CN106755073A - A kind of pocket orchid plasmid Ovary injection transgenic method - Google Patents
A kind of pocket orchid plasmid Ovary injection transgenic method Download PDFInfo
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Abstract
The invention discloses a kind of pocket orchid plasmid Ovary injection transgenic method.It is that the plasmid parenteral solution containing the plant expression vector for carrying genes of interest is expelled to after the blue pollination of pocket and is completed in the ovary before double fertilization, then the seed that will be developed into carries out aseptic seeding as explant, and transgenosis pocket orchid plant is obtained through the induction of protocorm, the screening of resistance protocorm and Molecular Detection.Pocket of the invention orchid plasmid Ovary injection transgenic method has the advantages that simple to operate, fruiting rate is high and high conversion rate, is conducive to the blue molecular breeding of pocket and accelerates the blue breeding process of pocket.
Description
Technical field：
The invention belongs to plant biotechnology field, and in particular to a kind of pocket orchid plasmid Ovary injection transgenic method.
Background technology：
Pocket orchid (Paphiopedilum) due to its unique flower moulding, gorgeous flower color, lasting view and admire the florescence
And there is high ornamental value, it is International Flower in the market top grade flower all the fashion.But because people are to the blue mistake of pocket
Degree excavation and the destruction in habitat, pocket orchid have turned into one of plant species most in imminent danger in the world, and all wild species are put into
《CITS》(CITES) annex I and be prohibited trade.By various breeding method energy seed selections
Go out free transaction in the international market, ornamental value orchid new varieties high to meet market needs.The transgenosis of orchid
Method have agrobacterium-mediated transformation and particle bombardment etc., main species include dendrobium, iris, oncidiumLuridum and epidendrum
Deng, but agrobacterium-mediated transformation and particle bombardment etc. be both needed to set up on the basis of effective regenerating system.However, cypripedium
Regenerating system be difficult to set up, both at home and abroad also without pocket orchid agrobacterium-mediated transformation and particle bombardment transgenic method report.Profit
This problem can be efficiently solved with plasmid Ovary injection.
Orchid Seeds enormous amount, in a general Fruit pod grain weight more than tens thousand of to hundreds thousand of, using matter
When grain Ovary injection carries out live body genetic transformation, even if transformation efficiency is relatively low, more transformed plant can be also obtained.But orchid family
Plant generally requires tens days from pollination to fertilization process, and in the of short duration period of prefecundation, due to reproduction cell or fertilization
Ovum does not have cell membrane or cell membrane weaker, in more sensitive competence, is easier to be converted, therefore selection is suitable
Injection time be that Ovary injection transgenosis is successfully crucial.
The content of the invention：
It is an object of the invention to overcome deficiency of the prior art, there is provided a kind of pocket orchid plasmid Ovary injection transgenosis
Method.
Pocket orchid plasmid Ovary injection transgenic method of the invention, it is characterised in that comprise the following steps：
1) plasmid Ovule injection：Carry out artificial pollination to pocket orchid, the ovary of 45~70 days carries out surface and disappears after selection pollination
, be expelled to plasmid parenteral solution inside ovary along ovary is longitudinally perpendicular then by poison, and injection volume is 20~30 μ L, to injection after injection
Position is sealed；Described plasmid parenteral solution contain 200~400 μ g/mL carrying genes of interest plant expression vector and
The dimethyl sulfoxide (DMSO) of volume fraction 1%~3%, remaining is water；Described plant expression vector carries resistant gene；
2) resistant plant screening and detection：The capsule that winning the injection of 180~210 days after pollinating has plasmid parenteral solution is carried out
Aseptic seeding, capsule is carried out disinfection treatment, cuts capsule under aseptic condition open, is taken out seed and is simultaneously carried out disinfection, then by seed
It is suspended in sterilized water and is made seed suspension liquid and is seeded on improvement H26 culture mediums, 26~30 DEG C of cultivation temperature, light application time
14~18h/d, 1600~2000lx of intensity of illumination, seed is sprouted to form protocorm, by protocorm be forwarded to containing with plant table
Step sizing, cultivation temperature 26 are carried out on up to the improvement H26 culture mediums of the corresponding antibiotic of the resistant gene of carrier carrying
~30 DEG C, 14~18h/d of light application time, 1600~2000lx of intensity of illumination obtain resistance protocorm, by the switching of resistance protocorm
Onto improvement H26 culture mediums, 26~30 DEG C of cultivation temperature, 14~18h/d of light application time, 1600~2000lx of intensity of illumination,
Culture obtains transgenosis pocket orchid plant to seedling is formed through Molecular Detection；
Described improvement H26 culture mediums are：Every liter of culture medium contains spends precious No. 1 1~2g, 10~20g of sucrose, peptone
1~3g, 1~3g of tyrosine, 5~6g of agar, 1.0~2.0g of activated carbon, vitamin B110mg, vitamin B61mg, nicotinic acid
1mg, glycine 2mg, inositol 0.1mg, 150~200mg of sodium dihydrogen phosphate, 50~100mL of 0.5~1mg of methyl α-naphthyl acetate and coconut milk,
Balance of water.
Described resistant gene is preferably hygromycin gene.
Described plant expression vector is preferably pCAMBIA1301.
It is dream pocket orchid (Paphiopedilum SCBG Dream) that described pocket is blue.
Described genes of interest is preferably sword-leaved cymbidium CeFT genes.
Described treatment that capsule carries out disinfection is specially alcohol-pickled the 1 of capsule volume fraction 70%~80%~
2 minutes, then with the mercuric chloride solution 10~20min of soaking disinfection of mass fraction 0.1%~0.2%, aseptic water washing 3~5 times.
Described be forwarded to protocorm contains the antibiotic corresponding with the resistant gene that plant expression vector is carried
Improveing carries out step sizing on H26 culture mediums, 26~30 DEG C of cultivation temperature, light application time 16h/d, and intensity of illumination 1600~
2000lx, obtains resistance protocorm and is specially：Protocorm is forwarded to the improvement H26 culture mediums containing 30~50mg/L hygromycin
On, 26~30 DEG C of cultivation temperature, 14~18h/d of light application time, 1600~2000lx of intensity of illumination will be deposited during 50~65d of culture
Protocorm living is forwarded on the improvement H26 culture mediums containing 40~60mg/L hygromycin, 26~30 DEG C of cultivation temperature, during illumination
Between 14~18h/d, 1600~2000lx of intensity of illumination, during 50~65d of culture by the protocorm of survival be forwarded to it is new containing 40~
On the improvement H26 culture mediums of 60mg/L hygromycin, 26~30 DEG C of cultivation temperature, 14~18h/d of light application time, intensity of illumination
1600~2000lx, the protocorm of survival is forwarded to No. H26 training of improvement containing 30~50mg/L hygromycin after 50~65d of culture
Support on base, 26~30 DEG C of cultivation temperature, 14~18h/d of light application time, 1600~2000lx of intensity of illumination, cultivate 50~65d days
Resistance protocorm is obtained afterwards.
The present invention is using orchid (Complex standard hybrids of Paphiopedilum) the kind dream of standard type pocket
Think pocket orchid (Paphiopedilum SCBG Dream) for material carries out plasmid Ovary injection transgenosis, due to dream pocket it is blue from
Plant to bloom and needed for 4 years, it is desirable to make that dream pocket is blue to bloom ahead of time by being transferred to sword-leaved cymbidium integrator gene (CeFT genes) of blooming.
Pocket orchid plasmid Ovary injection transgenic method of the invention, by the way that the plant table for carrying genes of interest will be contained
Plasmid parenteral solution up to carrier is expelled to after the blue pollination of pocket and completes in the ovary before double fertilization, and the seed that then will be developed into is made
For explant carries out aseptic seeding, transgenosis pocket is obtained through the induction of protocorm, the screening of resistance protocorm and Molecular Detection blue
Plant.Pocket orchid plasmid Ovary injection transgenic method of the invention has simple to operate, fruiting rate high and high conversion rate excellent
Point, is conducive to the blue molecular breeding of pocket and accelerates the blue breeding process of pocket.
Specific embodiment：
Following examples are further illustrated to of the invention, rather than limitation of the present invention.
Embodiment 1：
1. prepared by plasmid parenteral solution
Structure containing destination gene expression carrier：Bloomed integrator gene (CeFT) (GenBank accession number with sword-leaved cymbidium：
HM803115) it is purpose gene, designs primer：CeFT-F：5’-TCTAGAATGAATAGAGAGAGAGACTC-3 ' (underscore portions
It is divided into Xba I restriction enzyme sites), CeFT-R：5’-GGATCC(underscore part is BamH I to TCAATCCTGCATCCTTCTTC-3 '
Restriction enzyme site), using the cDNA of sword-leaved cymbidium bud as template, using CeFT-F and CeFT-R as primer amplified band restriction enzyme site Xba
The sword-leaved cymbidium CeFT genes of I and BamH I, are connected on the PMD18-T carriers of Takara, further according to J. Pehanorm Brookers etc.《Molecule
Cloning experimentation guide》Converted and identified.The positive plasmid that will be identified is using restriction enzyme site Xba I and BamH I from carrier T
On cut CeFT genes, with plant expression vector pCAMBIA1301 (the GenBank accession number with 35s promoters：
AF234297) same to be connected with after Xba I and BamH I digestions, construction of expression vector pCAMBIA1301-CeFT is tested through sequencing
Card, CeFT genes insert plant expression vector pCAMBIA1301.The sweet enzyme base of β-gluconic acid is carried on pCAMBIA1301
Because of (Gus A), hygromycin gene (Hpt) and kalamycin resistance gene (kan).
The preparation of plasmid parenteral solution：Engineering bacteria containing recombinant plasmid pCAMBIA1301-CeFT is being contained into 50mg/L Kan
LB fluid nutrient mediums in shaken cultivation, 37 DEG C of shaken cultivations to OD600Be worth is 0.6.Then further according to《Molecular Cloning: A Laboratory refers to
South》Using alkaline lysis method of extracting recombinant plasmid pCAMBIA1301-CeFT, by the plasmid dimethyl sulfoxide (DMSO) of volume fraction 1%
(DMSO) aqueous dissolution, makes the concentration of plasmid for 200 μ g/mL, as plasmid parenteral solution.
2. plasmid Ovule injection
Artificial pollination is carried out to dream pocket orchid (Paphiopedilum SCBG Dream) in greenhouse, after pollination is completed
45 days (complete double fertilizations before) carry out Ovule injection, the injection volume of each ovary is 20 μ L.During injection, alcohol swab is first used
(70% alcohol) wipes ovary surface and the debris on ovary surface is attached to remove, then is slowly inhaled with sterilized micro syringe
Plasmid parenteral solution is taken to physically well develop, behind the ovary inside that growing way is consistent, extract needle tubing alcohol out along longitudinally perpendicular being injected into of ovary
Cotton rub is wiped at injection and sealed at injection with the paper adhesive plaster sterilized, fruiting rate 65%.Win fruit and carry out within 210 days after pollination
Aseptic seeding.
3. resistant plant screening
The capsule that winning the injection of 210 days after pollinating has plasmid parenteral solution carries out aseptic seeding.During sowing, first capsule is put
Rinsed under running water and stick to the supracutaneous debris of capsule removing, then with 1 point of the alcohol-pickled capsule of volume fraction 70%
After clock, then with taking out capsule after the mercuric chloride solution soaking disinfection 20min of mass fraction 0.1%, aseptic water washing 3 times, capsule is blotted
Fruit surface moisture, on the aseptic operating platform with the scalpel after sterilization along capsule in suture longitudinally cut open, take out seed and general
It is put into the triangular flask of sterilizing, adds sodium hypochlorite (NaClO) solution soaking disinfection of the existing mass fraction 0.5% matched somebody with somebody
After 40min, aseptic water washing filter 23 time, seed is placed in sterilized water and is made suspension and is seeded into improvement H26 culture mediums
On, 26 DEG C of cultivation temperature, light application time 14h/d, intensity of illumination 1600lx, seed is sprouted to form protocorm, and protocorm is transferred
Cultivated on to the improvement H26 culture mediums of hygromycin containing 30mg/L (Hyg), every bottle is inoculated with 50,26 DEG C of cultivation temperature, during illumination
Between 14h/d, intensity of illumination 1600lx, the protocorm of survival is transferred to the improvement of hygromycin containing 40mg/L (Hyg) during culture 50d
On H26 culture mediums, 26 DEG C of cultivation temperature, light application time 14h/d, intensity of illumination 1600lx, the protocorm that will be survived during culture 50d
Stem is transferred on the improvement H26 culture mediums containing 40mg/L hygromycin and cultivates, 26 DEG C of cultivation temperature, light application time 14h/d, illumination
Intensity 1600lx, the protocorm of survival is transferred to the improvement H26 culture mediums of hygromycin containing 30mg/L (Hyg) during culture 50d
On carry out renewal cultivation, 26 DEG C of cultivation temperature, light application time 14h/d, intensity of illumination 1600lx, it is mould that culture 50d screenings obtain tide
Plain resistance protocorm, the hygromycin resistance protocorm filtered out by 4 wheels is transferred on improvement H26 culture mediums again, cultivation temperature
26 DEG C, light application time 14h/d, intensity of illumination 1600lx, culture form seedling after 90d days.It is calculated as follows conversion ratio：Turn
Rate=(seedling number/for the protocorm number of screening) × 100%.Conversion ratio is 0.30%.
Improveing H26 culture mediums is：Every liter of culture medium contain spend precious No. 1 (Hyponex I) 1g, sucrose (sugar) 10g,
Peptone (peptone) 1g, tyrosine (tyrosine) 1g, agar (agar) 5.0g, activated carbon (AC) 1.0g, vitamin B1
10mg, vitamin B61mg, nicotinic acid 1mg, glycine 2mg, inositol 0.1mg, sodium dihydrogen phosphate (NaH2PO4) 150mg, methyl α-naphthyl acetate
(NAA) 0.5mg and coconut milk 50mL, balance of water.
The compound method for improveing H26 culture mediums is that 1g is spent into precious No. 1,10g sucrose, 1g peptones, 1g tyrosine, 5.0g
Agar, 1.0g activated carbons, 10mg vitamin Bs1, 1mg vitamin Bs6, 1mg nicotinic acid, 2mg glycine, 0.1mg inositols, 150mg phosphoric acid
Sodium dihydrogen, 0.5mg methyl α-naphthyl acetates and 50mL coconut milk, are added in a small amount of water, then with water polishing to 1L, are well mixed, and sterilize standby
With.
4. resistant plant detection
Seedling to obtaining carries out GUS dyeing, PCR detections and Southern blot detections.GUS dyeing is in blueness,
PCR is detected and Southern blot detect simultaneously there is Gus A genes and CeFT genes, it is determined that the seedling for obtaining is and turns base
Because of pocket orchid plant.
Embodiment 2：
1. prepared by plasmid parenteral solution
Structure containing destination gene expression carrier：Bloomed integrator gene (CeFT) (GenBank accession number with sword-leaved cymbidium：
HM803115) it is purpose gene, designs primer：CeFT-F：5’-TCTAGAATGAATAGAGAGAGAGACTC-3 ' (underscore portions
It is divided into Xba I restriction enzyme sites), CeFT-R：5’-GGATCC(underscore part is BamH I to TCAATCCTGCATCCTTCTTC-3 '
Restriction enzyme site), using the cDNA of sword-leaved cymbidium bud as template, using CeFT-F and CeFT-R as primer amplified band restriction enzyme site Xba
The sword-leaved cymbidium CeFT genes of I and BamH I, are connected on the PMD18-T carriers of Takara, further according to J. Pehanorm Brookers etc.《Molecule
Cloning experimentation guide》Converted and identified.The positive plasmid that will be identified is using restriction enzyme site Xba I and BamH I from carrier T
On cut CeFT genes, with plant expression vector pCAMBIA1301 (the GenBank accession number with 35s promoters：
AF234297) same to be connected with after Xba I and BamH I digestions, construction of expression vector pCAMBIA1301-CeFT is tested through sequencing
Card, CeFT genes insert plant expression vector pCAMBIA1301.The sweet enzyme base of β-gluconic acid is carried on pCAMBIA1301
Because of (Gus A), hygromycin gene (Hpt) and kalamycin resistance gene (kan).
The preparation of plasmid parenteral solution：Engineering bacteria containing recombinant plasmid pCAMBIA1301-CeFT is being contained into 50mg/L Kan
LB fluid nutrient mediums in shaken cultivation, 37 DEG C of shaken cultivations to OD600Be worth is 0.6.Then further according to《Molecular Cloning: A Laboratory refers to
South》Using alkaline lysis method of extracting recombinant plasmid pCAMBIA1301-CeFT, by the plasmid dimethyl sulfoxide (DMSO) of volume fraction 2%
(DMSO) aqueous dissolution, makes the concentration of plasmid for 300 μ g/mL, as plasmid parenteral solution.
2. plasmid Ovule injection
Artificial pollination is carried out to dream pocket orchid (Paphiopedilum SCBG Dream) in greenhouse, after pollination is completed
60 days (complete double fertilizations before) carry out Ovule injection, the injection volume of each ovary is 25 μ L.During injection, alcohol swab is first used
(75% alcohol) wipes ovary surface and the debris on ovary surface is attached to remove, then is slowly inhaled with sterilized micro syringe
Plasmid parenteral solution is taken to physically well develop, behind the ovary inside that growing way is consistent, extract needle tubing alcohol out along longitudinally perpendicular being injected into of ovary
Cotton rub is wiped at injection and sealed at injection with the paper adhesive plaster sterilized, fruiting rate 70%.Win fruit and carry out within 195 days after pollination
Aseptic seeding.
3. resistant plant screening
The capsule that winning the injection of 195 days after pollinating has plasmid parenteral solution carries out aseptic seeding.During sowing, first capsule is put
Rinsed under running water and the supracutaneous debris of capsule are sticked to removal, then with the alcohol-pickled Fruit pod 1.5 of volume fraction 75%
Minute, then with taking out capsule after the mercuric chloride solution soaking disinfection 15min of mass fraction 0.15%, after aseptic water washing 4 times, blot
Capsule surface moisture, on the aseptic operating platform with the scalpel after sterilization along capsule in suture longitudinally cut open, take out seed simultaneously
Put it into the triangular flask of sterilizing, add sodium hypochlorite (NaClO) solution soaking disinfection of the existing mass fraction 1.0% matched somebody with somebody
30min, aseptic water washing filter 4 times after, seed is placed in sterilized water be made suspension be seeded into improvement H26 culture mediums
On, 28 DEG C of cultivation temperature, light application time 16h/d, intensity of illumination 1800lx, seed is sprouted to form protocorm, and protocorm is transferred
To the improvement H26 culture mediums of hygromycin containing 40mg/L (Hyg), every bottle is inoculated with 50,28 DEG C of cultivation temperature, light application time
16h/d, intensity of illumination 1800lx, the protocorm of survival is transferred to the improvement of hygromycin containing 50mg/L (Hyg) during culture 60d
On H26 culture mediums, 28 DEG C of cultivation temperature, light application time 16h/d, intensity of illumination 1800lx, the protocorm that will be survived during culture 60d
Stem is transferred on the improvement H26 culture mediums containing 50mg/L hygromycin, 28 DEG C of cultivation temperature, light application time 16h/d, intensity of illumination
1800lx, the improvement H26 culture mediums that the protocorm of survival is transferred into hygromycin containing 40mg/L (Hyg) during culture 60d are enterprising
Row renewal cultivation, 28 DEG C of cultivation temperature, light application time 16h/d, intensity of illumination 1800lx, it is former that culture 60d obtains hygromycin resistance
Bulb, the hygromycin resistance protocorm filtered out by 4 wheels is transferred on improvement H26 culture mediums again, 28 DEG C of cultivation temperature, illumination
Time 16h/d, intensity of illumination 1800lx, culture form seedling after 75d days.It is calculated as follows conversion ratio：Conversion ratio=(small
Seedling number/for the protocorm number of screening) × 100%.Conversion ratio is 1.0%.
Improveing H26 culture mediums is：Every liter of culture medium contains spends precious No. 1 (Hyponex I) 1.5g, sucrose (sugar)
15g, peptone (peptone) 2g, tyrosine (tyrosine) 2g, agar (agar) 5.5g, activated carbon (AC) 1.5g, vitamin
B110mg, vitamin B61mg, nicotinic acid 1mg, glycine 2mg, inositol 0.1mg, sodium dihydrogen phosphate (NaH2PO4) 170mg, naphthalene second
Acid (NAA) 0.75mg and coconut milk 75mL, balance of water.
The compound method for improveing H26 culture mediums be by 1.5g spend precious No. 1,15g sucrose, 2g peptones, 2g tyrosine,
5.5g agar, 1.5g activated carbons (AC), 10mg vitamin Bs1, 1mg vitamin Bs6, 1mg nicotinic acid, 2mg glycine, 0.1mg inositols,
170mg sodium dihydrogen phosphates, 0.75mg methyl α-naphthyl acetates and 75mL coconut milk, are added in a small amount of water, and then with water polishing to 1L, mixing is equal
It is even, sterilize standby.
4. resistant plant detection
Seedling to obtaining carries out GUS dyeing, PCR detections and Southern blot detections.GUS dyeing is in blueness,
PCR is detected and Southern blot detect simultaneously there is Gus A genes and CeFT genes, it is determined that the seedling for obtaining is and turns base
Because of pocket orchid plant.
Embodiment 3：
1. prepared by plasmid parenteral solution
Structure containing destination gene expression carrier：Bloomed integrator gene (CeFT) (GenBank accession number with sword-leaved cymbidium：
HM803115) it is purpose gene, designs primer：CeFT-F：5’-TCTAGAATGAATAGAGAGAGAGACTC-3 ' (underscore portions
It is divided into Xba I restriction enzyme sites), CeFT-R：5’-GGATCC(underscore part is BamH I to TCAATCCTGCATCCTTCTTC-3 '
Restriction enzyme site) using the cDNA of sword-leaved cymbidium bud as template, using CeFT-F and CeFT-R as primer amplified band restriction enzyme site Xba I
With the sword-leaved cymbidium CeFT genes of BamH I, it is connected on the PMD18-T carriers of Takara, further according to J. Pehanorm Brookers etc.《Molecule
Cloning experimentation guide》Converted and identified.The positive plasmid that will be identified is using restriction enzyme site Xba I and BamH I from carrier T
On cut CeFT genes, with plant expression vector pCAMBIA1301 (the GenBank accession number with 35s promoters：
AF234297) same to be connected with after Xba I and BamH I digestions, construction of expression vector pCAMBIA1301-CeFT is tested through sequencing
Card, CeFT genes insert plant expression vector pCAMBIA1301.The sweet enzyme base of β-gluconic acid is carried on pCAMBIA1301
Because of (Gus A), hygromycin gene (Hpt) and kalamycin resistance gene (kan).
The preparation of plasmid parenteral solution：Engineering bacteria containing recombinant plasmid pCAMBIA1301-CeFT is being contained into 50mg/L Kan
LB fluid nutrient mediums in shaken cultivation, 37 DEG C of shaken cultivations to OD600Be worth is 0.6.Then further according to《Molecular Cloning: A Laboratory refers to
South》Using alkaline lysis method of extracting recombinant plasmid pCAMBIA1301-CeFT, by the plasmid dimethyl sulfoxide (DMSO) of volume fraction 3%
(DMSO) aqueous dissolution, makes the concentration of plasmid for 400 μ g/mL, as plasmid parenteral solution.
2. plasmid Ovule injection
Artificial pollination is carried out to dream pocket orchid (Paphiopedilum SCBG Dream) in greenhouse, after pollination is completed
70 days (complete double fertilizations before) carry out Ovule injection, the injection volume of each ovary is 30 μ L.During injection, alcohol swab is first used
(80% alcohol) wipes ovary surface and the debris on ovary surface is attached to remove, then is slowly inhaled with sterilized micro syringe
Plasmid parenteral solution is taken to physically well develop, behind the ovary inside that growing way is consistent, extract needle tubing alcohol out along longitudinally perpendicular being injected into of ovary
Cotton rub is wiped at injection and sealed at injection with the paper adhesive plaster sterilized, fruiting rate 75%.Win fruit and carry out within 180 days after pollination
Aseptic seeding.
3. resistant plant screening
The capsule that winning the injection of 180 days after pollinating has the injection of plasmid parenteral solution carries out aseptic seeding.During sowing, first will
Capsule is placed under running water to rinse and stick to the supracutaneous debris of capsule removing, then with the alcohol-pickled capsule of volume fraction 80%
Really 2 minutes, then with taking out capsule after the mercuric chloride solution soaking disinfection 10min of mass fraction 0.2%, after aseptic water washing 5 times, inhale
Dry capsule surface moisture, on the aseptic operating platform with the scalpel after sterilization along capsule in suture longitudinally cut open, take out seed
And in putting it into the triangular flask of sterilizing, add sodium hypochlorite (NaClO) solution soaking disinfection of the existing mass fraction 2.0% matched somebody with somebody
20min, aseptic water washing filter 5 times after, seed is placed in sterilized water be made suspension be seeded into improvement H26 culture mediums
On, 30 DEG C of cultivation temperature, light application time 18h/d, intensity of illumination 2000lx, seed is sprouted to form protocorm, and protocorm is transferred
To the improvement H26 culture mediums of hygromycin containing 50mg/L (Hyg), every bottle is inoculated with 50,30 DEG C of cultivation temperature, light application time
18h/d, intensity of illumination 2000lx, the protocorm of survival is transferred to the improvement of hygromycin containing 60mg/L (Hyg) during culture 65d
On H26 culture mediums, 30 DEG C of cultivation temperature, light application time 18h/d, intensity of illumination 2000lx, the protocorm that will be survived during culture 65d
Stem is transferred on the improvement H26 culture mediums containing 60mg/L hygromycin, 30 DEG C of cultivation temperature, light application time 18h/d, intensity of illumination
2000lx, the improvement H26 culture mediums that the protocorm of survival is transferred into hygromycin containing 50mg/L (Hyg) during culture 65d are enterprising
Row renewal cultivation, 30 DEG C of cultivation temperature, light application time 18h/d, intensity of illumination 2000lx, culture 65d screenings obtain hygromycin and resist
Property protocorm, be transferred to again on improvement H26 culture mediums by the 4 hygromycin resistance protocorms that filter out of wheel, 30 DEG C of cultivation temperature,
Light application time 18h/ days, intensity of illumination 2000lx, culture forms seedling after 60d days.It is calculated as follows conversion ratio：Conversion ratio
=(seedling number/for the protocorm number of screening) × 100%.Conversion ratio is 1.50%.
Improveing H26 culture mediums is：Every liter of culture medium contain spend precious No. 1 (Hyponex I) 2g, sucrose (sugar) 20g,
Peptone (peptone) 3g, tyrosine (tyrosine) 3g, agar (agar) 6g, activated carbon (AC) 2g, vitamin B1 10mg、
Vitamin B61mg, nicotinic acid 1mg, glycine 2mg, inositol 0.1mg, sodium dihydrogen phosphate (NaH2PO4) 200mg, methyl α-naphthyl acetate (NAA)
1mg and coconut milk 100mL, balance of water.
The compound method for improveing H26 culture mediums is that 2g is spent into precious No. 1,20g sucrose, 3g peptones, 3g tyrosine, 6g fine jades
Fat, 2g activated carbons, 10mg vitamin Bs1, 1mg vitamin Bs6, 1mg nicotinic acid, 2mg glycine, 0.1mg inositols, 200mg biphosphates
Sodium, 1mg methyl α-naphthyl acetates and 100mL coconut milk, are added in a small amount of water, then with water polishing to 1L, are well mixed, and sterilize standby.
4. resistant plant detection
Seedling to obtaining carries out GUS dyeing, PCR detections and Southern blot detections.GUS dyeing is in blueness,
PCR is detected and Southern blot detect simultaneously there is Gus A genes and CeFT genes, it is determined that the seedling for obtaining is and turns base
Because of pocket orchid plant.
Claims (7)
1. a kind of pocket orchid plasmid Ovary injection transgenic method, it is characterised in that comprise the following steps：
1) plasmid Ovule injection：Artificial pollination is carried out to pocket orchid, the ovary of 45~70 days carries out surface sterilization after selection pollination, so
Plasmid parenteral solution is expelled to inside ovary along ovary is longitudinally perpendicular afterwards, injection volume is 20~30 μ L, to injection site after injection
Sealed；Described plasmid parenteral solution contains the plant expression vector and volume of the carrying genes of interest of 200~400 μ g/mL
The dimethyl sulfoxide (DMSO) of fraction 1%~3%, remaining is water；Described plant expression vector carries resistant gene；
2) resistant plant screening and detection：The capsule that winning the injection of 180~210 days after pollinating has plasmid parenteral solution carries out aseptic
Sowing, capsule is carried out disinfection treatment, cuts capsule under aseptic condition open, and taking-up seed simultaneously carries out disinfection, then by seed suspension
It is made seed suspension liquid in sterilized water to be seeded on improvement H26 culture mediums, 26~30 DEG C of cultivation temperature, light application time 14~
18h/d, 1600~2000lx of intensity of illumination, seed is sprouted to form protocorm, by protocorm be forwarded to containing with plant express carry
Step sizing, cultivation temperature 26~30 are carried out on the improvement H26 culture mediums of the corresponding antibiotic of resistant gene that body is carried
DEG C, 14~18h/d of light application time, 1600~2000lx of intensity of illumination obtain resistance protocorm, resistance protocorm is transferred to and is changed
On good H26 culture mediums, 26~30 DEG C of cultivation temperature, 14~18h/d of light application time, 1600~2000lx of intensity of illumination, culture
To seedling is formed, transgenosis pocket orchid plant is obtained through Molecular Detection；
Described improvement H26 culture mediums are：Every liter of culture medium contain spend precious No. 1 1~2g, 10~20g of sucrose, peptone 1~
3g, 1~3g of tyrosine, 5~6g of agar, 1.0~2.0g of activated carbon, vitamin B110mg, vitamin B6It is 1mg, nicotinic acid 1mg, sweet
Propylhomoserin 2mg, inositol 0.1mg, 150~200mg of sodium dihydrogen phosphate, 0.5~1mg of methyl α-naphthyl acetate and coconut milk 50~100mL, it is balance of
Water.
2. pocket according to claim 1 orchid plasmid Ovary injection transgenic method, it is characterised in that described resistance base
Because hygromycin gene.
3. the blue plasmid Ovary injection transgenic method of pocket according to any one of claims 1 to 3, it is characterised in that institute
The plant expression vector stated is plasmid pCAMBIA1301.
4. pocket according to claim 1 orchid plasmid Ovary injection transgenic method, it is characterised in that described pocket orchid is
Dream pocket is blue.
5. pocket according to claim 1 orchid plasmid Ovary injection transgenic method, it is characterised in that described purpose base
Because sword-leaved cymbidium CeFT genes.
6. pocket according to claim 1 orchid plasmid Ovary injection transgenic method, it is characterised in that described by capsule
The treatment that carries out disinfection is specially alcohol-pickled 1~2 minute of capsule volume fraction 70%~80%, then uses mass fraction
0.1%~0.2% mercuric chloride solution 10~20min of soaking disinfection, aseptic water washing 3~5 times.
7. pocket according to claim 2 orchid plasmid Ovary injection transgenic method, it is characterised in that described by protocorm
Stem is forwarded on the improvement H26 culture mediums containing the antibiotic corresponding with the resistant gene that plant expression vector is carried and carries out
Step sizing, 26~30 DEG C of cultivation temperature, 14~18h/d of light application time, 1600~2000lx of intensity of illumination obtain resistance protocorm
Stem is specially：Protocorm is forwarded on the improvement H26 culture mediums containing 30~50mg/L hygromycin, cultivation temperature 26~30
DEG C, be forwarded to for the protocorm of survival during 50~65d of culture contain by 14~18h/d of light application time, 1600~2000lx of intensity of illumination
On the improvement H26 culture mediums of 40~60mg/L hygromycin, 26~30 DEG C of cultivation temperature, 14~18h/d of light application time, illumination is strong
1600~2000lx of degree, the protocorm of survival is forwarded to the improvement H26 containing 40~60mg/L hygromycin during 50~65d of culture
On culture medium, 26~30 DEG C of cultivation temperature, 14~18h/d of light application time, 1600~2000lx of intensity of illumination cultivate 50~65d
The protocorm of survival is forwarded on the improvement H26 culture mediums containing 30~50mg/L hygromycin afterwards, 26~30 DEG C of cultivation temperature,
14~18h/d of light application time, 1600~2000lx of intensity of illumination, culture obtain resistance protocorm after 50~65d days.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107177628A (en) * | 2017-07-26 | 2017-09-19 | 黔西南州绿缘动植物科技开发有限公司 | A kind of utilization During Agrobacterium method causes the method for spending the blue gene conversion of pocket in vain |
| CN107326044A (en) * | 2017-07-28 | 2017-11-07 | 黔西南州绿缘动植物科技开发有限公司 | A kind of transgenosis spends the blue implantation methods of pocket in vain |
-
2016
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Non-Patent Citations (2)
| Title |
|---|
| 王佳等: "子房注射法转化五唇兰的初步研究", 《热带作物学报》 * |
| 邓莹: "子房注射法转化金钗石斛初步研究", 《中国硕士学位论文全文数据库》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107177628A (en) * | 2017-07-26 | 2017-09-19 | 黔西南州绿缘动植物科技开发有限公司 | A kind of utilization During Agrobacterium method causes the method for spending the blue gene conversion of pocket in vain |
| CN107326044A (en) * | 2017-07-28 | 2017-11-07 | 黔西南州绿缘动植物科技开发有限公司 | A kind of transgenosis spends the blue implantation methods of pocket in vain |
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