CN116686716B - Tissue culture seedling raising method for blueberries - Google Patents
Tissue culture seedling raising method for blueberries Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/002—Culture media for tissue culture
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- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a tissue culture seedling raising method for blueberries, and belongs to the technical field of plant tissue culture. The method comprises the following steps: s01, preparation and induction culture of explants: obtaining an explant, and culturing the explant on an induction culture medium to obtain an induction-cultured bud; s02, subculture: culturing the buds subjected to induction culture on a subculture medium to obtain subculture seedlings; s03, rejuvenation culture: culturing the subculture seedlings on a rejuvenation culture medium to obtain rejuvenation seedlings. The method has the advantages of high relay propagation coefficient, high and thick buds, short period, contribution to rooting and seedling formation, 15-20 d reduction of the relay period by utilizing the tissue culture seedling technology, reduction of energy consumption, improvement of space utilization rate, improvement of production efficiency, reduction of production cost and the like.
Description
Technical Field
The invention belongs to the technical field of plant tissue culture, and particularly relates to a tissue culture seedling method for blueberries.
Background
Blueberry, the academic name of cowberry, the fruit is blue, moderate in sweetness and sourness and fine in pulp, and is the only fruit in the ten products with the best human nutrition value which are recognized by the world health organization. The anthocyanin with the antioxidant function is rich in the anthocyanin, can help human bodies to neutralize harmful particles 'free radicals' generated by metabolism, has the health care effects of protecting eyes, delaying aging, enhancing skin elasticity, enhancing human immunity, reducing the incidence of cardiovascular diseases, reducing the incidence of various cancers and the like, and is classified as one of five healthy fruits of human beings by international grain and agriculture organizations.
In view of the wide blueberry market, the demand for blueberry seedlings is more and more vigorous. The traditional seedling raising methods such as seed propagation seedling raising, cutting seedling raising, grafting seedling raising and the like often have the problems of low propagation coefficient, low survival rate, low germination rate and the like. By the tissue culture seedling method, a large number of virus-free healthy seedlings can be bred in a relatively short time, and the production period and the production cost of enterprises are reduced. The tissue culture seedling raising technology becomes a main production mode of seedling breeding in the blueberry industry and is widely applied to the production practice of blueberry seedlings. CN101869078A discloses a method for tissue culture and micro propagation of blueberry, which comprises collecting new shoots of blueberry from field in growing season, sterilizing the surface under aseptic condition, flushing with aseptic water, shearing into stem segments with 1-2 buds, inoculating the stem segments with 1-2 buds to primary induction culture medium wpm+zt 3.0 mg/l+naa0.02 mg/l+sucrose 30 g/l+agar 6.5g/L, and the pH of the culture medium is 5.4; culturing for 2-3 generations to obtain sterile, vigorous and branched tissue culture clone; in blueThe subculture proliferation culture stage of the tissue culture seedling of the strawberry adopts a proliferation culture medium WPM+ZT 0.5-1.5 mg/L+CPPU 0.05-1.0 mg/L+sucrose 30 g/L+agar 6.5g/L and pH 5.4; every 50-60 days, the breeding system is improved by 5-10 times. CN102870680a discloses a high-efficiency rapid propagation technology of a suitable detoxified rabbit eye blueberry, which adopts an explant material as current young shoots, and adopts the following steps to obtain high-quality detoxified seedlings: a. b, induction and proliferation of cluster buds, c, establishment of a leaf regeneration system, d, rooting culture of test tube seedlings, e, hardening seedlings; the high-quality detoxified seedling is improved by adopting the following method: f. inducing and multiplying cluster buds, optimizing a leaf regeneration system, optimizing roots in bottles of test-tube seedlings, and optimizing seedling hardening matrixes. The method optimizes a series of links from regeneration of leaves, multiplication culture, rooting culture to hardening off, and establishes a technology capable of efficiently, quickly and continuously obtaining excellent detoxified rabbit eye tissue culture seedlings. CN103583368A discloses a construction method of a tissue culture and rapid propagation system of rabbit eye blueberries, which comprises the steps of selecting stem segments of water culture seedlings as explants, treating the stem segments with alcohol and mercuric chloride, inoculating the treated stem segments into a stem bud induction culture medium, vertically inserting the stem segments subjected to stem bud induction culture into stem bud multiplication culture, and taking IBA concentration of 2.0mgL in rooting culture -1 The rooting rate is higher than 85%. CN102812905a discloses a tissue culture and rapid propagation process of blueberry tender stem segments, which comprises the following steps: s1, selecting nonlignified tender stems from fields, cleaning, sterilizing and cutting into stem segments with buds; s2, performing induction culture, wherein an induction culture medium is MS+ZT3.0 mg/L+IBA0.1mg/L+30 g/L sucrose+6.5 g/L agar powder; s3, subculturing, wherein the adopted subculture proliferation medium is MS+ZT 0.5-1.5 mg/L+IBA 0.05-0.1 mg/L+sucrose 30 g/L+agar 6.5g/L; and (3) before the blueberry is transplanted out of the bottle, adopting an MS+ZT culture medium of 0.3-0.7 mg/L to rejuvenate the seedlings.
Disclosure of Invention
The invention aims to provide a blueberry tissue culture seedling raising method, which has the advantages of obtaining higher propagation coefficient and thick buds, shortening the subculture period, ensuring the quality of seedlings, improving the production efficiency and further reducing the production cost.
The invention discloses a tissue culture seedling method for blueberries, which is characterized by comprising the following steps of:
s01, preparation and induction culture of explants: obtaining an explant, and culturing the explant on an induction culture medium to obtain an induction-cultured bud;
s02, subculture: culturing the buds subjected to induction culture on a subculture medium to obtain subculture seedlings;
s03, rejuvenation culture: culturing the subculture seedlings on a rejuvenation culture medium to obtain rejuvenation seedlings.
In some embodiments of the invention, S01, the induction medium comprises basal medium, hormone, sucrose; the hormone has ZT of 1.5-2.5mg/L, preferably 1.8-2.2mg/L.
In some embodiments of the invention, in S02, the secondary medium comprises basal medium, hormone, sucrose; the hormone has ZT of 0.5-1.5mg/L, preferably 0.8-1.2mg/L.
In some embodiments of the invention, in S03, the rejuvenation medium comprises basal medium, hormone, sucrose; the hormone is ZT of 0.2-0.8mg/L, preferably 0.4-0.8mg/L.
In some embodiments of the invention, the basal medium is WPM, modified WPM, or optimized WPM;
the optimized WPM is EDDHAFe of 154mg/L 6 Replacing ferrous sulfate and sodium ethylenediamine tetraacetate in the original WPM culture medium;
the modified WPM is prepared from 0.05-0.15mg/L VB1, 650-700mg/LCa (NO) 3 ) 2 ·4H 2 O、70-75mg/LC 10 H 13 FeN 2 NaO 8 、160-210mg/L KNO 3 Replace K in the original WPM culture medium 2 SO 4 、CaCl 2 、FeSO 4 、EDTA-Na。
In some embodiments of the invention, sucrose in the induction medium, the secondary medium and the rejuvenation medium is 20-30mg/L.
In some embodiments of the invention, the induction medium, the secondary medium and the rejuvenation medium each further comprise 4-6mg/L agar.
In some embodiments of the invention, S01, S02 and S03 are all dark and then light cultured;
preferably, in S01, dark culture is performed for 8-12d, and then light culture is performed for 50-60d;
preferably, in S02, the dark culture is performed for 1-3d, and then the light culture is performed for 60-70d;
preferably, in S03, dark culture is performed for 1-3d, and then light culture is performed for 60-70d;
preferably, in S01, S02 and S03, the illumination time length is 12h/d or 16h/d, and more preferably 12h/d in the illumination culture.
Preferably, in S01, S02 and S03, the temperature is 23-28 ℃ in the dark culture and the light culture.
In some embodiments of the invention, the method further comprises the steps of:
s04, hardening: transplanting the rejuvenation seedlings to outdoor culture, and continuously culturing the rejuvenation seedlings after uncovering to obtain bottle seedlings;
s05, rooting outside the bottle: and dipping the bottle seedlings with the rooting agent, then cutting the bottle seedlings into a seedbed, spraying fixed root water, shading, and spraying water periodically.
In some embodiments of the invention, in S01, collecting newly drawn tender shoots within two weeks of growing healthy and strong in the virus-free region, sterilizing, and cutting stem segments with an axillary bud as explants;
preferably, the sterilization is carried out by soaking for 15S with 75% alcohol, washing with sterile water once, soaking for 10min with 1% sodium hypochlorite, and washing with sterile water for 5 times.
In some embodiments of the invention, in S04, transplanting the rejuvenating seedlings to an outdoor shade place under natural scattered light condition for 7-10 d, and then uncovering and continuing to culture for 1-2 d;
preferably, the height of the rejuvenating seedlings is 6-7cm.
In some embodiments of the invention, in S05, the flask is removed and washed to remove the enlarged portion of the base, cut into stem segments about 3.5cm long and remove leaves 1.5cm below, and then dip in rooting agent.
In some embodiments of the invention, in S05, the air relative humidity is 80-90% and the temperature is 25-28 ℃.
In some embodiments of the invention, in S05, the seedbed is a tray;
preferably, the matrixes of the plug are coconut husk, peat soil and perlite with the volume ratio of 3:2:1.
The beneficial effects are that:
the method has the advantages of high relay propagation coefficient, high and thick buds, short period, contribution to rooting and seedling formation, 15-20 d reduction of the relay period by utilizing the tissue culture seedling technology, reduction of energy consumption, improvement of space utilization rate, improvement of production efficiency, reduction of production cost and the like.
Detailed Description
Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.
The examples and comparative examples are parallel runs of the same components, component contents, preparation steps, preparation parameters, unless otherwise specified.
The ZT is Zeatin, i.e. Zeatin.
The optimized WPM is EDDHAFe of 154mg/L 6 Replacing ferrous sulfate and sodium ethylenediamine tetraacetate in the original WPM culture medium. The EDDHAFe 6 Commercially available commodity, chemical name: sodium iron edetate di-o-hydroxyphenyl, appearance: dark red to black particles, molecular formula: c (C) 18 H 16 N 2 O 6 FeNa, chelate iron content is more than or equal to 99%, and Fe content is more than or equal to 6%.
The expression mode of the culture medium is basal culture medium, hormone, sucrose and agar, and the contents of the hormone, the sucrose and the agar are all the concentrations in the whole system. For example, in example 1, the induction medium was optimized WPM+ZT2.0 mg/L+25 g/L of sucrose+5 g/L of agar, i.e., ZT concentration in the induction medium was 2.0mg/L, sucrose concentration was 25g/L, and agar was used in an amount of 5g/L.
Example 1:
a tissue culture seedling method of blueberries comprises the following steps:
(1) Preparation and induction culture of explants: selecting 9 am of sunny weather: 00-10: 00, cutting tender branches with vigorous growth vigor, subtracting all leaves, washing by tap water, slightly wiping the surfaces of stems by a soft toothbrush, washing by flowing water for half an hour, and transferring to an ultra-clean workbench for disinfection. Sterilization of explants: placing the explant into a container, soaking with 75% alcohol for 15S, washing with sterile water for one time, soaking with 1% sodium hypochlorite for 10min, washing with sterile water for 5 times, cutting off stem segment with an axillary bud, and inoculating into induction culture medium. The inoculated culture flask is placed under the dark condition of 25 ℃ for 10d, and then is placed under the illumination time of 12h/d of 25 ℃ for culturing for 50-60d, and the induction culture medium is optimized WPM+ZT2.0mg/L+25 g/L of sucrose+5 g/L of agar.
(2) Subculturing; inoculating the bud after induction culture to a secondary culture medium for dark culture for 2 days, and then culturing at the constant temperature of 25 ℃ for 60-70 days after 12h/d of illumination. The secondary culture medium is optimized WPM+ZT0.8mg/L+25 g/L of sucrose+5 g/L of agar.
(3) Rejuvenation culture, inoculating the subculture seedlings to a rejuvenation culture medium for dark culture for 2d, and then culturing for 60-70d under constant temperature of illumination time of 12h/d25 ℃; obtaining the robustly tissue culture seedlings. Rejuvenation medium is optimized WPM+ZT0.6mg/L+sucrose 20g/L+agar 5g/L.
(4) Hardening seedlings: after the rejuvenation tissue culture seedlings grow to about 6-7cm in height, removing the tissue culture seedlings from the tissue culture chamber, culturing the tissue culture seedlings in an outdoor shade place with natural scattered light conditions for 7-10 days, and then opening the cover for culturing for 1-2 days;
(5) Rooting outside the bottle: taking out the bottle seedlings after seedling hardening, washing, removing the expansion part of the base part, shearing into stem sections with the length of about 3.5cm, removing leaves with the length of 1.5cm at the lower part, and cutting into prepared plug trays. Spraying foot-setting water, shading, spraying water regularly, wherein the relative humidity of air is 85%, and the temperature is 27 ℃; the matrix in the plug is formed by coconut husk: peat soil: perlite is uniformly mixed according to the volume ratio of 3:2:1, and the matrix is sprayed and sterilized by 800 times of carbendazim for use.
Example 2:
a tissue culture seedling method of blueberries comprises the following steps:
(1) Preparation and induction culture of explants: selecting 9 am of sunny weather: 00-10: 00, cutting tender branches with vigorous growth vigor, subtracting all leaves, washing by tap water, slightly wiping the surfaces of stems by a soft toothbrush, washing by flowing water for half an hour, and transferring to an ultra-clean workbench for disinfection. Sterilization of explants: placing the explant into a container, soaking with 75% alcohol for 15S, washing with sterile water for one time, soaking with 1% sodium hypochlorite for 10min, washing with sterile water for 5 times, cutting off stem segment with an axillary bud, and inoculating into induction culture medium. The inoculated culture flask is placed under the dark condition of 25 ℃ for 10d, and then is placed under the illumination for 12h/d of 25 ℃ for culturing for 50-60 d. The induction culture medium is optimized WPM+ZT2.0mg/L+25 g/L of sucrose+5 g/L of agar.
(2) Subculturing; inoculating the bud after induction culture to a secondary culture medium for dark culture for 2d, and then culturing for 60-70d under the constant temperature of the illumination time length of 12h/d and 25 ℃. The secondary culture medium is optimized WPM+ZT1.0mg/L+25 g/L of sucrose+5 g/L of agar.
(3) The subculture seedlings are inoculated to a rejuvenation culture medium for dark culture for 2 days, and then are placed under constant temperature of illumination for 12h/d and 25 ℃ for culture for 60-70 days; obtaining a robustly tissue culture seedling; rejuvenation medium is optimized WPM+ZT0.6mg/L+sucrose 20g/L+agar 5g/L.
(4) Hardening seedlings: after the rejuvenation tissue culture seedlings grow to about 6-7cm in height, removing the tissue culture seedlings from the tissue culture chamber, culturing the tissue culture seedlings in an outdoor shade place with natural scattered light conditions for 7-10 days, and then opening the cover for culturing for 1-2 days;
(5) Rooting outside the bottle: taking out the bottle seedlings after seedling hardening, washing, removing the expansion part of the base part, shearing into stem sections with the length of about 3.5cm, removing leaves with the length of 1.5cm at the lower part, and cutting into prepared plug trays. Spraying foot-setting water, shading, spraying water regularly, wherein the relative humidity of air is 85%, and the temperature is 27 ℃; the matrix in the plug is formed by coconut husk: peat soil: perlite is uniformly mixed according to the volume ratio of 3:2:1, and the matrix is sprayed and sterilized by 800 times of carbendazim for use.
Example 3:
a tissue culture seedling method of blueberries comprises the following steps:
(1) Preparation and induction culture of explants: selecting 9 am of sunny weather: 00-10: 00, cutting tender branches with vigorous growth vigor, subtracting all leaves, washing by tap water, slightly wiping the surfaces of stems by a soft toothbrush, washing by flowing water for half an hour, and transferring to an ultra-clean workbench for disinfection. Sterilization of explants: placing the explant into a container, soaking with 75% alcohol for 15S, washing with sterile water for one time, soaking with 1% sodium hypochlorite for 10min, washing with sterile water for 5 times, cutting off stem segment with an axillary bud, and inoculating into induction culture medium. The inoculated culture flask is placed under the dark condition of 25 ℃ for 10d, and then is placed under the constant temperature of the illumination time length of 12h/d and 25 ℃ for culturing for 50-60d, and the induction culture medium is optimized WPM+ZT2.0mg/L+25 g/L of sucrose+5 g/L of agar.
(2) Subculturing; inoculating the bud after induction culture to a secondary culture medium for dark culture for 2 days, and then culturing at the constant temperature of 25 ℃ for 60-70 days after 12h/d of illumination. The secondary culture medium is optimized WPM+ZT1.2mg/L+25 g/L of sucrose+5 g/L of agar.
(3) The subculture seedlings are inoculated to a rejuvenation culture medium for dark culture for 2 days, and then are placed under constant temperature of illumination for 12h/d and 25 ℃ for culture for 60-70 days; obtaining a robustly tissue culture seedling; rejuvenation medium is optimized WPM+ZT0.6mg/L+25 g/L of sucrose+5 g/L of agar.
(4) Hardening seedlings: after the rejuvenation tissue culture seedlings grow to about 6-7cm in height, removing the tissue culture seedlings from the tissue culture chamber, culturing the tissue culture seedlings in an outdoor shade place with natural scattered light conditions for 7-10 days, and then opening the cover for culturing for 1-2 days;
(5) Rooting outside the bottle: taking out the bottle seedlings after seedling hardening, washing, removing the expansion part of the base part, shearing into stem sections with the length of about 3.5cm, removing leaves with the length of 1.5cm at the lower part, and cutting into prepared plug trays. Spraying foot-setting water, shading, spraying water regularly, wherein the relative humidity of air is 85%, and the temperature is 27 ℃; the matrix in the plug is formed by coconut husk: peat soil: perlite is uniformly mixed according to the volume ratio of 3:2:1, and the matrix is sprayed and sterilized by 800 times of carbendazim for use.
Example 4: effect of the method of the invention
The formulation of 3 different minimal media and 2 light duration gradients were set to optimize WPM, modified WPM (VB 1 at 0.1mg/L, 684mg/LCa (NO) 3 ) 2 ·4H 2 O、73.4mg/L C 10 H 13 FeN 2 NaO 8 、190mg/L KNO 3 Replace K in original WPM 2 SO 4 、CaCl 2 、FeSO 4 EDTA-Na), 12h/d, 16h/d. 6 different basic culture mediums and treatment combinations with different illumination durations are set, and the treatment combinations are respectively used for optimizing WPM+illumination duration of 12h/d (the invention), optimizing WPM+illumination duration of 16h/d (contrast 1), WPM+illumination duration of 12h/d (contrast 2), WPM+illumination duration of 16h/d (contrast 3), improving WPM+illumination duration of 12h/d (contrast 4) and improving WPM+illumination duration of 16h/d (contrast 5).
The method of the invention has the effects on the height of the buds, the thickness of stems and the propagation coefficient of the secondary seedlings:
the statistical results of shoot height, shoot thickness and propagation coefficient and leaf characteristics at 70 days of the subculture seedlings are shown in Table 1.
TABLE 1
The results show that the culture medium formula, the culture conditions and the method have remarkable progress and positive effects on bud height, stem thickness, propagation coefficient, leaf characteristics and the like through comparison of the data in the table. Along with the continuous expansion of the blueberry industry, the improved variety seedlings are deficient, the propagation technology is in shortage, the level of the construction of an industrial base is severely restricted, and the method has very important reality and long-term significance for the industrialized promotion of the blueberries.
While the preferred embodiments and examples of the present invention have been described in detail, the present invention is not limited to the above-described embodiments and examples, and various changes may be made within the knowledge of those skilled in the art without departing from the spirit of the present invention.
Claims (13)
1. The tissue culture seedling raising method for blueberries is characterized by comprising the following steps of:
s01, preparation and induction culture of explants: obtaining an explant, and culturing the explant on an induction culture medium to obtain an induction-cultured bud;
s02, subculture: culturing the buds subjected to induction culture on a subculture medium to obtain subculture seedlings;
s03, rejuvenation culture: culturing the subculture seedlings on a rejuvenation culture medium to obtain rejuvenation seedlings;
s01, the induction culture medium comprises a basal culture medium, hormone and sucrose; the hormone is ZT of 1.5-2.5 mg/L;
s02, the secondary culture medium comprises a basal culture medium, hormone and sucrose; the hormone is ZT of 0.5-1.5 mg/L;
s03, a rejuvenation culture medium comprises a basal culture medium, hormone and sucrose; the hormone is ZT of 0.2-0.8 mg/L;
the basal medium is optimized WPM;
the optimized WPM is EDDHAFe of 154mg/L 6 Replacing ferrous sulfate and sodium ethylenediamine tetraacetate in the original WPM culture medium;
s01, S02 and S03 are all dark culture and then illumination culture; s01, dark culturing for 8-12d, and then light culturing for 50-60d; s02, dark culturing for 1-3d, and then light culturing for 60-70d; s03, performing dark culture for 1-3d, and performing illumination culture for 60-70d;
in S01, S02 and S03, the illumination duration is 12h/d in the illumination culture;
the explant is a stem segment with an axillary bud.
2. The tissue culture seedling raising method of blueberries according to claim 1, wherein in S01, hormone in an induction medium is 1.8-2.2mg/L ZT;
and/or, in S02, hormone in the secondary culture medium is ZT of 0.8-1.2 mg/L;
and/or in S03, the hormone in the rejuvenation medium is ZT of 0.4-0.8mg/L.
3. The tissue culture seedling raising method of blueberries according to claim 1 or 2, wherein sucrose in the induction medium, the secondary medium and the rejuvenation medium is 20-30 g/L;
and/or, the induction medium, the secondary culture medium and the rejuvenation medium also comprise 4-6mg/L agar.
4. The tissue culture seedling raising method of blueberries according to claim 1 or 2, wherein in S01, S02 and S03, the temperature is 23-28 ℃ in the dark culture and the illumination culture.
5. The tissue culture seedling raising method of blueberries according to claim 1 or 2, further comprising the steps of:
s04, hardening: transplanting the rejuvenation seedlings to outdoor culture, and continuously culturing the rejuvenation seedlings after uncovering to obtain bottle seedlings;
s05, rooting outside the bottle: and dipping the bottle seedlings with the rooting agent, then cutting the bottle seedlings into a seedbed, spraying fixed root water, shading, and spraying water periodically.
6. The tissue culture seedling raising method of blueberries according to claim 1 or 2, wherein in S01, newly drawn buds within two weeks of growing healthy and strong virus-free areas are collected, sterilized, and stem segments with an axillary bud are cut as explants.
7. The method for tissue culture and seedling raising of blueberry according to claim 6, wherein the sterilization is carried out by soaking in 75% alcohol for 15S, washing with sterile water once, soaking in 1% sodium hypochlorite for 10min, and washing with sterile water for 5 times.
8. The method for tissue culture and seedling raising of blueberry according to claim 5, wherein in S04, the rejuvenating seedling is transplanted to an outdoor shade place under natural scattered light condition for 7-10 d, and then the cultivation is continued for 1-2 d after opening the cover.
9. The method for tissue culture and seedling raising of blueberry according to claim 8, wherein the height of the rejuvenated seedling is 6-7cm.
10. The method for tissue culture and seedling raising of blueberry according to claim 5, wherein in S05, the bottle seedlings are taken out, washed and then the basal expansion part is removed, the bottle seedlings are cut into stem sections with the length of 3.5cm, leaves with the length of 1.5cm at the lower part are removed, and then rooting agent is dipped.
11. The method for tissue culture and seedling raising of blueberry according to claim 5, wherein in S05, the relative humidity of air is 80-90%, and the temperature is 25-28 ℃.
12. The method for tissue culture and seedling raising of blueberry according to claim 5, wherein in S05, the seedbed is a plug.
13. The tissue culture seedling raising method of blueberries according to claim 12, wherein the substrate of the plug is coconut coir, peat soil and perlite in a volume ratio of 3:2:1.
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| CN103875529A (en) * | 2014-02-18 | 2014-06-25 | 杭州佑国生物科技有限公司 | Blueberry tissue culture propagation and ex-vitro rooting method |
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| CN115005105A (en) * | 2022-07-26 | 2022-09-06 | 辽宁省果树科学研究所 | Blueberry tissue culture method |
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| SU1127550A1 (en) * | 1982-02-18 | 1984-12-07 | Научно-производственное объединение "Биохимреактив" | Nutrient medium for growing carnations in vitro |
| CN103875529A (en) * | 2014-02-18 | 2014-06-25 | 杭州佑国生物科技有限公司 | Blueberry tissue culture propagation and ex-vitro rooting method |
| CN104823857A (en) * | 2015-05-18 | 2015-08-12 | 江苏农林职业技术学院 | Seedling raising method for promoting ex-vitro rooting of blueberry tissue culture seedlings |
| CN115005105A (en) * | 2022-07-26 | 2022-09-06 | 辽宁省果树科学研究所 | Blueberry tissue culture method |
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