CN107494274A - A kind of stripping bud sterilization method of alpine rose explant - Google Patents
A kind of stripping bud sterilization method of alpine rose explant Download PDFInfo
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- CN107494274A CN107494274A CN201710947077.3A CN201710947077A CN107494274A CN 107494274 A CN107494274 A CN 107494274A CN 201710947077 A CN201710947077 A CN 201710947077A CN 107494274 A CN107494274 A CN 107494274A
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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
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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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Abstract
The present invention discloses a kind of stripping bud sterilization method of alpine rose explant.This method mainly includes：Remove and prefetch leaf bud, bud at the top of bud branch, promote leaf bud at the top of its branch, sprouting of lateral bud at petiole；Treat that branch grows 10~30 days on its maternal plant, at top leaf bud, petiole after sprouting of lateral bud, but when blade is not fully deployed, its leaf bud, lateral bud is taken to carry out disinfection, it is each before and after sterilization to carry out a stripping bud processing, that is, peel off the explant that 1~2 layer of blade of bract, sprout from outside to inside is stripping bud sterilization.The explant of gained stripping bud sterilization; its tissue culture pollution rate is not higher than 30%; bud induction survival rate is higher than 63%; the close a kind of alpine rose by fine hair and/or body of gland of body surface is broken; the difficult technical bottleneck of explant sterilization during its plant regeneration system is established; the speed that this kind of alpine rose plant regeneration system is established is accelerated, the protection for azalea basic research, seedling factorial praluction and Precious, Rare, Endangered species provides technical support.
Description
Technical field
The invention belongs to field of plant tissue culture technique, and in particular to close by fine hair or/and the high mountain of body of gland to body surface
A kind of sterilization method of cuckoo explant, it can be widely applied to plant regeneration in azalea basic research and seedling factorial praluction
The explant sterilizing part of Establishing.
Background technology
The foundation of efficient plant regeneration system is that azalea genetic engineering breeding, filial generation stably expand the basis such as numerous and ground
Study carefully, and azalea seedling factorial praluction, the technical foundation of Precious, Rare, Endangered species child care.Use above is required to azalea plant
Strain regenerating system for explant, is raised up seed consistent with maternal inheritance character with nutrition organs (such as bud and stem section) with holding
Property.However, the stem and blade surface of azalea major part species are close by fine hair or/and body of gland, such as Rhododendron delavayi
(Rhododendron delavayi), rhododendron bullatum (Rhododendron edgeworthii), thick leaf cuckoo
(Rhododendron pachyphyllum), spur cuckoo (Rhododendron sinogrande), typhon cuckoo
(Rhododendron excellens) etc., causes its plant regeneration to stop at explant sterilization, and plant regeneration system establishes speed
Degree is slow.The foundation of this kind of alpine rose plant regeneration system, it is conventional that stem apex and stem section are directly removed from maternal plant as explant
Body, 75% 0.1% mercury chloride of alcohol-pickled 30s~1min combinations, 15~25min of immersion or 2% sodium hypochlorite immersion 10~
20min, which combines 0.1% mercury chloride and soaks 15~25min, carries out explant sterilization, or as a result because sterilizing not thorough and pollution rate
High (70%~100%)；Because sterilizing excessively and death rate height (60%~100%), or even explant is dead, also has
Pollution.The trial of multiple explant sterilization, which need to often be carried out, can just obtain a small amount of free of contamination surviving explant.Explant sterilization into
The close bottleneck established by the alpine rose plant regeneration system of fine hair or/and body of gland of body surface.At present, although having with Rhododendron delavayi
Stem apex, stem section etc. species induce the report of plant regeneration for explant, but specific explant pollution rate and the death rate do not have
Conclusion is specified, Disinfection Effect is unknown could.
To solve this bottleneck problem, we are with the close Yunnan Wild cuckoo by fine hair or/and body of gland of stem and blade：Ma Ying
Cuckoo and thick leaf cuckoo and cultivar ' Polaris ' are material, from azalea growth rhythm, when being drawn materials by studying
Between, branch pretreatment, stripping bud method, sterilization method etc. to reducing the effect of explant pollution rate and the death rate, obtain one kind
The stripping bud sterilization method of alpine rose explant.
The content of the invention
To solve, body surface is close to sterilize pollution rate and death rate height, obstruction by the alpine rose explant of fine hair or/and body of gland
The bottleneck problem that its plant regeneration system is established, the present invention provide a kind of stripping bud sterilization method of azalea explant.This method
Comprise the following steps：
(1) pretreatment of bud branch is taken
4~May or 7~August, growth selection is healthy and strong, the full alpine rose branch of lateral bud, and it is raw on maternal plant to retain it
It is long, only remove leaf bud and/or selected branch top bud at the top of selected branch；
(2) bud and first time stripping bud are taken
Treat that step (1) branch after pretreatment grows 10~30 days on its maternal plant, and the branch at the top of leaf bud and/or
Sprouting of lateral bud at petiole, but when blade is not fully deployed, the side that will be sprouted at the branch upper top leaf bud and/or petiole sprouted
Bud is removed, and two layers of the blade peelled off the bract of leaf bud and/or lateral bud and then peelled off from outside to inside obtains sprout；
(3) sprout sterilizes
By sprout, with 0.25% washing powder water oscillation cleaning, afterwards after the washing powder water of clear water rinsed clean sprout surface, use
2% liquor natrii hypochloritis 6~8min of soaking disinfection, then with 0.1% mercuric chloride solution 8~12min of soaking disinfection, sprout is taken out,
The solution of residual is cleaned up with sterilized water；
(4) second of stripping bud
Sprout peels off its 1~2 layer of blade from outside to inside on superclean bench, cuts sprout base portion 0.2~0.5mm stems
Section obtains the alpine rose explant of stripping bud sterilization.
Compared with prior art, the beneficial effects of the invention are as follows：
The present invention is front and rear common by being pre-processed, being sterilized to the branch of bud to be taken from alpine rose growth rhythm
The technical combinations of stripping bud twice are carried out, cleaner explant is obtained, reduces thimerosal usage time, reduce thimerosal pair
The injury of explant, the Disinfection Effect achieved.The pollution rate of azalea sprout tissue culture through the inventive method sterilization is not high
In 30%, bud induction survival rate is higher than 63%, and it is close dirty by the alpine rose Explants In Tissue Culture of fine hair or/and body of gland to have broken body surface
The technical bottleneck that dye rate is high, survival rate is low, the speed that this kind of azalea plant regeneration system is established is accelerated, be azalea basis
The protection of research, seedling factorial praluction and Precious, Rare, Endangered species provides technical support.
Brief description of the drawings
Fig. 1：Leaf bud at the top of alpine rose branch.Arrow is signified for leaf bud at the top of alpine rose branch in Fig. 1.
Fig. 2：Bud at the top of alpine rose branch.Arrow is signified for bud at the top of alpine rose branch in Fig. 2.
Fig. 3：Take lateral bud that bud branch is sprouted after pre-processing and that blade is not deployed.It is to take bud branch that arrow is signified in Fig. 3
Lateral bud sprouted after pretreatment and not deployed blade.
Fig. 4：The sprout obtained through first time stripping bud removed on alpine rose branch.Scheme in Fig. 4 A be the bract that peels and
Blade, it is the sprout obtained through first time stripping bud that B is schemed in Fig. 4.
Fig. 5：Sprout through second of stripping bud after sterilization.
Fig. 6：The sprout that the alpine rose explant of stripping bud sterilization of the present invention survives in bud inducement cultivation base.Arrow in Fig. 6
Head meaning is the sprout that it is survived.
Embodiment
Following examples are conventional method without specified otherwise.
The inventive method of embodiment 1 sterilizes to the stripping bud of Rhododendron delavayi explant
(1) pretreatment of bud branch is taken
In April, growth selection is healthy and strong, the full Rhododendron delavayi branch of lateral bud, retains its branch and is grown on maternal plant, only gone
Leaf bud and/or branch top bud (are removing leaf bud and/or the Ma Ying at the top of the Rhododendron delavayi branch at the top of the branch
After bud at the top of cuckoo branch, the Rhododendron delavayi branch still continued growth on its maternal plant), to promote leaf bud at the top of its branch
Sprouting of lateral bud at sprouting and/or petiole (see Fig. 1, Fig. 2).
(2) bud and first time stripping bud are taken
Treat that step (1) Rhododendron delavayi branch after pretreatment grows 15 days on its maternal plant, and the pretreated branch
The top leaf bud of bar sprout and/or petiole at sprouting of lateral bud, but when the blade that enclose its top leaf bud is not fully deployed and/or bag
When the blade of its lateral bud is not fully deployed (see Fig. 3), at the leaf bud and/or petiole that will be sprouted at the top of the pretreated branch
The lateral bud of sprouting is removed, and after the bract for peelling off the leaf bud removed and/or lateral bud, then two layers of the blade peelled off from outside to inside obtains bud
Body (see Fig. 4).
(3) sprout sterilizes
The sprout obtained by step (2), it is that 0.25% washing powder water slight oscillatory cleans with mass fraction, clear water floats afterwards
Wash clean.Then sterilize, be first 2% liquor natrii hypochloritis soaking disinfection 8min with mass fraction, then with mass fraction be 0.1%
Mercuric chloride solution soaking disinfection 10min, after taking out sprout, the previous solu of residual is cleaned up with sterilized water, is sterilized
Sprout.
(4) second of stripping bud and inoculation
The sprout that will be sterilized through step (3) peels off its 1~2 layer of blade from outside to inside, cuts sprout on superclean bench
Base portion 0.2~0.5mm stem sections obtain the Rhododendron delavayi explant (see Fig. 5) of stripping bud sterilization, afterwards by the sterilization of obtained stripping bud
Routinely method for plant tissue culture is inoculated on conventional alpine rose bud inducement cultivation base and lured Rhododendron delavayi explant
Culture (see Fig. 6) is led, it is 30% that pollution rate is counted after two weeks, and induction survival rate is 63.33%.
The inventive method of embodiment 2 sterilizes to the stripping bud of thick leaf cuckoo explant
For embodiment 2 in addition to following measures are different, remaining measure is same as Example 1, repeats no more.
(1) pretreatment of bud branch is taken
The bud branch that takes of pretreatment is thick leaf cuckoo branch.
(2) bud and first time stripping bud are taken
Treat that step (1) thick leaf cuckoo branch after pretreatment grows 25 days on its maternal plant, by the step of embodiment 1 (2)
Operation take bud and carry out first time stripping bud.
(3) sprout sterilizes
Mass fraction be the time of 0.1% mercuric chloride solution soaking disinfection be 12min.
(4) second of stripping bud and inoculation
Cut sprout base portion 0.2~0.5mm stem sections and obtain the thick leaf cuckoo explant of stripping bud sterilization, the stripping that will be obtained afterwards
Routinely method for plant tissue culture is inoculated into conventional alpine rose bud inducement cultivation base to the thick leaf cuckoo explant of bud sterilization
Upper carry out Fiber differentiation, it is 20% that pollution rate is counted after two weeks, and induction survival rate is 70%.
Implement the inventive method of example 3 to sterilize the stripping bud of ' Polaris ' explant
Implement example 3 in addition to following measures are different, remaining measure is same as Example 1, repeats no more.
(1) pretreatment of bud branch is taken
The bud branch that takes of pretreatment is ' Polaris ' branch.
(2) bud and first time stripping bud are taken
Treat that step (1) ' Polaris ' branch after pretreatment grows 21 days on its maternal plant, by the step of embodiment 1 (2)
Operation take bud and carry out first time stripping bud.
(3) sprout sterilizes
Mass fraction be the time of 2% liquor natrii hypochloritis's soaking disinfection be 6min, the mercuric chloride solution of mass fraction 0.1%
The soaking disinfection time is 8min.
(4) second of stripping bud and inoculation
Cut sprout base portion 0.2~0.5mm stem sections and obtain ' Polaris ' explant of stripping bud sterilization, will obtain afterwards
Routinely method for plant tissue culture is inoculated into conventional alpine rose bud inducement cultivation to ' Polaris ' explant of stripping bud sterilization
Fiber differentiation is carried out on base, it is 23.33% that pollution rate is counted after two weeks, and induction survival rate is 63.33%.
Compare 1 conventional method
Key step is as follows：
(1) Rhododendron delavayi children tender stem point or robust growth, the full semi-lignified Rhododendron delavayi branch of lateral bud, by branch are selected
Bar is cut into the stem section of 1~2 lateral bud of band, is that the concussion of 0.25% washing powder water is cleaned and after rinsed clean ultra-clean with mass fraction
Sterilized on workbench.
(2) the Rhododendron delavayi stem section or stem apex cleaned up, it is 2% sodium hypochlorite soaking disinfection 12min with mass fraction
Afterwards, it is 0.1% mercuric chloride solution soaking disinfection 18min with mass fraction, is rinsed 3 times with clear water afterwards.
(3) Rhododendron delavayi stem section or Rhododendron delavayi stem apex after the completion of sterilizing are inoculated into conventional alpine rose bud induction training
Base is supported, it is 80% that pollution rate is counted after two weeks, and it is 13.33% that bud, which lures survival rate,.
Control 2
In addition to following measures are different, remaining method is identical with control 1, repeats no more for control 2.
Step (1) selects thick leaf cuckoo children tender stem point or robust growth, the full semi-lignified thickness leaf cuckoo branch of lateral bud.
The thick leaf cuckoo stem section or stem apex that step (2) cleans up, with 2% liquor natrii hypochloritis soaking disinfection 10min, use
0.1% mercuric chloride solution soaking disinfection 20min,
Thick leaf cuckoo stem section or thick leaf cuckoo stem apex after the completion of step (3) sterilization are inoculated into conventional alpine rose bud and lured
Culture medium is led, it is 76.67% that pollution rate is counted after two weeks, and it is 10% that bud, which lures survival rate,.
Control 3
In addition to following measures are different, remaining method is identical with control 1, repeats no more for control 3.
The young tender stem point of step (1) selection ' Polaris ' or robust growth, the full semi-lignified of lateral bud ' Polaris ' branch
Bar.
' Polaris ' stem section or stem apex that step (2) cleans up, with 2% liquor natrii hypochloritis soaking disinfection 10min,
With 0.1% mercuric chloride solution soaking disinfection 16min.
' Polaris ' stem section or ' Polaris ' stem apex after the completion of step (3) sterilization are inoculated into conventional alpine rose bud
Inducing culture, it is 70% that pollution rate is counted after two weeks, and it is 20% that bud, which lures survival rate,.
Above-mentioned each experiment Disinfection Effect relatively refers to table 1.
The alpine rose explant of 1 embodiment of table 1~3 is sterilized compared with the effect of control sterilization
Table 1 shows：Disappeared by the inventive method is close to body surface by the alpine rose explant of fine hair or/and body of gland progress stripping bud
After poison, routinely the pollution rate of alpine rose tissue culture is 20-30%, and bud induction survival rate is 63.33-70%, and prior art
After (control) sterilization by the alpine rose explant of fine hair or/and body of gland close to body surface, the dirt of alpine rose tissue culture routinely
Dye rate is 70-80%, and bud induction survival rate is 10-20%.The present invention is close to body surface by outside the alpine rose of fine hair or/and body of gland
The stripping bud sterilization method of the implant sterilization side by fine hair or/and the alpine rose explant of body of gland closeer to body surface than prior art
Method, its tissue culture pollution rate reduce 62.5-71.43%, and bud induction survival rate improves 250-533.3%, generates expectation not
The technique effect arrived.Compared with prior art section, the inventive method uses sodium hypochlorite on the premise of aforementioned techniques effect is obtained
Solution disinfection saving of time 4 minutes, with mercuric chloride solution disinfecting time has saved 6-8 minutes, when reducing thimerosal and using
Between, reduce injury of the thimerosal to explant, follow-up tissue-cultured seedling growth is had laid a good foundation, also improved simultaneously
Operating efficiency.
Claims (1)
1. a kind of stripping bud sterilization method of alpine rose explant, it is characterised in that comprise the following steps：
(1) pretreatment of bud branch is taken
4~May or 7~August, growth selection is healthy and strong, the full alpine rose branch of lateral bud, retains it and is grown on maternal plant, only
Remove leaf bud and/or selected branch top bud at the top of selected branch；
(2) bud and first time stripping bud are taken
Treat that step (1) branch after pretreatment grows 10~30 days on its maternal plant, and leaf bud and/or petiole at the top of the branch
Locate sprouting of lateral bud, but when blade is not fully deployed, the lateral bud sprouted at leaf bud and/or petiole that the branch upper top is sprouted is taken
Under, two layers of the blade peelled off the bract of leaf bud and/or lateral bud and then peelled off from outside to inside obtains sprout；
(3) sprout sterilizes
By sprout, with 0.25% washing powder water oscillation cleaning, afterwards after the washing powder water of clear water rinsed clean sprout surface, with 2%
Liquor natrii hypochloritis 6~8min of soaking disinfection, then with 0.1% mercuric chloride solution 8~12min of soaking disinfection, sprout is taken out, with nothing
Bacterium water cleans up the solution of residual；
(4) second of stripping bud
Sprout peels off its 1~2 layer of blade from outside to inside on superclean bench, cuts sprout base portion 0.2~0.5mm stem sections and obtains
To the alpine rose explant of stripping bud sterilization.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112450071A (en) * | 2020-11-25 | 2021-03-09 | 贵州大学 | Disinfection method of rhododendron lapponicum seed explant |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112450071A (en) * | 2020-11-25 | 2021-03-09 | 贵州大学 | Disinfection method of rhododendron lapponicum seed explant |
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