CN104885943B - A kind of oncidiumLuridum chemical disinfection tissue culture method - Google Patents
A kind of oncidiumLuridum chemical disinfection tissue culture method Download PDFInfo
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- CN104885943B CN104885943B CN201510274108.4A CN201510274108A CN104885943B CN 104885943 B CN104885943 B CN 104885943B CN 201510274108 A CN201510274108 A CN 201510274108A CN 104885943 B CN104885943 B CN 104885943B
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Abstract
The present invention relates to a kind of oncidiumLuridum chemical disinfection tissue culture method.OncidiumLuridum chemical disinfection tissue culture method, including culture vessel sterilization, culture medium preparation, Fiber differentiation, Multiplying culture, culture of rootage and bottle seedling are transplanted.The oncidiumLuridum chemical disinfection tissue culture method of the present invention, sterilizing or antibacterial purpose are reached using chemical reagent, during culture medium is prepared, culture vessel and culture medium are all without autoclave sterilization, workload and energy resource consumption are reduced, oncidiumLuridum tissue culture link is simplified；And it is simple to operate, as long as being prepared by different culture medium prescriptions；Practical, generalization is good, can also effectively reduce the tissue culture cost of oncidiumLuridum, can typically reduce cost more than 10%.
Description
Technical field
The present invention relates to a kind of method for plant tissue culture, and in particular to a kind of oncidiumLuridum chemical disinfection tissue culture method, category
Biological technical field.
Background technology
OncidiumLuridum belongs to orchid family oncidiumLuridum category (Oncidium), also known as knurl lip Cymbidium, and full category has initial species more than 400.It spends
Small and various, color is gorgeous colorful, and metamorphosis is various, and flower is successively opened, sustainable 1~3 month, is first-class basin
Cultivation is viewed and admired and cut-flower material.The traditional division propagation speed of oncidiumLuridum is slow, and one is only point 2~3 buds, and breeding coefficient is low.Pass through
Tissue culture technology, can accelerate reproduction speed, carry out factorial praluction.Practice have shown that, oncidiumLuridum is endangered in production by virosis, is led
Cause quality and yield to decline, lose commodity value, cause quality deterioration, oncidiumLuridum kind is quickly bred using tissue culture technique
Seedling, beneficial to the good characteristic for keeping oncidiumLuridum, effectively control virus harm, therefore explore the quick breeding by group culture mode of oncidiumLuridum
It is significant.
OncidiumLuridum tissue cultures, on the one hand can expand its breeding coefficient, so as to realize biological control；On the other hand,
Germ-plasma resources protection and transgenic research of oncidiumLuridum etc. can be realized on the basis of its original merit is preserved.Text
The cost of heart orchid tissue cultures mainly includes culture medium needed for tissue-cultured seedling is produced, facilities and equipment, power supply cost, consumptive material and artificial
Cost.Conventional oncidiumLuridum method for tissue culture requires that explant is seeded in ability in the aseptic culture medium after autoclave sterilization
Normal growth, power consumption is big, and artificial running cost is high.If culture medium can be transformed by the method for chemical disinfection, make culture medium
Autoclave sterilization need not carried out, it becomes possible to obtain the normal growth of oncidiumLuridum.So, oncidiumLuridum can on the one hand be reduced
Requirement of the tissue cultures to facilities and equipment, the pressure cooker equipment of configuration, reduces throwing needed for autoclave sterilization is not needed especially
Cost is provided, power consumption will also be greatly lowered；On the other hand, oncidiumLuridum tissue cultures, tissue culture are carried out using this culture medium
Link is greatly simplified, and manually-operated speed is greatly improved, so as to reduce cost of labor.Further, since improved culture
Base itself has sterilization, antibacterial or bacteriostasis, can effectively control the pollution problem in tissue culture procedures, again will not be to the literary heart
Orchid growth, which is produced, to be poisoned or suppresses, i.e., do not influence the normal growth of oncidiumLuridum, radically simplify oncidiumLuridum tissue cultures.
The content of the invention
It is an object of the invention to provide a kind of oncidiumLuridum chemical disinfection tissue culture method.
The purpose of the present invention is realized by the following method.
A kind of oncidiumLuridum chemical disinfection tissue culture method of the present invention, including culture vessel sterilization, culture medium are prepared, induction training
Support, Multiplying culture, culture of rootage and bottle seedling are transplanted, it is characterised in that：
1. culture vessel is sterilized：The stainless steel disc of bottle cap and inoculation will be cultivated in 100mg/L sodium hypochlorite+10mg/L
Immersion is no less than 10h in the aqueous solution of nisin+100mg/L calcium propionates, saves backup；
2. culture medium is prepared：Inducing culture is MS+1~5mg/L 6-BA+0.1~1.0mg/L NAA+40~100mg/
L sodium hypochlorite+5~10mg/L nisin+50~100mg/L calcium propionate+20g/L sucrose+3.0g/L agar powders,
pH5.8；Proliferated culture medium is：MS+1.0~3mg/L 6-BA+0.1~1.0mg/L NAA+40~100mg/L sodium hypochlorite+5
~10mg/L nisin+50~100mg/L calcium propionate+20g/L sucrose+3.0g/L agar powders, pH5.8；Strong seedling culture
Base is MS+0.1~0.5mg/L 6-BA+0.5~1.0mg/L NAA+40~100mg/L sodium hypochlorite+5~10mg/L lactic acid chains
Coccus element+50~100mg/L calcium propionate+20g/L sucrose+3.0g/L agar powders, pH5.8；Root media be 1/2MS+0.5~
1.5mg/L NAA+40~100mg/L sodium hypochlorite+5~10mg/L nisin+50~100mg/L calcium propionates+20g/L
Sucrose+3.0g/L agar powders, pH5.8；The MS culture mediums are 1962, MS culture mediums disclosed in Murashige and Skoog；
The 6-BA, refers to the fast cry of certain animals of 6- benzyls amino；The NAA , Zhi 〆-methyl α-naphthyl acetate；When preparing culture medium, first claim sugarcane in each culture medium prescription
Sugar and agar powder, plus culture medium cumulative volume 1/2 running water, be heated to agar powder and be completely dissolved, then match somebody with somebody by each culture medium prescription
After other neat raw materials, constant volume and then the HCl with 1mol/L NaOH or 1mol/L adjust pH value to 5.8, are dispensed into the training sterilized
Support in container, sealing, it is standby after cooled and solidified；
3. Fiber differentiation：The seedling that blade not yet deploys is cut from maternal plant, outer blade is removed, is rinsed with running water
30min, peels off outmost one layer of leaf sheath, and 2~3s is soaked in volume ratio is 75% alcohol, with the liter that volume ratio is 0.1%
Mercury solution sterilizes 10min, takes out with after aseptic water washing 3 times, then aseptically strips terminal bud or lateral bud, cut 0.5
~1mm, stem apex, be inoculated on inducing culture, culture 25d after form a large amount of protocorms；It is 26 DEG C, illumination to cultivate room temperature
12h, intensity of illumination is 1400lx；
4. Multiplying culture：The protocorm of robust growth is transferred in proliferated culture medium, starts differentiation after 30d, progressively induces
Go out Multiple Buds and develop into the young shoot for not having root；Per 30d, switching once, obtains a large amount of young shoots；It is 26 DEG C, illumination to cultivate room temperature
12h, intensity of illumination is 1400lx；
5. strong seedling culture：The young shoot of Multiplying culture is inoculated on strong seedling culture base, 30d turns into height and grown thickly for 6~8cm
Sprout；It is 26 DEG C to cultivate room temperature, and illumination 12h, intensity of illumination is 1400lx；
6. culture of rootage：It is 6~8cm crowd shoots to take height, is cut into individual plant, is inoculated on root media, cultivates 25d
Turn into intact plant afterwards；It is 25 DEG C to cultivate room temperature, and illumination 12h, intensity of illumination is 1500lx；
7. bottle seedling is transplanted：The intact plant that culture of rootage is obtained takes out, and cleans the culture medium of root, many with 800 times
After bacterium spirit immersion 30min, transplant into the pasture and water of good water permeability, greenhouse upper strata is covered with shading net, and the environment temperature of transplanting is
22~28 DEG C.
The application effect of the present invention：
Due to the chemical disinfection tissue culture method of the present invention, it is to be added to using chemical reagent in various culture mediums, reaches and go out
Bacterium or antibacterial effect, so, the culture medium of preparation is without autoclave sterilization.Therefore, in oncidiumLuridum Fiber differentiation, propagation
Culture, strong seedling culture, the influence in each stage of culture of rootage, in addition to considering conventional evaluation index, it is also contemplated that pollution
The problem of rate.
1. Fiber differentiation：Experiments prove that, a kind of chemical disinfection tissue culture method of oncidiumLuridum of the invention with it is conventional
OncidiumLuridum tissue culture method is compared, as a result as shown in table 1, the survival rate of the explant culture in Fiber differentiation stage, pollution rate and
The death rate is all without too big difference.
Influence of the chemical disinfection tissue culture method of the present invention of table 1 to oncidiumLuridum Fiber differentiation
| Tissue culture mode | Average survival (%) | Average pollution rate (%) | Average mortality (%) |
| Conventional tissue culture method | 58 | 35 | 7 |
| Chemical disinfection tissue culture method | 62 | 32 | 6 |
2. Multiplying culture：The proliferation times and pollution rate of the chemical disinfection tissue culture method of the present invention and conventional tissue culture oncidiumLuridum
Comparison, as a result as shown in table 2, two indexs of proliferation times and pollution rate are all without too big difference.In the upgrowth situation of bottle seedling
See, the culture medium of chemical disinfection tissue culture method of the invention is due to without autoclave sterilization, hormone and nutrient loss
Less, its oncidiumLuridum tissue-cultured seedling is greener, more strong.
Influence of the chemical disinfection tissue culture method of the present invention of table 2 to oncidiumLuridum proliferation times and pollution rate
| Tissue culture mode | Average proliferation multiple | Average pollution rate (%) | Upgrowth situation |
| Conventional tissue culture method | 3.5 | 0 | Seedling is light green, weak |
| Chemical disinfection tissue culture method | 3.4 | 0 | It is Miao Lv, strong |
3. strong seedling culture：The result of the test of strong seedling culture is as shown in table 3, oncidiumLuridum chemical disinfection tissue culture method of the invention
Every bottle of average strong sprout number apparently higher than conventional oncidiumLuridum tissue culture method, bottle seedling is also more strong, greener.Do not have in terms of pollution rate
Difference.
Influence of the chemical disinfection tissue culture method of the present invention of table 3 to oncidiumLuridum strong sprout number and pollution rate
| Tissue culture mode | Average strong sprout number/bottle | Average pollution rate (%) | Upgrowth situation |
| Conventional tissue culture method | 15.2 | 0 | Seedling is light green, weak |
| Chemical disinfection tissue culture method | 20.5 | 0 | It is Miao Lv, strong |
* the bottle seedling that height of seedling is more than 2cm is strong sprout
Influence of the chemical disinfection tissue culture method of 4 present invention to oncidiumLuridum rooting rate and pollution rate：In rooting process, this
The chemical disinfection tissue culture method of invention is compared with conventional oncidiumLuridum tissue culture method, as a result as shown in table 4, rooting rate and pollution rate
All without too big difference；In terms of the upgrowth situation of bottle seedling, with the method for the present invention, oncidiumLuridum tissue-cultured seedling stem is thicker, and blade is bigger.
The oncidiumLuridum rooting situation of the chemical disinfection tissue culture method of the present invention of table 4
5. cost analysis：The chemical disinfection tissue culture method of the present invention and the cost analysis of conventional tissue culture are shown in Table 5.
Tissue culture of the present invention eliminates autoclaving link, simplifies tissue culture program, improves operating efficiency.From can be direct
The expense calculated is calculated, and every year can be saved 50,000 yuan or so (to prepare the calculating of 50L culture mediums daily).In addition, also one
A little projects for being not easy to calculate, such as pressure cooker are repaired, and safety saves space etc..
The easy tissue culture method culture of oncidiumLuridum of the present invention of table 5 and the cost analysis table of conventional tissue culture
The present invention has the advantages that：
1. in the oncidiumLuridum chemical disinfection tissue culture method of the present invention, culture vessel and culture medium all go out without HTHP
Bacterium, reduces workload and energy resource consumption, simplifies oncidiumLuridum tissue culture link, reduces oncidiumLuridum tissue culture cost.
2. the oncidiumLuridum chemical disinfection tissue culture method of the present invention, simple to operate, as long as being prepared by different culture medium prescriptions
Afterwards, practical, generalization is good.
3. the oncidiumLuridum chemical disinfection tissue culture method of the present invention is contrasted with conventional oncidiumLuridum tissue culture method, it can reduce into
This more than 10%.
Embodiment
In order to which the present invention is furture elucidated rather than the limitation present invention, it is illustrated with reference to embodiments.
Embodiment one：A kind of oncidiumLuridum chemical disinfection tissue culture method
A kind of oncidiumLuridum chemical disinfection tissue culture method, comprises the following steps：
1. culture vessel is sterilized：The stainless steel disc of bottle cap and inoculation will be cultivated in 100mg/L sodium hypochlorite+10mg/L
10h is soaked in the aqueous solution of nisin+100mg/L calcium propionates, is saved backup；
2. culture medium is prepared：Inducing culture be MS+4mg/L 6-BA+1.0mg/L NAA+70mg/L sodium hypochlorite+
5mg/L nisin+50mg/L calcium propionate+20g/L sucrose+3.0g/L agar powders, pH5.8；Proliferated culture medium is：MS+
3mg/L 6-BA+1.0mg/L NAA+60mg/L sodium hypochlorite+5mg/L nisin+50mg/L calcium propionate+20g/L sugarcanes
Sugar+3.0g/L agar powders, pH5.8；Strong seedling culture base be MS+0.5mg/L 6-BA+1.0mg/L NAA+60mg/L sodium hypochlorite+
5mg/L nisin+50mg/L calcium propionate+20g/L sucrose+3.0g/L agar powders, pH5.8；Root media is 1/2MS
+ 1.0mg/L NAA+60mg/L sodium hypochlorite+8mg/L nisin+50mg/L calcium propionate+20g/L sucrose+3.0g/L fine jades
Cosmetics, pH5.8；When preparing culture medium, first claim sucrose and agar powder in each culture medium prescription, plus culture medium cumulative volume 1/2 from
Water, is heated to agar powder and is completely dissolved, then is assorted by each culture medium prescription after other raw materials, constant volume and then with 1mol/L's
NaOH or 1mol/L HCl adjusts pH value to 5.8, is dispensed into the culture vessel sterilized, seals, standby after cooled and solidified；
3. Fiber differentiation：The seedling that blade not yet deploys is cut from maternal plant, outer blade is removed, is rinsed with running water
30min, peels off outmost one layer of leaf sheath, and 2~3s is soaked in volume ratio is 75% alcohol, with the liter that volume ratio is 0.1%
Mercury solution sterilizes 10min, takes out with after aseptic water washing 3 times, aseptically strips terminal bud or lateral bud, cut 0.5~1mm
Stem apex be inoculated on inducing culture, a large amount of protocorms can be formed after 25d；It is 26 DEG C, illumination 12h, illumination to cultivate room temperature
Intensity is 1400lx；
4. Multiplying culture：Take the protocorm of robust growth to be transferred in proliferated culture medium, start differentiation after 30d, progressively induce
Go out Multiple Buds and develop into the young shoot for not having root.Per 30d, switching once, can reach the effect largely bred；Cultivate room temperature
For 26 DEG C, illumination 12h, intensity of illumination is 1400lx；
5. strong seedling culture：The young shoot of Multiplying culture is inoculated on strong seedling culture base, 30d can obtain the high clumps of 6~8cm
Sprout seedling；It is 26 DEG C to cultivate room temperature, and illumination 12h, intensity of illumination is 1400lx；
6. culture of rootage：The crowd shoots for taking 6~8cm of strong seedling culture acquisition high, are cut into individual plant, are inoculated into culture of rootage
On base, intact plant is formed after 25d；It is 25 DEG C to cultivate room temperature, and illumination 12h, intensity of illumination is 1500lx；
7. bottle seedling is transplanted：The intact plant that culture of rootage is obtained takes out, and cleans the culture medium of root, many with 800 times
Transplanted after bacterium spirit immersion 30min into the pasture and water of good water permeability, greenhouse upper strata is covered with shading net, the environment temperature of transplanting is 22
~28 DEG C.
Claims (5)
1. a kind of oncidiumLuridum chemical disinfection tissue culture method, including culture vessel sterilization, culture medium preparation, Fiber differentiation, propagation training
Support, culture of rootage and bottle seedling are transplanted, it is characterised in that：
(1) culture vessel is sterilized：By blake bottle, bottle cap and the stainless steel disc of inoculation in the sodium hypochlorite+10mg/ of 1ml/L 10%
Immersion is no less than 10h in the aqueous solution of L nisin+100mg/L calcium propionates, saves backup；
(2) culture medium is prepared：Inducing culture is MS+1~5mg/L 6-BA+0.1~1.0mg/L NAA+40~100mg/L times
Sodium chlorate+5~10mg/L nisin+50~100mg/L calcium propionate+20g/L sucrose+3.0g/L agar powders, pH5.8；
Proliferated culture medium is：MS+1.0~3mg/L 6-BA+0.1~1.0mg/L NAA+40~+5~10mg/ of 100mg/L sodium hypochlorite
L nisin+50~100mg/L calcium propionate+20g/L sucrose+3.0g/L agar powders, pH5.8；Strong seedling culture base is MS+
0.1~0.5mg/L 6-BA+0.5~1.0mg/L NAA+40~100mg/L+5~10mg/L of sodium hypochlorite nisins+
50~100mg/L calcium propionate+20g/L sucrose+3.0g/L agar powders, pH5.8；Root media is 1/2MS+0.5~1.5mg/L
NAA+40~100mg/L sodium hypochlorite+5~10mg/L nisin+50~100mg/L calcium propionate+20g/L sucrose+
3.0g/L agar powders, pH5.8；The MS, is MS culture mediums disclosed in Murashige and Skoog in 1962；The 6-BA, refers to
6-benzyl aminopurine；The NAA , Zhi 〆-methyl α-naphthyl acetate；When preparing culture medium, first claim sucrose and agar powder in each culture medium prescription,
Plus the running water of culture medium cumulative volume 1/2, it is heated to agar powder and is completely dissolved, then other raw materials is assorted by each culture medium prescription
Afterwards, constant volume, then adjusts pH value to 5.8, is dispensed into the culture vessel after sterilization with 1mol/L NaOH or 1mol/L HCl,
Sealing, it is standby after solidification；
(3) Fiber differentiation：The seedling that blade not yet deploys is cut from maternal plant, outer blade is removed, is rinsed with running water
30min, peels off outmost one layer of leaf sheath, and 2~3s is soaked in volume ratio is 75% alcohol, with the liter that volume ratio is 0.1%
Mercury solution sterilizes 10min, takes out with aseptic water washing 3 times, then aseptically strips terminal bud or lateral bud, cut 0.5~
1mm stem apex is inoculated on inducing culture, and a large amount of protocorms are formed after 25d；It is 26 DEG C, illumination 12h, illumination to cultivate room temperature
Intensity is 1400lx；
(4) Multiplying culture：Take the protocorm of robust growth to be transferred in proliferated culture medium, start differentiation after 30d, progressively induce clump
Sprout and develop into the young shoot for not having root；Per 30d, once, culture room temperature is 26 DEG C, illumination 12h for switching, and intensity of illumination is
1400lx；
(5) strong seedling culture：The young shoot of Multiplying culture is taken to be inoculated on strong seedling culture base, 30d can reach the high sprouts of 6~8cm；Training
It is 26 DEG C to support room temperature, and illumination 12h, intensity of illumination is 1400lx；
(6) culture of rootage：Take the sprout after strong seedling culture highly for 6~8cm to be cut into individual plant, be inoculated on root media, it is raw
Intact plant is formed after root culture 25d；It is 25 DEG C to cultivate room temperature, and illumination 12h, intensity of illumination is 1500lx；
(7) bottle seedling is transplanted：The intact plant formed after culture of rootage is taken, the culture medium of root is cleaned, with many bacterium of 800 times of volumes
After spirit immersion 30min, transplant into the pasture and water of good water permeability, greenhouse upper strata is covered with shading net, the environment temperature of transplanting is 22
~28 DEG C.
2. a kind of oncidiumLuridum chemical disinfection tissue culture method according to claim 1, it is characterised in that the inducing culture
For：MS+4mg/L 6-BA+1.0mg/L NAA+70mg/L sodium hypochlorite+5mg/L nisin+50mg/L calcium propionates+
20g/L sucrose+3.0g/L agar powders, pH5.8.
3. a kind of oncidiumLuridum chemical disinfection tissue culture method according to claim 1, it is characterised in that the proliferated culture medium
For：MS+3mg/L 6-BA+1.0mg/L NAA+60mg/L sodium hypochlorite+5mg/L nisin+50mg/L calcium propionates+
20g/L sucrose+3.0g/L agar powders, pH5.8.
4. a kind of oncidiumLuridum chemical disinfection tissue culture method according to claim 1, it is characterised in that the strong seedling culture base
For：MS+0.5mg/L 6-BA+1.0mg/L NAA+60mg/L sodium hypochlorite+5mg/L nisin+50mg/L calcium propionates+
20g/L sucrose+3.0g/L agar powders, pH5.8.
5. a kind of oncidiumLuridum chemical disinfection tissue culture method according to claim 1, it is characterised in that the root media
For：1/2MS+1.0mg/L NAA+60mg/L sodium hypochlorite+8mg/L nisin+50mg/L calcium propionate+20g/L sucrose+
3.0g/L agar powders, pH5.8.
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| CN105165634A (en) * | 2015-11-03 | 2015-12-23 | 海南出入境检验检疫局热带植物隔离检疫中心 | Oncidium non-toxic germchit production method |
| CN106718929A (en) * | 2017-01-18 | 2017-05-31 | 福建农林大学 | A kind of utilization micro-organisms base cultivates the tissue culture method of romaine lettuce |
| CN112772420A (en) * | 2021-03-22 | 2021-05-11 | 龙岩市禾康生物科技有限公司 | Tissue culture method of oncidium odoratum |
| CN115644057A (en) * | 2022-09-22 | 2023-01-31 | 广东省农业科学院环境园艺研究所 | Culture medium combination and method for rapid propagation of oncidium glaucescens |
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