CN106069789B - One kind is carried disease germs in vitro vegetable material tissue culture medium (TCM) and method for tissue culture - Google Patents
One kind is carried disease germs in vitro vegetable material tissue culture medium (TCM) and method for tissue culture Download PDFInfo
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- CN106069789B CN106069789B CN201610693912.0A CN201610693912A CN106069789B CN 106069789 B CN106069789 B CN 106069789B CN 201610693912 A CN201610693912 A CN 201610693912A CN 106069789 B CN106069789 B CN 106069789B
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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
- A01H4/001—Culture apparatus for tissue culture
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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
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- A01H4/008—Methods for regeneration to complete plants
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Abstract
It carries disease germs in vitro vegetable material tissue culture medium (TCM) and method for tissue culture the invention discloses one kind.The in vitro vegetable material tissue culture medium (TCM) that carries disease germs of the present invention, includes the cephaloridnum solution of a concentration of 2000 2500mg/L of vegetable solid culture medium and layer disposed thereon.During practical tissue culture, the in vitro vegetable material that will carry disease germs is inoculated on vegetable solid culture medium, then cephaloridnum solution is added in vegetable solid media surface, tissue cultures are carried out again, this training method is reaching Efficient antibacterial while avoiding the toxic side effect to cultured in vitro material, it successfully solves bottleneck problem in the tissue-cultured seedling production process of the vegetable material with bacterium, efficiently accomplishes proliferation, rooting process, produce required Plant tissue culture seedling；This method can be applied to the cultured in vitro vegetable material of a variety of band bacteriums, and antibacterial efficient, production safety is reliable, have many advantages, such as strong operability, and production efficiency is high, has a extensive future.
Description
Technical field
The invention belongs to field of plant tissue culture technique, and in particular to one kind is carried disease germs in vitro vegetable material tissue culture medium (TCM)
And method for tissue culture.
Background technology
Plant Tissue Breeding is widely used to the multiple fields in agricultural production, in seedling detoxification production, seedling
Quick breeding, the scale application of plant new resources, new variety of plant selection and breeding etc. produce far-reaching shadow to modern agriculture
It rings, is that agricultural biotechnologies apply upper model in modern agriculture, is the important component of modern kind of industry.Currently, China is planted
Object tissue-cultured seedling has achieved the popularization and application of large area in flowers, fruit tree, shade plant, medicinal plant etc., and the whole world is planted
Object tissue-cultured seedling year the volume of trade more than 15,000,000,000 dollars, also, every year with increasing more than 10% speed.Using based on from
The plant seedling tissue culture and rapid propagation method of body culture technique produces seedling, than Sterile culture method fast ten thousand times or even 100,000 times with
On, a plant can breed tens of thousands of to millions of a plant for 1 year.It is non-come the cost for breeding seedling using group culturation rapid propagating technology
It is often cheap, than low 10 times of the seeling industry method production cost or more with routine.Also, it is low to some coefficients of nourishing and generating and not
It can be by the agricultural plant (such as banana) of seminal propagation or seed is difficult to plant (such as iron sheet stone sprouted under field conditions (factors)
Dry measure used in former times etc.) or the excellent material (the certain excellent variation single plants found in such as production process) of rare numbers for, if do not had
Group culturation rapid propagating technology, to realize that its scale industrialization production is very difficult, to some by seed come agriculture when breeding
For skill character will appear the crop (such as papaya) of separation, if the not tissue culture detoxifying fast breeding technique of seedling, into
It is also very difficult that one step, which improves its productivity effect,.Seedling group culturation rapid propagating technology is based in reproduction speed height and at low cost
Honest and clean etc. advantage, plant seedling tissue-culturing rapid propagation has been developed into a huge industry in agriculture field at present, and obtains
Huge economic results in society.The seedling tissue-culturing rapid propagation industry annual value of production and technology of our province maintain the leading position in the whole nation, mesh
Preceding our province is related to having more than 300 kinds using tissue-culturing rapid propagation to produce the agricultural plant of seedling, and estimation tissue culture industry annual value of production is 10
Hundred million yuan or more.
At present in tissue-cultured seedling production process, pollution rate is higher, some plant brownings are serious, seedling vitrifying and aberration rate
The problems such as high, still seriously exists in many seeling industry enterprises, and the presence of these problems seriously hinders tissue-cultured seedling production cost
Further decrease.Especially many tissue-cultured seedling manufacturing enterprises are after production for many years, due to production environment variation and its life
The reasons such as production tool aging influence, and the probability of happening of germ contamination greatly increases.
Currently, domestic have the antibacterial report for going to addition antibiotic in culture medium in culture medium high-temperature sterilization, this side
Method one is that have certain destruction to its antibacterial power by high-temperature sterilization, while being dissolved in the antibiotic in culture medium to cultured in vitro material
Expect that the toxic side effect generated is bigger.Control pollution is one of its key technology in Plant tissue culture seedling production process, carefully
The probability of happening of bacterium pollution is relatively high, causes manufacturing enterprise's cost excessively high or even failure.Therefore, exploitation one kind is in vitro plant material
It can tissue effectively antibacterial and that culture materials are not generated with toxic side effect (or minimum toxic side effect) in material tissue-cultured seedling production process
Culture medium and method for tissue culture are particularly important.
The sterile cephaloridnum solution first by medical cephaloridnum compounding high concentration is yet there are no, under aseptic condition
It is added in media surface and forms solid-liquid two-phase training method to reach Efficient antibacterial while avoid to cultured in vitro material
The report of toxic side effect and patent application.
Invention content
It is an object of the invention to solve because occur with the vegetable material after bacterium not only expand germ contamination range but also
Lead to the unavailable problem of germ contamination material, develops a kind of be effectively directed in tissue-cultured seedling production and have occurred and that band bacterium
In vitro plant culture materials remain valid utilize its proliferation, take root complete tissue-cultured seedling production tissue culture medium (TCM) and tissue training
The method of supporting.
It carries disease germs in vitro vegetable material tissue culture medium (TCM) the first purpose of the invention is to provide one kind, including, vegetable solid
The cephaloridnum solution of culture medium and layer disposed thereon.
It is preferred that a concentration of 2000-2500mg/L of the cephaloridnum solution.
It is preferred that the thickness that the cephaloridnum solution is formed in vegetable solid culture epibasal tier is 1-2mm.
It is preferred that medical cephaloridnum is dissolved in sterile water and is prepared by the cephaloridnum solution.
It is preferred that the vegetable solid culture medium is the solid medium for plant tissue proliferation or culture of rootage.
It is preferred that the plant is dendrobium candidum, roxburgh anoectochilus terminal bud, banana, papaya, the white palm or climing green suede.
Second object of the present invention is to provide one kind and carries disease germs in vitro vegetable material method for tissue culture, and will carry disease germs in vitro plant
Object material is inoculated on vegetable solid culture medium, cephaloridnum solution then is added in vegetable solid culture epibasal tier, then carry out
Tissue cultures.
It is preferred that the cephaloridnum solution concentration is 2000-2500mg/L, the cephaloridnum solution is solid in plant
The thickness that body culture epibasal tier is formed is 1-2mm.
Third object of the present invention is to provide the in vitro vegetable material tissue culture medium (TCM)s that carries disease germs in vitro plant of carrying disease germs
Application in object material culture.
It is preferred that the in vitro vegetable material tissue culture medium (TCM) that carries disease germs is in vitro vegetable material proliferation, the culture of rootage of carrying disease germs
In application.
The present invention selects medical cephaloridnum injection as the antibiotic used, and it is highly concentrated to be configured to its under aseptic condition
The sterile cephaloridnum solution of (2000-2500mg/L) is spent, the plant of sterilized cooled and solidified is added under aseptic condition
On object solid culture primary surface, the training method of solid-liquid two-phase is formd to reach Efficient antibacterial while avoid to cultured in vitro material
The toxic side effect of material successfully solves bottleneck problem in the tissue-cultured seedling production process of the vegetable material with bacterium, efficiently accomplishes
Proliferation, rooting process, produce required Plant tissue culture seedling.The present invention utilizes the sterile cephaloridnum solution of high concentration, can be with
It, can be extremely low to cultured in vitro material proliferation and growth effect of taking root applied to the cultured in vitro vegetable material of a variety of band bacteriums
Under conditions of complete its tissue-cultured seedling be factory produced process, antibacterial efficient, production safety is reliable, especially to some economic valences
The tissue-cultured seedling production for being worth the material that carries disease germs of high orchid is largely effective, and has strong operability, and production efficiency height etc. is excellent
Point, has a extensive future.
Specific implementation mode
The following examples are further illustrations of the invention, rather than limiting the invention.
Embodiment 1
1. material：The experiment material of this embodiment is dendrobium candidum (the Dendrobium officinale to carry disease germs
Kimura et Migo) cultured in vitro material.
Dendrobium candidum is that China often uses rare Chinese medicine, and due to long-term immoderate excavation, low reproduction rate itself, natural in addition
Resource exhaustion is now the medicinal material kind of special-protection-by-the-State.With nourishing Yin and clearing heat.Promote the production of body fluid beneficial stomach, moisten the lung and relieve the cough and other effects, it is used for
Consumption of body fluid caused by febrile disease, dry is fidgety, after being ill the various diseases such as abnormal heat.Modern pharmacological studies have shown that the stem of noble dendrobium is also with antitumor, anti-ageing
Always, enhance human immunity and expand the effect of blood vessel.Dendrobium candidum originates in area and is in temperate zone and subtropical zone, annual weather mostly
Warm, moistening, winter temperature is at 0 DEG C or more.It, be according to the growth of dendrobium candidum when carrying out the large-scale planting of dendrobium candidum
Habit considers that a variety of natural causes such as illumination, temperature, humidity, the ventilation in place carry out precision management.The rule of dendrobium candidum at present
The seedling of modelling production is coming substantially from aseptic seeding.
2. the preparation of sterile cephaloridnum solution
Buy several bottles of cephaloridnum injection (every bottle of 1g dress) of medical injection.Select the 250mL drop bottles with suction pipe and
The graduated cylinder of 1000mL cleans up, in 1.06kg/cm after being packaged with newspaper after drying2It sterilizes under the conditions of (121 DEG C)
The deionized water of 20min, the another orchid bottle injection 300mL with 500mL, in 1.06kg/cm2It sterilizes under the conditions of (121 DEG C)
30min obtains sterile water.Above drug, utensil, sterile water are positioned on superclean bench, are configured under aseptic condition
The sterile cephaloridnum solution of 2500mg/L, and the sterile cephaloridnum solution is sub-packed in sterile drop bottle, with sterile ox
Mulberry paper is placed under 4 DEG C of refrigerated conditions after sealing the neck part of drop bottle and is preserved for use.
3. culture medium is prepared
According to the requirement that culture materials candidum tissue culturing seedling produces, its proliferated culture medium PB3 is prepared respectively and training of taking root
Support base
PR2.Specific formula is：Proliferated culture medium PB3：No. 1 1.5g/L+ of treasured is spent to spend No. 2 1.5g/L+ coconut milks 200mL/ of treasured
L+ activated carbon 2g/L+6-BA 2.0mg/L+NAA 0.2mg/L+ sucrose 15g/L+ agar 6g/L；Root media PR2：Spend treasured 1
Number 1g/L+ spends precious No. 2 10% (final concentration) bananas of 1g/L+ peptone 2g/L+NAA1.0mg/L+ activated carbon 2g/L+ volume fractions
Juice+sucrose 15g/L+ agar 6g/L.It is 5.4 that both the above culture medium, which adjusts pH, then in 1.06kg/cm2(121 DEG C) condition
Lower sterilizing 20min, it is cooling for use.
It is used to spend precious No. 1 (HYPONeX 1) limited for Taiwan produced in USA and gardening enterprise stock action
Company dispenses product.It is U.S.'s Haponex Products, N to spend precious No. 2 (HYPONeX 2)：P：K=20：20：20.
4. the material that carries disease germs inoculation operation and culture
Sterile cephaloridnum solution is taken out from refrigerator and is placed on superclean bench, the iron to carry disease germs by normal operation cutting
Skin stem of noble dendrobium cultured in vitro material, transplanting then inject 5mL on proliferated culture medium PB3 toward the surfaces proliferated culture medium PB3 (upper layer)
Sterile cephaloridnum solution, which is about 1mm, forms the state of solid-liquid two-phase, finally covers bottle cap, complete
Inoculation operation.Multiplying culture condition is that cultivation temperature is (25 ± 2) DEG C, intensity of illumination 1 500-2 000Lx, illumination 12h/d.One
As 45 days be 1 shoot proliferation period, the proliferation times after every 1 shoot proliferation period are 2~4 times.When one subculture of culture
After period, the bacterium of dendrobium candidum cultured in vitro material is significantly suppressed under the action of cephaloridnum solution, trains simultaneously
The proliferation and growth for supporting material are not influenced significantly.Above inoculation operation is repeated again as differentiation material and adds
Add cephaloridnum solution in the surface of culture medium, cover bottle cap, cultivates；So cycle, can reach the group of usual sterilizable material
Train seedling production effect.
Cut the dendrobium candidum cultured in vitro material of pending culture of rootage to carry disease germs by normal operation, transplanting is in taking root
On culture medium PR2, the sterile cephaloridnum solution of 5mL, the solution thickness are then injected toward the surfaces root media PR2 (upper layer)
Degree is about 1mm, forms the state of solid-liquid two-phase, finally covers bottle cap, completes inoculation operation.Rooting and hardening-off culture condition is training
It is (26 ± 2) DEG C, illuminance 2 000-3 000Lx, illumination 12h/d to support temperature.The Rooting and hardening-off culture period is 60-90 days.It is raw
Bud on root culture medium can normal long root and growth, until bottle outlet is transplanted.
5. tissue culture transplantation of seedlings：When tissue-cultured seedling of taking root is grown it is high to 4~8 centimetres when, in natural lighting lower refining seedling 10 days.Then it beats
Corkage plug from culture bottle takes out tissue-cultured seedling with tweezers, washes off root culture medium, plant by bark and sawdust mixed in equal amounts at
Matrix in.Pay attention to watering, shade, heat preservation and moisturizing, survival rate is up to 95% or more.
Embodiment 2
1. material：The experiment material of this embodiment is roxburgh anoectochilus terminal bud (the Anoectochilus roxburghii to carry disease germs
(wall) lindl.) cultured in vitro material.
Roxburgh anoectochilus terminal bud alias Shorthairy Antenoron, rough melic herb are that orchid family (Orchidaceae) opens lip plant flowers aspidistra category
(Anoectochilus Bl.) perennial rare herbaceous plant, China have 23 kinds or so, and which part kind herb is civil to make medicine
With for the rare rare traditional Chinese medicine of tradition, it is wider in civil use scope, is chiefly used in treating diabetes, hyperlipidemia, hepatitis B
Etc. diseases, be known as " king of medicine ", " gold grass ", " god's medicine ", the laudatory titles such as " black ginseng ", be referred to as " king in medicine ".
2. the preparation of sterile cephaloridnum solution
Buy several bottles of cephaloridnum injection (every bottle of 1g dress) of medical injection.Select the 250mL drop bottles with suction pipe and
The graduated cylinder of 1000mL cleans up, in 1.06kg/cm after being packaged with newspaper after drying2It sterilizes under the conditions of (121 DEG C)
The deionized water of 20min, the another orchid bottle injection 300mL with 500mL, in 1.06kg/cm2It sterilizes under the conditions of (121 DEG C)
30min obtains sterile water.Above drug, utensil, sterile water are positioned on superclean bench, are configured under aseptic condition
The sterile cephaloridnum solution of 2000mg/L, and the sterile cephaloridnum solution is sub-packed in sterile drop bottle, with sterile ox
Mulberry paper is placed under 4 DEG C of refrigerated conditions after sealing the neck part of drop bottle and is preserved for use.
3. culture medium is prepared
According to the requirement that culture materials roxburgh anoectochilus terminal bud tissue-cultured seedling produces, its proliferated culture medium and root media are prepared respectively.
Specific formula is：Proliferated culture medium：MS+6-BA 5.0mg/L+NAA 0.5mg/L+ peptone 2g/L+ sucrose 30g/L+ agar
6g/L；Root media：10% (final concentration) bananas juice of MS+NAA1.0mg/L+ activated carbon 2g/L+ volume fractions+sucrose 30g/L
+ agar 6g/L.It is 5.4 that both the above culture medium, which adjusts pH, then in 1.06kg/cm2Sterilize 20min under the conditions of (121 DEG C),
It is cooling for use.
4. the material that carries disease germs inoculation operation and culture
Sterile cephaloridnum solution is taken out from refrigerator and is placed on superclean bench, the gold to carry disease germs by normal operation cutting
Line lotus cultured in vitro material, transplanting then inject the pioneer of 10mL on proliferated culture medium toward Multiplying culture primary surface (upper layer)
Mycin solution, which is about 2mm, forms the state of solid-liquid two-phase, finally covers bottle cap, completes inoculation operation.
Multiplying culture condition is that cultivation temperature is (23 ± 2) DEG C, intensity of illumination 1 500-2 000Lx, illumination 10h/d.General 45 days are 1
A shoot proliferation period, the proliferation times after every 1 shoot proliferation period are 3~5 times.After cultivating a subculture cycle, gold
The bacterium of line lotus cultured in vitro material is significantly suppressed under the action of cephaloridnum solution, while the proliferation of culture materials
It is not influenced significantly with growth.Above inoculation operation is repeated again as differentiation material and addition cephaloridnum is molten
Liquid covers bottle cap in the surface of culture medium, culture；So cycle, can reach the tissue-cultured seedling production effect of usual sterilizable material.
The roxburgh anoectochilus terminal bud cultured in vitro material of pending culture of rootage to carry disease germs is cut by normal operation, transplanting is in training of taking root
It supports on base, the cephaloridnum solution of 10mL is then injected toward culture of rootage primary surface (upper layer), which is about 2mm,
The state for foring solid-liquid two-phase finally covers bottle cap, completes inoculation operation.Rooting and hardening-off culture condition is that cultivation temperature is
(25 ± 2) DEG C, illuminance 2 000-3 000Lx, illumination 10h/d.The Rooting and hardening-off culture period is 60 days.On root media
Bud can normal long root and growth, until bottle outlet is transplanted.
5. tissue culture transplantation of seedlings：When tissue-cultured seedling of taking root is grown it is high to 5~8 centimetres when, in natural lighting lower refining seedling 10 days.Then it beats
Corkage plug from culture bottle takes out tissue-cultured seedling with tweezers, washes off root culture medium, plant by peat soil and vermiculite mixed in equal amounts
At matrix in, drenched with 800 times of carbendazim after transplanting.Pay attention to watering, shade, heat preservation and moisturizing, survival rate up to 95% with
On.
Embodiment 3
Using similar to the method in embodiment 1 and embodiment 2, by the sterile cephaloridnum solution of 2000-2500mg/L
For banana, the cultured in vitro material of papaya, the white palm or climing green suede to carry disease germs, it is also attained by normal sterile cultured in vitro
Proliferation, the culture of rootage effect of material, the survival rate after tissue culture transplantation of seedlings can reach 95% or more.
Claims (7)
- The in vitro vegetable material tissue culture medium (TCM) 1. one kind is carried disease germs, which is characterized in that including vegetable solid culture medium and disposed thereon The cephaloridnum solution of layer, a concentration of 2000-2500mg/L of the cephaloridnum solution, the cephaloridnum solution exist The thickness that vegetable solid culture epibasal tier is formed is 1-2mm.
- 2. the in vitro vegetable material tissue culture medium (TCM) according to claim 1 that carries disease germs, which is characterized in that the cephaloridnum Medical cephaloridnum is dissolved in sterile water and is prepared by solution.
- 3. the in vitro vegetable material tissue culture medium (TCM) according to claim 1 that carries disease germs, which is characterized in that the vegetable solid Culture medium is the solid medium for plant tissue proliferation or culture of rootage.
- 4. the in vitro vegetable material tissue culture medium (TCM) according to claim 1 that carries disease germs, which is characterized in that the plant is iron The skin stem of noble dendrobium, roxburgh anoectochilus terminal bud, banana, papaya, the white palm or climing green suede.
- The in vitro vegetable material method for tissue culture 5. one kind is carried disease germs, which is characterized in that include the following steps：To carry disease germs in vitro plant Material is inoculated on vegetable solid culture medium, is then added a concentration of 2000-2500mg/L's in vegetable solid culture epibasal tier Cephaloridnum solution, the thickness that the cephaloridnum solution is formed in vegetable solid culture epibasal tier is 1-2mm, then carries out group Knit culture.
- 6. in vitro vegetable material tissue culture medium (TCM) answering in vitro vegetable material culture of carrying disease germs described in claim 1 of carrying disease germs With.
- 7. application according to claim 6, which is characterized in that the in vitro vegetable material tissue described in claim 1 that carries disease germs Application of the culture medium in carry disease germs in vitro vegetable material proliferation, culture of rootage.
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| CN116349604A (en) * | 2023-05-12 | 2023-06-30 | 西昌海越生物科技有限公司 | Orange stem cranberry green velvet tissue culture method |
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