CN107603885A - A kind of rubber tree powdery mildew in vitro culture method and its culture medium - Google Patents
A kind of rubber tree powdery mildew in vitro culture method and its culture medium Download PDFInfo
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- CN107603885A CN107603885A CN201710899573.6A CN201710899573A CN107603885A CN 107603885 A CN107603885 A CN 107603885A CN 201710899573 A CN201710899573 A CN 201710899573A CN 107603885 A CN107603885 A CN 107603885A
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- rubber tree
- powdery mildew
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Abstract
The invention discloses a kind of rubber tree powdery mildew in vitro culture method and its culture medium, belongs to plant pathology technical field.Rubber tree powdery mildew in vitro culture method of the present invention, including step：A) nutrient solution and culture plate prepare：B) excised leaf prepares：C) by PCR plate hole of the excised leaf insertion equipped with nutrient solution after processing, gently brushed on Powdery Mildew to the excised leaf handled by nutrient solution on susceptible rubber tree blade with writing brush or hairbrush after sterilization is dried in 75% alcohol, it is placed in 23 DEG C, in the growth cabinet that humidity is 90%, 16 hours optical cultures, 8 hours light cultures are carried out daily.Rubber tree powdery mildew in vitro culture method of the present invention is easy, long-term efficient, energy large-scale culture rubber tree powdery mildew bacterium, realizes cultured in vitro of the rubber tree powdery mildew bacterium on blade, solves the culture of rubber tree powdery mildew bacterium and preserves problem.
Description
Technical field
The invention belongs to plant pathology technical field, more particularly, to a kind of rubber tree powdery mildew in vitro culture method and
Its culture medium.
Background technology
Rubber tree powdery mildew is as caused by rubber Oidiodendron (oidium heveae B.A.Steinm) fungal infection
One of most important leaf diseases of Para rubber tree, since 1918 since Indonesia Java is found first, time so far
Each rubber planting area in the cloth whole world.When falling ill serious, powdery mildew causes rubber tree fallen leaves, fallen flowers, makes the latex underproduction, rubber tree growing situation
Weak, young sprout is withered, very big to rubber tree growth and yield effect.
Rubber tree powdery mildew cools in spring weather as a kind of weather disease, mainly, it is dark and damp under conditions of eruption and prevalence,
, can only be in fresh and alive plant group and rubber tree Erysiphe can not be grown on the culture medium manually prepared in obligate parasite
Knit and cultivate and preserve on material.Rubber tree powdery mildew material needed for research, only when spring powdery mildew breaks out from big Tanaka
Collection.Therefore, rubber tree powdery mildew is present by effect of natural conditions is big, existence time is short, is not easily controlled the defects of preservation, gives
Research is made troubles.
In the experimental studies such as Disease identification, breeding for disease resistance in rubber tree powdery mildew, artificial culture and preservation rubber tree are white
Powder disease method mainly under conditions of humiture is ensured, by rubber tree powdery mildew spore inoculating rubber tree bagged seedlings children
On leaflet tablet, preserve its morbidity.But bagged seedlings preservation method has that floor space is larger, and the workable less grade of young leaflet tablet lacks
Point, it is extremely difficult to experiment demand.
The content of the invention
The present invention provides a kind of method that easy, long-term efficient, larger rubber tree powdery mildew bacterium culture preserves, profit
Rubber tree blade is kept long period viability with fresh-keeping nutrient solution, it is in vitro on blade to realize rubber tree powdery mildew bacterium
Culture, solve the culture of rubber tree powdery mildew bacterium and preserve problem.
To achieve the above object, the present invention adopts the following technical scheme that：
A kind of rubber tree powdery mildew in vitro culture method, including step：
A) nutrient solution and culture plate prepare：
A1, prepare nutrient solution：100~200mg/L of calcium chloride, 5~10mg/L of 6-benzyl aminopurine, salicylic acid 10~
80mg/L, 0.2~0.8mg/L of vitamin C, 0.2~0.8g/L of sodium thiosulfate, 1~5g/L of activated carbon, 0.2 times of MS culture medium
A great number of elements, 0.2 times of MS culture medium a great number of elements include：Ammonium nitrate 3.3g/L, potassium nitrate 3.8g/L, calcium chloride dihydrate
0.88g/L, epsom salt 0.74g/L, potassium dihydrogen phosphate 0.34g/L；
A2, the fine sand for loading in sterile culture disk 40 mesh or following dusting cover, to be kept no more than the sterilized water of Shamian Island
Fine sand moistens, and PCR plate is placed in sand, turning nutrient solution with liquid-transfering gun enters in PCR plate hole；
B) excised leaf prepares：
B1, selection rubber tree are in the young leaflet tablet of discoloration phase；
B2, by the virgin rubber leaf piece of collection with after aseptic water washing two to three times, it is molten in 0.3-0.5% sodium hypochlorite
Soak in liquid 3-5 minutes, then with aseptic water washing two to three times, drain；
C) by PCR plate hole of the excised leaf insertion equipped with nutrient solution after processing, sterilize and dry in 75% alcohol
Writing brush or hairbrush are gently brushed on Powdery Mildew to the excised leaf by nutrient solution processing on susceptible rubber tree blade afterwards, are placed in 23
DEG C, humidity be 90% growth cabinet in, daily carry out 16 hours optical cultures, 8 hours light cultures.
The two of the object of the invention are to provide a kind of culture medium for rubber tree powdery mildew bacterium cultured in vitro, including：
100~200mg/L of calcium chloride, 5~10mg/L of 6-benzyl aminopurine, 10~80mg/L of salicylic acid, vitamin C 0.2
~0.8mg/L, 0.2~0.8g/L of sodium thiosulfate, 1~5g/L of activated carbon, 0.2 times of MS culture medium a great number of elements (ammonium nitrate
3.3g/L, potassium nitrate 3.8g/L, calcium chloride dihydrate 0.88g/L, epsom salt 0.74g/L, potassium dihydrogen phosphate 0.34g/L).
The present invention compared with prior art, has the following advantages that and beneficial effect：
For the present invention by substantial amounts of exploratory development, configuring can make rubber tree excised leaf keep long period viability
Fresh-keeping nutrient solution (culture medium), cooperateed with by 6-benzyl aminopurine, salicylic acid, vitamin C with 0.2 times of MS culture medium a great number of elements
Effect, rubber tree powdery mildew bacterium optimum growh environment is simulated, realizes cultured in vitro of the rubber tree powdery mildew bacterium on blade, solved
Determined rubber tree powdery mildew bacterium culture and preserve problem.The method of the invention is simple, easy to operate, occupies little space, and leads to
Cross rubber tree excised leaf and carry out Powdery Mildew culture, substantial amounts of Powdery Mildew can be bred in a short time and be used for rubber tree germplasm
The artificial infection of resource, solve the major issue of rubber tree powdery mildew Resistance Identification, and promote rubber tree powdery mildew morphology, life
The research of thing and molecular biology, laid a good foundation immediately, efficiently to carry out the research of rubber tree powdery mildew bacterium.
Embodiment
The present invention is described in further details with reference to specific embodiment.
1st, excised leaf prepares
A, selection rubber tree is in the young leaflet tablet of discoloration phase；
B, the virgin rubber leaf piece of collection is soaked 5 with after aseptic water washing three times in 0.5% liquor natrii hypochloritis
Minute, then with aseptic water washing three times, drain；
2nd, strain selects：Select the asexual conidium of rubber tree powdery mildew fresh on rubber tree disease leaf；
3rd, nutrient solution and culture plate prepare
A, nutrient solution is prepared using following formula：100~200mg/L of calcium chloride, 5~10mg/L of 6-benzyl aminopurine, water
10~80mg/L of poplar acid, 0.2~0.8mg/L of vitamin C, 0.2~0.8g/L of sodium thiosulfate, 1~5g/L of activated carbon, 0.2 times
MS culture mediums a great number of elements (ammonium nitrate 3.3g/L, potassium nitrate 3.8g/L, calcium chloride dihydrate 0.88g/L, epsom salt 0.74g/
L, potassium dihydrogen phosphate 0.34g/L)；
B, the fine sand of 40 mesh sieves was loaded in white disk, to keep fine sand moistening no more than the sterilized water of Shamian Island, by PCR
Plate is placed in sand, and turning nutrient solution with liquid-transfering gun enters in PCR plate hole；
4th, inoculated and cultured
By in PCR plate hole of the excised leaf insertion equipped with nutrient solution after processing, writing brush sterilizes in 75% alcohol to be dried
Afterwards, gently brush Powdery Mildew on susceptible rubber tree blade on the excised leaf by nutrient solution processing, be placed in 23 DEG C, humidity be
In 90% growth cabinet, 16 hours optical cultures, 8 hours light cultures are carried out daily.
Rubber tree powdery mildew in vitro culture method described in the present embodiment：
1st, realize Powdery Mildew cultured in vitro, can quickly, long-term cultivation preserve rubber tree powdery mildew bacterium.Rubber tree white powder
Germ is a kind of obligate parasite, can only be colonized on fresh and alive rubber tree tender leaf, tender shoots, tender tip and inflorescence, and growth conditions
Strictly, usually because the reason such as temperature, humidity and rubber tree phenological period is difficult to obtain substantial amounts of rubber tree white powder in nature
Germ.This method overcomes the difficulty of the external conditions such as rubber tree powdery mildew disease research climate, phenology restriction, Ke Yichang
Phase, quickly rubber tree powdery mildew germ of the culture available for research.
2nd, the pilot scale culture of Powdery Mildew is realized.This method is simple to operate, occupies little space, and gathers Para rubber tree
Tender leaf, a large amount of Powdery Mildews can be bred in short term using artificial incubator, the germ cultured in vitro of such scale is to carry out
The effective means of rubber tree powdery mildew research.
Claims (2)
1. a kind of rubber tree powdery mildew in vitro culture method, including step：
A) nutrient solution and culture plate prepare：
A1, prepare nutrient solution：100~200mg/L of calcium chloride, 5~10mg/L of 6-benzyl aminopurine, 10~80mg/L of salicylic acid,
0.2~0.8mg/L of vitamin C, 0.2~0.8g/L of sodium thiosulfate, 1~5g/L of activated carbon, 0.2 times of MS culture medium a great number of elements
(ammonium nitrate 3.3g/L, potassium nitrate 3.8g/L, calcium chloride dihydrate 0.88g/L, epsom salt 0.74g/L, potassium dihydrogen phosphate
0.34g/L)；
A2, the fine sand for loading in sterile culture disk 40 mesh or following dusting cover, to keep fine sand no more than the sterilized water of Shamian Island
Moistening, PCR plate is placed in sand, turning nutrient solution with liquid-transfering gun enters in PCR plate hole；
B) excised leaf prepares：
B1, selection rubber tree are in the young leaflet tablet of discoloration phase；
B2, by the virgin rubber leaf piece of collection with after aseptic water washing two to three times, in 0.3-0.5% liquor natrii hypochloritises
Soak 3-5 minutes, then with aseptic water washing two to three times, drain；
C) by PCR plate hole of the excised leaf insertion equipped with nutrient solution after processing, sterilized in 75% alcohol and dry rear hair
Pen or hairbrush gently brush Powdery Mildew on susceptible rubber tree blade to the excised leaf by nutrient solution processing, be placed in 23 DEG C,
Humidity is in 90% growth cabinet, carries out 16 hours optical cultures, 8 hours light cultures daily.
2. a kind of culture medium for rubber tree powdery mildew bacterium cultured in vitro, including：
100~200mg/L of calcium chloride, 5~10mg/L of 6-benzyl aminopurine, 10~80mg/L of salicylic acid, vitamin C 0.2~
0.8mg/L, 0.2~0.8g/L of sodium thiosulfate, 1~5g/L of activated carbon, 0.2 times of MS culture medium a great number of elements, 0.2 times of MS
Culture medium a great number of elements includes：Ammonium nitrate 3.3g/L, potassium nitrate 3.8g/L, calcium chloride dihydrate 0.88g/L, epsom salt
0.74g/L, potassium dihydrogen phosphate 0.34g/L.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108641962A (en) * | 2018-03-23 | 2018-10-12 | 湖北省农业科学院植保土肥研究所 | A method of improving the wheat powdery mildew Plantlet in vitro time |
| CN109439551A (en) * | 2018-12-17 | 2019-03-08 | 靖西市秀美边城农业科技有限公司 | A method of using Isolated leaf inoculation grey leaf spot disease of maize and morbidity |
-
2017
- 2017-09-28 CN CN201710899573.6A patent/CN107603885A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108641962A (en) * | 2018-03-23 | 2018-10-12 | 湖北省农业科学院植保土肥研究所 | A method of improving the wheat powdery mildew Plantlet in vitro time |
| CN109439551A (en) * | 2018-12-17 | 2019-03-08 | 靖西市秀美边城农业科技有限公司 | A method of using Isolated leaf inoculation grey leaf spot disease of maize and morbidity |
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Application publication date: 20180119 |