selling fresh fruit. The main factor affecting the sustainable development of the strawberry industry is the seedling issue. my country has not yet formed an orderly strawberry seedling production system, and currently, both the supply and demand sides lack guarantees in terms of seedling quality and quantity.

[0003] As strawberry farmers say, "good seedlings account for 70% of the harvest," healthy seedlings are the fundamental guarantee for strawberry production. The three major diseases affecting strawberry production are gray mold, powdery mildew, and anthracnose. Gray mold mainly occurs in winter, and it is highly prevalent under low temperature and high humidity conditions. Powdery mildew mainly occurs during the fruiting period or in strawberry seedlings grown in cooler regions; its pathogen is not resistant to high temperatures. Anthracnose is the most important disease in the strawberry seedling stage, occurring in strawberry-growing areas worldwide and having plagued the strawberry industry for decades without a successful solution. The pathogens are *Anthracnose spp.*, *Anthracnose bacillus*, or *Anthracnose spp.*, with an optimal temperature of 28–32℃ and relative humidity above 90%. It is a typical high-temperature, high-humidity pathogen. In the middle and lower reaches of the Yangtze River in my country, July and August are the peak season for anthracnose. If control measures are not timely, it can cause large-scale infection and crop failure within 1–2 weeks. Infected strawberry seedlings will continue to die after transplanting. The pathogen can spread through stolons, water, soil, and contact. In recent years, there have been many reports of diseases such as red leaf disease and hollow heart disease. Red leaf disease mainly occurs in 'Sweet Charlie' and related varieties, while hollow heart disease is a bacterial disease.

[0004] For decades, strawberry cultivation in my country has primarily relied on soil-based seedlings. In recent years, to prevent the spread of soil-borne diseases, substrate-based seedling cultivation has been developed. This involves inserting or propagating seedlings into plug trays filled with substrate. Plug tray seedlings isolate the plant's root system and offer advantages such as high survival rates, short establishment periods, and high early-stage yields. Currently, substrate-based seedling cultivation bases are mainly located in high-altitude, cool regions. However, high-altitude strawberry seedlings often carry powdery mildew pathogens, making them susceptible to powdery mildew during the fruiting period. Strawberry seedling cultivation is primarily limited by local climatic conditions. In northern regions, slow spring warming and late planting of mother plants, coupled with a dry climate, result in a low propagation coefficient. In the subtropical monsoon region south of the middle and lower reaches of the Yangtze River, the warm and humid climate in spring and early summer results in a high propagation coefficient for strawberry seedlings. However, high temperatures of ≥35℃ are common in July and August, and when the root temperature of strawberry seedlings reaches ≥32℃, physiological disorders occur. Sustained high temperatures damage the root system, and wound infections easily lead to root rot. The optimal seedling age for plug seedlings is 45-60 days. In southern regions, runners begin to emerge from late April, with multiple daughter seedlings growing on each runner. The peak propagation period is from May to June. After the high temperatures begin in July, fewer new daughter seedlings are generated, resulting in the common problem of overly old seedlings and aging root systems. Under high temperature conditions, water evaporation is high, and the daughter seedlings are in the extremely limited capacity of the plug trays for a long time, with fluctuating water supply, which easily induces Verticillium wilt.

[0005] There is an imbalance in the number of strawberry seedlings in different years and regions: (1) The yield fluctuates greatly in different years. my country's agriculture still relies on the weather. Taking the strawberry seedling situation in the past three years as an example: In the summer of 2021, the summer temperature in the Jiangnan area was low, and the supply of strawberry seedlings was relatively sufficient; In the summer of 2022, the Jiangnan area experienced rare high temperatures, and the number of days with the highest temperature ≥35℃ in Hangzhou reached 56 days. Due to high temperature obstacles, water shortage and diseases, the strawberry seedling production was reduced, and the strawberry seedlings were in short supply; In 2023, the area of ​​strawberry seedling cultivation increased, and the summer of that year was cool, and the strawberry seedling production was high. The nationwide sluggish sales phenomenon occurred, and by the end of September, they had to be removed, resulting in serious losses for seedling farmers. (2) Imbalance in seedling supply and demand between regions: The planting time for strawberry seedlings is determined by the degree of flower bud differentiation and local temperature. Planting too early or too late will affect the ripening period and yield. Case 1: Planting too early: In 2021, a farmer planted strawberry seedlings on August 25th, which delayed the flowering time by about 20 days compared to planting on August 30th, and delayed the ripening period by about one month. Case 2: Planting too late: Due to the cool climate conditions, highland strawberry seedlings have always had the problem of premature flower bud differentiation. Often, the roots are not well developed after planting, and due to insufficient plant growth, the first batch of strawberries is small, resulting in low total yield. The climate conditions in different regions determine the differences in planting time. For example, Donggang, Liaoning, is usually planted in mid-to-late August; Changfeng, Anhui, in late August to early September; Jiande, Zhejiang, in early to mid-September; and Taizhou, Zhejiang and Fuzhou, Fujian, in mid-to-late September. The survival rate is affected by factors such as physiological suitability and potential issues like overheating and dehydration during transportation. Therefore, it is best to purchase strawberry seedlings locally, which to some extent limits the cross-regional circulation of seedlings.

[0006] The price of fresh strawberries in my country has a clear seasonality, with high prices in winter and low prices in spring. Prices are usually high when strawberries begin to ripen in November; January to the Spring Festival is also the peak period for prices, and demand is high; after the Spring Festival, especially after March, prices drop significantly. Therefore, the income of strawberry farmers mainly depends on the early production.

[0007] Strawberry seedlings can only be planted when they enter the flower bud differentiation stage. Flower bud differentiation is related to temperature, day length, and nitrogen level. Currently, the main measures to promote early cultivation are: (1) Night cooling seedling raising: taking advantage of the fact that strawberry seedlings require chilling for flower bud differentiation, the seedlings in the plug trays are gathered together at night for cooling treatment, and then moved out for daytime light treatment. This method has high costs in terms of manpower, power, and equipment; (2) Short-day treatment: the strawberry seedlings are fully shaded in the morning and evening. Short-day treatment promotes flower bud differentiation; (3) Nitrogen cut-off treatment: flower bud differentiation is negatively correlated with nitrogen level. Starting from August, nitrogen fertilizer supply is stopped, and strawberry seedlings can enter the reproductive growth stage earlier. This is the lowest cost treatment method. Although strawberries are on the market earlier, the yield during the Spring Festival is affected. The existing methods to promote early cultivation will generally result in reduced yield due to the weak growth of the plants.

[0008] In summary, my country is the largest strawberry producer, but there are still many problems in seedling production and fresh fruit supply and demand: (1) Strawberries have a high propagation coefficient in the south, but are prone to anthracnose outbreaks in the hot and humid climate of July and August; (2) In addition to problems such as high temperature and dehydration, plug seedlings also have problems such as excessively long seedling age and aging root system; (3) Seedling production is more blind and there is a lack of supply and demand control methods; (4) Fresh strawberries are usually in short supply before the Spring Festival; (5) Existing early cultivation methods have the disadvantages of high cost or impact on total yield; (6) Flower bud differentiation of seedlings in high-altitude areas is too early.

[0009] The most common method for preserving strawberry seedlings is refrigeration at 2-8℃, with a safe storage period of 7 days. During this period, the plants gradually weaken, and the maximum storage time is 14 days. The preservation method for imported European strawberry seedlings involves pre-cooling the seedlings in winter, removing all leaves, and then storing them at a controlled temperature of 0℃. + Preservation at 1℃ (with an extreme temperature range of -2 to 2℃) is also used in northern my country, but it is limited to storing strawberry seedlings in winter for use as mother seedlings in spring. Because the seedlings are less vigorous after freezing, lack functional leaves, and have a long recovery period, user acceptance is low. Current technology cannot solve these problems. Summary of the Invention

[0010] This application provides a method and application for whole-plant cryopreservation of strawberry seedlings, which can preserve the entire plant, including all functional leaves. After thawing, the original functional leaves continue to perform photosynthesis.

[0011] A method for cryopreservation of whole strawberry seedlings, comprising:

[0012] The strawberry seedlings were pretreated and all functional leaves were retained. The pretreated strawberry seedlings were then subjected to pre-cooling treatment and sprayed with a compound protective agent. After spraying with the compound protective agent, the seedlings were subjected to programmed cooling treatment to induce them to enter a state of full dormancy before being frozen for preservation.

[0013] This application retains all functional leaves of the strawberry seedlings. After pre-cooling treatment, a compound protective agent is sprayed on the seedlings. After a programmed cooling process, the seedlings enter a state of full dormancy. The entire plant is then frozen. After revival, the original functional leaves will continue to perform photosynthesis.

[0014] The functional leaves can be understood as leaves that have the functions of photosynthesis and transpiration. On a strawberry plant, there are usually 6 functional leaves arranged in a circle.

[0015] The aforementioned dormant state can be understood as the absence of visible growth signs at the rootstock production points of the strawberry seedlings. The preservation period using the method described in this application is 27 to 501 days. The 501-day period refers to the longest preservation test time, during which the plants remain viable; it does not mean the maximum storage period is 501 days. The strawberry seedling revival method involves natural thawing at room temperature or accelerated thawing by rinsing with water, followed by planting in the substrate or soil and watering thoroughly on the same day.

[0016] Preventing the formation of ice crystals during the cooling process is the key to achieving cryopreservation of strawberry seedlings. This application combines protective agents and cooling treatment: (1) to prevent the formation of ice crystals that damage the cell membrane structure, DMSO is added as an antifreeze agent; (2) DMSO also has the effect of inhibiting cell growth. Under the combined effect of low temperature, the cells enter a state of complete dormancy and no longer undergo metabolic activity, thus not consuming nutrients; (3) when the growth conditions are suitable, the seedlings are revived and the plants at the optimal seedling age quickly resume growth under the photosynthesis of the functional leaves.

[0017] Optionally, the pretreatment process includes removing yellow leaves, disabled and hardened old leaves, and runners from the strawberry seedlings. Leaves at the bottom should be promptly removed when they become disabled and brittle, as their respiration exceeds photosynthesis, and older leaves tend to have more insect eggs; leaving them may also become infection points for pathogens.

[0018] Optionally, the strawberry seedlings are preferably strawberry seedlings that have reached the bifurcation stage of flower bud differentiation and are 45-60 days old. These seedlings can be strawberry seedlings cultivated in different seasons or in different regions.

[0019] Optionally, the precooling treatment includes: precooling the strawberry seedlings at 6℃ to 25℃ for 4 to 24 hours (this step can be omitted when the temperature is low in winter). Further, the precooling treatment includes: precooling the strawberry seedlings at 6℃ to 15℃ for 4 to 6 hours.

[0020] Optionally, the composite protective agent contains 1.5–3.0 wt% dimethyl sulfoxide (DMSO) and 1.0–2.0 wt% polyvinylpyrrolidone (PVP K30); the pH of the composite protective agent is 6.0–6.8.

[0021] Optionally, the solvent for the composite protective agent is an aqueous phosphate solution.

[0022] Optionally, the composite protective agent should be stored at 2–4°C after preparation and used within 48 hours.

[0023] Optionally, the application process of the compound protective agent includes: uniformly spraying the protective agent onto the plant, specifically spraying the compound protective agent evenly onto the roots, stems, and leaves of the strawberry seedlings. The aim is to ensure that the leaves, shortened stems, and roots exposed on the outside of the plant are covered with uniform, fine droplets.

[0024] Optionally, the programmed cooling steps include: first placing at 4–10℃ for 30 min–4 h; then placing at -18–-20℃ for 30–60 min; and finally storing at -2.0–-8.2℃ for long-term storage. Further optionally, placing at 4–10℃ for 30 min–60 min.

[0025] This application also provides an application of the whole-plant cryopreservation method in strawberry substrate seedling cultivation or strawberry soil seedling cultivation.

[0026] This application also provides an application of the whole-plant cryopreservation method in strawberry cultivation.

[0027] This application also provides a strawberry cultivation method, comprising thawing strawberry seedlings preserved using the whole-plant cryopreservation method and then transplanting them into a substrate or soil. Strawberry seedlings cryopreserved using this method, after thawing and transplanting into a substrate or soil, exhibit vigorous early growth, strong disease and stress resistance, and high yield and fruit production. They are less prone to excessive vegetative growth later on and can maintain good fruit production. Therefore, field management is relatively labor-saving, requiring only conventional field management methods.

[0028] Optionally, the thawing method involves allowing the strawberry seedlings to thaw naturally at room temperature or quickly thawing them by rinsing them with water.

[0029] Optionally, it also includes a treatment process of soaking the revived strawberry seedlings in a root-soaking agent. Optionally, one method for preparing the root-soaking agent is as follows: take 5 ml of Amistar, 5 ml of Bright Shield, and 10 ml of Yishibang, and dilute with water to 10 L to prepare the root-soaking agent.

[0030] All reagents used in this application are commercially available products.

[0031] Compared with the prior art, this application has at least one of the following beneficial effects:

[0032] Firstly, its application in seedling cultivation: (1) enabling strawberry seedlings to safely pass through the high-temperature period of July to August; (2) preventing the root system of strawberry seedlings from aging and maintaining the optimal seedling age; (3) preserving strawberry seedlings for use in the next season or the following year.

[0033] Secondly, its application in fresh fruit production: (1) it enables fresh fruit to be marketed earlier; (2) strawberries mature during the Spring Festival, and the output value is increased by leveraging the price advantage of strawberries in the early period and during the Spring Festival; (3) after the dormant strawberry seedlings recover, the plants grow vigorously in the early stage, have strong disease resistance and stress resistance, and produce a large amount of fruit. They are not prone to excessive growth in the later stage and can continue to maintain a good fruit production state. Therefore, it is more labor-saving in field management. Attached Figure Description

[0034] Figure 1A picture of 'Pink Jade No. 1' cultivated over two years;

[0035] Figure 2 Image of 'Pink Jade No. 2' cultivated across seasons;

[0036] Figure 3 Image showing fruit set on frozen 'Xiangye' seedlings;

[0037] Figure 4 Image showing the ripening of frozen seedlings of 'Xiangye';

[0038] Figure 5 Image of 'Pink Jade No. 1', which was frozen for 501 days;

[0039] Figure 6 for Figure 5 Image of frozen seedlings transplanted 14 days after planting;

[0040] Figure 7 Photo of the greenhouse used for batch seedling collection experiments (July 21, 2023);

[0041] Figure 8 A picture of 'Beijing Suburbs Little White' frozen for 27 days.

[0042] Figure 9 Image of the 'Black Pearl' frozen for 63 days;

[0043] Figure 10 Photo of 'Black Pearl' seedlings frozen 15 days after transplanting;

[0044] Figure 11 This is a picture of a 'Xiangye' seedling frozen for 32 days after transplanting. Detailed Implementation

[0045] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0047] Example 1: Trans-year Preservation Experiment of Seedlings

[0048] (1) Seedling raising: From April to August 2021, substrate seedling raising experiments were conducted on strawberry varieties such as 'Fenyu No.1', 'Fenyu No.2', 'Hongyu', 'Hongyan', and 'Xiangye' at the Zhijiang Base of Hangzhou Academy of Agricultural Sciences.

[0049] (2) Flower bud differentiation observation: From the end of August to the beginning of September 2021, flower bud differentiation of plug seedlings and bare-root seedlings was observed under a dissecting microscope. 'Fenyu No. 1' plug seedlings entered the thickening period on August 30 and were transplanted on September 5.

[0050] (3) Sampling and pre-cooling: On September 6, 2021, from 8:00 to 10:00 a.m., 'Fenyu No. 1' plug seedlings were collected and stored at 8-10℃ for 4 hours. Yellow leaves, disabled hardened old leaves and runners were removed.

[0051] (4) Spraying compound protectant (PB): Weigh 1.5 g potassium dihydrogen phosphate and 2.0 g dipotassium hydrogen phosphate, dissolve them in 1 L of water, add 20 ml dimethyl sulfoxide (DMSO) and 20 g polyvinylpyrrolidone (PVP K30) (i.e., protectant containing 2.0 wt% DMSO), and store at 2-4℃. Spray it on the leaves, shortened stems and roots of strawberry seedlings (it is advisable to make the leaves, shortened stems and roots exposed on the outside of the plant have uniform fine droplets), and neatly put them in a cardboard box, with each layer separated by kraft paper.

[0052] (5) Cooling: Place the cardboard box filled with strawberry seedlings in a refrigerator at 4-10℃ for 30 minutes, and then transfer it to a freezer at -20℃ for 60 minutes.

[0053] (6) Storage: Adjust the refrigerator temperature to -4 to -6℃, use FreshShield electronic thermometer and hygrometer to remotely monitor the actual temperature inside the refrigerator via mobile phone to be -2.0 to -8.2℃.

[0054] (7) Recovery and Planting: On September 17, 2022, 30 frozen 'Pink Jade No. 1' strawberry seedlings were taken out. After they were completely thawed, 3 leaves and 1 bud were left, and 30% of the roots were cut off. 5 ml of Amistar, 5 ml of Bright Shield, and 10 ml of Yishibang were diluted with water to 10L to prepare a root soaking solution. The roots were soaked in the solution for 5 minutes. The frozen strawberry seedlings (T) were planted in the soil at a spacing of 20 cm. At the same time, 'Pink Jade No. 1' plug seedlings (CK) and bare-root seedlings (B) were planted.

[0055] (8) Fertilizer and water management: Water once in the morning and once in the evening within 6 days after transplanting; on the 7th day, drench the roots with 6000 times diluted 'Annorui' water-soluble fertilizer (nitrogen:phosphorus:potassium ratio of 20:10:20), 100ml per plant; on the 9th day, drench the roots with 3000 times diluted Trichoderma harzianum, 100ml per plant; on the 14th day, drench the roots with 5000 times diluted balanced water-soluble fertilizer (nitrogen:phosphorus:potassium ratio of 20:20:20) + 2000 times diluted 'Futawang' amino acid fertilizer, 100ml per plant. On the 20th day after transplanting, install drip irrigation tape and drip mixed water-soluble fertilizer with EC of 0.6-0.7ms / cm twice a week; remove runners immediately.

[0056] (9) Field observation: The field characteristics of each group of 'Fenyu No. 1' strawberry plants are shown in Table 1. The experimental group showed faster rooting, earlier flowering and fruiting, and larger fruit size. The first harvest period was on November 7, 2022. Figure 1 T); the leaves are large and thick, bright green, and the plant growth is significantly stronger than the control group (T). Figure 1 CK) and bare-root seedlings ( Figure 1 B); During the twelfth lunar month (December 23, 2022 to January 20, 2023), the yield per plant in the experimental group increased by 13% and 58% compared with the control group and bare-root seedlings, respectively.

[0057] Table 1 Growth Indicators of 'Pink Jade No. 1'

[0058]

[0059] In summary, strawberry seedlings treated with dormancy (Group T) showed high survival rates, vigorous plant growth, early maturity, large fruit size, and high early yields. Therefore, this treatment method is suitable for early-maturing and high-yield cultivation.

[0060] Example 2: Cross-season preservation experiment of mother seedlings for production seedlings

[0061] (1) Cutting propagation: From late October to early November 2021, a cutting propagation experiment of 'Fenyu No. 2' was carried out at the Zhijiang Base of Hangzhou Academy of Agricultural Sciences.

[0062] (2) Sampling and preservation: On December 13, 2021, 'Fenyu No. 2' seedlings were collected and sprayed with a composite protective agent containing 1.5% DMSO (that is, the amount of dimethyl sulfoxide (DMSO) in Example 1 was adjusted to 15 mL (1.5 wt% DMSO) and 10 g of polyvinylpyrrolidone (PVP K30), and other components and contents were the same as in Example 1); they were packed into cardboard boxes; placed in a freezer at 4-10℃ for 4 h; stored at -20℃ for 30 min; the temperature was set to -5℃ and stored for a long time under remote monitoring.

[0063] (3) Recovery and planting: On September 17, 2022, 60 frozen 'Pink Jade No. 2' strawberry seedlings were taken out and planted directly in the soil at a spacing of 20cm after they were completely thawed. At the same time, 'Pink Jade No. 2' strawberry seedlings from 2022 were planted as a control.

[0064] (4) Fertilizer and water management: Water twice a day during the first week after transplanting; starting from the 20th day, apply water-soluble fertilizer with EC of 0.6-0.7 ms / cm twice a week.

[0065] (5) Plant management: adopt a mixed management model of seedling production + fruit production. Remove the first inflorescence in mid-October, collect the stolons in early November for cuttings, and retain the second to fourth inflorescences for fruit production.

[0066] (6) Seedling production: Under the condition of early nutrient deficiency, the experimental group showed fast rooting, dark green leaves and strong plant growth, while the control group had yellowish leaves; the number of stolons produced was 4.6 per plant in the experimental group (6.7 offspring per plant), and 1.2 per plant in the control group (2.3 offspring per plant).

[0067] (7) Fruit production before the Spring Festival: In the experimental group, the 2nd to 4th inflorescences set fruit relatively concentratedly in mid-to-late November, with a total of 20.6 flowers and fruits per plant, a flower stalk length of 18.2 cm, and uniform inflorescences. Figure 2 (Right); The control group showed single inflorescences with successive fruit set, a total of 9.3 flowers and fruits per plant, a pedicel length of 32.2 cm, and uneven inflorescences. Figure 2 (Left). In the twelfth lunar month (December 23, 2022 to January 20, 2023), the yield per plant in the experimental group was 89.7 grams; the yield per plant in the control group was 57.6 grams.

[0068] (8) Field performance after the Spring Festival: After mid-February, the plants in the control group began to grow vigorously, with an average plant height of 29.2 cm; the plants in the experimental group maintained reproductive growth, with an average plant height of 26.9 cm. The total yield was 437 g / plant in the experimental group and 345 g / plant in the control group.

[0069] In summary, the 'Fenyu No. 2' strawberry seedlings treated with dormancy exhibited good early-stage resistance, vigorous plant growth, and were less prone to excessive growth in spring. Growth performance showed a 2.9-fold increase in the propagation coefficient; a 56% increase in yield during the Spring Festival; and a 27% increase in total yield. Therefore, when there is a surplus of mother seedlings, the method described in this application can be used to store them for dual-purpose seedling production (both seedling and fruit production).

[0070] Example 3: Highland Strawberry Seedling Preservation Experiment

[0071] (1) Experimental materials: 'Xiangye' and 'Qianmei No. 1' plug seedlings came from Kunming Xundian Kusen Agricultural Development Co., Ltd. When they arrived in Hangzhou on July 21, 2022, the plants in the middle of the boxes were slightly heated due to the high temperature.

[0072] (2) Treatment and preservation: Pre-cool the strawberry seedlings at 6-15℃ for 24 hours; take them out and air-dry their surface moisture at room temperature; spray with a composite protective agent containing 2.5% DMSO (that is, adjust the amount of dimethyl sulfoxide (DMSO) in Example 1 to 25 mL (2.5 wt% DMSO), and the other components and contents are the same as in Example 1); pack them into a cardboard box; place them in a refrigerator at 4-10℃ for 60 minutes, and then freeze them at -20℃ for 30 minutes; set the temperature to -5℃ for long-term storage.

[0073] (3) Recovery and planting: On September 17, 2022, 90 seedlings each of 'Xiangye' and 'Qianmei' were taken out of the frozen storage trays and planted in the soil at a spacing of 20cm. At the same time, a control group was planted.

[0074] (4) Fertilizer and water management: water once in the morning and once in the evening during the first week; starting from the 20th day after transplanting, apply water-soluble fertilizer with EC of 0.6-0.7 ms / cm twice a week.

[0075] (5) Plant management: 'Xiangye' adopts a mixed management model of seedling production and fruit production. The first inflorescence is removed at the end of September; runner seedlings are collected once each in late October and mid-November for cuttings, retaining the 2nd to 5th inflorescences; flower branches are removed in early March, leaving runners, and runner seedlings are harvested for the third time in early May. 'Qianmei No. 1' conducts fruit production trials, removing runners immediately and retaining all inflorescences.

[0076] (6) Experimental results:

[0077] The first two batches of the 'Xiangye' experimental group produced a total of 5.5 viable seedlings. From mid-October to mid-November, the second to fourth inflorescences successively budded, and a large number of fruits set in December. Figure 3 ), entering its peak fruiting period in January 2023. Figure 4 The yield reached 146 grams per plant in the first month; the number of seedlings per plant was 4.3 in the third harvest. The control group of 'Xiangye' that was not frozen did not produce any seedlings before the Spring Festival; the fruits matured in batches in single inflorescences, with a yield of 74.7 grams per plant in January and 0.7 runner seedlings per plant in May.

[0078] The number of runners produced in the 'Qianmei No. 1' experimental group was 3 per plant, while that in the control group was 0.5 per plant; the fruit ripening period was December 10th in both groups; the average weight of a single first-order fruit was 36.3g / fruit in the experimental group and 29.1g / fruit in the control group; the total yield was 429g / plant in the experimental group and 365g / plant in the control group.

[0079] In summary, highland strawberry seedlings stored under dormancy conditions showed significantly improved stolon growth and fresh fruit yield, thus this treatment method is beneficial for promoting the circulation of strawberry seedlings from different regions.

[0080] Example 4 Seedling Storage Experiment

[0081] (1) Seedling selection: Based on the field characteristics of 'Fenyu No. 1', select plants with excellent performance and use the stolons as seedlings by inserting them into plug trays.

[0082] (2) Processing and storage: On the morning of May 2, 2022, strawberry seedlings were collected and placed in a sorting box. At 10:00, they were placed in a refrigerator with a set temperature of 10°C. During the pre-cooling period, the temperature was remotely monitored to be 10-15°C. At 14:00, the strawberry seedlings were taken out, old leaves were removed, and 3 leaves and 1 bud were left. A composite protective agent containing 3.0% DMSO was sprayed (that is, the amount of dimethyl sulfoxide (DMSO) in Example 1 was adjusted to 30 mL (3.0 wt% DMSO), and other components and contents were the same as in Example 1). The seedlings were then packed into a cardboard box. The seedlings were placed in a chromatography refrigerator at 4-10°C for 30 min, and then transferred to a freezer at -20°C for 30 min. The seedlings were stored in a freezer with a set temperature of -5°C for a long time. The remote measured temperature was -3°C to -8°C.

[0083] (3) Recovery performance: On September 15, 2023, the 'Fenyu No. 1' seedlings, which had been frozen for 501 days, were retrieved. Figure 5 They were cultivated separately using substrate and soil. Field observations on September 29, 2023, showed an average of 1.5 new leaves per leaf, a 100% survival rate, a 57% budding rate, and a 12% flowering rate. Figure 6 ).

[0084] In summary, strawberry seedlings stored during dormancy have a high survival rate and grow quickly, effectively reducing the number of generations and the rate of variation in seedlings.

[0085] Example 5: Application of Strawberry Seedling Preservation Method in Substrate Seedling Cultivation

[0086] Technical challenges of substrate seedling cultivation: I. Overly old seedlings; II. Anthracnose, root rot, and wilt are prone to outbreaks under high temperature and humidity conditions in July and August; III. As early-grown leaves and roots age, the corresponding stolons degenerate, turn black in some areas, and become entry points for pathogens; IV. Seedlings at different stages crowd each other, causing poor ventilation; V. When the number of stolons reaches a certain level, the stolon growth rate slows down or even stops due to the excessive burden on the mother plant.

[0087] Since 2021, experiments have been conducted on substrate seedling cultivation combined with methods for preserving strawberry seedlings in full dormancy. After continuous improvements, the experimental content and results in 2023 are as follows:

[0088] (1) Experimental materials: 'Pink Jade No. 1', 'Red Beauty', 'Fragrant Wild', 'Sweet Charlie', 'Red Jade', 'Black Treasure Plant', 'Beijing Xiaobai', etc.

[0089] (2) Field management: transplant mother seedlings in mid-March and introduce daughter seedlings in late April; fertilize both mother plants and daughter seedlings by drip irrigation with water and fertilizer, with a fertilizer concentration of EC0.3~0.6ms / cm.

[0090] (3) Seedling collection: Seedlings were collected in batches from early June to early July, and areas where seedlings were not collected were used as controls.

[0091] (4) Preservation method: Pre-cool strawberry seedlings in an air-conditioned room at 20-25℃ for 2-6 hours; clean old leaves, spray with compound protective agent (2.0wt% DMSO, same as in Example 1), pack into cardboard boxes or foam boxes; place in a freezer at 4-10℃ for 30-40 minutes, then transfer to a freezer at -20℃ for 30-60 minutes; set the storage temperature to -4℃, and the remote measured temperature is -2℃ to -6℃.

[0092] (5) Performance in the later stages of seedling cultivation: Observations were conducted on the experimental and control groups from mid-July to August. Field performance on July 21 (see...) Figure 7 276T1 represents the area where 'Fenyu No. 1' seedlings were collected on June 5th. New seedlings comprised approximately 70% of the planting holes, and the seedlings were healthy, robust, and had white roots. 276T2 represents the area where 'Fenyu No. 1' seedlings were collected in late June. New seedlings comprised approximately 40% of the planting holes, and the seedling roots were white. 276CK represents the control group of 'Fenyu No. 1' seedlings for which no seedlings were collected. These seedlings had long petioles, yellow roots (indicating root aging), and many were floating. In August, the stolons of the aging seedlings in the 'Fenyu No. 1' control group showed localized blackening, and three plants developed wilt disease. The stolons in the experimental group remained green and showed no disease. XY T1 was the area where 'Xiangye' seedlings were collected on June 5th. New seedlings comprised approximately 60% of the planting holes, were healthy and robust, and had white roots. XY T2 was the area where 'Xiangye' seedlings were collected on July 10th. New seedlings had just taken root and comprised approximately 10% of the planting holes. The control group stopped producing new runners after an average of 9.2 runners were generated. The experimental group continued to produce new runners after seedling collection. HX T1 was the area where 'Hongyan' seedlings were collected on July 8th. New seedlings comprised approximately 20% of the planting holes, were healthy and robust, and had white roots. The control group seedlings had long petioles, yellow roots (indicating root aging), and more floating seedlings. In early August, approximately 15% of the 'Hongyan' control group developed anthracnose.

[0093] (6) Performance of frozen seedlings: Frozen strawberry seedlings maintain bright green leaves and white roots. Figure 8 The 'Jingxiao Xiaobai' was frozen on August 7, 2023, and retrieved on September 3. Figure 9 The 'Black Rare Plant' was frozen on July 2, 2023, and retrieved on September 3. Figure 10The performance of the substrate 15 days after transplanting; Figure 11 The 'Xiangye' seedlings that were planted on August 30th and frozen have already flowered and formed the second inflorescence by October 1st. The experimental group has produced 4 new leaves, and its leaf area is 1.5 to 3 times that of the control group. In addition, the 2 leaves that were frozen before freezing still have photosynthetic function, which is sufficient to support the fruiting of the first inflorescence.

[0094] In summary, the method of preserving strawberry seedlings in full dormancy can be combined with substrate seedling cultivation. By preserving the seedlings at their optimal age in a timely manner, it has a good effect on improving seedling quality, propagation coefficient, and reducing disease occurrence.

[0095] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. A method for cryopreservation of whole strawberry seedlings, characterized in that, include: Pre-treat the strawberry seedlings and retain all functional leaves; The pretreated strawberry seedlings were then subjected to pre-cooling treatment and sprayed with a compound protective agent. After spraying the compound protective agent, the strawberry seedlings were subjected to programmed cooling to induce them into a state of full dormancy before being frozen for storage. The composite protective agent contains 1.5-3.0 wt% dimethyl sulfoxide and 1.0-2.0 wt% polyvinylpyrrolidone; the pH of the composite protective agent is 6.0-6.

8. The cooling process includes: first placing the sample at 4~10℃ for 30min~4h; then placing it at -18~-20℃ for 30~60min; and finally storing it at -2.0~-8.2℃ for long-term storage. The precooling treatment includes: precooling the strawberry seedlings at 6℃~25℃ for 4~24 hours; Spray the compound protective agent evenly onto the roots, stems, and leaves of the strawberry seedlings.

2. The whole-plant cryopreservation method as described in claim 1 is applied in strawberry substrate seedling cultivation or strawberry soil seedling cultivation.

3. The whole-plant cryopreservation method as described in claim 1 is applied in strawberry cultivation.

4. A method for cultivating strawberries, characterized in that, This includes thawing strawberry seedlings preserved using the whole-plant cryopreservation method as described in claim 1 and then transplanting them into a substrate or soil; the thawing method is to thaw the strawberry seedlings naturally at room temperature or to thaw them quickly by rinsing them with water.

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