CN103820489A - Method for improving conversion rate of foreign gene of stem tip meristem of yellowing seedling of cotton - Google Patents
Method for improving conversion rate of foreign gene of stem tip meristem of yellowing seedling of cotton Download PDFInfo
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Abstract
The invention discloses a method for improving conversion rate of a foreign gene of a stem tip meristem of a yellowing seedling of cotton. According to the method, the stem tip meristem of the yellowing seedling of cotton is taken as a receptor, the apical meristem of the stem tip is scratched in a 'cross' manner or in a shape like a Chinese character 'mi' (means rice) with depth of 0.6-1.0mm, and the target gene is introduced into the cotton by a agrobacterium-mediated method, so as to obtain a transgenic plant. The method has advantages of high conversion rate and good repeatability. Experiments prove that the average conversion rate of the cotton converted by the method is improved by 30% in comparison with that of the cotton converted by the unimproved method, so that the method has very important value for genetic improvement of the cotton.
Description
Technical field
The present invention relates to a kind of method that improves gene transformation rate, relate in particular to a kind of method that improves cotton etiolated seedling shoot apical meristem gene transformation rate; Platymiscium genetically engineered field.
Background technology
From (Umbeck et al.Bio/Technology such as Umbeck in 1987,1987,5:263-266) successfully obtain after the cotton transfer-gen plant of jade-like stone word by agrobacterium-mediated transformation, agrobacterium-mediated transformation becomes in the world one of the most general conversion means gradually, and have made great progress (Wu et al, 2005, Plant Breeding, 124 (2): 142-146; Guo et al.Biologia Plantarum.2007,51 (2): 242-248).Up to the present, deliver about the article of Cotton Transformation also few in number.Major cause is that after carrying out transgenosis, cotton healing tissue is generally relatively difficult to regeneration, and the cotton somatic embryos fetal hair tissue culture time raw and plant regeneration is longer.From existing report, utilizing optimum genotype cotton variety to carry out genetic transformation also needs 8～10 months (Umbeck et al, Bio/Technology, 1987,5:263-266).Often there is more serious heritable variation in the regeneration plant that in addition, long-time isolated culture obtains.Some scholars attempt to utilize the direct converting cotton stem apex of particle gun cell, but transformation efficiency extremely low (McCabe et al.Bio/Technology, 1993,11:596-598; Zapata et al.Theor.Appl.Genet., 1999,98:252-256); Also have some scholars to utilize particle gun bombardment cotton cells suspension cell line (Fine et al.Plant Cell Rep.1990,8:586-589; Rajasekaran et al.Plant Cell Rep.2000,19:539-545), limited by genotype but set up cotton cells suspension system.Jade-like stone word cotton 312 (Coker312G), jade-like stone word cotton 201 (Coker201) are developed into the pattern kind of numerous laboratories cotton transgenic at present, but jade-like stone word cotton 312 and jade-like stone word cotton 201 are all out-of-date cotton varieties produced in USA, the transgenic cotton floral material obtaining will could be applied through selection cross and systematic breeding conventionally on producing, and wastes time and energy.Therefore, set up suitable Cotton Transformation technology and remain the important foundation of cotton gene engineering research.
Plant regeneration difficulty, genotype dependency is strong, the transformation period is long and efficiency low be the principal element that restricts at present Cotton Transformation.It is that acceptor carries out genetic transformation that the stem apex of yellow seedling is selected in laboratory, has avoided plant regeneration process, has overcome genotype restriction and has set up a new Cotton Transformation system (Lv Sulian etc., hi-tech communication, 2004,11:20-25).But owing to affecting the many factors of cotton yellow seedling stem apex transformation system, having in actual applications that transformation efficiency is unstable, the shortcoming of poor repeatability, is urgent problem therefore improve transformation efficiency stability, the repeatability of cotton yellow seedling stem apex transformation system.
Summary of the invention
For the deficiencies in the prior art, the problem to be solved in the present invention is further to improve to utilize cotton etiolated seedling shoot apical meristem to carry out efficiency and the repeatability of genetic transformation, and a kind of effective method that improves cotton etiolated seedling shoot apical meristem gene transformation rate is provided.
The method of raising cotton etiolated seedling shoot apical meristem gene transformation rate of the present invention, step is as follows:
A) cultivation of cotton etiolated seedling, obtains transgene receptor plant;
Cotton seeds, after sulfuric acid lint, soaks 10～15min with 70% alcohol immersion 0.5～1.5min, 0.1% mercuric chloride, then with sterilized water washing 3～5 times; After sterilization, seed is put into the aseptic bottle that is covered with three metafiltration paper, adds appropriate sterilized water, and in 25～28 ℃ of incubators, sprout dark place; After 2～3 days, after hypocotyl grows to 1～2cm, proceed in the CoM solid medium of pH5.8～6.0, put dark place and grow to the etiolated seedling that plant height is 8～10cm under 25～28 ℃, relative humidity 40～60% conditions, transform for stem apex;
Wherein, above-mentioned CoM solid medium refers to and in CoM nutrient solution, has added the substratum that agar that mass percent is 6% makes; CoM nutrient solution refers to the nutrient solution containing solute described in table 1 with distilled water preparation.
The solute composition of table 1, CoM nutrient solution
| Macroelement | Concentration in substratum (mg/L) |
| KNO 3 | 1900 |
| NH 4NO 3 | 1650 |
| CaCl 2·2H 2O | 440 |
| MgSO 4·7H 2O | 370 |
| KH 2PO 4·H 2O | 170 |
| Trace element | Concentration in substratum (mg/L) |
| FeSO 4·7H 2O | 27.8 |
| ZnSO 4·7H 2O | 8.6 |
| MnSO 4·4H 2O | 22.30 |
| H 3BO 3 | 8.70 |
| Na 2MoO 4·2H 2O | 0.25 |
| CuSO 4·5H 2O | 0.025 |
| CoCl 2·6H 2O | 0.025 |
| KI | 0.8 |
| ? | ? |
| Organic composition | Concentration in substratum (mg/L) |
| Vitamin | 10 |
| |
1 |
| |
1 |
| |
100 |
| Other | Concentration in substratum (g/L) |
| Sucrose | 30 |
B) seed coat and a slice cotyledon that remove etiolated seedling expose shoot apical meristem, then scratch stem apex top;
The method of above-mentioned scuffing stem apex is: the meristematic tissue on stem apex top is done to " ten " font or the scuffing of " rice " font, and the degree of depth of control scuffing wound is 0.6～1.0mm;
C) dip in suction dip-dyeing solution with rayon balls, and place it in the position, etiolated seedling stem apex top after scuffing, suck the unnecessary residual bacterium liquid of infection site with aseptic thieving paper, under condition of negative pressure, implement to contaminate;
The method that above-mentioned enforcement is contaminated is: be under 0.05～0.06MPa condition in negative pressure of vacuum, there is the rayon balls of dip-dyeing solution to be placed in the position, etiolated seedling stem apex top after scuffing with suction, under the condition of 25～28 ℃ of temperature, relative air humidity 40～60%, contaminate 10～15min; Wherein, described dip-dyeing solution composition is: on CoM nutrient solution basis, added the Syringylethanone of 100～200mg/L and the tensio-active agent Silwet-L77 that mass percent concentration is 0.01～0.02%, and the cell concentration that contains the foreign gene of recombinating is OD
600=0.6～0.8 Agrobacterium, described dip-dyeing solution pH is 5.8～6.0;
D) by contaminate after etiolated seedling in 25～28 ℃, secretly cultivate 3 ± 0.5 days, then light cultivate 2～3 days;
E) chrysanthemum seedling replanting after cultivation is entered to flowerpot, under 25～28 ℃ of temperature, humidity 40～60% conditions, cultivate, to growing 2～3 true leaves;
F) dip in suction selective agent with rayon balls, and place it in the position, transformation seedlings stem apex top that grows 2～3 true leaves, implement resistance screening, obtain resistant plant;
Above-mentioned selective agent is weedicide, the method of above-mentioned enforcement resistance screening is: under day 25～28 ℃ of temperature, night 20～22 ℃ of temperature, relative humidity 40～60% conditions, there is the rayon balls of selective agent to be placed in transformation seedlings stem apex top position 12～24h with suction, after 10～15d, observe the merismatic survival condition of plant bud pointed tip and select resistant plant, it is green that transfer-gen plant stem end vegetative point keeps, well-grown, non-transformed plant strain growth point blackout is dead.
In the method for above-mentioned raising cotton etiolated seedling shoot apical meristem gene transformation rate:
Described cotton can be kind, strain, breeding intermediate materials, can be conventional kind or cross-fertilize seed.
The degree of depth of described scuffing wound is preferably 0.6～1.0mm.
Preferably 10～15min of described immerged time.
Described weedicide is that chlorine sulphur is grand, and further, preferred concentration is that the chlorine sulphur of 20～40mg/L is grand.
The present invention is according to the principal element that affects Shoot Apex of Cotton genetic transformation efficiency, tensio-active agent Silwet-L77 concentration and shoot apical meristem damage mode in cell concentration in dip-dyeing solution, immerged time, vacuum infiltration, dip-dyeing solution are chosen, each factor is selected respectively 3 levels, utilizes L
9(3
4) orthogonal table, by tests multiple batches of, different cotton varieties, obtain the combination that improves Cotton Transformation efficiency.
In the method for raising disclosed by the invention cotton etiolated seedling shoot apical meristem gene transformation rate, key is stem apex apical meristem to be carried out to the scuffing of " ten " font or " rice " font, 0.6～1.0mm degree of depth.This damage main purpose is effectively to damage shoot apical meristem, improves the dip-dye of Agrobacterium, improves transformation efficiency and repeatability, and in actual production, contributes to applied for machines hand programming operations.Experiment confirms that the merismatic damage of Shoot Apex of Cotton can not be excessive, otherwise destroys stem apex apical meristem, and acceptor cotton growth is worked the mischief, therefore the optimal conditions in the inventive method is that screening obtains through lot of experiments, creative than existing methods.Further experiment confirms, utilizes method for transformation cotton average conversion of the present invention than the average conversion before improving and has improved 30%, and the genetic improvement of cotton is had to important value.
Accompanying drawing explanation
Fig. 1: be the structural representation of recombinant vectors pCAMBIA1300-TsVP-als.
Fig. 2: for utilizing Agrobacterium to carry out direct conversion process to cotton etiolated seedling shoot apical meristem:
1 sprouts for the cotton seeds after sterilizing in the aseptic plate that is covered with three metafiltration paper;
2 for to peel off yellow seedling seed coat and a slice cotyledon, scratches exposed stem apex growing tip with knife blade;
3 for to be placed in the sterilized rayon balls that is moistened with Agrobacterium bacterium liquid on stem apex, is aided with negative pressure and contaminates 10～15min;
4 transplant afterwards for secretly cultivating 3 days after contaminating;
5 is plant size when selective agent screens after 2～3 weeks.
Fig. 3: be the PCR result of transfer-gen plant
Swimming lane M is DNA Marker DL2000;
Swimming lane P is plasmid DNA;
Swimming lane WT is wild-type contrast;
Swimming lane CP-1, CP-2, CP-3, CP-4 are transfer-gen plant.
Embodiment
Describe the present invention in detail below in conjunction with embodiment.Should be noted that, embodiments of the invention only play illustration and there is no restriction the present invention.
1, the acquisition of restructuring Agrobacterium
By goal gene TsVP(genbank number of registration AY436553) be reconstituted in plant binary expression vector pCAMBIA1300-TsVP-als.
PCPA) and pCAMBIA1300-Pubi-TsVP-Tnos-als(abbreviation building plant expression vector plasmid used is: pCAMBIA1300-P35SMCSTnos – als(is called for short:: pCUA-TsVP), wherein pCPA contains CaMV35S promotor, antiweed selection markers gene als and a multiple clone site (MCS), and pCUA-TsVP contains TsVP gene.By XbaI enzyme cutting pCUA-TsVP plasmid DNA, reclaim TsVP fragment, be connected in pCPA multiple clone site and identify direction of insertion, obtain expressing plant expression vector pCAMBIA1300-P35S-TsVP-Tnos – als(Fig. 1 of goal gene).
PCAMBIA1300-P35S-TsVP-Tnos – als carrier is proceeded to agrobacterium tumefaciens LBA4404, obtain the restructuring agrobacterium tumefaciens LBA4404 that contains TsVP-als gene.
2, Agrobacterium is cultivated and activation
Will with binary vector, (Mini-Ti plasmid be with chlorsulfuron resistant gene als, genbank number of registration X51514.1) agrobacterium tumefaciens LBA4404 28 ℃ of concussions in additional antibiotic YEP substratum cultivate, concussion speed is 180～200r/min, make bacterium in logarithmic phase, bacterium liquid OD value (as 0.6) between 0.6～0.8.Then centrifugal 10min under 5000r/min, abandons supernatant liquor, obtains thalline.
CoM nutrient solution washing for thalline, centrifugal collection, for subsequent use.
On CoM nutrient solution basis, add the preferred 150mg/L of 100～200mg/L() Syringylethanone and mass percent concentration be 0.01～0.02%(preferably 0.15%) tensio-active agent Silwet-L77, and then to add the thalline that step 2 makes and make cell concentration be OD
600=0.6～0.8, make the dip-dyeing solution for transforming, described dip-dyeing solution pH is 5.8～6.0.
3, the cultivation of cotton etiolated seedling, obtains transgene receptor plant
Cotton seeds, after sulfuric acid lint, soaks 15min with 70% alcohol immersion 0.5～1.5min, 0.1% mercuric chloride, then with sterilized water washing 3～5 times.
After sterilization, seed is put into the aseptic bottle that is covered with three metafiltration paper, adds appropriate sterilized water, in 25～28 ℃ of incubators, sprouts.Hypocotyl elongation to 1 after 2～3 days～2cm(Fig. 2-1).
Germinating seed is inserted in CoM solid medium (pH5.8～6.0) and continues dark cultivate (25～28 ℃, 5～6d), get plant height 8～10cm seedling (preferably 8cm) and transform (Fig. 2-2) for stem apex.
4, cotton etiolated seedling meristematic tissue transforms
Get the cotton yellow seedling that plant height 8～10cm is high, carefully remove black seed coat and a slice cotyledon, expose cotton etiolated seedling shoot apical meristem;
Scratch shoot apical meristem from etiolated seedling stem apex top, that is: with knife blade, the meristematic tissue on stem apex top is done to " ten " font or the scuffing of " rice " font, and the degree of depth of control scuffing wound is 0.6～1.0mm;
After scuffing etiolated seedling shoot apical meristem (but can not destroy shoot apical meristem), dip in suction dip-dyeing solution with sterilizing rayon balls, and place it in the position, etiolated seedling stem apex top (Fig. 2-3) after scuffing, suck the unnecessary residual bacterium liquid of infection site with aseptic thieving paper.Be under 0.05～0.06MPa condition in negative pressure of vacuum, have the rayon balls of dip-dyeing solution to be placed in the position, etiolated seedling stem apex top after scuffing with inhaling, under the condition of 25～28 ℃ of temperature, relative air humidity 40～60%, contaminate the preferred 10min of 10～15min().
5, the transplanting of transformed plant
Cotton etiolated seedling bud point after dip-dye blots bacterium liquid with aseptic filter paper and in dark, cultivates 3 days, and culture temperature is 25～28 ℃, relative humidity 40～60%.Then aseptic seedling is placed under scattered light and cultivates 2 days, culture temperature is 25～28 ℃, relative humidity 40～60%.
Aseptic seedling after irradiation is cultivated is transplanted in the flowerpot that is covered with upper strata vermiculite and lower floor's loam, and vermiculite covers top (Fig. 2-4).Then allow plant grow under natural lighting, day temperature 25～28 ℃, night 20～22 ℃ of temperature, the next day water 1/2 modified MS medium inorganic salt, to growing true leaf.
6, the resistance screening of transgene cotton
When having grown to after 2～3 true leaves (Fig. 2-5), utilization is moistened with the grand rayon balls of selective agent chlorine sulphur and is placed in position, transformation seedlings stem apex top, carries out resistance screening, obtains resistant plant.
(25～28 ℃ of day temperature in greenhouse, night 20～22 ℃ of temperature, relative humidity 40～60%) plant to be transformed grows after 2～3 true leaves, to contain 20～40mg/l(factor receptor genotype and variant) grand (Shenyang Agricultural Chemicals Factory produces chlorine sulphur, effective constituent 25%) rayon balls of the aqueous solution is placed on plant bud pointed tip 12～24h, take unconverted plant as contrast.After 10～15d, observe the merismatic survival condition of plant bud pointed tip.Non-transformed plant strain growth point blackout is dead, and it is green that transfer-gen plant stem end vegetative point keeps, and upgrowth situation is good.
Plant to be survived grew to for 5～6 leaf phases, and its field planting is arrived to land for growing field crops.Seed in results resistant plant cotton boll.
7, Molecular Detection
With CTAB method trace extraction survival plant genomic dna.Adopt round pcr to detect foreign gene.The PCR primer of amplification TsVP gene: P1,5 '-CAGAACTCGCCGTAAAGACT-3 '; P2,5 '-GCAGAAACCGAAGATAACG-3 '.The PCR result of transfer-gen plant: transformed plant amplifies the fragment (see figure 3) of expection 550bp size.
8, transformation efficiency
Obtain Herbicid resistant plant from step (3)～(6) and complete PCR checking.Calculate transformation efficiency (n is as the plant number of PCR checking, N are as transformation seedlings number) take n/N* (100%).Positive plant with every group of PCR checking that transforms 2000 strain etiolated seedlings, repeat for 5 times to obtain calculates transformation efficiency, utilize the method for transformation after improving, Shandong cotton is ground No. 21, Shandong cotton and grinds No. 19, Shandong cotton and grind the transformation efficiency of No. 35 and be respectively 7.56 ± 0.45%, 15.47% ± 0.66 and 6.82 ± 0.37%, and before improving, the average conversion of method for transformation is respectively 5.51 ± 1.29%, 11.8 ± 3.21% and 5.2 ± 1.11%.The transformation efficiency of cotton improves more than 30%, and reproducible between group.
Claims (5)
1. a method that improves cotton etiolated seedling shoot apical meristem gene transformation rate, step is as follows:
A) after cotton seeds sterilizing, in incubator, sprout dark place, proceeds in CoM solid medium after hypocotyl grows to 1～2cm, puts dark place and grow to the etiolated seedling that plant height is 8～10cm, transforms for stem apex;
B) seed coat and a slice cotyledon that remove etiolated seedling expose shoot apical meristem, then scratch stem apex top;
C) dip in suction dip-dyeing solution with rayon balls, and place it in the position, etiolated seedling stem apex top after scuffing, under condition of negative pressure, implement to contaminate;
D) by contaminate after etiolated seedling in 25～28 ℃, secretly cultivate 3 ± 0.5 days, then light cultivate 2～3 days;
E) chrysanthemum seedling replanting after cultivation is entered to flowerpot, under 25～28 ℃ of temperature, humidity 40～60% conditions, cultivate, to growing 2～3 true leaves;
F) dip in suction selective agent with rayon balls, and place it in the position, transformation seedlings stem apex top that grows 2～3 true leaves, implement resistance screening, obtain resistant plant;
It is characterized in that:
The method of described scuffing stem apex is: the meristematic tissue on stem apex top is done to " ten " font or the scuffing of " rice " font, and the degree of depth of control scuffing wound is 0.6～1.0mm;
The method that described enforcement is contaminated is: be under 0.05～0.06MPa condition in negative pressure of vacuum, there is the rayon balls of dip-dyeing solution to be placed in the position, etiolated seedling stem apex top after scuffing with suction, under the condition of 25～28 ℃ of temperature, relative air humidity 40～60%, contaminate 10～15min; Wherein, described dip-dyeing solution composition is: on CoM nutrient solution basis, added the Syringylethanone of 100～200mg/L and the tensio-active agent Silwet-L77 that mass percent concentration is 0.01～0.02%, and the cell concentration that contains the foreign gene of recombinating is OD
600=0.6～0.8 Agrobacterium, described dip-dyeing solution pH is 5.8～6.0;
Described selective agent is weedicide, the method of described enforcement resistance screening is: under day 25～28 ℃ of temperature, night 20～22 ℃ of temperature, relative humidity 40～60% conditions, there is the rayon balls of selective agent to be placed in transformation seedlings stem apex top position 12～24h with suction, after 10～15d, observe the merismatic survival condition of plant bud pointed tip and select resistant plant, it is green that transfer-gen plant stem end vegetative point keeps, well-grown, non-transformed plant strain growth point blackout is dead.
2. the method that improves according to claim 1 cotton etiolated seedling shoot apical meristem gene transformation rate, is characterized in that: the degree of depth of described scuffing wound is 0.6～0.8mm.
3. the method that improves according to claim 1 cotton etiolated seedling shoot apical meristem gene transformation rate, is characterized in that: described immerged time 10～12min.
4. the method that improves according to claim 1 cotton etiolated seedling shoot apical meristem gene transformation rate, is characterized in that: described weedicide is that chlorine sulphur is grand.
5. the method that improves according to claim 4 cotton etiolated seedling shoot apical meristem gene transformation rate, is characterized in that: the grand concentration of described chlorine sulphur is 20～40mg/L.
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| CN105420274A (en) * | 2015-12-07 | 2016-03-23 | 中国农业大学 | Agrobacterium-mediated mature maize embryo shoot apex transformation method |
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