Rapid breeding process for saline-alkali-tolerant high-flavone sea-buckthorn improved variety

Abstract

The rapid breeding process is characterized by comprising the following steps that S1, after a proper land parcel is selected, sundries are removed, the land is deeply ploughed to 22 cm, 2.5 tons of decomposed farmyard manure is evenly applied to each mu, and planting holes with the diameter being 50 cm and the depth being 40 cm are dug according to the design density; s2, performing winnowing and water separation on the seeds, soaking the seeds in a 0.2% potassium permanganate solution for 20 minutes, performing clean water cleaning and cold plasma treatment, remarkably improving the performance of the seeds on the premise of not damaging the seeds through the interaction of low-temperature plasma and the surfaces and the interiors of the seeds, then soaking the seeds in a mixed solution of an aqueous solution and a sodium chloride aqueous solution for 18 hours, and drying the seeds. Finally washing with distilled water for three times; s3, the cuttings are collected in winter, the sand humidity is 60% during cellaring, and the cellaring temperature is 2 DEG C; through technology integration and innovation, the problems that sea-buckthorn is difficult to breed in the saline-alkali soil, the period is long and the benefit is low are systematically solved.

Description

Technical Field

[0001] The invention relates to the technical field of seabuckthorn breeding, in particular to a rapid breeding process for salt-alkali-tolerant and high-flavonoid seabuckthorn varieties. Background Art

[0002] As a perennial shrub with extremely high ecological and economic value, seabuckthorn has a well-developed root system and is resistant to drought and barrenness. It plays a vital role in ecological restoration, such as windbreak and sand fixation, and soil and water conservation. Its fruit, rich in active ingredients such as flavonoids and vitamins, is widely used in the food, pharmaceutical, and cosmetics industries. However, in adverse soil environments such as saline-alkali land, seabuckthorn's natural reproduction efficiency is low, resulting in poor seedling survival rates. Traditional breeding methods also suffer from long production cycles and the degradation of improved seedling characteristics, making it difficult to meet the demand for high-quality seedlings for large-scale ecological management and industrial development.

[0003] To this end, we proposed a rapid breeding process for salt- and alkali-tolerant, high-flavonoid seabuckthorn varieties to solve the problem. Summary of the Invention

[0004] (1) Technical problems solved

[0005] In view of the deficiencies in the prior art, the present invention provides a rapid breeding process for salt-alkali-tolerant and high-flavonoid seabuckthorn varieties, which solves the problems in the above-mentioned background technology.

[0006] (2) Technical solution

[0007] To achieve the above object, the present invention provides the following technical solution: a rapid breeding process for salt-alkali-tolerant and high-flavonoid seabuckthorn varieties, comprising the following steps:

[0008] S1: After selecting a suitable plot, clear away debris, deep-plow the soil to 22 cm, evenly apply 2.5 tons of decomposed farmyard manure per mu, and dig planting holes with a diameter of 50 cm and a depth of 40 cm according to the designed density;

[0009] S2: The seeds are air-sorted and water-sorted, and then soaked in a 0.2% potassium permanganate solution for 20 minutes. After being washed with clean water and treated with cold plasma, the low-temperature plasma interacts with the surface and interior of the seeds, significantly improving their performance without damaging the seeds. The seeds are then soaked in a mixture of an aqueous solution and a sodium chloride aqueous solution for 18 hours, and finally rinsed with distilled water three times.

[0010] S3: Collect cuttings in winter and store them in a cellar with sand humidity of 60% and cellar temperature of 2°C. Before spring cuttings, soak the base of the cuttings in 0.02% ABT1 rooting powder for 2.5 hours. This can regulate plant endogenous hormones, promote cell division and differentiation, and enhance metabolic activity. After cuttings, cover with mulch to keep them moist. Collect cuttings in mid-June, treat them, and insert them into the seedbed. Maintain air humidity of 90% and temperature of 23-25°C. Spray with carbendazim solution.

[0011] S4: Select explants and sterilize them. Culture them in the priming medium for 2-3 weeks. When the stem segments sprout, transfer them to the proliferation medium and subculture them every 2-3 weeks until the proliferation coefficient reaches 3-4. Transfer them to the rooting medium and culture them for 2-3 weeks. The rooting rate exceeds 90%.

[0012] S5: Transplant when the roots of the seedlings grow to about 4 cm. Maintain the air humidity at 95% and the temperature at 22-24°C for 3 days before transplanting. Then gradually ventilate and reduce humidity. Apply 0.2% potassium dihydrogen phosphate foliar fertilizer every 5 days. Transplant into the field after acclimatization for 2 months.

[0013] S6: Lay planting trenches in layers, plant the seedlings in improved soil, water them promptly after planting, and subsequently manage water and fertilizer appropriately based on their growth conditions.

[0014] Preferably, the pH value of the soil in the plot is 7.5-8.0, and the salt content is less than 0.5%.

[0015] Preferably, the design density is 50 cm × 50 cm × 50 cm.

[0016] Preferably, the aqueous solution is a PEG6000 aqueous solution, and the concentration is 200 g / kg.

[0017] Preferably, the carbendazim solution is sprayed at 0.2% every 7 days.

[0018] Preferably, young stem segments or buds of salt- and alkali-tolerant high-flavonoid sea buckthorn are selected as explants.

[0019] Preferably, an isolation layer, a water storage layer, a filtration layer, a first planting soil layer, a composite bacterial agent layer, a second planting soil layer and an organic fertilizer layer are laid in sequence at the bottom of the planting ditch.

[0020] (3) Beneficial effects

[0021] Compared with the existing technology, the present invention provides a rapid breeding process for salt-alkali-tolerant and high-flavonoid seabuckthorn varieties, which has the following beneficial effects:

[0022] 1. The present invention can activate seed activity through cold plasma treatment, significantly improve germination rate and stress resistance without damaging the embryo, and lay the foundation for planting in saline-alkali land. The present invention can induce the seed osmotic regulation mechanism by mixing PEG6000 and sodium chloride to soak the seeds, thereby enhancing their germination ability in saline-alkali soil.

[0023] 2. Through technological integration and innovation, the present invention systematically solves the problems of sea buckthorn breeding in saline-alkali land, such as difficulty, long breeding cycle and low efficiency, and provides an efficient and feasible technical solution for saline-alkali land ecological restoration and sea buckthorn industrialization development. DETAILED DESCRIPTION

[0024] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0025] The rapid breeding process of salt-alkali-tolerant and high-flavonoid seabuckthorn varieties includes the following steps:

[0026] S1: Site selection and land preparation

[0027] S11: Land requirements: Sandy soil with a pH value between 7.5-8.0 and a salt content of less than 0.5% is preferred. The terrain must be flat or a gentle slope with a slope not exceeding 5°. It must be away from pollution sources such as chemical plants and landfills. At the same time, ensure that the land is no more than 500 meters away from the water source and has convenient transportation conditions to meet production needs such as irrigation and transportation.

[0028] S12: Land preparation operation:

[0029] After clearing the plots of land, deep tillage to a depth of 22 centimeters was performed to enhance soil aeration and activity. Subsequently, 2.5 tons of decomposed farmyard manure was evenly applied per mu to enrich the soil with organic matter and nutrients, improving soil structure. Finally, planting holes with a diameter of 50 cm and a depth of 40 cm were dug at a designed density of 50 cm × 50 cm × 50 cm to provide ample growth space for the sea buckthorn seedlings and ensure root expansion and development.

[0030] S2: Seed treatment

[0031] S21: Screening: Through the dual screening process of wind selection and water selection, empty grains, impurities and inferior seeds are removed, and only high-quality seeds with full grains and sound development are retained.

[0032] S22: Disinfection: Soak the seeds in 0.2% potassium permanganate solution for 20 minutes. After sufficient disinfection, thoroughly wash away the residual solution with clean water.

[0033] S23: Cold plasma treatment: In the cold plasma treatment process, helium is used as the medium, and the seeds are precisely treated for 15-20 seconds in a vacuum-sealed environment with a power of 100-200W to activate the seed activity and improve its resistance to stress.

[0034] S24: Seed soaking and germination: Soak the seeds in a solution of 200g / kg PEG6000 aqueous solution and 35mmol / L sodium chloride aqueous solution in a 1:1 ratio. After soaking for 18 hours, rinse with distilled water three times to complete the osmotic adjustment and salt treatment of the seeds.

[0035] S3: Cutting propagation

[0036] S31: Hardwood Cuttings

[0037] S311: Cuttings should be collected during the winter dormancy period. Select one-year-old strong branches with a diameter of 0.5-1.5 cm and cut them into 15-20 cm long cuttings. Each cutting should retain 3-4 full buds.

[0038] S312: Cellar storage: Lay 10-15 cm thick wet sand (humidity maintained at 60%) on the bottom of the cellar, place the cuttings in layers in an orderly manner, and separate each layer of cuttings with wet sand. The temperature in the cellar is controlled at 0-5°C, with 2°C as the best storage temperature.

[0039] S313: Pre-cutting treatment: Before spring cutting, immerse the base of the cuttings in 0.02% ABT1 rooting powder solution for 2.5 hours to promote rooting of the cuttings.

[0040] S314: Cutting: Use sandy soil or sandy loam as the cutting medium, insert the cuttings vertically into the soil, the insertion depth is 2 / 3 of the cutting length, keep the plant spacing 10cm×20cm, and cover the soil with film after the cutting is completed to maintain soil moisture.

[0041] S32: Softwood Cuttings

[0042] S322: Cutting collection: In mid-June, select semi-lignified new shoots and cut them into 8-12 cm long cuttings, retaining 2-3 leaves on each cutting.

[0043] S323: Seedbed preparation: The seedbed needs to adopt a high bed structure (bed height 20cm, width 1m). First, lay a 15cm thick mixture of coarse river sand and forest soil on the bottom layer, then cover the upper layer with 5cm thick fine river sand, and finally use 0.5% potassium permanganate solution to fully disinfect the seedbed.

[0044] S324: Cutting treatment: During the cutting treatment, immerse the base of the cutting in 0.03% indolebutyric acid (IBA) or naphthaleneacetic acid (NAA) solution for 2 hours, and then insert it into the seedbed with an insertion depth of 2-3 cm, maintaining a plant spacing of 5 cm × 10 cm.

[0045] S325: Environmental Control: For environmental control, use an automatic intermittent spray device to maintain air humidity at 85%-95% and temperature at 20-28°C, with 23-25°C being optimal. Also, spray with a 0.2% carbendazim solution every seven days to create a suitable environment for cuttings and prevent diseases.

[0046] S4: Tissue Culture

[0047] S41: Explant selection: Select young stem segments or buds of salt- and alkali-tolerant sea buckthorn with high flavonoid content.

[0048] S42: Explant disinfection: The explant disinfection process is as follows: first, rinse with running water for 30 minutes to remove surface impurities; then immerse in 75% alcohol for 30-60 seconds for preliminary sterilization; then soak in 0.1% mercuric chloride solution for 5-10 minutes to enhance the disinfection effect; finally, rinse with sterile water 3-5 times to thoroughly remove residual liquid medicine.

[0049] S43: Initiation of culture: When initiating culture, the sterilized explants are inoculated into 1 / 3 MS medium containing 0.2 mg / L IBA, 30 g / L sucrose, and 7 g / L agar, and cultured under conditions of light intensity of 1100-1300 lx, photoperiod of 10-12 hours / day, and temperature of 26±1°C for 2-3 weeks until the stem segments sprout.

[0050] S44: Proliferation Culture: During the proliferation culture phase, germinated explants are transferred to MS medium containing 1.0-2.0 mg / L 6-BA, 0.1-0.3 mg / L NAA, 30 g / L sucrose, and 7 g / L agar. The culture environment should maintain a light intensity of 1500-1800 lx and a photoperiod of 14-16 hours per day. Transfers should be performed every 2-3 weeks to achieve a stable proliferation coefficient of 3-4.

[0051] S45: Rooting culture: The proliferated seedlings are transferred to 1 / 2 MS medium containing 0.5-1.0 mg / L IBA, 20 g / L sucrose, and 7 g / L agar. They are cultured under a light intensity of 1800-2000 lx and a photoperiod of 16 hours / day for 2-3 weeks to achieve a rooting rate of >90%.

[0052] S5: Transplantation and Acclimation

[0053] S51: Transplanting time: When the roots of tissue culture seedlings or cuttings grow to 3-5cm (4cm is better), transplant them into nutrient pots filled with mixed substrate, which is made of vermiculite, perlite and peat soil in a ratio of 1:1:1.

[0054] S52: Acclimation management: During the first 3-5 days after transplanting, the air humidity should be maintained at >90% (preferably 95%), the temperature should be controlled at 20-25°C (preferably 22-24°C), and plastic film should be covered to maintain moisture, creating a high-humidity and stable transition environment for the seedlings;

[0055] S53: Follow-up: 5-10 days after transplanting (5 days is the best interval), gradually ventilate and reduce air humidity while increasing light intensity. During this period, spray 0.2% potassium dihydrogen phosphate foliar fertilizer every 5-10 days to promote healthy growth of seedlings;

[0056] S54: Acclimation period: 2-3 months (preferably 2 months), transplanting into the field after the seedlings are strong.

[0057] S6: Saline-alkali land improvement and planting

[0058] S61 Soil improvement: Lay the following at the bottom of the planting trench:

[0059] Isolation layer (crushed stone / ceramsite) → aquifer (water-retaining agent / humus) → filtration layer (fine sand / perlite) → first planting soil layer (improved soil) → composite bacterial agent layer (salt-alkali tolerant microbial agent) → second planting soil layer (further improved soil) → organic fertilizer layer (decomposed farmyard manure), among which, isolation layer: paving crushed stone or ceramsite to block impurities in the bottom layer and enhance air permeability; aquifer: filling with water-retaining agent or humus to improve the soil's water retention and water supply balance capacity; filtration layer: covering with fine sand or perlite to filter water and optimize soil pore structure; first planting soil layer: filling with improved soil to lay the foundation for basic fertility and aggregate structure; composite bacterial agent layer: evenly spreading salt-alkali tolerant microbial agents to improve rhizosphere microecology; second planting soil layer: covering with further improved soil to enhance nutrients and stress resistance; organic fertilizer layer: spreading decomposed farmyard manure to provide long-term nutrition and enhance soil activity.

[0060] S62: Planting: Plant the seedlings in the improved soil at a depth that ensures the root collar is level with the ground. Immediately after planting, water thoroughly to allow the roots to establish. During the growing season, water and apply compound fertilizers in a timely manner based on soil moisture conditions and the growth needs of the seedlings to ensure healthy growth.

[0061] Typical cases:

[0062] Seabuckthorn cultivation has been successful in many areas, achieving both ecological and economic benefits. After years of hard work, the 170th Regiment of the 9th Division of the Xinjiang Production and Construction Corps has completed large-scale seabuckthorn forest planting, promoted hardwood cuttings, established cooperatives and companies, and expanded sales channels. Dagele Township in Golmud City, Qinghai Province, is encouraging residents to cultivate seabuckthorn, providing industrial support for rural revitalization. Wushi County in Aksu Prefecture is integrating seabuckthorn cultivation with sand control, building a comprehensive industrial chain and increasing farmers' incomes.

[0063] 1. The 170th Regiment of the 9th Division of the Xinjiang Production and Construction Corps: Located on the edge of the Gurbantunggut Desert, this regiment faces harsh natural conditions. In 2010, Wang Junyang was appointed Deputy Director of the Forestry Station, responsible for sea buckthorn seedling cultivation and planting. Through continuous experimentation, he and his colleagues selected the locally suitable "Deep Autumn Red" variety from 47 varieties, increasing the seedling survival rate from 30% to over 80%. After two years of hard work, they completed the planting of 14,000 mu (approximately 1,000 mu) of sea buckthorn forest, with a survival rate exceeding 90%. They also promoted the sea buckthorn hardwood cutting technique, producing 600,000 sea buckthorn seedlings annually. In 2018, Wang Junyang established the Jinfu Sea Buckthorn Planting Professional Cooperative in Emin County. Using a "enterprise + cooperative + grower" model, this cooperative helped the company sell 846 tons of sea buckthorn fruit, generating sales of 5.4 million yuan. In 2019, Wang Junyang founded Silk Road Seabuckthorn Biotechnology Co., Ltd. The company has sold more than 14,000 tons of seabuckthorn fruit so far. It is one of the largest seabuckthorn raw material suppliers in China and has launched 24 seabuckthorn product series in 6 categories.

[0064] 2. Dagele Township, Golmud City, Qinghai Province: Since 2019, in addition to growing goji berries, some residents have been encouraged to cultivate sea buckthorn. Wang Lieyong, a farmer in Chana Village, Dagele Township, has planted over 20 mu (approximately 16 acres) of sea buckthorn. Since the fruit began to set, yields have increased annually, reaching 4,000 kilograms (approximately 16,000 jin) per mu. Dried fruit fetches 23 yuan per kilogram, while fresh fruit fetches 4,000 yuan per ton. Liang Deng, a farmer in Longyang Village, converted 30 mu (approximately 16,000 jin) of goji berry field to sea buckthorn. This year, he experienced a bumper harvest, with five people able to harvest the fruit in just ten days. Liang Deng believes that sea buckthorn is easy to plant and harvest, and offers a good return. The total planting area in the township has now reached over 300 mu (approximately 16,000 jin) of sea buckthorn, making it a popular choice among farmers and providing industrial support for rural revitalization.

[0065] Wushi County, Aksu Prefecture: Since 2016, seabuckthorn has been designated a core species for ecological governance and incorporated into the county's land and space planning, creating an innovative "ecological bank" mechanism. Aktohai Town, once a site of severe sandstorms, saw staff from the Forestry and Grassland Bureau planted seabuckthorn in 2019. By establishing micro-reservoirs, deploying a percolation irrigation network, and implementing misting and moisturizing techniques, they increased the survival rate from less than 30% to 85%. Today, Wushi County boasts over 160,000 mu (approximately 160,000 mu) of seabuckthorn cultivation, with expansion into Qiemo County. Local companies, such as Xinjiang Daer Biotechnology Co., Ltd., have mastered key technologies such as pulp separation and low-temperature extraction, developing 32 products across seven categories. Their seabuckthorn oil boasts a purity of 99.7% and sells for over 2,000 yuan per kilogram. Wushi County has established a "zero-waste" seabuckthorn production system, achieving a resource utilization rate of 98%. By 2024, the seabuckthorn industry is expected to generate over 10,000 seasonal jobs and over 3,000 permanent positions. The county is also exploring seabuckthorn ecotourism projects.

[0066] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A rapid breeding process for salt-alkali-tolerant and high-flavonoid seabuckthorn varieties, characterized in that: The following steps are involved: S1: After selecting a suitable plot, clear away debris, deep-plow the soil to 22 cm, evenly apply 2.5 tons of decomposed farmyard manure per mu, and dig planting holes with a diameter of 50 cm and a depth of 40 cm according to the designed density; S2: The seeds are air-sorted and water-sorted, and then soaked in a 0.2% potassium permanganate solution for 20 minutes. After being washed with clean water and treated with cold plasma, the low-temperature plasma interacts with the surface and interior of the seeds, significantly improving their performance without damaging the seeds. The seeds are then soaked in a mixture of an aqueous solution and a sodium chloride aqueous solution for 18 hours, and finally rinsed with distilled water three times. S3: Collect cuttings in winter and store them in a cellar with sand humidity of 60% and cellar temperature of 2°C. Before spring cuttings, soak the base of the cuttings in 0.02% ABT1 rooting powder for 2.5 hours. This can regulate plant endogenous hormones, promote cell division and differentiation, and enhance metabolic activity. After cuttings, cover with mulch to keep them moist. Collect cuttings in mid-June, treat them, and insert them into the seedbed. Maintain air humidity of 90% and temperature of 23-25°C. Spray with carbendazim solution. S4: Select explants and sterilize them. Culture them in the priming medium for 2-3 weeks. When the stem segments sprout, transfer them to the proliferation medium and subculture them every 2-3 weeks until the proliferation coefficient reaches 3-4. Transfer them to the rooting medium and culture them for 2-3 weeks. The rooting rate exceeds 90%. S5: Transplant when the roots of the seedlings grow to about 4 cm. Maintain the air humidity at 95% and the temperature at 22-24°C for 3 days before transplanting. Then gradually ventilate and reduce humidity. Apply 0.2% potassium dihydrogen phosphate foliar fertilizer every 5 days. Transplant into the field after acclimatization for 2 months. S6: Lay planting trenches in layers, plant the seedlings in improved soil, water them promptly after planting, and subsequently manage water and fertilizer appropriately based on their growth conditions.

2. The rapid breeding process for salt-alkali-tolerant and high-flavonoid seabuckthorn varieties according to claim 1, characterized in that: The soil pH value in the plot is 7.5-8.0, and the salt content is less than 0.5%.

3. The rapid breeding process for salt-alkali-tolerant and high-flavonoid seabuckthorn varieties according to claim 1, characterized in that: The design density is 50 cm × 50 cm × 50 cm.

4. The rapid breeding process for salt-alkali-tolerant and high-flavonoid seabuckthorn varieties according to claim 1, characterized in that: The aqueous solution used was a PEG6000 aqueous solution, and the concentration was 200 g / kg.

5. The rapid breeding process of salt-alkali-tolerant and high-flavonoid seabuckthorn varieties according to claim 1, characterized in that: Spray 0.2% carbendazim solution every 7 days.

6. The rapid breeding process for salt-alkali-tolerant and high-flavonoid seabuckthorn varieties according to claim 1, characterized in that: Select young stem segments or buds of salt- and alkali-tolerant high-flavonoid sea buckthorn as explants.

7. The rapid breeding process for salt-alkali-tolerant and high-flavonoid seabuckthorn varieties according to claim 1, characterized in that: The bottom of the planting ditch is laid in sequence with an isolation layer, a water storage layer, a filtration layer, a first planting soil layer, a compound bacterial agent layer, a second planting soil layer and an organic fertilizer layer.