CN109937885A - A kind of sterile seedling fast reproducing method of wrinkle leaf cuckoo - Google Patents
A kind of sterile seedling fast reproducing method of wrinkle leaf cuckoo Download PDFInfo
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- CN109937885A CN109937885A CN201910375533.0A CN201910375533A CN109937885A CN 109937885 A CN109937885 A CN 109937885A CN 201910375533 A CN201910375533 A CN 201910375533A CN 109937885 A CN109937885 A CN 109937885A
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Abstract
The present invention provides a kind of sterile seedling fast reproducing methods of wrinkle leaf cuckoo, are related to plant tissue culture fast breeding technique field.The method of the invention will be inoculated in induced medium, Fiber differentiation 30d or more obtains wrinkle leaf cuckoo aseptic seedling the following steps are included: using the tender tissue for the leaf cuckoo that wrinkles as explant after explant disinfection；The induced medium is using 1/2WPM as minimal medium, further includes: 1.5mg/L ZT, 1.0mg/L NAA, 50mg/L caseinhydrolysate, 30g/L sucrose and 8g/L agar powder, pH value 5.0.The method of the invention is easy to operate, and emergence rate can be made to be up to 80%, solve the problems, such as presently, there are wrinkle leaf cuckoo reproductive efficiency it is low.
Description
Technical field
The invention belongs to plant tissue culture fast breeding technique fields, and in particular to a kind of sterile seedling fast reproducing method of wrinkle leaf cuckoo.
Background technique
Wrinkle leaf cuckoo (Rhododendron denudatum Levl.) shrub or dungarunga, high about 3~6m；Sturdy branch
Item, sprout are become hairless afterwards by starlike villus.Leaf keratin, long ovum multiform, such as long ovum lanceolar, elliptic-lanceolate etc. have above leaf
Apparent wrinkle, it is hairless, there is loose faint yellow villus below.The total shape umbrella shape of inflorescence, spends 8~12；Corolla mitriform, long 3.5
~4cm, rose color inside have darkviolet spot, and 5 split, and sliver is bordering on circle.The cuckoo maximum feature is that branches and leaves are luxuriant, and flower is dazzled
Beautiful colourful, resistance to trimming, pile is peculiar, is the optimal selection of potted landscape material.Most preferably it is planted in gardens border, small stream side, Chi Pan and rock
It by stone, can also transplant under sparse woods, as the good material of flower hedge, often cultivate by trimming into various forms.The cuckoo adolescence
When bursting forth, flower appearance is glamorous fascinating, after spending the adolescence, bottle-green blade be still parapet in flower garden or barrier most
Suitable selection.In addition to this, the species are because of flower beauty, bright-colored, artificial cultivation, horticultural importance with higher.
Cuckoo is born in high altitude localities, likes wet weather of feeling nice and cool, and suitable growth temperature is 15~30 DEG C, and 3~May opens
Flower, there are certain requirements light, and intolerant to exposure, summer and autumn should have deciduous tree or cool canopy blocks burning sun, and often spray ground with water,
Can normal growth, but in extremely hot drying, arid, barren, soil pH middle growth and development between 7~8 is suppressed.Current text
Offer research in it is more for the research of other tissue culture of Rhododendron simsii Planch, but for wrinkle leaf cuckoo tissue culture technology research
There is not been reported.
Summary of the invention
In view of this, establishing wrinkle leaf cuckoo the purpose of the present invention is to provide a kind of sterile seedling fast reproducing method of wrinkle leaf cuckoo
Aseptic seedling quick breeding technology system can solve the problems, such as that wrinkle leaf cuckoo reproductive efficiency is low.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides a kind of sterile seedling fast reproducing methods of wrinkle leaf cuckoo, comprising the following steps: tender with the children for the leaf cuckoo that wrinkles
Tissue is explant, will be inoculated in induced medium after explant disinfection, Fiber differentiation 30d or more, must wrinkle leaf cuckoo
Aseptic seedling；The induced medium is using 1/2WPM as minimal medium, further includes: 1.5mg/L ZT, 1.0mg/L NAA, 50mg/
L caseinhydrolysate, 30g/L sucrose and 8g/L agar powder, pH value 5.0.
Preferably, the tender tissue includes petal, sepal, stem section or bud.
It preferably, further include rinsing the stem section or bud with tap water respectively when making explant with the stem section or bud
The segment of 1~2cm long is cut into after 4h.
Preferably, when using the petal as explant, further include rinsing 4h with tap water, peel off the outer layer of cuckoo petal
The petal for peelling off sepal is cut into the segment of 1~2cm long by sepal.
It preferably, further include the outer layer sepal distilled water immersion 5h that will be peeled when using sepal as explant.
Preferably, the method for the disinfection includes impregnating the 10~15min of explant with 0.1% mercuric chloride.
Preferably, the light application time of the Fiber differentiation is 14h/d, intensity of illumination 1500lx；The Fiber differentiation
Temperature is 22 ± 2 DEG C.
The present invention provides a kind of sterile seedling fast reproducing methods of wrinkle leaf cuckoo, using the tender tissue for the leaf cuckoo that wrinkles as explant,
It will be inoculated in induced medium after explant disinfection, Fiber differentiation 30d or more, must wrinkle leaf cuckoo aseptic seedling；It is described to lure
Culture medium is led using 1/2WPM as minimal medium, further includes: 1.5mg/L ZT, 1.0mg/L NAA, 50mg/L caseinhydrolysate,
30g/L sucrose and 8g/L agar powder, pH value 5.0.In embodiments of the present invention, emergence rate reaches as high as 80%.Institute of the present invention
Method simplicity is stated, it is easy to operate, it is easy to implement, and a large amount of wrinkle leaf cuckoo aseptic seedlings can be obtained in a short time.
Specific embodiment
The present invention provides a kind of sterile seedling fast reproducing methods of wrinkle leaf cuckoo, comprising the following steps: tender with the children for the leaf cuckoo that wrinkles
Tissue is explant, will be inoculated in induced medium after explant disinfection, Fiber differentiation 30d or more, must wrinkle leaf cuckoo
Aseptic seedling；The induced medium is using 1/2WPM as minimal medium, further includes: 1.5mg/L ZT, 1.0mg/L NAA, 50mg/
L caseinhydrolysate, 30g/L sucrose and 8g/L agar powder, pH value 5.0.
In the method for the invention, using the tender tissue for the leaf cuckoo that wrinkles as explant, the tender tissue is preferably included
Petal, sepal, stem section and bud, more preferably sepal.There is no particular determinations in source of the present invention to the tender tissue, utilize
The parent of growing way stalwartness.Sepal of the present invention is preferably the outer layer sepal of the petal.The present invention is stripping the flower
Before the outer layer sepal of bud, it is also preferable to include petal tap water is rinsed 4h, strip up to the sepal.The present invention is to institute
Stating the method that strips, there is no particular determinations, carry out preferably in gnotobasis.
In the present invention, when making explant with the stem section, bud, it is also preferable to include cut after rinsing 4h with tap water respectively
At the segment of 1~2cm long, the disinfection is then carried out again；It further include being rinsed with tap water when using the petal as explant
4h strips the outer layer sepal of cuckoo petal, the petal for peelling off sepal is cut into the segment of 1~2cm long, then carries out described disappear again
Poison；It further include then carrying out the outer layer sepal distilled water immersion 5h stripped described again when using sepal as explant
Disinfection.
The method of disinfection of the present invention preferably includes to impregnate the 10~15min of explant with 0.1% mercuric chloride, more excellent
It is selected as impregnating 12min.
The explant is inoculated in induced medium and carries out Fiber differentiation by the present invention, when the illumination of the Fiber differentiation
Between preferably 14h/d, intensity of illumination is preferably 1500lx.The temperature of Fiber differentiation of the present invention is preferably 22 DEG C ± 2 DEG C.This
It invents in the induced medium, WPM can provide a great number of elements, microelement, organic element and molysite as basic culture medium
Etc. basal needs；ZT and NAA, which is used in mixed way, can promote callus germination；Caseinhydrolysate contains hydrolysis free amino acid, by
In the complex effect of a variety of amino acid, being absorbed and utilized for other nutriments can be enhanced；Sucrose provides glycogen for material；Agar mentions
For supporting role.There is no particular determinations in source of the present invention to each ingredient in the induced medium, utilize the normal of this field
Advise reagent.
The sterile seedling fast reproducing method of wrinkle leaf cuckoo provided by the invention is described in detail below with reference to embodiment, still
They cannot be interpreted as limiting the scope of the present invention.
Embodiment 1
1) petal for acquiring the wrinkle leaf cuckoo acquired from Guizhou Baili azalea, is rinsed into 4h with tap water first, strips Du
The outer layer sepal of cuckoo petal uses distilled water immersion 5h as explant afterwards.
2) pretreated sepal 12min, is impregnated with 0.1% mercuric chloride, is during which stirred continuously, explant is carried out
Sterilizing, after with sterile distilled water rinse 4~5 times, sterile distilled water is poured out completely, with make inoculation it is spare.
3) explant that sterilization treatment, is clamped with the tweezers for having been subjected to autoclave sterilization, is inoculated in induced medium
(caseinhydrolysate+30g/L sucrose+8g/L agar powder of 1/2WPM+1.5mg/LZT+1.0mg/LNAA+50mg/L, pH 5.0)
On, 14h/d illumination is incubated at later, in 22 DEG C of culturing room.
Culture 30d after grow macroscopic aseptic seedling, measure its emergence rate be 81.35%, breeding coefficient be 300~
465。
Embodiment 2
1.1. experimental material
Petal, sepal, the spire of the wrinkle leaf cuckoo acquired from Guizhou Baili azalea, the explants such as bud, stem section, root.
1.2. the pretreatment of material
Make explant with petal, sepal, stem section, bud, the root of the leaf cuckoo that wrinkles, rinses 4h with tap water first, strip cuckoo
The outer layer sepal of petal uses distilled water immersion 5h afterwards, will peel off immature petal, stem section, the root of sepal as explant
It is cut to the segment of 1-2 centimeter length, it is spare to make.
1.3. the sterilizing of explant
With three kinds of 0.1% mercuric chloride, 10%NaClO and 70% alcohol bactericidal agents in this invention, explant is carried out
Sterilizing, is respectively set different sterilization times, specific experiment scheme is as shown in table 1:
The wrinkle leaf cuckoo explant sterilization method experimental program of table 1
Above 12 kinds of modes are respectively adopted to wrinkle leaf cuckoo explant and carry out sterilization treatment, by the explant after sterilizing
It is inoculated with.In addition to sterilizing methods are different, other factors are all identical as embodiment 1.
After being inoculated with 5d, the contamination rate of 14 kinds of sterilization methods is counted, the results are shown in Table 2 for specific experiment:
The wrinkle leaf cuckoo explant sterilization method experimental result of table 2
As shown in Table 2,12min is sterilized with 0.1% mercuric chloride, pollution rate is minimum, although 0.1% mercuric chloride mixes sterilizing with alcohol
Effect is also fine, but alcohol causes further damage to explant.
Embodiment 3
In addition to explant species are different, remaining factor is same as Example 1:
Explant species: petal, sepal, the spire of the wrinkle leaf cuckoo acquired from Guizhou Baili azalea, bud, stem section, root etc.
Explant.
After being inoculated with 30d, the emergence of different explants is counted, the results are shown in Table 3 for specific experiment:
The wrinkle leaf cuckoo difference explant emergence experimental result of table 3
| Explant | Inoculation number | Number of emerging | Emergence rate (%) |
| Sepal | 5 | 4 | 80 |
| Petal | 5 | 2 | 40 |
| Stem section | 5 | 1 | 20 |
| Bud | 5 | 2 | 30 |
| Root | 5 | 0 | 0 |
As shown in Table 3, the wrinkle optimum explant of leaf cuckoo is sepal, and emergence rate is up to 80%.
Embodiment 4
In addition to induced medium is different from embodiment 1, remaining factor is all the same.
Induced medium: using 1/2WPM culture medium as minimal medium, ZT, α-NAA and caseinhydrolysate three are selected
Minimal medium and three plant growth regulators are formed the training of following 7 various concentration levels by plant growth regulators
Feeding base is tested, and detailed experimental protocols are as shown in table 4:
The experimental program of the wrinkle leaf cuckoo induced medium of table 4
After inoculation 30 days, wrinkle leaf cuckoo emergence in various culture mediums is counted, the results are shown in Table 5 for specific experiment:
There is callus situation experimental result in the wrinkle leaf cuckoo different culture medium of table 5
As shown in Table 5, it can reach highest emergence rate using induced medium of the present invention, can reach 80%.
It is easy to operate the present invention provides a kind of sterile seedling fast reproducing method of wrinkle leaf cuckoo, and emergence rate can be made to be up to
80%, solve the problems, such as presently, there are wrinkle leaf cuckoo reproductive efficiency it is low.
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 (7)
1. a kind of sterile seedling fast reproducing method of wrinkle leaf cuckoo, which comprises the following steps: with the tender tissue for the leaf cuckoo that wrinkles
It for explant, will be inoculated in induced medium after explant disinfection, Fiber differentiation 30d or more, the leaf cuckoo that must wrinkle is sterile
Seedling；The induced medium is using 1/2WPM as minimal medium, further includes: 1.5mg/L ZT, 1.0mg/L NAA, 50mg/L water
Solve casein, 30g/L sucrose and 8g/L agar powder, pH value 5.0.
2. method according to claim 1, which is characterized in that the tender tissue includes petal, sepal, stem section or bud.
3. method according to claim 2, which is characterized in that further include respectively with certainly when making explant with the stem section
Water rinses after the stem section 4h segment for being cut into 1~2cm long.
4. method according to claim 2, which is characterized in that further include using tap water when using the petal as explant
4h is rinsed, the outer layer sepal of cuckoo petal is peelled off, the petal for peelling off sepal is cut into the segment of 1~2cm long.
5. method according to claim 4, which is characterized in that further include described by what is peeled when using sepal as explant
Outer layer sepal distilled water immersion 5h.
6. method according to claim 1, which is characterized in that the method for the disinfection includes impregnating institute with 0.1% mercuric chloride
State 10~15min of explant.
7. method according to claim 1, which is characterized in that the light application time of the Fiber differentiation is 14h/d, intensity of illumination
For 1500lx；The temperature of the Fiber differentiation is 22 ± 2 DEG C.
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Citations (4)
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| CN102919125A (en) * | 2012-11-13 | 2013-02-13 | 云南省农业科学院花卉研究所 | Method for building efficient regeneration system of Yunnan rhododendron |
| CN103039367A (en) * | 2013-01-18 | 2013-04-17 | 通化师范学院 | Basal culture medium formula suitable for azalea tissue culture |
| CN103636492A (en) * | 2013-11-14 | 2014-03-19 | 石家庄市农林科学研究院 | Rhododendron hybrides cosmopolitan tissue culture rapid propagation method |
| CN107079815A (en) * | 2017-05-25 | 2017-08-22 | 贵州师范大学 | A kind of method for inducing charming cuckoo and Rhododendron delavayi cenospecies callus |
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2019
- 2019-05-07 CN CN201910375533.0A patent/CN109937885A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102919125A (en) * | 2012-11-13 | 2013-02-13 | 云南省农业科学院花卉研究所 | Method for building efficient regeneration system of Yunnan rhododendron |
| CN103039367A (en) * | 2013-01-18 | 2013-04-17 | 通化师范学院 | Basal culture medium formula suitable for azalea tissue culture |
| CN103636492A (en) * | 2013-11-14 | 2014-03-19 | 石家庄市农林科学研究院 | Rhododendron hybrides cosmopolitan tissue culture rapid propagation method |
| CN107079815A (en) * | 2017-05-25 | 2017-08-22 | 贵州师范大学 | A kind of method for inducing charming cuckoo and Rhododendron delavayi cenospecies callus |
Non-Patent Citations (4)
| Title |
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| 汪玲敏等: "云南杜鹃(Rhododendron yunnanense Franch.)无菌叶片高频再生体系的建立", 《分子植物育种》 * |
| 顾地周等: "牛皮杜鹃一步成苗技术", 《林业实用技术》 * |
| 顾地周等: "短果杜鹃组培快繁及其种质试管保存培养基的筛选1）", 《东北林业大学学报》 * |
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