CN106718872B - It is a kind of to expand breeding culture medium and the tissue culture method of wave Rana for raspberry wave Rana - Google Patents
It is a kind of to expand breeding culture medium and the tissue culture method of wave Rana for raspberry wave Rana Download PDFInfo
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- CN106718872B CN106718872B CN201610998572.2A CN201610998572A CN106718872B CN 106718872 B CN106718872 B CN 106718872B CN 201610998572 A CN201610998572 A CN 201610998572A CN 106718872 B CN106718872 B CN 106718872B
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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
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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Abstract
Invention provides a kind of expansion breeding culture medium of culture raspberry wave Rana, contains 1.0~2.0mg/L kinetins, 0.1~0.5mg/L methyl α-naphthyl acetates and 0.5~2.0mg/L basic elements of cell division, the MS culture mediums of 18~25g/L sucrose and 5.0~6.0g/L agar.A kind of tissue culture method of raspberry wave Rana, includes the following steps：1) the band lateral bud wave Rana stem section after sterilizing is seeded on basal medium and is cultivated；2) it when the lateral bud of wave Rana stem section grows to 1.5~2.0cm, lateral bud is cut to be seeded on expansion breeding culture medium as claimed in claim 1 or 2 carries out expanding numerous culture, obtain expanding numerous seedling；3) it when expanding numerous seedling and growing to 2.0~3.0cm, is inoculated on root media and carries out culture of rootage, obtain rooted seedling.The expansion breeding culture medium and tissue culture method of tissue culture provided by the invention can make the survival rate of wave Rana reach 96% or more.
Description
Technical field
The present invention relates to quick breeding fields, and in particular to a kind of group of culture medium and wave Rana that cultivating raspberry wave Rana
Culture method.
Background technology
Raspberry also known as Rubus corchorifolius, bubble in March, bubble in April, mountain throwing, thorn cucurbit, raspberry, steamed bun spinach, high foot spinach, dragon-boat
Bubble, milk bubble, bubble youngster's thorn, are the perennial machaka of rose family rubus, there is 750 kinds or more, because of its fruit type, color, taste
It is similar to strawberry but it is long in the tree, therefore named raspberry is widely distributed in other in addition to northeast, the Inner Mongol, Xinjiang, Tibet of China
Each provinces, municipalities and autonomous regions are grown in the endroit of 300~1500m of height above sea level, small stream side, mountain valley, wasteland and bushes.Raspberry
Fruit nature and flavor micro-sweet, acid, temperature, Zhejiang and Fujian one are used as medicine to do and be drawn with its common immature fruit replacement raspberry, have puckery essence
Improving eyesight that kidney-nourishing is supporing yang, sober up quench the thirst, resolving sputum removing toxic substances the effect of, cure mainly the diseases such as kidney deficiency, seminal emission, drunk, erysipelas.The natural of raspberry resists
Cancer substance " ellagic acid " content is more than blueberry, is occupied first of all kinds of comestibles；The sugar content of fruit of raspberry is 5.58-10.67%, with
Apple, pears, three people's fruit of citrus are similar；Acid content 0.62-2.17%；Additionally contain abundant vitamin C, B1, B2, B12
And minerals, amino acid content is higher than apple, grape；In raspberry plant SOD superoxide dismutase contents occupy various fruit it
The content of head, vitamin E are also very abundant, often it is edible can anti-aging, there are beauty functions and immunity can be improved, prevent from swelling
Tumor.
Raspberry kind is numerous, the kind of common raspberry have plentiful red, Ha Ruitaizi, Qiu Fu, red precious jade, Fil Dodd,
European red and wave Rana etc..The raspberry of different cultivars has species the advantage, such as plentiful red strong, anti-with yield height, early fruiting character
The characteristics of high and cold, impoverishment tolerant；Qiu Fu has the characteristics that big fruit, high yield, precocity, kind are leading, and plant strain growth is healthy and strong.Branch is compared with it
His kind is sturdy, and orthostatic is strong；Red precious jade is with bright-colored, sugar content is high, flavor is dense, thick flavor, adaptable, flower is awarded certainly
The features such as powder ability is strong, disease and insect resistance.From the point of view of quickly breeding angle, although tissue cultures are this field, realization is quickly numerous
The effective means grown, but since there are larger Differences for raspberry species, cause the training of tissue cultures between different cultivars
It supports base and tissue culture method cannot be general.And in the prior art without the tissue culture method suitable for raspberry wave Rana kind, therefore carry
Rapid propagation method for a kind of raspberry wave Rana kind is highly important.
Invention content
In view of this, it is an object of the invention to a kind of culture medium and its cultural method of culture raspberry wave Rana, make institute
It states culture medium and disclosure satisfy that the growth in raspberry wave Rana tissue cultures stage, while can ensure to cultivate the tissue culture of raspberry wave Rana
Method is simple, efficiently, fast breeding, and higher survival rate and rooting efficiency can be obtained.
In order to achieve the above-mentioned object of the invention, the present invention provides following technical scheme：
The present invention provides a kind of expansion breeding culture medium of culture raspberry wave Rana, the expansions breeding culture medium for containing 1.0~
2.0mg/L kinetins, 0.1~0.5mg/L methyl α-naphthyl acetates, 0.5~2.0mg/L basic elements of cell division, 18~25g/L sucrose and 5.0~
The MS culture mediums of 6.0g/L agar.
Preferably, the expansion breeding culture medium is containing 1.5mg/L kinetins, 0.2mg/L methyl α-naphthyl acetates, 1.0mg/L cells point
Split element, the MS culture mediums of 20g/L sucrose and 5.6g/L agar.
The present invention also provides a kind of tissue culture methods of raspberry wave Rana, include the following steps：
1) the band lateral bud wave Rana stem section after sterilizing is seeded on basal medium and is cultivated；
2) when the lateral bud of wave Rana stem section grows to 1.5~2.0cm, cut lateral bud be seeded to it is as claimed in claim 1 or 2
Expand on breeding culture medium and carry out expanding numerous culture, obtains expanding numerous seedling；
3) it when expanding numerous seedling and growing to 2.0~3.0cm, is inoculated on root media and carries out culture of rootage, obtain tissue-cultured seedling.
4, tissue culture method according to claim 1, which is characterized in that the condition of culture is in the step 1)：Temperature
Degree is 23~27 DEG C, and intensity of illumination is 1800~2200Lx, and light application time is 11~13h/d.
Preferably, the time cultivated in the step 1) is 20~30d.
Preferably, 25~35d of time of the numerous culture of expansion of the step 2).
Preferably, the root media of the step 3) includes 0.5~1.5mg/L IBA, 18~25g/L sucrose and 5.0
The 1/2MS culture mediums of~6.0g/L agar.
Preferably, further include after the culture of rootage of the step 3)：The tissue-cultured seedling that the step 3) obtains is placed in room temperature
Progress hardening growth in matrix is transplanted in environment after 3~4d of adaptive training.
Preferably, it is 2 that the matrix, which includes volume ratio,:The vermiculite and turf of (0.8~1.5).
Preferably, the environment temperature of the hardening is 22~27 DEG C, and the ambient humidity of the hardening is 75%~90%.
The present invention provides a kind of expansion breeding culture medium of culture raspberry wave Rana, for contain 1.0~2.0mg/L kinetins,
0.1~0.5mg/L methyl α-naphthyl acetates and 0.5~2.0mg/L basic elements of cell division, the MS of 18~25g/L sucrose and 5.0~6.0g/L agar
Culture medium.In the present invention, the kinetin, methyl α-naphthyl acetate and the basic element of cell division cooperate with to play as plant growth regulator and make
With kinetin and the basic element of cell division have the morphogenesis for promoting cell division, controlling cultured tissue, delay senility and trophotrophic object
Matter is mobile, promote cell expansion, promote lateral bud development, participates in the important roles such as chloroplaset biology generation；Methyl α-naphthyl acetate and kinetin and
The basic element of cell division, which is used together, can stimulate tissue growth, adapt to new culture environment, and raspberry wave Rana is made to reach quick breeding
Effect.
The present invention also provides a kind of tissue culture methods of raspberry wave Rana, by the band lateral bud wave Rana stem section inoculation after sterilizing
It is cultivated on to basal medium；When the lateral bud of wave Rana stem section grows to 1.5~2.0cm, by the lateral bud inoculation expansion
It carries out expanding numerous culture on breeding culture medium, obtains expanding numerous seedling；When expanding numerous seedling and growing to 2.0~3.0cm, it is inoculated on root media
Culture of rootage is carried out, tissue-cultured seedling is obtained.Tissue culture method provided by the invention selects lateral bud for induced material, on expanding breeding culture medium
After culture when expanding numerous seedling a length of 2.0~3.0cm, it is seeded to culture of rootage and obtains rooted seedling, can successfully cultivate wave Rana
Tissue-cultured seedling, and reach 96% or more using the survival rate of the cultural method tissue culture.
Specific implementation mode
The present invention provides a kind of expansion breeding culture medium of culture raspberry wave Rana, for contain 1.0~2.0mg/L kinetins,
0.1~0.5mg/L methyl α-naphthyl acetates and 0.5~2.0mg/L basic elements of cell division, the MS of 18~25g/L sucrose and 5.0~6.0g/L agar
Culture medium.
In the present invention, the expansion breeding culture medium is preferably containing 0.5~1.5mg/L kinetins, 0.2~0.4mg/L naphthalene second
Acid and 1.0~1.5mg/L basic elements of cell division, the MS culture mediums of 20~23g/L sucrose and 5.6~5.8g/L agar.
In the present invention, the kinetin, methyl α-naphthyl acetate, the basic element of cell division, sucrose and agar source be not particularly limited, adopt
With the source of above-mentioned substance well-known to those skilled in the art.In the present invention, the kinetin, methyl α-naphthyl acetate and cell point
It splits element and is purchased from Sigma-Aldrich Co.LLC..In the present invention, the sucrose is purchased from Tianjin good fortune morning chemical reagent factory；Institute
It states agar and is purchased from JAPAN companies.
In the present invention, the MS culture mediums preferably include constituent content below：Potassium nitrate 1900mg/L, ammonium nitrate
1650mg/L, potassium dihydrogen phosphate 700mg/L, magnesium sulfate 370mg/L, calcium chloride 440mg/L, potassium iodide 0.83mg/L, boric acid
6.2mg/L, manganese sulfate 22.3mg/L, zinc sulfate 8.6mg/L, sodium molybdate 0.25mg/L, copper sulphate 0.025mg/L, cobalt chloride
0.025mg/L, disodium ethylene diamine tetraacetate 37.3mg/L, ferrous sulfate 27.8mg/L, inositol 100mg/L, glycine 2mg/L,
Thiamine hydrochloride 0.1mg/L, puridoxine hydrochloride 0.5mg/L, niacin 0.5mg/L.The pH value of the MS culture mediums is preferably 5.6~
6.0, more preferably 5.8.
In the present invention, the preparation method for expanding breeding culture medium is not particularly limited, ripe using those skilled in the art institute
The preparation method for the culture medium known.In the present invention in embodiment, by kinetin, methyl α-naphthyl acetate and the basic element of cell division, sucrose and
Agar is mixed with MS culture mediums comprising each component.
The present invention provides a kind of tissue culture methods of raspberry wave Rana, include the following steps：
1) the band lateral bud wave Rana stem section after sterilizing is seeded on basal medium and is cultivated；
2) when the lateral bud of wave Rana stem section grows to 1.5~2.0cm, lateral bud is cut and is seeded to described in claims 1 or 2
Expansion breeding culture medium on carry out expanding numerous culture, obtain expanding numerous seedling；
3) it when expanding numerous seedling and growing to 2.0~3.0cm, is inoculated on root media and carries out culture of rootage, obtain tissue-cultured seedling.
Band lateral bud wave Rana stem section after sterilizing will be seeded on basal medium and cultivates by the present invention.The present invention
In, wave Rana stem section is preferably current year raw stem section.The length with lateral bud wave Rana stem section is preferably 1.5~2cm, more
Preferably 1.8cm.The quantity of the lateral bud is preferably greater than 1.
Band lateral bud wave Rana stem section of the present invention intercepts one section preferably through band lateral bud wave Rana branch.The present invention is excellent
Band lateral bud wave Rana branch is carried out pre-treatment by choosing.The method of the pre-treatment is preferably：It will be with lateral bud branch in flowing water
20~30min of middle flushing, then with 5~8min of distilled water immersion, carried out at sterilizing after the branch with lateral bud is cut into stem section
Reason.In the present invention, the time of the flushing is preferably 25min；The time of the distilled water immersion is preferably 6min.
The present invention is not particularly limited the method for the sterilizing, using tissue culture field well-known to those skilled in the art
Sterilizing methods.In embodiments of the present invention, the sterilizing is as follows：It is with volumetric concentration by stem segment with lateral bud
70%~75% ethanol water washs 50~70s；Stem section after the washing is subjected to the first flushing 1~2 time with sterile water；
The mercuric chloride that stem section volumetric concentration after described first is rinsed is 0.05~0.15% impregnates 5~10min；After the immersion
Stem section with sterile water carry out second flushing 4~5 times；Stem section surface moisture after blotting second flushing with aseptic filter paper.
In the present invention, the volumetric concentration of the ethanol water is more preferably 72%；The time of ethanol solution washing is preferably
60s.In the present invention, the volumetric concentration of the mercuric chloride is preferably 0.1%；The time that the mercuric chloride impregnates is preferably 8min.
The present invention is not particularly limited the method for the inoculation, using inoculation method well-known to those skilled in the art
.In the present invention, preferably the both ends surface of a wound is cut before the stem section inoculation of band lateral bud wave Rana.
In the present invention, the basal medium is preferably (thin comprising 0.08~0.12mg/LNAA, 0.3~0.8mg/L6BA
Born of the same parents' mitogen), the MS culture mediums of 15~25g/L sucrose and 5.0~6.0g/L agar, more preferably include 0.1mg/LNAA,
The MS culture mediums of 0.5mg/L 6BA, 20g/L sucrose and 5.6g/L agar.The composition content and said program of the MS culture mediums
Described in MS culture mediums it is consistent.
In the present invention, the condition of the culture is preferably：22~27 DEG C, 1800~2200Lx of intensity of illumination of temperature, illumination
11~13h/d of time；More preferably 25 DEG C of temperature, intensity of illumination 2000Lx, light application time 12h/d.It is described in the present invention
The time of culture is preferably 20~30d, more preferably 25d.The purpose of heretofore described culture is that lateral bud is made to grow.
After the lateral bud cultivated, lateral bud is inoculated with by the present invention when lateral bud of wave Rana stem section grows to 1.5~2.0cm
It carries out expanding numerous culture on to the expansion breeding culture medium described in above-mentioned technical proposal, obtains expanding numerous seedling.Specifically, the present invention will be in stem section
Lateral bud cut after be seeded to expand breeding culture medium on.The present invention preferably when the lateral bud of wave Rana stem section grows to 1.8cm, incites somebody to action
Lateral bud, which is seeded to, to be expanded on breeding culture medium.
In the present invention, the length of the lateral bud for inoculation is preferably 0.8~1.5cm, more preferably 1.0~1.2cm.
In the present invention, the time for expanding numerous culture is preferably 25~35d, more preferably 30d.The temperature for expanding numerous culture
Preferably 23~26 DEG C, more preferably 25 DEG C of degree；The humidity for expanding numerous culture is preferably 70%~80%, more preferably
75%.The intensity of the illumination for expanding numerous culture is preferably 180~2200Lx, more preferably 2000Lx.
When expanding numerous seedling and growing to 2.0~3.0cm, the present invention, which will expand numerous seedling and be inoculated on root media, carries out training of taking root
It supports, obtains tissue-cultured seedling.In the present invention, the root media is preferably comprising 0.5~1.5mg/LIBA, 18~25g/L sucrose
More preferably include 1.0mg/LIBA, 20g/L sucrose and 5.6g/L agar with the 1/2MS culture mediums of 5.0~6.0g/L agar
1/2MS culture mediums.
In the present invention, the 1/2MS nutrient media components are preferably potassium nitrate 950mg/L, ammonium nitrate 825mg/L, di(2-ethylhexyl)phosphate
Hydrogen potassium 350mg/L, magnesium sulfate 185mg/L, calcium chloride 220mg/L, potassium iodide 0.83mg/L, boric acid 6.2mg/L, manganese sulfate
22.3mg/L, zinc sulfate 8.6mg/L, sodium molybdate 0.25mg/L, copper sulphate 0.025mg/L, cobalt chloride 0.025mg/L, ethylenediamine
Tetraacethyl disodium 37.3mg/L, ferrous sulfate 27.8mg/L, inositol 100mg/L, glycine 2mg/L, thiamine hydrochloride 0.1mg/
L, puridoxine hydrochloride 0.5mg/L, niacin 0.5mg/L.
In the present invention, the time of the culture of rootage is preferably 30d；The temperature of the culture of rootage is preferably 23~26
DEG C, more preferably 25 DEG C；The humidity of the culture of rootage is preferably 70%~80%, and more preferably 75%.The culture of rootage
The intensity of illumination be preferably 180~2200Lx, more preferably 2000Lx.
After the culture of rootage obtains tissue-cultured seedling, the present invention will preferably transplant after 3~4d of the tissue-cultured seedling adaptive training
To hardening growth is carried out in matrix, the La Bona seedlings suitable for outdoor growth are obtained.In the present invention, the adaptive training
It is preferred that being carried out in room temperature environment.
In the present invention, it is 2 that the matrix, which preferably includes volume ratio,:The vermiculite and turf of (0.8~1.5), more preferably body
Product is than being 2:1 vermiculite and turf.The present invention is not particularly limited the source of the vermiculite and turf, using art technology
Vermiculite known to personnel and turf.In the present invention, the vermiculite is preferably carried out disinfection using preceding；The disinfection is used
Disinfectant is preferably the potassium permanganate solution that mass concentration is 0.1~0.3%.The present invention is to the method for the disinfection without spy
Different limitation, using the technical solution well-known to those skilled in the art to be carried out disinfection using liquor potassic permanganate.
In the present invention, the environment temperature of the hardening is preferably 22~27 DEG C, more preferably 25 DEG C；The environment of the hardening
Humidity is preferably 75%~90%, and more preferably 85%.In the present invention, the mesh of the adaptive training is tissue culture transplantation of seedlings to room
One transition culture of outer offer.
In the present invention, the method for the transplanting is not particularly limited using method for transplanting well-known to those skilled in the art
.
With reference to embodiment to a kind of culture medium and wave Rana for cultivating raspberry wave Rana provided by the invention
Tissue culture method is described in detail, but they cannot be interpreted as limiting the scope of the present invention.
Embodiment 1
Raspberry wave Rana current year green tape lateral bud branch is taken, 20min is rinsed in flowing water, then with bubble 5min is distilled, by band
There is the stem section of lateral bud to be cut into 1.5cm, sterilization treatment is carried out on superclean bench.60S is washed with 70% ethyl alcohol, is rushed with sterile water
It washes 1 time, then 5min, aseptic water washing 4 times is impregnated with 0.1% mercuric chloride, then surface moisture is blotted with aseptic filter paper.
Stem section after sterilizing cuts the both ends surface of a wound, is seeded to additional NAA0.1mg/L, 6BA0.5mg/L, addition sucrose 20g/
L, sterile culture is carried out on the MS culture mediums of agar 5.6g/L 20 days.Condition of culture is：25 DEG C of temperature, intensity of illumination are
2000Lx, light application time 12h/d.
When lateral bud grows to 1.5cm, cuts and be seeded to additional 1.0mg/LKT, 0.5mg/LNAA and 0.5mg/L 6-BA, add
Add and carries out expanding numerous culture on the MS culture mediums of sucrose 20g/L, agar 5.6g/L.
When tissue-cultured seedling is grown to 2.0cm, 25 days, the tissue culture plant inoculation for cutting 2.0cm added 0.5mg/ to 1/2MS culture mediums
LIBA, sucrose 25g/L, agar 6.0g/L culture medium on carry out culture of rootage.
The tissue-cultured seedling bottle that will take root takes greenhouse and takes off lid adaptive training 4 days, then from tissue culture bottle it is with blunt nosed tweezers that seedling is small
The heart presss from both sides out taking-up, and the volume ratio that clean culture medium is transplanted to vermiculite and turf after being sterilized with 0.2% potassium permanganate is 2:1 base
Hardening in matter, 27 degree of temperature, humidity 75% obtain wave Rana seedling.The wave Rana seedling obtained using this method is survived
Rate 96%.
Embodiment 2
Raspberry wave Rana current year green tape lateral bud branch is taken, 30min is rinsed in flowing water, then with bubble 10min is distilled, by band
There is the stem section of lateral bud to be cut into 2cm, sterilization treatment is carried out on superclean bench.70s is washed with 75% ethyl alcohol, uses aseptic water washing
1 time, then 10min, aseptic water washing 4 times are impregnated with 0.08% mercuric chloride, then blot surface moisture, preferably 7min with aseptic filter paper.
Stem section after sterilizing cuts the both ends surface of a wound, is seeded to additional NAA0.1mg/L, 6BA0.5mg/L, addition sucrose 15g/
L, sterile culture is carried out on the MS culture mediums of agar 5.0g/L 30 days.Condition of culture is：25 DEG C of temperature, intensity of illumination are
2000Lx, light application time 12h/d.
When lateral bud grows to 2.0cm, cuts and be seeded to additional 2.0mg/L KT, 0.1mg/LNAA and 2.0mg/L6-BA, add
Add and carries out expanding numerous culture on the MS culture mediums of sucrose 15g/L, agar 5.0g/L.
When tissue-cultured seedling is grown to 3.0cm, 35 days, the tissue culture plant inoculation for cutting 2.0cm added 1.5mg/ to 1/2MS culture mediums
LIBA, sucrose 25g/L, agar 6.0g/L culture medium on carry out culture of rootage.
The tissue-cultured seedling bottle that will take root takes greenhouse and takes off lid adaptive training 4 days, then from tissue culture bottle it is with blunt nosed tweezers that seedling is small
The heart presss from both sides out taking-up, and the volume ratio that clean culture medium is transplanted to vermiculite and turf after being sterilized with 0.1% potassium permanganate is 2:1 base
Hardening in matter, 22 DEG C of temperature, humidity 75% obtain wave Rana seedling.The wave Rana seedling obtained using this method is survived
Rate 99%.
Embodiment 3
Raspberry wave Rana current year green tape lateral bud branch is taken, 25min is rinsed in flowing water, then with bubble 8min is distilled, by band
There is the stem section of lateral bud to be cut into 1.8cm, sterilization treatment is carried out on superclean bench.50s is washed with 72% ethyl alcohol, is rushed with sterile water
It washes 1 time, then 7min, aseptic water washing 5 times is impregnated with 0.15% mercuric chloride, then surface moisture, preferably 7min are blotted with aseptic filter paper.
Stem section after sterilizing cuts the both ends surface of a wound, is seeded to additional NAA0.1mg/L, 6BA0.5mg/L, addition sucrose 20g/
L, sterile culture is carried out on the MS culture mediums of agar 5.6g/L 25 days.Condition of culture is：25 DEG C of temperature, intensity of illumination are
2000Lx, light application time 12h/d.
When lateral bud grows to 1.8cm, cuts and be seeded to additional 1.5mg/LKT, 0.2mg/LNAA and 1.0mg/L 6-BA, add
Add and carries out expanding numerous culture 30 days on the MS culture mediums of sucrose 20g/L, agar 5.6g/L.
When tissue-cultured seedling is grown to 2.5cm, cut the tissue culture plant inoculation of 2.0cm to 1/2MS culture mediums addition 1.0mg/LIBA,
Sucrose 20g/L, agar 5.6g/L culture medium on carry out culture of rootage.
The tissue-cultured seedling bottle that will take root takes greenhouse and takes off lid adaptive training 3.5 days, then from tissue culture bottle with blunt nosed tweezers by seedling
Careful to press from both sides out taking-up, the volume ratio that clean culture medium is transplanted to vermiculite and turf after being sterilized with 0.3% potassium permanganate is 2:1
Hardening in matrix, 25 DEG C of temperature, humidity 85% obtain wave Rana seedling.The wave Rana seedling obtained using this method at
Motility rate 97%.
Comparative example
Raspberry wave Rana current year green tape lateral bud branch is taken, 20min is rinsed in flowing water, then with bubble 5min is distilled, by band
There is the stem section of lateral bud to be cut into 1.5-2cm, sterilization treatment is carried out on superclean bench.60s is washed with 70% ethyl alcohol, uses sterile water
It rinses 1 time, then 5min, aseptic water washing 5 times is impregnated with 0.1% mercuric chloride, then surface moisture is blotted with aseptic filter paper.
Stem section after sterilizing cuts the both ends surface of a wound, is seeded to additional NAA0.1mg/L, 6BA0.5mg/L, addition sucrose 20g/
L, sterile culture is carried out on the MS culture mediums of agar 5.6g/L 20 days.Condition of culture is：25 DEG C of temperature, intensity of illumination are
2000Lx, light application time 12h/d.
It when lateral bud grows to 1.5cm, cuts and is seeded on additional autumn good fortune culture medium, autumn good fortune culture medium includes
On the MS culture mediums of 0.1mg/L IBA, 1.0mg/L 6BA and 0.5mg/LGA3, wave Rana grows on this culture medium and gradually declines
It is weak, smaller and smaller, 20 days or so yellow leafs, death in 40 days or so.
As seen from the above embodiment, a kind of expansion breeding culture medium of culture raspberry wave Rana and its cultural method, use are above-mentioned
The survival rate of amplification culture medium and the La Bona of cultural method culture reach 96% or more, using autumn good fortune medium culture La Bona
As a result death in 40 days or so illustrates plant modifying agent and nutrients that raspberry La Bona kinds of the present invention carry out tissue culture to culture medium
Matter is selective, and the raspberry kind tissue culture amplification culture medium of other kinds, which cultivates La Bona, to be not suitable for.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (6)
1. a kind of tissue culture method of raspberry wave Rana, includes the following steps：
1) the band lateral bud wave Rana stem section after sterilizing is seeded on basal medium and is cultivated；
The condition of the culture is：Temperature is 23~27 DEG C, and intensity of illumination is 1800~2200Lx, and light application time is 11~13h/
d；
The basal medium is only comprising 0.08~0.12mg/L NAA, 0.3~0.8mg/L 6-BA, 15~25g/L sucrose
With the MS culture mediums of 5.0~6.0g/L agar；
2) when the lateral bud of wave Rana stem section grows to 1.5~2.0cm, cut lateral bud be seeded to expand breeding culture medium on carry out expanding numerous training
It supports, obtains expanding numerous seedling；
The expansion breeding culture medium is only to contain 1.0~2.0mg/L kinetins, 0.1~0.5mg/L methyl α-naphthyl acetates, 0.5~2.0mg/L
The MS culture mediums of 6-BA, 18~25g/L sucrose and 5.0~6.0g/L agar；
The temperature for expanding numerous culture is 23~26 DEG C；The humidity for expanding numerous culture is 70%~80%；It is described to expand numerous culture
Illumination intensity be 180~2200Lx；
3) it when expanding numerous seedling and growing to 2.0~3.0cm, is inoculated on root media and carries out culture of rootage, obtain tissue-cultured seedling；
The root media be only include 0.5~1.5mg/L IBA, 18~25g/L sucrose and 5.0~6.0g/L agar 1/
2MS culture mediums；
The temperature of the culture of rootage is 23~26 DEG C；The humidity of the culture of rootage is 70%~80%；The culture of rootage
Illumination intensity be 180~2200Lx；
The MS culture mediums are grouped as by below group：Potassium nitrate 1900mg/L, ammonium nitrate 1650mg/L, potassium dihydrogen phosphate
700mg/L, magnesium sulfate 370mg/L, calcium chloride 440mg/L, potassium iodide 0.83mg/L, boric acid 6.2mg/L, manganese sulfate 22.3mg/
L, zinc sulfate 8.6mg/L, sodium molybdate 0.25mg/L, copper sulphate 0.025mg/L, cobalt chloride 0.025mg/L, ethylenediamine tetra-acetic acid two
Sodium 37.3mg/L, ferrous sulfate 27.8mg/L, inositol 100mg/L, glycine 2mg/L, thiamine hydrochloride 0.1mg/L, hydrochloric acid pyrrole
Tremble alcohol 0.5mg/L, niacin 0.5mg/L；
The 1/2MS culture mediums are grouped as by below group：Potassium nitrate 950mg/L, ammonium nitrate 825mg/L, potassium dihydrogen phosphate
350mg/L, magnesium sulfate 185mg/L, calcium chloride 220mg/L, potassium iodide 0.83mg/L, boric acid 6.2mg/L, manganese sulfate 22.3mg/
L, zinc sulfate 8.6mg/L, sodium molybdate 0.25mg/L, copper sulphate 0.025mg/L, cobalt chloride 0.025mg/L, ethylenediamine tetra-acetic acid two
Sodium 37.3mg/L, ferrous sulfate 27.8mg/L, inositol 100mg/L, glycine 2mg/L, thiamine hydrochloride 0.1mg/L, hydrochloric acid pyrrole
Tremble alcohol 0.5mg/L, niacin 0.5mg/L.
2. tissue culture method according to claim 1, which is characterized in that the time cultivated in the step 1) is 20~30d.
3. tissue culture method according to claim 1, which is characterized in that the time 25 of the numerous culture of expansion of the step 2)~
35d。
4. tissue culture method according to claim 1, which is characterized in that further include after the culture of rootage of the step 3)：It will
It is transplanted in matrix after 3~4d of tissue-cultured seedling adaptive training that the step 3) obtains and carries out hardening.
5. tissue culture method according to claim 4, which is characterized in that the matrix includes that volume ratio is 2: (0.8~1.5)
Vermiculite and turf.
6. tissue culture method according to claim 4 or 5, which is characterized in that the environment temperature of the hardening is 22~27 DEG C,
The ambient humidity of the hardening is 75%~90%.
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