CN1214858A - Saccharide-free box-type plant tissue culturing method - Google Patents
Saccharide-free box-type plant tissue culturing method Download PDFInfo
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- CN1214858A CN1214858A CN 98121867 CN98121867A CN1214858A CN 1214858 A CN1214858 A CN 1214858A CN 98121867 CN98121867 CN 98121867 CN 98121867 A CN98121867 A CN 98121867A CN 1214858 A CN1214858 A CN 1214858A
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Abstract
The sugarless incubator type plant tissue culture method uses incubator as culture container instead of culture bottole, and is favourable for plant to fully absorb CO2 and gas exchange. Said invented optimum culture method is superior to existent technology obviously in plant tissue growth indexes, the plant growth quantity can be multiplied, seedling quality is good, commodity seedling rate is high, and its cost is low. Said invention is applicable to several plants of Caixing. Carnation, potato, Africa chrysanthemum, Lujuren and rugosa rose, etc.
Description
The present invention relates to a kind of culture technique of plant tissue, especially relate to a kind of sugar-free culture technique.
The culture technique of plant tissue began one's study from 1930's, had been widely used in the preservation of quick breeding, detoxification and the germ plasm resource of plant corpus, thereby had promoted developing by leaps and bounds of biological breeding technique.Traditional for a long time method for plant tissue culture is with the carbon source of sugar as plant corpus, and replenishes corresponding organic substance.Conventional method exists pollution rate height in the incubation, and the rooting of vitro seedling rate is low, and the transition shoot survival percent is low, defectives such as production cost height.
Existing sugar free tissue culture technology (list of references: 1.Kozait, Fujiwarak, Hagashim.Collected papers on Environmaental Control in MicropropagationVolumel.JoPan:Chiba Vniversity press, 1991; 2. Xiao yulan etc.The research of flameray gerbera sugar free tissue culture The Application of Technology.The gardening journal, 1998,25 (4)) be to adopt bottle to make culture vessel, it is low to have a pollution rate, robust plant, the advantage that the transition shoot survival percent is high.But bottle is because the space is too little, and gas circulation is not smooth, even replenished CO
2, also have only air-vent just can enter in the bottle for plant absorption, CO by sealing on the film
2Quantity delivered still not enough, plant strain growth speed is slow, and operating difficulties, cost is higher.
The objective of the invention is to eliminate the defective of prior art, provide a kind of and replace bottle to make culture vessel, adopt sugar-free culture-medium, suitably illumination and feeding CO with incubator
2Cultural method, can overcome the problems referred to above of prior art effectively, easy and simple to handle, can promote that plant grows fast, further reduce production costs, increase economic efficiency.
Saccharide-free box-type plant tissue cultural method provided by the invention comprises the following steps:
One, make incubator: adopt lucite to make incubator, the thickness of lucite can be 3～5 millimeters, the size of casing: length * height * wide=500～1000 millimeter * 200～250 millimeters * 500 millimeters.
Two, the regulation and control of culture environment: to incubator configuration carbonic acid gas aerating system, illumination system, the control condition of culture is convenient to regulate in temperature control system and wet control system.
Three, preparation medium:
1, preparation culture fluid:
1. the present invention has done important improvement to MS culture fluid of the prior art, adopts the present invention to prepare especially
Sugar-free MS culture fluid, promptly the prescription in do not add sugar, organic matter and vitamin, kept other composition, the prescription of sugar-free MS culture fluid (unit: mg/litre) be:
NH
4NO
3?????????1650
KNO
3????????????1900
KH
2PO
4?????????170
MgSO
4·7H
2O????370
CaCl·2H
2O??????440
MnSO
4·H
2O?????22.3
ZnSO
4·7H
2O????8.6
H
3BO
3??????????6.2
KI????????????????0.83
NaMoO
4·2H
2O???0.25
CuSO
4·5H
2O????0.025
FeSO
4·7H
2O????27.8
Na
2·EDTA???????37.3
2. the present invention serves as a basis preparation enrichment culture liquid with the sugar-free culture fluid, its prescription (unit: mg/litre) be: MS (not sugaring, organic matter and vitamin)+(0～0.5) 6-benzylaminopurine
3. the present invention serves as a basis preparation culture of rootage liquid with the sugar-free culture fluid, and its prescription is (unit: mg/litre) MS (not sugaring, organic matter and vitamin)+(0～0.1) methyl+(0～5000) activated carbon.
2, medium matrix kind:, can select following material as matrix respectively: 1. 2. 3. 4. vermiculite of sand of perlite of agar according to the biological property of cultivation plant;
3, the production process of medium:
1. by prescription culture fluid is prepared;
2. as selecting agar as culture matrix, can add culture fluid in agar dissolving back, after sterilizing, it is stand-by to put into incubator;
3. as when selecting perlite or sand or vermiculite to make matrix, then need to sterilize respectively, add culture fluid in the matrix stand-by again.
Four, sterilization:
1, culturing room's sterilization: weighing 6 gram potassium permanganate are put into a container, add 10 milliliters of formaldehyde again and fumigate ultra violet lamp;
2, incubator sterilization: after earlier incubator being cleaned up, the ethanol with 75% carries out cabinet wall wiping sterilization, and then uses 6 gram potassium permanganate and 10 milliliters of formaldehyde to carry out fumigation;
3, medium sterilization: 1. culture fluid+agar: through 120 ℃, 20 minutes autoclaving sterilizations or scalding in the container of lid is arranged; 2. the sterilization of culture fluid+perlite or sand or vermiculite: culture fluid is through 120 ℃, 20 minutes autoclave sterilizations or boiled in the container of lid is arranged 20 minutes; Perlite, sand, vermiculite install through 120 ℃ 30 minutes autoclaving with a container.
Five, saccharide-free box-type is cultivated
1, under aseptic condition, test-tube plantlet segmentation or plant division are transferred in the incubator that installs medium;
2, the low light level is according to being period 7～10 days (time length is determined according to floristics or growing state), and its illumination is 2500～2700 luxs, and every day, light application time was 10～12 hours, and cultivation temperature is 22～25 ℃;
3, its illumination was adjusted into 6000～8000 luxs to intense light irradiation since the 8th～11 day period, illumination every day 14～16 hours;
4, the 8th day input CO
2, input CO
2Time and illumination carry out synchronously, its concentration is 2000～3500PPm:
5, humidity control: the humidity in 0～3 day or 0～5 day incubator is to be 70% after reducing to 75～85%, 20 days gradually after 100%; Then can emerge in 20～25 days, but direct transplanting just can become commercial seedling in 18～25 days in seedbed and nutritious bag.
The present invention utilizes sugar-free to cultivate the low advantage of pollution rate, adopts big box culture vessel to cultivate, and is beneficial to plant to CO
2Abundant absorption and gas exchange, can promote the quick growth of plant effectively, further reduce production costs, through to winner in lottery, carnation, potato, flameray gerbera, various plants such as rose and The Incredible Hulk has been carried out test of many times, and favorable reproducibility has good popularizing application prospect.
Embodiment and comparative trial:
For trying material: winner in lottery (Limonium latifolium), potato (Potato) test-tube plantlet
Culture vessel: 1. bottle (250 milliliters of volumes)
2. incubator (1000 millimeters * 500 millimeters * 250 millimeters)
Test is handled: No. 1 sample sugar-free+bottle+CO
2
No. 2 sample sugar-free+incubator+CO
2
Concrete operations:
Before changeing seedling, at first incubator and culturing room are carried out strict disinfecting, then prepared culture medium is poured in the incubator, treat to connect seedling after the medium cooling.The every case of winner in lottery is inserted seedling 1500 strains, and potato is cut into stem section of a leaf, and every case is inserted 3000 seedlings, no matter be that the seedling that winner in lottery or potato are all chosen uniformity is transferred.The employing bottle is cultivated, 100 bottles of every plant species inoculations, and winner in lottery is inserted 10 seedlings for every bottle, and potato inserts 18 seedlings.The bottle cultivation is all adopted to breathe freely and is sealed film, and the air-vent area is 157mm
2
Sugar-free was cultivated in 10 days, the quantity of illumination 2700 luxs, 12 hours every days of time; The quantity of illumination increases to 8000 luxs after 10 days; Light application time is 14 hours every days of winner in lottery, 16 hours every days of potato; Replenish CO
23920 milligrams/mm of agricultural degree
3, replenish CO
2Time and illumination carry out synchronously.Saccharide-free box-type is cultivated directly CO
2Be input in the incubator.The sugar-free bottle is cultivated to be placed on the culturing rack in 10 days and is cultivated, and after 10 days bottle is put into incubator, imports CO again
2Potato is cultivated after 20 days and emerges, and winner in lottery is cultivated after 28 days and emerged.Directly seedling is planted on the nutrition soil after emerging, carry out the transition hardening, 20 days " Invest, Then Investigate " survival rates.
Result of the test:
1, the size of culture vessel is very obvious to the influence of plant strain growth.With regard to incubator and bottle, bottle is because the space is too little, and gas circulation is not smooth, even replenished CO
2, but have only air-vent just can enter in the bottle for plant absorption, CO by sealing on the film
2Quantity delivered still not enough, plant strain growth is slow.Be both sugar-free as can be seen from Table 1 and cultivate and feed CO
2, the height of the plant of cultivating with incubator is apparently higher than cultivating with bottle.Winner in lottery on average exceeds 1.97 centimetres, and potato on average exceeds 9.03 centimetres, and robust plant, and every growth indexes all is better than cultivating with bottle.Wherein the difference of potato is particularly evident.Reason is that the incubator space is big, is convenient to gas exchange, CO
2Absorb fully.But incubator can only be used for sugar-free to be cultivated, and has sugared cultivation can only be used for bottle, because the increase of sugar cultivation along with culture vessel arranged, pollution rate increases gradually.Have only the big or small wide adaptability of sugar-free cultivation to the culture vessel volume.
2, CO
2It is the key factor that influences plant strain growth.Carbon nutrition is the life basis of plant, and plant will absorb CO
2Carry out photosynthesis, have sugar to cultivate by the carbon source of sugar as plant, sugar-free is cultivated and must be replenished CO
2With enhancing illumination, make plant self carry out photosynthesis and make organic matter.So CO
2Supply be vital.If only by airborne CO
2Replenish and to satisfy the needs of plant far away.As can be seen from Table 1, even in large-scale culture vessel, under the strong illumination of 8000LX, if do not replenish CO
2, plant still grows bad.Under same condition of culture, replenish CO
2Can make winner in lottery weight increase by 35%, plant height increases by 14%, and the number of blade increases by 31%, and leaf area increases by 66%, and the increase of potato growth amount is more, and weight increases by 303%, and plant height increases by 147%, and the number of blade increases by 82%, and leaf area increases by 11.91 times.As seen CO
2Importance to plant strain growth.
3, there is sugar to cultivate and sugar-free cultivation cost analysis
Table 2 shows employing sugar-free cultivation of the present invention cost analysis, and the average every strain cost of winner in lottery reduces by 0.038 yuan, and potato reduces by 0.028 yuan, has good economic benefit.Handle Trea tment fresh weight plant height number of blade leaf area transplanted seedling number and survive several transplanting survival rate examination material medium sugar culture vessel CO
2Fresh Shoot Leaf number leaf area transplant live number transplantplantlets dium Sucrocse Vessel weight length number live percent
(%) (mg/m
3) (mg/Pant) (cm) (leaf/plant) (cm
3/ plant) (Plants) (Plants) (%) winner in lottery MS 3 bottles 559 5.68 11.10 17.65 950 831 87.5% (Limonium MS 0 (bottle) 3,920 561 5.71 9.83 18.35 978 881 90.1latifolum) MS 0 incubator 563 7.54 9.77 18.10 1,418 1,270 89.6
MS 0 box 3,920 758 8.57 12.8 30.08 1,497 1,424 95.1 potato MS 3 bottles 135 6.76 6.15 0.73 1,600 1450 90.6Poaoto MS 0 (bottle) 3,920 113 6.11 6.01 0.71 1,358 1,153 84.90
MS 0 incubator 127 6.12 5.92 0.71 2,245 2,018 89.8
MS 0 box 3,920 512 15.14 10.81 8.46 2,972 2970 99.9* investigate when changeing seedling: plant weight plant height number of blade leaf area
(mg/ strain) be (leaf/strain) (cm (cm)
2/ strain)
Winner in lottery 237 5.52 7.14 4.67
Potato 22 1.10 1.00 0.12
The different training method of table 1. is to the influence of plant strain growth
| Expenditure | There is sugar to cultivate production cost (unit) | Saccharide-free box-type is cultivated production cost (unit) | ||
| Winner in lottery 1000 seedlings | Potato 1800 seedlings | Winner in lottery 1500 seedlings | Potato 3000 seedlings | |
| Medium sterilization electricity charge sealed membrane labour cost is polluted and is cultivated electricity charge CO2Take | ????7.18 ????6.00 ????5.50 ????22.00 ????7.03 ????29.76 ????- | ????7.18 ????6.00 ????5.50 ????27.00 ????7.68 ????23.36 ????- | ????8.83 ????2.00 ????- ????14.00 ????- ????49.92 ????3.90 | ????8.83 ????2.00 ????- ????19.00 ????- ????39.98 ????3.50 |
| Add up to commercial seedling average: (unit/strain) | ????77.47 ????831 ????0.093 | ????76.72 ????1450 ????0.053 | ????78.65 ????1424 ????0.055 | ????73.31 ????2970 ????0.025 |
The contrast of table 2. production cost
Claims (7)
1. the cultural method of a saccharide-free box-type plant tissue comprises and 1. makes culture vessel; 2. the regulation and control of culture environment; 3. prepare medium; 4. sterilization; 5. sugar-free is cultivated, and it is characterized in that: 1. culture vessel is a box-type; 2. prepare the used MS culture fluid of medium and do not add sugar, organic matter and vitamin; 3. carbonic acid gas is imported incubator.
2. according to the process of claim 1 wherein that described culture vessel is to adopt lucite to make incubator, the thickness of lucite can be 3～5 millimeters, the size of casing: length * height * wide=500～1000 millimeter * 200～250 millimeters * 500 millimeters.
3. according to the process of claim 1 wherein that described sugar-free MS culture fluid further is mixed with enrichment culture liquid, its prescription (unit: mg/litre) be: MS (not sugaring, organic matter and vitamin)+(0～0.5) 6-benzylaminopurine; Further prepare culture of rootage liquid, its prescription is (unit: mg/litre) MS (not sugaring, organic matter and vitamin)+(0～0.1) methyl+(0～5000) activated carbon.
4. according to the process of claim 1 wherein that the matrix of described medium can be agar or perlite or sand or vermiculite.
5. according to the method for claim 4, the production process of wherein said medium is: 1. by prescription culture fluid is prepared; 2. as selecting agar as culture matrix, can add culture fluid in agar dissolving back, after sterilizing, it is stand-by to put into incubator; 3. as when selecting perlite or sand or vermiculite to make matrix, then need to sterilize respectively, add culture fluid in the matrix stand-by again.
6. according to the process of claim 1 wherein that described sterilization comprises: 1. culturing room's sterilization: weighing 6 gram potassium permanganate are put into a container, add 10 milliliters of formaldehyde again and fumigate ultra violet lamp; 2. incubator sterilization: after earlier incubator being cleaned up, the ethanol with 75% carries out cabinet wall wiping sterilization, and then uses 6 gram potassium permanganate and 10 milliliters of formaldehyde to carry out fumigation; 3. medium sterilization: culture fluid ten agar: through 120 ℃, 20 minutes autoclaving sterilizations or scalding in the container of lid is arranged; The sterilization of culture fluid ten perlites or sand or vermiculite: culture fluid is through 120 ℃, 20 minutes autoclave sterilizations or boiled in the container of lid is arranged 20 minutes; Perlite, sand, vermiculite install through 120 ℃ 30 minutes autoclaving with a container.
7. according to the process of claim 1 wherein that described sugar-free cultivation operation comprises: 1. under aseptic condition, test-tube plantlet segmentation or plant division are transferred in the incubator that installs medium; 2. the low light level is 7～10 days according to period, and its illumination is 2500～2700 luxs, and every day, light application time was 10～12 hours, and cultivation temperature is 22～25 ℃; 3. its illumination was adjusted into 6000～8000 luxs to intense light irradiation since the 8th～11 day period, illumination every day 14～16 hours; 4. imported CO on the 8th day
2, input CO
2Time and illumination carry out synchronously, its concentration is 2000～3500PPm; 5. humidity control: the humidity in 0～3 day or 0～5 day incubator is to be 70% after reducing to 75～85%, 20 days gradually after 100%; Then can emerge in 20～25 days, but direct transplanting just can become commercial seedling in 18～25 days in seedbed and nutritious bag.
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|---|---|---|---|
| CN 98121867 CN1214858A (en) | 1998-11-20 | 1998-11-20 | Saccharide-free box-type plant tissue culturing method |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 98121867 CN1214858A (en) | 1998-11-20 | 1998-11-20 | Saccharide-free box-type plant tissue culturing method |
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| CN1214858A true CN1214858A (en) | 1999-04-28 |
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Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005058022A1 (en) * | 2003-12-19 | 2005-06-30 | Yongtai Zhang | Combination-type plant sugarless tissue culture propagation device and method thereof |
| CN102084813A (en) * | 2010-12-23 | 2011-06-08 | 福建永安天奇健金线莲生态实业有限公司 | Method for culturing sugar-free tissue of jewel orchid |
| CN102668990A (en) * | 2012-06-13 | 2012-09-19 | 贵州省亚热带作物研究所 | Plant tissue culturing method and device |
| CN104969862A (en) * | 2015-06-30 | 2015-10-14 | 柳州市天姿园艺有限公司 | Matrix for Chinese orchid tissue culture |
| CN106016490A (en) * | 2016-07-13 | 2016-10-12 | 大连民族大学 | Ecotype air purifier |
| CN106172009A (en) * | 2016-08-15 | 2016-12-07 | 上海离草科技有限公司 | Plant sugar-free culture systems and plant cultivation container |
| CN106577219A (en) * | 2016-11-11 | 2017-04-26 | 浙江师范大学 | Plant transgenic seed screening method |
| CN106954551A (en) * | 2017-04-28 | 2017-07-18 | 上海离草科技有限公司 | Sugar Free Plant Tissue Culture special culture media |
| CN109122322A (en) * | 2018-09-28 | 2019-01-04 | 西北农林科技大学 | A kind of Chinese catalpa tissue culture and rapid propagation method and device |
| CN109169275A (en) * | 2018-09-13 | 2019-01-11 | 甘肃源宜生物科技有限公司 | A kind of pale reddish brown trident bletilla striata tissue-culturing rapid propagation culture medium and method |
| CN109863996A (en) * | 2018-10-28 | 2019-06-11 | 陕西海棠生态农林股份有限公司 | A kind of sugar-free tissue culture method improving plant establishment rate |
| CN109952869A (en) * | 2019-04-28 | 2019-07-02 | 华中农业大学 | Potato detoxicating micro potato breeding technique |
| CN110089430A (en) * | 2019-04-28 | 2019-08-06 | 江门市新会区林业科学研究所（江门市新会区林业科技推广站） | Cortex Eucommiae method for culturing seedlings |
| CN113016592A (en) * | 2021-01-22 | 2021-06-25 | 四川省农业科学院作物研究所 | Efficient autotrophic water culture seedling strengthening method for potato virus-free test-tube seedlings |
| CN115702630A (en) * | 2021-08-17 | 2023-02-17 | 江门市新会区林业科学研究所（江门市新会区林业科技推广站） | Sugar-free tissue culture method for pithecellobium clypearia |
-
1998
- 1998-11-20 CN CN 98121867 patent/CN1214858A/en active Pending
Cited By (20)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2005058022A1 (en) * | 2003-12-19 | 2005-06-30 | Yongtai Zhang | Combination-type plant sugarless tissue culture propagation device and method thereof |
| CN102084813A (en) * | 2010-12-23 | 2011-06-08 | 福建永安天奇健金线莲生态实业有限公司 | Method for culturing sugar-free tissue of jewel orchid |
| CN102668990A (en) * | 2012-06-13 | 2012-09-19 | 贵州省亚热带作物研究所 | Plant tissue culturing method and device |
| CN102668990B (en) * | 2012-06-13 | 2013-08-28 | 贵州省亚热带作物研究所 | Plant tissue culturing method and device |
| CN104969862A (en) * | 2015-06-30 | 2015-10-14 | 柳州市天姿园艺有限公司 | Matrix for Chinese orchid tissue culture |
| CN106016490A (en) * | 2016-07-13 | 2016-10-12 | 大连民族大学 | Ecotype air purifier |
| CN106172009A (en) * | 2016-08-15 | 2016-12-07 | 上海离草科技有限公司 | Plant sugar-free culture systems and plant cultivation container |
| CN106172009B (en) * | 2016-08-15 | 2018-08-21 | 上海离草科技有限公司 | Plant sugar-free culture systems and plant cultivation container |
| CN106577219A (en) * | 2016-11-11 | 2017-04-26 | 浙江师范大学 | Plant transgenic seed screening method |
| CN106954551A (en) * | 2017-04-28 | 2017-07-18 | 上海离草科技有限公司 | Sugar Free Plant Tissue Culture special culture media |
| CN109169275A (en) * | 2018-09-13 | 2019-01-11 | 甘肃源宜生物科技有限公司 | A kind of pale reddish brown trident bletilla striata tissue-culturing rapid propagation culture medium and method |
| CN109122322A (en) * | 2018-09-28 | 2019-01-04 | 西北农林科技大学 | A kind of Chinese catalpa tissue culture and rapid propagation method and device |
| CN109863996A (en) * | 2018-10-28 | 2019-06-11 | 陕西海棠生态农林股份有限公司 | A kind of sugar-free tissue culture method improving plant establishment rate |
| CN109952869A (en) * | 2019-04-28 | 2019-07-02 | 华中农业大学 | Potato detoxicating micro potato breeding technique |
| CN110089430A (en) * | 2019-04-28 | 2019-08-06 | 江门市新会区林业科学研究所（江门市新会区林业科技推广站） | Cortex Eucommiae method for culturing seedlings |
| CN109952869B (en) * | 2019-04-28 | 2021-05-28 | 华中农业大学 | Breeding process of detoxicated miniature potato |
| CN113016592A (en) * | 2021-01-22 | 2021-06-25 | 四川省农业科学院作物研究所 | Efficient autotrophic water culture seedling strengthening method for potato virus-free test-tube seedlings |
| CN113016592B (en) * | 2021-01-22 | 2023-12-12 | 四川省农业科学院作物研究所 | Efficient autotrophic water culture seedling strengthening method for potato virus-free test-tube seedlings |
| CN115702630A (en) * | 2021-08-17 | 2023-02-17 | 江门市新会区林业科学研究所（江门市新会区林业科技推广站） | Sugar-free tissue culture method for pithecellobium clypearia |
| CN115702630B (en) * | 2021-08-17 | 2023-11-24 | 江门市新会区林业科学研究所（江门市新会区林业科技推广站） | Sugar-free tissue culture method for pithecellobium clypearia |
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