CN108901851A - A kind of method of broad-leaved epiphyllum tissue culture detoxification nursery - Google Patents
A kind of method of broad-leaved epiphyllum tissue culture detoxification nursery Download PDFInfo
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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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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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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Abstract
The present invention relates to field of plant tissue culture technique, specifically provide a kind of method of broad-leaved epiphyllum tissue culture detoxification nursery, include the following steps：（1）The selection and cycle heat treatment of broad-leaved epiphyllum plant,（2）Explant disinfection is removed with stem apex,（3）Induction of callus,（4）Callus differentiation culture,（5）Viral diagnosis.Method provided by the invention can be avoided the problem of broad-leaved epiphyllum Sterile culture method susceptible virus leads to variety deterioration, and stem apex high survival rate, callus induction differentiation effect is good, virus elimination rate is high, so as to greatly improve the success rate of broad-leaved epiphyllum virus-elimination seedlings cultivation, conducive to the market demand is met.
Description
Technical field
The present invention relates to a kind of methods of broad-leaved epiphyllum tissue culture detoxification nursery, belong to plant tissue culture and gardening technical field.
Background technique
Broad-leaved epiphyllum（Scientific name：Epiphyllum oxypetalum （DC.）Haw）, alias rare flower, below the moon beauty belong to cactus
Section, broad-leaved epiphyllum category shrub shape Woody plant.Stem is in thin cylinder, and branch metamorphosis is in flat lobate, flower mostly white, large size, leakage
It is bucket-shaped, have fragrance.Originate in Mexico and in, in the Tropical forests of South America, property likes warm and moist and half shade environment, the present world
Each department are cultivated extensively, in each provinces and regions also common cultivation of China.The general Chang Xia of broad-leaved epiphyllum, night in autumn are open, and the duration is shorter,
Generally 2 ~ 3h, so there is saying for " short-lived ".Broad-leaved epiphyllum plant type is stood upright, and the big plumpness of leaf is bud green, spends big beauty graceful without losing,
Because flowering time is short, it is difficult to enjoy, outstanding aobvious preciousness, therefore be liked by many common people and flower-grower.
Not only for ornamental, Er Qiehua, cauline leaf can be used as medicine broad-leaved epiphyllum, and cauline leaf has soft stool detoxification, and the function of asthma is treated in heat-clearing
Effect cures mainly big enteric fever, constipation hematochezia, swollen sore, pneumonia, has the diseases such as the trace of blood, asthma in phlegm.The very strong treatment function that flower has
Effect, can treat the diseases such as palpitaition, tuberculosis, insomnia, cough with lung heat, while hypertension and fatty excessively high symptom can be made to be delayed
Solution.Furthermore broad-leaved epiphyllum also has the function of improving environment, and broad-leaved epiphyllum can increase indoor anion concentration, allow indoor with fresh air
It is pleasant.Therefore broad-leaved epiphyllum cultivation has a wide range of applications and very high economic value in fields such as ornamental, medicine, environmental protection.
Broad-leaved epiphyllum Planting pattern currently on the market is mainly cutting propagation, and this method belongs to conventional asexual reproduction method,
Its drawback is that breeding coefficient is low, is unable to satisfy the requirement of industrialization production, and with the increase of reproductive order of generation, virus is in plant
Cylinder accumulation makes germplasm seriously degenerate, and yield and quality declines to a great extent, and commodity value is greatly reduced.Plant virus distribution is wide,
Breeding is fast, prevention and treatment is difficult, there is the title of plant " cancer ".It is known to have cactus virus X, cucumber to the biggish virus of broad-leaved epiphyllum harm
Mosaic virus（CMV）, tobacco mosaic virus (TMV)（TMV）Deng the plant of virus infection is usually expressed as floral leaf system or local necrosis, base
The symptoms such as portion's browning or complete stool wilt, downgrade, deformity or system are mottled.
Tissue cultural seedlings of free is produced by plant tissue culture detoxification technology may be implemented prevention and treatment purpose, can eliminate and endanger plant
The harm to plant of virus, phytoplasma, class bacterium, some merits for keeping plant original show again,
Simultaneously because eliminating the influence of virus, the growth performance of plant is greatly enhanced, and can effectively improve plant growth index and economy
Index.Methods of virus elimination by plant tissue culture has Shoot Tip Culture detoxification, heat treatment detoxification, cold treatment detoxification, Chemical treatment de-
Poison, Anther Culture detoxification, callus detoxification, megarchidium embryo culture detoxification, stem apex micro-graft detoxification etc., wherein since stem apex is trained
It is good to support detoxification efficiency, is that the virus-free seedling of current plant cultivates a most widely used, most important approach.
The problem of can solve the accumulation of broad-leaved epiphyllum virosis and Rapid Popularization using technologies such as tissue cultures, rapid propagation in vitro.Mesh
The research and application of the preceding domestic tissue culture technology to broad-leaved epiphyllum are still at an early stage, and the detoxification technology of broad-leaved epiphyllum is also rarely reported, because
This has seriously affected broad-leaved epiphyllum seedling large-scale production.The present invention is directed to establish a kind of method of broad-leaved epiphyllum tissue culture detoxification nursery, to rush
It is mass produced into broad-leaved epiphyllum virus-elimination seedlings batch production, the popularization of broad-leaved epiphyllum excellent variety is had a vast market foreground.
Summary of the invention
It is increasingly recognized for the economic value of broad-leaved epiphyllum, the market demand is also gradually increased, by traditional cuttage
Nursery is unable to satisfy the demand of reality production from quality and quantity.The present invention provides one kind in order to solve problem above
The method of broad-leaved epiphyllum tissue culture detoxification nursery, this method have filled up the blank of broad-leaved epiphyllum detoxification technology, can satisfy batch production detoxification production
Needs.
Specific technical solution of the present invention is as follows：
A kind of method of broad-leaved epiphyllum tissue culture detoxification nursery, which is characterized in that including following operating procedure：
（1）The selection and cycle heat treatment of broad-leaved epiphyllum plant：
The broad-leaved epiphyllum plant for choosing robust growth, no disease and pests harm, is put into culturing room, daily in 36~38 DEG C, 2000-2200Lux
It is cultivated under illumination condition 10~12 hours, is then cultivated 12 ~ 14 hours under 22 ~ 25 DEG C, dark condition, return 36 ~ 38 DEG C
Environment, such Cyclic culture 30 ~ 35 days；
（2）Explant disinfection is removed with stem apex：
Take step（1）Middle plant cuts long 2cm tender stem segments of its branch containing growing point, first dips in a small amount of cleaning solution hand gently
It washes by rubbing with the hands 2 ~ 3 times, then with after 30 ~ 60 min of flowing water flushing, which is transferred on superclean bench, uses volume fraction first
For 75% 3 ~ 5min of alcohol disinfecting, mass fraction is then used to drip the disinfection of S106 fungicide for 0.5% bromogeramine+2
10min, then mass fraction is used to drip Tween-80/liter sterilizing liquid disinfectant 10min for 0.6% o-phthalaldehyde+2-3, finally use
Aseptic filter paper blots surface moisture；The stem section for retaining the 0.5cm from stem apex is cut with scalpel in aseptic processing environment, is placed on
It after handling 20s in 0.6% o-phthalaldehyde, places it in 75% alcohol and disinfects 10s, then with sterile water repeated flushing 3
Time, it drains away the water；Its stem section is cut into sterile petri dish with sterilized blade, tweezers again the little particle of 0.4 ~ 0.5mm,
These little particles are stem-tip tissue；
（3）Induction of callus：
The stem-tip tissue of above-mentioned acquisition is quickly inoculated into callus inducing medium, it is put into 3 in each tissue culture flasks ~
5 stem-tip tissue particles, seal bottleneck and are put into artificial climate incubator, and condition of culture is 20 ~ 25 DEG C of temperature, relative humidity 55
~ 65%, 30 ~ 40 days available granular callus of yellow green are cultivated in the daily illumination of 1000-1500Lux 10 ~ 12 hours；
（4）Callus differentiation culture：
By step（3）The fresh callus obtained is aseptically cut into fritter of the same size, is transferred to callus point
Change and continue to cultivate in culture medium, the control of incubator temperature is 22 ~ 26 DEG C, intensity of illumination control is 1000-1500Lux, relatively wet
Degree control be 55 ~ 65%, daily illumination 12 ~ 14 hours, culture 13 ~ 16 days after callus can differentiate successively grow thickly it is indefinite
Bud, be further cultured for 18 ~ 21 days sprout plant heights it is long to 2 ~ 3cm when be the test tube seedling obtained；
（5）Viral diagnosis：
Using enzyme linked immunosorbent assay（ELISA）Viral diagnosis is carried out to the test tube seedling seedling of above-mentioned acquisition, if detection qualification is
Broad-leaved epiphyllum virus-elimination seedlings.
The step（3）The formula of middle callus inducing medium is：1850 ~ 2000mg/L of potassium nitrate, biphosphate
120 ~ 140mg/L of ammonium, 310 ~ 330mg/L of magnesium sulfate, 295 ~ 315mg/L of anhydrous calcium chloride, 0.8 ~ 0.9mg/L of potassium iodide, boric acid
4.0 ~ 5.0mg/L, 10.0 ~ 12.0mg/L of manganese sulfate, 0.8 ~ 1.2mg/L of zinc sulfate, 0.12 ~ 0.14mg/L of sodium molybdate, copper sulphate
0.23 ~ 0.27mg/L, 0.09 ~ 0.12mg/L of cobalt chloride, 17.5 ~ 19.5mg/L of sodium iron ethylene diamine tetra acetate, inositol 90 ~ 110
Mg/L, 0.4 ~ 0.6 mg/L of vitamin B6,0.2 ~ 0.4 mg/L of vitamin B1,0.4 ~ 0.6mg/L of vitamin C, niacin
0.2 ~ 0.5 mg/L, 1.3 ~ 1.5 mg/L of proline, 2.7 ~ 3.3 mg/L of glutathione, 8 ~ 11g/L of active carbon, tomato juice 30 ~
40ml/L, 0.3 ~ 0.5mg/L of nucleosides, 0.2 ~ 0.4mg/L of triacontanol, 0.4 ~ 0.6mg/L of methyl α-naphthyl acetate, 1.5 ~ 1.7mg/ of kinetin
L, 0.9 ~ 1.1mg/L of cupreol, 0.08 ~ 0.11 mg/L of BAST antivirotic, 25 ~ 30g/L of sucrose, 7 ~ 10g/ of plant gel
L, pH are adjusted to 5.6 ~ 6.0.
The step（4）The formula of middle callus differential medium is：1850 ~ 2000mg/L of potassium nitrate, biphosphate
120 ~ 140mg/L of ammonium, 310 ~ 330mg/L of magnesium sulfate, 295 ~ 315mg/L of anhydrous calcium chloride, 0.8 ~ 0.9mg/L of potassium iodide, boric acid
4.0 ~ 5.0mg/L, 10.0 ~ 12.0mg/L of manganese sulfate, 0.8 ~ 1.2mg/L of zinc sulfate, 0.12 ~ 0.14mg/L of sodium molybdate, copper sulphate
0.23 ~ 0.27mg/L, 0.09 ~ 0.12mg/L of cobalt chloride, 17.5 ~ 19.5mg/L of sodium iron ethylene diamine tetra acetate, inositol 90 ~ 110
Mg/L, 0.4 ~ 0.6 mg/L of vitamin B6,0.2 ~ 0.4 mg/L of vitamin B1,0.4 ~ 0.6mg/L of vitamin C, niacin 0.2
~ 0.5 mg/L, 1.3 ~ 1.5 mg/L of proline, 2.7 ~ 3.3 mg/L of glutathione, 8 ~ 11g/L of active carbon, tomato juice 30 ~
40ml/L, 0.1 ~ 0.3mg/L of triacontanol, 0.08 ~ 0.12mg/L of methyl α-naphthyl acetate, 0.6 ~ 0.9mg/L of kinetin, rare earth 0.1 ~ 0.3
Mg/L, 0.3 ~ 0.5mg/L of nucleosides, 0.9 ~ 1.1mg/L of cupreol, 0.08 ~ 0.11 mg/L of BAST antivirotic, sucrose 25 ~
30g/L, plant gel 7 ~ 10g/L, pH are adjusted to 5.6 ~ 6.0.
The step（5）The type and method of middle viral diagnosis be：Using the double-antibody method in ELISA to cactus X
Virus and tobacco mosaic virus (TMV) are detected, and direct method detects cucumber mosaic virus.
The beneficial effects of the invention are as follows：
1. present invention incorporates different types of environment-friendlydisinfectant disinfectants to carry out disinfection to explant, Disinfection Effect is good, pollution rate
It is low；It avoids using damage of the strong stimulations disinfectant to explant such as mercuric chloride solution, sodium hypochlorite, is beneficial to explant living body
Success induces.
2. the present invention using by cycle heat treatment, Shoot Tip Culture and in the medium addition anti-bacteria and anti-virus agent combine
Poison-removing method, compared with conventional poison-removing method, this method pollution rate is low, and high survival rate, detoxification efficiency are significant, can obtain high-quality
The broad-leaved epiphyllum virus-elimination seedlings of amount.
3. a kind of method that hair establishes broad-leaved epiphyllum tissue culture detoxification nursery for the first time, had not only improved the kind of broad-leaved epiphyllum, but also into one
Step is stripped of broad-leaved epiphyllum virus, and this method process is simple, easy to operate, is easily formed the virus-elimination seedlings of large-scale production, fits
Preferably promote the use of.
Detailed description of the invention
Attached drawing 1 is the result figure that the dedifferentiation of broad-leaved epiphyllum stem apex generates callus；
Attached drawing 2 is the result figure that broad-leaved epiphyllum callus differentiates adventitious bud；
Attached drawing 3 is the broad-leaved epiphyllum virus-elimination seedlings picture obtained.
Specific embodiment
It is compared below in conjunction with each embodiment, further proves effect of the invention.The test of each embodiment below
Time is 2017 to 2018, and test site is Jiangsu Province Donghai County flowers proving ground.
Embodiment 1
The method of broad-leaved epiphyllum tissue culture detoxification nursery, including following operating procedure：
（1）The selection and cycle heat treatment of broad-leaved epiphyllum plant：
The broad-leaved epiphyllum plant for choosing robust growth, no disease and pests harm, is put into culturing room, daily in 36 ~ 38 DEG C, 2000-2200Lux light
It is cultivated according under the conditions of 10 ~ 12 hours, is then cultivated 12 ~ 14 hours under 22 ~ 25 DEG C, dark condition, return 36 ~ 38 DEG C of rings
Border, such Cyclic culture 33 days；
（2）Explant disinfection is removed with stem apex：
Take step（1）Middle plant cuts long 2cm tender stem segments of its branch containing growing point, first dips in a small amount of cleaning solution hand gently
It washes by rubbing with the hands 2 ~ 3 times, then with after 30 ~ 60 min of flowing water flushing, which is transferred on superclean bench, uses volume fraction first
For 75% 3 ~ 5min of alcohol disinfecting, mass fraction is then used to drip the disinfection of S106 fungicide for 0.5% bromogeramine+2
10min, then mass fraction is used to drip Tween-80/liter sterilizing liquid disinfectant 10min for 0.6% o-phthalaldehyde+2-3, finally use
Aseptic filter paper blots surface moisture；The stem section for retaining the 0.5cm from stem apex is cut with scalpel in aseptic processing environment, is put
Handle 20s in 0.6% o-phthalaldehyde, after place it in 75% alcohol and disinfect 10s, then it is anti-with sterile water
It rinses 3 times, drains away the water again；Its stem section is cut into 0.4 ~ 0.5mm's in sterile petri dish with sterilized blade, tweezers again
Little particle, these little particles are stem-tip tissue；
（3）Induction of callus：
The stem-tip tissue of above-mentioned acquisition is quickly inoculated into callus inducing medium（Callus inducing medium is matched
Fang Wei：Potassium nitrate 1950mg/L, ammonium dihydrogen phosphate 130mg/L, magnesium sulfate 315mg/L, anhydrous calcium chloride 300mg/L, potassium iodide
0.85mg/L, boric acid 4.7mg/L, manganese sulfate 11.0mg/L, zinc sulfate 1.0mg/L, sodium molybdate 0.13mg/L, copper sulphate
0.25mg/L, cobalt chloride 0.11mg/L, sodium iron ethylene diamine tetra acetate 18.0mg/L, 100 mg/L of inositol, vitamin B6 0.5mg/
L, vitamin B1 0.3mg/L, vitamin C 0.5mg/L, niacin 0.4mg/L, proline 1.4mg/L, 2.9 mg/ of glutathione
L, active carbon 10g/L, tomato juice 40ml/L, nucleosides 0.4mg/L, triacontanol 0.4mg/L, methyl α-naphthyl acetate 0.5mg/L, kinetin
1.5mg/L, cupreol 1.0mg/L, BAST antivirotic 0.1mg/L, sucrose 26g/L, plant gel 8g/L, pH are adjusted to
5.8）, 3 ~ 5 stem-tip tissue particles are put into each tissue culture flasks, bottleneck is sealed and is put into artificial climate incubator, are cultivated
Condition is 20 ~ 25 DEG C of temperature, relative humidity 55 ~ 65%, the daily illumination of 1000-1500Lux 10 ~ 12 hours, and cultivating 37 days can get
The callus of Bai Lvse discrete particles shape；
（4）Callus differentiation culture：
By step（3）The fresh callus obtained is aseptically cut into fritter of the same size, is transferred to callus point
Change culture medium（The formula of callus differential medium is：Potassium nitrate 1950mg/L, ammonium dihydrogen phosphate 130mg/L, magnesium sulfate
315mg/L, anhydrous calcium chloride 300mg/L, potassium iodide 0.85mg/L, boric acid 4.7mg/L, manganese sulfate 11.0mg/L, zinc sulfate
1.0mg/L, sodium molybdate 0.13mg/L, copper sulphate 0.25mg/L, cobalt chloride 0.11mg/L, sodium iron ethylene diamine tetra acetate 18.0mg/
L, 100 mg/L of inositol, vitamin B6 0.5mg/L, vitamin B1 0.3mg/L, vitamin C 0.5mg/L, niacin 0.4mg/
L, proline 1.4mg/L, 2.9 mg/L of glutathione, active carbon 10g/L, tomato juice 40ml/L, triacontanol 0.2mg/L, naphthalene
Acetic acid 0.1mg/L, kinetin 0.8mg/L, rare earth 0.1mg/L, nucleosides 0.4mg/L, cupreol 1.0mg/L, BAST are disease-resistant
0.1 mg/L of toxic agent, sucrose 26g/L, plant gel 7g/L, pH are adjusted to 5.8）In continue to cultivate, incubator temperature control be 22
~ 26 DEG C, intensity of illumination control be 1000-1500Lux, relative humidity control be 55 ~ 65%, daily illumination 12 ~ 14 hours, cultivate
Callus can differentiate the adventitious bud grown thickly successively after 14 days, be further cultured for 20 days sprout plant heights it is long to 2 ~ 3cm when be to obtain
Test tube seedling；
（5）Viral diagnosis：
Using enzyme linked immunosorbent assay（ELISA）Viral diagnosis is carried out to the test tube seedling seedling of above-mentioned acquisition, if detection qualification is
Broad-leaved epiphyllum virus-elimination seedlings.Cactus virus X and tobacco mosaic virus (TMV) are detected using the double-antibody method in ELISA, directly
Method detects cucumber mosaic virus.
Embodiment 2
Operating method is with embodiment 1, wherein cancellation step（1）In to broad-leaved epiphyllum plant carry out cycle heat treatment scheme.
Embodiment 3
Operating method is with embodiment 1, wherein by step（2）In to broad-leaved epiphyllum explant disinfection treatment method be changed to routine disinfection
Method uses volume fraction for 75% 3 ~ 5min of alcohol disinfecting first, then uses mass fraction molten for 0.1% mercuric chloride
Liquid disinfectant 10min, finally blots surface moisture with aseptic filter paper；Reservation is cut from stem apex with scalpel in aseptic processing environment
The stem section for playing 0.5cm is placed on 0.1% mercuric chloride solution processing 20s, after place it in 75% alcohol and disinfect 10s.
Embodiment 4
Operating method is with embodiment 1, wherein cancellation step（3）,（4）In each culture medium antiviral agent, i.e., cancellation β-paddy steroid
The use of pure and mild BSAT antivirotic.
Test result statistics：
1, the comparison of detoxification efficiency：Every group takes 70 explant samples, and embodiment 1-4 method is respectively adopted and is operated, is training
Stem apex inoculation survival rate is counted during supporting, pollution rate, progress viral diagnosis counts virus elimination rate after obtaining virus-elimination seedlings.
From the results shown in Table 1, using poison-removing method of the present invention, high-temperature process detoxicity method, stem apex are taken off
Poison and the three kinds of modes of detoxicity method for adding antivirotic in the medium combine, to the tissue cultures of broad-leaved epiphyllum nursery, quickly numerous
It educates with good toxic action of preventing or cure a disease, the detoxic seedling of acquisition is through viral diagnosis, and virus elimination rate is up to 95% or more, and in the kind in later period
During plant, nursery growing way, in terms of have a clear superiority.
A kind of method that the present invention establishes broad-leaved epiphyllum tissue culture detoxification nursery for the first time, this method can be with by tissue culture detoxification technology
The high-quality detoxic seedling for obtaining neat and consistent in a short time, so that the seedling large-scale production for enterprise provides technical support.
For the ordinary skill in the art, specific embodiment is only exemplarily described the present invention,
Obviously the present invention specific implementation is not subject to the restrictions described above, as long as use the inventive concept and technical scheme of the present invention into
The improvement of capable various unsubstantialities, or not improved the conception and technical scheme of the invention are directly applied to other occasions
, it is within the scope of the present invention.
Claims (4)
1. a kind of method of broad-leaved epiphyllum tissue culture detoxification nursery, which is characterized in that including following operating procedure：
（1）The selection and cycle heat treatment of broad-leaved epiphyllum plant：
The broad-leaved epiphyllum plant for choosing robust growth, no disease and pests harm, is put into culturing room, daily in 36~38 DEG C, 2000-2200Lux
It is cultivated under illumination condition 10~12 hours, is then cultivated 12 ~ 14 hours under 22 ~ 25 DEG C, dark condition, return 36 ~ 38 DEG C
Environment, such Cyclic culture 30 ~ 35 days；
（2）Explant disinfection is removed with stem apex：
Take step（1）Middle plant cuts long 2cm tender stem segments of its branch containing growing point, first dips in a small amount of cleaning solution hand gently
It washes by rubbing with the hands 2 ~ 3 times, then with after 30 ~ 60 min of flowing water flushing, which is transferred on superclean bench, uses volume fraction first
For 75% 3 ~ 5min of alcohol disinfecting, mass fraction is then used to drip the disinfection of S106 fungicide for 0.5% bromogeramine+2
10min, then mass fraction is used to drip Tween-80/liter sterilizing liquid disinfectant 10min for 0.6% o-phthalaldehyde+2-3, finally use
Aseptic filter paper blots surface moisture；The stem section for retaining the 0.5cm from stem apex is cut with scalpel in aseptic processing environment, is placed on
It after handling 20s in 0.6% o-phthalaldehyde, places it in 75% alcohol and disinfects 10s, then with sterile water repeated flushing 3
Time, it drains away the water；Its stem section is cut into sterile petri dish with sterilized blade, tweezers again the little particle of 0.4 ~ 0.5mm,
These little particles are stem-tip tissue；
（3）Induction of callus：
The stem-tip tissue of above-mentioned acquisition is quickly inoculated into callus inducing medium, it is put into 3 in each tissue culture flasks ~
5 stem-tip tissue particles, seal bottleneck and are put into artificial climate incubator, and condition of culture is 20 ~ 25 DEG C of temperature, relative humidity 55
~ 65%, 30 ~ 40 days available granular callus of yellow green are cultivated in the daily illumination of 1000-1500Lux 10 ~ 12 hours；
（4）Callus differentiation culture：
By step（3）The fresh callus obtained is aseptically cut into fritter of the same size, is transferred to callus point
Change and continue to cultivate in culture medium, the control of incubator temperature is 22 ~ 26 DEG C, intensity of illumination control is 1000-1500Lux, relatively wet
Degree control be 55 ~ 65%, daily illumination 12 ~ 14 hours, culture 13 ~ 16 days after callus can differentiate successively grow thickly it is indefinite
Bud, be further cultured for 18 ~ 21 days sprout plant heights it is long to 2 ~ 3cm when be the test tube seedling obtained；
（5）Viral diagnosis：
Using enzyme linked immunosorbent assay（ELISA）Viral diagnosis is carried out to the test tube seedling seedling of above-mentioned acquisition, if detection qualification is
Broad-leaved epiphyllum virus-elimination seedlings.
2. a kind of method of broad-leaved epiphyllum tissue culture detoxification nursery as described in claim 1, which is characterized in that the step（3）In more
The formula of injured tissue induced medium is：1850 ~ 2000mg/L of potassium nitrate, 120 ~ 140mg/L of ammonium dihydrogen phosphate, magnesium sulfate 310 ~
330mg/L, 295 ~ 315mg/L of anhydrous calcium chloride, 0.8 ~ 0.9mg/L of potassium iodide, 4.0 ~ 5.0mg/L of boric acid, manganese sulfate 10.0 ~
12.0mg/L, 0.8 ~ 1.2mg/L of zinc sulfate, 0.12 ~ 0.14mg/L of sodium molybdate, 0.23 ~ 0.27mg/L of copper sulphate, cobalt chloride 0.09
~ 0.12mg/L, 17.5 ~ 19.5mg/L of sodium iron ethylene diamine tetra acetate, 90 ~ 110 mg/L of inositol, 0.4 ~ 0.6 mg/ of vitamin B6
L, 0.2 ~ 0.4 mg/L of vitamin B1,0.4 ~ 0.6mg/L of vitamin C, 0.2 ~ 0.5 mg/L of niacin, proline 1.3 ~ 1.5
Mg/L, 2.7 ~ 3.3 mg/L of glutathione, 8 ~ 11g/L of active carbon, 30 ~ 40ml/L of tomato juice, 0.3 ~ 0.5mg/L of nucleosides, 30
0.2 ~ 0.4mg/L of alkanol, 0.4 ~ 0.6mg/L of methyl α-naphthyl acetate, 1.5 ~ 1.7mg/L of kinetin, 0.9 ~ 1.1mg/L of cupreol, BAST
0.08 ~ 0.11 mg/L of antivirotic, 25 ~ 30g/L of sucrose, plant gel 7 ~ 10g/L, pH are adjusted to 5.6 ~ 6.0.
3. a kind of method of broad-leaved epiphyllum tissue culture tissue culture detoxification nursery as described in claim 1, which is characterized in that the step（4）
The formula of middle callus differential medium is：1850 ~ 2000mg/L of potassium nitrate, 120 ~ 140mg/L of ammonium dihydrogen phosphate, magnesium sulfate
310 ~ 330mg/L, 295 ~ 315mg/L of anhydrous calcium chloride, 0.8 ~ 0.9mg/L of potassium iodide, 4.0 ~ 5.0mg/L of boric acid, manganese sulfate
10.0 ~ 12.0mg/L, 0.8 ~ 1.2mg/L of zinc sulfate, 0.12 ~ 0.14mg/L of sodium molybdate, 0.23 ~ 0.27mg/L of copper sulphate, chlorination
0.09 ~ 0.12mg/L of cobalt, 17.5 ~ 19.5mg/L of sodium iron ethylene diamine tetra acetate, 90 ~ 110 mg/L of inositol, vitamin B6 0.4 ~
0.6 mg/L, 0.2 ~ 0.4 mg/L of vitamin B1,0.4 ~ 0.6mg/L of vitamin C, 0.2 ~ 0.5 mg/L of niacin, proline
1.3 ~ 1.5 mg/L, 2.7 ~ 3.3 mg/L of glutathione, 8 ~ 11g/L of active carbon, 30 ~ 40ml/L of tomato juice, triacontanol 0.1 ~
0.3mg/L, 0.08 ~ 0.12mg/L of methyl α-naphthyl acetate, 0.6 ~ 0.9mg/L of kinetin, 0.1 ~ 0.3 mg/L of rare earth, nucleosides 0.3 ~
0.5mg/L, 0.9 ~ 1.1mg/L of cupreol, 0.08 ~ 0.11 mg/L of BAST antivirotic, 25 ~ 30g/L of sucrose, plant gel 7
~ 10g/L, pH are adjusted to 5.6 ~ 6.0.
4. a kind of method of broad-leaved epiphyllum tissue culture tissue culture detoxification nursery as described in claim 1, which is characterized in that the step（5）
The type and method of middle viral diagnosis be：Using the double-antibody method in ELISA to cactus virus X and tobacco mosaic virus (TMV) into
Row detection, direct method detect cucumber mosaic virus.
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