CN107125135A - A kind of method that utilization rachis efficiently induces garlic embryonic callus - Google Patents
A kind of method that utilization rachis efficiently induces garlic embryonic callus Download PDFInfo
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- CN107125135A CN107125135A CN201710417756.XA CN201710417756A CN107125135A CN 107125135 A CN107125135 A CN 107125135A CN 201710417756 A CN201710417756 A CN 201710417756A CN 107125135 A CN107125135 A CN 107125135A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
Abstract
The invention discloses a kind of method that utilization rachis efficiently induces garlic embryonic callus.This technology is first that explant induces somatic embryo using garlic rachis, and construct the optimization system that a large amount of garlic somatic embryos are obtained in a set of short time, it is extensive with explant materials, body embryo induced efficiency is high, quantity is more, speed is fast, structural integrity and the good advantage of genetic stability, in addition, somatic embryo suspend cultivating and can not only shorten cultivation cycle, set up rapid propagation system, it can also be breed improvement, transgene receptor and screening mutant etc. provide good vegetative propagation test system, artificial seed is made in body embryo, realize seedling large-scale production, application value and development prospect are wide.
Description
Technical field
The present invention relates to plant tissue culture technique, specific design one kind efficiently induces garlic embryo callus subculture using rachis
The method of tissue.
Background technology
Garlic (Allium sativum L.) alias garlic, giant garlic, are Liliaceae (Liliaceae) allium (Allium) two
Year raw herbaceous plant, mainly with tender leaf (garlic bolt or garlic sprouts), scape (garlic stems), bulb (garlic) for consumable products, nutritive value
It is high, it is one of long Important Economic crop of China's cultivation history, is also important foreign exchange earning vegetables.China's overwhelming majority
Cultivation garlic can not form seed, and vegetative propagation is carried out mainly by bulb, and its sterility is also caused to kind genetic improvement
Difficulty, is unfavorable for breeding of new variety, while long-term invisible breeding also causes kind of a sexual involution, cause the yield and quality of garlic by
Year declines.
Using tissue culture technique, the problem of being effectively improved kind of sexual involution greatly increases reproductive efficiency, and then improve
Garlic yield and quality.Garlic Tissue culture mainly has 2 kinds of approach：Adventitious organogenesis and somatic embryogenesis pathway.Compare
Adventitious organogenesis, have that quantity is more, speed fast by somatic embryogenesis pathway formation regeneration plant, structural integrity, heredity
The advantages of stability is strong, wherein somatic embryo, which suspend cultivating, can not only shorten cultivation cycle, sets up quick brood body
System, can also provide good vegetative propagation test system, by body embryo system for breed improvement, transgene receptor and screening mutant etc.
Into artificial seed, seedling large-scale production is realized, application value and development prospect are wide.
At present, garlic somatic embryo occur system generally existing both domestic and external the problem of body embryo occurrence frequency is low.China
A set of perfect embryonic callus induction technical system is not yet formed, body embryo is made a difference about kind and different explants
Research go deep into system not enough, the problems such as existing system has low body embryo occurrence frequency, recovery time length and high fast numerous cost
It is badly in need of establishing the regenerating system that a kind of efficient induced embryonic callus and body embryo occur.
The content of the invention
It is an object of the invention to provide a kind of method that utilization rachis efficiently induces garlic embryonic callus, solve existing
The problems such as having low callus induction rate present in technology, induction time length and high fast numerous cost.
The purpose of the present invention can be reached by following measures：
A kind of method that utilization rachis efficiently induces garlic embryonic callus, comprises the following steps：
Step (1)：In rachis upper surface diameter 0.3-0.5cm, the scape development relative altitude of garlic stems be scape height/
When false stem is highly 0.5-3.0, garlic stems is harvested successively, is then cut involucre climing section of 4-6cm of band, is immersed in saturation washing powder solution
Middle 25-35min, then rinses 25-35min with flowing water；
Step (2)：By the material fully rinsed on superclean bench, with 70-75% ethanol surface sterilization 60-90s,
After 2% liquor natrii hypochloritis's soaking disinfection 10-15min, with aseptic water washing 4-5 times, finally it is placed on aseptic filter paper and blots table
Face moisture carries out healing tissue inducting test；
Step (3)：Peel off sterile rachis outer layer bract, remove surface degradation flower primordium residue and film quality bract and
Scape part, is 4 pieces by cross rip cutting, is seeded in embryonic callus induction culture medium；
Step (4)：Select that yellow-white graininess, quality be hard and breakable embryo callus, accessed 20-
Suspend shaken cultivation in 40mL callus proliferation mediums, vibration rotating speed be 160-200r/min, per 6-8d change 1 time it is fresh
Culture medium；
Step (5)：Embryo callus of the same size is material after selection propagation, in addition 6.0-7.0gL-1Agar
Somatic embryo germination medium on cultivated, every bottle of inoculation 4-6g callus group, per 4-5 weeks subculture 1 time.
Rachis upper surface diameter 0.3-0.5cm, the scape development relative altitude (flower of garlic stems are taken in step (1) of the present invention
Stem height/false stem height) garlic rachis when being about 0.5-3.0 is as explant, and preferably surface diameter is 0.5cm and garlic stems
The rachis that scape development relative altitude is 1, from rachis, it goes out more time and inductivity and is significantly better than other explants
Type, and applicable variety is extensive.
During the sterilization treatment of step (2) rachis of the present invention, 70~75% alcohol surface disinfecting times are 60~90 seconds,
2% liquor natrii hypochloritis's processing time was 10-15 minutes, to ensure that sterilizing is thorough.The 90s it is preferred that 75% alcohol sterilizes, 2%
Sodium hypochlorite sterilizes 12 minutes.
After being sterilized in step (3) of the present invention, sterile rachis outer layer bract is peelled off, the flower primordium residual of surface degradation is removed
The parts such as thing, can promote the induction of rachis surface callus.
The minimal medium used in embryonic callus induction culture medium employed in step (3) of the present invention can be group
One of basal medium commonly used in training or its mixing, such as MS, B5Or MS+B5, but in order to obtain higher embryo callus
Inductivity, it is preferred to use B5Culture medium.One kind is more preferably matched：1 times of B5Culture medium, 2.0-3.0mgL-12,4-D,
0.3-0.6mg·L-1KT, 30-40gL-1Sucrose, 6.0-7.0gL-1Agar, pH5.8~6.2, more preferably proportioning are：1
Times B5Culture medium, 2.5mgL-12,4-D, 0.5mgL-1KT, 30gL-1Sucrose, 6.5gL-1Agar, pH5.8.Work as selection
2.5mg·L-12,4-D, 0.5mgL-1During KT, callus induction rate is higher.
Step (4) callus of the present invention culture of rising in value in liquid suspension culture can quickly increase weight, after four weeks, callus group
Rate of body weight gain is knitted up to 44.30%.
The proportioning of embryo callus proliferated culture medium employed in step (4) of the present invention is 1 times of B5 medium, 1.0-
1.5mg·L-16-BA, 0-0.5mgL-1KT, 20-40gL-1Sucrose, pH is 5.8-6.0 or 1 times of B5 medium,
1.0-1.5mg·L-16-BA, 0-0.1mgL-1NAA, 20-40gL-1Sucrose, pH is 5.8-6.0；It is preferred that proportioning
For 1.0mgL-16-BA, 0.5mgL-1KT, 30gL-1Sucrose, 6.5gL-1Agar, pH5.8, on this condition, rate of body weight gain
It is maximum.
23-27 DEG C is placed in step (4) of the present invention in embryo callus breeding, intensity of illumination is 50-200 μ
mol·m-2·s-1, photoperiod 12-14hd-1Cultivated under illumination condition.
Included in step (5) of the present invention in somatic embryo germination medium：6-BA concentration is 1.5-2.5mgL-1, KT
Concentration be 0.5-1.0mgL-1, proportioning preferably is 2.0mgL-16-BA, 0.5mgL-1KT.Every gram of callus is sprouted
Bud number up to 6.67.
The minimal medium used in somatic embryo germination medium in step (5) of the present invention can be to commonly use in tissue culture
One of basal medium or its mixing, such as MS, B5Or MS+B5, but in order to obtain higher germination rate, it is preferred to use MS culture mediums.
Further preferably include containing 25-35gL in somatic embryo germination medium described in step (5)-1Sucrose.
Beneficial effects of the present invention：
1st, draw materials extensive：It is first that explant induces rachis on somatic embryo, garlic garlic stems using garlic rachis
Typically not as edible organs, explant abundance further increases the utilization rate of garlic product organ.
2nd, somatic embryo inducement rate is high：4 kinds Callus induction rate be 88%-90%, wherein early with ' two water ' be
Explant highest, callus induction rate is up to 90.18%.
3rd, the recovery time is short：The present invention is explant using rachis, and the more time that averages out is 9d.
4th, genetic stability is strong：Compare with general callus, somatic embryo can keep preferable mother cell shape, become
Different coefficient is low.
5th, cultivation cycle is short：The time of the direct somatic embryos of rachis is most short only 9 days, and utilizes other explants
The incubation time of induction is up to more than 1 month, and what is had even needs 2-3 months；Culture week is substantially reduced using suspension culture
Phase.
6th, derived cost is low：Explant wide material sources, and the difference such as garlic clove, the harvesting of rachis are sold to garlic product organ
Less, body embryo inductivity is high for the influence sold, and the recovery time is short, greatly reduce tissue cultures to the controls such as temperature, illumination into
This.
7th, applicable variety is extensive：Be applicable to ' two water early ', ' Xuzhou is white ', ' first month of the lunar year is early ', ' Portugal's garlic ' and ' precocity
Garlic ' etc. a variety of kinds.
8th, somatic embryo suspend cultivating and can not only shorten cultivation cycle, set up rapid propagation system, can also be
Breed improvement, transgene receptor and screening mutant etc. provide good vegetative propagation test system, and body embryo is made into people's work post
Son, realizes seedling large-scale production, and application value and development prospect are wide.
Brief description of the drawings
Fig. 1 suspends culture to embryo callus proliferative effect；A. before breeding；B. after breeding；
The influence that Fig. 2 different hormone combinations are developed to garlic somatic embryo；A.6-BA+KT；B.6-BA+NAA；C.KT+
NAA；
Fig. 3 garlic somatic embryo occurs；A. strumae；B. loosely organized embryo callus；C. globular embryo, peltate
Embryo；D. mature embryo；
The embryo callus form of Fig. 4 garlics rachis formation；A. the white formation in bottom；B. projection is expanded at top；
C.D.E. ' two water are early ' embryo callus；F. hairy root；
Fig. 5 garlic somatic embryo sprouting situations；A. body embryo center greening；B.C. sprout root and bud at green point；D. it is normal
Seedling.
Embodiment
By following examples, the present invention is described in further detail, but present disclosure is not limited thereto.
Embodiment 1：
In order to contrast influence of the different explant types to different cultivars garlic embryonic callus induction, test with inflorescence
Axle, plateau (with stem apex), the leaf base of test tube seedling and the tip of a root are material, and kind is that ' two water are early ', ' Xuzhou is white ', ' first month of the lunar year
Early ', ' Portugal's garlic ' and ' precocious garlic '.Rachis is peelled off into outer layer bract, the flower primordium residue and film of surface degradation is removed
Matter bract and scape part, are 4 pieces by cross rip cutting, are seeded on same culture dish；The garlic clove that sterilization treatment is crossed removes
Storage leaf and trophophyll, cut the false stem and blade at the 6mm of plateau top, cut the about l mm of stem tray bottom cork
Change tissue, obtain the stem disk (band stem apex) of about 5mm thicker strip germination phyllopodium, 4 are inoculated with per culture dish；The germination that test tube seedling is turned white
Leaf base is cut into about 0.5cm2Fritter, in the middle part of the vein it is crosscutting it is several under, blade back is inoculated into downwards callus inducing medium
On；The tip of a root is cut into 1cm segment, and leaf base and the tip of a root connect 10 respectively per culture dish.3 repetitions, per 10 trainings of repeated inoculation
Support ware.Growing state is observed daily and counts callus induction rate.
Callus induction rate (%)=generation callus explant number/(inoculation explant number-death toll-pollution
Number) × 100.
It the results are shown in Table 1 and table 2：Different garlic cultivars and materials position more have a significant impact to going out.Same kind is not
With more time and inductivity between explant, is gone out, there were significant differences.
The influence of the different cultivars of table 1 and explant to garlic embryonic callus induction
Note:Different letters represent the significant difference in 0.05 level in table.
As shown in table 1, the tip of a root and leaf base are early with ' two water ' go out faster and inductivity highest, next to that ' Xuzhou
In vain ', ' first month of the lunar year is early ', ' precocious garlic ', ' Portugal's garlic ' is most slow and inductivity is minimum；Plateau is then gone out faster with ' Xuzhou is white '
(16d), next to that ' precocious garlic ', ' first month of the lunar year is early ', ' two water morning ', ' Portugal's garlic ' is most slow, needs 30d, each other significant difference,
But most fast more time that goes out is also required at least 16 days above, and inductivity is early with ' two water ', ' Xuzhou is white ', ' first month of the lunar year is early ' it is higher,
With ' precocious garlic ' and ' Portugal's garlic ' significant difference, inductivity can reach 25.01%.
As shown in table 2, it is explant rachis of the present invention, early with ' two water ' and ' Xuzhou is white ' go out sooner, be
7-9d, ' first month of the lunar year is early ' and ' precocious garlic ' is 12-14d；Different cultivars inductivity is 90% or so, and it goes out more time and inductivity
Other explant types are significantly better than, and applicable variety is extensive.The water of garlic ' two is early ' rachis induction garlic somatic embryo is shown in
Fig. 3.
Influence of the different cultivars of table 2 to garlic rachis embryonic callus induction
Note:Different letters represent the significant difference in 0.05 level in table.
Embodiment 2：
In order to contrast influence of the different minimal mediums to different cultivars garlic embryonic callus induction, from MS, B5
And MS+B5Organic culture medium, experiment kind has ' two water early ', ' Xuzhou is white ', ' first month of the lunar year is early ' and ' precocious garlic ', hormone selection
2.5mg·L-1 2,4-D+0.5mg·L-1KT.Rachis calculates for the method and inductivity of explant induced embryonic callus
Method be the same as Example 1.
As a result such as table 3：' two water are early ', ' Xuzhou is white ', ' first month of the lunar year is early ' and ' precocious garlic ' be in B5In minimal medium and in MS
And MS+B5Going out the more time in organic two kinds of minimal mediums, there was no significant difference, but 4 kinds are in B5Callus on culture medium is lured
Conductance is above other basal mediums, is 88%-90%, wherein early with ' two water ' highest is 90.18%.
The influence of the different cultivars of table 3 and minimal medium to garlic embryonic callus induction
Note:Different letters represent the significant difference in 0.05 level in table.
Embodiment 3：
In order to contrast the influence of hormon species and concentration to garlic embryonic callus induction, the cell point of this experiment
Split element from KT (6- glycosyls adenine phosphate), if 0.1,0.5,1.0mgL-13 concentration, auxin 2,4-D sets 1.5,2.5,
3.5mg·L-13 concentration complete combination designs, totally 9 processing often handle 3 repetitions, per 10 culture dishes of repeated inoculation, often
Individual culture dish is inoculated with 4 explants.Growing state is observed daily and counts callus induction rate.Rachis induces for explant
The method and inductivity computational methods of embryo callus are with case study on implementation 1.
As a result such as table 4：The present invention preferentially uses 2.5mgL-1 2,4-D+0.5mg·L-1KT callus induction rates
Highest, is 90.18%, and illustrating that 2,4-D and KT acts synergistically on garlic rachis at this concentration better than other combined concentrations is cured
Wound induction.Under this processing, the embryo callus form of garlic rachis formation is shown in Fig. 4.
Influence of the hormon of table 4 to garlic embryonic callus induction
Note:Different letters represent the significant difference in 0.05 level in table.
Embodiment 4：
In order to contrast the influence of different sucrose and pH to garlic embryonic callus induction, sucrose concentration sets 20,30
And 40gL-1, pH sets 5.4,5.8 and 6.2 3 gradients, and totally 9 processing often handle 3 repetitions, per 10 cultures of repeated inoculation
Ware, each culture dish is inoculated with 4 explants.Method and inductivity calculating side of the rachis for explant induced embryonic callus
Method is with case study on implementation 1.
As a result it is as shown in table 5：Different sucrose and pH have an impact to garlic embryonic callus induction, and the present invention is selected
Sucrose 30-40gL-1, pH value be 5.8-6.2 effects preferably, particularly sucrose be 30gL-1, pH5.8 when, callus
Inductivity highest, is 90.18%.
The influence of the different sucrose of table 5 and pH to garlic embryonic callus induction
Note:Different letters represent the significant difference in 0.05 level in table.
Embodiment 5：
In order to contrast the shadow that hormon species and concentration and different training methods are bred to garlic embryonic callus
Ring, select that yellow-white graininess, quality be hard and breakable embryo callus, accessed solid medium and 50mL tri-
In the bottle of angle (suspend culture), fluid nutrient medium volume is 30mL.Hormone selects 6-BA, KT, TDZ, NAA, specific concentration and combination
It is shown in Table 6.5 bottles are often handled, the blake bottle weight before and after callus access is determined.The culture that suspends is used with the speed governing of HY-4A digital displays
Oscillator (production of Changzhou Whiskas instrument manufacturing Co., Ltd), speed setting is 180r/min, and 1 fresh cultured is changed per 7d
Base.Callus is determined after 4 weeks and produces front and rear blake bottle weight, the increment and proliferation times of embryo callus is counted.
The garlic embryonic callus proliferated culture medium of table 6
As a result it is as shown in table 7：Minimum L3 (the 1.5mgL of rate of body weight gain in Liquid Culture selected by the present invention-1 6-BA+
0.5mg·L-1TDZ rate of body weight gain highest S1 in solid culture, i.e. Liquid Culture callus group selected by the present invention) are still higher than
Knit cultivation effect more preferable；Requirement from fluid nutrient medium to hormone kind and concentration is lower slightly, such as L1 or L2 combination have compared with
Good effect, L1 (1.0mgL preferably-1 6-BA+0.5mg·L-1KT Liquid Cultures) rate of body weight gain maximum, up to 44.30%.It is outstanding
After floating culture 4 weeks, callus Gain weight is shown in Fig. 1.
The influence that the different training methods of table 7 and hormone are bred to garlic embryonic callus
Note:Different letters represent the significant difference in 0.05 level in table.
Embodiment 6：
In order to contrast embryo of the same size after the influence that hormon species and concentration are sprouted to garlic somatic embryo, selection propagation
Property callus be material, addition 6.5gL-1Cultivated on the solid medium of agar, specific formula is shown in Table 8.Often handle 3 times
Repeat, often repeatedly 10 bottles, every bottle of inoculation 5g or so callus group, every 4 weeks subcultures 1 time, culture 16 weeks under illumination.Observe body embryo hair
Situation is educated, statistics regenerated root, seedling number calculate germination rate.
Germination rate (seedling number/g)=sprouting seedling number/embryo callus fresh weight.
The garlic somatic embryo of table 8 develops culture medium
As a result such as table 9 and Fig. 2：6-BA+KT body embryo germination rate highests selected by the present invention, are combined better than contrast, and seedling
Normally；And not only germination rate is relatively low for 6-BA+NAA combination, and there is curling seedling；KT+NAA assembly embryo germination rate it is same compared with
It is low, and part body embryo seedling is weak and slightly vitrifying.Garlic somatic embryo sprouting situation is shown in Fig. 5.
The influence that the different hormone combinations of table 9 are developed to garlic somatic embryo
Claims (10)
1. a kind of method that utilization rachis efficiently induces garlic embryonic callus, it is characterised in that comprise the following steps：
Step (1)：In rachis upper surface diameter 0.3-0.5cm, the scape development relative altitude of garlic stems is scape height/false stem
When being highly 0.5-3.0, garlic stems is harvested successively, is then cut involucre climing section of 4-6cm of band, is immersed in saturation washing powder solution
25-35min, then rinses 25-35min with flowing water；
Step (2)：By the material fully rinsed on superclean bench, with 70-75% ethanol surface sterilizations 60-90s, 2% time
After sodium chlorate solution's soaking disinfection 10-15min, with aseptic water washing 4-5 times, finally it is placed on aseptic filter paper and blots surface moisture
Carry out healing tissue inducting test；
Step (3)：Sterile rachis outer layer bract is peelled off, flower primordium residue and film quality bract and the scape of surface degradation is removed
Part, is 4 pieces by cross rip cutting, is seeded in embryonic callus induction culture medium；
Step (4)：Select that yellow-white graininess, quality be hard and breakable embryo callus, accessed 20-40mL and be cured
Suspend shaken cultivation in injured tissue proliferated culture medium, and vibration rotating speed is 160-200r/min, and 1 fresh cultured is changed per 6-8d
Base；
Step (5)：Embryo callus of the same size is material after selection propagation, in addition 6.0-7.0gL-1The body of agar
Cultivated on blast germination medium, every bottle of inoculation 4-6g callus group, every 4-5 weeks subculture 1 time.
2. the method that utilization rachis according to claim 1 efficiently induces garlic embryonic callus, it is characterised in that
In step (1) rachis surface diameter be 0.5cm and garlic stems scape development relative altitude be 1 when harvest garlic stems.
3. the method that utilization rachis according to claim 1 efficiently induces garlic embryonic callus, it is characterised in that
The material fully rinsed is sterilized 90s on superclean bench with 75% alcohol, 2% sodium hypochlorite sterilizes 12 minutes.
4. the method that utilization rachis according to claim 1 efficiently induces garlic embryonic callus, it is characterised in that
Embryonic callus induction Medium Proportion described in step (3) is：1 times of B5 medium, 2.0-3.0mgL-12,4-D,
0.3-0.6mg·L-1KT, 30-40gL-1Sucrose, 6.0-7.0gL-1Agar, pH5.8~6.2.
5. the method that utilization rachis according to claim 4 efficiently induces garlic embryonic callus, it is characterised in that
Embryonic callus induction Medium Proportion described in step (3) is：1 times of B5 medium, 2.5mgL-12,4-D,
0.5mg·L-1KT, 30gL-1Sucrose, 6.5gL-1Agar, pH5.8.
6. the method that utilization rachis according to claim 1 efficiently induces garlic embryonic callus, it is characterised in that
The mode of embryo callus Multiplying culture is liquid suspension shaken cultivation in step (4).
7. the method that utilization rachis according to claim 1 efficiently induces garlic embryonic callus, it is characterised in that
The proportioning of callus proliferation medium described in step (4) is：1 times of B5 medium, 1.0-1.5mgL-16-BA, 0-
0.5mg·L-1KT, 20-40gL-1Sucrose, pH is 5.8-6.0 or 1 times of B5 medium, 1.0-1.5mgL-16-BA,
0-0.1mg·L-1NAA, 20-40gL-1Sucrose, pH is 5.8-6.0；It is preferred that 1 times of B5 medium, 1.0mgL-16-BA,
0.5mg·L-1KT, 30gL-1Sucrose, 6.5gL-1Agar, pH5.8.
8. the method that utilization rachis according to claim 1 efficiently induces garlic embryonic callus, it is characterised in that
23-27 DEG C is placed in step (4) in embryo callus breeding, intensity of illumination is 50-200 μm of olm-2·s-1, light week
Phase 12-14hd-1Cultivated under illumination condition.
9. the method that utilization rachis according to claim 1 efficiently induces garlic embryonic callus, it is characterised in that
Included in step (5) in somatic embryo germination medium：1.5-2.5mg·L-16-BA and 0.5-1.0mgL-1KT.
10. the method that utilization rachis according to claim 1 efficiently induces garlic embryonic callus, its feature exists
In the minimal medium of somatic embryo germination medium is MS culture mediums in step (5)；In the somatic embryo germination medium
Also contain 25-35gL-1Sucrose.
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| CN110268979A (en) * | 2019-06-03 | 2019-09-24 | 南京农业大学 | A kind of method and application of the foundation without pollen lily high-efficiency regeneration system |
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| CN101851601A (en) * | 2010-05-06 | 2010-10-06 | 江苏徐淮地区徐州农业科学研究所 | Culture medium for inducing somatic embryos from garlic embryonic suspension culture system |
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| 龙玉娟: "大蒜（Allium Sativum L.）试管苗不同再生途径及特征的研究", 《CNKI中国优秀硕士学位论文全文数据库 农业科技辑》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110268979A (en) * | 2019-06-03 | 2019-09-24 | 南京农业大学 | A kind of method and application of the foundation without pollen lily high-efficiency regeneration system |
| CN110268979B (en) * | 2019-06-03 | 2022-03-11 | 南京农业大学 | Method for establishing efficient regeneration system of pollen-free lily and application |
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