CN106613976A - Method for carrying out detoxification and rapid propagation by virtue of garlic rachis and application of method - Google Patents
Method for carrying out detoxification and rapid propagation by virtue of garlic rachis and application of method Download PDFInfo
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- CN106613976A CN106613976A CN201611163996.3A CN201611163996A CN106613976A CN 106613976 A CN106613976 A CN 106613976A CN 201611163996 A CN201611163996 A CN 201611163996A CN 106613976 A CN106613976 A CN 106613976A
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- 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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- 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 method for carrying out detoxification and rapid propagation by virtue of a garlic rachis. The method mainly comprises the following steps: inducing adventitious buds into test tube bulbs by virtue of garlic rachises as an explant, carrying out low-temperature domestication on the test tube bulbs, and planting the test tube bulbs into soil, so as to obtain a complete system of a detoxified seed garlic, wherein the number of the adventitious buds induced by each garlic rachis can reach 11, a culture medium for inducing the test tube bulbs is MS+120g.L<1>soft sugar+6.8g.L<1>agar, and pH is 7.5. According to the method, the garlic rachises of Baodi garlic-''Liubanhong'' are taken as the explant, a complete garlic detoxification and rapid propagation method is constructed through technical links of high-frequency induction of the test tube bulbs, the low-temperature domestication of the test tube bulbs, the optimization of planting conditions and the like, the induction rate of the test tube bulbs induced by the adventitious buds can be as high as 98.78%, the number of the adventitious buds induced by each garlic rachis can be as high as 10.64, and the propagation coefficient is increased by above two times relative to that of a direct planting manner of the bulbs; technical supports are provided for the improvement of the detoxification and rapid propagation efficiencies of the garlic, the saving of the cultivation time and the lowering of the production cost.
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 fast numerous method, more specifically
Say it is to build a kind of new method that detoxifying fast breeding is carried out with garlic rachis.
Background technology
Garlic (Allium sativum L.) it is Liliaceae allium annual plant, can not be formed more than garlic cultivar
Seed, generally using vegetative propagation.This is just restricted the bio-diversity and hereditary capacity of garlic, causes serious kind
Degenerate, have a strong impact on the yield and quality of garlic.Long-term vegetative propagation also results in the continuous accumulation of Garlic Virus, has a strong impact on big
The growth of garlic plant and the commodity value of bulb.Currently, many times of Plant Tissue Breeding, induced mutations, genetic engineering and garlic
The application of the technologies such as physical culture kind, largely improves the seed selection work of garlic improved seeds.Wherein Plant Tissue Breeding skill
Art as a kind of effective detoxifying fast breeding technique, into garlic breeding research focus.
In the Plant Tissue Breeding research of garlic, the different explants of garlic such as, stem apex, the tip of a root, the tender leaf of children, storage
Hide leaf, phyllopodium, immature rachis, bulb and aerosol index etc. to be all applied to be used to induce in tissue cultures
With regeneration.Adventitious bud proliferation approach is directly to induce the adventitious bud hidden in explant body to sprout, with stabilization characteristics of genetics, mutation
Rate is low, the advantage such as the recovery time is shorter.At present, from stem apex and stem disk more than garlic adventitious bud proliferation mode（Deng for explant, its
Breeding coefficient is averagely between 3 to 7.Rachis is the reproductive organs of garlic, is in growth top, and viral level is relatively low, and it is certainly
So detoxification efficiency can reach 77.6%；Petal base portion floral disc has the potentiality of very high evoking adventive bud, Wang Xiuzhi etc. with big garlic flower
Sequence axle is explant, and evoking adventive bud, each rachis adventitious bud sum can reach 19.2.It can be seen that garlic rachis can be with
As the important explant of garlic detoxifying fast breeding.
However, although many researchers obtain more adventitious bud, but the successful related report of detoxification kind garlic high-efficient culture
Road is more rare.Because the induction of high-frequency test tube seedling is only one of speed limit condition of detoxification kind garlic production, how to ensure this
It is another essential condition that a little rooting of vitro seedling, transplant survival further obtain detoxification kind garlic.Many researchers adopt cultured in vitro
Technology obtains a large amount of regeneration plants from callus, but in root induction and transplants large losses plant in link, reduces
Reproductive efficiency, and take time and effort, it is restricted in application aspect.There is researcher to pass through the transplanting of optimization garlic regeneration plant
Condition, improves its survival rate, but plant domestication is difficult, and effect is not satisfactory.Xiong Zhengqin etc. is by test tube seedling induced synthesis test tube squama
Stem, its transplanting survival rate can be significantly improved.Additionally, test tube bulbs be easy to store, convenient transport, with higher resistance and nothing
The advantage that need to be tamed, the mode for inducing into test tube bulbs is more applicable for Garlic Tissue culture.And there are some researches show, test tube squama
Stem is similar to the plantation of Common garlic garlic clove, through its rest period, just can directly plant and normal growth.
The present invention is matched somebody with somebody using the rachis of Baodi garlic " six lobes are red " as explant by optimizing hormone kind and concentration
Than, obtain efficient evoking adventive bud culture medium, zygotic induction test tube bulbs, Cultural practices are improved, construct from explant to de-
The complete method of seed culture of viruses garlic production, the system be garlic detoxification, it is fast it is numerous, keep excellent genetic stability, improve bulb yield and
Quality etc. is there is provided important technical basis.
The content of the invention
It is an object of the invention to disclose a kind of method for carrying out detoxifying fast breeding with garlic rachis, it is by as follows
Step is carried out:
（1）Rachis evoking adventive bud：Baodi garlic " six lobes are red " is selected, when garlic enters reproductive stage, its scape：False stem
During=1-1.2, pluck the rachis of garlic and preserve two weeks in 4 DEG C.To be placed in originally Jing the rachis after low-temperature storage two weeks
30 min, 0.1% HgCl are rinsed under water2 Sterilize 20 min, distilled water flushing 3-4 time, then with 75% the min of alcohol disinfecting 10,
Distilled water flushing 3-4 time.The flower primordium residue and film quality bract of outer layer bract and surface degradation are peeled, and removes scape portion
Point, rachis is accessed in adventitious bud induction culture base, 2 rachises of per bottle of access.The culture medium of evoking adventive bud is: MS+
2.0 mg L-1 KT+0.1 mg L-1 NAA+ 30.0 g· L-1 The g L of sucrose+7.5-1Agar, pH=6.2.Each inflorescence
The adventitious bud number of axle induction can reach 11；The inductivity of adventitious bud inducing test tube bulbs can reach more than 90%
98.78%, and the test tube bulbs number of each rachis induction can reach 10.64, its breeding coefficient is direct relative to bulb
Plant high more than 2 times；
（2）Adventitious bud inducing test tube bulbs：After rachis grows six weeks, when the adventitious bud of formation highly reaches 3-5cm, connect
Enter and induce in inducing culture test tube bulbs, described inducing culture refers to test tube bulbs inducing culture：MS+120
g· L-1The g L of soft white sugar+6.8-1Agar, pH=7.5, its detailed method is：When the adventitious bud growth six of rachis induction
Zhou Hou, when the adventitious bud of formation highly reaches 3-5cm, is accessed in another test tube bulbs inducing culture and is induced test tube bulbs；
（3）The results of test tube bulbs and storage：After adventitious bud induces six weeks in the culture medium of induction test tube bulbs, test tube bulbs
Maturation, is drawn off and washes away culture medium, dries at lucifuge, after drying, plants after preserving 3 weeks at 4 DEG C.
（4）Regeneration plant and again bulb bearing acquisition：After test tube bulbs spend rest period, plant in by the end of October, overlay film.The
In May, 2, film is opened, and carry out weeding, work of watering and loosen the soil.In late June, tissue culture garlic is ripe and harvests；
（5）Tissue culture Garlic Virus are detected and chromosome detection：Using Enzyme-multiplied immune technique and root tip cell chromosome tabletting technology
Tissue culture garlic detoxification efficiency and chromosomal variation situation are detected respectively, tissue culture garlic reaches part detoxification efficiency, do not observe dye
Colour solid anomaly.
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：KNO3 1900mg,NH4NO3 1650mg, MgSO4∙7H2O 370 mg, KH2PO4
170 mg, CaCl2∙2H2O 440 mg, MnSO4∙4H2O 22.3 mg, ZnSO4∙7H2O 8.6 mg, H3BO3 6.2 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∙7H2The mg of O 27.8, the mg of glycine 2.0, the mg of thiamine hydrochloride 0.4, pyridoxine hydrochloride
0.5 mg, the mg of nicotinic acid 0.5, the mg of inositol 100.
The present invention further discloses detoxifying fast breeding method is carried out with garlic rachis is improving garlic detoxifying fast breeding efficiency
The application of aspect, experimental result shows：The method of the present invention is effective, can shorten the garlic production of hybrid seeds time, increases line of breeding
Number, due to without dedifferentiation and atomization again, can to greatest extent ensure that the genetic stability of excellent garlic cultivar.
The present invention is constructed from explant selection, rachis adventitious bud inducing, adventitious bud with garlic rachis as explant
Induction test tube bulbs, test tube bulbs breaking dormancy, field production plantation, detoxification efficiency detection, genetic stability detection, until de-
The complete method that seed culture of viruses garlic is obtained.
Below the present invention is with fast numerous as representative, the more specifically bright embodiment of typical Tianjin Baodi garlic：
1st, rachis evoking adventive bud optimal medium screening：Garlic used by the present invention be Baodi garlic " six lobes are red ", material
In coming from Tianjin Normal University's biotechnology garden.Select the Baodi garlic " six lobes are red " planted by the end of October in First Year, garlic
During into reproductive stage, its scape：During false stem=1-1.2, pluck the rachis of garlic and preserve two weeks in 4 DEG C.Jing is low
Rachis after temperature storage two weeks is placed under running water and rinses 30 min, 0.1% HgCl2 Sterilize 20 min, distilled water flushing 3-4
It is secondary, then with 75% the min of alcohol disinfecting 10, distilled water flushing 3-4 time.Peel the flower primordium residual of outer layer bract and surface degradation
Thing and film quality bract, and scape part is removed, rachis is accessed in screening and culturing medium, 2 rachises of per bottle of access.Screening training
Foster base adds variety classes with MS as minimal medium（KT, 6-BA and NAA）And the hormone of concentration, additional saccharose 30gL-1, agar 7.5gL-1, pH=6.2,121 DEG C (1.03 × 105 Pa) use after 20 min of sterilizing.It is below rachis induction
The screening and culturing medium of adventitious bud：
The hormone in medium proportioning of table 1
2nd, adventitious bud inducing test tube bulbs：After rachis grows six weeks, when the adventitious bud of formation highly reaches 3-5cm, connect
In entering another kind of solid medium（MS+120 g· L-1The g L of soft white sugar+6.8-1Agar, pH=7.5）Induction test tube bulbs,
Observe and record test tube bulbs upgrowth situation.
3rd, the results of test tube bulbs and storage：After adventitious bud induces six weeks in the culture medium of induction test tube bulbs, test tube
Bulb is ripe, is drawn off and washes away culture medium, dries at lucifuge, after drying, plants after preserving 3 weeks at 4 DEG C.
4th, the morphological observation of regeneration plant and bulb：After test tube bulbs spend rest period, plant in by the end of October, and cover
Film.In Second Year May, film is opened, and carry out weeding, work of watering and loosen the soil.In late June, garlic is ripe and harvests.
Measurement bulb diameter, is divided into three groups, ten data of each group of measurement, by obtain three by garlic bulb according to large, medium and small standard
Ten data carry out averagely, obtaining the average diameter size of test tube bulbs.
5th, Viral diagnosis：The DAS-ELISA detection kits of ELISA reagent OYDV, LYSV and GMV are purchased from the U.S.
Agdia companies.Detection antibody used, enzyme labelled antibody, 4-NPP（PNP）Substrate, positive control, GEB samples are carried
Take buffer solution, polyethylene micropore plate and be purchased from Agdia companies, enzyme conjugates is alkaline phosphatase（AP）The OYDV of mark, LYSV,
GMV3 kind antiviral antibodies, negative control is GEB Extraction buffers, and concrete detection method is with reference to Agdia companies specification.With dual anti-
Body sandwich ELISA method detects the detoxification efficiency of garlic, and, with reference to kit specification, each sample is in triplicate for method.
6th, regeneration plant chromosome detection：The garlic rachis test tube bulbs regeneration plant tip of a root is chosen, is fixed with fixer
After 24h, with distilled water flushing 3-4 time, it is put into 70% ethanol and is preserved.The tip of a root for preserving is chosen, distilled water flushing 3- is used
5 times, add 1M HCl that 8 min are hydrolyzed in 60 DEG C of water-baths, Root apical meristem area is placed in load glass by distilled water flushing 3-5 time
Piece central authorities, are added dropwise 2-3 drop carbolfuchsin solution, dye 8min, and with filter paper unnecessary dye liquor is sucked, and examine under a microscope the tip of a root point
Raw tissue area chromosome morphology.
It is positive that the method for carrying out detoxifying fast breeding with garlic rachis disclosed by the invention has compared with prior art
Effect is：
（1）The core of the method is by high-frequency induction test tube bulbs, test tube bulbs low temperature vernalization, facility cultivation and then is realizing
Detoxification kind garlic is produced.Tradition utilizes stem apex detoxifying fast breeding, needs through dedifferentiation, breaks up again, induce test tube seedling, test tube seedling to give birth to
The complicated processes such as root, acclimatization and transplantses, lasting 2 years could obtain detoxification kind garlic.The method that this laboratory builds, substantially increases
Garlic detoxifying fast breeding efficiency, saves the cultivation time, reduces production cost.
（2）Garlic rachis is in garlic plant strain growth top, and itself band poison amount is few, and is directly induced by rachis
Adventitious bud, not through dedifferentiation and atomization again, plant genetic stability is protected.
（3）Process two weeks at 4 DEG C, break rachis rest period, advantageously in adventitious bud inducing.
（4）Present invention optimizes links, construct from explant to detoxification kind garlic and tissue culture garlic detoxification efficiency and
The complete method of genetic stability detection, the method is at home and abroad there is not yet relevant report.
Description of the drawings
Fig. 1 is garlic rachis adventitious bud inducing, and wherein a. rachises have just accessed culture medium, b-f. rachises growth about 1
Upgrowth situation when week, 2 weeks, 3 weeks, 4 weeks and 6 weeks；
Fig. 2 is garlic titbit axle adventitious bud inducing test tube bulbs, and wherein a. adventitious buds are accessed in induction test tube bulbs culture medium, b-
F. the adventitious bud inducing test tube bulbs upgrowth situation of 1 week, 2 weeks, 3 weeks, 4 weeks and 6 weeks；
Fig. 3 is the adventitious bud to be formed sum and the test tube bulbs number sum correlation analysis for being formed；
Fig. 4 is the outward appearance of test tube bulbs, size and internal anatomy, the test tube bulbs of the cluster that wherein a-b. is harvested from blake bottle
C. all test tube bulbs for harvesting, d. compares Baodi garlic clove and is dissecting Microscopic observation test tube bulbs with test tube bulbs size e-f.
Growing point（Arrow indication）（Figure medium scale is 1.9cm）；
Fig. 5 is Through Bulblet Formation In Garlic gained regeneration plant and the form of bulb, wherein a. regeneration plants growing state in the ground,
The form of regeneration plant when b. harvesting bulb, the only head bulb d. point of lobe bulb e. diauxic growth bulb of c.（Figure medium scale is
1.9cm）；
Fig. 6 is detected for the chromosome in garlic titbit axle test tube bulbs regeneration plant Root apical meristem area.
Specific embodiment
With reference to the embodiment explanation present invention, the scheme of embodiment described here does not limit the present invention, this area it is special
Industry personnel can make improvements and change according to the spirit of the present invention, 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 are commercially available.
Embodiment 1
（1）Rachis evoking adventive bud：Baodi garlic " six lobes are red " is selected, when garlic enters reproductive stage, its scape：False stem
During=1-1.2, pluck the rachis of garlic and preserve two weeks in 4 DEG C.To be placed in originally Jing the rachis after low-temperature storage two weeks
30 min, 0.1% HgCl are rinsed under water2 Sterilize 20 min, distilled water flushing 3 times, then with 75% the min of alcohol disinfecting 10, steam
Distilled water is rinsed 3 times.The flower primordium residue and film quality bract of outer layer bract and surface degradation are peeled, and removes scape part, will
Rachis is accessed in adventitious bud induction culture base, 2 rachises of per bottle of access.The culture medium of evoking adventive bud is: MS+2.0
mg L-1 KT+0.1 mg L-1 NAA+ 30.0 g· L-1 The g L of sucrose+7.5-1Agar, pH=6.2.Each rachis is lured
The adventitious bud number led can reach 11；The inductivity of adventitious bud inducing test tube bulbs can reach more than 90%
98.78%, and the test tube bulbs number of each rachis induction can reach 10.64, its breeding coefficient is direct relative to bulb
Plant high more than 2 times；
（2）Adventitious bud inducing test tube bulbs：After rachis grows six weeks, when the adventitious bud of formation highly reaches 3-5cm, connect
Enter and induce in inducing culture test tube bulbs, described inducing culture refers to test tube bulbs inducing culture：MS+120
g· L-1The g L of soft white sugar+6.8-1Agar, pH=7.5, its detailed method is：When the adventitious bud growth six of rachis induction
Zhou Hou, when the adventitious bud of formation highly reaches 3-5cm, is accessed in another test tube bulbs inducing culture and is induced test tube bulbs.
（3）The results of test tube bulbs and storage：After adventitious bud induces six weeks in the culture medium of induction test tube bulbs, test tube
Bulb is ripe, is drawn off and washes away culture medium, dries at lucifuge, after drying, plants after preserving 3 weeks at 4 DEG C.
（4）Regeneration plant and again bulb bearing acquisition：After test tube bulbs spend rest period, plant in by the end of October, overlay film.The
In May, 2, film is opened, and carry out weeding, work of watering and loosen the soil.In late June, tissue culture garlic is ripe and harvests；
（5）Tissue culture Garlic Virus are detected and chromosome detection：Using Enzyme-multiplied immune technique and root tip cell chromosome tabletting technology
Tissue culture garlic detoxification efficiency and chromosomal variation situation are detected respectively, tissue culture garlic reaches part detoxification efficiency, do not observe dye
Colour solid anomaly.
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：KNO3 1900mg,NH4NO3 1650mg, MgSO4∙7H2O 370 mg, KH2PO4
170 mg, CaCl2∙2H2O 440 mg, MnSO4∙4H2O 22.3 mg, ZnSO4∙7H2O 8.6 mg, H3BO3 6.2 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∙7H2The mg of O 27.8, the mg of glycine 2.0, the mg of thiamine hydrochloride 0.4, pyridoxine hydrochloride
0.5 mg, the mg of nicotinic acid 0.5, the mg of inositol 100.
Embodiment 2
1st, garlic rachis evoking adventive bud induction
Due to hormon species（KT, 6-BA and NAA）And concentration has a certain impact to adventitious shoot regeneration efficiency, we design
12 kinds of screening and culturing mediums, the garlic titbit axle after Cord blood two weeks is accessed in above-mentioned screening and culturing medium, counts adventitious bud
Inductivity, it is determined that optimal inducing culturing condition, its result is as shown in the table：
The hormon of table 2 is with the impact for comparing Baodi garlic rachis adventitious bud proliferation
From Table 2, it can be seen that in culture medium 1 to 6, the inductivity of culture medium 2 is higher, the adventitious bud of each rachis induction
Number reaches 6.12.It is higher without the culture medium inductivity of NAA when 6- BA concentration is identical, illustrate 6- during evoking adventive bud
Impacts of the BA than NAA is big.In culture medium 7 to 12,11 inductivity is higher, and the adventitious bud number of each rachis induction reaches
11.00, when KT concentration is identical, the culture medium inductivity for adding NAA is higher, shows KT with NAA collective effects rachis not
The induction of normal bud.And result finds from table, culture medium 7 to 12 is high than the inductivity of culture medium 1 to 6, illustrates that KT compares 6-BA
It is more suitable for the induction of garlic titbit axle adventitious bud, therefore, the best medium of garlic titbit axle evoking adventive bud is：Culture medium
11, i.e. MS+2.0 mg L-1 KT+0.1 mg L-1 NAA+ 30.0 g· L-1 The g L of sucrose+7.5-1Agar, pH=6.2,
Its inductivity reaches 11.
Fig. 1 is each link of evoking adventive bud on optimal inducing culture：Have no when just having accessed culture medium and substantially expand（Figure
1-a）.Through or so week, there is projection in its surface, and rachis substantially expands, and diced facets are thickened, and is formed translucent
Multiple Buds, great majority are grown on middle part and bottom, and dense growth（Fig. 1-b）.Adventitious bud continued propagation, after about two weeks,
Translucent accidental sport reaches 2 centimetres of length into peak green（Fig. 1-c）.After 4 weeks, intensive adventitious bud clump is formed（Fig. 1-
e）.After about 6 weeks, adventitious bud can be transplanted in induction test tube bulbs culture medium（Fig. 1-f）.
2nd, garlic rachis adventitious bud inducing test tube bulbs
Because numerous adventitious buds is grown thickly in same titbit axle base portion, it is difficult to ensure that each seedling can induce the root that grows directly from seeds and enter
Row is transplanted, and this research is by inducing test tube bulbs approach to improve reproductive efficiency.Induction test tube bulbs culture medium be：MS+
120.0 g· L-1 The g L of soft white sugar+6.8-1 Agar, pH=7.5.From fig. 2 it can be seen that after inducing about 1 week, no
Normal bud root starts to expand（Fig. 2-b）；After 2 weeks, test tube seedling occurs withered（Fig. 2-c）；When 3 weeks, bulb exocuticle gradually becomes
Purple（Fig. 2-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. 2-e）；After 6 weeks, test tube seedling is withered, and test tube bulbs are mature on the whole（Fig. 2-f）.Now, test tube bulbs are taken from blake bottle
Go out, wash away surface medium, dry in shady place.
Can see from table 3, the inductivity of adventitious bud inducing test tube bulbs can reach more than 90%
98.78%, and the test tube bulbs number of each rachis induction can reach 10.64, its breeding coefficient is direct relative to bulb
Plantation（Breeding coefficient is 5）It is high more than 2 times.From figure 3, it can be seen that the adventitious bud sum that garlic rachis is formed is tried with being formed
Pipe bulb sum is in notable positive correlation（p<0.05）.Therefore, when needing to obtain the test tube bulbs of high inductivity, evoking adventive bud
Then need to be grown on the higher culture medium of inductivity.
The garlic titbit axle adventitious bud inducing test tube bulbs of table 3
Note：Lowercase significant difference in 0.05 level, capitalization significant difference in 0.01 level.
The results of 3 test tube bulbs and storage
Garlic rachis induced synthesis test tube bulbs amount to 1260.A diameter of 6.65mm of larger test tube bulbs is medium big
Little a diameter of 4.23mm, and relatively small a diameter of 1.68mm.The test tube bulbs harvested from blake bottle are arranged into tufted,
25 are at most can reach per cluster（See Fig. 4-a-b）.Test tube bulbs exocuticle after drying is in purple（See Fig. 4-c）, it is big with Baodi
Garlic bulb exocuticle is consistent.Microscopic observation is being dissected, test tube bulbs all have growing point（See Fig. 4-e-f）, therefore, tiding over not
After the dormancy phase, transplant to ground, test tube bulbs can be with normal growth.
The morphology statistical analysis of 4 Through Bulblet Formation In Garlics gained regeneration plant and bulb
The regeneration plant quantity of Through Bulblet Formation In Garlic is 539 plants, and in the bulb of results, single clove garlic has 530 plants, is divided into having for two lobes
8 plants, 1 plant of pintongs.The maximum gauge of bulb is 22.14mm, and minimum diameter is 3.16mm, 1 plant of diauxic growth.
5 Viral diagnosis
Respectively DAS- is carried out with OYDV, LYSV and GMV kit to the regeneration plant blade as obtained by test tube bulbs plantation land for growing field crops
ELISA detections, with ELIASA 405nm absorbances are read, and are compared with negative control, positive control absorbance.Knot
Fruit shows, the infection of non-detoxification Baodi Garlic Virus than more serious, and for compound infection（It is shown in Table 4）.
As can be seen from Table 4, detect the above-mentioned three kinds of virus of infection, but virus to planting in non-detoxification Baodi garlic
Degree difference is infected in strain, and this is a kind of mode of Combined Infection.Non- detoxification garlic samples OYDV, LYSV and GMV testing result point
Not Wei 3.10,6.11 and 3.11 times of negative control, infection conditions are quite serious.This is probably to cause Garlic quality to decline,
One of the reason for breeding coefficient is reduced.Regeneration plant sample OYDV, LYSV and GMV detection after rachis detoxifying fast breeding approach
As a result 2.9,4.3 and 1.2 times of negative control are respectively, certain detoxification efficiency is reached.
The regeneration plant blade ELISA Viral diagnosis results of table 4
6 cytological observations
Garlic is eucaryotic organism, and the normal morphology and structure of chromosome ensure that the stability of gene genetic, observe garlic
The chromosome morphology and structure in rachis test tube bulbs regeneration plant Root apical meristem area, there is 16 chromosomes in cell, and
And form is normal, does not find chromosome abnormality phenomenon, therefore ensure that the genetic stability of garlic germplasm.
The garlic detoxifying fast breeding method that the present invention builds is as follows with the comparative test result of conventional method：
Contrast test：
Conclusion：Compared with conventional method, the present invention is constructed from explant selection, rachis with garlic rachis as explant
Adventitious bud inducing, adventitious bud inducing test tube bulbs, test tube bulbs breaking dormancy, field production plantation, detoxification efficiency detection, heredity
Detection of Stability, until detoxification kind garlic obtain complete method, save rooting of vitro seedling and transplanting, solve test tube seedling and survive
The low problem of rate.The system is garlic detoxification, fast numerous, the excellent genetic stability of holding, improves the offer such as bulb yield and quality
Important technical basis.
Claims (2)
1. a kind of method for carrying out detoxifying fast breeding with garlic rachis, it is characterised in that carry out by the steps：
（1）Rachis evoking adventive bud：Baodi garlic " six lobes are red " is selected, garlic enters reproductive stage, its scape：False stem=
During 1-1.2, the rachis for plucking garlic is simultaneously preserved two weeks in 4 DEG C, will be placed in originally Jing the rachis after low-temperature storage two weeks
Rinse 30 min under water, 0.1%（w/w） HgCl2 Sterilize 20 min, distilled water flushing 3-4 time, then with 75%（w/w）Alcohol disappear
10 min of poison, distilled water flushing 3-4 time；The flower primordium residue and film quality bract of outer layer bract and surface degradation are peeled, and is gone
Except scape part, rachis is accessed in adventitious bud induction culture base, 2 rachises of per bottle of access；The culture of evoking adventive bud
Base is: MS+2.0 mg L-1 KT+0.1 mg L-1 NAA+ 30.0 g· L-1 The g L of sucrose+7.5-1Agar, pH=
6.2；
（2）Adventitious bud inducing test tube bulbs：After rachis grows six weeks, when the adventitious bud of formation highly reaches 3-5cm, connect
Enter in test tube bulbs inducing culture and induce test tube bulbs；
（3）The results of test tube bulbs and storage：After adventitious bud induces six weeks in the culture medium of induction test tube bulbs, test tube bulbs
Maturation, is drawn off and washes away culture medium, dries at lucifuge, after drying, plants after preserving 3 weeks at 4 DEG C；
（4）Regeneration plant and again bulb bearing acquisition：After test tube bulbs spend rest period, plant in by the end of October, overlay film, Second Year
In May, film is opened, and carry out weeding, work of watering and loosen the soil, in late June, tissue culture garlic is ripe and harvests；
（5）Tissue culture Garlic Virus are detected and chromosome detection：Using Enzyme-multiplied immune technique and root tip cell chromosome tabletting technology
Tissue culture garlic detoxification efficiency and chromosomal variation situation are detected respectively.
2. described in claim 1 with garlic rachis carry out detoxifying fast breeding method improve garlic detoxifying fast breeding efficiency in terms of should
With.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107125135A (en) * | 2017-06-06 | 2017-09-05 | 南京农业大学 | A kind of method that utilization rachis efficiently induces garlic embryonic callus |
| 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 |
| CN111436372A (en) * | 2019-12-23 | 2020-07-24 | 山东农业大学 | Nutrient solution for inducing garlic aerial bulbs, method and application |
-
2016
- 2016-12-16 CN CN201611163996.3A patent/CN106613976B/en active Active
Non-Patent Citations (2)
| Title |
|---|
| 刘伟伟等: "大蒜花序轴离体再生体系的建立", 《北方园艺》 * |
| 张昌伟等: "太仓大蒜根尖离体培养直接诱导不定芽及其试管鳞茎的形成", 《植物生理学通讯》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107125135A (en) * | 2017-06-06 | 2017-09-05 | 南京农业大学 | A kind of method that utilization rachis efficiently induces garlic embryonic callus |
| CN107125135B (en) * | 2017-06-06 | 2020-08-04 | 南京农业大学 | Method for efficiently inducing garlic embryonic callus by using rachis |
| 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 |
| CN114403007A (en) * | 2017-07-17 | 2022-04-29 | 成都市农林科学院 | Culture medium group for garlic virus-free tissue culture, application thereof and method for rapidly obtaining virus-free garlic |
| CN111436372A (en) * | 2019-12-23 | 2020-07-24 | 山东农业大学 | Nutrient solution for inducing garlic aerial bulbs, method and application |
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