CN103461141A - Method for improving culturing efficiency of japonica rice anther - Google Patents
Method for improving culturing efficiency of japonica rice anther Download PDFInfo
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Abstract
The invention discloses a method for improving the culturing efficiency of japonica rice anther. The method comprises the steps that firstly, culture medium mother solutions of the japonica rice anther are prepared according to formulas of the culture medium mother solutions of the japonica rice anther, then the various mother solutions are utilized to prepare an induction culture medium, a differentiation culture medium and a rooting culture medium all of which are used in different periods, and the culture media in the different periods are correspondingly applied to induction, green plantlet differentiation and rooting of calluses of the japonica rice anther respectively. By the adoption of the method, the percentage of seeding emergency of culturing of the japonica rice anther can be improved, the rice breeding progress is quickened, and the seed selection efficiency of good variety of rice is improved.
Description
Technical field
The present invention relates to japonica rice anther culture field, is a kind of method that improves the japonica rice Anther Culture Efficiency specifically
Background technology
Anther culture is a kind of means of effective fast and stable mutative material, and since 20 century 70s are carried out Rice Anther Culture Breeding research, anther culture has developed into a kind of maturation and practical rice breeding supplementary means; But simultaneously anther culture exists callus induction rate and green seedling differentiation rate is low, planting percent is not high shortcoming.
Summary of the invention
In order to overcome problems of the prior art with not enough, the first purpose of the present invention is the inducing culture, differential medium, the root media that a kind of japonica rice anther culture medium mother liquor are provided and utilize this mother liquor to be mixed with.
Another object of the present invention is to provide a kind of utilizes above-mentioned each medium to improve the method for japonica rice Anther Culture Efficiency.
In order to solve above-mentioned the first purpose, the technical solution adopted in the present invention is as follows:
A kind of japonica rice anther culture medium mother liquor, comprise mother liquor A, mother liquor B, mother solution C, mother liquor D; Wherein:
Mother liquor A comprises following component: by 10 times of concentration (be about to mother liquor A dilute with water 10 times after mother liquor A working solution concentration), calculate KNO
330g/L, NH
4nO
316.5g/L, KH
2pO
45g/L, MgSO
47H
2o3.7g/L, CaCl
24.4g/L;
Mother liquor B comprises following component: by 100 times of concentration (be about to mother liquor B dilute with water 100 times after mother liquor B working solution concentration), calculate ZnSO
47H
2o500mg/L, MnSO
4h
2o1000mg/L, H
3bO
3500mg/L, CuSO
45H
2o2.5mg/L, KI83mg/L, Na
2moO
42H
2o25mg/L, CoCl
26H
2o2.5mg/L;
Mother solution C comprises following component: by 100 times of concentration (be about to mother solution C dilute with water 100 times after mother solution C working solution concentration), calculate Na
2-EDTA5.6g/L, Fe
2(SO
4)
37H
2o4.3g/L;
Mother liquor D comprises following component: by 100 times of concentration (be about to mother liquor D dilute with water 100 times after mother liquor D working solution concentration), calculate inositol 10g/L, nicotinic acid 0.05g/L, thiamine hydrochloride 0.05g/L, puridoxine hydrochloride 0.05g/L, glycine 0.2g/L.
The inducing culture that utilizes above-mentioned japonica rice anther culture medium mother liquor to be made into, the mother solution C, the mother liquor D of 10ml of mother liquor B, 10ml that comprises mother liquor A, the 10ml of following component: 100ml, the zeatin 0.8ml of the 2.4-D that 5ml concentration is 1mg/ml, sucrose 40g/L, agar 6g/L, caseinhydrolysate 1g/L, sorbitol 20g/L, 0.1mg/mL; PH is 5.8.
The preparation method of above-mentioned inducing culture, specific as follows: get the mother solution C, the mother liquor D of 10ml of mother liquor B, 10ml of mother liquor A, the 10ml of 100ml, the 2.4-D that the concentration of 5ml is 1mg/ml, pour in the Erlenmeyer flask of 1000ml, adds water to 600-700ml, mixes; By caseinhydrolysate 1g, agar 6g, sucrose 40g, sorbitol 20g, pour in Erlenmeyer flask, heating mixes, and is settled to 1000ml again, regulates pH=5.8, obtains mixed solution; Seal the laggard row sterilizing of tapered bottleneck with glassine paper; After sterilizing finishes, above-mentioned mixed solution is moved on the clean work station that passes through uv disinfection, solution temperature to be mixed is down to 60-70 ℃, add the 0.1mg/mL zeatin 0.8ml through filtration sterilization, mix, slowly pour in the culture dish that has passed through sterilizing, treat that its natural coagulation has just completed the preparation of inducing culture.
The differential medium that utilizes above-mentioned japonica rice anther culture medium mother liquor to be made into, the mother solution C, the mother liquor D of 10ml of mother liquor B, 10ml that comprises mother liquor A, the 10ml of following component: 100ml, the NAA that the 6-BA that 2ml concentration is 2mg/ml, 0.5ml concentration are 0.5mg/ml, the phenylacetic acid that 1ml concentration is 2mg/ml, sucrose 30g/L, agar 6g/L, caseinhydrolysate 1g/L, sorbitol 10g/L; PH is 5.8.
The preparation method of above-mentioned differential medium, specific as follows: the mother solution C, the mother liquor D of 10ml of mother liquor B, 10ml of getting mother liquor A, the 10ml of 100ml, the NAA that the 6-BA that 2ml concentration is 2mg/ml, 0.5ml concentration are 0.5mg/ml, the phenylacetic acid that 1ml concentration is 2mg/ml is poured in the Erlenmeyer flask of 1000ml, add the position of water to 600-700ml, mix; Then sucrose 30g, agar 6g, caseinhydrolysate 1g, sorbitol 10g are poured in Erlenmeyer flask, heating mixes, and is settled to 1000ml, regulates pH=5.8; Seal the laggard row sterilizing of tapered bottleneck with glassine paper; Sterilizing moves to differential medium on the clean work station that passes through uv disinfection after finishing, and pours in the culture dish that passes through sterilizing, treats that its natural coagulation has just completed the preparation of differential medium.
The root media that utilizes above-mentioned japonica rice anther culture medium mother liquor to be made into, comprise mother solution C, mother liquor D, the sucrose 30g/L of 10ml, agar 6g/L, caseinhydrolysate 1g/L, sorbitol 10g/L, the paclobutrazol 2mg/L of mother liquor B, 10ml of mother liquor A, the 10ml of following component: 100ml; PH is 5.8.
The preparation method of above-mentioned root media, specific as follows: the mother solution C, the mother liquor D of 10ml of mother liquor B, 10ml of getting mother liquor A, the 10ml of 100ml poured in the Erlenmeyer flask of 1000ml, adds the position of water to 600-700ml, mixes; Sucrose 30g, agar 6g, caseinhydrolysate 1g, sorbitol 10g, paclobutrazol 2mg are poured in Erlenmeyer flask, heating mixes, and is settled to 1000ml again, regulates pH=5.8; Seal the laggard row sterilizing of tapered bottleneck with glassine paper; Sterilizing moves to root media on the clean work station that passes through uv disinfection after finishing, and pours in the blake bottle that passes through sterilizing, treats that its natural coagulation has just completed the preparation of root media.
In order to solve above-mentioned the second purpose, the applicant, through repetition test for many years, finds in many anther culture schemes, and following methods can be realized goal of the invention well, specific as follows:
A kind of method that improves the japonica rice Anther Culture Efficiency, the method comprises the following steps:
1) according to above-mentioned japonica rice anther culture medium mother liquor formulated japonica rice anther culture medium mother liquor; Standby;
2) get booting stage sword-like leave pulvinus apart from the young fringe of 2-5cm, at 8～10 ℃ of pretreatment 7-10d, sterilization; Getting the flower pesticide of pollen in monokaryon period is seeded on above-mentioned inducing culture and carries out cultured in vitro; Inoculation will be stated inducing culture after finishing and move to intelligent illumination box, close all fluorescent tubes in incubator, and hide incubator printing opacity place with newspaper or black cloth, prevent that extraneous light from injecting, setting the temperature inside the box is 24-26 ℃, and the dark place reason that schedules to last 21-35 days, formed callus;
3) by step 2) in the callus that obtains be transferred on above-mentioned differential medium; The intelligence illumination box, design temperature is 24-26 ℃, setting light application time is 14 hours/day, dark place 8 hours/day time of reason;
4) callus on above-mentioned differential medium through 15 days be differentiated to form root and bud, grow green seedling, by green seedling, from differential medium, be transferred on root media; The intelligence illumination box, design temperature is 27 ℃, setting light application time is 14 hours/day, dark place 8 hours/day time of reason, obtains test-tube plantlet;
5) hardening is transplanted: green seedling proceeds to above-mentioned root media and cultivates after 3 days, and all test-tube plantlets all start root of hair, when the green seedling of test-tube plantlet grows to 7～8cm,, there are 5～10 while being about the long root of 0.5～1cm, take out green seedling from test tube, wash away the root medium, clear water is cultivated; The next day, transplant to the set rice seedling bed in the Hua Pei nursery, uses sunshade net to hide; Culture transferring land for growing field crops after one week.
The present invention has following beneficial effect: the inventive method is applicable to the inducing of conventional japonica rice anther callus, the differentiation of green seedling, the three step seedling processes of taking root, can improve the anther cultural planting percent of japonica rice, accelerate the rice breeding process, improve the Breeding Efficiency of paddy rice improved seeds.
Embodiment
A kind of method that improves the japonica rice Anther Culture Efficiency of the present invention comprises the following steps:
(1) preparation of japonica rice anther culture medium mother liquor
The medium mother liquor that anther culture is used mainly comprises four parts: mother liquor A, mother liquor B, mother solution C, mother liquor D comprise some plant growth regulator in addition.Following table is its main component and concentration thereof:
Table 1: mother liquor A: macroelement, calculate by 10 times of concentration
In table 1,10 times of MS medium mother liquid concentrations as a control group.10 times of mother liquor A concentration refers to the mother liquor A working solution concentration after 10 times of mother liquor A dilute with waters.
Table 2 mother liquor B: trace element, calculate by 100 times of concentration
In table 2,100 times of MS medium mother liquid concentrations as a control group.100 times of mother liquor B concentration refers to mother liquor B working solution concentration after 100 times of mother liquor B dilute with waters.
Table 3 mother solution C: molysite, calculate by 100 times of concentration
In table 3,100 times of MS medium mother liquid concentrations as a control group.100 times of mother solution C concentration refers to mother solution C working solution concentration after 100 times of mother solution C dilute with waters.
Table 4 mother liquor D: organic element, calculate by 100 times of concentration
In table 4,100 times of MS medium mother liquid concentrations as a control group.100 times of mother liquor D concentration refers to mother liquor D working solution concentration after 100 times of mother liquor D dilute with waters.
The service condition of plant growth regulator in medium:
| Plant growth regulator | Type of culture medium of the present invention | The MS medium |
| 2.4-D | Inducing culture | The MS medium |
| Zeatin | Inducing culture | Nothing |
| 6-BA | Differential medium | The MS medium |
| Methyl α-naphthyl acetate（NAA） | Differential medium | The MS medium |
| Phenylacetic acid | Differential medium | Nothing |
In inducing culture, differential medium, root media, mother liquor A, mother liquor B, mother solution C, mother liquor D used is the mother liquor of undiluted mistake.
(2) preparation of inducing culture and use
Get the mother solution C, the mother liquor D of 10ml of mother liquor B, 10ml of mother liquor A, the 10ml of 100ml, the 2.4-D(1mg/ml of 5ml), pour in the Erlenmeyer flask of 1000ml, add water to 600-700ml, mix.Weigh in the balance and get CH1g, Agar6g, Sugar40g, Sorbitol20g, pour in Erlenmeyer flask, heating mixes, and is settled to 1000ml, regulates PH=5.8.Seal tapered bottleneck with glassine paper, put into the vertical pressure steam sterilizer sterilizing.After sterilizing finishes, inducing culture is moved on the clean work station that passes through uv disinfection, treat that the medium temperature is down to 60-70 ℃, add the 0.1mg/mL zeatin 0.8ml through filtration sterilization, mix, slowly pour in the culture dish that has passed through sterilizing, treat that its natural coagulation has just completed the preparation of inducing culture.
Get booting stage sword-like leave pulvinus apart from the young fringe of 2-5cm, at 8～10 ℃ of pretreatment 7-10d, sterilization; Getting the flower pesticide of pollen in monokaryon period is seeded on above-mentioned inducing culture and carries out cultured in vitro; Inoculation will be stated inducing culture after finishing and move to intelligent illumination box, close all fluorescent tubes in incubator, and hide incubator printing opacity place with newspaper or black cloth, prevent that extraneous light from injecting, setting the temperature inside the box is 24-26 ℃, and the dark place reason that schedules to last 21-35 days, formed callus;
Plant growth regulator composition contrast in inducing culture
| ? | Inducing culture | The MS medium |
| Sucrose (sucrose) | 40g/L | 30g/L |
| Agar (agar) | 6g/L | 7g/L |
| Caseinhydrolysate (CH) | 1g/L | Nothing |
| Sorbitol (sorbitol) | 20g/L | Nothing |
(3) preparation of differential medium and use
Get the NAA (0.5mg/ml) of 6-BA (2mg/ml), 0.5ml of mother liquor D, 2ml of mother solution C, 10ml of mother liquor B, 10ml of mother liquor A, the 10ml of 100ml, the phenylacetic acid of 1ml (2mg/ml) is poured in the Erlenmeyer flask of 1000ml, add the position of water to 600-700ml, mix.Weigh in the balance and get CH1g, Agar6g, Sugar30g, Sorbitol10g, pour in Erlenmeyer flask, heating mixes, and is settled to 1000ml, regulates PH=5.8.Seal tapered bottleneck with glassine paper, put into the vertical pressure steam sterilizer sterilizing.Sterilizing moves to differential medium on the clean work station that passes through uv disinfection after finishing, and pours in the culture dish that passes through sterilizing, treats that its natural coagulation has just completed the preparation of differential medium.
Flower pesticide, through the inducing culture cultured in vitro of 21-35 days, has formed callus, and callus is transferred on differential medium.The intelligence illumination box, design temperature is 24-26 ℃, setting light application time is 14 hours/day, dark place 8 hours/day time of reason.
The contrast of differential medium plant growth regulator composition
| ? | Differential medium | The MS medium |
| Sucrose (sucrose) | 30g/L | 30g/L |
| Agar (agar) | 6g/L | 7g/L |
| Caseinhydrolysate (CH) | 1g/L | Nothing |
| Sorbitol (sorbitol) | 10g/L | Nothing |
(4) preparation of root media
Get the mother solution C, the mother liquor D of 10ml of mother liquor B, 10ml of mother liquor A, the 10ml of 100ml, pour in the Erlenmeyer flask of 1000ml, add the position of water to 600-700ml, mix.Weigh in the balance and get CH1g, Agar6g, Sugar30g, Sorbitol10g, paclobutrazol 2mg, pour in Erlenmeyer flask, and heating mixes, and is settled to 1000ml, regulates PH=5.8.Seal tapered bottleneck with glassine paper, put into the vertical pressure steam sterilizer sterilizing.Sterilizing moves to root media on the clean work station that passes through uv disinfection after finishing, and pours in the blake bottle that passes through sterilizing, treats that its natural coagulation has just completed the preparation of root media.
The contrast of root media plant growth regulator composition
| ? | Root media | The MS medium |
| Sucrose (sucrose) | 30g/L | 30g/L |
| Agar (agar) | 6g/L | 7g/L |
| Caseinhydrolysate (CH) | 1g/L | Nothing |
| Sorbitol (sorbitol) | 10g/L | Nothing |
| Paclobutrazol | 2mg/L | Nothing |
(5) callus on above-mentioned differential medium through 15 days be differentiated to form root and bud, grow green seedling, by green seedling, from differential medium, be transferred on root media; The intelligence illumination box, design temperature is 27 ℃, setting light application time is 14 hours/day, dark place 8 hours/day time of reason, obtains test-tube plantlet;
(6) hardening is transplanted: green seedling proceeds to above-mentioned root media and cultivates after 3 days, and all test-tube plantlets all start root of hair, when the green seedling of test-tube plantlet grows to 7～8cm,, there are 5～10 while being about the long root of 0.5～1cm, take out green seedling from test tube, wash away the root medium, clear water is cultivated; The next day, transplant to the set rice seedling bed in the Hua Pei nursery, uses sunshade net to hide; Culture transferring land for growing field crops after one week.
Comparing result by above the present invention and MS medium is further set forth beneficial effect of the present invention, and result is as follows:
1. compare with the MS medium, added caseinhydrolysate (CH), these 2 kinds of compositions of sorbitol in inducing culture, differential medium.Caseinhydrolysate (CH) is the mixture of several amino acids, and the differentiation of embryoid, indefinite bud is had to good facilitation, can improve the efficiency of inducing differentiation; Sorbitol can improve the quality of callus, and green seedling differentiation rate is significantly improved.By test, find, under the condition of common MS medium, inoculate 1000 parts of flower pesticide, the medium healing rate that does not add caseinhydrolysate (CH), sorbitol is 12.5%, and green seedling differentiation rate is 16.1%; The healing rate that adds the medium of caseinhydrolysate (CH), sorbitol is 17.3%, and green seedling differentiation rate is 21.6%.
2. compare with the MS medium, added zeatin in inducing culture, zeatin, under the condition mixed with plant hormone 2.4-D, has promoted inducing of callus fast.By test, find, under the condition of common MS medium, inoculate 1000 parts of flower pesticide, the medium healing rate that does not add zeatin is 12.5%, and the healing rate that adds the medium of zeatin is 14.7%.
3. compare with the MS medium, added phenylacetic acid in differential medium, phenylacetic acid, under the condition mixed with plant hormone 6BA, NAA, has promoted the differentiation of callus fast.Find by test, under the condition of common MS medium, inoculate 1000 parts of flower pesticide, the green seedling differentiation rate of medium of not adding phenylacetic acid is 16.1%, and the healing rate that adds the medium of zeatin is 18.7%.
4. well developed root system, after transplanting, survival rate is high.Added 2mg/L paclobutrazol (triazole type plant growth regulator in the root media used in the present invention, be the synthetic inhibitor of endogenous gibberellins, can obviously weaken the apical growth advantage, promote that lateral bud grows, the stem chap, plant is downgraded compact), can overcome in tissue cultivation the test-tube plantlet growing way a little less than, the underdeveloped shortcoming of root system, there is the reduction height of seedling, promote root system development, the effect of healthy and strong seedling, after transplanting, survival rate is high.
5. use three step seedling methods, nutrient component and the concentration of three different developmental phases of inducing, break up, take root are carried out to meticulous adjustment and quantification, more be conducive to inducing, breaking up of callus, anther culture seedling, strong sprout.
Claims (8)
1. a japonica rice anther culture medium mother liquor, is characterized in that, comprises mother liquor A, mother liquor B, mother solution C, mother liquor D; Wherein
Mother liquor A comprises following component: calculate KNO by 10 times of concentration
330 g/L, NH
4nO
316.5g/L, KH
2pO
45g/L, MgSO
47H
2o 3.7g/L, CaCl
24.4g/L;
Mother liquor B comprises following component: calculate ZnSO by 100 times of concentration
47H
2o 500mg/L, MnSO
4h
2o 1000mg/L, H
3bO
3500mg/L, CuSO
45H
2o 2.5mg/L, KI 83mg/L, Na
2moO
42H
2o 25mg/L, CoCl
26H
2o 2.5mg/L;
Mother solution C comprises following component: calculate Na by 100 times of concentration
2-EDTA 5.6 g/L, Fe
2(SO
4)
37H
2o 4.3g/L;
Mother liquor D comprises following component: calculate inositol 10g/L, nicotinic acid 0.05g/L, thiamine hydrochloride 0.05g/L, puridoxine hydrochloride 0.05g/L, glycine 0.2g/L by 100 times of concentration.
2. the inducing culture that japonica rice anther culture medium mother liquor according to claim 1 is made into, it is characterized in that, the mother solution C, the mother liquor D of 10ml of mother liquor B, 10ml that comprises mother liquor A, the 10ml of following component: 100ml, the zeatin 0.8ml of the 2.4-D that 5ml concentration is 1mg/ml, sucrose 40g/L, agar 6g/L, caseinhydrolysate 1 g/L, sorbitol 20g/L, 0.1mg/mL; PH is 5.8.
3. the preparation method of inducing culture according to claim 2, is characterized in that, gets the mother solution C, the mother liquor D of 10ml of mother liquor B, 10ml of mother liquor A, the 10ml of 100ml, the 2.4-D that the concentration of 5ml is 1mg/ml, pour in the Erlenmeyer flask of 1000ml, add water to 600-700ml, mix; By caseinhydrolysate 1g, agar 6g, sucrose 40g, sorbitol 20g, pour in Erlenmeyer flask, heating mixes, and is settled to 1000ml again, regulates pH=5.8, obtains mixed solution; Seal the laggard row sterilizing of tapered bottleneck with glassine paper; After sterilizing finishes, above-mentioned mixed solution is moved on the clean work station that passes through uv disinfection, solution temperature to be mixed is down to 60-70 ℃, add the 0.1mg/mL zeatin 0.8ml through filtration sterilization, mix, slowly pour in the culture dish that has passed through sterilizing, treat that its natural coagulation has just completed the preparation of inducing culture.
4. the differential medium that japonica rice anther culture medium mother liquor according to claim 1 is made into, it is characterized in that, the mother solution C, the mother liquor D of 10ml of mother liquor B, 10ml that comprises mother liquor A, the 10ml of following component: 100ml, the NAA that the 6-BA that 2ml concentration is 2mg/ml, 0.5 ml concentration are 0.5 mg/ml, the phenylacetic acid that 1 ml concentration is 2 mg/ml, sucrose 30g/L, agar 6g/L, caseinhydrolysate 1 g/L, sorbitol 10g/L; PH is 5.8.
5. the preparation method of differential medium according to claim 4, it is characterized in that, get the mother solution C, the mother liquor D of 10ml of mother liquor B, 10ml of mother liquor A, the 10ml of 100ml, the NAA that the 6-BA that 2ml concentration is 2mg/ml, 0.5 ml concentration are 0.5 mg/ml, the phenylacetic acid that 1 ml concentration is 2 mg/ml is poured in the Erlenmeyer flask of 1000ml, add the position of water to 600-700ml, mix; Then sucrose 30g, agar 6g, caseinhydrolysate 1 g, sorbitol 10g are poured in Erlenmeyer flask, heating mixes, and is settled to 1000ml, regulates pH=5.8; Seal the laggard row sterilizing of tapered bottleneck with glassine paper; Sterilizing moves to differential medium on the clean work station that passes through uv disinfection after finishing, and pours in the culture dish that passes through sterilizing, treats that its natural coagulation has just completed the preparation of differential medium.
6. the root media that japonica rice anther culture medium mother liquor according to claim 1 is made into, it is characterized in that, comprise mother solution C, mother liquor D, the sucrose 30g/L of 10ml, agar 6g/L, caseinhydrolysate 1 g/L, sorbitol 10g/L, the paclobutrazol 2mg/ L of mother liquor B, 10ml of mother liquor A, the 10ml of following component: 100ml; PH is 5.8.
7. the preparation method of root media according to claim 6, is characterized in that, gets the mother solution C, the mother liquor D of 10ml of mother liquor B, 10ml of mother liquor A, the 10ml of 100ml and pour in the Erlenmeyer flask of 1000ml, adds the position of water to 600-700ml, mixes; Sucrose 30g, agar 6g, caseinhydrolysate 1 g, sorbitol 10g, paclobutrazol 2mg are poured in Erlenmeyer flask, heating mixes, and is settled to 1000ml again, regulates pH=5.8; Seal the laggard row sterilizing of tapered bottleneck with glassine paper; Sterilizing moves to root media on the clean work station that passes through uv disinfection after finishing, and pours in the blake bottle that passes through sterilizing, treats that its natural coagulation has just completed the preparation of root media.
8. a method that improves the japonica rice Anther Culture Efficiency, is characterized in that, the method comprises the following steps:
1) according to the described japonica rice anther culture medium of claim 1 mother liquor formulated japonica rice anther culture medium mother liquor; Standby;
2) get booting stage sword-like leave pulvinus apart from the young fringe of 2-5cm, at 8～10 ℃ of pretreatment 7-10d, sterilization; Getting the flower pesticide of pollen in monokaryon period is seeded on described inducing culture and carries out cultured in vitro; Inoculation will be stated inducing culture after finishing and move to intelligent illumination box, close all fluorescent tubes in incubator, and hide incubator printing opacity place with newspaper or black cloth, prevent that extraneous light from injecting, setting the temperature inside the box is 24-26 ℃, and the dark place reason that schedules to last 21-35 days, formed callus; Wherein said inducing culture comprises the mother solution C, the mother liquor D of 10ml of mother liquor B, 10ml of mother liquor A, the 10ml of following component: 100ml, the zeatin 0.8ml of the 2.4-D that 5ml concentration is 1mg/ml, sucrose 40g/L, agar 6g/L, caseinhydrolysate 1 g/L, sorbitol 20g/L, 0.1mg/mL; PH is 5.8;
3) by step 2) in the callus that obtains be transferred on differential medium; The intelligence illumination box, design temperature is 24-26 ℃, setting light application time is 14 hours/day, dark place 8 hours/day time of reason; Wherein said differential medium comprises the mother solution C, the mother liquor D of 10ml of mother liquor B, 10ml of mother liquor A, the 10ml of following component: 100ml, the NAA that the 6-BA that 2ml concentration is 2mg/ml, 0.5 ml concentration are 0.5 mg/ml, the phenylacetic acid that 1 ml concentration is 2 mg/ml, sucrose 30g/L, agar 6g/L, caseinhydrolysate 1 g/L, sorbitol 10g/L; PH is 5.8;
4) callus on differential medium through 15 days be differentiated to form root and bud, grow green seedling, by green seedling, from differential medium, be transferred on root media; The intelligence illumination box, design temperature is 27 ℃, setting light application time is 14 hours/day, dark place 8 hours/day time of reason, obtains test-tube plantlet; Wherein said root media comprises mother solution C, mother liquor D, the sucrose 30g/L of 10ml, agar 6g/L, caseinhydrolysate 1 g/L, sorbitol 10g/L, the paclobutrazol 2mg/ L of mother liquor B, 10ml of mother liquor A, the 10ml of following component: 100ml; PH is 5.8;
5) hardening is transplanted: green seedling proceeds to root media and cultivates after 3 days, and all test-tube plantlets all start root of hair, when the green seedling of test-tube plantlet grows to 7 ~ 8cm,, there are 5 ~ 10 while being about the long root of 0.5 ~ 1cm, take out green seedling from test tube, wash away the root medium, clear water is cultivated; The next day, transplant to the set rice seedling bed in the Hua Pei nursery, uses sunshade net to hide; Culture transferring land for growing field crops after one week.
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| CN104686372A (en) * | 2015-04-04 | 2015-06-10 | 黎建波 | Indica rice anther induction culture improved bouillon medium formula |
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| CN108781659B (en) * | 2018-06-20 | 2021-06-15 | 湖北省农业科学院粮食作物研究所 | Remote transportation and transplanting method for rice tissue culture seedlings |
| CN117721143A (en) * | 2023-12-19 | 2024-03-19 | 湖北省农业科学院粮食作物研究所 | Gene editing method |
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