Tissue culture, rapid propagation and rooting culture method for blueberry yunnanensis

Abstract

The invention discloses a tissue culture, rapid propagation and rooting culture method for blueberry yunnanensis and application of the tissue culture, rapid propagation and rooting culture method. The culture method disclosed by the invention comprises the following steps of: cleaning and disinfecting an explant of a stem segment of the blueberry yunnanensis, carrying out primary callus culture, carrying out primary callus induction culture, carrying out subculture differentiation culture, carrying out rooting culture and transplanting rooted seedlings. Through disinfection with 15% sodium hypochlorite, the contamination rate can be 0, the germination rate in the callus primary culture process reaches 88.89%, the browning rate in primary callus induction culture is only 13.89%, the browning rate is reduced by more than 30%, the callus yield reaches 96.77%, the callus yield is improved by more than 10%, the callus buds and differentiates in the callus subculture, buds with good growth vigor are formed, and the germination rate of the callus is increased by more than 10%. The rooting rate in rooting culture reaches 80%, and the survival rate after transplanting reaches 83%.

Description

Technical Field

[0001] The invention belongs to the technical field of plant tissue culture, and particularly relates to a tissue culture, rapid propagation and rooting culture method for skylark blueberry. Background Art

[0002] Blueberries (Vaccinium spp.) are small, deciduous berry trees or shrubs belonging to the genus Vaccinium in the Ericaceae family. Rich in anthocyanins, blueberries are highly nutritious and have health benefits, including boosting immunity, providing antioxidants, inhibiting bacteria, and softening blood vessels. They are particularly effective in preventing cellular aging, earning them the nickname "a new generation of functional fruit."

[0003] Currently, there are three main types of blueberry varieties cultivated both domestically and internationally: the lowbush blueberry (Vaccinium angustifolium) is highly cold-resistant (tolerating extreme temperatures of -40°C) and is suited to sandy soils in cold zones; the highbush blueberry (Vaccinium corymbsum) is divided into three subspecies: northern highbush (requiring 800-1200 hours of chilling), southern highbush (requiring 150-600 hours of chilling), and semi-highbush (requiring 400-800 hours of chilling); and the rabbiteye blueberry (Vaccinium ashei) is highly drought-tolerant and suited to acidic reddish-yellow soils with a pH of 4.5-5.5. The southern highbush blueberry variety "Skylark," developed at the University of Florida in 2010, is an upright southern highbush blueberry with a sweet and sour taste. It is a medium-early maturing variety characterized by high yields and good storage properties. Its fruits are large and firm, medium blue in color, with small and dry stems, good flavor, and a crisp and sweet taste. Market demand continues to grow, but its traditional breeding efficiency is low, which restricts its large-scale application.

[0004] This study used the southern highbush blueberry "Skylark" as the material, and constructed an efficient tissue culture and rapid propagation system by optimizing disinfection, hormone regulation and rooting technology to achieve seedling breeding. Compared with traditional seedling cultivation methods, in vitro culture can significantly shorten the breeding cycle, and the single-batch seedling proliferation efficiency is increased several times; this technology can completely inherit the maternal genotype, ensuring the stable inheritance of economic traits such as fruit quality and stress resistance; breaking through the natural climate limitations, achieving year-round continuous production under artificial control environment, providing stable seedling guarantees for large-scale planting, and promoting the development of the blueberry industry towards intensive and standardized directions. Summary of the Invention

[0005] The purpose of the present invention is to propose a tissue culture, rapid propagation and rooting culture method for skylark blueberry. By optimizing disinfection, hormone regulation and rooting technology, an efficient tissue culture and rapid propagation system is constructed to achieve seedling breeding. Compared with traditional seedling breeding methods, in vitro culture can significantly shorten the breeding cycle and increase the single batch seedling proliferation efficiency several times.

[0006] The technical solution of the present invention is a method for tissue culture, rapid propagation and rooting of blueberry, which is carried out according to the following steps:

[0007] (1) Explant collection: In the spring growing season, select sunny weather with an air humidity of ≤45% and collect new shoots from healthy, disease-free Lark Blueberry plants in the morning. These shoots are quickly placed in a fresh-keeping bag and placed in an ice pack to keep them fresh. Grade A is obtained and set aside.

[0008] (2) Explant pretreatment: Take product A, remove the 1.8-2.2 cm tender part at the top of the shoot and the lignified part at the bottom, retain the middle 5-12 cm length, cut off the leaves, add detergent and purified water to the shoot, scrub, and soak for 8-12 minutes, rinse with purified water, and hang to wash under clean water for 1-3 hours to obtain product B;

[0009] (3) Explant disinfection: After ventilation and UV sterilization in a clean bench, place product B on the clean bench and disinfect it with 75% alcohol for 20-40 seconds, then rinse it with sterile water 1-3 times, and then disinfect it with 15% sodium hypochlorite for 10-25 minutes. After disinfection, rinse it with sterile water 3-6 times, and use filter paper to absorb the moisture on the surface of the material to obtain callus tissue, namely product C, for later use;

[0010] (4) Primary callus culture: Take sample C and inoculate it into primary callus culture medium and culture it for 25-35 days to obtain sample D;

[0011] The primary callus culture medium is WPM+25-35g / L sucrose+6-8g / L agar powder+0.5-1.5mg / L ZT, and the pH is adjusted to 5.2-5.4 using 1mol / L NaOH and 1mol / L HCl;

[0012] The culture conditions are as follows: temperature of 17-25°C, light intensity of 2000-3500 lx, and light duration of 10-14 h / d;

[0013] (5) Primary callus induction culture: Take product D and inoculate it into the primary callus induction medium and culture it for 30-40 days to obtain product E;

[0014] The primary callus induction medium is WPM+0.5-1.5 mg / L TDZ+0.2-0.4 mg / L NAA+150-250 mg / L VC, and the pH is adjusted to 5.2-5.4 using 1 mol / L NaOH and 1 mol / L HCl;

[0015] The culture conditions are as follows: temperature of 17-25°C, light intensity of 2000-3500 lx, and light duration of 10-14 h / d;

[0016] (6) Subculture differentiation culture: Take product E and inoculate it into subculture differentiation medium and culture it for 30-40 days to obtain differentiated buds;

[0017] The subculture differentiation medium is 1 / 2 MS + 0.4-0.6 mg / L 6-BA, and the pH is adjusted to 5.2-5.4 using 1 mol / L NaOH and 1 mol / L HCl;

[0018] The culture conditions are: temperature 17-25°C, light intensity 2000-3500lx, photoperiod 10-14h / d;

[0019] (7) Rooting culture: The differentiated buds were inoculated into a rooting medium and cultured for 85-95 days to obtain rooted seedlings; the rooting medium consisted of 1 / 2 MS + 0.4-0.6 mg / L 6-BA, and the pH was adjusted to 5.2-5.4 using 1 mol / L NaOH and 1 mol / L HCl;

[0020] The culture conditions are as follows: temperature of 17-25°C, light intensity of 2000-3500 lx, and light duration of 10-14 h / d;

[0021] (8) Transplantation of rooted seedlings: The rooted seedlings were acclimated using the hydroponic transition method. The rooted seedlings with the culture medium were completely immersed in sterile water at 20-30°C. The agar matrix was gradually separated by water oscillation, and the water surface was kept submerged in the roots. A soft brush was used to gently scrub along the growth direction of the root tip to clean the root primordium attached to the culture medium. Then, 0.8-1.2% rooting powder was used to soak for 0.5-1.5 minutes and the seedlings were transplanted into a ternary composite matrix, which was peat soil, gravel and perlite in a volume ratio of 2. -4:0.5-1.5:0.5-1.5 are mixed together. Immediately after transplanting, use a fine-hole nozzle to irrigate the rooting water three times. The EC value of the rooting water is ≤0.5mS / cm. When the surface 1.5-2.5cm matrix is dry, water is added to maintain the matrix humidity at 55-75%. Shade treatment is carried out within 6-8 days after transplanting, and the shading rate is 65-75%. After 6-8 days, the light intensity is gradually increased as the plant grows. The light intensity is controlled in the range of 6000-14000lux during the seedling growth period.

[0022] In the aforementioned step (1), explant harvesting: During the spring growing season, select sunny weather with an air humidity of ≤45%, and between 9:00 and 11:00 in the morning, collect new shoots from healthy, disease-free skylark blueberry plants. These shoots are quickly placed in a fresh-keeping bag and placed in an ice pack to keep them fresh, thereby obtaining product A for future use.

[0023] In the aforementioned step (2), the explants are pretreated by removing the tender 1.9-2.1 cm part at the top of the shoot and the lignified part at the bottom of the shoot, leaving the middle 6-10 cm part, cutting off the leaves, scrubbing the shoot with detergent and purified water, and soaking for 10 minutes. After rinsing with purified water, the shoot is hung under running water for 1-2 hours to obtain the shoot B.

[0024] In the above step (3), explant disinfection: after ventilation and ultraviolet sterilization in a clean bench, product B is placed in the clean bench and disinfected with 75% alcohol for 30 seconds, then rinsed twice with sterile water, and then disinfected with 15% sodium hypochlorite for 15 minutes. After disinfection, rinse with sterile water 4-5 times, and use filter paper to absorb the moisture on the surface of the material to obtain callus tissue, i.e. product C, for later use.

[0025] In the above step (4), the primary callus culture: take product C and inoculate it into the primary callus culture medium, culture it for 30 days, and obtain product D;

[0026] The primary callus culture medium is WPM+30g / L sucrose+7g / L agar powder+1mg / L ZT, and the pH is adjusted to 5.2-5.4 using 1mol / L NaOH and 1mol / L HCl;

[0027] The culture conditions are: temperature of 19-23° C., light intensity of 2500-3000 lx, and light duration of 12 h / d.

[0028] In the aforementioned step (5), the primary callus induction culture is as follows: the D product is inoculated into the primary callus induction medium and cultured for 35 days to obtain the E product;

[0029] The primary callus induction medium is WPM+1 mg / L TDZ+0.3 mg / L NAA+200 mg / L VC, and the pH is adjusted to 5.2-5.4 using 1 mol / L NaOH and 1 mol / L HCl;

[0030] The culture conditions are: temperature of 19-23° C., light intensity of 2500-3000 lx, and light duration of 12 h / d.

[0031] In the aforementioned step (6), subculture differentiation culture: take product E and inoculate it into subculture differentiation medium, culture it for 35 days, and obtain differentiated buds;

[0032] The subculture differentiation medium is 1 / 2 MS + 0.5 mg / L 6-BA, and the pH is adjusted to 5.2-5.4 using 1 mol / L NaOH and 1 mol / L HCl;

[0033] The culture conditions are: temperature 19-23℃, light intensity 2500-3000lx, and light time 12h / d.

[0034] In the aforementioned step (7), rooting culture: taking differentiated buds and inoculating them in rooting medium and culturing them for 90 days to obtain rooted seedlings;

[0035] The rooting medium is 1 / 2 MS + 0.5 mg / L 6-BA, and the pH is adjusted to 5.2-5.4 using 1 mol / L NaOH and 1 mol / L HCl;

[0036] The culture conditions are: temperature of 19-23° C., light intensity of 2500-3000 lx, and light duration of 12 h / d.

[0037] In the above step (8), the rooted seedlings are transplanted: the rooted seedlings are acclimated by the hydroponic transition method, the rooted seedlings with the culture medium are completely immersed in 22-28 ° C sterile water, the agar matrix is gradually separated by water oscillation, the water surface is kept submerged in the root system, and the root primordium is gently scrubbed along the growth direction of the root tip with a soft brush to clean the culture medium; then 1% rooting powder is used to dip for 0.7-1.2 minutes, and the root primordium is transplanted into a ternary composite matrix, wherein the ternary composite matrix is peat soil, gravel and Perlite is mixed in a volume ratio of 2-4:1:1. Immediately after transplanting, use a fine-hole sprinkler to water the roots three times. The EC value of the rooting water is ≤0.5mS / cm. When the surface 1.8-2.2cm matrix is dry, water is added to maintain the matrix humidity at 68-72%. Shade treatment is carried out within 7 days after transplanting, and the shading rate is 60-70%. After 7 days, the light intensity is gradually increased as the plant grows. The light intensity is controlled in the range of 7000-13000 lux during the seedling growth period.

[0038] Specifically, in the aforementioned step (8), the rooted seedlings are transplanted: the rooted seedlings are acclimated using the hydroponic transition method, the rooted seedlings with the culture medium are completely immersed in 25°C sterile water, the agar matrix is gradually separated by water oscillation, the water surface is kept submerged in the root system, and a soft brush is used to gently scrub along the root tip growth direction to clean the root primordium attachment culture medium; then 1% rooting powder is used to dip for 1 minute, and the root is transplanted into a ternary composite matrix, the ternary composite matrix is a mixture of peat soil, gravel and perlite in a volume ratio of 3:1:1, and immediately after transplanting, a fine-hole nozzle is used to water the rooting water three times, the EC value of the rooting water is ≤0.5mS / cm, and when the surface 2cm of the matrix is dry, water is added to maintain the matrix humidity at 60-70%, and shade treatment is performed within 7 days after transplanting, with a shading rate of 70%. After 7 days, the light intensity is gradually increased as the plant grows, and the light intensity is controlled in the range of 8000-12000lux during the seedling growth period.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] 1. The culture method of the present invention comprises cleaning, disinfecting, primary callus culture, primary callus induction culture, subculture differentiation culture, rooting culture, and transplanting rooted seedlings of blueberry stem explants. The contamination rate can be reduced to zero by disinfecting with 15% sodium hypochlorite.

[0041] 2. The germination rate during the primary callus culture process reached 88.89%.

[0042] 3. In the primary callus induction culture, the browning rate was only 13.89%, a decrease of more than 30%, and the callus induction rate reached 96.77%, an increase of more than 10%.

[0043] 4. In the subculture of callus tissue, the callus tissue sprouted and differentiated, forming young shoots with good growth. The rooting rate in rooting culture reached 80%, and the survival rate after transplantation reached 83%. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 : Effects of different air humidity on explants at harvest (A: 15% sodium hypochlorite treatment for 5 min at an air humidity of 90%; B: 15% sodium hypochlorite treatment for 10 min at an air humidity of 90%; C: 15% sodium hypochlorite treatment for 5 min at an air humidity of 45%; D: 15% sodium hypochlorite treatment for 10 min at an air humidity of 45%);

[0045] Figure 2 : Effects of different disinfection concentrations on explants (A: 5% sodium hypochlorite treatment for 10 min; B: 10% sodium hypochlorite treatment for 10 min; C: 15% sodium hypochlorite treatment for 10 min; D: 20% sodium hypochlorite treatment for 10 min);

[0046] Figure 3 : Effects of different disinfection times on explants (A: 15% sodium hypochlorite treatment for 5 min; B: 15% sodium hypochlorite treatment for 10 min; C: 15% sodium hypochlorite treatment for 15 min; D: 15% sodium hypochlorite treatment for 20 min);

[0047] Figure 4 : Schematic diagram of plant tissue manipulation;

[0048] Figure 5 :The effects of different tissue site selection on the germination rate of experimental seedlings;

[0049] Figure 6 :Effects of different plant hormones on the germination rate of experimental seedlings (A is 1 mg / L ZT; B is 1 mg / L 6-BA; C is 1 mg / L 2,4-D);

[0050] Figure 7: Effects of different concentrations of the same hormone on the germination rate of test seedlings (A is the control group without hormones; B is 1 mg / L ZT; C is 2 mg / L ZT);

[0051] Figure 8 :Effects of different plant hormones on the callus rate of test seedling leaves (A: 2 mg / L ZT; B: 4 mg / L ZT; C: 1 mg / L TDZ + 0.1 mg / L NAA; D: 1 mg / L TDZ + 0.2 mg / L NAA; E: 1 mg / L TDZ + 0.5 mg / L NAA; F: 1 mg / L TDZ + 0.8 mg / L NAA);

[0052] Figure 9 : Effects of different plant hormones on the callus rate of test seedling stem segments (A: 1 mg / L TDZ + 0.1 mg / L NAA; B: 1 mg / L TDZ + 0.3 mg / L NAA; C: 1 mg / L TDZ + 0.5 mg / L NAA; D: 1 mg / L TDZ + 0.8 mg / L NAA);

[0053] Figure 10 : Effects of adding subsequent hormone 2,4-D on callus tissue (A: 1 mg / L TDZ + 0.2 mg / L NAA; B: 1 mg / L TDZ + 0.2 mg / L NAA + 2 mg / L 2,4-D; C: 1 mg / L TDZ + 0.8 mg / L NAA; D: 1 mg / L TDZ + 0.8 mg / L NAA + 2 mg / L 2,4-D);

[0054] Figure 11 :Effects of 0.5mg / L 6-BA on the budding and differentiation rate of callus tissue of experimental seedlings

[0055] Figure 12 :The effects of different plant hormones on the rooting rate in experimental seedling bottles (A and B are 0.5 mg / L 6-BA; C is 0.2 mg / L 6-BA; D is 0.05 mg / L 6-BA; E is 0.5 mg / L NAA; F is 0.2 mg / L NAA (no rooting));

[0056] Figure 13 : Transplanting of rooted seedlings. DETAILED DESCRIPTION

[0057] The present invention will be further described below with reference to the examples, but they are not intended to limit the present invention.

[0058] Example 1: Tissue culture rapid propagation and rooting method of blueberry

[0059] (1) Explant collection: During the spring growing season, select sunny weather with an air humidity of ≤45%, and between 9:00 and 11:00 in the morning, collect new shoots from healthy, disease-free Lark Blueberry plants. Quickly place these shoots in a fresh-keeping bag and place an ice pack to keep them fresh. Obtain Grade A for future use.

[0060] (2) Explant pretreatment: Take product A, remove the 2.0 cm tender part at the top of the shoot and the lignified part at the bottom, retain the middle 8 cm length, cut off the leaves, add detergent and purified water to the shoot, scrub it, and soak it for 10 minutes. Rinse it with purified water, and hang it in clean water for 1.5 hours to obtain product B;

[0061] (3) Explant disinfection: After ventilation and UV sterilization in a clean bench, place product B on the clean bench and disinfect it with 75% alcohol for 30 seconds, then rinse it twice with sterile water, and then disinfect it with 15% sodium hypochlorite for 15 minutes. After disinfection, rinse it with sterile water five times, and use filter paper to absorb the moisture on the surface of the material to obtain callus tissue, namely product C, for later use;

[0062] (4) Primary callus culture: Sample C was inoculated into primary callus culture medium and cultured for 30 days to obtain sample D; the primary callus culture medium was WPM + 30 g / L sucrose + 7 g / L agar powder + 1 mg / L ZT, and the pH was adjusted to 5.2-5.4 using 1 mol / L NaOH and 1 mol / L HCl; the culture conditions were: temperature 19-23°C, light intensity 3000 lx, and light duration 12 h / d;

[0063] (5) Primary callus induction culture: Take product D and inoculate it into primary callus induction medium and culture it for 35 days to obtain product E; the primary callus induction medium is WPM + 1 mg / L TDZ + 0.3 mg / L NAA + 200 mg / L VC, and the pH is adjusted to 5.2-5.4 using 1 mol / L NaOH and 1 mol / L HCl; the culture conditions are: temperature 19-23 °C, light intensity 2500-3000 lx, and light duration 12 h / d;

[0064] (6) Subculture differentiation culture: Take product E and inoculate it into subculture differentiation medium and culture it for 35 days to obtain differentiated buds; the subculture differentiation medium is 1 / 2 MS + 0.5 mg / L 6-BA, and the pH is adjusted to 5.2-5.4 using 1 mol / L NaOH and 1 mol / L HCl; the culture conditions are: temperature 19-23°C, light intensity 2500-3000 lx, and light duration 12 h / d;

[0065] (7) Rooting culture: Differentiated buds were inoculated into rooting medium and cultured for 90 days to obtain rooted seedlings; the rooting medium consisted of 1 / 2 MS + 0.5 mg / L 6-BA, and the pH was adjusted to 5.2-5.4 using 1 mol / L NaOH and 1 mol / L HCl; the culture conditions were: temperature 19-23°C, light intensity 2500-3000 lx, and photoperiod 12 h / d;

[0066] (8) Transplantation of rooted seedlings: The rooted seedlings were acclimated using the hydroponic transition method. The rooted seedlings with the culture medium were completely immersed in 25°C sterile water. The agar matrix was gradually separated by water oscillation, and the water surface was kept submerged in the root system. The root primordium was gently scrubbed along the growth direction of the root tip with a soft brush to clean the culture medium. Then, 1% rooting powder was used to dip the seedlings for 1 minute and the seedlings were transplanted into a ternary composite matrix. The ternary composite matrix was a mixture of peat soil, gravel and perlite in a volume ratio of 3:1:1. After transplanting, the seedlings were immediately irrigated with rooting water three times using a fine-hole nozzle. The EC value of the rooting water was ≤0.5mS / cm. When the surface 2cm of the matrix was dry, water was added to maintain the matrix humidity at 60-70%. Shading was performed within 7 days after transplanting, with a shading rate of 70%. After 7 days, the light intensity was gradually increased as the plant grew. The light intensity was controlled in the range of 8000-12000 lux during the seedling acclimatization period.

[0067] Example 2: Tissue culture rapid propagation and rooting method of skylark blueberry

[0068] (1) Explant collection: During the spring growing season, select sunny weather with an air humidity of ≤45%, and between 9:00 and 11:00 in the morning, collect new shoots from healthy, disease-free Lark Blueberry plants. Quickly place these shoots in a fresh-keeping bag and place an ice pack to keep them fresh. Obtain Grade A for future use.

[0069] (2) Explant pretreatment: Take product A, remove the 1.8 cm tender part at the top of the shoot and the lignified part at the bottom, retain the middle 12 cm length, cut off the leaves, add detergent and purified water to the shoot, scrub it, and soak it for 12 minutes. Rinse it with purified water, and hang it in clean water for 1 hour to obtain product B.

[0070] (3) Explant disinfection: After ventilation and UV sterilization in a clean bench, place product B on the clean bench and disinfect it with 75% alcohol for 40 seconds, then rinse it once with sterile water, and then disinfect it with 15% sodium hypochlorite for 25 minutes. After disinfection, rinse it with sterile water three times and dry the surface moisture of the material with filter paper to obtain callus tissue, namely product C, for later use;

[0071] (4) Primary callus culture: Take sample C and inoculate it into primary callus culture medium and culture it for 35 days to obtain sample D;

[0072] The primary callus culture medium is WPM+25g / L sucrose+6g / L agar powder+0.5mg / L ZT, and the pH is adjusted to 5.2-5.4 using 1mol / L NaOH and 1mol / L HCl;

[0073] The culture conditions are as follows: temperature of 23-25°C, light intensity of 3000-3500 lx, and light duration of 14 h / d;

[0074] (5) Primary callus induction culture: Take product D and inoculate it into the primary callus induction medium and culture it for 30 days to obtain product E;

[0075] The primary callus induction medium is WPM+0.5 mg / L TDZ+0.2 mg / L NAA+150 mg / L VC, and the pH is adjusted to 5.2-5.4 using 1 mol / L NaOH and 1 mol / L HCl;

[0076] The culture conditions are as follows: temperature of 23-25°C, light intensity of 2000-2500 lx, and light duration of 10 h / d;

[0077] (6) Subculture differentiation culture: Take product E and inoculate it into subculture differentiation medium and culture it for 30 days to obtain differentiated buds;

[0078] The subculture differentiation medium is 1 / 2 MS + 0.6 mg / L 6-BA, and the pH is adjusted to 5.2-5.4 using 1 mol / L NaOH and 1 mol / L HCl;

[0079] The culture conditions are: temperature 23-25°C, light intensity 2000-2500 lx, and photoperiod 14 h / d;

[0080] (7) Rooting culture: Differentiated buds were inoculated into rooting medium and cultured for 95 days to obtain rooted seedlings;

[0081] The rooting medium is 1 / 2 MS + 0.6 mg / L 6-BA, and the pH is adjusted to 5.2-5.4 using 1 mol / L NaOH and 1 mol / L HCl;

[0082] The culture conditions are as follows: temperature of 17°C, light intensity of 2000 lx, and light duration of 10 h / d;

[0083] (8) Transplantation of rooted seedlings: The rooted seedlings were acclimated using the hydroponic transition method. The rooted seedlings with the culture medium were completely immersed in 30°C sterile water. The agar matrix was gradually separated by water oscillation, and the water surface was kept submerged in the root system. The root primordium was gently scrubbed along the growth direction of the root tip with a soft brush to clean the culture medium. Then, 0.8% rooting powder was used to dip for 0.5 minutes and the root primordium was transplanted into a ternary composite matrix. The ternary composite matrix was a mixture of peat soil, gravel and perlite in a volume ratio of 4:1.5:1.5. After transplanting, the rooting water was immediately irrigated three times with a fine-hole nozzle. The EC value of the rooting water was ≤0.5mS / cm. When the surface 2.5cm of the matrix was dry, water was added to maintain the matrix humidity at 70-75%. Shading was performed within 6 days after transplanting, with a shading rate of 75%. After 6 days, the light intensity was gradually increased as the plant grew. The light intensity was controlled within the range of 14,000 lux during the seedling acclimatization period.

[0084] Example 3: Tissue culture rapid propagation and rooting method of blueberry

[0085] (1) Explant collection: During the spring growing season, select sunny weather with an air humidity of ≤45%, and between 9:00 and 11:00 in the morning, collect new shoots from healthy, disease-free Lark Blueberry plants. Quickly place these shoots in a fresh-keeping bag and place an ice pack to keep them fresh. Obtain Grade A for future use.

[0086] (2) Explant pretreatment: Take product A, remove the 2.2 cm tender part at the top of the shoot and the lignified part at the bottom, retain the middle 5 cm length, cut off the leaves, add detergent and purified water to the shoot, scrub, and soak for 8 minutes, rinse with purified water, and hang it in clean water for 3 hours to obtain product B;

[0087] (3) Explant disinfection: After ventilation and UV sterilization in a clean bench, place product B on the clean bench and disinfect it with 75% alcohol for 20 seconds, then rinse it with sterile water three times, and then disinfect it with 15% sodium hypochlorite for 10 minutes. After disinfection, rinse it with sterile water six times, and use filter paper to absorb the moisture on the surface of the material to obtain callus tissue, namely product C, for later use;

[0088] (4) Primary callus culture: Take sample C and inoculate it into primary callus culture medium and culture it for 25 days to obtain sample D;

[0089] The primary callus culture medium is WPM+35g / L sucrose+8g / L agar powder+1.5mg / L ZT, and the pH is adjusted to 5.2-5.4 using 1mol / L NaOH and 1mol / L HCl;

[0090] The culture conditions are as follows: temperature of 17-19°C, light intensity of 2000-2500 lx, and light duration of 10 h / d;

[0091] (5) Primary callus induction culture: Take product D and inoculate it into the primary callus induction medium and culture it for 40 days to obtain product E;

[0092] The primary callus induction medium is WPM+1.5 mg / L TDZ+0.4 mg / L NAA+250 mg / L VC, and the pH is adjusted to 5.2-5.4 using 1 mol / L NaOH and 1 mol / L HCl;

[0093] The culture conditions are as follows: temperature of 17-19° C., light intensity of 3000-3500 lx, and light duration of 10 h / d;

[0094] (6) Subculture differentiation culture: Take product E and inoculate it into subculture differentiation medium and culture it for 40 days to obtain differentiated buds;

[0095] The subculture differentiation medium is 1 / 2 MS + 0.4 mg / L 6-BA, and the pH is adjusted to 5.2-5.4 using 1 mol / L NaOH and 1 mol / L HCl;

[0096] The culture conditions are: temperature 17-19°C, light intensity 3000-3500lx, and photoperiod 10h / d;

[0097] (7) Rooting culture: Differentiated buds were inoculated into rooting medium and cultured for 85 days to obtain rooted seedlings;

[0098] The rooting medium is 1 / 2 MS + 0.4 mg / L 6-BA, and the pH is adjusted to 5.2-5.4 using 1 mol / L NaOH and 1 mol / L HCl;

[0099] The culture conditions are as follows: temperature of 23-25°C, light intensity of 3000-3500 lx, and light duration of 14 h / d;

[0100] (8) Transplantation of rooted seedlings: The rooted seedlings were acclimated using the hydroponic transition method. The rooted seedlings with the culture medium were completely immersed in 20°C sterile water. The agar matrix was gradually separated by water oscillation, and the water surface was kept submerged in the root system. The root primordium was gently scrubbed along the growth direction of the root tip with a soft brush to clean the culture medium. Then 1.2% rooting powder was used to dip for 1.5 minutes and the root primordium was transplanted into a ternary composite matrix. The ternary composite matrix was a mixture of peat soil, gravel and perlite in a volume ratio of 2:0.5:0.5. After transplanting, the rooting water was immediately irrigated three times with a fine-hole nozzle. The EC value of the rooting water was ≤0.5mS / cm. When the surface 1.5cm of the matrix was dry, water was added to maintain the matrix humidity at 55-60%. Shading was performed within 8 days after transplanting, with a shading rate of 65%. After 8 days, the light intensity was gradually increased as the plant grew. The light intensity was controlled within the range of 6000 lux during the seedling acclimatization period.

[0101] Example 4: Tissue culture rapid propagation and rooting method of skylark blueberry

[0102] (4) Explant collection: During the spring growing season, select sunny weather with an air humidity of ≤45% and collect new shoots from healthy, disease-free Lark Blueberry plants between 9:00 and 11:00 a.m. These shoots are quickly placed in a fresh-keeping bag and placed in an ice pack to keep them fresh. Grade A is obtained and set aside.

[0103] (5) Explant pretreatment: Take product A, remove the 1.9 cm tender part at the top of the shoot and the lignified part at the bottom, retain the middle 6 cm length, cut off the leaves, add detergent and purified water to the shoot, scrub it, and soak it for 10 minutes. After rinsing it with purified water, hang it in clean water for 2 hours to obtain product B;

[0104] (6) Explant disinfection: After ventilation and UV sterilization in a clean bench, place product B on the clean bench and disinfect it with 75% alcohol for 30 seconds, then rinse it twice with sterile water, and then disinfect it with 15% sodium hypochlorite for 15 minutes. After disinfection, rinse it with sterile water four times, and use filter paper to absorb the moisture on the surface of the material to obtain callus tissue, namely product C, for later use;

[0105] (4) Primary callus culture: Sample C was inoculated into primary callus culture medium and cultured for 28 days to obtain sample D; the primary callus culture medium was WPM + 30 g / L sucrose + 7 g / L agar powder + 1 mg / L ZT, and the pH was adjusted to 5.2-5.4 using 1 mol / L NaOH and 1 mol / L HCl; the culture conditions were: temperature 19-23°C, light intensity 2500-3000 lx, and photoperiod 12 h / d;

[0106] (5) Primary callus induction culture: Take product D and inoculate it into primary callus induction medium and culture it for 32 days to obtain product E; the primary callus induction medium is WPM + 1 mg / L TDZ + 0.3 mg / L NAA + 200 mg / L VC, and the pH is adjusted to 5.2-5.4 using 1 mol / L NaOH and 1 mol / L HCl; the culture conditions are: temperature 19-23 °C, light intensity 2500-3000 lx, and light duration 12 h / d;

[0107] (6) Subculture differentiation culture: Take product E and inoculate it into subculture differentiation medium and culture it for 32 days to obtain differentiated buds; the subculture differentiation medium is 1 / 2 MS + 0.5 mg / L 6-BA, and the pH is adjusted to 5.2-5.4 using 1 mol / L NaOH and 1 mol / L HCl; the culture conditions are: temperature 19-23°C, light intensity 2500-3000 lx, and light duration 12 h / d;

[0108] (7) Rooting culture: Differentiated buds were inoculated into rooting medium and cultured for 92 days to obtain rooted seedlings; the rooting medium consisted of 1 / 2 MS + 0.5 mg / L 6-BA, and the pH was adjusted to 5.2-5.4 using 1 mol / L NaOH and 1 mol / L HCl; the culture conditions were: temperature 19-23°C, light intensity 2500-3000 lx, and photoperiod 12 h / d;

[0109] (8) Transplantation of rooted seedlings: The rooted seedlings were acclimated using the hydroponic transition method. The rooted seedlings with the culture medium were completely immersed in 23°C sterile water. The agar matrix was gradually separated by water oscillation, and the water surface was kept submerged in the root system. The root primordium was gently scrubbed along the growth direction of the root tip with a soft brush to clean the culture medium. Then 1% rooting powder was used to soak for 1 minute and the seedlings were transplanted into a ternary composite matrix. The ternary composite matrix was a mixture of peat soil, gravel and perlite in a volume ratio of 3:1:1. Immediately after transplanting, the seedlings were watered three times with a fine-hole nozzle. The EC value of the rooting water was ≤0.5mS / cm. When the surface 2cm of the matrix was dry, water was added to maintain the matrix humidity at 60-70%. Shading was performed within 7 days after transplanting, with a shading rate of 68%. After 7 days, the light intensity was gradually increased as the plant grew. The light intensity was controlled in the range of 8000-12000 lux during the seedling acclimatization period.

[0110] Example 5: Tissue culture rapid propagation and rooting method of skylark blueberry

[0111] (7) Explant collection: During the spring growing season, select sunny weather with an air humidity of ≤45% and collect new shoots from healthy, disease-free Lark Blueberry plants between 9:00 and 11:00 a.m. These shoots are quickly placed in a fresh-keeping bag and placed in an ice pack to keep them fresh. Grade A is obtained and set aside.

[0112] (8) Explant pretreatment: Take product A, remove the 2.1 cm tender part at the top of the shoot and the lignified part at the bottom, retain the middle 10 cm length, cut off the leaves, add detergent and purified water to the shoot, scrub it, and soak it for 10 minutes. Rinse it with purified water, and hang it in clean water for 1 hour to obtain product B.

[0113] (9) Explant disinfection: After ventilation and UV sterilization in a clean bench, place product B on the clean bench and disinfect it with 75% alcohol for 30 seconds, then rinse it twice with sterile water, and then disinfect it with 15% sodium hypochlorite for 15 minutes. After disinfection, rinse it with sterile water five times and dry the surface moisture of the material with filter paper to obtain callus tissue, namely product C, for later use;

[0114] (4) Primary callus culture: Sample C was inoculated into primary callus culture medium and cultured for 32 days to obtain sample D; the primary callus culture medium was WPM + 30 g / L sucrose + 7 g / L agar powder + 1 mg / L ZT, and the pH was adjusted to 5.2-5.4 using 1 mol / L NaOH and 1 mol / L HCl; the culture conditions were: temperature 19-23°C, light intensity 2500-3000 lx, and photoperiod 12 h / d;

[0115] (5) Primary callus induction culture: Take product D and inoculate it into primary callus induction medium and culture it for 37 days to obtain product E; the primary callus induction medium is WPM + 1 mg / L TDZ + 0.3 mg / L NAA + 200 mg / L VC, and the pH is adjusted to 5.2-5.4 using 1 mol / L NaOH and 1 mol / L HCl; the culture conditions are: temperature 19-23 °C, light intensity 2500-3000 lx, and light duration 12 h / d;

[0116] (6) Subculture differentiation culture: Take product E and inoculate it into subculture differentiation medium and culture it for 37 days to obtain differentiated buds; the subculture differentiation medium is 1 / 2 MS + 0.5 mg / L 6-BA, and the pH is adjusted to 5.2-5.4 using 1 mol / L NaOH and 1 mol / L HCl; the culture conditions are: temperature 19-23°C, light intensity 2500-3000 lx, and light duration 12 h / d;

[0117] (7) Rooting culture: Differentiated buds were inoculated into rooting medium and cultured for 88 days to obtain rooted seedlings; the rooting medium consisted of 1 / 2 MS + 0.5 mg / L 6-BA, and the pH was adjusted to 5.2-5.4 using 1 mol / L NaOH and 1 mol / L HCl; the culture conditions were: temperature 19-23°C, light intensity 2500-3000 lx, and photoperiod 12 h / d;

[0118] (8) Transplantation of rooted seedlings: The rooted seedlings were acclimated using the hydroponic transition method. The rooted seedlings with the culture medium were completely immersed in 27°C sterile water. The agar matrix was gradually separated by water oscillation, and the water surface was kept submerged in the root system. The root primordium was gently scrubbed along the growth direction of the root tip with a soft brush to clean the culture medium. Then 1% rooting powder was used to dip for 1 minute and the seedlings were transplanted into a ternary composite matrix. The ternary composite matrix was a mixture of peat soil, gravel and perlite in a volume ratio of 3:1:1. Immediately after transplanting, the seedlings were watered three times with a fine-hole nozzle. The EC value of the rooting water was ≤0.5mS / cm. When the surface 2cm of the matrix was dry, water was added to maintain the matrix humidity at 60-70%. Shading was performed within 7 days after transplanting, with a shading rate of 72%. After 7 days, the light intensity was gradually increased as the plant grew. The light intensity was controlled in the range of 8000-12000 lux during the seedling acclimatization period.

[0119] The inventors conducted the following experiments to verify the effects of the present invention in order to better understand how the present invention achieves its objectives and solves related technical problems:

[0120] 1 Experimental materials and methods

[0121] 1.1 Experimental Materials

[0122] This experiment selected the southern highbush blueberry variety "Skylark" as the research material, and the picking site was the nursery base of Huizhou University.

[0123] 2 Screening of explant humidity and disinfection methods

[0124] During the spring growing season, new shoots were collected from healthy, disease-free plants between 9:00 AM and 11:00 AM on clear, low-humidity days. These shoots were quickly placed in a fresh-keeping bag with an ice pack to maintain freshness and then brought back to the laboratory for further processing.

[0125] Pre-treatment: Remove the tender, approximately 2 cm top section of the shoot and the heavily lignified lower section, retaining the central section (approximately 6-10 cm). Trim the leaves and place the shoot in a clean beaker. Add detergent and purified water to the beaker, scrub, and soak for 10 minutes. Rinse thoroughly with purified water and hang under running water for 1-2 hours.

[0126] After ventilation and UV sterilization in a clean bench, the inoculated materials were disinfected with 75% alcohol for 30 seconds, rinsed twice with sterile water, and then disinfected with sodium hypochlorite at varying concentrations, with the disinfection time determined by the concentration. After disinfection, the materials were rinsed four to five times with sterile water and the surface moisture was blotted with filter paper. For inoculation, the materials, treated at different concentrations and for different times, were inoculated into the culture medium. After inoculation, the materials were placed in an incubation room at (21±2)°C, with a light intensity of 2500-3000 lx for 12 hours per day.

[0127] Culture medium: WPM medium was used as the basal medium for primary culture, sucrose 30 g·L-1, agar powder 7 g·L-1, pH adjusted to 5.2-5.4, and auxin 2 mg / L ZT were added.

[0128] 2.1 Effects of different air humidity on explants during harvesting

[0129] Air humidity has a great influence on the establishment of sterile system for explants. Figure 1 It can be seen that the contamination rate ranges from 0 to 69.57%. High humidity promotes microbial reproduction (for example, the germination rate of fungal spores increases by 2-3 times), while weakening plant immune defenses (for example, inhibiting the salicylic acid signaling pathway), increasing the risk of pathogen invasion. At the same humidity, the contamination rate after 10 minutes of treatment is lower than that after 5 minutes. Based on the above, explants should be harvested on sunny days with low air humidity to significantly reduce the contamination rate.

[0130] Table 1 Effects of different air humidity on explants during harvesting

[0131]

[0132] 2.2 Effects of different disinfection concentrations on explants

[0133] From March 2024, the explants of blueberry "Skylark" were collected. Sodium hypochlorite was used as disinfectant. The concentration of sodium hypochlorite ranged from 5% to 20%. Four gradients (5%, 10%, 15%, 20%) were set. The treatment time was controlled to be 10 minutes. Figure 2 It can be seen that the contamination rate of sodium hypochlorite treatment is in the range of 19.23% to 44.44%; the browning rate is between 8.33% and 21.15%; and the germination rate is between 12.90% and 35.00%.

[0134] Among them, the 15% concentration had the best overall performance, with a higher germination rate, a contamination rate of 23.26%, and a browning rate of 11.63%, which were significantly lower than the 21.15% browning rate of the 20% group. The number of surviving samples was large, which could provide sufficient materials for large-scale culture of explants; although the germination rate of the 10% concentration was high, the number of surviving samples was limited, and the browning rate was relatively high; the 20% concentration was limited, and the browning rate increased sharply, which may be due to the high concentration of sodium hypochlorite causing cell membrane lipid peroxidation, and excessive disinfection significantly inhibited germination.

[0135] Based on the above disinfection results, sodium hypochlorite achieves disinfection by destroying microbial cell walls, but high concentrations (≥20%) may also damage plant cell membranes, leading to increased browning. A concentration of 15% is optimal, achieving a balance between sterilization efficacy and cell viability protection, effectively killing microorganisms while maintaining the physiological activity of explants.

[0136] Table 2 Effects of different disinfection concentrations on explants

[0137]

[0138]

[0139] 2.3 Effects of different disinfection times on explants

[0140] Add 15% sodium hypochlorite and set four different time gradients (5min, 10min, 15min, 20min). Figure 3 It can be seen that the contamination rate is between 0 and 8.33%; the browning rate is between 0 and 12.50%; and the germination rate is between 26.32 and 56.00%.

[0141] The sterilization threshold for contamination is 15 minutes. Exceeding this threshold may damage the explants, exposing internal tissues and increasing the risk of contamination. The browning rate shows a decreasing-then-increasing trend over time, with minimal oxidative damage at 15 minutes and severe damage to the cell membrane system at 20 minutes, leading to increased phenolic oxidation. A 15-minute treatment for germination effectively sterilizes while preserving cell viability to the greatest extent possible, while a 20-minute treatment causes irreversible damage to the meristem.

[0142] Based on the above situation, 15% sodium hypochlorite concentration should be selected for treatment for 15 minutes, which can completely sterilize the plants and keep the meristem intact, and significantly improve the germination rate.

[0143] Table 3 Effects of different disinfection times on explants

[0144]

[0145] 2.4 Conclusion

[0146] The explants were picked under the condition of 45% humidity and treated with 15% sodium hypochlorite for 15 minutes. The germination rate reached up to 56% and the contamination rate was 0, which was the best disinfection scheme.

[0147] 3. Screening of different tissue sites and culture hormones in primary callus culture

[0148] Primary callus culture medium: WPM medium was selected as the basal culture medium, sucrose 30 g·L-1, agar powder 7 g·L-1 were added, the pH was adjusted to 5.2-5.4, and different plant growth hormones were added and different concentrations were set as controls.

[0149] The culture conditions are: temperature 19-23℃, light intensity 2500-3000lx, photoperiod 12h / d

[0150] See Figure 4 The stem segment processing method is to remove the leaves on the stem segment, cut off the tender yellow part about 2-4 cm at the top of the stem segment and the adult branches at the bottom, and retain the tender green stem segment in the middle. Cut the stem segment into about 1-2 cm long, retain the axillary buds, and inoculate it into the culture medium.

[0151] See Figure 4 The leaf processing method is material processing (leaves). The leaves are wounded, the tips and bases of the leaves are cut off to form wounds, and then inoculated into the culture medium to observe the leaf germination and callus growth effects.

[0152] 3.1 Effects of different tissue site selection on the germination rate of experimental seedlings

[0153] From Table 4, Figure 5 It can be seen that under the same plant growth regulator (ZT concentration 2 mg / L) conditions, the germination rate of stem segments as explants (75.61%) was significantly higher than that of leaves (0%), and the browning rate was lower. This shows that stem segments are a better choice for explants for tissue culture and rapid propagation of skylark blueberry. The reason may be that stem segments are rich in meristems and vascular bundles, have strong cell division ability, and have high levels of endogenous hormones (such as auxin), which are more responsive to the induction of exogenous ZT and initiate bud differentiation. In contrast, leaves are mainly composed of mature thin-walled cells with a high degree of differentiation and weak dedifferentiation ability. In addition, the leaves have a large surface area and a loose structure, which are easily damaged by oxidative damage during disinfection, resulting in a surge in browning rate.

[0154] In the stem segments, ZT (zeatin riboside) may be efficiently transported to the bud primordium through the vascular bundles, promoting cell division; while in the leaves, the hormone may induce callus formation rather than bud differentiation, but the high browning rate inhibits this process.

[0155] The stem segments are significantly superior to leaves in budding and differentiation potential, disinfection tolerance and hormone response efficiency, and are ideal experimental materials for the tissue culture and rapid propagation system of skylark blueberry.

[0156] Table 4 Effects of different tissue site selection on the germination rate of test seedlings

[0157]

[0158] 3.2 Effects of different plant hormones on the germination rate of experimental seedlings

[0159] According to Table 5, Figure 6 When stem segments were used as explants for germination, the browning rate ranged from 3.33% to 38.70% after 30 days of culture, and the germination rate ranged from 0% to 88.89%. Zeatin (ZT), a cytokinin, effectively activated meristem activity and promoted shoot primordium formation at a concentration of 1 mg / L, but this increased the browning rate to 22.86%, likely through the promotion of polyphenol oxidase activity. 6-BA (6-benzylaminoadenine) at a concentration of 1 mg / L may disrupt cell membrane integrity, leading to electrolyte leakage and phenolic oxidation (browning rate 38.70%). Excessive cytokinin signaling may interfere with auxin transport, resulting in impaired bud development (germination rate only 5.26%). 2,4-D (2,4-dichlorophenoxyacetic acid), a potent auxin, significantly inhibited bud differentiation at a concentration of 1 mg / L (germination rate 0%), possibly inducing root primordium formation, inhibiting cell division and reducing polyphenol synthesis.

[0160] When inducing the test seedlings to germinate, ZT should be applied. This hormone has a positive promoting effect on the germination of the test seedlings.

[0161] Table 5 Effects of different plant hormones on the germination rate of experimental seedlings

[0162]

[0163] 3.3 Effects of different concentrations of the same hormone on the germination rate of experimental seedlings

[0164] According to Table 6, Figure 7 Using stem segments as explants, the germination rate ranged from 10.86% to 42.86% when treated with different concentrations of the plant hormone ZT. The germination rate ranged from 40.00% to 88.89%. In the control group, lacking cytokinin stimulation, cell senescence accelerated, leading to membrane lipid peroxidation, accumulation of polyphenols, insufficient endogenous hormone levels, and inactivated meristem activity. At 1 mg / L, ZT binds to cytokinin receptors, activating transcription factors and promoting shoot primordium formation. At 2 mg / L, the high concentration resulted in overactivation of the receptors, triggering cell cycle disruption and programmed cell death.

[0165] Based on the above situation, hormone ZT should be selected as the most suitable germination hormone, and when the concentration is 1 mg / L, the germination effect is the best, and the germination rate is 88.89%.

[0166] Table 6 Effects of different concentrations of the same hormone on the germination rate of test seedlings

[0167]

[0168]

[0169] 3.4 Conclusion

[0170] The best primary culture medium for germination was WPM+30g / L sucrose+7g / L agar powder+1mg / L ZT. After 30 days of culture, the germination rate of the stem segments reached 88.89%.

[0171] 4. Screening of different tissue locations and culture hormones in the induction culture of primary callus

[0172] For primary culture, WPM medium was used as the basal medium and sucrose 30 g·L was added. -1 , agar powder 7g·L -1 , adjust the pH to 5.2-5.4, add different plant growth hormones, set different concentrations as controls, and culture for 35 days.

[0173] Material treatment: Callus tissue with good growth was inoculated into WPM medium.

[0174] 4.1 Effects of different plant hormones on the healing rate of test seedling leaves

[0175] There are many factors that promote the formation of callus tissue in the leaves of the test seedlings, so experiments with different hormones (ZT, TDZ, NAA) at different concentrations were set up. Figure 8 It can be seen that the browning rate is between 4.08% and 54.29%; the healing rate is between 0% and 96.77%. When the TDZ concentration is 1 mg / L and the NAA concentration is between 0.1 and 1 mg / L, the healing rate of all combinations reaches more than 85%.

[0176] The combined use of TDZ and NAA significantly improved the healing rate, particularly when the TDZ concentration was 1 mg / L and the NAA concentration was between 0.1 and 0.5 mg / L. In contrast, the healing rate was low or insignificant when either NAA or TDZ was used alone. ZT, when used alone, had limited effect on improving the healing rate.

[0177] Therefore, when inducing callus formation, the hormone combination of TDZ concentration of 1 mg / L and NAA concentration of 0.1-0.5 mg / L can be given priority to further improve the callus rate.

[0178] Among them, the formula with the highest leaf healing rate (96.77%) was WPM+1mg / LTDZ+0.1mg / LNAA.

[0179] Table 7 Effects of different plant hormones on the healing rate of test seedling leaves

[0180]

[0181]

[0182] 4.2 Effects of different plant hormones on the callus rate of experimental seedling stem segments

[0183] There are certain differences in callus induction in different parts of plants. To discuss the callus induction rate of stem segments, we will mainly focus on different hormones (ZT, TDZ, NAA) and different concentrations to find the most suitable combination formula. Figure 9 It can be seen that the browning rate is between 0% and 63.89%; the healing rate is between 7.69% and 96.67%. TDZ and NAA have a significant synergistic effect. When the TDZ concentration is 1 mg·L -1 At NAA concentrations of 0.1 to 0.3 mg / L, the healing rate exceeded 96%, with the combination of 1 mg / L TDZ and 0.3 mg / L NAA achieving the highest healing rate (96.67%). Using hormones alone has limited effectiveness; healing rates with single hormones (such as TDZ, NAA, or 2,4-D) are all below 60%, and the browning rate fluctuates significantly. 2,4-D is less effective, with no significant improvement in healing rate observed alone or at low concentrations, requiring its combination with other hormones.

[0184] In summary, the synergistic effect of TDZ and NAA is also critical in stem callus induction. The optimal formula is 1 mg / L TDZ + 0.3 mg / L NAA, which is better than the optimal combination in the leaf experiment (WPM + 1 mg / L TDZ + 0.1 mg / L NAA), which may be related to the endogenous hormone level or cell response characteristics of the stem segment.

[0185] Table 8 Effects of different plant hormones on the callus rate of test seedling stem segments

[0186]

[0187]

[0188] 4.3 Effects of subsequent hormone 2,4-D addition on callus

[0189] In the study of callus induction system, the subsequent hormone addition strategy showed a complex multi-dimensional effect on the regulation of tissue growth state. Figure 10The browning rate ranged from 10.81% to 72.97%, while the callus induction rate ranged from 80.00% to 96.77%. In a basal medium containing 1 mg / L TDZ and 0.1 mg / L NAA, increasing the 2,4-D concentration from 0 to 2 mg / L resulted in a significant quantitative change in the browning rate, while the callus induction efficiency (callus induction rate) decreased by 3.91%. This inverse trend clearly indicates that the introduction of 2,4-D not only failed to achieve the expected growth-promoting effect, but instead caused severe physiological disturbances in the tissue culture. The antioxidant protection provided by NAA (naphthaleneacetic acid) was completely suppressed, typified by the uncontrolled development of browning, which is likely closely related to the abnormal accumulation of phenolic compounds and activation of the programmed cell death (PCD) pathway induced by 2,4-D.

[0190] The addition of 2,4-D resulted in a significant increase in browning without significantly improving callus formation, indicating a strong negative effect in the callus induction system. This result contrasts sharply with the synergistic effect of TDZ / NAA reported previously, further highlighting the importance of dynamic equilibrium in hormone combination selection.

[0191] Table 9 Effects of adding subsequent hormone 2,4-D on callus tissue

[0192]

[0193]

[0194] 4.4 Effect of adding vitamin C on preventing callus browning

[0195] During callus cultivation, browning was severe. Therefore, 200 mg / L VC was added as a control to investigate its effectiveness in preventing callus browning. Table 10 shows that the addition of 200 mg / L VC significantly reduced the browning rate and improved the callus initiation rate. Combined with 1 mg / L TDZ and 0.1 mg / L NAA, the browning rate decreased by 28.96% and the callus initiation rate increased by 16.77%.

[0196] In summary, the addition of vitamin C (VC) significantly reduced the browning rate of callus tissue and increased the callus formation rate, indicating that it has a positive effect on inhibiting browning and promoting callus formation.

[0197] Table 10 Effect of adding vitamin C on preventing callus browning

[0198]

[0199] 4.5 Conclusion

[0200] The best primary callus induction culture medium is WPM+1mg / LTDZ+0.1mg / L NAA+200mg / L VC. The browning rate is only 13.89%, which is a decrease of more than 30%. The callus induction rate reaches 96.77%, which is an increase of more than 10%.

[0201] 5. Subculture of callus tissue

[0202] 5.1 Effects of different plant hormones on the budding and differentiation rate of callus tissue of experimental seedlings

[0203] The budding and differentiation of callus tissue is an important stage in the rapid propagation technology of blueberry tissue culture. Whether the budding and differentiation can be carried out well is the key to the establishment of the subculture system. The basic culture medium uses 1 / 2MS and adds 0.5mg / L 6-BA, which can effectively promote the budding and differentiation of callus tissue and form young shoots with good growth. Figure 11 .

[0204] 6. Rooting Culture

[0205] For primary culture, 1 / 2MS medium was used as the basal medium and sucrose 30 g·L was added. -1 , agar powder 7g·L -1 , adjust the pH to 5.2-5.4, add different plant growth hormones, and set different concentrations as controls.

[0206] 6.1 Effects of different plant hormones on the rooting rate of experimental seedlings in bottles

[0207] The effects of different plant hormones on rooting of tissue culture seedlings in bottles, using 1 / 2MS as the basic medium, the culture time needs to be more than 90 days. Figure 12 As can be seen, the rooting rate in the bottle ranged from 0 to 80%. When NAA = 0, increasing the 6-BA concentration from 0.05 mg / L to 0.5 mg / L increased the rooting rate from 40% to 80%, showing a dose-dependent effect. The 0.5 mg / L 6-BA treatment group showed the best rooting effect (80% rooting rate + 3-fold proliferation), suggesting that higher concentrations of cytokinin in this system may indirectly promote rooting by regulating bud differentiation.

[0208] The 0.2 mg / L NAA treatment group completely inhibited rooting (0% rooting rate), but recovered to 40% rooting rate at 0.5 mg / L, presenting an atypical dose-effect curve. This may be because low concentrations of NAA induced ethylene synthesis to inhibit rooting or the experimental materials had a special sensitivity threshold to auxin.

[0209] The data analysis shows that in the single hormone treatment system, 0.5 mg / L 6-BA is the current optimal rooting formula. Its ability to achieve both high rooting rate and stable proliferation may be due to the special mechanism of action of cytokinin in promoting organ regeneration by regulating the balance of endogenous hormones.

[0210] Table 11 Effects of different plant hormones on the rooting rate of experimental seedlings in bottles

[0211]

[0212] 7. Transplanting rooted seedlings

[0213] Pre-transplantation: Use sterile tweezers to grasp the base of the tissue culture flask and remove the rooted plantlet along with the culture medium. Acclimation should be performed using the hydroponic transition method. Completely immerse the plantlet with the culture medium in 25°C sterile water. The agar matrix will gradually separate through the water flow. Keep the roots submerged in water and gently scrub them with a soft-bristled brush along the root tip, focusing on cleaning the root primordium attached to the culture medium. Ensure that the newly formed capillary roots remain intact at >95%.

[0214] Cultivation medium preparation:

[0215] (1) A ternary composite matrix system was used: peat soil, gravel, and perlite were mixed in a volume ratio of 3:1:1.

[0216] (2) Water management: Immediately after transplanting, water the plants to establish roots (EC value ≤ 0.5mS / cm). Use a fine-hole sprinkler to water thoroughly three times. Maintain the substrate humidity at 60% to 70% (determined by weight). Rehydrate the top 2 cm of substrate when it becomes dry.

[0217] (3) Light management: Shade treatment (shading rate 70%) is carried out within 7 days after transplanting, and the light intensity is gradually increased as the plant grows. During the transition period, the light intensity is controlled in the range of 8000-12000 lux.

Claims

1. A method for tissue culture, rapid propagation and rooting of blueberry, characterized by: The method is carried out according to the following steps: (1) Explant collection: In the spring growing season, select sunny weather with an air humidity of ≤45% and collect new shoots from healthy, disease-free Lark Blueberry plants in the morning. These shoots are quickly placed in a fresh-keeping bag and placed in an ice pack to keep them fresh. Grade A is obtained and set aside. (2) Explant pretreatment: Take product A, remove the 1.8-2.2 cm tender part at the top of the shoot and the lignified part at the bottom, retain the middle 5-12 cm length, cut off the leaves, add detergent and purified water to the shoot, scrub, and soak for 8-12 minutes, rinse with purified water, and hang to wash under clean water for 1-3 hours to obtain product B; (3) Explant disinfection: After ventilation and UV sterilization in a clean bench, place product B on the clean bench and disinfect it with 75% alcohol for 20-40 seconds, then rinse it with sterile water 1-3 times, and then disinfect it with 15% sodium hypochlorite for 10-25 minutes. After disinfection, rinse it with sterile water 3-6 times, and use filter paper to absorb the moisture on the surface of the material to obtain callus tissue, namely product C, for later use; (4) Primary callus culture: Take sample C and inoculate it into primary callus culture medium and culture it for 25-35 days to obtain sample D; The primary callus culture medium is WPM+25-35g / L sucrose+6-8g / L agar powder+0.5-1.5mg / L ZT, and the pH is adjusted to 5.2-5.4 using 1mol / L NaOH and 1mol / L HCl; The culture conditions are as follows: temperature of 17-25°C, light intensity of 2000-3500 lx, and light duration of 10-14 h / d; (5) Primary callus induction culture: Take product D and inoculate it into the primary callus induction medium and culture it for 30-40 days to obtain product E; The primary callus induction medium is WPM+0.5-1.5 mg / L TDZ+0.2-0.4 mg / L NAA+150-250 mg / L VC, and the pH is adjusted to 5.2-5.4 using 1 mol / L NaOH and 1 mol / L HCl; The culture conditions are as follows: temperature of 17-25°C, light intensity of 2000-3500 lx, and light duration of 10-14 h / d; (6) Subculture differentiation culture: Take product E and inoculate it into subculture differentiation medium and culture it for 30-40 days to obtain differentiated buds; The subculture differentiation medium is 1 / 2 MS + 0.4-0.6 mg / L 6-BA, and the pH is adjusted to 5.2-5.4 using 1 mol / L NaOH and 1 mol / L HCl; The culture conditions are: temperature 17-25°C, light intensity 2000-3500lx, photoperiod 10-14h / d; (7) Rooting culture: The differentiated buds were inoculated into rooting medium and cultured for 85-95 days to obtain rooted seedlings; The rooting medium is 1 / 2 MS + 0.4-0.6 mg / L 6-BA, and the pH is adjusted to 5.2-5.4 using 1 mol / L NaOH and 1 mol / L HCl; The culture conditions are as follows: temperature of 17-25°C, light intensity of 2000-3500 lx, and light duration of 10-14 h / d; (8) Transplantation of rooted seedlings: The rooted seedlings were acclimated using the hydroponic transition method. The rooted seedlings with the culture medium were completely immersed in sterile water at 20-30°C. The agar matrix was gradually separated by water oscillation, and the water surface was kept submerged in the roots. A soft brush was used to gently scrub along the growth direction of the root tip to clean the root primordium attached to the culture medium. Then, 0.8-1.2% rooting powder was used to soak for 0.5-1.5 minutes and the seedlings were transplanted into a ternary composite matrix, which was peat soil, gravel and perlite in a volume ratio of 2. -4:0.5-1.5:0.5-1.5 are mixed together. Immediately after transplanting, use a fine-hole nozzle to irrigate the rooting water three times. The EC value of the rooting water is ≤0.5mS / cm. When the surface 1.5-2.5cm matrix is dry, water is added to maintain the matrix humidity at 55-75%. Shade treatment is carried out within 6-8 days after transplanting, and the shading rate is 65-75%. After 6-8 days, the light intensity is gradually increased as the plant grows. The light intensity is controlled in the range of 6000-14000lux during the seedling growth period.

2. The method for tissue culture, rapid propagation and rooting of blueberry according to claim 1, wherein: In the step (1), explant picking: in the spring growing season, select sunny weather with air humidity ≤45%, and collect new shoots from healthy and disease-free skylark blueberry plants between 9:00 and 11:00 in the morning. These shoots are quickly placed in a fresh-keeping bag and placed in an ice pack to keep them fresh, thereby obtaining product A for future use.

3. The method for tissue culture, rapid propagation and rooting of blueberry according to claim 1, wherein: In the step (2), the explant is pretreated by taking product A, removing the tender part (1.9-2.1 cm) at the top of the tender branch and the lignified part at the bottom, retaining the middle part (6-10 cm in length), cutting off the leaves, adding detergent and purified water to the tender branch, scrubbing it, and soaking it for 10 minutes, rinsing it with purified water, and hanging it in clean water for 1-2 hours to obtain product B.

4. The method for tissue culture, rapid propagation and rooting of blueberry according to claim 1, wherein: In step (3), explant disinfection: after ventilation and ultraviolet sterilization in a clean bench, product B is placed in the clean bench and disinfected with 75% alcohol for 30 seconds, then rinsed twice with sterile water, and then disinfected with 15% sodium hypochlorite for 15 minutes. After disinfection, rinse with sterile water 4-5 times, and use filter paper to absorb the moisture on the surface of the material to obtain callus tissue, i.e. product C, for later use.

5. The method for tissue culture, rapid propagation and rooting of blueberry according to claim 1, wherein: In the step (4), the primary callus culture is as follows: the product C is inoculated into the primary callus culture medium and cultured for 30 days to obtain the product D; The primary callus culture medium is WPM+30g / L sucrose+7g / L agar powder+1mg / L ZT, and the pH is adjusted to 5.2-5.4 using 1mol / L NaOH and 1mol / L HCl; The culture conditions are: temperature of 19-23° C., light intensity of 2500-3000 lx, and light duration of 12 h / d.

6. The method for tissue culture, rapid propagation and rooting of blueberry according to claim 1, characterized in that: In step (5), primary callus induction culture: take product D and inoculate it into primary callus induction medium, culture it for 35 days, and obtain product E; The primary callus induction medium is WPM+1 mg / L TDZ+0.3 mg / L NAA+200 mg / L VC, and the pH is adjusted to 5.2-5.4 using 1 mol / L NaOH and 1 mol / L HCl; The culture conditions are: temperature of 19-23° C., light intensity of 2500-3000 lx, and light duration of 12 h / d.

7. The method for tissue culture, rapid propagation and rooting of blueberry according to claim 1, characterized in that: In the step (6), subculture differentiation culture: take product E and inoculate it into subculture differentiation medium, culture it for 35 days, and obtain differentiated buds; The subculture differentiation medium is 1 / 2 MS + 0.5 mg / L 6-BA, and the pH is adjusted to 5.2-5.4 using 1 mol / L NaOH and 1 mol / L HCl; The culture conditions are: temperature of 19-23° C., light intensity of 2500-3000 lx, and light duration of 12 h / d.

8. The method for tissue culture, rapid propagation and rooting of blueberry according to claim 1, characterized in that: In step (7), rooting culture: taking differentiated buds and inoculating them into rooting medium and culturing them for 90 days to obtain rooted seedlings; The rooting medium is 1 / 2 MS + 0.5 mg / L 6-BA, and the pH is adjusted to 5.2-5.4 using 1 mol / L NaOH and 1 mol / L HCl; The culture conditions are: temperature of 19-23° C., light intensity of 2500-3000 lx, and light duration of 12 h / d.

9. The method for tissue culture, rapid propagation and rooting of skylark blueberry according to claim 1, characterized in that: In the step (8), the rooted seedlings are transplanted: the rooted seedlings are acclimated by the hydroponic transition method, the rooted seedlings with the culture medium are completely immersed in 22-28°C sterile water, the agar matrix is gradually separated by water oscillation, the water surface is kept submerged in the root system, and the root primordium is gently scrubbed along the growth direction of the root tip with a soft brush to clean the culture medium; then 1% rooting powder is used to dip for 0.7-1.2 minutes, and the rooted seedlings are transplanted into a ternary composite matrix, wherein the ternary composite matrix is peat soil, gravel and Perlite is mixed in a volume ratio of 2-4:1:

1. Immediately after transplanting, use a fine-hole sprinkler to water the roots three times. The EC value of the rooting water is ≤0.5mS / cm. When the surface 1.8-2.2cm matrix is dry, water is added to maintain the matrix humidity at 68-72%. Shade treatment is carried out within 7 days after transplanting, and the shading rate is 60-70%. After 7 days, the light intensity is gradually increased as the plant grows. The light intensity is controlled in the range of 7000-13000 lux during the seedling growth period.

10. The method for tissue culture, rapid propagation and rooting of skylark blueberry according to claim 9, characterized in that: In the step (8), the rooted seedlings are transplanted: the rooted seedlings are acclimated by a hydroponic transition method, the rooted seedlings with the culture medium are completely immersed in 25°C sterile water, the agar matrix is gradually separated by water flow oscillation, the water surface is kept to submerge the root system, and the root primordium is gently scrubbed along the root tip growth direction with a soft brush to clean the root primordium attachment culture medium; then 1% rooting powder is used to dip for 1 minute, and the seedlings are transplanted into a ternary composite matrix, wherein the ternary composite matrix is obtained by mixing peat soil, gravel and perlite in a volume ratio of 3:1:1, and immediately after transplanting, a fine-hole nozzle is used to water the rooting water three times, and the EC value of the rooting water is ≤0.5mS / cm; when the surface 2cm of the matrix is dry, water is added to maintain the matrix humidity at 60-70%, and shade treatment is performed within 7 days after transplanting, with a shading rate of 70%. After 7 days, the light intensity is gradually increased as the plant grows, and the light intensity is controlled in the range of 8000-12000lux during the seedling acclimatization period.

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