CN105580731A - Seed disinfection method in mature embryo tissue culture - Google Patents
Seed disinfection method in mature embryo tissue culture Download PDFInfo
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- CN105580731A CN105580731A CN201410560780.5A CN201410560780A CN105580731A CN 105580731 A CN105580731 A CN 105580731A CN 201410560780 A CN201410560780 A CN 201410560780A CN 105580731 A CN105580731 A CN 105580731A
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Abstract
The invention relates to a seed disinfection method in mature embryo tissue culture. The method comprises the following steps: 1) selecting the corn seeds for standby; and 2) flushing the corn seeds in the step 1) for 20-30 minutes, then placing the flushed corn seeds on a super clean bench and rinsing the material with alcohol with mass fraction being 75% for 0.5-1 minute, disinfecting the rinsed corn seeds with sodium hypochlorite with mass fraction being 2% for 10 minutes, and finally flushing the corn seeds for 3-5 times with sterile water. Compared with the prior art by using mercuric chloride, the method provided in the invention has the advantages of easy removal, no residues, less damage to tissue culture materials, and no environment pollution.
Description
Technical field
The invention belongs to biotechnology field of tissue culture, be specifically related to a kind of seed disinfection method in corn mature embryo tissue cultivation.
Background technology
Corn (ZeamaysL.) is grass family Zea annual herb plant, is also important grain, feed and insutrial crop. According to " Chinese countryside Forecast and Analysis of Economic Situation (2013) ", corn total output in 2012 exceedes paddy first becomes the first grain variety of China. The Study on tissue culture of corn is the basic work of Breeding of Biotechnology of Maize, is deeply paid attention to always. With respect to maize immature embryos, corn mature seed is easy to a large amount of preservations, is not subject to the season of growth and restricted number, can take at any time, and therefore, ripe corn seed is a kind of comparatively ideal explant that corn tissue cultivates that is applicable to. Corn seed sterilization is the first step that Mature Embryo Tissue is cultivated, and the success that corn mature embryo tissue is cultivated has material impact.
In existing report, in most of corn seed disinfection experiment, use 0.1% mercuric chloride (HgCl2) as disinfectant (as Xiang Yan etc., 2007; Jia Xiuping etc., 2008; Cheng Li etc., 2009; Kong Weiping, 2010; Pei Dongli etc., 2011; Piao Lianyu etc., 2013), also there are the report (He Ting etc. that use 0.2% mercuric chloride, 2009), above-mentioned report disinfectant used has the removal of being difficult for, injury to group training material is larger, environment is had to the shortcomings such as pollution, is that the method that 2% clorox carries out corn seed sterilization rarely has report and adopt mass fraction in corn mature embryo is cultivated.
Summary of the invention
For above-mentioned the deficiencies in the prior art, the invention provides a kind of seed disinfection method in corn mature embryo tissue cultivation.
The present invention is achieved in that
A seed disinfection method in the cultivation of corn mature embryo tissue, comprises the steps:
1) select size evenly, without obviously corn seed heterochromatic, full seed is for subsequent use;
2) getting the corn seed running water that step 1) selects rinses, washing time is 20-30 minute, then the corn seed after rinsing being placed on superclean bench is 75% alcohol rinsing with mass fraction, the time of rinsing is 0.5-1 minute, corn seed after the clorox sterilization rinsing that rinsing is then 2% with mass fraction later again, the time of sterilization is 10 minutes, finally uses aseptic water washing 3-5 time.
Described step 2) in running water washing time be 10-30 minute, be preferably 20 minutes.
Described step 2) in be 2% with mass fraction clorox disinfecting time is 8-14 minute, be preferably 10 minutes.
Technique effect: compared with the mercury chloride that the method for the invention adopts with the seed disinfection method in existing corn mature embryo tissue cultivation, there is easy removal, do not stay residual hazard, less to the injury of group training material, there is no the advantages such as environmental pollution.
Detailed description of the invention
Embodiment 1
1) employing is flat educates 2B corn seed, selects big or small corn seed even, full seed and is divided into five equal portions, is respectively charged in 50ml triangular flask for subsequent use;
2) getting the corn seed running water that step 1) selects rinses, washing time is 30 minutes, then the corn seed after rinsing being placed on superclean bench is 75% alcohol rinsing with mass fraction, the time of rinsing is 1 minute, the clorox that rinsing is then 2% with mass fraction later is again sterilized the corn seed after alcohol rinsing respectively, the time of sterilization is followed successively by 4min, 6min, 8min, 10min, 12min, finally uses aseptic water washing 3-5 time
Then respectively be equipped with disinfect after in the 50ml triangular flask of corn seed, add sterilized water, can submergence be as the criterion with firm. Corn seed after the sterilization that sterilized water is soaked is put into incubator and is secretly cultivated, and temperature is made as 26 DEG C, observes seed growth situation every day, becomes muddiness as pollution norms, Continuous Observation 4 days taking the long bacterium of seed and sterilized water.
Experiment conclusion, is that 2% liquor natrii hypochloritis processes and flat educates 2B corn seed more than 8 minutes with mass fraction, and contamination phenomenon does not appear in corn seed, and Disinfection Effect is better.
In superclean bench, corn seed sterilized water being soaked after the sterilization of 72h strips mature embryo, along plumular axis rip cutting two halves, otch is inoculated in downwards on MS calli induction media, calli induction media is that MS culture medium adds: Glu(L-glutamic acid) (300mg/L)+CH(caseinhydrolysate) (500mg/L)+Pro(L-proline) (700mg/L)+KT(kinetin) (0.5mg/L)+3% sucrose+agar (7.5g/L), 2, 4-D(2, 4-dichlorphenoxyacetic acid) concentration is 1mg/L, pH5.8. on this basis, dark culturing in 26 DEG C of intelligent illumination boxs, after three weeks, statistics corn mature embryo callus induction rate is 39.3%.
Embodiment 2
1) adopt Zheng Dan 958 corn seeds, select big or small corn seed even, full seed and be divided into five equal portions, be respectively charged in 50ml triangular flask for subsequent use;
2) getting the corn seed running water that step 1) selects rinses, washing time is 30 minutes, then the corn seed after rinsing being placed on superclean bench is 75% alcohol rinsing with mass fraction, the time of rinsing is 1 minute, the clorox that rinsing is then 2% with mass fraction later is again sterilized the corn seed after alcohol rinsing respectively, the time of sterilization is followed successively by 4min, 6min, 8min, 10min, 12min, finally uses aseptic water washing 3-5 time
Then respectively be equipped with disinfect after in the 50ml triangular flask of corn seed, add sterilized water, can submergence be as the criterion with firm. Corn seed after the sterilization that sterilized water is soaked is put into incubator and is secretly cultivated, and temperature is made as 26 DEG C, observes seed growth situation every day, becomes muddiness as pollution norms, Continuous Observation 4 days taking the long bacterium of seed and sterilized water.
Experiment conclusion, is that 2% liquor natrii hypochloritis processes Zheng Dan 958 corn seeds more than 8 minutes with mass fraction, and contamination phenomenon does not appear in corn seed, and Disinfection Effect is better.
In superclean bench, the later corn seed of sterilization that sterilized water is soaked to 72h strips mature embryo, along plumular axis rip cutting two halves, otch is inoculated in downwards on MS calli induction media, calli induction media is that MS culture medium adds: Glu (300mg/L)+CH (500mg/L)+Pro (700mg/L)+KT (0.5mg/L)+3% sucrose+agar (7.5g/L), 2,4-D concentration is 2mg/L, pH5.8. on this basis, dark culturing in 26 DEG C of intelligent illumination boxs, after three weeks, statistics corn mature embryo callus induction rate is 44.4%.
Embodiment 3
1) adopt refreshing 56 corn seeds, select big or small corn seed even, full seed and be divided into five equal portions, be respectively charged in 50ml triangular flask for subsequent use;
2) getting the corn seed running water that step 1) selects rinses, washing time is 30 minutes, then the corn seed after rinsing being placed on superclean bench is 75% alcohol rinsing with mass fraction, the time of rinsing is 1 minute, the clorox that rinsing is then 2% with mass fraction later is again sterilized the corn seed after alcohol rinsing respectively, the time of sterilization is followed successively by 4min, 6min, 8min, 10min, 12min, finally uses aseptic water washing 3-5 time
Then respectively be equipped with disinfect after in the 50ml triangular flask of corn seed, add sterilized water, can submergence be as the criterion with firm. Corn seed after the sterilization that sterilized water is soaked is put into incubator and is secretly cultivated, and temperature is made as 26 DEG C, observes seed growth situation every day, becomes muddiness as pollution norms, Continuous Observation 4 days taking the long bacterium of seed and sterilized water.
Experiment conclusion, is that 2% liquor natrii hypochloritis processes refreshing 56 corn seeds more than 10 minutes with mass fraction, and contamination phenomenon does not appear in corn seed, and Disinfection Effect is better.
In superclean bench, corn seed sterilized water being soaked after the sterilization of 72h strips mature embryo, along plumular axis rip cutting two halves, otch is inoculated in downwards on MS calli induction media, calli induction media is that MS culture medium adds: Glu (300mg/L)+CH (500mg/L)+Pro (700mg/L)+KT (0.5mg/L)+3% sucrose+agar (7.5g/L), 2,4-D concentration is 2mg/L, pH5.8. on this basis, dark culturing in 26 DEG C of intelligent illumination boxs, after three weeks, statistics corn mature embryo callus induction rate is 54.3%.
Embodiment 4
1) adopt Zheng's 58 corn seeds, select big or small corn seed even, full seed and be divided into five equal portions, be respectively charged in 50ml triangular flask for subsequent use;
2) getting the corn seed running water that step 1) selects rinses, washing time is 30 minutes, then the corn seed after rinsing being placed on superclean bench is 75% alcohol rinsing with mass fraction, the time of rinsing is 1 minute, the clorox that rinsing is then 2% with mass fraction later is again sterilized the corn seed after alcohol rinsing respectively, the time of sterilization is followed successively by 4min, 6min, 8min, 10min, 12min, finally uses aseptic water washing 3-5 time
Then respectively be equipped with disinfect after in the 50ml triangular flask of corn seed, add sterilized water, can submergence be as the criterion with firm. Corn seed after the sterilization that sterilized water is soaked is put into incubator and is secretly cultivated, and temperature is made as 26 DEG C, observes seed growth situation every day, becomes muddiness as pollution norms, Continuous Observation 4 days taking the long bacterium of seed and sterilized water.
Experiment conclusion, is that 2% liquor natrii hypochloritis processes Zheng's 58 corn seeds more than 10 minutes with mass fraction, and contamination phenomenon does not appear in corn seed, and Disinfection Effect is better.
In superclean bench, corn seed sterilized water being soaked after the sterilization of 72h strips mature embryo, along plumular axis rip cutting two halves, otch is inoculated in downwards on MS calli induction media, calli induction media is that MS culture medium adds: Glu (300mg/L)+CH (500mg/L)+Pro (700mg/L)+KT (0.5mg/L)+3% sucrose+agar (7.5g/L), 2,4-D concentration is 1mg/L, pH5.8. on this basis, dark culturing in 26 DEG C of intelligent illumination boxs, after three weeks, statistics corn mature embryo callus induction rate is 60%.
Claims (3)
1. the seed disinfection method in the cultivation of corn mature embryo tissue, is characterized in that:
Comprise the steps:
1) select size evenly, without obviously corn seed heterochromatic, full seed is for subsequent use;
2) getting the corn seed running water that step 1) selects rinses, washing time is 20-30 minute, then the corn seed after rinsing being placed on superclean bench is 75% alcohol rinsing with mass fraction, the time of rinsing is 0.5-1 minute, corn seed after the clorox sterilization rinsing that rinsing is then 2% with mass fraction later again, the time of sterilization is 10 minutes, finally uses aseptic water washing 3-5 time.
2. the seed disinfection method of a kind of corn mature embryo tissue according to claim 1 in cultivating, is characterized in that: step 2) in running water washing time be 10-30 minute, be preferably 20 minutes.
3. the seed disinfection method of a kind of corn mature embryo tissue according to claim 1 in cultivating, is characterized in that: step 2) in be 2% with mass fraction clorox disinfecting time is 8-14 minute, be preferably 10 minutes.
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| CN201410560780.5A CN105580731A (en) | 2014-10-21 | 2014-10-21 | Seed disinfection method in mature embryo tissue culture |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109061057A (en) * | 2018-07-12 | 2018-12-21 | 安徽省农业科学院作物研究所 | A kind of non-destructive monitoring method of crops seedling stage root pathogens colonization status |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US6114603A (en) * | 1998-03-27 | 2000-09-05 | John Innes Center | Genetic engineering of sugarbeet plants |
| CN1763207A (en) * | 2005-08-30 | 2006-04-26 | 广东省农业科学院作物研究所 | A kind of corn borer resistant transgenetic ultra-sweet corn regeneration system and establishment method thereof |
| CA2438633C (en) * | 2001-02-19 | 2014-05-13 | Bayer Bioscience N.V. | Method and means for determining fitness in plants |
| CN104082132A (en) * | 2014-06-24 | 2014-10-08 | 广东省农业科学院作物研究所 | Chemical speed-soaking haploid doubling method for sweet corn |
-
2014
- 2014-10-21 CN CN201410560780.5A patent/CN105580731A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6114603A (en) * | 1998-03-27 | 2000-09-05 | John Innes Center | Genetic engineering of sugarbeet plants |
| CA2438633C (en) * | 2001-02-19 | 2014-05-13 | Bayer Bioscience N.V. | Method and means for determining fitness in plants |
| CN1763207A (en) * | 2005-08-30 | 2006-04-26 | 广东省农业科学院作物研究所 | A kind of corn borer resistant transgenetic ultra-sweet corn regeneration system and establishment method thereof |
| CN104082132A (en) * | 2014-06-24 | 2014-10-08 | 广东省农业科学院作物研究所 | Chemical speed-soaking haploid doubling method for sweet corn |
Non-Patent Citations (1)
| Title |
|---|
| 王洪振等: ""玉米成熟胚组织培养的研究进展"", 《种子》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109061057A (en) * | 2018-07-12 | 2018-12-21 | 安徽省农业科学院作物研究所 | A kind of non-destructive monitoring method of crops seedling stage root pathogens colonization status |
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Application publication date: 20160518 |