CN109220788A - A kind of sterilizing methods of tissue culture rape seed - Google Patents
A kind of sterilizing methods of tissue culture rape seed Download PDFInfo
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- CN109220788A CN109220788A CN201810874260.XA CN201810874260A CN109220788A CN 109220788 A CN109220788 A CN 109220788A CN 201810874260 A CN201810874260 A CN 201810874260A CN 109220788 A CN109220788 A CN 109220788A
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- sterilizing
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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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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
- A61L2/16—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor using chemical substances
- A61L2/18—Liquid substances or solutions comprising solids or dissolved gases
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- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
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- Veterinary Medicine (AREA)
- Pretreatment Of Seeds And Plants (AREA)
Abstract
The invention discloses a kind of sterilizing methods of tissue culture rape seed, first seed is put and impregnates 1 h in water, remove the seed to float on the surface, tap water rinses 2 times, it is transferred in superclean bench, 75% ethanol postincubation sterilizes 1 min, keeps shaking transition with during 3%NaClO sterilizing seed 15min(), it uses aseptic water washing 5-6 times again, finally seed is transferred on aseptic filter paper and is dried.Wherein, 3%NaClO is the best sterilizing concentration filtered out from 0%-30%NaClO sterilizing seed effect, and sterilizing 15min is with the best sterilization time screened in optium concentration 3%NaClO sterilizing seed 10min-30min.The present invention can obtain production of most healthy seedlings for transgene rape, to improve the success rate of rapeseed gene conversion.
Description
Technical field
The present invention relates to field of plant tissue culture, and in particular to a kind of sterilizing methods of tissue culture rape seed.
Background technique
Rape category Cruciferae Brassica plants, are a kind of important oil crops, and product can both be processed into the mankind
Edible oil, industrial lubricant, can also be used as green manure and compost.It is reported that global rapeseed oil produces between 2014-2015
Amount is the third-largest oil-produced vegetable more than 269.8 ten thousand tons.It is quickly obtained ideal character rape cultivation kind by transgenic technology, it is existing
It is being widely used, is being counted according to International Agriculture biotechnology applications Servers Organization (ISAAA), genetically modified crops plantation in 2016
Area is up to 1.851 hundred million hectares, and the rape of grown worldwide has 24% for transformed variety (http://www.isaaa.org), obtains
The common practice for obtaining transgene rape is Agrobacterium_mediated method, obtains transgene rape by tissue cultures, and organizes training
The first step for the technology of supporting is exactly the sterilization processing of rape seed, and Seed sterilization is that transgene rape is successfully crucial.
Current most commonly used rape seed bactericidal agent is sodium hypochlorite (NaClO), it should be noted that if use
NaClO concentration is too low or sterilization time is inadequate, and Seed sterilization is just not thorough, after planting pathogen easy to breed, and seedling is easy sick
Opportunistic pathogen infection；Excessive concentration or sterilization time too long, then can damage seed, enable germination abnormal or even can not germinate, nothing
Method obtains enough seedlings and is used for transgenic research, and the NaClO reported at present using concentration from 0.1% to 50%, sterilization time is from 6
Min is differed to 45 min, this brings great difficulty for the application of transgene rape research.
Summary of the invention
In view of the deficiencies of the prior art, the present invention is intended to provide a kind of sterilizing methods of tissue culture rape seed, with difference
The NaClO of concentration sterilizes rape seed different time to filter out most suitable Seed sterilization concentration and sterilization time, improves oil
The sub- sterilizing efficiency of colza.
To achieve the above object, the present invention adopts the following technical scheme:
A kind of sterilizing methods of tissue culture rape seed, include the following steps:
(1) first tissue culture is put with rape seed and impregnates 1 h in water, remove the seed to float on the surface；
(2) it is rinsed 2 times with tap water；
(3) 1 min is sterilized with 75% ethanol postincubation；
(4) with 0%-30% NaClO sterilizing, 10-30 min；
(5) it uses aseptic water washing 5-6 times, the moisture content on seed is finally sucked with aseptic filter paper；
(6) seed is seeded on MS seed germination medium；
(7) after sowing 5, statistics percentage of seedgermination, healthy bud rate and clump count.
Preferably, in step (4), NaClO concentration is 3%, and sterilizing seed time is 15 min.
In step (6), MS seed germination medium are as follows: 2.22 g/L of MS culture medium+30 g/L of sucrose+agar powder 8
G/L, pH value 5.8.
In step (6), culture environment are as follows: 25 DEG C, relative humidity 60% -70%, preceding full dark treatment on the 2nd, latter 3 days 2000
Lx illumination 16 h/ days.
The present invention has the advantages that
Processing method of the present invention has obtained the rape that sterilized with NaClO by comparing the NaClO sterilizing concentration reported at present and time
The optium concentration of seed and time, while the use cost for having saved NaClO, and when saving the operation of Seed sterilization
Between, on the basis of not influencing percentage of seedgermination, and most healthy bud rates can be obtained, obtain enough healthy seedlings for oil
The transgenic research of dish.The present invention is that the Study on Genetic Transformation of rape has established solid foundation, and rape transgenosis can be improved
Explant quantity, to improve the efficiency of genetic transformation.
Specific embodiment
Technical solution of the present invention is described further below by way of specific implementation example.
1 0% -30% NaClO of embodiment sterilizing seed screens on a large scale
It selects full and without obvious scar seed and puts and impregnate 1h in water, after removing the seed to float on the surface, tap water punching
It washes several times, is transferred in superclean bench and operates later, 75% alcohol disinfecting 1 minute, aseptic water washing 2 times, then use various concentration
NaClO(0%, 0.1%, 1%, 5%, 10%, 20%, 30%；It separately sets one and does not have to 75% alcohol, only with sterile H2The control treatment group of O) it goes out
10 min of bacterium is used rinsed with sterile water 6 times later, is transferred on the filter paper of sterile drying and dries, and is seeded in MS seed and is sprouted culture
Base, 32 seeds of every group of processing, totally 4 repetitions.
Above-mentioned seed is seeded in MS seed germination medium are as follows: 2.22 g/L of MS culture medium+30 g/L of sucrose+fine jade
8 g/L of cosmetics, pH value 5.8.Culture environment: 25 DEG C, relative humidity 60% -70%, preceding full dark treatment on the 2nd, rear 2000 Le on the 3rd
Ke Si (lx) illumination 16 h/ days.After sowing 5 days, percentage of seedgermination is counted, (stalk is higher than 5 cm to healthy bud rate, and cotyledon is normally opened up
Open and the seedling of not raw bacterium be healthy bud), clump count.
Table 1:0% -30% NaClO sterilizing a wide range of the selection result of seed
Percentage of seedgermination=(seed number of germination/sowing seed number) × 100%
Seed health bud rate=(healthy bud number/sowing seed number) × 100%
Germination percentage and healthy bud rate data carry out subduplicate dazzling (arcsin anyway) Dan Yin of SPSS software is used after conversion
Plain variance analysis (carrying out Multiple range test with Duncan), clump count directlys adopt the one-way analysis of variance of SPSS software, different
Letter indicate have significant difference (P=0.05).
Experimental result discovery, various concentration NaClO sterilizing after percentage of seedgermination have significant difference (df 1,2 = 7,
24, F = 222.644, P< 0.001), highest 1% NaClO of processing group of germination percentage, percentage of seedgermination 99.2%, health
Bud rate is 94.5%, and the clump count of generation is less.
2 1% -5% NaClO of embodiment sterilizing seed small range screening
From embodiment 1 it is found that 1% concentration NaClO is best to Seed sterilization effect, it will be appreciated that 1% and 5% NaClO
The germination percentage and healthy bud rate significant difference that sterilizing seed obtains, therefore have reason to suspect that there are one between 1% -5% concentration
It is more suitable the concentration of Seed sterilization, specific as follows in order to verify this it is assumed that we have done embodiment 2:
It selects full and without obvious scar seed and puts and impregnate 1h in water, after removing the seed to float on the surface, tap water punching
It washes several times, is transferred in superclean bench and operates later, 75% alcohol disinfecting 1 minute, aseptic water washing 2 times, then use various concentration
NaClO(1%, 2%, 3%, 4%, 5%) sterilize 10 min, uses rinsed with sterile water 6 times later, is transferred on the filter paper of sterile drying and dries in the air
It is dry, it is seeded in MS seed germination medium, 32 seeds of every group of processing, totally 4 repetitions.
Above-mentioned seed is seeded in MS seed germination medium are as follows: 2.22 g/L of MS culture medium+30 g/L of sucrose+fine jade
8 g/L of cosmetics, pH value 5.8.Culture environment: 25 DEG C, relative humidity 60% -70%, preceding full dark treatment on the 2nd, rear 2000 Le on the 3rd
Ke Si (lx) illumination 16 h/ days.After sowing 5 days, percentage of seedgermination is counted, (stalk is higher than 5 cm to healthy bud rate, and cotyledon is normally opened up
Open and the seedling of not raw bacterium be healthy bud), clump count.
Table 2:1% -5% NaClO sterilizing seed small range the selection result
Percentage of seedgermination=(seed number of germination/sowing seed number) × 100%
Seed health bud rate=(healthy bud number/sowing seed number) × 100%
Germination percentage and healthy bud rate data carry out subduplicate dazzling (arcsin anyway) Dan Yin of SPSS software is used after conversion
Plain variance analysis (carrying out Multiple range test with Duncan), clump count directlys adopt the one-way analysis of variance of SPSS software, different
Letter indicate have significant difference (P=0.05).
The experimental results showed that 1% -5% NaClO to the germination percentage after Seed sterilization do not have significant difference (df 1,2 = 4,
15, F = 2.190, P=0.12), but healthy bud rate there is significant difference (df 1,2 = 4,15, F = 21.877,P< 0.001), 2%, 3% and 1% NaClO sterilizes the obtained healthy bud rate highest of seed, the bacterium generated between various concentration processing group
Fall number there is also significant difference (df 1,2 = 4,16, F = 7.045, P=0.002), 1% NaClO processing group generates
Clump count it is most, be significantly higher than other processing groups.Therefore, the effect of 2% or 3% NaClO sterilizing seed is best known to us,
But in view of high concentration sterilization effect more preferably finally can think that optimal concentration is 3%.
The screening of 3 optium concentration NaClO sterilization time of embodiment
It selects full and without obvious scar seed and puts and impregnate 1h in water, after removing the seed to float on the surface, tap water punching
It washes several times, is transferred to operation in superclean bench later, 75% alcohol disinfecting 1 minute, aseptic water washing 2 times, then with 3% NaClO
Sterilize 10 min of seed, 15 min, 20 min, 25 min and 30 min, uses rinsed with sterile water 6 times later, is transferred to sterile dry
It is dried on dry filter paper, is seeded in MS seed germination medium, every group of 65 seeds, 3 repetitions.
Above-mentioned seed is seeded in MS seed germination medium are as follows: 2.22 g/L of MS+30 g/L of sucrose+agar powder 8
G/L, pH value 5.8.Culture environment: 25 DEG C, relative humidity 60% -70%, preceding full dark treatment on the 2nd, rear 2000 luxs on the 3rd
(lx) illumination 16 h/ days.After sowing 5 days, count percentage of seedgermination, healthy bud rate (stalk is higher than 5 cm, cotyledon be normally unfolded and
The seedling of not raw bacterium is healthy bud), clump count.
Table 3: optium concentration NaClO sterilization time screening
Percentage of seedgermination=(seed number of germination/sowing seed number) × 100%
Seed health bud rate=(healthy bud number/sowing seed number) × 100%
Germination percentage and healthy bud rate data carry out subduplicate dazzling (arcsin anyway) list of SPSS software is used after conversion
Analysis of variance (carries out Multiple range test with Duncan), and clump count directlys adopt the one-way analysis of variance of SPSS software, no
With letter indicate have significant difference (P=0.05).
The experimental results showed that 3% NaClO sterilizes 10-30 min of seed to the germination percentage of seed (df 1,2 = 4,10, F
= 2.018, P=0.168) and generate clump count (df 1,2 = 4,10, F = 1.238, P =0.355) without significant
Difference, but healthy bud rate there are the difference of conspicuousness (df 1,2 = 4,15, F = 3.875, P=0.037), sterilizing 15
The healthy bud rate that min, 20 min, 30 min and 25 min are obtained is significantly higher than 10 min processing groups, wherein 15 min that sterilize are obtained
The healthy bud rate highest arrived is 81.5%, and therefore, 3% NaClO sterilizes seed Best Times as 15 min.
But above-mentioned specific embodiment is only exemplary, and is to preferably those skilled in the art be enable to manage
Solve this patent, be not to be construed as include to this patent range limitation；As long as the spirit according to disclosed in this patent is made
Any equivalent change or modification, each fall within the range that this patent includes.
Claims (4)
1. a kind of sterilizing methods of tissue culture rape seed, which comprises the steps of:
(1) first tissue culture is put with rape seed and impregnates 1 h in water, remove the seed to float on the surface；
(2) it is rinsed 2 times with tap water；
(3) 1 min is sterilized with 75% ethanol postincubation；
(4) with 0%-30% NaClO sterilizing, 10-30 min；
(5) it uses aseptic water washing 5-6 times, the moisture content on seed is finally sucked with aseptic filter paper；
(6) seed is seeded on MS seed germination medium；
(7) after sowing 5, statistics percentage of seedgermination, healthy bud rate, clump count.
2. a kind of sterilizing methods of tissue culture rape seed according to claim 1, which is characterized in that in step (4),
NaClO concentration is 3%, and sterilizing seed time is 15 min.
3. a kind of sterilizing methods of tissue culture rape seed according to claim 1, which is characterized in that in step (6), MS
Seed germination medium are as follows: 2.22 g/L of MS+30 g/L of sucrose+agar powder, 8 g/L, pH value 5.8.
4. a kind of sterilizing methods of tissue culture rape seed according to claim 1, which is characterized in that in step (6), training
Support environment are as follows: 25 DEG C, relative humidity 60%-70%, preceding full dark treatment on the 2nd, latter 2000 lx illumination on the 3rd 16 h/ days.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112335544A (en) * | 2020-10-30 | 2021-02-09 | 天津农学院 | Method for improving seed germination rate and seedling survival rate of ice vegetable seeds and application |
| CN113287526A (en) * | 2021-07-05 | 2021-08-24 | 甘肃农业大学 | Chinese cabbage type rape regeneration system establishment and anther culture method |
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| CN101044838A (en) * | 2007-04-30 | 2007-10-03 | 中国农业科学院油料作物研究所 | Method for breeding hybrid seeds of rape of heterogenesis cytoplasm male sterility line |
| CN101946689A (en) * | 2010-07-21 | 2011-01-19 | 江苏里下河地区农业科学研究所 | Method for extracting yellow-seeded rapeseed |
| CN105918104A (en) * | 2016-04-27 | 2016-09-07 | 西南大学 | Method for widening genetic diversity of Brassica napus L. by using cabbage |
| CN107475174A (en) * | 2017-08-16 | 2017-12-15 | 北京大北农生物技术有限公司 | The method for converting rape |
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| CN101044838A (en) * | 2007-04-30 | 2007-10-03 | 中国农业科学院油料作物研究所 | Method for breeding hybrid seeds of rape of heterogenesis cytoplasm male sterility line |
| CN101946689A (en) * | 2010-07-21 | 2011-01-19 | 江苏里下河地区农业科学研究所 | Method for extracting yellow-seeded rapeseed |
| CN105918104A (en) * | 2016-04-27 | 2016-09-07 | 西南大学 | Method for widening genetic diversity of Brassica napus L. by using cabbage |
| CN107475174A (en) * | 2017-08-16 | 2017-12-15 | 北京大北农生物技术有限公司 | The method for converting rape |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112335544A (en) * | 2020-10-30 | 2021-02-09 | 天津农学院 | Method for improving seed germination rate and seedling survival rate of ice vegetable seeds and application |
| CN113287526A (en) * | 2021-07-05 | 2021-08-24 | 甘肃农业大学 | Chinese cabbage type rape regeneration system establishment and anther culture method |
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Application publication date: 20190118 |