CN108668903A - A kind of processing method of rye winter explant - Google Patents
A kind of processing method of rye winter explant Download PDFInfo
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- CN108668903A CN108668903A CN201810746734.2A CN201810746734A CN108668903A CN 108668903 A CN108668903 A CN 108668903A CN 201810746734 A CN201810746734 A CN 201810746734A CN 108668903 A CN108668903 A CN 108668903A
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- explant
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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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Abstract
The present invention relates to plant tissue cultivation fields, and in particular to a kind of processing method of rye winter explant.The processing method, step include：1) selection of explant：With rye winter above-ground plant parts, blade is removed, as explant；2) pretreatment of explant：Clean the explant surface smut；3) disinfection of explant：The liquor natrii hypochloritis that using volumetric concentration be 70%~75% ethanol solution and mass concentration is 0.1~0.3% to the explant carry out infiltration disinfection to get.The processing method of explant provided by the invention is made that improvement compared with the existing technology, can turn out ideal explant, has good adaptability, survival rate high in tissue cultures.
Description
Technical field
The present invention relates to plant tissue cultivation fields, and in particular to a kind of processing method of rye winter explant.
Background technology
Rye winter, also known as black dwarf lilyturf, scientific name were Liliaceae Ophiopogon, are that nature is rare black in the rye winter
Color plant.Plant is short and small, and high 5-10 centimetres general, overgrown, stockless, leaf is linear, blackish green, the month at florescence 5-7.Blue berry.
Phyllopodium generates clump, and standing grain is lobate, 10-50 centimetres long, 1.5-3.5 millimeters wide.The long 6-15 (- 27) centimetre of scape；The long 2-5 of raceme
Centimetre, tool several Hua Dansheng or is born in bract armpit, bract lanceolar is nethermost 7-8 millimeters long in pairs to more than 10 flowers.Flower
Obstruct long 3-4 millimeters, middle part or more or nearly middle part are born in joint；Tapel is often slightly sagging not to be carried out, and lanceolar is about 5 millimeters, in vain
Or lavender；Anther triangular shape lanceolar, long 2.5-3 millimeters；Style is about 4 millimeters, and about 1 millimeter wide, base portion is wide, upwards gradually
It is narrow.Seed is spherical, 7-8 millimeters of diameter.The month at florescence 5-8, the fruiting period 8-9 months.
Invention content
The object of the present invention is to provide a kind of processing methods of rye winter explant.
A kind of processing method of rye winter explant, step include：
1) selection of explant：With rye winter above-ground plant parts, blade is removed, as explant；
2) pretreatment of explant：Clean the explant surface smut；
3) disinfection of explant：Using volumetric concentration be 70%~75% ethanol solution and mass concentration is 0.1~0.3%
Liquor natrii hypochloritis to the explant carry out infiltration disinfection to get.
Processing method of the present invention preferably selects the rye winter healthy plant of current year life, selects aerial part, removes
Blade selects current year stem of the raw plant with growing point as explant.
Preferably, the stem with growing point of 1cm long is chosen as explant, and such selection contributes to sprouting for growing point bud
The acquisition of hair and detoxification plant.
Step 2) explant pretreatment of the present invention, cleans explant surface smut, it is preferred to use suitable concentration is washed
Clothing powder water impregnates 10-20min, then rinses to cleaning in tap water down-flow water, was advisable with 1-2 hours.Washing powder water concentration is excellent
It is selected as 10ppm.
Step 3) explant of the present invention disinfection, preferably the specific steps are：
3.1) it uses the ethanol solution that volumetric concentration is 70%~75% to infiltrate the explant to sterilize；
3.2) the use of volumetric concentration is successively 70%~75% ethanol solution and secondary chlorine that mass concentration is 0.1~0.3%
Acid sodium solution carries out infiltration disinfection to the explant.
Step 3.1) the preferred operations are：The ethanol solution for the use of volumetric concentration being 70%~75% is to the explant
Infiltration disinfection；
Preferred disinfecting time is 40-80s,
The preferred concentration of the ethanol solution is 70%, and disinfecting time is preferably 60~65s.
The step 3.2) preferably concrete operations be：Explant obtained by step 3.1) is peelled off into surface layer leaf sheath, then is made successively
It is carried out disinfection with ethanol solution and liquor natrii hypochloritis.The base portion tissue contacted with sodium hypochlorite is cut off after hypochlorite disinfectant.
The liquor natrii hypochloritis is the aqueous solution of sodium hypochlorite, and every liter of liquor natrii hypochloritis is preferably added to tween 3-4
Drop, the tween are polysorbas20-80.
Disinfecting time using ethanol solution is preferably 20-40s；
And/or using liquor natrii hypochloritis disinfecting time be disinfection 400-800s.
Preferably, the volumetric concentration of the ethanol solution is preferably 70%, uses 30~35s of ethanol solution disinfecting time；
The mass concentration of the liquor natrii hypochloritis is preferably 0.1% (3-4 drops polysorbas20-80 is added in every liter of liquor natrii hypochloritis), is disappeared
Malicious 600~650s of time.
Processing method of the present invention, step A) and step B) it is to be disinfected to explant, but it is used
Reagent treatment and time are different, and such processing mode improves the sterile rate of rye winter explant, while to the damage of tissue
Small, sterile rate and survival rate have reached optimal balance.
Aseptic water washing will be used after disinfection every time, thoroughly to remove sodium hypochlorite.It will fully be stirred when sterilizing and rinsing
It mixes, ensures that material each section comes into full contact with disinfectant or sterile water.It is preferred that being rinsed 5-7 times with sterile water, every time no less than
3min。
Sterilizing operation is carried out in desinfection chamber superclean bench.
Rinse water need to pass through and disinfect after water and disinfection in disinfectant, and those skilled in the art can carry out conventional choosing
It selects, provides a kind of sterilization method herein, 121 DEG C, the disinfection with high pressure steam of 28min is handled.
The present invention provides a kind of tissue cultural method of rye winter explant together, as follows：
Using the rye winter explant by pre-processing and disinfecting, it is seeded in culture medium.At 20-25 DEG C
13h·d-1Illumination cultivation, preferably 25 DEG C.
The processing method of explant provided by the invention is made that improvement compared with the existing technology, can turn out ideal
Explant, have good adaptability in tissue cultures, survival rate is high.
Specific implementation mode
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..
Sterilizing utensil prepares：
250ml plastics tissue culture bottles are chosen, are cleaned up, disinfection with high pressure steam is spare.
The preparation of disinfectant：
The sterile water of autoclave sterilization
0.1% liquor natrii hypochloritis (every liter is added 3 drop Tween-20s)
0.2% liquor natrii hypochloritis (every liter is added 3 drop Tween-20s)
0.3% liquor natrii hypochloritis (every liter is added 3 drop Tween-20s)
70% ethyl alcohol
75% ethyl alcohol.
The preparation of instrument：
Sterilized filter paper, scalpel, scissors, tweezers.
Embodiment 1
The present embodiment provides a kind of processing method of rye winter explant, concrete operations are：
1) selection of explant：Rye winter above-ground plant parts is acquired, blade is removed, 1cm is taken to carry the stem conduct of tender shoots
Explant；
2) pretreatment of explant：20min is impregnated with the washing powder water of a concentration of 10ppm, is then carried out under tap water
Flowing water rinses 2h and is cleaned to thorough；
3) disinfection of explant：
A) infiltration is carried out using volumetric concentration for 70% ethanol solution to the explant pre-processed in superclean bench to disappear
Poison, disinfecting time 60s are rinsed 5 times, each 3min using sterile water；
B it is 75% ethanol solution infiltration disinfection 30s) to use volumetric concentration, reuses the hypochlorous acid that mass concentration is 0.1%
Sodium solution (every liter be added 3 drop polysorbas20s) carries out infiltration disinfection 600s to the explant, rinses 7 times using sterile water, every time
3min.The part that is contacted with thimerosal of excision wound to get.
Embodiment 2
The present embodiment provides a kind of processing method of rye winter explant, concrete operations are：
1) selection of explant：Rye winter above-ground plant parts is acquired, blade is removed, 1cm is taken to carry the stem conduct of tender shoots
Explant；
2) pretreatment of explant：20min is impregnated with the washing powder water of a concentration of 10ppm, is then carried out under tap water
Flowing water rinses 2h and is cleaned to thorough；
3) disinfection of explant：
A) infiltration is carried out using volumetric concentration for 75% ethanol solution to the explant pre-processed in superclean bench to disappear
Poison, disinfecting time 60s are rinsed 5 times, each 3min using sterile water；
B it is 70% ethanol solution infiltration disinfection 30s) to use volumetric concentration, reuses the hypochlorous acid that mass concentration is 0.1%
Sodium solution (every liter be added 3 drop polysorbas20s) carries out infiltration disinfection 600s to the explant, rinses 7 times using sterile water, every time
3min.The part that is contacted with thimerosal of excision wound to get.
Embodiment 3
The present embodiment provides a kind of processing method of rye winter explant, concrete operations are：
1) selection of explant：Rye winter above-ground plant parts is acquired, blade is removed, 1cm is taken to carry the stem conduct of tender shoots
Explant；
2) pretreatment of explant：20min is impregnated with the washing powder water of a concentration of 10ppm, is then carried out under tap water
Flowing water rinses 2h and is cleaned to thorough；
3) disinfection of explant：
A) infiltration is carried out using volumetric concentration for 70% ethanol solution to the explant pre-processed in superclean bench to disappear
Poison, disinfecting time 60s are rinsed 5 times, each 3min using sterile water；
B it is 70% ethanol solution infiltration disinfection 30s) to use volumetric concentration, reuses the hypochlorous acid that mass concentration is 0.1%
Sodium solution (every liter be added 3 drop polysorbas20s) carries out infiltration disinfection 600s to the explant, rinses 7 times using sterile water, every time
3min.The part that is contacted with thimerosal of excision wound to get.
Embodiment 4
The present embodiment provides a kind of processing method of rye winter explant, concrete operations are：
1) selection of explant：Rye winter above-ground plant parts is acquired, blade is removed, 1cm is taken to carry the stem conduct of tender shoots
Explant；
2) pretreatment of explant：20min is impregnated with the washing powder water of a concentration of 10ppm, is then carried out under tap water
Flowing water rinses 2h and is cleaned to thorough；
3) disinfection of explant：
A) infiltration is carried out using volumetric concentration for 70% ethanol solution to the explant pre-processed in superclean bench to disappear
Poison, disinfecting time 60s are rinsed 5 times, each 3min using sterile water；
B it is 75% ethanol solution infiltration disinfection 30s) to use volumetric concentration, reuses the hypochlorous acid that mass concentration is 0.3%
Sodium solution (every liter be added 3 drop polysorbas20s) carries out infiltration disinfection 600s to the explant, rinses 7 times using sterile water, every time
3min.The part that is contacted with thimerosal of excision wound to get.
Embodiment 5
The present embodiment provides a kind of processing method of rye winter explant, concrete operations are：
1) selection of explant：Rye winter above-ground plant parts is acquired, blade is removed, 1cm is taken to carry the stem conduct of tender shoots
Explant；
2) pretreatment of explant：20min is impregnated with the washing powder water of a concentration of 10ppm, is then carried out under tap water
Flowing water rinses 2h and is cleaned to thorough；
3) disinfection of explant：
A) infiltration is carried out using volumetric concentration for 70% ethanol solution to the explant pre-processed in superclean bench to disappear
Poison, disinfecting time 80s are rinsed 5 times, each 3min using sterile water；
B it is 75% ethanol solution infiltration disinfection 20s) to use volumetric concentration, reuses the hypochlorous acid that mass concentration is 0.1%
Sodium solution (every liter be added 3 drop polysorbas20s) carries out infiltration disinfection 600s to the explant, rinses 7 times using sterile water, every time
3min.The part that is contacted with thimerosal of excision wound to get.
Embodiment 6
The present embodiment provides a kind of processing method of rye winter explant, concrete operations are：
1) selection of explant：Rye winter above-ground plant parts is acquired, blade is removed, 1cm is taken to carry the stem conduct of tender shoots
Explant；
2) pretreatment of explant：20min is impregnated with the washing powder water of a concentration of 10ppm, is then carried out under tap water
Flowing water rinses 2h and is cleaned to thorough；
3) disinfection of explant：
A) infiltration is carried out using volumetric concentration for 70% ethanol solution to the explant pre-processed in superclean bench to disappear
Poison, disinfecting time 60s are rinsed 5 times, each 3min using sterile water；
B it is 70% ethanol solution infiltration disinfection 30s) to use volumetric concentration, reuses the hypochlorous acid that mass concentration is 0.2%
Sodium solution (every liter be added 3 drop polysorbas20s) carries out infiltration disinfection 600s to the explant, rinses 7 times using sterile water, every time
3min.The part that is contacted with thimerosal of excision wound to get.
Test example 1
The preparation-obtained explant of the above various embodiments is inoculated into culture medium, the 13hd at 25 DEG C-1Illumination is trained
It supports.It is each to repeat 50, to ensure the repeatability of experiment.
After culture 10 days, observation culture situation.
Culture medium is MS+2.0mg/L 6-BA+0.1mg/L IAA+3.0g/L sucrose+0.7g/L agar, pH=5.8.
After testing, as a result as follows：
| Embodiment 1 | Embodiment 2 | Embodiment 3 | Embodiment 4 | Embodiment 5 | Embodiment 6 | |
| Sterile rate | 99% | 92% | 92% | 100% | 88% | 95% |
| Survival rate | 96% | 89% | 91% | 82% | 84% | 88% |
Specifically, the equal thorough in sterilization of explant of 1 gained of embodiment, is easily obtained the regeneration plant of health, growth
Point or bud point are damaged relatively light or do not have, and are conducive to the sprouting of bud and the extension growth of growing point.
Embodiment 4 occurs explantation tissue's browning necrosis phenomena, can not obtain detoxification plant；Embodiment 2,3,5 has
Existing explant disinfection is not thorough phenomenon, and the regeneration plant that carries disease germs germination is slow, forms runt seedling.
Although above having used general explanation, specific implementation mode and experiment, the present invention is made to retouch in detail
It states, but on the basis of the present invention, it can be made some modifications or improvements, this is apparent to those skilled in the art
's.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to claimed
Range.
Claims (10)
1. a kind of processing method of rye winter explant, step include：
1) selection of explant：With rye winter above-ground plant parts, blade is removed, selects the stem with growing point as explant
Body；
2) pretreatment of explant：Clean the explant surface smut；
3) disinfection of explant：Time that using volumetric concentration be 70%~75% ethanol solution and mass concentration is 0.1~0.3%
Sodium chlorate solution to the explant carry out infiltration disinfection to get.
2. processing method according to claim 1, it is characterised in that：The disinfection of step 3) explant, including：
3.1) it uses the ethanol solution that volumetric concentration is 70%~75% to infiltrate the explant to sterilize；
3.2) the use of volumetric concentration is successively 70%~75% ethanol solution and sodium hypochlorite that mass concentration is 0.1~0.3%
Solution carries out infiltration disinfection to the explant.
3. processing method according to claim 1 or 2, it is characterised in that：The rye winter of step 1) the selection current year life
Healthy plant.
4. according to claim 1-3 any one of them processing methods, which is characterized in that chooses 1-2cm long carries growing point
Stem as explant.
5. according to claim 2-4 any one of them processing methods, it is characterised in that：Step 3.1) the ethanol solution it is dense
Degree is 70%, and infiltration disinfecting time is 40-80s.
6. according to claim 2-5 any one of them processing methods, it is characterised in that：In step 3.2), the ethanol solution
A concentration of 75%, the use of liquor natrii hypochloritis's disinfecting time is 400-800s using ethanol disinfection time 20-40s.
7. according to claim 2-6 any one of them processing methods, it is characterised in that：Step 3) the liquor natrii hypochloritis is dense
Degree is 0.1%；
Every liter of addition 3-4 drops polysorbas20 or Tween 80 in the liquor natrii hypochloritis.
8. according to claim 2-7 any one of them processing methods, it is characterised in that：In the step 3.1), ethyl alcohol is used
The disinfecting time of solution is 60-65s；
In the step 3.2), the disinfecting time using ethanol solution is 30-35s；Use the disinfecting time of liquor natrii hypochloritis
To sterilize 600-650s.
9. processing method according to claim 1, it is characterised in that：The step 3) is：
3.1) it uses ethanol solution to infiltrate the explant to sterilize；
3.2) explant obtained by step 3.1) is peelled off into surface layer leaf sheath, uses ethanol solution and mass concentration and sodium hypochlorite successively
Solution carries out infiltration disinfection to the explant.
10. processing method according to claim 1, it is characterised in that：Include the following steps：
1) selection of explant：Rye winter above-ground plant parts is acquired, blade is removed, takes stems of the 1cm with tender shoots as explant
Body；
2) pretreatment of explant：Thoroughly clean；
3) disinfection of explant：
A the use of volumetric concentration is) that 70% ethanol solution carries out infiltration disinfection, disinfecting time 60s rinses 5 times, often using sterile water
Secondary 3min；
B it is 75% ethanol solution infiltration disinfection 30s) to use volumetric concentration, reuses every liter that mass concentration is 0.1% and is added 3
The liquor natrii hypochloritis for dripping polysorbas20 carries out infiltration disinfection 600s to the explant, rinses 7 times using sterile water, every time
3min；The part that is contacted with thimerosal of excision wound to get.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201810746734.2A CN108668903A (en) | 2018-07-09 | 2018-07-09 | A kind of processing method of rye winter explant |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201810746734.2A CN108668903A (en) | 2018-07-09 | 2018-07-09 | A kind of processing method of rye winter explant |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101869061A (en) * | 2009-04-24 | 2010-10-27 | 上海上房园林植物研究所 | Tissue culture method of ophiopogon planiscapus |
| CN104186312A (en) * | 2014-08-04 | 2014-12-10 | 江苏农林职业技术学院 | Tissue culture and rapid propagation method for ophiopogon japonicus |
-
2018
- 2018-07-09 CN CN201810746734.2A patent/CN108668903A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101869061A (en) * | 2009-04-24 | 2010-10-27 | 上海上房园林植物研究所 | Tissue culture method of ophiopogon planiscapus |
| CN104186312A (en) * | 2014-08-04 | 2014-12-10 | 江苏农林职业技术学院 | Tissue culture and rapid propagation method for ophiopogon japonicus |
Non-Patent Citations (6)
| Title |
|---|
| JING-WEN ZHAO等: "Evaluation of anti-inflammatory activity of compounds isolated from the rhizome of Ophiopogon japonicas", 《BMC COMPLEMENTARY AND ALTERNATIVE MEDICINE》 * |
| 孙晓萍: "《观赏植物良种繁育技术》", 31 August 2009, 浙江人民出版社 * |
| 李秀霞: "《植物组织培养》", 30 June 2014, 东北大学出版社 * |
| 王华宇等: "黑麦冬工厂化育苗关键技术研究", 《江苏农业科学》 * |
| 郑志仁等: "黑麦冬的组织培养和快速繁殖", 《植物生理学通讯》 * |
| 雷加容等: "麦冬组培与快速繁殖技术研究", 《西南农业学报》 * |
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