Efficient rapid propagation method for blueberry mycorrhizal seedlings

Abstract

The invention provides an efficient rapid propagation method for blueberry mycorrhizal seedlings, and relates to the technical field of blueberry mycorrhizal seedling propagation. The efficient rapid propagation method of the blueberry mycorrhizal seedlings comprises the following specific steps: S1, selection of explants, S2, disinfection of the explants, S3, bud induction of the explants, S4, subculture multiplication culture, and S5, rooting of tissue culture seedlings outside a bottle and inoculation of blueberry mycorrhizal fungi. In the disinfection process, when the explant is soaked in 70% ethyl alcohol for disinfection for 3 min and soaked in 0.1% mercury bichloride for disinfection for 7 min, the pollution rate of the Lackxi explant is low, the survival rate is high, in the explant bud induction process, when the concentration of zeatin hormone (ZT) is 1.5 mg / L, the bud induction rate, the proliferation multiple and the bud growth vigor reach the best, and the survival rate is high. In the subculture multiplication culture process, when the concentration of zeatin hormone (ZT) is 0.6 mg / L, the Lackxi plant has many clumpy buds, and the plant is high and robust and grows fast.

Description

Technical Field

[0001] This invention relates to the field of blueberry mycorrhizal seedling propagation technology, specifically to a highly efficient and rapid propagation method for blueberry mycorrhizal seedlings. Background Technology

[0002] Blueberries, scientifically known as Vaccinium bracteatum, are perennial deciduous fruit trees, growing as shrubs. The berries are blue, nearly round, with delicate flesh, tiny seeds, a balanced sweet and sour taste, and a pleasant aroma. Blueberries are highly nutritious, rich in superoxide dismutase (SOD), an enzyme that eliminates excess free radicals in the body. They not only offer excellent nutritional and health benefits but also help prevent brain aging, strengthen the heart, fight cancer, soften blood vessels, and enhance the immune system. Blueberries possess both high nutritional and economic value, making them one of the fastest-growing third-generation fruit tree varieties worldwide in recent years, combining nutritional and health benefits. Furthermore, the introduction of mycorrhizae during blueberry cultivation promotes the absorption of nutrients, especially nitrogen and phosphorus, and also enhances plant growth, fruit yield, and quality.

[0003] However, during the culture process, the explants have a high contamination rate and low survival rate. In addition, the bud induction rate, proliferation multiple and bud growth are not very good during the explant bud induction process. During the subculture and proliferation culture, the plants grow slowly and are thin and weak. Summary of the Invention

[0004] (a) Technical problems to be solved To address the shortcomings of existing technologies, this invention provides a highly efficient and rapid propagation method for blueberry mycorrhizal seedlings, solving the problems mentioned in the background section.

[0005] (II) Technical Solution To achieve the above objectives, the present invention provides the following technical solution: a highly efficient and rapid propagation method for blueberry mycorrhizal seedlings, comprising the following specific steps: S1. Selection of explants: Collect tender shoots of one-year-old Lexis with unsprouted axillary buds as explants. Remove the leaves, leaving a few petioles. After removing the terminal bud, cut the explants into stem segments with one unsprouted axillary bud and a length of 1.5-3cm. S2. Explant disinfection: First, soak the explants in detergent solution, then rinse them with running water until there is no foam. Rinse them three times with sterile water and transfer them to a clean bench. Soak them in 70% ethanol. After soaking, rinse them five times with sterile water. Then, soak them in 0.1% mercuric chloride solution for disinfection. After disinfection, rinse them five times with sterile water before inoculation. S3. Explant bud induction: WPM medium was used as the basic medium, and zeatin hormone was added to the medium. After sterilization, the explants were inoculated into the medium for induction culture. S4. Subculture proliferation: WPM medium was used as the basic medium, and zeatin hormone was added to the medium. The induced axillary buds were then inoculated into the medium for proliferation culture to obtain tissue culture seedlings. S5. Inoculation of blueberry mycorrhizal fungi for exogenous rooting of tissue culture seedlings: Select high-quality and robust tissue culture seedlings, and after hardening, transplant them into nutrient pots containing sterilized perlite, peat moss, and vermiculite as the culture substrate for mycorrhizal seedlings. Cover the substrate with sterilized moss, and then root the seedlings under the conditions of rapid dipping and soaking in IBA at a concentration of 1000 mg / L. Then, evenly irrigate each seedling with 10 ml of the target mycorrhizal fungus C1-GP.

[0006] Preferably, in step S2, the soaking time is 10 minutes with laundry detergent, 2-4 minutes with 70% ethanol, and 6-8 minutes with 0.1% mercuric chloride.

[0007] Preferably, the concentration of zeatin hormone in S3 is 0.5-2.0 mg / L, and the induced budding of explants is observed and counted after 15 days of culture, and the count is completed after 60 days.

[0008] Preferably, the concentration of zeatin hormone in S4 is 0.2-1.0 mg / L, and the growth of axillary buds is observed and counted after 15 days of culture, and the count ends after 60 days.

[0009] Preferably, the mass ratio of perlite: peat moss: vermiculite in S5 is 1:1:1.

[0010] Preferably, in step S5, the IBA concentration is 1000 mg / L, the soaking time is 1-5 min, and the target bacterial strain C1-GP is poured in during the 0-14 day of cultivation.

[0011] (III) Beneficial Effects This invention provides a highly efficient and rapid propagation method for blueberry mycorrhizal seedlings. It has the following beneficial effects: 1. According to the present invention, when explants are disinfected by soaking in 70% ethanol for 3 minutes and 0.1% mercuric chloride for 7 minutes during the disinfection process, the contamination rate of Lexi explants is low and the survival rate is high.

[0012] 2. In this invention, the bud induction rate, proliferation rate, and bud growth are optimal when using zeatin hormone (ZT) at a concentration of 1.5 mg / L during the explant bud induction process.

[0013] 3. In this invention, when using zeatin hormone (ZT) at a concentration of 0.6 mg / L during subculture, Lexi plants have more shoots, are taller and stronger, and grow faster. Detailed Implementation

[0014] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0015] Example: This invention provides a highly efficient and rapid propagation method for blueberry mycorrhizal seedlings, including the following specific steps: S1, selection of explants: collect tender shoots of one-year-old Lexis with unsprouted axillary buds as explants, cut off the leaves, leave a few petioles, remove the terminal bud, and cut the explants into stem segments with one unsprouted axillary bud and a length of 1.5-3cm. S2. Explant disinfection: Soak in detergent solution for 10 minutes, then rinse with running water until no foam remains. Rinse three times with sterile water and transfer to a clean bench. Soak in 70% ethanol for 2 minutes, 3 minutes, and 4 minutes, then rinse five times with sterile water. Disinfect by soaking in 0.1% mercuric chloride for 6 minutes, 7 minutes, and 8 minutes, then rinse five times with sterile water. Inoculate with two stem segments per bottle, for a total of 50 bottles. After 7 days of culture, observe and record the contamination rate and survival rate of explants under each treatment daily. S3. Explant bud induction: Using WPM medium as the basal medium, two hormones, zeatin (ZT) and 6-benzylaminopurine (6-BA), were selected for comparative treatment. ZT concentrations were set at 0.5 mg / L, 1.0 mg / L, 1.5 mg / L, and 2.0 mg / L, while 6-BA concentrations were set at 0.5 mg / L, 1.0 mg / L, 1.5 mg / L, and 2.0 mg / L. Sterilized explants were inoculated into different treatments, with 10 stem segments inoculated per bottle of medium, and 10 bottles inoculated per treatment. Bud induction was observed and statistically analyzed after 15 days of culture for each treatment, and statistical analysis was completed after 60 days. S4. Subculture and Proliferation: Using WPM medium as the basal medium, two hormones, zeatin (ZT) and 6-benzylaminopurine (6-BA), were selected for comparative treatment. The concentrations of ZT were set at 0.2 mg / L, 0.4 mg / L, 0.6 mg / L, 0.8 mg / L, and 1.0 mg / L, and the concentrations of 6-BA were set at 0.2 mg / L, 0.4 mg / L, 0.6 mg / L, 0.8 mg / L, and 1.0 mg / L. The induced axillary buds were inoculated into different treatments, with 10 axillary buds inoculated into each bottle of medium, and 10 bottles inoculated into each treatment. After 15 days of culture, the growth of axillary buds under each treatment was observed and statistically analyzed, and the statistical analysis was completed after 60 days. S5. Inoculation of blueberry mycorrhizal fungi into the exogenous rooting of tissue culture seedlings: High-quality and robust tissue culture seedlings were selected and, after hardening, transplanted into nutrient pots containing a sterilized perlite: peat moss: vermiculite culture substrate in a mass ratio of 1:1:1. One group of substrates was covered with sterilized moss, while the other group was not covered with moss. The rooting of the tissue culture seedlings was compared under the conditions of IBA concentration of 1000 mg / L and quick dipping and soaking for 1 min, 3 min, and 5 min. At 0 days, 7 days, and 14 days after transplanting, each seedling was evenly watered with 10 ml of the target mycorrhizal fungus C1-GP. Another group was not watered as a control. After cultivation, the inoculation time and infection rate of blueberry mycorrhizal fungi in seedlings in different culture substrates after watering were compared, as well as the growth of seedlings without watering and those with watering.

[0016] Experimental Results and Analysis Table 1. Effects of the same disinfection method on Lexi explants. According to the results in Table 1, when explants were disinfected with 70% ethanol and 0.1% mercuric chloride, the contamination rate of the explants decreased significantly with the increase of disinfection time. However, after the disinfection time was too long, the survival rate and budding rate of the explants also decreased significantly. Considering the contamination rate, budding rate and growth of the explants, the contamination rate of Lexi explants was lower and the survival rate was higher when they were disinfected by soaking in 70% ethanol for 3 minutes and 0.1% mercuric chloride for 7 minutes. This is considered to be the most suitable method for disinfecting Lexi explants.

[0017] Table 2 Effects of different hormones and concentrations on shoot induction in Lexi explants Table 2 shows that ZT and 6-BA had significantly different effects on the induction and proliferation of axillary buds in Lexicamibora lexicamii. With increasing 6-BA concentration, the bud proliferation rate and axillary bud induction rate increased, and the number and growth of induced buds improved, with the best induction effect observed at a 6-BA concentration of 2 mg / L. Conversely, as the ZT concentration increased from 0.5 mg / L to 2 mg / L, the bud induction and proliferation rate initially increased significantly before slightly decreasing, with the same trend observed in the number and proliferation rate of induced buds. The optimal bud induction rate, proliferation rate, and bud growth were achieved at a ZT concentration of 1.5 mg / L. Comparing the induction effects of the two hormones revealed that 6-BA significantly reduced the induction effect on Lexicamibora lexicamii explants compared to ZT.

[0018] Table 3. Effects of different hormones and concentrations on the proliferation of Lexigenous offspring. Table 3 shows that both ZT and 6-BA had some effect on the subculture proliferation of Lexicam, but the effects differed significantly. As the ZT concentration increased from 0.2 mg / L to 1 mg / L, the average height of the Lexicam plants gradually increased. When the concentration exceeded 0.6 mg / L, the plants formed numerous clusters of buds, plant growth slowed, and the plants became thinner with smaller leaves. With increasing 6-BA concentration, the behavior of the Lexicam plants was largely consistent with that under ZT-induced growth. At a 6-BA concentration of 0.4 mg / L, the average height of the Lexicam plants reached its peak, and the plants exhibited the best growth. As the concentration increased further, numerous clusters of buds formed, growth slowed, and the plants became thin and weak. Comparing the proliferation effects of the two hormones, the effect of 6-BA on the subculture proliferation of Lexicam was significantly lower than that of ZT.

[0019] Table 4. Results of exogenous rooting and inoculation with blueberry mycorrhizal fungi in tissue culture seedling bottles. Table 4 shows that when seedlings were dipped in a 1000 mg / L solution for 1 minute and then transplanted into a substrate of perlite: peat moss: vermiculite = 1:1:1, covered with a small amount of moss, and irrigated with 10 ml of the target fungal inoculum 7 days after transplanting, the blueberry seedlings exhibited the highest rooting rate, infection rate, and best growth. Seedlings inoculated with the target blueberry mycorrhizal fungus showed better growth than uninoculated seedlings, with inoculated seedlings covered with moss showing superior growth compared to uninoculated seedlings.

[0020] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A highly efficient and rapid propagation method for blueberry mycorrhizal seedlings, characterized in that: The specific steps include the following: S1. Selection of explants: Collect tender shoots of one-year-old Lexis with unsprouted axillary buds as explants. Remove the leaves, leaving a few petioles. After removing the terminal bud, cut the explants into stem segments with one unsprouted axillary bud and a length of 1.5-3cm. S2. Explant disinfection: First, soak the explants in detergent solution, then rinse them with running water until there is no foam. Rinse them three times with sterile water and transfer them to a clean bench. Soak them in 70% ethanol. After soaking, rinse them five times with sterile water. Then, soak them in 0.1% mercuric chloride solution for disinfection. After disinfection, rinse them five times with sterile water before inoculation. S3. Explant bud induction: WPM medium was used as the basic medium, and zeatin hormone was added to the medium. After sterilization, the explants were inoculated into the medium for induction culture. S4. Subculture proliferation: WPM medium was used as the basic medium, and zeatin hormone was added to the medium. The induced axillary buds were then inoculated into the medium for proliferation culture to obtain tissue culture seedlings. S5. Inoculation of blueberry mycorrhizal fungi for exogenous rooting of tissue culture seedlings: Select high-quality and robust tissue culture seedlings, and after hardening, transplant them into nutrient pots containing sterilized perlite, peat moss, and vermiculite as the culture substrate for mycorrhizal seedlings. Cover the substrate with sterilized moss, and then root the seedlings under the conditions of rapid dipping and soaking in IBA at a concentration of 1000 mg / L. Then, evenly irrigate each seedling with 10 ml of the target mycorrhizal fungus C1-GP.

2. The efficient and rapid propagation method for blueberry mycorrhizal seedlings according to claim 1, characterized in that: The S2 solution uses laundry detergent for 10 minutes, 70% ethanol for 2-4 minutes, and 0.1% mercuric chloride for 6-8 minutes.

3. The efficient and rapid propagation method for blueberry mycorrhizal seedlings according to claim 1, characterized in that: The concentration of zeatin hormone in S3 was 0.5-2.0 mg / L, and the induced shoot emergence of explants was observed and counted after 15 days of culture, and the count was completed after 60 days.

4. The efficient and rapid propagation method for blueberry mycorrhizal seedlings according to claim 1, characterized in that: The concentration of zeatin hormone in S4 was 0.2-1.0 mg / L, and the growth of axillary buds was observed and counted after 15 days of culture, and the count was completed after 60 days.

5. The efficient and rapid propagation method for blueberry mycorrhizal seedlings according to claim 1, characterized in that: The mass ratio of perlite, peat moss, and vermiculite in S5 is 1:1:

1.

6. The efficient and rapid propagation method for blueberry mycorrhizal seedlings according to claim 1, characterized in that: The IBA concentration in S5 is 1000 mg / L, the soaking time is 1-5 min, and the target strain C1-GP is poured in during the 0-14 days of cultivation.

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