CN106489745A - A kind of garlic Root apical meristem regeneration method and application - Google Patents
A kind of garlic Root apical meristem regeneration method and application 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
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Abstract
The invention discloses a kind of garlic Root apical meristem regeneration method and application.Mainly using Baodi garlic root as explant evoked callus, inducing culture is：MS+1mg·L‑12,4‑D+1mg·L‑16‑BA+30.0g·L‑1Sucrose+7.5g L‑1Agar, pH=5.8, callus induction rate is up to 97.5%.Callus is broken up after twice again through subculture, and after differentiation 7 weeks, adventitious bud carries out the induction of test tube bulbs when growing to 5cm, test tube bulbs inducing culture is：MS+120g·L‑1Soft white sugar+6.8g L‑1Agar, pH=7.5.The method carries out detoxifying fast breeding for garlic callus approach and provides new technical method, the survival rate that improve plantlet in vitro by the regeneration of test tube bulbs approach.
Description
The present invention obtains Tianjin Urban Committee on Agriculture emphasis Funded Projects（201302030）；Tianjin Normal University's research for application and development
Fund key project is subsidized（52XK1210,52XK1505）.
Technical field
The invention belongs to agro-biological engineering technical field, is related to Garlic Tissue culture and method numerous soon, more specifically
Say it is to build a kind of garlic Root apical meristem regeneration method.
Background technology
How garlic is planted with garlic bulblet as seed in production as asexually propagated crop, and production cost is high, line of breeding
The problems such as low, the viral accumulation of number, kind sexual involution, has a strong impact on garlic production.Find numerous soon garlic, detoxification and blastogenesis to change
Good technology is extremely urgent.Multiple garlics numerous detoxification technology system soon is constructed in previous work, such as uses garlic spire, full lamina root
Point, stem apex, rachis etc. are that explant builds Garlic Tissue cultivating system；Zhang Suzhi etc. carries out callus using garlic stem disk
Induction, and obtain be suitable for callus proliferation differentiation culture medium；Luciani etc. is lured with Allium fistulosum stem tips as explant
Derive the stronger callus of power of regeneration；Zhang Changwei etc. then by the direct evoking adventive bud of the garlic tip of a root, obtains test tube seedling simultaneously
Test tube bulbs have successfully been obtained., in growth top, meristem cell division is fast for garlic Root apical meristem, and band poison amount is few
Not even band poison, and garlic clove can send out even tens roots several, and experiment is drawn materials conveniently, compared compared with Allium fistulosum stem tips, each
Garlic clove only one of which stem apex, the stem apex for callus induction need to peel off under anatomical lens, the degree of disinfection pair in later stage
Callus induction also has a certain impact.
Using after garlic garlic clove overall disinfection, root of hair under aseptic condition, with Root apical meristem as explant, leads to the present invention
High frequency evoked callus, regeneration induction adventitious bud and test tube bulbs are crossed, regeneration techniques system is built, gained test tube bulbs are equal
There is storage leaf, can directly be inoculated in land for growing field crops, it is easy to manage, the transplanting survival rate of test tube seedling can be significantly improved.
Content of the invention
It is an object of the invention to disclosing a kind of garlic Root apical meristem regeneration method, it is characterised in that by as follows
The step of carry out：
（1）With garlic Root apical meristem as explant evoked callus：
Choose running water after not having the garlic bulb of scab to peel off crust and rinse 30 min, then the ultra-clean work after ultraviolet sterilization
Make in platform with 0.1%（w/w）HgCl2Soak 20 min, sterile distilled water washs 2-3 time, 75%（w/w）Alcohol-pickled 10
Garlic bulb after is accessed and is taken root in sterile culture flask by min, sterile distilled water washing 3-5；When root length is to 2-3cm, cut
Garlic root, is drawn with dissecting needle is vertical, is accessed；Inducing culture is MS+1
mg·L-12,4-D+ 1 mg·L-16-BA+30.0 g· L-1+ 7.5 g L of sucrose-1Agar, pH=5.8；Callus
Inductivity is up to 97.5%；
（2）Garlic tip of a root callus subculture and break up again：
After garlic tip of a root callus grows 4 weeks on inducing culture, subculture can be carried out, during squamous subculture about 2 weeks, callus group
Knit granular sensation substantially, in light yellow, during squamous subculture 4 weeks, callus increment is obvious, and subculture is broken up twice afterwards again, after
Culture base is identical with inducing culture, and redifferential medium is：
MS+3 mg·L-16-BA+30g·L-1Sucrose+7.5g L-1Agar, pH=5.8；
（3）Test tube bulbs induction and results
During dedifferentiation culture 1 week, callus surface starts substantial amounts of green point occur, 2 weeks it is observed that green point constantly grows,
Adventitious bud is gradually formed, when 3 weeks, most of green point forms adventitious bud, 4 weeks adventitious bud length to about 2cm, adventitious bud growth after 7 weeks
Cluster life test tube seedling is moved into induction test tube bulbs culture medium by the long cluster life test tube seedling to 5-8 cm, and its culture medium is MS+120
g · L-1+ 6.8 g L of soft white sugar-1Agar, pH=7.5 are taken out after test tube bulbs maturation, after cleaning top layer culture medium
Dry in the shade, put 4 DEG C of refrigerator low-temperature storages.
The present invention further discloses garlic Root apical meristem regeneration method is in terms of tissue culture shoot survival percent is improved
Application.The results show：It is explant evoked callus with garlic sterilized root, inductivity is up to 97.5%；Callus regeneration
Up to 85%, the inductivity of test tube bulbs is up to more than 95% for rate.Environment is adapted to by the test tube bulbs of tissue culture approach induced development
Property strong, storage tolerance is easily survived.The callus that the present invention is induced by garlic Root apical meristem, regeneration induction plant is further
Test tube bulbs are obtained, these test tube bulbs are respectively provided with storage leaf, that is, are respectively provided with the potentiality of germination.De- for Garlic Tissue ways for training
Poison is numerous there is provided new technical method soon.
With garlic Root apical meristem as explant evoked callus, callus is lured the present invention through squamous subculture
Regeneration test tube seedling is led, test tube seedling directly induces into the test tube bulbs with sprouting ability, it is to avoid test tube seedling is through taking root, refining
The complicated processes such as seedling, transplanting, improve test tube shoot survival percent.Simultaneously compared with by Shoot-tip Culture approach, although the tip of a root and stem
Point has the effect of natural detoxification, but each garlic bulblet only one of which stem apex, for callus induction all in growing point
Stem apex need to peel off under anatomical lens, the degree of disinfection in later stage also has a certain impact to callus induction；And one big
Garlic clove can send several or tens roots, and garlic bulblet can be sprouted with carrying out the induction of root after overall disinfection again, test the side of drawing materials
Just, quantity of material is sufficient.
More detailed description of the present invention is as follows：
（1）Garlic Root apical meristem is obtained：
Choose running water after not having the garlic bulb of scab to peel off crust and rinse 30 min, then the ultra-clean work after ultraviolet sterilization
Make in platform with 0.1%（w/w）HgCl2Soak 20 min, sterile distilled water washs 2-3 time, 75%（w/w）Alcohol-pickled 10
Garlic bulb after is accessed and is taken root in sterile culture flask by min, sterile distilled water washing 3-5；
（2）With garlic Root apical meristem as explant evoked callus：
Treat（1）When the sterilized root length of middle culture is to 2-3cm, garlic root is cut, for callus induction.Inducing culture is
MS+1 mg·L-12,4-D+ 1 mg·L-16-BA+30.0 g· L-1+ 7.5 g L of sucrose-1Agar, pH=5.8 are lured
Conductance is up to 97.5%.Its specific method is as follows：Garlic root will be cut and manufacture wound will longitudinally be scratched with aseptic dissecting needle, access will be healed
Callus induction is carried out in injured tissue inducing culture.Through 1 week or so, garlic tip of a root meristem zone occurred expanding phenomenon
And it is changed into light brown；After about 2 weeks；Garlic tip of a root meristem zone persistently expands and is changed into light yellow；The garlic tip of a root point after 3 weeks
There is obvious institutional framework in raw tissue area, in light yellow translucent；After about 4 weeks, garlic Meristernatic zone has substantially
Block callus generate.
（3）Garlic tip of a root callus subculture and break up again：
After garlic tip of a root callus grows 4 weeks on inducing culture, subculture can be carried out.Concrete grammar is：Squamous subculture about 2
Zhou Shi, callus granular sensation are obvious, in light yellow.During squamous subculture about 4 weeks, callus increment is substantially.Subculture twice after
Broken up again.Subculture medium is identical with inducing culture, and redifferential medium is：MS+3 mg·L-16-BA+30g·L-1
Sucrose+7.5g L-1Agar, pH=5.8.
（4）Test tube bulbs induction and results：
During dedifferentiation culture 1 week or so, callus surface starts substantial amounts of green point occur, and about 2 weeks or so it is observed that green point
Constantly grow, gradually form adventitious bud, when 3 weeks or so, most of green point forms adventitious bud, and adventitious bud length is to about within 4 weeks or so
2cm, after 7 weeks, cluster life test tube seedling is moved into induction test tube bulbs culture medium by the cluster life test tube seedling of adventitious bud length to 5-8 cm, and which is trained
Foster base is+120 g L of MS-1+ 6.8 g L of soft white sugar-1Agar, pH=7.5 are taken out after test tube bulbs maturation, clearly
Dry in the shade after washing top layer culture medium, put 4 DEG C of refrigerator low-temperature storages.
NAA of the present invention is a- methyl α-naphthyl acetates（1-naphthlcetic acid）, abbreviation NAA；KT is kinetin
（Kinetin）, abbreviation KT；
Described MS minimal mediums formula：
Contain in per 1L minimal mediums：KNO31900mg, NH4NO31650mg, MgSO4∙7H2O 370 mg, KH2PO4
170 mg, CaCl2∙2H2O 440 mg, MnSO4∙4H2O 22.3 mg, ZnSO4∙7H2O 8.6 mg, H3BO36.2 mg,
KI 0.83 mg, Na2MO3∙2H2O 0.25 mg, CuSO4∙5H2O 0.025 mg, CoCl2∙6H2O 0.025 mg, Na2-
EDTA 37.7 mg, FeSO4∙7H227.8 mg of O, 2.0 mg of glycine, 0.4 mg of thiamine hydrochloride, pyridoxine hydrochloride
0.5 mg, 0.5 mg of nicotinic acid, 100 mg of inositol.
, more specifically bright embodiment numerous as representative soon with typical Tianjin Baodi garlic of the invention below：
（1）Garlic Root apical meristem is obtained：
Choosing does not have Baodi garlic " six lobes the are red " bulb of scab, and after peelling off crust, running water rinses 30 min, then in ultraviolet
With 0.1% in superclean bench after sterilizing（w/w）HgCl2Soak 20 min, sterile distilled water washs 2-3 time, 75%（w/
w）Alcohol-pickled 10 min, sterile distilled water washing 3-5 all over after, will garlic bulb access sterile culture flask in take root；
（2）With garlic Root apical meristem as explant evoked callus：
Treat（1）When the sterilized root length of middle culture is to 2-3cm, garlic root is cut with aseptic operation knife, will cut garlic root nothing
Bacterium dissecting needle longitudinally scratches manufacture wound, and accessing in callus inducing medium carries out callus induction.Inducing culture
For MS+1 mg L-12,4-D+ 1 mg·L-16-BA+30.0 g· L-1+ 7.5 g L of sucrose-1Agar, pH=5.8.
Through 1 week or so, expanding phenomenon and being changed into light brown occurred in garlic tip of a root meristem zone；Callus continued propagation, about 2
Zhou Hou, garlic tip of a root meristem zone persistently expand and are changed into light yellow；After 3 weeks, garlic tip of a root meristem zone occurs substantially
Institutional framework, in light yellow translucent；After about 4 weeks, there is significantly bulk callus life garlic Meristernatic zone
Into.Statistics callus induction rate（Induce the total radical % of callus radical/access culture medium）, up to 97.5%.
（3）Garlic tip of a root callus subculture and break up again：
After garlic tip of a root callus grows 4 weeks on inducing culture, subculture can be carried out.Subculture medium and inducing culture
Identical.During squamous subculture about 2 weeks, callus granular sensation substantially, in light yellow.During squamous subculture about 4 weeks, callus rises in value
Substantially.Subculture is inoculated into callus on redifferential medium afterwards twice and carries out induction again.Redifferential medium is：MS
+3 mg·L-16-BA+30g·L-1Sucrose+7.5g L-1Agar, pH=5.8.
（4）Test tube bulbs induction and results
After cultivating about 7 weeks on redifferential medium, long cluster gives birth to test tube seedling to adventitious bud, and the height of cluster life test tube seedling reaches
5-8 cm, you can cluster life test tube seedling is moved into induction test tube bulbs culture medium, its test tube bulbs inducing culture is MS+120
g · L-1+ 6.8 g L of soft white sugar-1Agar, pH=7.5, after about 6 weeks, 95% test tube seedling induces into test tube bulbs, will
Ripe test tube bulbs take out, and dry in the shade, put 4 DEG C of refrigerator low-temperature storages after cleaning top layer culture medium.
（5）Test tube bulbs sprout potentiality detection：
The test tube bulbs of results are carried out longitudinal dissection, which is observed and be whether there is storage leaf, the examination of storage leaf can be clearly observable
Pipe bulb has sprouts potentiality, can form regeneration plant in field planting.
The good effect that garlic Root apical meristem regeneration method disclosed by the invention has compared with prior art
It is：
（1）It is explant evoked callus with garlic Root apical meristem that the core of the method is, callus is through subculture
Culture, then test tube seedling is differentiated, test tube seedling induces into test tube bulbs.Callus induction rate up to 97.5%, callus regeneration rate
Up to 85%, the inductivity of test tube bulbs is up to more than 95%.
（2）Test tube bulbs by tissue culture approach induced development are strong to environmental suitability, and storage tolerance is easily survived.The present invention
The test tube bulbs of acquisition are respectively provided with storage leaf, that is, be respectively provided with the potentiality of germination.There is provided for Garlic Tissue ways for training detoxifying fast breeding
New technical method.
（3）The present invention with garlic Root apical meristem as explant evoked callus, callus through squamous subculture,
Regeneration induction test tube seedling, test tube seedling directly induce into the test tube bulbs with sprouting ability, it is to avoid test tube seedling is through taking root, refining
The complicated processes such as seedling, transplanting, improve test tube shoot survival percent.Simultaneously compared with by Shoot-tip Culture approach, although the tip of a root and stem
Point has the effect of natural detoxification, but each garlic bulblet only one of which stem apex, for callus induction all in growing point
Stem apex need to peel off under anatomical lens, the degree of disinfection in later stage also has a certain impact to callus induction；And one big
Garlic clove can send several or tens roots, and garlic bulblet can be sprouted with carrying out the induction of root after overall disinfection again, test the side of drawing materials
Just, quantity of material is sufficient.
（4）The present invention build with garlic Root apical meristem as explant, by callus induction, again break up, test tube
Bulb induction and test tube bulbs sprout the complete method of potentiality detection, and the method is at home and abroad there is not yet relevant report.
Description of the drawings
Fig. 1 is taken root for garlic Aquaponic；
Fig. 2 is garlic tip of a root callus induction；Wherein a. garlics root is accessed in screening and culturing medium, and b-e. garlic ape xifications are healed
Injured tissue 1 week, 2 weeks, 3 weeks, the upgrowth situation culture of 4 weeks；
Fig. 3 is dissection Microscopic observation garlic tip of a root evoked callus；Wherein a. garlics root is accessed in screening and culturing medium, b-e.
Garlic tip of a root evoked callus 1 week, 2 weeks, 3 weeks, the upgrowth situation culture of 4 weeks；
Fig. 4 breaks up and regeneration test tube seedling again for garlic tip of a root callus；Wherein a. callus accesses b- in subculture medium
C. callus subculture 2 weeks, 5 weeks d. subcultures access afterwards in differential medium twice e-i. differentiation culture 1 week, 2 weeks, 3 weeks, 4
Week, the test tube seedling regenerative process of 7 weeks；
Fig. 5 is regeneration induction seedling test tube bulbs；Wherein a. access induction test tube bulbs culture medium, b-f. be respectively induce 1 week, 2
Week, 3 weeks, 4 weeks, the test tube bulbs of 6 weeks；
Fig. 6 is test tube bulbs form and growing point；A. test tube bulbs b. test tube bulbs growing point（Position shown in arrow in figure）.
Specific embodiment
The present invention is described with reference to embodiment, the scheme of embodiment described here does not limit the present invention, this area special
Industry personnel can make improvements according to the spirit of the present invention and change, and described such modifications and variations are regarded as at this
In the range of invention, the scope of the present invention and essence are defined by the claims.NAA wherein used in the present invention, KT, sugarcane
Sugar, 10% antiformin, agar, the red garlic of six lobes are commercially available.
Embodiment 1
A kind of garlic Root apical meristem regeneration method：
（1）With garlic Root apical meristem as explant evoked callus：
Choose running water after not having the garlic bulb of scab to peel off crust and rinse 30 min, then the ultra-clean work after ultraviolet sterilization
Make in platform with 0.1%（w/w）HgCl2Soak 20 min, sterile distilled water washs 2 times, 75%（w/w）Alcohol-pickled 10
Min, after sterile distilled water washs 3 times, garlic bulb is accessed and is taken root in sterile culture flask；When root length is to 2cm, garlic is cut
Root, is drawn with dissecting needle is vertical, is accessed；Inducing culture is MS+1 mg
L-12,4-D+ 1 mg·L-16-BA+30.0 g· L-1+ 7.5 g L of sucrose-1Agar, pH=5.8；
（2）Garlic tip of a root callus subculture and break up again：
After garlic tip of a root callus grows 4 weeks on inducing culture, subculture can be carried out, during squamous subculture about 2 weeks, callus group
Knit granular sensation substantially, in light yellow, during squamous subculture 4 weeks, callus increment is obvious, and subculture is broken up twice afterwards again, after
Culture base is identical with inducing culture, and redifferential medium is：
MS+3 mg·L-16-BA+30g·L-1Sucrose+7.5g L-1Agar, pH=5.8；
（3）Test tube bulbs induction and results
During dedifferentiation culture 1 week, callus surface starts substantial amounts of green point occur, 2 weeks it is observed that green point constantly grows,
Adventitious bud is gradually formed, when 3 weeks, most of green point forms adventitious bud, 4 weeks adventitious bud length to about 2cm, adventitious bud growth after 7 weeks
Cluster life test tube seedling is moved into induction test tube bulbs culture medium by the long cluster life test tube seedling to 6cm, and its culture medium is+120 g of MS
· L-1+ 6.8 g L of soft white sugar-1Agar, pH=7.5 are taken out after test tube bulbs maturation, cloudy after cleaning top layer culture medium
Dry, put 4 DEG C of refrigerator low-temperature storages.
Embodiment 2
（1）Garlic Root apical meristem is obtained：
Choosing does not have Baodi garlic " six lobes the are red " bulb of scab, and after peelling off crust, running water rinses 30 min, then in ultraviolet
With 0.1% in superclean bench after sterilizing（w/w）HgCl2Soak 20 min, sterile distilled water washs 2-3 time, 75%（w/
w）Alcohol-pickled 10 min, sterile distilled water washing 3-5 all over after, will garlic bulb access sterile culture flask in take root；Garlic
When bulb has just been accessed in culture of rootage bottle, base portion has grown the root of 0.2-0.3cm（See Fig. 1-a）, cultivate 3 days in water
Afterwards, garlic root minister is to 2-3cm（See Fig. 1-b）, can access in callus induction culture medium.
（2）With garlic Root apical meristem as explant evoked callus
When garlic root minister is to 2-3cm, garlic root is cut, drawn with dissecting needle is vertical, being accessed in screening and culturing medium is carried out
Induction, and observe its induction situation.
The present invention devises 16 kinds of screening and culturing mediums with MS as minimal medium by adding hormon species and concentration
（It is shown in Table 1）, while additional saccharose 30g L-1, agar 7.5g L-1, pH=5.8,121 DEG C (1.03 × 105Pa) sterilizing 20
Use after min.Table 1 is garlic tip of a root callus induction screening and culturing medium：
1 screening and culturing medium hormone combination of table
Due to hormon species（2,4-D and 6-BA）And concentration has a certain impact to adventitious shoot regeneration efficiency, we design
16 kinds of screening and culturing mediums, access during by garlic root minister to 2-3cm in above-mentioned screening and culturing medium, access 40 in per bottle
Root, goes out callus bar number/40 with 2 time-of-weeks to calculate callus induction rate, counts the inductivity of Callus formation, really
Fixed optimal inducing culturing condition, its result are as shown in the table：
2 hormon of table is with the impact for comparing Baodi garlic apexification callus
From table 2 it can be seen that in culture medium 1 to 8, only 2,4-D, its inductivity is relatively low, and in culture medium 9 to 16,
Under the conditions of 2,4-D of same concentrations, its inductivity is high than 1 to No. 8, illustrates that 2,4-D is lured to the callus of the garlic tip of a root
Effect is led, but when 2,4-D and 6-BA interacts, its inductivity is higher.Therefore, induction garlic root is pointed into callus
Optimal medium be：No. 10 culture mediums, i.e.,：MS+1 mg·L-12,4-D+ 1 mg·L-16-BA+30.0 g· L-1Sugarcane
+ 7.5 g L of sugar-1Agar, pH=5.8, inductivity reach 97.5%.Fig. 1 is evoked callus on optimal inducing culture
Each link：Have no when just having accessed culture medium and substantially expand（Fig. 2-a）.Through 1 week or so, garlic tip of a root meristem zone occurred swollen
Big phenomenon is simultaneously changed into light brown（Fig. 2-b）.Callus continued propagation, after about 2 weeks, garlic tip of a root meristem zone continues swollen
Big and be changed into light yellow（Fig. 2-c）.After 3 weeks, there is obvious institutional framework in garlic tip of a root meristem zone, in light yellow semi-transparent
Bright state（Fig. 2-d）.After about 4 weeks, garlic Meristernatic zone has significantly bulk callus to generate（Fig. 2-e）.Final true
Healing rate highest culture medium is determined for MS+1 mg L-12,4-D+ 1 mg·L-16-BA+30.0 g· L-1Sucrose+7.5
g ·L-1Agar, pH=5.8.Tip of a root calli induction process under the anatomical lens that Fig. 3 shows：Dissecting Microscopic observation garlic
Apexification callus process is shown in Fig. 3, and when just accessing inducing culture, the garlic tip of a root is unchanged.In inducing culture
1 week or so the time of middle growth, garlic tip of a root meristem zone expands, and is faint yellow（Fig. 3-a）.2 weeks or so time, separate living tissue
Area persistently expands, and is light yellow, and be translucent state（Fig. 3-b）.After 3 weeks, there is obvious bulk tissue in meristem zone（Fig. 3-
d）.After 4 weeks, meristem zone's block structure increase can carry out squamous subculture（Fig. 3-e）.
（3）Garlic tip of a root callus subculture and break up again：
After the callus that garlic tip of a root meristem zone is formed grows 4 weeks on inducing culture, subculture can be carried out（Fig. 4-
a）.During squamous subculture about 2 weeks, callus granular sensation substantially, in light yellow（Fig. 4-b）.During squamous subculture about 4 weeks, callus group
Knit increment substantially（Fig. 4-c）.Subculture twice after, callus can be broken up（Fig. 4-d）, callus table when 1 week or so
Face starts substantial amounts of green point occur（Fig. 4-e）, it is observed that within about 2 weeks or so that green point constantly grows, and gradually forms adventitious bud（Figure
4-f）, when 3 weeks or so, most of green point forms adventitious bud（Fig. 4-g）, 4 weeks or so adventitious bud length to about 2cm（Fig. 4-h）, 7 weeks
Adventitious bud grows vigorous, about 5 cm or so afterwards, can be used to induce test tube bulbs（Fig. 4-i）.
（4）Test tube bulbs induction and results
After cultivating about 7 weeks on redifferential medium, long cluster gives birth to test tube seedling to adventitious bud, and the height of cluster life test tube seedling reaches
5-8 cm, you can cluster life test tube seedling is moved into induction test tube bulbs culture medium, the culture medium for inducing test tube bulbs is：MS+
120g· L-1Soft white sugar+6.8g L-1Agar, pH=7.5.From fig. 5, it is seen that after inducing about 1 week, test tube seedling root
Portion starts to expand（Fig. 5-b）；After 2 weeks, test tube seedling occurs withered（Fig. 5-c）；When 3 weeks, bulb exocuticle gradually becomes purple（Figure
5-d）；When 4 weeks, the test tube bulbs major part exocuticle of induced synthesis is changed into purple, and test tube seedling wilt phenomenon is serious（Fig. 5-e）；6
Zhou Hou, test tube seedling are withered, and test tube bulbs are mature on the whole（Fig. 5-f）.Now, test tube bulbs are taken out from blake bottle, washes away table
Face culture medium, dries in shady place.
（5）Test tube bulbs sprout potentiality detection：
The test tube bulbs of results are longitudinally cut open under anatomical lens, carries out anatomic observation, observe which and whether there is storage leaf, can be clear
The clear test tube bulbs for observing storage leaf have sprouts potentiality.Fig. 6 is test tube bulbs longitudinal direction internal anatomy, can be clearly observable life
Long point（Fig. 6-a-b）, showing that the test tube bulbs for harvesting can regenerate plant in field, this is that later regeneration plant is normal in field
Growth and character observation are laid a good foundation.
Claims (2)
1. a kind of garlic Root apical meristem regeneration method, it is characterised in that carry out by the steps：
（1）With garlic Root apical meristem as explant evoked callus：
Choose running water after not having the garlic bulb of scab to peel off crust and rinse 30 min, then the ultra-clean work after ultraviolet sterilization
Make in platform with 0.1%（w/w）HgCl2Soak 20 min, sterile distilled water washs 2-3 time, 75%（w/w）Alcohol-pickled 10
Garlic bulb after is accessed and is taken root in sterile culture flask by min, sterile distilled water washing 3-5；When root length is to 2-3cm, cut
Garlic root, is drawn with dissecting needle is vertical, is accessed；Inducing culture is MS+1
mg·L-12,4-D+ 1 mg·L-16-BA+30.0 g· L-1+ 7.5 g L of sucrose-1Agar, pH=5.8；
（2）Garlic tip of a root callus subculture and break up again：
After garlic tip of a root callus grows 4 weeks on inducing culture, subculture can be carried out, during squamous subculture about 2 weeks, callus group
Knit granular sensation substantially, in light yellow, during squamous subculture 4 weeks, callus increment is obvious, and subculture is broken up twice afterwards again, after
Culture base is identical with inducing culture, and redifferential medium is：
MS+3 mg·L-16-BA+30g·L-1Sucrose+7.5g L-1Agar, pH=5.8；
（3）Test tube bulbs induction and results
During dedifferentiation culture 1 week, callus surface starts substantial amounts of green point occur, 2 weeks it is observed that green point constantly grows,
Adventitious bud is gradually formed, when 3 weeks, most of green point forms adventitious bud, 4 weeks adventitious bud length to about 2cm, adventitious bud growth after 7 weeks
Cluster life test tube seedling is moved into induction test tube bulbs culture medium by the long cluster life test tube seedling to 5-8 cm, and its culture medium is MS+120
g · L-1+ 6.8 g L of soft white sugar-1Agar, pH=7.5 are taken out after test tube bulbs maturation, after cleaning top layer culture medium
Dry in the shade, put 4 DEG C of refrigerator low-temperature storages.
2. application of the garlic Root apical meristem regeneration method described in claim 1 in terms of tissue culture shoot survival percent is improved.
Priority Applications (1)
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| CN107278896A (en) * | 2017-07-17 | 2017-10-24 | 成都市农林科学院 | A kind of method of quick acquisition detoxification garlic |
| CN109526743A (en) * | 2018-12-28 | 2019-03-29 | 中国农业科学院蔬菜花卉研究所 | The fast numerous and stock breeding method of garlic callus |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107278896A (en) * | 2017-07-17 | 2017-10-24 | 成都市农林科学院 | A kind of method of quick acquisition detoxification garlic |
| CN109526743A (en) * | 2018-12-28 | 2019-03-29 | 中国农业科学院蔬菜花卉研究所 | The fast numerous and stock breeding method of garlic callus |
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