Blueberry seedling method and application thereof

Abstract

The invention provides a blueberry seedling method and application thereof. The method comprises the following steps: selection and treatment of plants and explants, induction culture of axillary buds in an improved culture medium, zeatin (ZT) decreasing subculture, and further cultivation of subculture seedlings into commercial seedlings. According to the method provided by the invention, the basic culture medium prepared from the improved MS and WPM culture media is adopted, so that explants germinate quickly, the subculture period is short, seedlings grow well, branching is reasonable, and buds are thick and strong. The induction budding rate of the blueberries is increased to 92.5%, and the multiplication rate multiple after five times of subculture reaches 404.8 times. When commercial seedlings are cultured for 25 days by adopting a one-step seedling method, the rooting rate reaches 98.2%, the seedling culture time is averagely shortened by 19.4 days, and root systems are dispersed and uniform. According to the scheme, the seedling raising efficiency, the seedling quality and the plant growth vigor after planting are effectively improved, and the variation risk is reduced. Various root system problems which seriously affect survival, growth and yield after planting are fundamentally solved.

Description

Technical Field

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

[0002] With the rapid development of the blueberry industry, the tissue culture and rapid propagation technology of blueberry seedlings has become increasingly mature and applied on a large scale. The use of conventional tissue culture technology provides good technical support and guarantee for the breeding and industrial development of blueberry seedlings. However, in the past two years, the rapid increase in the planting area has led to a sharp increase in the demand for seedlings, especially in the supply of new and superior varieties of seedlings. In order to pursue high proliferation rates and seedling production, excessive use of cytokinins, failure to update seed sources in a timely manner, and unlimited increase in the number of successive generations to maintain seedling production, as well as the phenomenon of performing a second or even more topping bud cuttings on production tissue culture seedlings to cultivate second and third cuttings, have increased. As a result, the quality of seedlings has become increasingly poor, mutations, poor root systems and growth, low yields, and even plant death are difficult to avoid and can be seen everywhere, causing great losses to later planting.

[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 the propagation, 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 and rooting. In addition, there are many blueberry strains and varieties with great differences, which leads to long propagation cycle, uneven seedling quality, poor growth and rooting; (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 transplanted out of the bottle into a plug tray to cultivate into plug tray seedlings. The plug tray seedlings are then transplanted twice or three times to small cups or medium to large-sized seedling pots to cultivate into commercial seedlings according to demand. Not only is the production cycle of commercial seedlings long, but at all stages of tissue culture seedling cultivation, especially the plug tray seedling stage, there are prominent problems such as root entanglement, root entanglement, and old roots due to small holes and untimely cup changing, which affect the quality in the later stage.

[0004] 5-10 years ago, growers required blueberry seedlings to be 1.5-2 years old, 30-40 cm tall, and to bear fruit 2-3 years after planting. In the past 2-3 years, with the widespread application of blueberry soilless cultivation technology across the country, new requirements have been put forward for blueberry seedlings: seedlings within half a year, 2-4 branches, especially the root system can only grow to 2 / 3 of the pot body or full pot, and cannot age and entangle. Under the conditions of soilless cultivation controlled by water and fertilizer, it can bloom and bear fruit in 7-10 months of planting and have a high yield. It is common to see a single plant produce 1-2 kg of fruit in the first year, and 4-5 kg ​​in the second year.

[0005] In order to adapt to the efficient soilless cultivation model of blueberry with high investment, high yield, high quality and high return, 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 the research and development of blueberry seedling technology. Summary of the invention

[0006] The present invention provides a blueberry seedling method to solve the problems of long seedling raising period, mixed varieties, variation, root coiling, root entanglement, root aging and the resulting plant death, poor growth and low yield in the cultivation of blueberry tissue culture seedlings, and to meet the new demand for tissue culture seedlings in the efficient soilless cultivation mode.

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

[0008] 1) taking explants from the single plants with the preferred mark, cutting the explants into small segments of 1-2 nodes, washing, refrigerating at 4-6° C. for 15-20 hours, sterilizing in a 0.2% mercuric chloride solution for 30-40 minutes, and then rinsing with sterile water twice to obtain the treated explants;

[0009] 2) inoculating the explants 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 improved 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 N03400mg / L, CaCl2·2H2O 96mg / L;

[0012] Formula of modified MS: H3BO3 3.1mg / L, MgSO4·7H2O 370mg / L, Na 2- EDTA 37.3mg / L, FeSO4·7H2O 27.8mg / L, inositol 100mg / L, vitamin B6 0.5mg / L, niacin 0.5mg / L, glycine 2mg / L, vitamin B1 1.0mg / 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 the first subculture to obtain adventitious buds; after the first subculture for 35 days; 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 + ZT1.5-2.0mg / L + NAA0.2mg / L + agar 6.5g / L + white sugar 25g / L;

[0015] 4) Subculturing the adventitious bud segments in 3) for the 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 from the 4th to 5th subculture; after the culture time reaches 30 days, obtaining the subculture bottle seedlings.

[0016] Furthermore, the culture conditions in 2) are: temperature 25-28°C, light intensity 2000-3000Lx, 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 to repeat the steps in 3); the small segment has 1-2 branches and 1-3 nodes.

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

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

[0020] (2) Peat, perlite and coconut bran were mixed evenly in a volume ratio of 6:2:2 and placed in a nutrient cup, which 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 medium and large pot seedlings are cultivated, the peat has a particle size of 0-40 mm and 10-30 mm; the volume ratio of the peat with a particle size of 0-40 mm to the peat with a particle size of 0-30 mm is 4:2;

[0023] (3) After planting, first water the roots, then spray a broad-spectrum fungicide, then cover the shed to form a small arch, and cover the small arch with a film and a 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 maintained 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 is unfolded when the light intensity at noon is higher than 20000Lx, and the greenhouse temperature is maintained at 10-30℃ and the humidity is above 60%; after 40 days, normal water and fertilizer management is carried out, and 1000-1500 times of 17:17:17+TE compound fertilizer is sprayed, and the fertilization cycle is 10-15 days / time;

[0026] After 85-95 days of cultivation, the small cup seedlings can be transplanted 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, and the blueberry small cup commercial seedlings are obtained;

[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 the 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 for plant tissue culture and the WPM medium commonly used for 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 a fast explant germination time, a short subculture cycle, and good and strong seedling growth under the same hormone ratio conditions. It significantly improved the tissue culture efficiency of blueberry and the quality of tissue culture seedlings, and significantly increased the induced germination rate of blueberry to 92.5%.

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

[0037] The invention calibrates and verifies the mother plant for seed collection, and reduces zeatin (ZT) from generation to generation during the process of subculture and proliferation until the minimum ZT concentration required for maintaining the growth of strong seedlings is achieved, and the number of generations in the subculture and production stages is controlled within 10 generations. On the one hand, the late planting loss caused by impure seed sources is avoided, and consistent variety seedlings are provided for production. On the other hand, in the process of subculture, the concentration of ZT, which is easy to cause variation but is indispensable for blueberry tissue culture, is used in a generation-by-generation decreasing manner, so as to reduce 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, so as to reduce variation, weakened growth and seedling degeneration caused by too many subcultures, and technically ensure that the growth and branching of the seedlings are carried out in a normal state. The proliferation multiple after five subcultures is 404.8 times, so that the effective proliferation rate is guaranteed, the seedling quality is improved, and the risk of degeneration and variation is reduced and lowered.

[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, which reduces the rooting nodes in the bottle, shortens the seedling raising cycle, shortens the seedling raising time by 19.4 days on average, and the directly induced root system is evenly distributed and can continuously grow in all directions in the container, and there will be no root system problems after changing the cup due to the small hole of the hole plate, the root system quickly grows to the hole wall, and then ages, entangles, and coils in the hole, thereby ensuring the quality of the tissue culture commercial seedlings. In the existing tissue culture seedling production technology, on the one hand, after the rooting in the bottle is transplanted, the root system will pile up and cannot stretch out during transplanting, resulting in uneven root distribution; on the other hand, if the cup is not changed in time at the hole plate seedling stage, the root system of the seedling in the hole will be full of the hole, entangled in the hole wall, aged and yellowed, and the root system will float, etc., which affects the generation of new roots after transplanting to the nutrient pot or planting pot, and the death of the whole plant or part of the branches after coiling and winding.

[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 technical invention is mainly aimed at the outstanding problems in the existing blueberry tissue culture seedling production: (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, plug tray seedling cultivation, small cup seedling cultivation (or medium and large cup seedling cultivation) to obtain commercial seedlings that can be planted in the field. The rooting in the bottle takes 25-30 days. After the bottle is taken out, each domestication stage from plug tray seedling to cup (pot) seedling 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 entanglement, root entanglement, and root aging and yellowing.

[0043] The invention performs single plant calibration, explant selection treatment, basic culture medium formula development and preparation, axillary bud induction, zeatin (ZT) decreasing subculture, control of proliferation generation, matrix formula and environmental regulation in one-step seedling cultivation of commercial seedlings, variety and quality testing and verification on blueberry seed mother plants. The success rate of seed induction germination reaches 92.5%, the proliferation multiple after five subcultures reaches 404.8 times, the rooting rate of commercial seedlings after 25 days of cultivation reaches 98.2%, and the seedling cultivation time of commercial seedlings is shortened by 19.4 days.

[0044] Example 1

[0045] 1. Selection, disinfection and calibration of explants: Select the 'Misti' plants with good growth, high yield and no symptoms, number them and mark them for selection. Select the plants with the selection mark, cut the semi-lignified nutrient branches with a thickness of 2-4mm and full axillary buds, remove the leaves and cut them into small sections with 1-2 nodes, wash them, refrigerate them in a refrigerator at 4-6℃ for 20 hours, disinfect them in a 0.2% mercuric chloride solution for 32 minutes, and rinse them with sterile water twice.

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

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

[0048] Improved WPM culture 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] Improved MS medium: H3BO3 3.1mg / L, MgSO4·7H2O 370mg / L, Na 2- EDTA 37.3mg / L, FeSO4·7H2O 27.8mg / L, inositol 100mg / L, vitamin B6 0.5mg / L, niacin 0.5mg / L, glycine 2mg / L, vitamin B1 1.0mg / L.

[0050] The formula of the induction medium is: improved 1 / 4MS + improved 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, axillary buds germinate, and the culture is continued for 10-20 days. When most axillary buds grow into primary buds of more than 1cm, subculture transfer is carried out.

[0052] 3. Zeatin (ZT) decreasing subculture: Cut the primary buds in step 2 into small segments with 1-2 nodes, inoculate them into subculture proliferation medium (1 / 2MS modified MS + 1 / 2WPM modified + ZT2.5mg / L + NAA0.2mg / L + agar 6.5g / L + white sugar 25g / L), and after 35 days of culture, cut the adventitious buds into small segments with 1-2 branches and 1-3 nodes for continued subculture. Repeat the above steps for the second, third, fourth, and fifth subcultures, but the ZT concentration should be reduced by 0.5mg / L each time, and the ZT concentration in the subculture proliferation medium should be maintained at 0.3mg / L from the sixth subculture. The pH value of the medium in the subculture is 5.4, the culture temperature is 25-28℃, the light intensity is 3000-5000Lx, the light time 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 step 2 and step 3 are the same as those in step 2 above.

[0054] 4. One-step seedling 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] The subcultured bottle seedlings with 6-10 subculture times in step 3 are moved to a greenhouse with a temperature of 20-30°C and a light intensity of 5000-10000Lx for acclimatization and cultivation for 7 days, and then cut out of the bottle and further cultivated into commercial seedlings. During the acclimatization and cultivation, the temperature and light intensity are controlled by opening and closing the greenhouse top window, side film, shade net, heat preservation curtain and other measures.

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

[0058] (1) Pretreatment of subcultured seedlings: Take the acclimated tissue culture seedlings out of the bottle and cut them into small segments, where 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 dilution 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 bran in a volume ratio of 6:2:2, 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, and 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 establish roots, then spray them with 1000-1500 times of broad-spectrum fungicide, then cover the arch shed (forming a small arch) and cover it with film and shade net. In the first 20 days, keep the light in the greenhouse at 3000-12000Lx, the temperature at 20-30℃, and the humidity in the small arch above 95%. After 30 days, remove the shade net covering the small arch, and after 35 days, remove the film covering the small arch. Unfold the shade net when the light intensity at noon is higher than 20000Lx, and keep the temperature in the greenhouse at 10-30℃ and the 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 1000-1500 times of 17-17-17-TE compound fertilizer, and the fertilization cycle is 10-15 days / time. Small cup seedlings are cultivated for 85-95 days, and can be transplanted when the roots fill the nutrient cup, the seedling height is 15-25 cm, and it has more than 2 branches; medium and large pot seedlings are cultivated for 120-140 days, and the roots fill the nutrient pot, the seedling height is 25-40 cm, and it has more than 3 branches.

[0063] 5. Variety and quality test: When the number of tissue culture seedlings has exceeded 100, randomly select 5 seedlings to be cultivated in the above steps and then carry out planting test. 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 formula of induction culture medium 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 is maintained at 0.2 mg / L from the 5th subculture. The pH value of the medium in the subculture is 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 5 days, and 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 followed:

[0071] (1) Pretreatment of subculture bottle seedlings: The method was the same as that in Example 1 except that the disinfectant was 2000 times diluted Aierjia Haizao (registration number: Agricultural Fertilizer (2015), approval number 4481) and the seedlings were immersed in it for 4 minutes.

[0072] (2) Microbranch cuttings of commercial seedlings in medium and large pots: Mix 0-40 mm and 10-30 mm 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 cultivation time in the pot seedlings must reach more than 125 days, the root system must grow all over the nutrient pot, the seedling height must be 25-30 cm, and when there are more than 3 branches, it can be taken out of the nursery and directly moved 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 multiples; (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 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 adopted the method of the present invention to conduct experimental research starting from explant induction, using the same operating steps and hormone ratios as in Example 1, using varieties 'F6' and 'Faxin' as materials, and selecting 40 buds as explants for each variety. By statistically analyzing the induction success rate and the number of proliferation bottles after 5 subcultures, the effect of the basic culture medium ratio on the induction and subculture proliferation effects was studied.

[0079] As shown in Table 1, different culture medium ratios have significant differences in the induced germination rate after seeding and the proliferation multiples after five subcultures. The best effect of induced culture was achieved in the culture medium ratio of 1 / 4MS+3 / 4WPM, with an induced germination rate of 92.5%. The best effect of subculture proliferation was achieved in the culture medium ratio of 1 / 2MS+1 / 2WPM, with a proliferation multiple of 404.8 times after five subcultures, and the effect was second in 1 / 2MS+1 / 2WPM.

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

[0081]

[0082] Note: The number of seedlings per bottle in the 1st to 5th generation subculture is 10 buds / bottle.

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

[0084] This study used 1 / 2MS+1 / 2WPM+NAA0.3mg / L as the basic culture medium and the dosage of auxin, and the dosage of zeatin (ZT) was 0.5mg / L, 1.0mg / L, 1.5mg / L, 2.0mg / L, 2.5mg / L and 3.0mg / L, and took single buds with consistent growth as materials. After three consecutive generations in the same culture medium with the above ZT concentration, the proliferation times, average bud height, average number of branches, and the overall morphology of leaves and plants were calculated. From the results in Table 2, it can be seen that with the increase of zeatin (ZT) concentration, the number of branches increased, the height of plants became shorter, and the trend of thickening leaves was obvious. When the ZT concentration is 1.5mg / L-2.0mg / L, a high proliferation multiple can be guaranteed while the plant 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 leaves of the plant 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: Effect 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 of subculture were cut into individual plants, they were respectively subjected to in-bottle rooting culture and direct cutting outside the bottle. After the rooted plants were cultured in the bottle for 20-25 days to form root primordia, they were taken out of the bottle for transplanting. After the cutting outside the bottle was sheared and pre-treated according to the method of seedling raising steps (1) and (2) in Example 1, they were directly inserted into small cups or medium-sized pots for commercial seedling cultivation. When they reached the standard for going out to the nursery, 30 plants were randomly selected, the root system was cleaned, and the root winding and root entangling conditions were counted, as well as the time to cultivate into commercial seedlings and medium-sized pot seedlings for going out to the nursery. It can be seen from Table 3 below that by using the one-step seedling method, the seedling raising time of the small cup seedlings was 92.05 days, which was 16.0 days shorter than the original technology of 108 days, and the seedling raising time of the medium-sized pot seedlings was 131.2 days, which was 22.9 days shorter than the original technology of 154.1 days, and the average seedling raising time was shortened by 19.4 days, and the proportion of root winding and root entangling was less than 1%. The one-step seedling technology's roots grow around them after germination and are evenly distributed, effectively improving the seedling quality and later plant growth.

[0089] The above embodiments 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 for commercial seedlings and the original method on seedling raising time and root system

[0091]

[0092] The embodiments of the present application have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The selection of terms used herein is intended to best explain the principles of the embodiments, practical applications, or improvements to the technology in the market, or to enable other persons of ordinary skill 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) taking explants from the single plants with the preferred mark, cutting the explants into small segments of 1-2 nodes, washing, refrigerating at 4-6° C. for 15-20 hours, sterilizing in a 0.2% mercuric chloride solution for 30-40 minutes, and then rinsing with sterile water twice to obtain the treated explants; 2) inoculating the explants 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 3 0.3 mg / L + agar 6.5-7.0 g / L + white sugar 20-25 g / L; pH value is 5.2-5.6; The formula of the improved WPM: 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 N03 400mg / L, CaCl2·2H2O 96mg / L; The formula of the improved MS is: H3BO3 3.1mg / L, MgSO4·7H2O 370mg / L, Na 2- EDTA 37.3mg / L, FeSO4·7H2O 27.8mg / L, inositol 100mg / L, vitamin B6 0.5mg / L, niacin 0.5mg / L, glycine 2mg / L, vitamin B11.0mg / L; 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 the first subculture to obtain adventitious buds; after the first subculture for 35 days; 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 the 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 to 5th subculture; after the culture time reaches 30 days, obtaining the subculture bottle seedlings.

2. The method according to claim 1, characterized in that The culture conditions in 2) are: temperature 25-28°C, light intensity 2000-3000Lx, and illumination time 8 hours; 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); said small segment has 1-2 branches and 1-3 nodes.

4. A method for cultivating commercial blueberry seedlings in one step, characterized in that: The method comprises the following steps: (1) moving the subculture bottle seedlings obtained by the seedling raising method according to claim 1 into a greenhouse for acclimation and cultivation; taking out the acclimated tissue culture seedlings from the bottle, cutting them into small segments, and then soaking them in a 2000-fold dilution of a rooting agent for 5 minutes, taking them out and filtering the liquid, and storing them for later use; (2) Peat, perlite and coconut bran were mixed evenly in a volume ratio of 6:2:2 and placed in a nutrient cup, which was then watered thoroughly. The small segment processed in (1) was vertically inserted into the nutrient cup containing the culture medium; When small cup seedlings are cultivated, the particle size of the peat is 0-20 mm; When medium and large pot seedlings are cultivated, the peat has a particle size of 0-40 mm and 10-30 mm; the volume ratio of the peat with a particle size of 0-40 mm to the peat with a particle size of 0-30 mm is 4:2; (3) After planting, first water the roots, then spray a broad-spectrum fungicide, then cover the shed to form a small arch, and cover the small arch with a film and a shade net. For the first 20 days, keep the light in the shed at 3000-12000Lx and the temperature at 20-30℃; The humidity of the small arch is maintained at above 95%, and the sunshade net covering the small arch is removed after 30 days, and the film covering the small arch shed is removed after 35 days; The greenhouse shade net is unfolded when the light intensity at noon is higher than 20000Lx, and the greenhouse temperature is maintained at 10-30℃ and the humidity is above 60%; after 40 days, normal water and fertilizer management is carried out, and 1000-1500 times of 17:17:17+TE compound fertilizer is sprayed, and the fertilization cycle is 10-15 days / time; After 85-95 days of cultivation, the small cup seedlings can be transplanted 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, and the blueberry small cup commercial seedlings are obtained; 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.

5. The method according to claim 4, characterized in that The conditions of the greenhouse in (1) are: temperature 20-30°C, light 5000-10000Lx; the time of acclimatization and cultivation is 5-7 days.

6. The method according to claim 4, characterized in that 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.

7. The method according to claim 4, characterized in that The insertion depth in (2) is 1.0-1.2 cm.

8. The method according to claim 4, characterized in that The broad-spectrum fungicide in (3) is used at a concentration of 1000-1500 times.

9. Use of the method for growing blueberry seedlings according to claim 1 in cultivating commercial blueberry seedlings.

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