CN104195171A - Efficient, rapid and stable genetic transformation method for strawberries - Google Patents
Efficient, rapid and stable genetic transformation method for strawberries Download PDFInfo
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Abstract
The invention discloses an efficient, rapid and stable genetic transformation method for strawberries 'beauty'. The method comprises the steps of activation of culture, preparation of plant materials, infection, co-culture, screening culture and rooting culture, solves the problems of the pollution of agrobacterium and infectious microbes in a traditional agrobacterium-mediated method, low conversion efficiency of the strawberries 'beauty' and long period of the strawberries 'beauty' from transgenosis and transgenic detection to field transplanting, shortens the period, further solves the problems of large workload and heavy work of transgenic detection, simplifies a detection method, and increases the detection speed. On the basis of the established 'beauty' strawberry leaf regeneration system, the method researches various factors for influencing agrobacterium tumefaciens transforming the 'beauty' strawberry leaves, optimizes various parameters, simplifies the operation process, and determines the best transformation conditions.
Description
Technical field
The invention belongs to gene engineering technology field field, relate in particular to the transgenic technology of a kind of strawberry.
Background technology
Strawberry (
fragaria ananassa) be the crop that a kind of economic worth is higher, be of high nutritive value, Strawberry industry, in the many regional develop rapidlys of China, has become the important component part of some regional agriculture industrialization in recent years.Therefore China's researcher has also dropped into a large amount of energy in the research of strawberry in recent years, especially along with molecular biology and engineered develop rapidly in recent years, study hotspot turns to the degree of depth of strawberry germplasm excellent genes to excavate, and carries out qualification and the mensuration of molecule marker and expressed sequence.In succession report to some extent in the result of study of the aspects such as genetic improvement such as resistance, storage tolerance, flavor quality, Strwberry Breeding is just towards molecular breeding future development simultaneously.And most important means are exactly strawberry transgenosis in the process of strawberry molecular breeding.Since nineteen ninety James etc. has successfully obtained agriculture bacillus mediated Transformation of Strawberry plant, by the research of recent two decades, strawberry has become after walnut, apple, and the 3rd obtains the fruit tree of transfer-gen plant.So far, various countries scholar utilizes agrobacterium-mediated transformation that the multiple external source goal gene that has economic worth is imported in strawberry genome.On the other hand, for fruit tree, strawberry is short to the result cycle from blooming, and in 1 year, can repeatedly become colored, and inflorescence is many, can four seasons result, and plant is short and small, and plantation is convenient.Therefore concerning fruit tree crop, strawberry research is used as a kind of model study of fruit tree research, and especially, aspect the growing of research fruit, the fruit development mechanism of research strawberry is easier, has efficiently advantage fast.
Conventional strawberry gene transformation method has agrobacterium-mediated transformation, particle bombardment and electric shocking method at present, wherein agriculture bacillus mediated leaf dish method is the main method of Transformation of Strawberry, is also the most ripe the most frequently used a kind of transgenic method of research in plant transgene research up to now.Although the genetic transfoumation route maturation of strawberry, but transformation efficiency is very low, the transfer-gen plant obtaining very little, bring obstacle to the later stage qualification work of transfer-gen plant, and from transgenosis, transgenosis detect, the whole cycle of field-transplanting is longer, transgenosis testing amount is large, works heavy.Traditional agrobacterium-mediated transformation operating process is more loaded down with trivial details, in genetically modified process, easily there are many problems, such as selection, the pollution of miscellaneous bacteria, the Agrobacterium of the selection of suitable carrier, suitable bacterial strain cannot suppress, bacterial concentration is too high or too lowly cause that transgenosis is unsuccessful, the control of time of infection length etc., these problems all can affect the genetically modified efficiency of strawberry and speed, and the strawberry transgene efficiency that traditional method obtains is less than one of percentage.Therefore how obtaining an efficiently strawberry stable conversion system fast, is a problem urgently to be resolved hurrily in strawberry transgenosis.
Strawberry cultivars ' beauty ' yielding ability is good, best in quality, is suitable for facility cultivation, domestic this kind of large-scale cultivation, has also obtained larger economic benefit at present, but because this kind also exists many-sided defect, such as resistance is poor, easily infect not storage tolerance etc. of disease and pest and fruit.
Summary of the invention
For solving the problems of the technologies described above, the invention provides the efficient fast and stable gene transformation method of a kind of strawberry ' beauty ', not only solve Agrobacterium and the pollution problem of miscellaneous bacteria in traditional agrobacterium-mediated transformation, the problem that strawberry transformation efficiency is low; But also solve strawberry transgenosis from transgenosis, transgenosis detects, the field-transplanting cycle is long problem,, transgenosis testing amount is large, the heavy problem of working.
For achieving the above object, the present invention is achieved by the following technical solutions.
The substratum of the efficient fast and stable gene transformation of a kind of strawberry ' beauty ',
Strawberry subculture medium FMS1, the composition comprising has: MS, sucrose 30g/L, agar 5.5 g/L, 6-BA0.5mg/L, NAA0.1mg/L, and the pH value of substratum is 5.8;
Strawberry is culture medium FMS2 altogether, and the composition comprising has: MS, and sucrose 30g/L, agar 5.5 g/L, TDZ 2mg/L, 2,4-D 0.1mg/L, and the pH value of substratum is 5.8;
Strawberry screening culture medium FMS3, the composition comprising has: MS, sucrose 30g/L, agar 5.5 g/L, TDZ 2mg/L, 2,4-D 0.1mg/L, kantlex 0-50mg/L, Pyocianil 100-400 mg/L, and the pH value of substratum is 5.8;
The strawberry screening culture medium FMS4 of taking root, the composition comprising has: MS, sucrose 30g/L, agar 5.5 g/L, IBA 0.1mg/L, kantlex 0-50mg/L, Pyocianil 100-400 mg/L, and the pH value of substratum is 5.8.
Further described strawberry screening culture medium FMS3, the composition comprising has: MS, sucrose 30g/L, agar 5.5 g/L, TDZ 2mg/L, 2,4-D 0.1mg/L, kantlex 20mg/L, Pyocianil 400 mg/L; The strawberry screening culture medium FMS4 of taking root, the composition comprising has: MS, sucrose 30g/L, agar 5.5 g/L, IBA 0.1mg/L, kantlex 20mg/L, Pyocianil 300 mg/L.
Further in the every 1L of described each substratum, the consumption of MS substratum pulvis is 4.4g; After the common culture medium FMS2 autoclaving of strawberry, be cooled to 60 DEG C of packing flat boards for subsequent use; Strawberry screening culture medium FMS3 and strawberry take root and are cooled to 60 DEG C after screening culture medium FMS4 autoclaving and add kantlex, Pyocianil packing culturing bottle for subsequent use.
The efficient fast and stable gene transformation method of a kind of strawberry ' beauty ', the method comprises the steps:
1) actication of culture: will rule on solid medium containing plasmid pK7WG2D Agrobacterium EHA105, cultivate 2 days for 28 DEG C, be inoculated in liquid nutrient medium and shake and be cultured to OD600 value 0.4～0.6 in 28 DEG C of bacterium, after centrifugal bacterium liquid, remove supernatant liquid, collect bacterial sediment, the precipitation of collecting is resuspended with MS liquid nutrient medium suspension, and resuspended rear mensuration bacterial concentration is 0.2～0.6, the bacterium liquid after resuspended is put in to refrigerator for subsequent use;
2) vegetable material is prepared: be taken at the blade of upper subculture strawberry " beauty " tissue cultured seedling of strawberry subculture medium FMS1, cut off blade tip and leaf margin, be cut into 0.4 cm
2leaf piece,
3) infect: bacterium liquid is poured in the blade shearing, slightly shaken up, contaminate 40～60 minutes, after having infected, pour out bacterium liquid, blade is blotted on filter paper;
4) cultivate altogether: the blade inoculation after infecting is total in substratum FMS2 strawberry, dark culturing 2～3 days in 25 ± 2 DEG C of incubators;
5) screening and culturing: by the blade of the common cultivation strawberry of 3 days ' beauty ', be seeded on strawberry screening culture medium FMS3, in culturing room 25 ± 2 DEG C, illumination cultivation 20～30 days, From Strawberry Leaves edge has callus to grow, and after detecting, retains kanamycin-resistant callus tissue, remove non-resistance callus, cultivation by kanamycin-resistant callus tissue through 40～50 d, differentiates plant, again detects the resistant plant that the whole plant leaf of reservation and petiole are all resistance;
6) root culture: when resistant plant grows to 2-3 centimetre high, resistant plant is proceeded to strawberry and take root on screening culture medium FMS4 and take root, obtain complete resistance seedling, the blade, petiole and the root that retain resistance seedling after detecting are all the plant of resistance.
In further described step 1), solid medium is: LB solid medium 50 mL+ spectinomycin 50mg/L+ Rifampin 100mg/ L; Wherein LB solid culture based formulas: each rises in LB solid medium and comprises Tryptones 10g, yeast extract 5g, sodium-chlor 10g and Agar (agar) 15g;
Liquid nutrient medium is: LB liquid nutrient medium 50mL+ spectinomycin 50mg/L+ Rifampin 100mg/ L; Wherein LB liquid culture based formulas: each rises in LB liquid nutrient medium and comprises Tryptones 10g, yeast extract 5g, sodium-chlor 10g.
In further described step 1), resuspended rear mensuration bacterial concentration is 0.2～0.3.
In described step 5), step 6), use body formula fluorescent microscope to detect, observe green glow and be resistance.
beneficial effect:compared with prior art, the invention has the advantages that:
1) improve strawberry transgene efficiency, transformation efficiency can reach more than 30%;
2) bacterial concentration is reduced, time of infection lengthens, and avoids Agrobacterium to pollute, and has improved again and has infected efficiency;
3) the normal auxiliary medicaments that infect such as Syringylethanone that add in traditional method, can damage blade, reduce leaf regeneration ability, in bacterium liquid, do not add any auxiliary medicine medicament infecting and infect, the injury to From Strawberry Leaves be can reduce, surviving rate and regeneration rate that blade is cultivated improved;
4) use and carry the carrier of strongly expressed reporter gene, obtain transfer-gen plant by observing directly to screen, avoid loaded down with trivial details work in traditional detection, shortened the genetically modified cycle.
Brief description of the drawings
Fig. 1 is the fluoroscopic examination picture of Transgenic Strawberry ' beauty ', wherein:
FA: callus fluorescence FB: blade fluorescence FC: petiole fluorescence
FD: tissue cultured seedling fluorescence FE: flower fluorescence FF: fruit fluorescence
Fig. 2 is the impact of kantlex concentration on strawberry regeneration rate;
Fig. 3 is that Pyocianil is on the genetically modified impact of strawberry;
Fig. 4 is different bacterial concentrations and time of infection pair
egfpthe impact of genetic expression rate.
Embodiment
Below in conjunction with drawings and Examples, the present invention is described in further detail.
Embodiment 1:
One, material is prepared
1, vegetable material
Strawberry " beauty " tissue cultured seedling, on strawberry subculture medium, subculture is for experiment.
2, culture medium prescription and compound method:
Strawberry ' beauty ' subculture medium FMS1:
MS4.4g/L, sucrose 30g/L, agar 5.5 g/L, 6-BA0.5mg/L, NAA0.1mg/L, pH5.8, autoclaving, for subsequent use;
Strawberry ' beauty ' is culture medium FMS2 altogether:
MS4.4g/L, sucrose 30g/L, agar 5.5 g/L, TDZ 2mg/L, 2,4-D 0.1mg/L, pH5.8, autoclaving, is cooled to 60 DEG C of left and right packing flat boards for subsequent use;
Strawberry ' beauty ' screening culture medium FMS3:
MS4.4g/L, sucrose 30g/L, agar 5.5 g/L, TDZ 2mg/L, 2,4-D 0.1mg/L, pH5.8, autoclaving, is cooled to 60 DEG C and adds kantlex 0-50mg/L, and Pyocianil 100-400 mg/L packing culturing bottle is for subsequent use;
Strawberry ' beauty ' the screening culture medium FMS4 of taking root:
MS4.4g/L, sucrose 30g/L, agar 5.5 g/L, IBA 0.1mg/L, pH5.8, autoclaving, is cooled to 60 DEG C and adds kantlex 0-50mg/L, and Pyocianil 100-400 mg/L packing culturing bottle is for subsequent use.
MS:MS is MS substratum, that Murashige and Skoog were tobacco cell Training Design in 1962, be characterized in that inorganic salt and ionic concn are higher, more stable ionic equilibrium solution, its nitrate content is high, and quantity and the ratio of its nutrient are suitable, can meet nutrition and the physiological requirements of vegetable cell, thereby the scope of application is wider, most plants tissue-culturing quick-propagation uses it as the minimum medium of substratum.The present invention uses by sigma company of the U.S. and produces MS substratum pulvis (Murashige and Skoog basal salt mixture), this MS substratum pulvis requires one liter of substratum of every preparation to add 4.4 grams of pulvis, in the preparation of basal culture medium, one liter of substratum of every preparation need to add 4.4 grams, MS substratum pulvis.
TDZ:TDZ is a kind of new plant growth regulator, there is very strong cytokine activity, it can promote regeneration and the breeding of plant sprout, break the eye of stopping of bud, promote seed germination, promote callus growth, delay plant senescence etc., and can be to the growth and development process that is used for regulating plant of other plant hormone and physiologically active substance, it is a plant-growth regulator that reactive force is very strong, in agricultural, be widely used and be also in daily use in the tissue culture of strawberry with promotional value, the TDZ that adds 2.0mg/L in the present invention can finely induce ' beauty ' blade to form indefinite bud.TDZ is mixed with to the mother liquor of 1mg/mL for preparation substratum.
2,4-D:2,4-D is 2,4 dichlorophenoxyacetic acid, very effective for induction and the growth of callus, is plant-growth regulator conventional in tissue culture.In the present invention, add 2 of 0.1mg/L, 4-D just can be good at induction ' beauty ' adventitious bud formation.2,4-D is mixed with to the mother liquor of 1mg/mL for preparation substratum.
6-BA:6-BA is 6-benzyl aminoadenine, is a kind of material of cytokinin, has efficient, stable, cheap and to be easy to the features such as use be the favorite phytokinin of tissue culture person.The Main Function of 6-BA is to promote the formation of bud, in the present invention for strawberry ' beauty ' succeeding transfer culture.6-BA is mixed with to the mother liquor of 1mg/mL for preparation substratum.
IBA:IBA is indolebutyric acid, can promote the growth of plant main root, improves percentage of germination, surviving rate.The propagation that high density indolebutyric acid also can promotion division divides tissue cultured seedling.In the present invention, IBA is mainly used in taking root of transfer-gen plant.Be mixed with the mother liquor of 1mg/mL for preparation substratum.
Kantlex: be biological antibiotic class medicament, buy from sigma company of the U.S., medicine is white pulvis, and the concentration that is mixed with 50mg/mL is for experiment; Kantlex is the foliage filter screening mark in transgenosis, by add kantlex in substratum, can weed out a part of not genetically modified bud or plant.The anti-kantlex of transfer-gen plant therefore can survive in the substratum that adds kantlex, and the not anti-kantlex of common plant, can be dead.
Rif: Rifampin is biological antibiotic class medicament, buys from sigma company of the U.S., and medicine is scarlet pulvis, and the concentration that is mixed with 25mg/mL is for experiment.
Spectinomycin: be biological antibiotic class medicament, buy from sigma company of the U.S., medicine is white pulvis, and the concentration that is mixed with 50mg/mL is for experiment; Kantlex is the selection markers of the carrier in transgenosis, by add spectinomycin in LB substratum, can accurately screen single bacterium colony, reduces the probability of living contaminants.
Pyocianil: Pyocianil is for suppressing the growth of transgenosis later stage Agrobacterium.
3, bacterial strain and plant expression vector
The bacterial strain using in the present invention is agrobacterium strains EHA105, and this bacterial strain is conventional bacterial strain in strawberry transgenosis, and infection ability is stronger.This bacterial strain is bought in US mode culture collection warehousing ATCC (American type culture collection).
Plant expression vector is selected the pK7WG2D plant expression vector based on Gateway technology, Gateway technology has been simplified the step of gene clone and subclone widely, Gateway has utilized site-specific restructuring, so building after entry vector, no longer need to use restriction enzyme and ligase enzyme.In addition pK7WG2D plant expression vector carries the reporter gene Egfp of superpower expression, is convenient to the detection of transfer-gen plant.Carrier is bought the biotech company in Invitrogen.
Two, the efficient fast and stable gene transformation method of strawberry ' beauty '
1, actication of culture
1) will on solid medium, rule containing plasmid pK7WG2D Agrobacterium EHA105, cultivate 2 days for 28 DEG C; Wherein solid medium is: LB solid medium 50 mL+ spectinomycin 50mg/L+ Rifampin 100mg/ L; Wherein LB solid culture based formulas: each rises and comprises Tryptone (Tryptones) 10g in LB solid medium, Yeast Extract (yeast extract) 5g, NaCl (sodium-chlor) 10g and Agar (agar) 15g.。
2) long good single bacterium colony 2-3 on picking flat board, be inoculated in 50 mL liquid nutrient mediums, 28 DEG C, 220 rpm in shaking table shaking culture to the about 12-16 of OD600 value 0.4(h); Wherein liquid nutrient medium is: LB liquid nutrient medium 50mL+ spectinomycin 50mg/L+ Rifampin 100mg/ L; Wherein LB liquid culture based formulas: each rises in LB liquid nutrient medium and comprises Tryptone (Tryptones) 10g, Yeast Extract (yeast extract) 5g, NaCl (sodium-chlor) 10g.
3) under normal temperature, centrifugal 8 minutes of 5000rmp, removes supernatant liquor, back-off centrifuge tube on aseptic filter paper, supernatant liquor is removed as far as possible, with the resuspended thalline of 100 mLMS liquid, in shaking table, about 1 hour mensuration OD600 value of shaking culture is 0.2, and these data are that (this OD600 value is very important for the best, can not be lower than 0.2, can not be higher than 0.3, be controlled between 0.2～0.3, approach 0.2).The bacterium liquid having activated can be used for doing next step vegetable material and infect.
2, vegetable material is prepared
The blade that is taken at 30 days tender, open and flat leaf ages of children on upper subculture strawberry " beauty " tissue cultured seedling of strawberry subculture medium FMS1, cuts off blade tip and leaf margin, is cut into about 0.4 cm
2leaf piece infect for bacterium liquid.Make surrounding on From Strawberry Leaves have wound as far as possible, cut so easy long callus.
3, infect
The bacterium liquid of suspension is poured in the blade shearing, slightly shaken up, contaminate 40 minutes, in process, every shake in 5 minutes once, increase the contact area of bacterium liquid and blade, after having infected, pour out bacterium liquid, blade is blotted on aseptic filter paper to surperficial bacterium liquid.
4, cultivate altogether
Blade inoculation after infecting is total in substratum FMS2 strawberry, dark culturing 3 days in 25 ± 2 DEG C of incubators.
5, screening and culturing
By the common cultivation strawberry of 3 days ' beauty ' blade, be seeded in strawberry screening culture medium FMS3 upper, wound is fully contacted with substratum, in culturing room 25 ± 2 DEG C, illumination cultivation.Cultivate about 20 days, From Strawberry Leaves edge can grow by adularescent callus, now under body formula fluorescent microscope, observes, and can obviously find out that a part of callus has very bright green glow to send, this part callus is kanamycin-resistant callus tissue, retain, and that a part does not have is luminous, this part callus is non-resistance callus, get rid of, by this step, non-resistance callus is removed, greatly reduce further work amount.The kanamycin-resistant callus tissue retaining, after the cultivation of approximately 40 d left and right, can differentiate resistant plant.Now carry out again first order fluorescence detection, under body formula fluorescent microscope, observe resistant plant, all remaining of green light of whole resistant plant blade and petiole, what glow gets rid of.
6, root culture
When resistant plant grows to about 2-3 centimetre high, resistant plant is proceeded to strawberry and take root on screening culture medium FMS4 and take root, obtain complete resistance seedling.Resistance seedling now can't determine to be transgenic seedling completely, therefore needs to carry out first order fluorescence detection, the blade of resistance seedling in fluoroscopic examination again, petiole all shinny green glow can be defined as transfer-gen plant, as FB in Fig. 1, FC, shown in FD, the blade petiole of transfer-gen plant has fluorescence.
The present invention can also carry out the test of fruit to obtaining fruit: after transplanting, wait strawberry to yield positive results, after yielding positive results in order further to determine genetically modified stability, the fluorescence that can be under fluorescent microscope again detects flower and fruit different developmental phases as Fig. 1 in FE, shown in FF, genetically modified strawberry and fruit have hyperfluorescenceZeng Yongminggaoyingguang to send.
Three, further set forth beneficial effect of the present invention below by test.
1, determining of foliage filter screening mark kantlex concentration:
By explant be inoculated in respectively containing kantlex be 0,10,15,20,25, on the FMS2 substratum of 50mg/L, dark cultivation 14 days, go under light and cultivate again 20 days, the brown rate of statistics blade, callus rate of formation and adventitious shoot regeneration rate are determined the suitableeest kantlex selection pressure.
From Strawberry Leaves regeneration is more responsive to the concentration ratio of kantlex, and along with increase explant chlorosis, brownization, gauffer, the downright bad phenomenon of kantlex concentration in substratum increase the weight of, explant regeneration rate also declines thereupon, and albefaction bud increases.Therefore, determine that suitable kantlex concentration is the key that successfully obtains transfer-gen plant.As can be seen from Figure 2 in the time that kantlex concentration is 20mg/L, the brown rate of blade reaches 65.7%, there is the callus of some amount to form, but do not have indefinite bud to generate, when therefore kantlex concentration reaches 20mg/L, suppressed the regeneration of strawberry indefinite bud completely.(Fig. 2) pressed in the selection that can be used as the differentiation adventitious buds stage.
2, the selection of antibacterial microbiotic (Pyocianil) concentration
Blade after infecting after common cultivation is inoculated in respectively on the FMS2 substratum containing Pyocianil 100,200,300,400mg/L, cultivate 14 days through dark, after screening and culturing to 40 day, the brown rate of statistics blade, the bacteriostatic level of callus rate of formation and adventitious shoot regeneration rate and Agrobacterium is determined best antibacterial antibiotic concentration.
The most important effect that antibacterial microbiotic uses is the growth that suppresses Agrobacterium in transgenosis process, and therefore, antibacterial antibiotic concentration requirement can either suppress Agrobacterium growth, can not damage plant-growth again.So determine that antibacterial antibiotic concentration is very important in whole transgenic experiments.In this test, antibacterial antibiotic concentration can well suppress Agrobacterium growth in the time of 300mg/L, does not also injure the normal growth of plant simultaneously, as shown in Figure 3, do not have Agrobacterium to overflow, and plant can normal growth in substratum.
3, bacterial concentration, time of infection optimization
By the bacterial concentration OD600=0.6 in traditional method, time of infection 8min, with bacterial concentration OD600=0.2 in present method, immerged time 40min compares, compare two kinds of different methods infect efficiency, infect with rear blade surviving rate and the later stage Agrobacterium pollution level determine best bacterial concentration and time of infection.
With
egfpgenetic expression rate is weighed the selection of time of infection and bacterial concentration.Method is to be placed under body formula fluorescent microscope and to detect luciferase expression infecting the blade that grows callus after conversion, and statistics has very bright green glow and there is no the blade number of green glow.
egfpgenetic expression rate=(having the green light number of blade/total explant number) × 100%
The optimization of bacterial concentration and time of infection is most important with part in the present invention, be different from high time of bacterial concentration in traditional strawberry transgenic method short infect method, in the present invention, use lower concentration bacterium liquid, infect for a long time, prove through test of many times, lower concentration infects for a long time and can increase substantially transformation efficiency, and late stage of culture is not easy to produce Agrobacterium pollution.Adopt as shown in Figure 4 lower concentration bacterium liquid to infect for a long time and can significantly improve
egfpgenetic expression rate, improves transformation efficiency.
Embodiment 2: substantially the same manner as Example 1, difference is: the efficient fast and stable gene transformation method of strawberry ' beauty ', specific as follows:
Second step in step 1 actication of culture, long good single bacterium colony 2-3 on picking flat board, be inoculated in 50 mL LB liquid nutrient mediums, 28 DEG C, 220 rpm in shaking table shaking culture to OD600 value 0.6.
Step 3 infects operation: the bacterium liquid of suspension is poured in the blade shearing, slightly shaken up, contaminate 60 minutes, in process, shook once every 5 minutes, increase the contact area of bacterium liquid and blade, after having infected, pour out bacterium liquid, blade is blotted on aseptic filter paper to surperficial bacterium liquid.
Step 4 is cultivated operation altogether: the blade inoculation after infecting is total in substratum FMS2 strawberry, dark culturing 2 days in 25 ± 2 DEG C of incubators.
Step 5 screening and culturing operation:
By the common cultivation strawberry of 2 days ' beauty ' blade, be seeded in strawberry screening culture medium FMS3 upper, wound is fully contacted with substratum, in culturing room 25 ± 2 DEG C, illumination cultivation.Cultivate 30 days, From Strawberry Leaves edge can grow by adularescent callus, now under body formula fluorescent microscope, observes, and can obviously find out that a part of callus has very bright green glow to send, this part callus is kanamycin-resistant callus tissue, retain, and that a part does not have is luminous, this part callus is non-resistance callus, get rid of, by this step, non-resistance callus is removed, greatly reduce further work amount.The kanamycin-resistant callus tissue retaining, after the cultivation of approximately 50 d, can differentiate resistant plant.Now carry out again first order fluorescence detection, under body formula fluorescent microscope, observe resistant plant, all remaining of green light of whole resistant plant blade and petiole, what glow gets rid of.
Claims (9)
1. a substratum for the efficient fast and stable gene transformation of strawberry ' beauty ', is characterized in that, comprises following composition:
Strawberry ' beauty ' subculture medium FMS1, the composition comprising has: MS, sucrose 30g/L, agar 5.5 g/L, 6-BA0.5mg/L, NAA0.1mg/L, and the pH value of substratum is 5.8;
Strawberry ' beauty ' is culture medium FMS2 altogether, and the composition comprising has: MS, and sucrose 30g/L, agar 5.5 g/L, TDZ 2mg/L, 2,4-D 0.1mg/L, and the pH value of substratum is 5.8;
Strawberry ' beauty ' screening culture medium FMS3, the composition comprising has: MS, sucrose 30g/L, agar 5.5 g/L, TDZ 2mg/L, 2,4-D 0.1mg/L, kantlex 0-50mg/L, Pyocianil 100-400 mg/L, and the pH value of substratum is 5.8;
Strawberry ' beauty ' the screening culture medium FMS4 of taking root, the composition comprising has: MS, sucrose 30g/L, agar 5.5 g/L, IBA 0.1mg/L, kantlex 0-50mg/L, Pyocianil 100-400 mg/L, and the pH value of substratum is 5.8.
2. the substratum of the efficient fast and stable gene transformation of a kind of strawberry according to claim 1 ' beauty ', it is characterized in that: described strawberry ' beauty ' screening culture medium FMS3, the composition comprising has: MS, sucrose 30g/L, agar 5.5 g/L, TDZ 2mg/L, 2,4-D 0.1mg/L, kantlex 20mg/L, Pyocianil 400 mg/L;
The strawberry screening culture medium FMS4 of taking root, the composition comprising has: MS, sucrose 30g/L, agar 5.5 g/L, IBA 0.1mg/L, kantlex 20mg/L, Pyocianil 300 mg/L.
3. the substratum of the efficient fast and stable gene transformation of a kind of strawberry according to claim 1 and 2 ' beauty ', is characterized in that: in the every 1L of each substratum, the consumption of MS substratum pulvis is 4.4g; After the common culture medium FMS2 autoclaving of strawberry, be cooled to 60 DEG C of packing flat boards for subsequent use; Strawberry screening culture medium FMS3 and strawberry take root and are cooled to 60 DEG C after screening culture medium FMS4 autoclaving and add kantlex, Pyocianil packing culturing bottle for subsequent use.
4. utilize the substratum described in claim 1 to carry out the efficient fast and stable gene transformation method of strawberry ' beauty ', it is characterized in that, the method comprises the steps:
1) actication of culture: will rule on solid medium containing plasmid pK7WG2D Agrobacterium EHA105, cultivate 2 days for 28 DEG C, be inoculated in liquid nutrient medium and shake in 28 DEG C of bacterium that to be cultured to OD600 value be 0.4～0.6, after centrifugal bacterium liquid, remove supernatant liquid, collect bacterial sediment, the precipitation of collecting is resuspended with MS liquid nutrient medium suspension, and resuspended rear mensuration bacterium liquid OD600 value is 0.2～0.6, the bacterium liquid after resuspended is put in to refrigerator for subsequent use;
2) vegetable material is prepared: be taken at the blade of upper subculture strawberry ' beauty ' tissue cultured seedling of subculture medium FMS1, cut off blade tip and leaf margin, be cut into 0.4 cm
2leaf piece;
3) infect: bacterium liquid is poured in the blade shearing, slightly shaken up, contaminate 40～60 minutes, after having infected, pour out bacterium liquid, blade is blotted on filter paper;
4) cultivate altogether: the blade inoculation after infecting is total in substratum FMS2 strawberry ' beauty ', dark culturing 2～3 days in 25 ± 2 DEG C of incubators;
5) screening and culturing: by the blade of the common cultivation strawberry of 2～3 days ' beauty ', be seeded on screening culture medium FMS3, in culturing room 25 ± 2 DEG C, when illumination cultivation 20～30 days, blade edge has callus to grow, and after detecting, retains kanamycin-resistant callus tissue, remove non-resistance callus, cultivation by kanamycin-resistant callus tissue through 40～50 days, differentiates plant, again detects the resistant plant that the whole plant leaf of reservation and petiole are all resistance;
6) root culture: when resistant plant grows to 2-3 centimetre high, resistant plant is proceeded to strawberry and take root on screening culture medium FMS4 and take root, obtain complete resistance seedling, the blade, petiole and the root that retain resistance seedling after detecting are all the plant of resistance.
5. the efficient fast and stable gene transformation method of strawberry according to claim 4 ' beauty ', is characterized in that, in described step 1), solid medium is: LB solid medium 50 mL+ spectinomycin 50mg/L+ Rifampin 100mg/ L; Wherein LB solid culture based formulas: each rises in LB solid medium and comprises Tryptones 10g, yeast extract 5g, sodium-chlor 10g and agar 15g;
Liquid nutrient medium is: LB liquid nutrient medium 50mL+ spectinomycin 50mg/L+ Rifampin 100mg/ L; Wherein LB liquid culture based formulas: each rises in LB liquid nutrient medium and comprises Tryptones 10g, yeast extract 5g, sodium-chlor 10g.
6. the efficient fast and stable gene transformation method of strawberry according to claim 4 ' beauty ', is characterized in that, in described step 1), resuspended rear mensuration bacterial concentration is 0.2～0.3.
7. the efficient fast and stable gene transformation method of strawberry according to claim 4 ' beauty ', is characterized in that described step 2) in blade be the blade of 30 days tender, open and flat leaf ages of children.
8. the efficient fast and stable gene transformation method of strawberry according to claim 4 ' beauty ', is characterized in that, in described step 3) dip-dye process, shakes once every 5 minutes.
9. the efficient fast and stable gene transformation method of strawberry according to claim 4 ' beauty ', is characterized in that, uses body formula fluorescent microscope to detect in described step 5), step 6), observes green glow and is resistance.
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