CN103202226A - Quick and efficient rooting method for Chinese cabbage tissue culture seedlings - Google Patents
Quick and efficient rooting method for Chinese cabbage tissue culture seedlings Download PDFInfo
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Abstract
The invention discloses a quick and efficient rooting method for Chinese cabbage tissue culture seedlings. The method includes following steps: (1), preparing a proper rooting culture medium; (2), inoculating; (3), culturing; and (4), hardening and transplanting seedlings. By the method, ingredients and concentration of the quick and efficient rooting culture medium are determined, and by choosing the proper rooting culture medium, an efficient Chinese cabbage tissue culture seedling rooting system is established. The quick and efficient rooting method for the Chinese cabbage tissue culture seedlings has the advantages of simplicity in operation, low cost, stability and efficiency, greatly-increased survival rate of tissue culture seedlings, and the like.
Description
Technical field
The present invention relates to Chinese cabbage tissue culture sprout quick speed high-effective root-growing technology, specifically refer to a kind of Chinese cabbage tissue culture sprout quick speed high-effective root-growing method.
Background technology
It is reproductive speed propagation method faster that tissue is cultivated, and group training seedling rooting is the important step that tissue is cultivated, and group training seedling rooting quality and quantity is to be directly connected to the key that group training transplantation of seedlings survives; To organize the training seedling when shifting out from blake bottle, group training shoot root portion adheres to, and to contain that sugar culture-medium mass-energy rapidly and efficiently do not separate with root system under the prerequisite of not hindering root be that group training seedling is for mass-produced essential condition.
Curing agent play fixed supporting role in medium, plant tissue culture traditionally many with agar as curing agent, carragheen is because having advantages such as good coagulability and price medium relatively cheap, that preparation is come out is more transparent, using method is simple, also be widely used in Plant Tissue Breeding, Du Xueling etc. (2006) discover that import plant gel (phytagel) is better curing agent, but expensive.
The research of taking root of Chinese cabbage group training seedling at present mainly concentrates in hormone kind combined sorting and the concentration increase and decrease, and the screening of root media curing agent is not reported, and all is with agar, blocks that glue etc. as curing agent., because the solid agar medium very closely is attached to the surface of group training shoot root, be difficult to wash during as the curing agent of Chinese cabbage root media with agar etc.As the medium that adheres on the root not being cleaned, then because containing sucrose or white sugar in the medium, be easy to make root mouldy rotten, the survival rate of reduction group training seedling; Clean and then be easy to damage root system again, the survival rate of also reduction group training seedling, and time-consuming.Press for a kind of can playing a supporting role to explant in the production, be beneficial to and take root, again the root media of easy wash-out.
Summary of the invention
The purpose of this invention is to provide a kind of Chinese cabbage tissue culture sprout quick speed high-effective root-growing method, compare with present method in common, in having avoided taking root branch root few, be attached to the drawback that medium on the root is difficult for wash-out, utilize perlite and a small amount of agar as the solid support thing in the medium, the quality of taking root is good, elution speed is fast, low price can be saved a large amount of manpower and materials; And determined the agar concentration, the perlitic consumption that suit in the root media, and the kind of hormone and concentration.
For achieving the above object, Chinese cabbage tissue culture sprout quick speed high-effective root-growing method provided by the invention comprises the process of preparing root media, inoculation, cultivation and acclimatization and transplants successively, it is characterized in that:
(1) preparation root media: with the macroelement content in the conventional MS medium reduce by half, the trace element and other component contents constant, as minimal medium; In minimal medium, add agar, sucrose, methyl, heteroauxin and deionized water, be hybridly prepared into culture fluid; Then culture fluid is poured in the blake bottle that is loaded with perlite particle, perlite particle can be soaked into fully by culture fluid and do not have excess solution and overflow; Again it being carried out moist heat sterilization handles, naturally cooling forms root media, and the mass volume ratio of following component is controlled at agar 1 ~ 3g/L, sucrose 28 ~ 30g/L, methyl 0.05 ~ 0.15mg/L, heteroauxin 0.01 ~ 0.05mg/L, perlite particle 80 ~ 120g/L in root media;
(2) inoculation: the medium in blake bottle is inserted out duck eye, and the blade-carrying stem apex base portion that Chinese cabbage group training seedling downcuts is put into duck eye, tamps stem apex base portion space with the medium on next door;
(3) cultivate: will inoculate the blake bottle of getting well and be placed on and carry out culture of rootage in the culturing room;
(4) acclimatization and transplants: culture of rootage is after 21 ± 2 days, blake bottle is placed indoor place near the window, in the natural conditions lower refining seedling, make Chinese cabbage group training seedling adapt to external environment condition gradually, after the hardening seedling is taken out from bottle. wash the field planting after the sugar culture-medium matter that contains on the clean root with flowing water.
As preferred version, in the described step (1), the particle diameter of selecting perlite particle for use is 3 ~ 5mm; Control moist heat sterilization temperature is that 121 ℃, time are 15min.
As preferred version, in the described step (2), the medium in blake bottle is inserted out 2 ~ 3 duck eyes, and its size can be put Chinese cabbage group training seedling stem apex base portion just into, and Chinese cabbage group training seedling stem apex base portion has 2 ~ 3 leaves.
As preferred version, in the described step (3), the condition of culture of rootage is temperature (25 ± 2) ℃, intensity of illumination (2500 ± 150) Lx, light application time (14 ± 3) h.
As preferred version, in the described step (4), the hardening time opens and seals film hardening 3d for not loose film hardening 2d, loose film hardening 2d.
The invention has the advantages that:
One, the method for taking root is set by step compared with traditional root media, and the lateral root quantity that produces root is many, and the quality of root is better.
They are two years old, determined to match as the supporter of explant with the agar of perlite and debita spissitudo, medium wash-out from the root easily when transplanting, elution speed is fast, does not damage root system, has improved group training transplantation of seedlings survival rate, replace most of agar with perlite, the cleaning efficiency height has been saved a large amount of manpower and materials, makes to cultivate the cost reduction.
Embodiment
The present invention is described in further detail below in conjunction with specific embodiment.
For trying material:
Chinese cabbage for examination is rabbit ear cabbage white (be common green grass or young crops stalk dish, there is sale in market, and the public can obtain easily), the blue or green a variety of Chinese cabbage in Shanghai and the no offspring of purple flowering stalk group training.
Culture of rootage sill: agar, sucrose, 1/2MS, perlite (granularity 3～5mm), methyl, heteroauxin, deionized water.
The 1/2MS medium is exactly that the macroelement mother liquor reduces by half, MS is the minimal medium that we use always, and the concentration of its mineral salt and ion is higher, is comparatively stable balance culture fluid, but nitrate content is higher, is widely used in organ, flower pesticide, the cultivation of cell nucleus protoplast.And 1/2MS to be macroelement with MS reduce by half other constant and medium configuration, this mainly is to reduce inorganic salt concentration, effect and MS are roughly the same, just are fit to the different requirements of different plants.
Methyl
English common name 1-naphthlcetic acid, other titles a-methyl.Methyl toxicity is to people and animals' low toxicity.The oral LD50 of rat acute is 1000-5900mg/kg, and skin and mucous membrane are had spread effect.
Methyl is the broad spectrum type plant growth regulator, can promote cell division and expansion, and inducing adventitious root forms, and increase is beared fruit, and prevents shedding, changes female, male flower ratio etc.Can enter in the plant through tender epidermis, the seed of blade, branch, arrive complete stool with the nutritional flow transporting.Be applicable to cereal, increase is tillered, and improves the percentage of earbearing tiller and thousand kernel weight; Cotton reduces fruit abscission rate, increases the peach weightening finish, improves the quality.Fruit tree is short to bloom, anti-shedding, the volume increase of accelerating the ripening.Melon-fruit-like vegetable prevents fallen flowers, forms little seed fruit; Promote that the cuttage branch is taken root.Methyl is very much commonly used in plant tissue culture, is used for the differentiation of inducing cell division and root.
Heteroauxin
Have another name called indole-3-acetic acid, English common name indole-3-acetic acid is the plant hormone of a kind of energy coordinate plant growth and growth.
Heteroauxin is a kind of organic matter.Pure product are colourless lobate crystal or crystalline powder.Become rose-colored behind the chance light.Fusing point 165-166 ℃ (168-170 ℃).Be soluble in absolute ethyl alcohol, ethyl acetate, dichloroethane, dissolve in ether and acetone, be insoluble to benzene, toluene, gasoline and chloroform.Water insoluble, its aqueous solution can be decomposed by ultraviolet light, but stable to visible light.Its sodium salt, sylvite are stablized than acid itself, and is very easily water-soluble.Easily decarboxylation becomes 3-methyl indol (scatol).In tissue is cultivated, be used to the division of inducing cell and the differentiation of root.
Embodiment one:
Unrooted group training seedling is the bluish white dish group training in Shanghai bud seedling.The medium that reduces by half with the macroelement with the MS medium is minimal medium, adds: agar 1g/L, sucrose 28g/L, perlite (granularity 3mm) 80g/L, methyl 0.05mg/L, heteroauxin 0.01mg/L, deionized water.Add earlier the perlite that 4g cleans airing in blake bottle, add the 50ml culture fluid then, 121 ℃ of moist heat sterilizations 15 minutes cool off standby naturally.
Earlier insert out 3 duck eyes with the medium of tweezers in blake bottle, size is advisable to put explant stem apex base portion into, and 3 stem apex base portions that have 2 leaves are put into duck eye, tamps stem apex base portion space with peripheral medium and gets final product.To inoculate good vial then and be placed on culturing room and carry out culture of rootage, condition of culture is: 25 ℃ of temperature, intensity of illumination 2500Lx, light application time 14 hours.Cultivate the quality and quantity of statistics root after 21 days.One-level radical order is direct quantity of giving birth at the root of group training seedling base portion, and the secondary root is to send out roots on the one-level root, three grades of roots be on the secondary root the root of giving birth to, many, carefully be better with root white, root tubbiness and mostly be, be a bit darkish in color, less, thin and length be poor.
One-level root, two or three grades of radical orders of group training seedling are respectively 3.05 and 42.25/strain after the culture of rootage, and the root quality is good.On the group training shoot root contain the sugar culture-medium cleaning speed be 1.0 strains/minute, transplanting survival rate is 99%.
Embodiment two:
Unrooted group training seedling is for exempting from the white a variety of Chinese cabbage group of ear training bud seedling.The medium that reduces by half with the macroelement with the MS medium is minimal medium, adds: agar 2.0g/L, sucrose 29g/L, perlite (granularity 4mm) 100g/L, methyl 0.1mg/L, heteroauxin 0.03mg/L, deionized water.Add earlier the perlite that 5g cleans airing in blake bottle, add the 50ml culture fluid then, 121 ℃ of moist heat sterilizations 15 minutes cool off standby naturally.
Earlier insert out 2 duck eyes with tweezers at medium, size is advisable to put explant stem apex base portion into, and 2 stem apex base portions that have 3 leaves are put into duck eye, tamps stem apex base portion space with peripheral medium and gets final product.To inoculate good vial then and be placed on culturing room and carry out culture of rootage, condition of culture is: 25 ℃ of temperature, intensity of illumination 2500Lx, light application time 14 hours.Cultivate the quality and quantity of statistics root after 22 days.One-level radical order is direct quantity of giving birth at the root of group training seedling base portion, and the secondary root is to send out roots on the one-level root, and three grades of roots are the root of giving birth on the secondary root, and is many, carefully be better with root white, root tubbiness and mostly be, be a bit darkish in color, less, thin and length be poor.
One-level root, two or three grades of radical orders of group training seedling are respectively 3.58 and 43.78/strain after the culture of rootage, and the one-level root is sturdy, and secondary, three grades of roots are many.On the group training shoot root contain the sugar culture-medium cleaning speed be 1.2 strains/minute, transplanting survival rate is 99.2%.
Embodiment three:
Unrooted group training seedling is purple flowering stalk group training bud seedling.The medium that reduces by half with the macroelement with the MS medium is minimal medium, adds: agar 3.0g/L, sucrose 30.0g/L, perlite (granularity 5mm) 120g/L, methyl 0.15mg/L, heteroauxin 0.05mg/L, deionized water.Add earlier the perlite that 6g cleans airing in blake bottle, add the 50ml culture fluid then, 121 ℃ of moist heat sterilizations 15 minutes cool off standby naturally.
Earlier insert out 3 duck eyes with tweezers at medium, size is advisable to put explant stem apex base portion into, and 3 stem apex base portions that have 2 leaves are put into duck eye, tamps stem apex base portion space with peripheral medium and gets final product.To inoculate good vial then and be placed on culturing room and carry out culture of rootage, condition of culture is: 25 ℃ of temperature, intensity of illumination 2500Lx, light application time 14 hours.Cultivate the quality and quantity of statistics root after 23 days.One-level radical order is direct quantity of giving birth at the root of group training seedling base portion, and the secondary root is to send out roots on the one-level root, and three grades of roots are the root of giving birth on the secondary root, and is many, carefully be better with root white, root tubbiness and mostly be, be a bit darkish in color, less, thin and length be poor.Be contrast with pure agar as the ordinary culture medium (MS medium+agar 7.5g/L, sucrose 27/L, methyl 0.03mg/L) of curing agent.
Data are handled:
All data all adopt SPSS11.5(LSD, p＜0.05) carry out statistical analysis and handle.
Result and analysis:
Different medium are to the influence of the quality and quantity of taking root
Table one, the different root media quality and quantity statistical form of taking root
Annotate: same row lowercase is represented the difference of 0.05 level, and alphabetical identical person's difference is not remarkable.
Table one shows, be respectively 2.15 and 23.6/strain with pure agar as the one-level root of the root media of curing agent, two, three grades of radical orders, and be respectively 3.05 and 41.62/strain with medium of the present invention, be significantly higher than the former, and latter's root quality has been grade.
Medium cleaning speed and the survival rate of the test-tube plantlet that different root medias are cultivated
The test-tube plantlet medium cleaning speed of table two, different root medias and survival rate statistical form
Annotate: same row lowercase is represented the difference of 0.05 level, and alphabetical identical person's difference is not remarkable.
Table two shows, on the test tube shoot root of cultivating as the ordinary culture medium of curing agent with pure agar contain the sugar culture-medium cleaning speed be 0.3 strain/minute, survival rate is 85%, and with medium culture of the present invention be respectively 1.4 strains/minute and 97.8%, be significantly higher than the former.
Conclusion:
Result of study shows, as the significant difference that exists that contains sugar culture-medium cleaning speed and planting survival rates and medium culture of the present invention on the quality and quantity of the ordinary culture medium culture test tube seedling rooting of curing agent, the root, the latter significantly is better than the former with pure agar.Contain the sugar culture-medium cleaning speed and improve this method cost is greatly reduced, and the test-tube plantlet survival rate improved 12.8%, this lays a solid foundation for Chinese cabbage tissue culture technology large-scale application on producing.
3 embodiment show, contain the sugar culture-medium cleaning speed on the quality and quantity of the group training shoot root of cultivating with this root media, the root and planting survival rates is all very high.Contain the raising of sugar culture-medium cleaning speed and saved a large amount of labours, meaningful especially under the situation that labor cost increases fast now, also reduced culture medium cost with perlite instead of part agar in addition, group training seedling production cost is greatly reduced, and group training shoot survival percent is improved, and this lays a solid foundation for Chinese cabbage tissue culture technology large-scale application on producing.
Claims (5)
1. a Chinese cabbage tissue culture sprout quick speed high-effective root-growing method comprises the process of preparing root media, inoculation, cultivation and acclimatization and transplants successively, it is characterized in that:
(1) preparation root media: with the macroelement content in the conventional MS medium reduce by half, the trace element and other component contents constant, as minimal medium; In minimal medium, add agar, sucrose, methyl, heteroauxin and deionized water, be hybridly prepared into culture fluid; Then culture fluid is poured in the blake bottle that is loaded with perlite particle, perlite particle can be soaked into fully by culture fluid and do not have excess solution and overflow; Again it being carried out moist heat sterilization handles, naturally cooling forms root media, and the mass volume ratio of following component is controlled at agar 1 ~ 3g/L, sucrose 28 ~ 30g/L, methyl 0.05 ~ 0.15mg/L, heteroauxin 0.01 ~ 0.05mg/L, perlite particle 80 ~ 120g/L in root media;
(2) inoculation: the medium in blake bottle is inserted out duck eye, and the blade-carrying stem apex base portion that Chinese cabbage group training seedling downcuts is put into duck eye, tamps stem apex base portion space with the medium on next door;
(3) cultivate: will inoculate the blake bottle of getting well and be placed on and carry out culture of rootage in the culturing room;
(4) acclimatization and transplants: culture of rootage is after 21 ± 2 days, blake bottle is placed indoor place near the window, in the natural conditions lower refining seedling, make Chinese cabbage group training seedling adapt to external environment condition gradually, after the hardening seedling is taken out from bottle. wash the field planting after the sugar culture-medium matter that contains on the clean root with flowing water.
2. Chinese cabbage tissue culture sprout quick according to claim 1 speed high-effective root-growing method, it is characterized in that: in the described step (1), the particle diameter of selecting perlite particle for use is 3 ~ 5mm; Control moist heat sterilization temperature is that 121 ℃, time are 15min.
3. Chinese cabbage tissue culture sprout quick according to claim 1 speed high-effective root-growing method, it is characterized in that: in the described step (2), medium in blake bottle is inserted out 2 ~ 3 duck eyes, and its size can be put Chinese cabbage group training seedling stem apex base portion just into, and Chinese cabbage group training seedling stem apex base portion has 2 ~ 3 leaves.
4. Chinese cabbage tissue culture sprout quick according to claim 1 speed high-effective root-growing method is characterized in that: in the described step (3), the condition of culture of rootage is temperature (25 ± 2) ℃, intensity of illumination (2500 ± 150) Lx, light application time (14 ± 3) h.
5. Chinese cabbage tissue culture sprout quick according to claim 1 speed high-effective root-growing method, it is characterized in that: in the described step (4), the hardening time opens and seals film hardening 3d for not loose film hardening 2d, loose film hardening 2d.
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| CN103461143A (en) * | 2013-09-30 | 2013-12-25 | 中南林业科技大学 | Method for tissue culture and rapid propagation of camellia oleifera |
| CN103548657A (en) * | 2013-11-13 | 2014-02-05 | 湖北省农业科学院经济作物研究所 | Rooting and seedling-hardening method for raspberry tissue culture seedlings |
| CN111629585A (en) * | 2018-01-10 | 2020-09-04 | 瑞博泰克Ptc有限公司 | Nutrient medium for automatically cultivating plants |
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| CN103548657A (en) * | 2013-11-13 | 2014-02-05 | 湖北省农业科学院经济作物研究所 | Rooting and seedling-hardening method for raspberry tissue culture seedlings |
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| CN111629585A (en) * | 2018-01-10 | 2020-09-04 | 瑞博泰克Ptc有限公司 | Nutrient medium for automatically cultivating plants |
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| CN103202226B (en) | 2014-10-22 |
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