CN109042336A - With the method for the sterile callus of rice paddy seed culture rice containing endogenous bacterium - Google Patents
With the method for the sterile callus of rice paddy seed culture rice containing endogenous bacterium Download PDFInfo
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- CN109042336A CN109042336A CN201811154770.6A CN201811154770A CN109042336A CN 109042336 A CN109042336 A CN 109042336A CN 201811154770 A CN201811154770 A CN 201811154770A CN 109042336 A CN109042336 A CN 109042336A
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- callus
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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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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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
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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
- A01H4/001—Culture apparatus for tissue culture
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- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
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- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses the methods with the sterile callus of rice paddy seed culture rice containing endogenous bacterium, include the following steps: that the rice paddy seed containing endogenous bacterium is exposed to the sun by (1) under daylight；(2) aseptic water washing, drying are used；(3) ethanol water impregnates, and takes out, uses aseptic water washing；(4) it is impregnated, is taken out with liquor natrii hypochloritis, with aseptic water washing, drying；(5) seed that step (4) obtain is swung in into NB minimal medium；(6) seed of no microbiological contamination sign was transferred in new NB minimal medium by 27-29 DEG C of dark culture every 3 days；To callus is grown, picking callus, in 27-29 DEG C of dark culture, obtains the sterile callus of rice in new NB minimal medium.Method of the invention makes to obtain sterile callus containing endogenous bacterium rice paddy seed, not only at low cost, easy to operate, but also seed is saved in breeding process, at low cost, easy to operate, can adopt and carry out rescue culture to precious germ plasm resource.
Description
Technical field
The present invention relates to molecular biology and agricultural technology field, and in particular to a kind of rice paddy seed containing endogenous bacterium
The method for cultivating the sterile callus of rice.
Background technique
It is total almost to account for grain in Chinese rice yield as one of China or even the very important cereal crops in the world for rice
The half of yield.Over the years, breeder and agricultural technology expert are dedicated to improving rice yield and improve rice resistance, realize
One and another target.But as population is continuously increased the raising with economic level, cultivation yield is higher, tolerance is more preferable, battalion
Supporting new rice variety more abundant is the target that needs are constantly explored and pursued, and needs constantly further to send out in terms of breeding
Exhibition.In breeding process, some germ plasm resources being of great rarity will be taken because of reasons such as implantation times in storing process
The phenomenon that with various bacterium, causes the case where occurring being different from operation microbiological contamination in seed callus incubation, by seed
Portion and send out, grow up to the microbiological contamination situation of bacterium colony around seed, this influences callus tissue culture very big, is highly desirable to containing
There is the germplasm of endogenous bacterium to carry out rescue culture.It is current rare to the method for containing endogenous bacterium rice paddy seed callus culture, often
The method of rule can not cultivate to obtain sterile callus.
Summary of the invention
The purpose of the present invention is overcome the deficiencies of the prior art and provide a kind of rice paddy seed culture rice containing endogenous bacterium
The method of sterile callus.
Technical solution of the present invention is summarized as follows:
With the method for the sterile callus of rice paddy seed culture rice containing endogenous bacterium, include the following steps:
(1) rice paddy seed containing endogenous bacterium is exposed to the sun 2-3 days under daylight；
(2) it uses aseptic water washing 2-3 times, is dried up in sterile；
(3) 4-5min is impregnated with 75% ethanol water of volumetric concentration, taken out, with aseptic water washing 2-3 times；
(4) in a reservoir, 40-45min is impregnated with the liquor natrii hypochloritis that distilled water dilutes 0.8 times, every 8-10min rocks
Hold
Device takes out, with aseptic water washing, dries up in sterile；
(5) in sterile, the seed that step (4) obtain is swung in the culture bottle equipped with NB minimal medium；
(6) seed of no microbiological contamination sign was transferred to new NB minimal medium every 3 days by 27-29 DEG C of dark culture
In；To callus is grown, picking callus, in 27-29 DEG C of dark culture, obtains the sterile callus of rice in new NB minimal medium.
Method of the invention quickly can obtain sterile callus, not only at low cost, behaviour with containing endogenous bacterium rice paddy seed
Make simply, and saves seed in breeding process, it is at low cost, easy to operate, it can adopt and precious germ plasm resource is rescued
Property culture.
Detailed description of the invention
Fig. 1 is to grow callus photo on seed.
Fig. 2 is the sterile callus photo of a large amount of rice for continuing culture and obtaining.
Specific embodiment
Liquor natrii hypochloritis is commercially available, and in terms of effective chlorine 10%.
The signing of rice paddy seed containing endogenous bacterium: it is existing to will appear two kinds of microbiological contaminations in Rice seed callus incubation
As: one is as there is no a good sterile working of strict control and caused by microbiological contamination, general microbiological contamination type is uncertain, in culture base table
Each position in face grows up to bacterium colony；Another kind is that rice paddy seed the case where carrying various bacterium occurs in storing process, and then in rice
Occurs the case where being sent out by Interior Seed, growing up to bacterium colony around seed during callus tissue culture, rice paddy seed is known as at this time
Rice paddy seed containing endogenous bacterium.
Former fragrant 98 rice paddy seeds of saliva are cultivated callus 4 times in a conventional manner, always occur by Interior Seed and send out,
The case where growing up to bacterium colony around seed, and with batch, other rice varieties do not occur around seed microbiological contamination such case, therefore reflect
It makes former fragrant 98 rice paddy seeds of saliva used and contains endogenous bacterium.
Below by specific embodiment, the present invention is further illustrated.
The rice varieties that the present invention uses are commercially available for former fragrant 98 rice of saliva.
Embodiment 1
With the method for the sterile callus of rice paddy seed culture rice containing endogenous bacterium, include the following steps:
(1) rice paddy seed containing endogenous bacterium is exposed to the sun 2.5 days under daylight；
(2) it uses aseptic water washing 2 times, is dried up in sterile；
(3) 5min is impregnated with 75% ethanol water of volumetric concentration, taken out, with aseptic water washing 3 times；
(4) in a reservoir, 43min being impregnated with the liquor natrii hypochloritis that distilled water dilutes 0.8 times, every 9min rocks container,
It takes
Out, it with aseptic water washing, is dried up in sterile；
(5) in sterile, the seed that step (4) obtain is swung in the culture bottle equipped with NB minimal medium；
The seed of no microbiological contamination sign was transferred in new NB minimal medium by (6) 28 DEG C of dark cultures every 3 days；Extremely
Grow callus (7 days) (Fig. 1), picking callus is in new NB minimal medium, in 28 DEG C of dark cultures, obtain a large amount of rice without
Bacterium callus (Fig. 2).
Embodiment 2
With the method for the sterile callus of rice paddy seed culture rice containing endogenous bacterium, include the following steps:
(1) rice paddy seed containing endogenous bacterium is exposed to the sun 2 days under daylight；
(2) it uses aseptic water washing 2 times, is dried up in sterile；
(3) 4min is impregnated with 75% ethanol water of volumetric concentration, taken out, with aseptic water washing 2 times；
(4) in a reservoir, 45min being impregnated with the liquor natrii hypochloritis that distilled water dilutes 0.8 times, every 10min rocks container,
It takes out, with aseptic water washing, is dried up in sterile；
(5) in sterile, the seed that step (4) obtain is swung in the culture bottle equipped with NB minimal medium；
The seed of no microbiological contamination sign was transferred in new NB minimal medium by (6) 27 DEG C of dark cultures every 3 days；Extremely
Callus (9 days) are grown, picking callus, in 27 DEG C of dark cultures, it is sterile more to obtain a large amount of rice in new NB minimal medium
Wound.
Embodiment 3
With the method for the sterile callus of rice paddy seed culture rice containing endogenous bacterium, include the following steps:
(1) rice paddy seed containing endogenous bacterium is exposed to the sun 3 days under daylight；
(2) it uses aseptic water washing 3 times, is dried up in sterile；
(3) 5min is impregnated with 75% ethanol water of volumetric concentration, taken out, with aseptic water washing 3 times；
(4) in a reservoir, 40min being impregnated with the liquor natrii hypochloritis that distilled water dilutes 0.8 times, every 8min rocks container,
It takes
Out, it with aseptic water washing, is dried up in sterile；
(5) in sterile, the seed that step (4) obtain is swung in the culture bottle equipped with NB minimal medium；
The seed of no microbiological contamination sign was transferred in new NB minimal medium by (6) 29 DEG C of dark cultures every 3 days；Extremely
Callus (8 days) are grown, picking callus, in 29 DEG C of dark cultures, it is sterile more to obtain a large amount of rice in new NB minimal medium
Wound.
NB minimal medium, formula such as table 1.
1 NB minimal medium of table
After 2,4-D 2mg/L, 121 DEG C of sterilizing 20min is added.
N6 is a large amount of (20 ×)
2 N6 of table is a large amount of (20 ×)
(1)KNO3,(NH4)2SO4,KH2PO4Beaker is poured into simultaneously, adds water and stirs, is allowed to be completely dissolved；
(2)MgSO4.7H2O is first dissolved in the beaker for having added 100mL water and is poured slowly into 1 again after dissolution, and side bevelling stirs, no
There can be precipitating；
(3)CaCl2.2H2The same MgSO of the preparation of O4.7H2O；
(4) 4 DEG C of brown bottle preservations are put in after preparing.
B5 micro (100 ×)
3 B5 of table micro 1 (100 ×)
4 B5 of table micro 2 (100 ×)
Note: each reagent pours into beaker in 1, adds water and stirs, and is allowed to be completely dissolved.Each reagent is individually made into mother liquor in 2, respectively takes
50mL is added in 1, is settled to 500mL and is put in 4 DEG C of brown bottle preservations
Fe salt (100 ×)
5 Fe salt (100 ×) of table
Note: FeSO4It is dissolved in water, Na2- EDTA is dissolved in hot water, mixes constant volume, heats, boil in micro-wave oven.It darkens, it is cold
But to room temperature, moisturizing, 4 DEG C of brown bottle preservations
B5 organic (100 ×)
6 VB6 of table (1000 ×)
7 VB3 of table (1000 ×)
Note: VB6, niacin are made into mother liquor respectively, and 50mL is respectively taken to be added in 400mL water, and 0.5gVB1 is added, is settled to
500mL is made into organic mother liquor.Inositol is current now to be added.
2,4-D 2mg/L(100×)
0.1g 2,4-D are dissolved in 500ul dehydrated alcohol, add 1N KOH to dissolve, are settled to 500mL.
The above content is being further elaborated with for the invention, is not intended to limit the invention creation, all in this hair
Made any modifications, equivalent replacements, and improvements etc., should be included in the invention within the spirit and principle of bright creation
Within protection scope.
Claims (1)
1. with the method for the sterile callus of rice paddy seed culture rice containing endogenous bacterium, it is characterised in that include the following steps:
(1) rice paddy seed containing endogenous bacterium is exposed to the sun 2-3 days under daylight；
(2) it uses aseptic water washing 2-3 times, is dried up in sterile；
(3) 4-5min is impregnated with 75% ethanol water of volumetric concentration, taken out, with aseptic water washing 2-3 times；
(4) in a reservoir, 40-45min is impregnated with the liquor natrii hypochloritis that distilled water dilutes 0.8 times, every 8-10min rocks appearance
Device takes out, with aseptic water washing, dries up in sterile；
(5) in sterile, the seed that step (4) obtain is swung in the culture bottle equipped with NB minimal medium；
(6) seed of no microbiological contamination sign was transferred in new NB minimal medium by 27-29 DEG C of dark culture every 3 days；Extremely
Callus is grown, picking callus, in 27-29 DEG C of dark culture, obtains the sterile callus of rice in new NB minimal medium.
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| CN201811154770.6A CN109042336A (en) | 2018-09-30 | 2018-09-30 | With the method for the sterile callus of rice paddy seed culture rice containing endogenous bacterium |
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| CN201811154770.6A CN109042336A (en) | 2018-09-30 | 2018-09-30 | With the method for the sterile callus of rice paddy seed culture rice containing endogenous bacterium |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110106066A (en) * | 2019-06-14 | 2019-08-09 | 华南农业大学 | A kind of extracting method of the paddy DNA sample suitable for high-throughput Genotyping |
| CN110169357A (en) * | 2019-06-18 | 2019-08-27 | 天津市农作物研究所(天津市水稻研究所) | A kind of production method of rice paddy seed |
| CN113637701A (en) * | 2021-09-10 | 2021-11-12 | 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) | Method for establishing wild rice genetic transformation system |
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| CN103609449A (en) * | 2013-11-29 | 2014-03-05 | 福建省农业科学院生物技术研究所 | Callus preparation method for rice 13C |
| CN103993038A (en) * | 2014-06-04 | 2014-08-20 | 安徽省农业科学院水稻研究所 | Method for guiding exogenous gene into cleistogamous indica rice by using PMI (phosphomannose isomerase) selection marker |
| CN108184375A (en) * | 2017-12-29 | 2018-06-22 | 青岛袁策生物科技有限公司 | A kind of sterilization method of rice paddy seed |
| CN108277233A (en) * | 2017-12-29 | 2018-07-13 | 青岛袁策生物科技有限公司 | The method for inducing and cultivating of Mature Embryos of Rice callus |
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| CN103609449A (en) * | 2013-11-29 | 2014-03-05 | 福建省农业科学院生物技术研究所 | Callus preparation method for rice 13C |
| CN103993038A (en) * | 2014-06-04 | 2014-08-20 | 安徽省农业科学院水稻研究所 | Method for guiding exogenous gene into cleistogamous indica rice by using PMI (phosphomannose isomerase) selection marker |
| CN108184375A (en) * | 2017-12-29 | 2018-06-22 | 青岛袁策生物科技有限公司 | A kind of sterilization method of rice paddy seed |
| CN108277233A (en) * | 2017-12-29 | 2018-07-13 | 青岛袁策生物科技有限公司 | The method for inducing and cultivating of Mature Embryos of Rice callus |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110106066A (en) * | 2019-06-14 | 2019-08-09 | 华南农业大学 | A kind of extracting method of the paddy DNA sample suitable for high-throughput Genotyping |
| CN110169357A (en) * | 2019-06-18 | 2019-08-27 | 天津市农作物研究所(天津市水稻研究所) | A kind of production method of rice paddy seed |
| CN113637701A (en) * | 2021-09-10 | 2021-11-12 | 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) | Method for establishing wild rice genetic transformation system |
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Application publication date: 20181221 |
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