CN103461141B - 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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- 240000008467 Oryza sativa Japonica Group Species 0.000 title claims abstract description 26
- 238000012258 culturing Methods 0.000 title claims abstract description 6
- 238000000034 method Methods 0.000 title abstract description 14
- 239000001963 growth medium Substances 0.000 claims abstract description 44
- 239000010413 mother solution Substances 0.000 claims abstract description 30
- 239000012452 mother liquor Substances 0.000 claims description 96
- 230000001954 sterilising effect Effects 0.000 claims description 32
- 108010079058 casein hydrolysate Proteins 0.000 claims description 26
- 238000002156 mixing Methods 0.000 claims description 25
- 230000001939 inductive effect Effects 0.000 claims description 24
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 claims description 22
- 239000000600 sorbitol Substances 0.000 claims description 22
- 239000000843 powder Substances 0.000 claims description 19
- 238000002360 preparation method Methods 0.000 claims description 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 19
- 229930006000 Sucrose Natural products 0.000 claims description 18
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 18
- 239000005720 sucrose Substances 0.000 claims description 18
- 229920001817 Agar Polymers 0.000 claims description 15
- 238000004659 sterilization and disinfection Methods 0.000 claims description 14
- 239000008272 agar Substances 0.000 claims description 12
- UZKQTCBAMSWPJD-UQCOIBPSSA-N trans-Zeatin Natural products OCC(/C)=C\CNC1=NC=NC2=C1N=CN2 UZKQTCBAMSWPJD-UQCOIBPSSA-N 0.000 claims description 11
- UZKQTCBAMSWPJD-FARCUNLSSA-N trans-zeatin Chemical compound OCC(/C)=C/CNC1=NC=NC2=C1N=CN2 UZKQTCBAMSWPJD-FARCUNLSSA-N 0.000 claims description 11
- 229940023877 zeatin Drugs 0.000 claims description 11
- 239000002253 acid Substances 0.000 claims description 9
- 230000015271 coagulation Effects 0.000 claims description 9
- 238000005345 coagulation Methods 0.000 claims description 9
- 239000011086 glassine Substances 0.000 claims description 9
- 238000010438 heat treatment Methods 0.000 claims description 9
- RMOGWMIKYWRTKW-UONOGXRCSA-N (S,S)-paclobutrazol Chemical compound C([C@@H]([C@@H](O)C(C)(C)C)N1N=CN=C1)C1=CC=C(Cl)C=C1 RMOGWMIKYWRTKW-UONOGXRCSA-N 0.000 claims description 7
- 239000005985 Paclobutrazol Substances 0.000 claims description 7
- 239000000243 solution Substances 0.000 claims description 7
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 4
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 3
- 239000004471 Glycine Substances 0.000 claims description 2
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 claims description 2
- 229960000367 inositol Drugs 0.000 claims description 2
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 claims description 2
- 229960003512 nicotinic acid Drugs 0.000 claims description 2
- 235000001968 nicotinic acid Nutrition 0.000 claims description 2
- 239000011664 nicotinic acid Substances 0.000 claims description 2
- ZUFQODAHGAHPFQ-UHFFFAOYSA-N pyridoxine hydrochloride Chemical compound Cl.CC1=NC=C(CO)C(CO)=C1O ZUFQODAHGAHPFQ-UHFFFAOYSA-N 0.000 claims description 2
- 229960004172 pyridoxine hydrochloride Drugs 0.000 claims description 2
- 235000019171 pyridoxine hydrochloride Nutrition 0.000 claims description 2
- 239000011764 pyridoxine hydrochloride Substances 0.000 claims description 2
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 claims description 2
- 239000011782 vitamin Substances 0.000 claims description 2
- 229940088594 vitamin Drugs 0.000 claims description 2
- 235000013343 vitamin Nutrition 0.000 claims description 2
- 229930003231 vitamin Natural products 0.000 claims description 2
- 150000003722 vitamin derivatives Chemical class 0.000 claims description 2
- 206010020649 Hyperkeratosis Diseases 0.000 abstract description 14
- 230000004069 differentiation Effects 0.000 abstract description 11
- 235000007164 Oryza sativa Nutrition 0.000 abstract description 7
- 235000009566 rice Nutrition 0.000 abstract description 7
- 230000006698 induction Effects 0.000 abstract description 6
- 238000009395 breeding Methods 0.000 abstract description 5
- 230000001488 breeding effect Effects 0.000 abstract description 5
- 241000209094 Oryza Species 0.000 abstract 2
- 238000010899 nucleation Methods 0.000 abstract 1
- 239000012883 rooting culture medium Substances 0.000 abstract 1
- 239000012530 fluid Substances 0.000 description 8
- 239000005648 plant growth regulator Substances 0.000 description 7
- 238000005286 illumination Methods 0.000 description 6
- 239000000575 pesticide Substances 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 240000007594 Oryza sativa Species 0.000 description 5
- 230000035876 healing Effects 0.000 description 5
- 238000011081 inoculation Methods 0.000 description 5
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- 230000009286 beneficial effect Effects 0.000 description 2
- 239000004744 fabric Substances 0.000 description 2
- 244000037666 field crops Species 0.000 description 2
- 239000003375 plant hormone Substances 0.000 description 2
- 238000002203 pretreatment Methods 0.000 description 2
- PRPINYUDVPFIRX-UHFFFAOYSA-N 1-naphthaleneacetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CC=CC2=C1 PRPINYUDVPFIRX-UHFFFAOYSA-N 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 229930191978 Gibberellin Natural products 0.000 description 1
- 241001464837 Viridiplantae Species 0.000 description 1
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- 238000005516 engineering process Methods 0.000 description 1
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- IXORZMNAPKEEDV-OBDJNFEBSA-N gibberellin A3 Chemical class C([C@@]1(O)C(=C)C[C@@]2(C1)[C@H]1C(O)=O)C[C@H]2[C@]2(C=C[C@@H]3O)[C@H]1[C@]3(C)C(=O)O2 IXORZMNAPKEEDV-OBDJNFEBSA-N 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 1
- 239000000463 material Substances 0.000 description 1
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- 239000002609 medium Substances 0.000 description 1
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- 230000010496 root system development Effects 0.000 description 1
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- 235000013619 trace mineral Nutrition 0.000 description 1
- 150000003852 triazoles Chemical class 0.000 description 1
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
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 improving 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 carry out Rice Anther Culture Breeding research, anther culture has developed into the rice breeding supplementary means of a kind of maturation and practicality; But the shortcoming that anther culture exists callus induction rate and plantlet differentiation rate is low, seedling rate is not high simultaneously.
Summary of the invention
In order to overcome problems of the prior art with not enough, the first object of the present invention is the inducing culture, division culture medium, the root media that provide a kind of japonica rice anther culture medium mother liquor and utilize this mother liquor to become.
Another object of the present invention is to provide a kind of utilizes above-mentioned each substratum to improve the method for japonica rice Anther Culture Efficiency.
In order to solve above-mentioned first object, the technical solution adopted in the present invention is as follows:
A kind of japonica rice anther culture medium mother liquor, comprises mother liquor A, mother liquor B, mother solution C, mother liquor D; Wherein:
Mother liquor A comprises following component: calculate by 10 times of concentration (by mother liquor A working fluid concentration after mother liquor A dilute with water 10 times), 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: calculate by 100 times of concentration (by mother liquor B working fluid concentration after mother liquor B dilute with water 100 times), 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: calculate by 100 times of concentration (by mother solution C working fluid concentration after mother solution C dilute with water 100 times), Na
2-EDTA5.6g/L, Fe
2(SO
4)
37H
2o4.3g/L;
Mother liquor D comprises following component: calculate by 100 times of concentration (by mother liquor D working fluid concentration after mother liquor D dilute with water 100 times), inositol 10g/L, nicotinic acid 0.05g/L, vitamin 0.05g/L, pyridoxine hydrochloride 0.05g/L, glycine 0.2g/L.
Utilize the inducing culture that above-mentioned japonica rice anther culture medium mother liquor is made into, comprise following component: the mother solution C of mother liquor B, 10ml of mother liquor A, 10ml of 100ml, the mother liquor D of 10ml, 5ml concentration is the zeatin 0.8ml of 2.4-D, the sucrose 40g/L of 1mg/ml, agar 6g/L, caseinhydrolysate 1g/L, Sorbitol Powder 20g/L, 0.1mg/mL; PH is 5.8.
The preparation method of above-mentioned inducing culture, specific as follows: to get the mother solution C of mother liquor B, 10ml of mother liquor A, 10ml of 100ml, the concentration of the mother liquor D of 10ml, 5ml is the 2.4-D of 1mg/ml, pour in the Erlenmeyer flask of 1000ml, add water to 600-700ml, mixing; Again by caseinhydrolysate 1g, agar 6g, sucrose 40g, Sorbitol Powder 20g, pour in Erlenmeyer flask, heating mixing, is settled to 1000ml, regulates pH=5.8, obtains mixing solutions; The laggard row sterilizing of tapered bottleneck is sealed with glassine paper; After sterilizing terminates, above-mentioned mixing solutions is moved on the clean work station of uv disinfection, solution temperature to be mixed is down to 60-70 DEG C, add the 0.1mg/mL zeatin 0.8ml through filtration sterilization, mixing, slowly pour in the culture dish of sterilizing, treat that its natural coagulation just completes the preparation of inducing culture.
Utilize the division culture medium that above-mentioned japonica rice anther culture medium mother liquor is made into, comprise following component: the mother solution C of mother liquor B, 10ml of mother liquor A, 10ml of 100ml, the mother liquor D of 10ml, toluylic acid, sucrose 30g/L, agar 6g/L, caseinhydrolysate 1g/L, the Sorbitol Powder 10g/L of 2ml concentration is 6-BA, 0.5ml concentration of 2mg/ml to be the NAA of 0.5mg/ml, 1ml concentration be 2mg/ml; PH is 5.8.
The preparation method of above-mentioned division culture medium, specific as follows: to get the mother solution C of mother liquor B, 10ml of mother liquor A, 10ml of 100ml, the mother liquor D of 10ml, 2ml concentration is 6-BA, 0.5ml concentration of 2mg/ml is the NAA of 0.5mg/ml, 1ml concentration is that the toluylic acid of 2mg/ml is poured in the Erlenmeyer flask of 1000ml, add water the position of 600-700ml, mixing; Then pour in Erlenmeyer flask by sucrose 30g, agar 6g, caseinhydrolysate 1g, Sorbitol Powder 10g, heating mixing, is settled to 1000ml, regulates pH=5.8; The laggard row sterilizing of tapered bottleneck is sealed with glassine paper; After sterilizing terminates, division culture medium is moved on the clean work station of uv disinfection, pour in the culture dish of sterilizing, treat that its natural coagulation just completes the preparation of division culture medium.
The root media utilizing above-mentioned japonica rice anther culture medium mother liquor to be made into, comprises following component: the mother solution C of mother liquor B, 10ml of mother liquor A, 10ml of 100ml, the mother liquor D of 10ml, sucrose 30g/L, agar 6g/L, caseinhydrolysate 1g/L, Sorbitol Powder 10g/L, paclobutrazol 2mg/L; PH is 5.8.
The preparation method of above-mentioned root media, specific as follows: to get the mother solution C of mother liquor B, 10ml of mother liquor A, 10ml of 100ml, the mother liquor D of 10ml pours in the Erlenmeyer flask of 1000ml, the position of the 600-700ml that adds water, mixing; Pour in Erlenmeyer flask by sucrose 30g, agar 6g, caseinhydrolysate 1g, Sorbitol Powder 10g, paclobutrazol 2mg again, heating mixing, is settled to 1000ml, regulates pH=5.8; The laggard row sterilizing of tapered bottleneck is sealed with glassine paper; After sterilizing terminates, root media is moved on the clean work station of uv disinfection, pour in the culturing bottle of sterilizing, treat that its natural coagulation just completes the preparation of root media.
In order to solve above-mentioned second object, applicant is through repetition test for many years, and find in many anther culture schemes, following methods can realize goal of the invention well, specific as follows:
Improve a method for 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; For subsequent use;
2) the young fringe of sword-like leave pulvinus distance 2-5cm in boot stage is got, at 8 ~ 10 DEG C of pre-treatment 7-10d, sterilization; Get flower pesticide that pollen is in monokaryon period to be seeded on above-mentioned inducing culture and to carry out isolated culture; Inducing culture will be stated after inoculation terminates and move to intelligent illumination box, close all fluorescent tubes in incubator, and hide incubator printing opacity place with newspaper or black cloth, prevent extraneous light from injecting, setting the temperature inside the box is 24-26 DEG C, carries out the dark treatment scheduling to last 21-35 days, defines callus;
3) by step 2) in the callus that obtains be transferred on above-mentioned division culture medium; Intelligence illumination box, design temperature is 24-26 DEG C, and setting light application time is 14 hours/day, 8 hours/day dark treatment time;
4) callus on above-mentioned division culture medium through 15 days be differentiated to form root and bud, grow green seedling, green seedling be transferred on root media from division culture medium; Intelligence illumination box, design temperature is 27 DEG C, and setting light application time is 14 hours/day, 8 hours/day dark treatment time, obtains test-tube plantlet;
5) hardening is transplanted: green seedling proceeds to after above-mentioned root media cultivates 3, 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 when being about the long root of 0.5 ~ 1cm, from test tube, take out green seedling, wash away root substratum, clear water is cultivated; The next day, transplants in the set rice seedling bed in bloom control garden, 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 induction, the green plant regeneration of conventional japonica rice anther callus, three step plumule emergence of taking root, the anther cultural seedling rate of japonica rice can be improved, accelerate rice breeding process, improve the Breeding Efficiency of paddy rice improved seeds.
Embodiment
A kind of method improving japonica rice Anther Culture Efficiency of the present invention comprises the following steps:
(1) preparation of japonica rice anther culture medium mother liquor
The substratum mother liquor that anther culture uses 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, calculates by 10 times of concentration
In table 1,10 times of MS substratum mother liquid concentrations as a control group.10 times of mother liquor A concentration refer to the mother liquor A working fluid concentration after mother liquor A dilute with water 10 times.
Table 2 mother liquor B: trace element, calculates by 100 times of concentration
In table 2,100 times of MS substratum mother liquid concentrations as a control group.100 times of mother liquor B concentration refer to mother liquor B working fluid concentration after mother liquor B dilute with water 100 times.
Table 3 mother solution C: molysite, calculates by 100 times of concentration
In table 3,100 times of MS substratum mother liquid concentrations as a control group.100 times of mother solution C concentration refer to mother solution C working fluid concentration after mother solution C dilute with water 100 times.
Table 4 mother liquor D: organic element, calculates by 100 times of concentration
In table 4,100 times of MS substratum mother liquid concentrations as a control group.100 times of mother liquor D concentration refer to mother liquor D working fluid concentration after mother liquor D dilute with water 100 times.
Plant-growth regulator service condition in the medium:
| Plant-growth regulator | Type of culture medium of the present invention | MS substratum |
| 2.4-D | Inducing culture | MS substratum |
| Zeatin | Inducing culture | Nothing |
| 6-BA | Division culture medium | MS substratum |
| Naphthylacetic acid（NAA） | Division culture medium | MS substratum |
| Toluylic acid | Division culture medium | Nothing |
Mother liquor A used in inducing culture, division culture medium, root media, mother liquor B, mother solution C, mother liquor D are the mother liquor of undiluted mistake.
(2) preparation of inducing culture and use
Get the mother solution C of mother liquor B, 10ml of mother liquor A, 10ml of 100ml, the mother liquor D of 10ml, the 2.4-D(1mg/ml of 5ml), pour in the Erlenmeyer flask of 1000ml, add water to 600-700ml, mixing.Weigh in the balance and get CH1g, Agar6g, Sugar40g, Sorbitol20g, pour in Erlenmeyer flask, heating mixing, is settled to 1000ml, regulates PH=5.8.Seal tapered bottleneck with glassine paper, put into vertical pressure steam sterilizer sterilizing.After sterilizing terminates, inducing culture is moved on the clean work station of uv disinfection, treat that substratum temperature is down to 60-70 DEG C, add the 0.1mg/mL zeatin 0.8ml through filtration sterilization, mixing, slowly pour in the culture dish of sterilizing, treat that its natural coagulation just completes the preparation of inducing culture.
Get the young fringe of sword-like leave pulvinus distance 2-5cm in boot stage, at 8 ~ 10 DEG C of pre-treatment 7-10d, sterilization; Get flower pesticide that pollen is in monokaryon period to be seeded on above-mentioned inducing culture and to carry out isolated culture; Inducing culture will be stated after inoculation terminates and move to intelligent illumination box, close all fluorescent tubes in incubator, and hide incubator printing opacity place with newspaper or black cloth, prevent extraneous light from injecting, setting the temperature inside the box is 24-26 DEG C, carries out the dark treatment scheduling to last 21-35 days, defines callus;
Plant-growth regulator composition contrast in inducing culture
| Inducing culture | MS substratum | |
| Sucrose (sucrose) | 40g/L | 30g/L |
| Agar (agar) | 6g/L | 7g/L |
| Caseinhydrolysate (CH) | 1g/L | Nothing |
| Sorbitol Powder (sorbitol) | 20g/L | Nothing |
(3) preparation of division culture medium and use
Get the mother solution C of mother liquor B, 10ml of mother liquor A, 10ml of 100ml, the 6-BA (2mg/ml) of mother liquor D, 2ml of 10ml, the NAA (0.5mg/ml) of 0.5ml, the toluylic acid (2mg/ml) of 1ml is poured in the Erlenmeyer flask of 1000ml, add water the position of 600-700ml, mixing.Weigh in the balance and get CH1g, Agar6g, Sugar30g, Sorbitol10g, pour in Erlenmeyer flask, heating mixing, is settled to 1000ml, regulates PH=5.8.Seal tapered bottleneck with glassine paper, put into vertical pressure steam sterilizer sterilizing.After sterilizing terminates, division culture medium is moved on the clean work station of uv disinfection, pour in the culture dish of sterilizing, treat that its natural coagulation just completes the preparation of division culture medium.
Flower pesticide, through the isolated culture of inducing culture 21-35 days, defines callus, is transferred on division culture medium by callus.Intelligence illumination box, design temperature is 24-26 DEG C, and setting light application time is 14 hours/day, 8 hours/day dark treatment time.
Division culture medium plant-growth regulator composition contrasts
| Division culture medium | MS substratum | |
| Sucrose (sucrose) | 30g/L | 30g/L |
| Agar (agar) | 6g/L | 7g/L |
| Caseinhydrolysate (CH) | 1g/L | Nothing |
| Sorbitol Powder (sorbitol) | 10g/L | Nothing |
(4) preparation of root media
Get the mother solution C of mother liquor B, 10ml of mother liquor A, 10ml of 100ml, the mother liquor D of 10ml, pour in the Erlenmeyer flask of 1000ml, the position of the 600-700ml that adds water, mixing.Weigh in the balance and get CH1g, Agar6g, Sugar30g, Sorbitol10g, paclobutrazol 2mg, pour in Erlenmeyer flask, heating mixing, is settled to 1000ml, regulates PH=5.8.Seal tapered bottleneck with glassine paper, put into vertical pressure steam sterilizer sterilizing.After sterilizing terminates, root media is moved on the clean work station of uv disinfection, pour in the culturing bottle of sterilizing, treat that its natural coagulation just completes the preparation of root media.
Root media plant-growth regulator composition contrasts
| Root media | MS substratum | |
| Sucrose (sucrose) | 30g/L | 30g/L |
| Agar (agar) | 6g/L | 7g/L |
| Caseinhydrolysate (CH) | 1g/L | Nothing |
| Sorbitol Powder (sorbitol) | 10g/L | Nothing |
| Paclobutrazol | 2mg/L | Nothing |
(5) callus on above-mentioned division culture medium through 15 days be differentiated to form root and bud, grow green seedling, green seedling be transferred on root media from division culture medium; Intelligence illumination box, design temperature is 27 DEG C, and setting light application time is 14 hours/day, 8 hours/day dark treatment time, obtains test-tube plantlet;
(6) hardening is transplanted: green seedling proceeds to after above-mentioned root media cultivates 3, 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 when being about the long root of 0.5 ~ 1cm, from test tube, take out green seedling, wash away root substratum, clear water is cultivated; The next day, transplants in the set rice seedling bed in bloom control garden, uses sunshade net to hide; Culture transferring land for growing field crops after one week.
Set forth beneficial effect of the present invention further by the comparing result of above the present invention and MS substratum, result is as follows:
1. compare with MS substratum, in inducing culture, division culture medium, with the addition of caseinhydrolysate (CH), these 2 kinds of compositions of Sorbitol Powder.Caseinhydrolysate (CH) is the mixture of multiple amino acids, has good promoter action to the differentiation of embryoid, indefinite bud, can improve differentiation-inducing efficiency; Sorbitol Powder can improve the quality of callus, and plantlet differentiation rate is significantly improved.Found by test, under the condition of common MS substratum, inoculation 1000 parts of flower pesticide, do not add caseinhydrolysate (CH), the substratum healing rate of Sorbitol Powder is 12.5%, plantlet differentiation rate is 16.1%; The healing rate of the substratum of interpolation caseinhydrolysate (CH), Sorbitol Powder is 17.3%, and plantlet differentiation rate is 21.6%.
2. compare with MS substratum, in inducing culture, with the addition of zeatin, zeatin, under the condition mixed with plant hormone 2.4-D, facilitates the induction of callus fast.Found by test, under the condition of common MS substratum, inoculation 1000 parts of flower pesticide, the substratum healing rate not adding zeatin is 12.5%, and the healing rate adding the substratum of zeatin is 14.7%.
3. compare with MS substratum, in division culture medium, with the addition of toluylic acid, toluylic acid, under the condition mixed with plant hormone 6BA, NAA, facilitates the differentiation of callus fast.Found by test, under the condition of common MS substratum, inoculation 1000 parts of flower pesticide, the substratum plantlet differentiation rate not adding toluylic acid is 16.1%, and the healing rate adding the substratum of zeatin is 18.7%.
4. well developed root system, after transplanting, surviving rate is high.2mg/L paclobutrazol (triazole species plant-growth regulator is with the addition of in the root media used in the present invention, be the inhibitor of endogenous gibberellins synthesis, can obviously weaken apical growth advantage, promote that lateral bud grows, stem is thicker, plant is downgraded compact), test-tube plantlet growing way in tissue culture can be overcome more weak, the underdeveloped shortcoming of root system, there is reduction height of seedling, promote root system development, the effect of healthy and strong seedling, after transplanting, surviving rate is high.
5. use three step seedling methods, to inducing, breaking up, take root the nutritive ingredient of three different developmental phases and concentration to carry out meticulous adjustment and quantification, advantageously in induction, the differentiation of callus, anther culture seedling, strong sprout.
Claims (4)
1. a japonica rice anther culture medium mother liquor, is characterized in that, is made up of mother liquor A, mother liquor B, mother solution C, mother liquor D; Wherein
Mother liquor A is made up of following component: calculate by 10 times of concentration, KNO
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 is made up of following component: calculate by 100 times of concentration, ZnSO
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 is made up of following component: calculate by 100 times of concentration, Na
2-EDTA 5.6 g/L, Fe
2(SO
4)
37H
2o 4.3g/L;
Mother liquor D is made up of following component: calculate by 100 times of concentration, inositol 10g/L, nicotinic acid 0.05g/L, vitamin 0.05g/L, pyridoxine hydrochloride 0.05g/L, glycine 0.2g/L.
2. the inducing culture that is made into of japonica rice anther culture medium mother liquor according to claim 1, it is characterized in that, comprise following component: the mother solution C of mother liquor B, 10ml of mother liquor A, 10ml of 100ml, the mother liquor D of 10ml, 5ml concentration is the zeatin 0.8ml of 2.4-D, the sucrose 40g/L of 1mg/ml, agar 6g/L, caseinhydrolysate 1 g/L, Sorbitol Powder 20g/L, 0.1mg/mL; PH is 5.8;
The preparation method of described inducing culture is: get the mother solution C of mother liquor B, 10ml of mother liquor A, 10ml of 100ml, the concentration of the mother liquor D of 10ml, 5ml is the 2.4-D of 1mg/ml, pour in the Erlenmeyer flask of 1000ml, add water to 600-700ml, mixing; Again by caseinhydrolysate 1g, agar 6g, sucrose 40g, Sorbitol Powder 20g, pour in Erlenmeyer flask, heating mixing, is settled to 1000ml, regulates pH=5.8, obtains mixing solutions; The laggard row sterilizing of tapered bottleneck is sealed with glassine paper; After sterilizing terminates, above-mentioned mixing solutions is moved on the clean work station of uv disinfection, solution temperature to be mixed is down to 60-70 DEG C, add the 0.1mg/mL zeatin 0.8ml through filtration sterilization, mixing, slowly pour in the culture dish of sterilizing, treat that its natural coagulation just completes the preparation of inducing culture.
3. the division culture medium that is made into of japonica rice anther culture medium mother liquor according to claim 1, it is characterized in that, comprise following component: the mother solution C of mother liquor B, 10ml of mother liquor A, 10ml of 100ml, the mother liquor D of 10ml, the NAA that the 6-BA that 2ml concentration is 2mg/ml, 0.5 ml concentration are 0.5 mg/ml, 1 ml concentration is the toluylic acid of 2 mg/ml, sucrose 30g/L, agar 6g/L, caseinhydrolysate 1 g/L, Sorbitol Powder 10g/L; PH is 5.8;
The preparation method of described division culture medium is: get the mother solution C of mother liquor B, 10ml of mother liquor A, 10ml of 100ml, the mother liquor D of 10ml, the NAA that the 6-BA that 2ml concentration is 2mg/ml, 0.5 ml concentration are 0.5 mg/ml, 1 ml concentration is that the toluylic acid of 2 mg/ml is poured in the Erlenmeyer flask of 1000ml, add water the position of 600-700ml, mixing; Then pour in Erlenmeyer flask by sucrose 30g, agar 6g, caseinhydrolysate 1 g, Sorbitol Powder 10g, heating mixing, is settled to 1000ml, regulates pH=5.8; The laggard row sterilizing of tapered bottleneck is sealed with glassine paper; After sterilizing terminates, division culture medium is moved on the clean work station of uv disinfection, pour in the culture dish of sterilizing, treat that its natural coagulation just completes the preparation of division culture medium.
4. the root media that is made into of japonica rice anther culture medium mother liquor according to claim 1, it is characterized in that, comprise following component: the mother solution C of mother liquor B, 10ml of mother liquor A, 10ml of 100ml, the mother liquor D of 10ml, sucrose 30g/L, agar 6g/L, caseinhydrolysate 1 g/L, Sorbitol Powder 10g/L, paclobutrazol 2mg/ L; PH is 5.8;
The preparation method of described root media is: get the mother solution C of mother liquor B, 10ml of mother liquor A, 10ml of 100ml, the mother liquor D of 10ml pours in the Erlenmeyer flask of 1000ml, the position of the 600-700ml that adds water, mixing; Pour in Erlenmeyer flask by sucrose 30g, agar 6g, caseinhydrolysate 1 g, Sorbitol Powder 10g, paclobutrazol 2mg again, heating mixing, is settled to 1000ml, regulates pH=5.8; The laggard row sterilizing of tapered bottleneck is sealed with glassine paper; After sterilizing terminates, root media is moved on the clean work station of uv disinfection, pour in the culturing bottle of sterilizing, treat that its natural coagulation just completes the preparation of root media.
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| CN103782908B (en) * | 2014-01-13 | 2015-12-02 | 浙江农科种业有限公司 | A kind of Indica-Japonica hybrid rice anther culture method |
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| CN104705189B (en) * | 2015-04-04 | 2016-07-20 | 安徽袁粮水稻产业有限公司 | A kind of japonica rice flower pesticide inducing culture based formulas |
| CN104719169B (en) * | 2015-04-04 | 2016-11-30 | 青岛玉兰祥商务服务有限公司 | One induces japonica rice anther cultural inducing culture based formulas efficiently |
| CN104686372B (en) * | 2015-04-04 | 2016-06-15 | 黄伟洪 | A kind of long-grained nonglutinous rice flower pesticide inducing culture improved culture medium |
| CN104705190B (en) * | 2015-04-04 | 2016-07-27 | 安徽袁粮水稻产业有限公司 | A kind of japonica rice flower pesticide differentiation culture based formulas |
| CN107410029B (en) * | 2017-08-25 | 2019-07-26 | 江苏沿海地区农业科学研究所 | A kind of culture medium and application for improving long-grained nonglutinous rice and Xian round-grained rice and handing over Anther Culture Efficiency |
| 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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