CN1561697A - Method for establishing lily gene transforming receptor system - Google Patents
Method for establishing lily gene transforming receptor system Download PDFInfo
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- CN1561697A CN1561697A CN 200410017688 CN200410017688A CN1561697A CN 1561697 A CN1561697 A CN 1561697A CN 200410017688 CN200410017688 CN 200410017688 CN 200410017688 A CN200410017688 A CN 200410017688A CN 1561697 A CN1561697 A CN 1561697A
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Abstract
A method for creating the gene transfer receptor system of lily includes disinfecting the surface of lily bulb or filament, cutting the become segments, culturing in differentiating culture medium to create a high-frequency regeneration system, antibiotic sensitivity test to determine the screening concentrations of cephamycin, hygromycin and kanamycin, inducing the rooting adventitious embryo, and transplanting.
Description
Technical field
The present invention relates to a kind of method for building up of lily genetic transformation receptor system, be used for plant gene transformation receptor biological technical field.
Background technology
Lily is world-renowned flowers, is widely used.The improvement of its quality for a long time all is to depend on traditional genetic and breeding method.Developing rapidly of molecular biology of plants, provide brand-brand-new way for improveing plant quality and proterties, utilize genetically modified means improvement lily proterties and quality to become practical, and good receptor system is a key technique of lily genetic transformation, directly determined the height of simple and easy, the transformation tissue culture frequency of method for transformation, efficient, the stable and foundation plant receptor system that regeneration capacity is strong is the prerequisite that realizes genetic transformation.The lily tissue culture once had many reports, but failed better to set up based on the lily high frequency regeneration system of genetic transformation always.The present invention is an object with the lily kind of extensive use, has set up lily genetic transformation embryo callus and has directly broken up receptor system, has established good basis for genetic transformation obtains the lily new varieties.
Summary of the invention
The objective of the invention is at the deficiencies in the prior art, a kind of method of setting up lily genetic transformation receptor system is provided.Make it pass through lily ex vivo differentiation condition optimizing, antibiotic susceptibility test, overcome the deficiency of lily tissue culture, provide the method for building up of two kinds of genetic transformation receptor systems, for genetic transformation cultivation lily new varieties or the strain that realizes lily provides the method with stronger operability.
The present invention is achieved through the following technical solutions, the present invention selects representational lily kind, optimizes its ex vivo differentiation condition, sets up the high frequency regeneration system and determine in the genetic transformation reasonably to select technology such as pressure to reach by antibiotic susceptibility test and set up the purpose that the lily gene genetic transforms best receptor system at different explants.The present invention is placed on cultured in vitro on the multiple differential medium that adds variety classes and variable concentrations hormone with materials such as lily bulb, filigrees earlier through surface sterilization, under the prerequisite of optimizing the ex vivo differentiation condition, selection suitable when determining genetic transformation is pressed.
Concrete grammar of the present invention comprises the steps:
1, material such as lily bulb or filigree carries out surface sterilization, is cut into the explant of 5-8mm segment as cultured in vitro, and is standby.
2, explant is seeded in MS, WS on the minimal mediums such as B5 and Nitsch medium, investigates its isolated growth situation after 30 days, and the minimal medium screening determines that minimal medium is enriched element balance medium MS;
3, best ex vivo differentiation medium determines.On the basis of determining minimal medium, it is the KT of 0.25-2mg/l that explant is seeded in additional concentration, BA, and 2,4-D, cultured in vitro on the differential medium of IAA and NAA is investigated its differentiation rate after 30-45 days.
4, be screening resistance tissue and cell when genetic transformation, set up in cultured in vitro on the basis of high frequency regeneration system and carry out antibiotic susceptibility test, determine the screening concentration of cephalosporin, hygromycin and kanamycin.Lily bulb or embryo callus are seeded in respectively on the selection medium of additional kanamycin (Km), hygromycin (Hyg) and cephalosporin (Cef), observe explant brown stain situation and bacteriostasis after 30-45 days, determine the screening concentration of hygromycin and kanamycin by the browning rate of explant, the selection of hygromycin and kanamycin is pressed when reaching 90% concentration when above as genetic transformation with browning rate, is used to screen resisting cell and tissue; By the screening concentration that the browning rate and the bacteriostasis of explant are determined cephalosporin, can suppress Agrobacterium and assorted bacterium, the concentration of browning rate＜10% is as the screening concentration of cephalosporin.The concentration gradient of kanamycin is 0,25,50,75,100,120,140mg/l, and the concentration gradient of hygromycin is 0,10,20,30,40mg/l, and the concentration gradient of cephalosporin is 0,100,250,500,750mg/l.
5, the indefinite bud that differentiates in will selecting to cultivate changes root induction in the root media over to.Test-tube plantlet grows to 7～8cm and transplants.
Method of the present invention is simple, has prominent matter characteristics and marked improvement.Can provide the good technical platform for the molecular breeding that will utilize particle bombardment, agrobacterium-mediated transformation etc. to carry out lily from now on technology of the present invention.Simultaneously, can select diverse ways to set up the genetic transformation receptor system, have stronger flexibility and broad applicability according to the specific requirement of different cultivars explant.
Embodiment
Below in conjunction with specific embodiment technical scheme of the present invention is further described.
Embodiment 1:
The experiment kind is an oriental hybrid lily, with its bulb as explant.
With bulb strip off from outside to inside, clean with flushing with clean water, detergent liquid soaks 30min, sterilize to the sterile working platform then: 75% alcohol-pickled 2min, aseptic water washing 3 times, the commercially available antiformin of 20% concentration soaks 15-20min, aseptic water washing 3 times, and it is standby as explant to be cut into the 5mm size.
Bulb is inoculated in MS respectively, and WS on the minimal mediums such as B5 and Nitsch medium, investigates growing state after 1 month.Determine that minimal medium is enriched element balance medium MS.
Bulb is seeded in MS+BA 0.5-2.0mg/l+NAA 0.5-2.0mg/l+2, cultivates on (pH5.8) solid differential medium of 4-D 0.1-1.0mg/l, per 2 all subcultures are once added up differentiation rate after 45 days.Determine that according to the result its best differential medium is MS+BA 1.0mg/l+NAA 0.5mg/l+2,4-D0.1mg/l (pH5.8).
Explant is seeded on the selection medium of additional variable concentrations kanamycin and cultivates.Promptly place MS+BA1.0mg/l+NAA 0.5mg/l+2, cultivate on 4-D 0.1mg/l+Km (0,25,50,75,100,120, the 140mg/l) solid culture medium, per 2 all subcultures once, statistics explant browning rate after 45 days.The result shows that the browning rate of explant when kanamycin is 100mg/l reaches more than 90%, and definite in view of the above kanamycin is advisable with 100mg/l to the screening concentration of oriental hybrid lily.
Get the part bulb and be inoculated in MS+BA 1.0mg/l+NAA 0.5mg/l+2, cultivate on the solid culture medium of 4-D 0.1mg/l+Cef (0,100,250,500,750mg/l), per 2 all subcultures once, statistics explant browning rate and bacteriostasis after 45 days.The result shows that cephalosporin is advisable with 250mg/l to the screening concentration of oriental hybrid lily.
Change the indefinite bud of selecting to differentiate in the cultivation in MS+NAA0.2mg/L (pH5.8) root media root induction.Test-tube plantlet grows to 7～8cm and transplants.
Embodiment 2:
The experiment kind is a Lilium longiflorum, with its filigree as explant.
The whole petal of Lilium longiflorum is cut, and clean with flushing with clean water, detergent liquid soaks the rearmounted sterile working platform sterilization of 30min.Method is as follows: material is put into sterile chamber, handle 10min with the commercially available liquor natrii hypochloritis with 20% concentration behind 75% the alcohol disinfecting 30s, with aseptic water washing 6-8 time, use aseptic blotting paper suck dry moisture at last, the strip off petal, it is standby as explant filigree to be isolated and is cut into the 5-8mm segment.
Filigree is inoculated in MS, and WS on the minimal mediums such as B5 and Nitsch medium, investigates growing state after 1 month.Determine that minimal medium is enriched element balance medium MS.
With explant be seeded in MS+BA (or KT) 0.25-2.0mg/l+NAA (or IAA or 2,4-D) (pH5.8) solid of 0.25-2.0mg/l induce with differential medium on cultivate, per 2 all subcultures are once.Statistics callus induction rate and differentiation adventitious buds rate after 30 days.Determine that according to the result its best inducing culture is MS+BA0.5mg/l+NAA 1.0mg/l, and best differential medium is MS+KT 1.0mg/l+NAA 0.2mg/l.
Embryo callus is inoculated on MS+KT 1.0mg/l+NAA 0.2mg/l+Km (0,25,50,75,100,120, the 140mg/l) solid culture medium cultivates, per 2 all subcultures once, statistics explant browning rate after 30 days.The result shows that the browning rate of explant when kanamycin is 75mg/l reaches 92%, and definite in view of the above kanamycin is advisable with 75mg/l to the screening concentration of Lilium longiflorum.
Embryo callus is inoculated on MS+KT 1.0mg/l+NAA 0.2mg/l+Hyg (0,10,20,30, the 40mg/l) solid culture medium cultivates, per 2 all subcultures once, statistics explant browning rate after 30 days.The result shows that the browning rate of explant when hygromycin is 20mg/l reaches 95.8%, determines that in view of the above hygromycin is 20mg/l to the screening concentration of Lilium longiflorum.
Get on the solid culture medium that the part embryo callus is inoculated in MS+KT 1.0mg/l+NAA 0.2mg/l+Cef (0,100,250,500,750mg/l) and cultivate, per 2 all subcultures once, statistics explant browning rate and bacteriostasis after 30 days.Determine that cephalosporin is 250-500mg/l to the screening concentration of Lilium longiflorum.
Change the indefinite bud of selecting to differentiate in the cultivation in MS+NAA0.2mg/L root media root induction.Test-tube plantlet grows to 7～8cm and transplants.
Claims (5)
1, a kind of method of setting up lily genetic transformation receptor system, it is characterized in that, place on the differential medium cultured in vitro to set up the high frequency regeneration system through segment after the surface sterilization lily bulb or filigree, the screening concentration of cephalosporin, hygromycin and kanamycin when determining genetic transformation by antibiotic susceptibility test, the adventitious bud inducing that differentiates is taken root, transplant.
2, the method for setting up lily genetic transformation receptor system according to claim 1 below further limits the present invention, and concrete steps are as follows:
1. lily bulb or filigree material carry out surface sterilization, are cut into the 5-8mm segment as explant, and be as cultured in vitro, standby;
2. explant is seeded in MS, WS on B5 and the Nitsch medium minimal medium, according to its isolated growth situation, through screening, determines that minimal medium is enriched element balance medium MS after 30 days;
3. on the basis of determining the best base basal culture medium, it is the KT of 0.25-2mg/l that explant is seeded in additional concentration, BA, and 2,4-D, cultured in vitro on the differential medium of IAA and NAA is investigated its differentiation rate after 30-45 days;
4. be screening resistance tissue and cell when genetic transformation, set up in cultured in vitro on the basis of high frequency regeneration system and carry out antibiotic susceptibility test;
5. the indefinite bud that will differentiate in will selecting to cultivate changes root induction in the root media over to, and test-tube plantlet grows to 7～8cm and transplants.
3, the method for setting up lily genetic transformation receptor system according to claim 2, it is characterized in that, described antibiotic susceptibility test, be meant lily bulb or embryo callus are seeded in respectively on the selection medium of kanamycin Km, hygromycin Hyg and cephamycin C ef, observe explant brown stain situation after 30-45 days, statistics explant browning rate.
4, the method for setting up lily genetic transformation receptor system according to claim 2, it is characterized in that, described antibiotic susceptibility test, be meant the screening concentration of coming hygromycin and kanamycin by the browning rate of explant, the selection of hygromycin and kanamycin is pressed when reaching 90% concentration when above as genetic transformation with browning rate, is used to screen resisting cell and tissue; By the screening concentration that the browning rate and the bacteriostasis of explant are determined cephalosporin, can suppress Agrobacterium and assorted bacterium, the concentration of browning rate＜10% is as the screening concentration of cephalosporin.
According to claim 3 or the 4 described methods of setting up lily genetic transformation receptor system, it is characterized in that 5, the concentration gradient of kanamycin is 0,25,50,75,100,120,140mg/l; The concentration gradient of hygromycin is 0,10,20,30,40mg/l; The concentration gradient of cephalosporin is 0,100,250,500,750mg/l.
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| CN 200410017688 CN1561697A (en) | 2004-04-15 | 2004-04-15 | Method for establishing lily gene transforming receptor system |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103563740A (en) * | 2012-07-23 | 2014-02-12 | 上海市农业科学院 | Method for utilizing lilium brownie roots to induce callus and embryoid |
| CN112544441A (en) * | 2020-11-10 | 2021-03-26 | 江苏省农业科学院 | Method for establishing tissue culture regeneration system of new acer palmatum variety |
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2004
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103563740A (en) * | 2012-07-23 | 2014-02-12 | 上海市农业科学院 | Method for utilizing lilium brownie roots to induce callus and embryoid |
| CN103563740B (en) * | 2012-07-23 | 2014-12-10 | 上海市农业科学院 | Method for utilizing lilium brownie roots to induce callus and embryoid |
| CN112544441A (en) * | 2020-11-10 | 2021-03-26 | 江苏省农业科学院 | Method for establishing tissue culture regeneration system of new acer palmatum variety |
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