A method for growing blueberry seedlings and its application

Abstract

The present application provides a method for blueberry seedling cultivation and its application. The method includes: selection and treatment of plants and explants, induction culture of axillary buds in an improved culture medium, and zeatin (ZT) decreasing subculture culture, and further cultivation of subculture seedlings into commercial seedlings. The method provided in the present application uses a basic culture medium prepared from improved MS and WPM culture media, so that the explants germinate quickly, the subculture cycle is short, the seedlings grow well, the branches are reasonable, and the buds are thick. The induced budding rate of blueberries is increased to 92.5%, and the proliferation rate after five subcultures reaches 404.8 times. The one-step seedling cultivation method used in commercial seedling cultivation has a rooting rate of 98.2% after 25 days of cultivation, shortens the seedling cultivation time by an average of 19.4 days, and the root system is dispersed and uniform. This solution effectively improves seedling cultivation efficiency, seedling quality and plant growth after planting and reduces the risk of variation. It fundamentally solves various root system problems that seriously affect survival, growth and yield after planting.

Description

Technical Field

[0001] The present invention relates to the technical field of plant breeding, and in particular to a method for blueberry seedling formation and application thereof. Background Art

[0002] With the rapid development of the blueberry industry, tissue culture rapid propagation technology for blueberry seedlings has become increasingly mature and widely used. Conventional tissue culture technology provides excellent technical support and guarantee for the breeding of blueberry seedlings and industrial development. However, in the past two years, the rapid increase in cultivated area has led to a sharp increase in the demand for seedlings, especially for new and superior seedling varieties, which are now in short supply. In pursuit of high proliferation rates and seedling yields, excessive use of cytokinins, failure to timely update seed sources, unlimited increase in the number of subcultures to maintain seedling production, and the increasing practice of using cuttings from production tissue culture seedlings twice or even more times to cultivate second or third crops of cuttings have resulted in increasingly unavoidable problems such as poor seedling quality, mutation, poor root system and growth, low yield, and even plant death, which are common and cause significant losses to later plantings.

[0003] At the same time, the existing tissue culture technology has many problems in seed selection, culture medium and hormone ratio, and production process, specifically: (1) Seed collection: multiple plants are mixed to collect buds, and farmers or enterprises arbitrarily collect seeds for tissue culture seedling merchants to expand and propagate, resulting in serious problems such as seedling mixing, disease and degeneration; (2) Culture medium: most of them use WPM culture medium as the basic culture medium for subculture proliferation and rooting. In addition, there are many blueberry strains and varieties, and they are very different, which leads to long propagation cycles, uneven seedling quality, poor growth and rooting, and other prominent problems; (3 ) In terms of tissue culture formula and technical process: in order to increase the proliferation rate, ultra-high or high amounts of zeatin (ZT) are used for continuous propagation and subculture. Rooting is first cultured in a bottle and then taken out of the bottle and transplanted into a plug tray to be cultivated into plug tray seedlings. The plug tray seedlings are then transplanted two or three times to small cups or medium or large-sized seedling pots to be cultivated into commercial seedlings according to demand. Not only is the production cycle of commercial seedlings long, but in various stages of tissue culture seedling cultivation, especially the plug tray seedling stage, problems such as root entanglement, root wrapping, and old roots that affect the later quality are prominent due to small holes and untimely cup changes.

[0004] Five to ten years ago, growers required blueberry seedlings to be 1.5-2 years old, 30-40 cm tall, and 2-3 years old before fruiting. In the past two to three years, with the widespread adoption of soilless blueberry cultivation technology nationwide, new requirements have emerged for blueberry seedlings: seedlings must be less than six months old, have 2-4 branches, and roots must grow only to two-thirds of the pot or fill it completely, without aging or becoming entangled. Under soilless cultivation conditions with controlled water and fertilizer, they can bloom and bear fruit within 7-10 months of planting, with high yields. It's common for a single plant to produce 1-2 kg of fruit in the first year, and 4-5 kg ​​in the second.

[0005] In order to adapt to the efficient soilless cultivation model of blueberries with high investment, high yield, high quality and high returns, it is necessary to provide blueberry seedlings with root distribution, plant type, branching level, seedling age, etc. that can meet higher requirements of cultivation methods to adapt to new planting needs. Therefore, it is urgent to carry out research and development of blueberry seedling technology. Summary of the Invention

[0006] To solve the problems existing in the cultivation of blueberry tissue culture seedlings, such as long seedling raising cycle, mixed varieties, variation, root entanglement, root entanglement, root aging, and the resulting plant death, poor growth and low yield, and to adapt to the new demand for tissue culture seedlings in efficient soilless cultivation models, the present invention provides a blueberry seedling raising method.

[0007] The first aspect of the present application provides a method for growing blueberry seedlings, comprising the following steps:

[0008] 1) Explants were taken from single plants with preferred markers and cut into small segments of 1-2 nodes. After washing, the explants were refrigerated at 4-6° C. for 15-20 hours, and then sterilized in a 0.2% mercuric chloride solution for 30-40 minutes. The explants were then rinsed twice with sterile water to obtain treated explants.

[0009] 2) inoculating the explant treated in 1) into an induction medium for culturing to obtain germinated axillary buds;

[0010] The formula of the induction medium is: 1 / 4 modified MS + 3 / 4 modified WPM + ZT 3.0-4.0 mg / L + GA 30.3 mg / L + agar 6.5-7.0 g / L + sugar 20-25 g / L; pH value is 5.2-5.6;

[0011] Formula of modified WPM: K2SO4 495mg / L, KH2PO4 340mg / L, CuSO4·5H2O0.25mg / L, ZnSO4·7H2O 8.6mg / L, MnSO4·H2O 22.4mg / L, Na2MoO4·2H2O 0.25mg / L, Ca(NO3)2·4H2O 139mg / L, NH4 N03 400mg / L, CaCl2·2H2O 96mg / L;

[0012] Formula of modified MS: H3BO3 3.1mg / L, MgSO4·7H2O 370mg / L, Na 2- EDTA 37.3 mg / L, FeSO4·7H2O 27.8 mg / L, inositol 100 mg / L, vitamin B6 0.5 mg / L, niacin 0.5 mg / L, glycine 2 mg / L, vitamin B1 1.0 mg / L;

[0013] 3) continuing to culture the axillary buds in 2) for 10-21 days to obtain primary buds; cutting the primary buds into small segments of 1-2 nodes, inoculating them into a subculture proliferation medium for a first subculture to obtain adventitious buds; after 35 days of the first subculture, cutting the adventitious buds into small segments to obtain adventitious bud small segments;

[0014] The formula of the subculture proliferation medium is: 1 / 2 modified MS + 1 / 2 modified WPM + ZT1.5-2.0 mg / L + NAA0.2 mg / L + agar 6.5 g / L + sugar 25 g / L;

[0015] 4) Subculturing the adventitious bud segments in 3) for a second time, with the ZT concentration decreasing by 0.5 mg / L each time, and maintaining the ZT concentration in the subculture proliferation medium at 0.2-0.3 mg / L starting from the 4th-5th subculture; obtaining subculture flask seedlings after 30 days of culture.

[0016] Furthermore, the culture conditions in 2) are: temperature 25-28° C., light intensity 2000-3000 Lx, and light duration 8 hours; and the culture time is 15-20 days.

[0017] Furthermore, the culturing steps for each generation after the second subculture in 4) are repeated by the steps in 3); the small segment has 1-2 branches and 1-3 nodes.

[0018] A second aspect of the present application provides a method for cultivating commercial blueberry seedlings in one step, the method comprising the following steps:

[0019] (1) The subcultured seedlings obtained by the above-mentioned seedling-forming method are moved to a greenhouse for acclimation and cultivation; the acclimated tissue culture seedlings are removed from the bottle, cut into small pieces, and then immersed in a 2000-fold dilution of a rooting agent for 5 minutes, removed, and the liquid is drained and stored for later use;

[0020] (2) Peat, perlite and coconut coir were mixed evenly in a volume ratio of 6:2:2 and placed in a nutrient cup. The mixture was then watered thoroughly. The small segment processed in (1) was vertically inserted into the nutrient cup containing the culture medium.

[0021] When small cup seedlings are cultivated, the particle size of the peat is 0-20 mm;

[0022] When cultivating medium and large pot seedlings, the peat has particle sizes of 0-40 mm and 10-30 mm; the volume ratio of 0-40 mm particle size peat to 0-30 mm particle size peat is 4:2;

[0023] (3) After planting, water the roots first, then spray with a broad-spectrum fungicide, then cover the shed to form a small arch, and cover the small arch with film and shade net. For the first 20 days, keep the light in the shed at 3000-12000Lx and the temperature at 20-30℃;

[0024] The humidity of the small arch is kept above 95%. After 30 days, the sunshade net covering the small arch is removed, and after 35 days, the film covering the small arch is removed.

[0025] The greenhouse shade net should be unfolded when the sunlight intensity at noon is higher than 20,000Lx, and the greenhouse temperature should be maintained at 10-30℃ and the humidity should be above 60%. After 40 days, normal water and fertilizer management should be carried out, and 1000-1500 times diluted compound fertilizer of 17:17:17+TE should be sprayed, and the fertilization cycle should be 10-15 days / time.

[0026] After 85-95 days of cultivation, when the root system has grown all over the nutrient cup, the seedling height is 15-25 cm, and there are more than 2 branches, the blueberry small cup commercial seedlings can be transplanted.

[0027] Medium and large seedlings are cultivated for 120 to 150 days. When the roots have fully grown in the nutrient pots and the seedlings are 25 to 40 centimeters tall and have more than three branches, they can be transplanted to obtain medium and large cup commercial blueberry seedlings.

[0028] Furthermore, (1) the conditions of the medium greenhouse are: temperature 20-30°C, light 5000-10000Lx; and acclimatization and cultivation time is 5-7 days.

[0029] Furthermore, the small segments in (1) are segments with terminal buds cut into 3.5-4.5 cm, and segments without terminal buds cut into 2.5-3.5 cm.

[0030] Furthermore, the insertion depth in (2) is 1.0-1.2 cm.

[0031] Furthermore, the broad-spectrum fungicide in (3) is used at a concentration of 1000-1500 times.

[0032] The third aspect of the present application provides an application of the above-mentioned seedling-forming method in cultivating commercial blueberry seedlings.

[0033] Beneficial effects

[0034] 1. Develop a special basic culture medium for blueberry tissue culture, which significantly improves the culture effect compared with WPM culture medium.

[0035] Based on the improvement of the MS medium commonly used in plant tissue culture and the WPM medium commonly used in blueberry, through further experiments on the mixing ratio of the two improved culture media, a basic culture medium for blueberry tissue culture was obtained, which has the characteristics of fast explant germination time, short subculture cycle, good and strong seedling growth under the same hormone ratio conditions. This significantly improves the tissue culture efficiency of blueberries and the quality of tissue culture seedlings, and significantly increases the induced germination rate of blueberries to 92.5%.

[0036] 2. Improve the quality of seedlings and prevent impure varieties, degeneration and mutation.

[0037] The present invention calibrates and verifies seed mother plants, reduces zeatin (ZT) from generation to generation during the subculture and proliferation process until the minimum ZT concentration required for maintaining strong seedling growth is achieved, and the number of generations in the subculture and production stages is controlled within 10 generations. On the one hand, this avoids late planting losses caused by impure seed sources and provides consistent variety seedlings for production. On the other hand, during the subculture process, ZT, which is prone to cause variation but is indispensable for blueberry tissue culture, is used in a concentration-decreasing manner from generation to generation, thereby reducing tissue culture variation and excessive nutrient branching caused by generation-by-generation accumulation. Thirdly, the number of tissue culture subcultures is strictly controlled within 10 generations, thereby reducing variation, weakened growth and seedling degeneration caused by excessive subcultures. From a technical perspective, it is ensured that the growth and branching of the seedlings are carried out in a normal state. The multiplication multiple after five subcultures is 404.8 times, thereby ensuring an effective multiplication rate, improving seedling quality, and reducing and lowering the risk of degeneration and variation.

[0038] 3. Reduce links, shorten time, improve efficiency, and fundamentally solve various root problems of seedlings.

[0039] The present invention adopts a one-step seedling cultivation method, eliminating the need for rooting nodes within the bottle, shortening the seedling raising cycle by an average of 19.4 days. The directly induced roots are evenly distributed and can continuously grow in all directions within the container. Root problems that persist after changing the pot due to the small holes in the pot tray and the rapid growth of roots to the hole walls, such as aging, entanglement, and root coiling, are avoided. This ensures the quality of commercial tissue culture seedlings. In existing tissue culture seedling production technologies, on the one hand, if the pot tray is transplanted after rooting in the bottle, the roots will cluster together and be unable to spread out, resulting in uneven root distribution. On the other hand, if the pot tray seedling stage is not changed in time, the roots of the seedlings in the pot will grow full of the hole, entangle with the hole walls, age and turn yellow, and float upward, affecting the formation of new roots after transplanting into a nutrient pot or planting pot, and causing the death of the entire plant or part of its branches due to root coiling and root coiling.

[0040] It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present application. DETAILED DESCRIPTION

[0041] The embodiments of the present application will be described in more detail below. However, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided to make the present application more thorough and complete, and to fully convey the scope of the present application to those skilled in the art.

[0042] This invention is mainly aimed at the outstanding problems in the existing production of blueberry tissue culture seedlings: (1) Due to the excessive amount of cytokinin used in the subculture proliferation stage, the vegetative growth is too vigorous, the flowering and fruiting are poor or delayed, and the mutation rate increases; (2) the tissue culture seedling cultivation cycle is long: it takes rooting in the bottle, cultivating the plug seedlings, and cultivating the small cup seedlings (or medium and large cup seedlings) to obtain commercial seedlings that can be planted in the field. The rooting in the bottle takes 25-30 days, and after the bottle is taken out, each acclimatization stage from the plug seedlings to the cup (pot) seedlings takes 50-70 days, and it takes a total of 5-6 months to cultivate into commercial seedlings that can be transplanted into the planting pot. (3) This technology uses the technology of cultivating commercial seedlings once to fundamentally solve the problems of root coiling, root entanglement, and root aging and yellowing.

[0043] The present invention involves individual plant calibration, explant selection, basic culture medium formulation development and preparation, axillary bud induction, zeatin (ZT)-decreasing subculture, control of proliferation generations, substrate formulation and environmental regulation during one-step cultivation of commercial seedlings, and variety and quality testing and verification. The invention achieves a 92.5% success rate for bud induction, a 404.8-fold proliferation after five subcultures, a 98.2% rooting rate for commercial seedlings after 25 days of cultivation, and a 19.4-day shortened cultivation time for commercial seedlings.

[0044] Example 1

[0045] 1. Explant Selection, Disinfection, and Calibration: Select individual 'Misti' plants with good growth, high yield, and no symptoms, number them, and mark them as preferred. Select the marked plants and cut semi-lignified vegetative branches with a diameter of 2-4 mm and plump axillary buds. Remove the leaves and cut them into small segments with 1-2 nodes. After washing, refrigerate at 4-6°C for 20 hours, disinfect them in a 0.2% mercuric chloride solution for 32 minutes, and rinse them twice with sterile water.

[0046] 2. Axillary bud induction culture: inoculate the material treated in step 1 into the induction culture medium made by mixing the modified MS and WPM basic culture media.

[0047] The basic culture medium of the present invention is based on modified MS medium and modified WPM medium. The modified medium is mixed in different proportions according to the different culture stages of blueberries to prepare the required culture medium. The specific components and contents of the MS and WPM modified basic culture media are as follows:

[0048] Modified WPM medium: K2SO4 495mg / L, KH2PO4 340mg / L, CuSO4·5H2O0.25mg / L, ZnSO4·7H2O 8.6mg / L, MnSO4·H2O 22.4mg / L, Na2MoO4·2H2O 0.25mg / L, Ca(NO3)2·4H2O 139mg / L, NH4 N03400mg / L, CaCl2·2H2O 96mg / L.

[0049] Modified MS medium: H3BO3 3.1mg / L, MgSO4·7H2O 370mg / L, Na 2- EDTA 37.3 mg / L, FeSO4·7H2O 27.8 mg / L, inositol 100 mg / L, vitamin B6 0.5 mg / L, niacin 0.5 mg / L, glycine 2 mg / L, vitamin B1 1.0 mg / L.

[0050] The induction medium formula is: modified 1 / 4MS + modified 3 / 4WPM + ZT3.0mg / L + GA30.3mg / L + agar 7.0g / L + sugar 20g / L, pH 5.2;

[0051] The culture temperature is 25-26℃, the light intensity is 2000-3000Lx, and the lighting time is 8 hours. After 20 days of culture, the axillary buds germinate, and the culture is continued for 10-20 days. When most axillary buds grow into primary buds of more than 1 cm, subculture transfer is carried out.

[0052] 3. Zeatin (ZT) decreasing subculture: Cut the primary buds from step 2 into small segments with 1-2 nodes and inoculate them into subculture proliferation medium (1 / 2 MS modified MS + 1 / 2 WPM modified + ZT 2.5 mg / L + NAA 0.2 mg / L + agar 6.5 g / L + sugar 25 g / L). After 35 days of culture, cut the adventitious buds into small segments with 1-2 branches and 1-3 nodes and continue subculture. Repeat the above steps for the second, third, fourth, and fifth subcultures, but reduce the ZT concentration by 0.5 mg / L each time. Starting from the sixth subculture, maintain the ZT concentration in the subculture proliferation medium at 0.3 mg / L. The subculture medium pH is 5.4, the culture temperature is 25-28°C, the light intensity is 3000-5000 Lx, the light duration is 8 hours, and the subculture cycle is 30-35 days.

[0053] The specific components and content compositions of the modified MS medium and modified WPM medium described in steps 2 and 3 are the same as those in step 2 above.

[0054] 4. One-step cultivation of commercial seedlings

[0055] Depending on the market demand for commercial blueberry seedlings, the specifications of commercial seedlings can be small cup seedlings (volume 0.2-0.5 liters), medium and large pot seedlings (volume 0.7-1.5 liters).

[0056] After 6-10 generations of subculture in step 3, transfer the subcultured seedlings to a greenhouse at 20-30°C and 5,000-10,000 Lx for acclimatization for 7 days. Then, take cuttings outside the jars and further cultivate them into commercial seedlings. During acclimatization, control the temperature and light intensity by opening and closing the greenhouse's top windows, using side films, using shade nets, and using thermal screens.

[0057] In the cultivation of commercial seedlings, the following steps are carried out:

[0058] (1) Pretreatment of subcultured seedlings: Take the acclimated tissue culture seedlings out of the bottle and cut them into small segments. The segments with terminal buds are cut into 3.5-4.5 cm, and the segments without terminal buds are cut into 2.5-3.5 cm. Then soak them in 2000 times the solution of Guoguang Rooting (registration number: PD20061509, production license number: Pesticide Shengxu (Chuan) 0001) for 5 minutes. After taking out and draining the liquid, put them into a sealed container and place them in a cool place.

[0059] (2) Cultivation of commercial seedlings

[0060] Cultivation of small cup seedlings: Mix 0-20mm peat with perlite and coconut coir in a volume ratio of 6:2:2, then put it into a 0.2-0.5 liter nutrient cup, water it thoroughly, and insert the previously processed small segments vertically into the nutrient cup at a depth of 1.0-1.2 cm.

[0061] Cultivation of medium and large pot seedlings: Mix 0-40mm and 10-30mm peat with perlite and coconut particles in a volume ratio of 4:2:2:2, then put them into a 0.7-1.5 liter nutrient pot, water it thoroughly, and insert the previously processed small segments vertically into the nutrient cup at a depth of 1.0-1.2 cm.

[0062] (3) Seedling environment control and management: After the seedlings are planted, water them to help them take root, then spray them with a 1000-1500 times concentration of a broad-spectrum fungicide. Cover the shed (forming a small shed) with film and shade nets. For the first 20 days, maintain a light intensity of 3000-12000 Lx, a temperature of 20-30°C, and humidity above 95% in the shed. Remove the shade nets covering the sheds after 30 days, and remove the film covering the sheds after 35 days. Unfold the shade nets when the midday light intensity is higher than 20000 Lx. Maintain the shed temperature at 10-30°C and humidity above 60%. The rooting rate is 98.2% after 25 days of cultivation. After 40 days, normal water and fertilizer management is carried out, spraying with a 1000-1500 times dilution of 17-17-17-TE compound fertilizer. Fertilization is repeated every 10-15 days. Small cup seedlings are cultivated for 85-95 days, and can be transplanted when the roots have filled the nutrient cup, the seedling height is 15-25 cm, and there are more than 2 branches; medium and large pot seedlings are cultivated for 120-140 days, and can be transplanted when the roots have filled the nutrient pot, the seedling height is 25-40 cm, and there are more than 3 branches.

[0063] 5. Variety and quality testing: When the number of tissue culture seedlings has exceeded 100, randomly select 5 seedlings to be cultivated in the above steps and then tested for planting. Compare the growth, branches, leaves and other botanical characteristics with the original mother plant 'Misty'. If the two are completely consistent, then production can be carried out.

[0064] Example 2

[0065] Except for the following steps, the remaining steps of Example 2 are the same as those of Example 1.

[0066] 1. Selection, disinfection and calibration of explants: The variety is named 'F6' and the refrigeration time in the refrigerator is 15 hours.

[0067] 2. Axillary bud induction culture: The induction culture medium formula is: improved 1 / 4MS + improved 3 / 4WPM + ZT4.0mg / L + GA30.3mg / L + agar 6.5g / L + white sugar 20g / L.

[0068] 3. ZT decreasing subculture: Same as Example 1, except that the ZT concentration in the subculture proliferation medium was maintained at 0.2 mg / L starting from the fifth subculture. The pH value of the subculture medium was 5.6.

[0069] 4. One-step cultivation of commercial seedlings in medium and large pots: Move the subculture bottle seedlings that have been subcultured for 6 generations and 32 days in step 3 to a greenhouse with a temperature of 20-30°C and a light intensity of 5000-10000Lx for acclimatization and cultivation for 5 days, then carry out cuttings outside the bottle and further cultivate them into commercial seedlings.

[0070] In the cultivation of commercial seedlings, the following steps are carried out:

[0071] (1) Pretreatment of subcultured bottle seedlings: The method was the same as that in Example 1, except that the disinfectant was immersion in a 2000-fold solution of Aierjia Haizao (registration number: Agricultural Fertilizer (2015), standard number 4481) for 4 minutes.

[0072] (2) Microbranch cuttings of commercial seedlings in medium and large pots: Mix 0-40mm and 10-30mm peat with perlite and coconut particles in a ratio of 5:1:2:2, and put them into a 1.0 liter nutrient pot. Water it thoroughly and insert the bud segment processed above into the nutrient cup at a depth of 1.0-1.2 cm.

[0073] (3) Seedling environment control and management: Same as Example 1. Except that the seedlings should be cultured for more than 125 days, the roots should be fully grown in the nutrient pot, the seedlings should be 25-30 cm tall, and have more than 3 branches, they can be transplanted directly into the planting pot.

[0074] 5. Variety and quality test: Same as Example 1, except that 5 plants were randomly selected for medium and large cup seedling cultivation and then planted for testing.

[0075] Example 3: Comparative experiment

[0076] The following experiments were conducted: (1) the effects of different improved culture medium ratios on induction rate and subculture proliferation times; (2) the effects of ZT concentration in subculture on tissue culture efficiency and seedling quality; and (3) the study of the effects of this application and the prior art on seedling raising time and quality.

[0077] (1) Effects of different improved culture medium ratios on induction rate and subculture proliferation

[0078] This experiment used the method of the present invention to conduct experimental research starting from explant induction. The same operating steps and hormone ratios as in Example 1 were adopted. The varieties 'F6' and 'Faxin' were used as materials. 40 buds of each variety were selected as explants. By statistically analyzing the induction success rate and the number of proliferation bottles after 5 generations of subculture, the effect of the basic culture medium ratio on the induction and subculture proliferation effects was studied.

[0079] As shown in Table 1, different medium ratios significantly affected the germination rate after seeding and the proliferation rate after five subcultures. The best induction culture was achieved with a medium ratio of 1 / 4 MS + 3 / 4 WPM, with a germination rate of 92.5%. Subculture was most effective with a medium ratio of 1 / 2 MS + 1 / 2 WPM, with a proliferation rate of 404.8 after five subcultures. The second-best effect was achieved with a medium ratio of 1 / 2 MS + 1 / 2 WPM.

[0080] Table 1 Effects of different culture medium ratios on induction rate and proliferation number

[0081]

[0082] Note: The number of seedlings in each bottle for 1-5 generations of subculture is 10 buds / bottle.

[0083] (2) Effect of subculture ZT concentration on tissue culture efficiency and seedling quality

[0084] This study used 1 / 2 MS + 1 / 2 WPM + 0.3 mg / L NAA as the basic culture medium and auxin dosages, with zeatin (ZT) dosages of 0.5 mg / L, 1.0 mg / L, 1.5 mg / L, 2.0 mg / L, 2.5 mg / L, and 3.0 mg / L, respectively. Single buds of consistent growth were used as materials. After three consecutive generations in the same culture medium with the aforementioned ZT concentrations, the proliferation rate, average bud height, and average number of branches were calculated, and the leaf and plant morphology were observed. The results in Table 2 show that with increasing ZT concentration, the number of branches increased, the plant height decreased, and the leaves became thicker. When the ZT concentration is 1.5mg / L-2.0mg / L, a high proliferation multiple can be guaranteed and the plant morphology and leaf morphology are normal; when the ZT concentration is lower than 1mg / L, the number of branches is small but the plant grows tall, and the plant and leaf morphology are normal; when the ZT concentration is higher than 2.5mg / L, the number of branches increases, the plant leaves become thicker, the leaves become shorter, the internodes shorten, and other morphological variation trends are obvious, and the trend of proliferation multiples increasing with the increase of ZT concentration is not obvious.

[0085] Table 2: Effects of ZT on tissue culture proliferation and variation in subculture

[0086]

[0087] (3) Effects of the one-step seedling method and the original method on seedling raising time and quality

[0088] After the strong seedlings obtained in the above experiment (1) after 5 generations were cut into individual plants, they were cultured in bottles for rooting and directly cut into plants outside the bottles. After the rooted plants were cultured in bottles for 20-25 days to form root primordia, they were removed from the bottles and transplanted. After the cuttings were cut and pre-treated according to the seedling raising steps (1) and (2) in Example 1, they were directly inserted into small cups or medium or large pots for commercial seedling cultivation. When they reached the standard for planting, 30 plants were randomly selected, the roots were cleaned, and the root entanglement and root coiling conditions were counted, as well as the time it took to cultivate them into commercial seedlings and medium or large pot seedlings for planting. As can be seen from Table 3 below: using the one-step seedling method, the seedling raising time of small cup seedlings was 92.05 days, which was 16.0 days shorter than the original technology of 108 days. The seedling raising time of medium and large pot seedlings was 131.2 days, which was 22.9 days shorter than the original technology of 154.1 days. The average seedling raising time was shortened by 19.4 days. In addition, the proportion of root entanglement and root coiling was less than 1%. The one-step seedling technology allows the roots to grow in all directions after germination and be evenly distributed, effectively improving the quality of the seedlings and the subsequent growth of the plants.

[0089] The above examples further verify that the present application can reduce the seedling raising steps and shorten the seedling raising cycle, improve the seedling raising efficiency and seedling quality, and reduce the risk of variation.

[0090] Table 3: Effects of the one-step seedling method and the original method on seedling raising time and root system

[0091]

[0092] The embodiments of the present application have been described above. The above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is selected to best explain the principles of the embodiments, their practical applications, or improvements to the technology in the market, or to enable other persons skilled in the art to understand the embodiments disclosed herein.

Claims

1. A method for growing blueberry seedlings, characterized in that: The following steps are involved: 1) Explants were taken from single plants with preferred markers and cut into small segments of 1-2 nodes. After washing, the explants were refrigerated at 4-6°C for 15-20 hours, sterilized in a 0.2% mercuric chloride solution for 30-40 minutes, and then rinsed twice with sterile water to obtain the treated explants. 2) inoculating the explant treated in 1) into an induction medium for culturing to obtain germinated axillary buds; The formula of the induction medium is: 1 / 4 modified MS + 3 / 4 modified WPM + ZT 3.0-4.0 mg / L + GA 30.3 mg / L + agar 6.5-7.0 g / L + white sugar 20-25 g / L; pH value is 5.2-5.6; 3) continuing to culture the axillary buds in 2) for 10-20 days to obtain primary buds, cutting the primary buds into small segments of 1-2 nodes, inoculating them into a subculture proliferation medium for a first subculture to obtain adventitious buds, and after 35 days of the first subculture, cutting the adventitious buds into small segments to obtain adventitious bud small segments; The formula of the subculture proliferation medium is: 1 / 2 modified MS + 1 / 2 modified WPM + ZT1.5-2.0 mg / L + NAA0.2 mg / L + agar 6.5 g / L + white sugar 25 g / L; 4) Subculturing the adventitious bud segments in 3) for a second time, with the ZT concentration in the subculture proliferation medium decreasing by 0.5 mg / L each time, and maintaining the ZT concentration in the subculture proliferation medium at 0.2-0.3 mg / L starting from the fourth or fifth subculture; the subculture cycle is 30 days, and subculture bottle seedlings are obtained; The formula of the improved WPM is: K2SO4 495mg / L, KH2PO4 340mg / L, CuSO4·5H2O 0.25mg / L, ZnSO4·7H2O 8.6mg / L, MnSO4·H2O 22.4mg / L, Na2MoO4·2H2O 0.25mg / L, Ca(NO3)2·4H2O 139mg / L, NH4 NO3 400mg / L, CaCl2·2H2O 96mg / L; The formula of the improved MS is: H3BO3 3.1 mg / L, MgSO4·7H2O 370 mg / L, Na2-EDTA 37.3 mg / L, FeSO4·7H2O 27.8 mg / L, inositol 100 mg / L, vitamin B6 0.5 mg / L, niacin 0.5 mg / L, glycine 2 mg / L, and vitamin B1 1.0 mg / L.

2. The method according to claim 1, characterized in that The culture conditions in 2) are: temperature 25-28° C., light intensity 2000-3000 Lx, and illumination time 8 hours; and the culture time is 15-20 days.

3. The method according to claim 1, characterized in that The culturing steps for each generation after the second subculture in said 4) are to repeat the steps in said 3); the adventitious bud segment in said 4) has 1-2 branches and 1-3 nodes.

4. Use of the method for establishing blueberry seedlings according to claim 1 in cultivating commercial blueberry seedlings.

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