CN104823857A - Seedling raising method for promoting ex-vitro rooting of blueberry tissue culture seedlings - Google Patents
Seedling raising method for promoting ex-vitro rooting of blueberry tissue culture seedlings Download PDFInfo
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Abstract
The invention provides a seedling raising method for promoting ex-vitro rooting of blueberry tissue culture seedlings. The seedling raising method comprises the following steps: (1) culturing blueberry tissues; (2) performing strong seedling culture; (3) cutting the tissue culture seedlings; and (4) performing management after cutting. Compared with the prior art, the seedling raising method provided by the invention has the advantages that the tissue culture seedling raising cost can be saved, namely, the cost can be saved by 30-40% averagely, the pollution mortality of the tissue culture seedlings can be significantly reduced, and the cutting rooting rate and the survive rate of rooted seedlings can be increased.
Description
Technical field
The present invention relates to a kind of seedling-cultivating method promoting blueberry tissue culture outside sprout-cultivating-bottle radication, belong to blueberry seedling growing process field.
Background technology
Blueberry has another name called blue berry, belong to Ericaceae Vaccinium plant, perennial fallen leaves or evergreen shrubs, its fruit and seed is little, and pulp is fine and smooth, fruity sweet and sour palatability, nutritious, containing protein, fat, carbohydrate, vitamin and phosphorus, calcium, magnesium, iron, copper, the mineral element such as zinc in fresh fruit, also prevent encephalasthenia containing anthocyanin etc., strengthen cardiac function, improving eyesight and the unique activity material such as anticancer, therefore, one of large healthy food of the mankind five is classified as by international food and agricultural organization.
The Sterile culture speed of excellent blueberry kind is slow, breeding scale is by the quantitative limitation of cutting number, need of production can not be met in a short time, utilizing tissue culture technique can obtain the clonal tissue culture seedling of a large amount of blueberry improved seeds quickly and efficiently, is the optimal path carrying out high-quality blueberry kind seedling breeding.
The fast numerous cultivation program of blueberry tissue culture is many, and technical requirement is high, and utilize plantlet in vitro outside sprout-cultivating-bottle technology can reduce plantlet in vitro and cultivate link, shorten growing-seedling period, reducing seedling cost, is the innovative technology of a fast numerous factorial seedling growth of blueberry tissue culture.But still there are some problems at present, as: (1) tissue culture propagating nursery incubation is complicated, incubation time is long; (2) plantlet in vitro domestication survival rate is low, easily pollutes death; (3) problems such as plantlet in vitro outside sprout-cultivating-bottle is difficult, survival rate is low.
Summary of the invention
Goal of the invention: in order to overcome the deficiencies in the prior art, the invention provides a kind of seedling-cultivating method promoting blueberry tissue culture outside sprout-cultivating-bottle radication.
Technical scheme: for achieving the above object, the invention provides a kind of seedling-cultivating method promoting blueberry tissue culture outside sprout-cultivating-bottle radication, comprises the following steps:
(1) blueberry tissue cultural method: choose the shoot tip meristem of blueberry plant as explant, segment, sterilization, be seeded in and be added with on the WPM medium of hormone, induced bundle is sprouted; Through the Fiber differentiation of 40-50 days, blueberry explant grew sprouting on inducing culture, and then adjust the concentration of hormone in medium, be reduced to original 1/2, carry out squamous subculture, 48-52 days subcultures once, carry out 6-7 for squamous subculture, obtain initial plantlet in vitro;
(2) strong seedling culture: be 24-26 degree Celsius in temperature, intensity of illumination is 2500-3500 lux, and light application time is under the condition of 13-15 hour every day, carries out strong seedling culture to above-mentioned initial plantlet in vitro, when plantlet in vitro height reaches 5-8 centimetre, obtain strong sprout;
(3) plantlet in vitro cuttage: get above-mentioned strong sprout, wash medium off, prunes, is then placed in taking root liquid and dips 1-2 minute, and insertion has in the cave dish of culture matrix, and compress, every cave 1 strain, the degree of depth of insertion is 2-3 centimetre;
(4) management after inserting: above-mentioned cave dish is placed in Small plastic shed, covered with plastic film above shed, upper cover sunshade net, sprays water 1 time immediately, shade in 15 days, keep temperature 23-28 degree Celsius, humidity more than 90%, every day sprays water 2-3 time in blade face, in time taking root and have young leaves to grow, remove the sunshade net on shed and plastic film gradually, and number of times of spraying water is reduced to 1 day every day, namely completes outside sprout-cultivating-bottle process.
As preferably, the WPM medium being added with hormone in described step (1), wherein said hormone is zeatin, and its concentration is 2-4mg/L.
Preferred as another kind, hardening domestication process is also comprised: be placed on greenhouse movable seedbed by described strong sprout between described step (2) and (3), cover 1 layer of sunshade net, place 6-8 days, then open sunshade net, bottle cap is unscrewed, hardening 6-8 days again, repeatedly rotates body, makes it the temperature, the illumination condition that progressively adapt to greenhouse, obtain hardening, and then hardening is carried out step (3) plantlet in vitro cuttage process;
Preferred as another kind, the taking root liquid in described step (4) is the methyl α-naphthyl acetate aqueous solution, and its concentration is (900-1100) mg/L.
Preferred as another kind, the culture matrix in described step (4) is made by liver moss, and described liver moss selects faintly acid liver moss, first soaks 11-13 hour before using, and then dries, and fills in the dish of cave, obtains described culture matrix.
After described drying, the humidity of liver moss is as the criterion slightly firmly can extrude water droplet and don't can be linked to be waterline; The size of described cave dish is 3cm × 5cm.
Preferred as another kind, the Small plastic shed in described step (5) is arranged on the moving nursery bed in greenhouse, and it is highly 48-52cm, and inner installation sprinkling irrigation equipment, is covered with layer of non-woven fabric for moisturizing above moving nursery bed.
WPM medium is that this area tissue cultures commonly uses medium.
Beneficial effect: relative to prior art, seedling-cultivating method of the present invention has following advantage;
(1) compared with simple tissue culture propagating seedling, adopt plantlet in vitro outside sprout-cultivating-bottle seedling-cultivating method, simplify group training program, shorten seedling raise period, save tissue culture cost, the cost-saving 30-40% of average energy;
(2) adopt transition type plantlet in vitro domesticating method of the present invention, tissue culture seedling pollution lethality can be reduced;
(3) adopt plantlet in vitro cuttage dedicated substrate of the present invention, utilize plantlet in vitro cuttage technique method of the present invention and HORMONE TREATMENT to improve cutting plantation;
(4) utilize and of the present inventionly breed integrated management measure after facility and cuttage, the survival rate of outside sprout-cultivating-bottle seedling can be improved.
Embodiment
Below in conjunction with embodiment, the present invention is further described.
Embodiment 1:
(1) blueberry tissue cultural method: select the stalwartness of excellent blueberry kind, without damage by disease and insect plant, select well-developed current-year branch young sprout, get the tender shoot tip meristem of its children as explant, segment, sterilization, be seeded in and be added with on the WPM medium of zeatin, the concentration of zeatin is 2mg/L, and induced bundle is sprouted; Through the Fiber differentiation of 40 days, blueberry explant grew sprouting on inducing culture, and then adjusting the concentration of zeatin in medium is original 1/2, carries out squamous subculture, 48 days subcultures once, carry out 6 generation squamous subculture, obtain initial plantlet in vitro;
(2) strong seedling culture: be 24 degrees Celsius in temperature, intensity of illumination is 2500 luxs, and light application time is under the condition of 13 hours every days, carries out strong seedling culture to above-mentioned initial plantlet in vitro, when plantlet in vitro height reaches 5 centimetres, obtains strong sprout;
(3) hardening domestication process: described strong sprout is placed on greenhouse movable seedbed, covers 1 layer of sunshade net, place 6 days, then open sunshade net, bottle cap is unscrewed, then hardening 6 days, repeatedly rotate body, make it the temperature, the illumination condition that progressively adapt to greenhouse, obtain hardening;
(4) plantlet in vitro cuttage: the above-mentioned hardening that growth selection is healthy and strong, degree of lignification is good, take out gently with tweezers, wash medium off, cut off cuttings shoot root portion enlargement with scissors, the blade of bud seedling bottom three/part is removed, retain the blade of middle and upper part, then be placed in taking root liquid and dip 1 minute, insertion has in the cave dish of culture matrix, compresses, every cave 1 strain, the degree of depth of insertion is 2 centimetres;
(5) management after inserting: above-mentioned cave dish is placed in Small plastic shed, covered with plastic film above shed, upper cover sunshade net, sprays water 1 time immediately, shade in 15 days, keep temperature 23 degrees Celsius, humidity more than 90%, every day sprays water 2 times in blade face, in time taking root and have young leaves to grow, remove the sunshade net on shed and plastic film gradually, and number of times of spraying water is reduced to 1 day every day, namely completes outside sprout-cultivating-bottle process.
Taking root liquid in described step (4) is the methyl α-naphthyl acetate aqueous solution, and its concentration is 900mg/L; Culture matrix is made by liver moss, and described liver moss selects faintly acid liver moss, first soaks 11 hours, then dry before using, and fills in the dish of cave, obtains described culture matrix.
After described drying, the humidity of liver moss is as the criterion slightly firmly can extrude water droplet and don't can be linked to be waterline; The size of described cave dish is 3cm × 5cm.
Small plastic shed in described step (5) is arranged on the moving nursery bed in greenhouse, and it is highly 48cm, and inner installation sprinkling irrigation equipment, is covered with layer of non-woven fabric for moisturizing above moving nursery bed.
Embodiment 2:
(1) blueberry tissue cultural method: select the stalwartness of excellent blueberry kind, without damage by disease and insect plant, select well-developed current-year branch young sprout, get the tender shoot tip meristem of its children as explant, segment, sterilization, be seeded in and be added with on the WPM medium of zeatin, the concentration of zeatin is 4mg/L, and induced bundle is sprouted; Through the Fiber differentiation of 50 days, blueberry explant grew sprouting on inducing culture, and then adjusting the concentration of zeatin in medium is original 1/2, carries out squamous subculture, 52 days subcultures once, carry out 7 generation squamous subculture, obtain initial plantlet in vitro;
(2) strong seedling culture: be 26 degrees Celsius in temperature, intensity of illumination is 3500 luxs, and light application time is under the condition of 15 hours every days, carries out strong seedling culture to above-mentioned initial plantlet in vitro, when plantlet in vitro height reaches 8 centimetres, obtains strong sprout;
(3) plantlet in vitro cuttage: the above-mentioned strong sprout that growth selection is healthy and strong, degree of lignification is good, take out gently with tweezers, wash medium off, cut off cuttings shoot root portion enlargement with scissors, the blade of bud seedling bottom three/part is removed, retain the blade of middle and upper part, then be placed in taking root liquid and dip 2 minutes, insertion has in the cave dish of culture matrix, compresses, every cave 1 strain, the degree of depth of insertion is 3 centimetres;
(4) management after inserting: above-mentioned cave dish is placed in Small plastic shed, covered with plastic film above shed, upper cover sunshade net, sprays water 1 time immediately, shade in 15 days, keep temperature 28 degrees Celsius, humidity more than 90%, every day sprays water 3 times in blade face, in time taking root and have young leaves to grow, remove the sunshade net on shed and plastic film gradually, and number of times of spraying water is reduced to 1 day every day, namely completes outside sprout-cultivating-bottle process.
Taking root liquid in described step (4) is the methyl α-naphthyl acetate aqueous solution, and its concentration is 1100mg/L; Culture matrix is made by liver moss, and described liver moss selects faintly acid liver moss, first soaks 13 hours, then dry before using, and fills in the dish of cave, obtains described culture matrix.
After described drying, the humidity of liver moss is as the criterion slightly firmly can extrude water droplet and don't can be linked to be waterline; The size of described cave dish is 3cm × 5cm.
Small plastic shed in described step (5) is arranged on the moving nursery bed in greenhouse, and it is highly 52cm, and inner installation sprinkling irrigation equipment, is covered with layer of non-woven fabric for moisturizing above moving nursery bed.
Embodiment 3:
(1) blueberry tissue cultural method: select the stalwartness of excellent blueberry kind, without damage by disease and insect plant, select well-developed current-year branch young sprout, get the tender shoot tip meristem of its children as explant, segment, sterilization, be seeded in and be added with on the WPM medium of zeatin, the concentration of zeatin is 3mg/L, and induced bundle is sprouted; Through the Fiber differentiation of 45 days, blueberry explant grew sprouting on inducing culture, and then adjusting the concentration of zeatin in medium is original 1/2, carries out squamous subculture, 50 days subcultures once, carry out 6 generation squamous subculture, obtain initial plantlet in vitro;
(2) strong seedling culture: be 25 degrees Celsius in temperature, intensity of illumination is 3000 luxs, and light application time is under the condition of 14 hours every days, carries out strong seedling culture to above-mentioned initial plantlet in vitro, when plantlet in vitro height reaches 7 centimetres, obtains strong sprout;
(3) hardening domestication process: described strong sprout is placed on greenhouse movable seedbed, covers 1 layer of sunshade net, place 7 days, then open sunshade net, bottle cap is unscrewed, then hardening 7 days, repeatedly rotate body, make it the temperature, the illumination condition that progressively adapt to greenhouse, obtain hardening;
(4) plantlet in vitro cuttage: the above-mentioned hardening that growth selection is healthy and strong, degree of lignification is good, take out gently with tweezers, wash medium off, cut off cuttings shoot root portion enlargement with scissors, the blade of bud seedling bottom three/part is removed, retain the blade of middle and upper part, then be placed in taking root liquid and dip 1 minute, insertion has in the cave dish of culture matrix, compresses, every cave 1 strain, the degree of depth of insertion is 2.5 centimetres;
(5) management after inserting: above-mentioned cave dish is placed in Small plastic shed, covered with plastic film above shed, upper cover sunshade net, sprays water 1 time immediately, shade in 15 days, keep temperature 25 degrees Celsius, humidity more than 90%, every day sprays water 2 times in blade face, in time taking root and have young leaves to grow, remove the sunshade net on shed and plastic film gradually, and number of times of spraying water is reduced to 1 day every day, namely completes outside sprout-cultivating-bottle process.
Taking root liquid in described step (4) is the methyl α-naphthyl acetate aqueous solution, and its concentration is 1000mg/L; Culture matrix is made by liver moss, and described liver moss selects faintly acid liver moss, first soaks 12 hours, then dry before using, and fills in the dish of cave, obtains described culture matrix.
After described drying, the humidity of liver moss is as the criterion slightly firmly can extrude water droplet and don't can be linked to be waterline; The size of described cave dish is 3cm × 5cm.
Small plastic shed in described step (5) is arranged on the moving nursery bed in greenhouse, and it is highly 50cm, and inner installation sprinkling irrigation equipment, is covered with layer of non-woven fabric for moisturizing above moving nursery bed.
Embodiment 4:
(1) blueberry tissue cultural method: select the stalwartness of excellent blueberry kind, without damage by disease and insect plant, select well-developed current-year branch young sprout, get the tender shoot tip meristem of its children as explant, segment, sterilization, be seeded in and be added with on the WPM medium of zeatin, the concentration of zeatin is 3mg/L, and induced bundle is sprouted; Through the Fiber differentiation of 45 days, blueberry explant grew sprouting on inducing culture, and then adjusting the concentration of zeatin in medium is original 1/2, carries out squamous subculture, 50 days subcultures once, carry out 6 generation squamous subculture, obtain initial plantlet in vitro;
(2) strong seedling culture: be 25 degrees Celsius in temperature, intensity of illumination is 3000 luxs, and light application time is under the condition of 13-15 hour every day, carries out strong seedling culture to above-mentioned initial plantlet in vitro, when plantlet in vitro height reaches 7 centimetres, obtains strong sprout;
(3) plantlet in vitro cuttage: the above-mentioned strong sprout that growth selection is healthy and strong, degree of lignification is good, take out gently with tweezers, wash medium off, cut off cuttings shoot root portion enlargement with scissors, the blade of bud seedling bottom three/part is removed, retain the blade of middle and upper part, then be placed in taking root liquid and dip 1 minute, insertion has in the cave dish of culture matrix, compresses, every cave 1 strain, the degree of depth of insertion is 2.5 centimetres;
(4) management after inserting: above-mentioned cave dish is placed in Small plastic shed, covered with plastic film above shed, upper cover sunshade net, sprays water 1 time immediately, shade in 15 days, keep temperature 25 degrees Celsius, humidity more than 90%, every day sprays water 2 times in blade face, in time taking root and have young leaves to grow, remove the sunshade net on shed and plastic film gradually, and number of times of spraying water is reduced to 1 day every day, namely completes outside sprout-cultivating-bottle process.
Taking root liquid in described step (4) is the methyl α-naphthyl acetate aqueous solution, and its concentration is 1000mg/L; Culture matrix is made by liver moss, and described liver moss selects faintly acid liver moss, first soaks 12 hours, then dry before using, and fills in the dish of cave, obtains described culture matrix.
After described drying, the humidity of liver moss is as the criterion slightly firmly can extrude water droplet and don't can be linked to be waterline; The size of described cave dish is 3cm × 5cm.
Small plastic shed in described step (5) is arranged on the moving nursery bed in greenhouse, and it is highly 50cm, and inner installation sprinkling irrigation equipment, is covered with layer of non-woven fabric for moisturizing above moving nursery bed.
Experimental example seedling-cultivating method of the present invention is for the impact of tissue culture seedling pollution lethality, cutting plantation and shoot survival percent of taking root
Experimental field select to carry out in Jiangsu Polytechnic College of Agriculture and Forestry's experiment greenhouse, select the strain of same batch of blueberry 60, be divided into control group at random, test 1 group and test 2 groups, control group is according to the embodiment of the present invention 4 method, after step (2) strong seedling culture is placed in step (3) plantlet in vitro cuttage, income approach carries out nursery; Test 1 group and carry out nursery according to the embodiment of the present invention 4 and embodiment 3 seedling-cultivating method respectively with test 2 groups, the results are shown in following table 1.
The effect detection of the different seedling-cultivating method of table 1
Note: compared with test 1 group, * P<0.05; Compared with control group, #P<0.05
As seen from the above table, compared with control group, namely be placed in compared with the method after plantlet in vitro cuttage with strong seedling culture, test 1 group, namely the embodiment of the present invention 4 seedling-cultivating method (without hardening domestication process) can significantly improve cutting plantation, meanwhile, embodiment 3 seedling-cultivating method (having hardening to tame process) also can significantly improve cutting plantation.
In addition, (process is tamed without hardening) compared with test 1 group, test 2 groups (having hardening to tame process) and can significantly reduce tissue culture seedling pollution lethality, show to adopt transition type plantlet in vitro domesticating method of the present invention, can significantly reduce tissue culture seedling pollution lethality.
Claims (6)
1. promote a seedling-cultivating method for blueberry tissue culture outside sprout-cultivating-bottle radication, it is characterized in that, comprise the following steps:
(1) blueberry tissue cultural method: choose the shoot tip meristem of blueberry plant as explant, segment, sterilization, be seeded in and be added with on the WPM medium of hormone, induced bundle is sprouted; Through the Fiber differentiation of 40-50 days, blueberry explant grew sprouting on inducing culture, and then adjust the concentration of hormone in medium, be reduced to original 1/2, carry out squamous subculture, 48-52 days subcultures once, carry out 6-7 for squamous subculture, obtain initial plantlet in vitro;
(2) strong seedling culture: be 24-26 degree Celsius in temperature, intensity of illumination is 2500-3500 lux, and light application time is under the condition of 13-15 hour every day, carries out strong seedling culture to above-mentioned initial plantlet in vitro, when plantlet in vitro height reaches 5-8 centimetre, obtain strong sprout;
(3) plantlet in vitro cuttage: get above-mentioned strong sprout, wash medium off, prunes, is then placed in taking root liquid and dips 1-2 minute, and insertion has in the cave dish of culture matrix, and compress, every cave 1 strain, the degree of depth of insertion is 2-3 centimetre;
(4) management after inserting: above-mentioned cave dish is placed in Small plastic shed, covered with plastic film above shed, upper cover sunshade net, sprays water 1 time immediately, shade in 15 days, keep temperature 23-28 degree Celsius, humidity more than 90%, every day sprays water 2-3 time in blade face, in time taking root and have young leaves to grow, remove the sunshade net on shed and plastic film gradually, and number of times of spraying water is reduced to 1 day every day, namely completes outside sprout-cultivating-bottle process.
2. seedling-cultivating method according to claim 1, is characterized in that, the WPM medium being added with hormone in described step (1), and wherein said hormone is zeatin, and its concentration is 2-4mg/L.
3. seedling-cultivating method according to claim 1, it is characterized in that, also comprise hardening domestication process between described step (2) and (3): be placed on greenhouse movable seedbed by described strong sprout, cover 1 layer of sunshade net, place 6-8 days, then open sunshade net, bottle cap is unscrewed, then hardening 6-8 days, repeatedly rotate body, make it the temperature, the illumination condition that progressively adapt to greenhouse, obtain hardening, and then hardening is carried out step (3) plantlet in vitro cuttage process.
4. seedling-cultivating method according to claim 1, is characterized in that, the taking root liquid in described step (4) is the methyl α-naphthyl acetate aqueous solution, and its concentration is (900-1100) mg/L.
5. seedling-cultivating method according to claim 1, is characterized in that, the culture matrix in described step (4) is made by liver moss, described liver moss selects faintly acid liver moss, first soaks 11-13 hour before using, and then dries, fill in the dish of cave, obtain described culture matrix.
6. seedling-cultivating method according to claim 1, it is characterized in that, the Small plastic shed in described step (5) is arranged on the moving nursery bed in greenhouse, and it is highly 48-52cm, inner installation sprinkling irrigation equipment, is covered with layer of non-woven fabric for moisturizing above moving nursery bed.
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| CN106171725A (en) * | 2016-07-07 | 2016-12-07 | 盐津福瑞欣远农业开发有限公司 | A kind of implantation methods of Region of Yunnan Plateau blue berry |
| CN106171988B (en) * | 2016-07-13 | 2018-06-22 | 吉林省普蓝高科技有限公司 | Improve quick reproductive survival rate culture medium of blueberry detoxic seedling and preparation method thereof |
| CN106171988A (en) * | 2016-07-13 | 2016-12-07 | 吉林省普蓝高科技有限公司 | Improve blue berry detoxic seedling Fast-propagation survival rate culture medium and preparation method thereof |
| CN106212237A (en) * | 2016-08-08 | 2016-12-14 | 天水市果树研究所 | A kind of outside sprout-cultivating-bottle method of Tissue-cultured apple seedling |
| CN106212237B (en) * | 2016-08-08 | 2021-05-04 | 天水市果树研究所 | Ex-bottle rooting method for apple tissue culture seedlings |
| CN107173223A (en) * | 2017-05-22 | 2017-09-19 | 金英子 | A kind of cultivation of plants method |
| CN107155849A (en) * | 2017-05-27 | 2017-09-15 | 遵义盛林农业发展有限公司 | A kind of method for culturing seedlings of blueberry |
| CN107494264B (en) * | 2017-08-31 | 2020-01-14 | 地缘（厦门）生物科技有限公司 | Method for rooting semiliquidambar cathayensis tissue culture seedling outside bottle |
| CN107494264A (en) * | 2017-08-31 | 2017-12-22 | 地缘（厦门）生物科技有限公司 | A kind of method of Pterospermi Heterophylli tissue culture outside sprout-cultivating-bottle radication |
| CN108307823A (en) * | 2018-02-05 | 2018-07-24 | 黑龙江省农业科学院园艺分院 | A method of carrying out cutting propagation using blueberry tissue culture seedling |
| CN108668659A (en) * | 2018-07-18 | 2018-10-19 | 苏州枫彩生态科技集团有限公司 | A kind of method of the outer cuttage root-taking of blueberry bottle |
| CN109349117A (en) * | 2018-12-17 | 2019-02-19 | 江苏省中国科学院植物研究所 | Blueberry tissue culture outside sprout-cultivating-bottle radication method |
| CN109349117B (en) * | 2018-12-17 | 2021-07-02 | 江苏省中国科学院植物研究所 | Blueberry tissue culture seedling ex-vitro rooting method |
| CN116686716A (en) * | 2023-07-25 | 2023-09-05 | 广州智源农业科技发展有限公司 | Tissue culture seedling raising method for blueberries |
| CN116686716B (en) * | 2023-07-25 | 2024-03-22 | 广州智源农业科技发展有限公司 | Tissue culture seedling raising method for blueberries |
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Application publication date: 20150812 |