CN101768604B - Efficient and rapid sugarcane genetically modified method - Google Patents
Efficient and rapid sugarcane genetically modified method Download PDFInfo
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- CN101768604B CN101768604B CN 201010110668 CN201010110668A CN101768604B CN 101768604 B CN101768604 B CN 101768604B CN 201010110668 CN201010110668 CN 201010110668 CN 201010110668 A CN201010110668 A CN 201010110668A CN 101768604 B CN101768604 B CN 101768604B
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Abstract
The invention belongs to the field of plant biotechnology, in particular to an efficient and rapid sugarcane genetically modified method, wherein spire tissues of sugarcane are taken as explants to induce embryonic callus; embryonic callus is taken as a receptor material infected by agrobacterium, and cultivates into embryoid, budlets and seedlings through differentiation, then resistance transformation is carried out to obtain the resistance transformation plants; then the DNA and RNA of resistance plants are extracted to carry out PCR and RT-PCR detection; the plants which are detected to be positive through PCR and RT-PCR detection are transplanted to a high tunnel, and then other molecular and physiology and biochemistry detections are carried out after the plants grow up. The invention can greatly improve transformation efficiency, shorten transformation time, obtain the transgenic plants which are detected to be positive by PCR and RT-PCR detections and only needs 4 months, and save transformation costs; and transplantation can be carried out after PCR and RT-PCR detections, thus reducing transplantation and management of a plurality of false resistance seedlings and lowering experimental cost.
Description
Technical field
The invention belongs to plant biotechnology field, relate to a kind of sugarcane genetically modified breeding method, specifically is a kind of efficiently sugarcane genetically modified method fast.
Technical background
Sugarcane is the whole world of paramount importance sugar material cash crop, also is China torrid zone, the important sugar material cash crop in subtropical zone.China is the third place in the world sugared state of big product at present, and 2008/2009 presses the total output of sugar in the season whole nation reaches 1,243 ten thousand tons, and wherein sucrose accounts for 94% of total output of sugar.Sugarcane is also developed into a kind of important energy crop in recent years, is to produce green fuel---the important source material of alcohol.
Because modern cultivation of sugar cane kind is the saccharum noble cane; Cut that hand is close to wait the interspecific hybrid of species with India kinds, in secular noble qualityization breeding process, maiotic feasible modern cultivation of sugar cane kind unusually forms a large amount of aneuploids; Its karyomit(e) quantity is many, genome structure is complicated, the karyomit(e) multiple is different in homophyletic; Simultaneously because sugarcane is responsive to photoperiodic reaction, and many important parents are difficult to bloom in subtropics and area, temperate zone, even the pollen quantity of blooming is also not enough; And the flowering asynchronism of frequent and kindred plant, this brings many difficulties for conventional breeding for stress tolerance.Therefore the genetically engineered transgenic technology has become the important channel of sugar cane breed genetic improvement.
The genetic transformation technology of sugarcane mainly contains particle gun conversion method and agrobacterium-mediated transformation at present.The acquisition of early stage sugarcane genetically modified plant mainly is to adopt the particle gun conversion method, but this method transformation efficiency is lower, and mosaic is many, and foreign gene inserts copy number than many, and genetic stability is poor, and it is high to transform cost.Since 1998, the world adopts agrobacterium-mediated transformation in a plurality of laboratories, obtains a variety of transfer-gen plants.The advantage of agrobacterium-mediated transformation is that the copy number of foreign gene insertion is low, and is little to the acceptor injury, and realized the conversion of large fragment DNA.In the conversion process of agrobacterium-mediated transformation; Though the correct employing of Syringylethanone has significantly improved the transformation efficiency of agrobacterium-mediated transformation; But its transformation efficiency is about 3%; Still can not satisfy the industrialized development demand of transgenic engineering; And agrobacterium-mediated transformation still exists not enough in the sugarcane genetic transformation: (1) is being followed the callus subculture of early gene rifle conversion method always and is being induced mode in the employing of sugarcane callus, the differentiation efficiency of callus own is on the low side, has directly influenced last transformation efficiency; (2) after Agrobacterium and callus are cultivated altogether, be difficult to Agrobacterium is thoroughly removed clean, cause differentiation culture and regenerating and culturing seriously polluted period; (3) the callus differentiation phase promptly screened after employing was cultivated altogether usually, but less because of the bud of sugarcane callus differentiation, resistant buds is difficult to grow tall, and caused in replacing substratum process, being difficult to resistant buds and the bud that is killed by selective agent are made a distinction.(4) transformation time is longer, reaches more than 6 months.
Summary of the invention
The objective of the invention is to overcome the deficiency of original agrobacterium-mediated transformation sugarcane genetic transformation technology and a kind of efficiently sugarcane genetically modified method fast is provided; Through changing transformation mode; Improve transformation efficiency greatly, shortened the time that transforms, practiced thrift the cost that transforms.
The technical scheme that the present invention adopted:
A kind of efficiently sugarcane genetically modified method fast; The process of inducing that comprises the sugarcane embryo callus; The activation of Agrobacterium and the conversion process of embryo callus, the differentiation regeneration and the screening process of embryo callus transform plantlet DNA/RNA trace extraction and PCR and RT-PCR qualification process:
1, the sugarcane embryo callus induce process: the spire with the apical growing point place of good cultivation of sugar cane kind is organized as explant; Explant is thinly sliced after sterilization, aseptic water washing, aseptic filter paper blot its surperficial moisture and is inoculated on the inducing culture, under the lucifuge condition, is cultured to and induces embryo callus.Said inducing culture is to be basic medium with the MS substratum, and adds 1-2mg/L 2,4-D, 30g/L sucrose, 8g/L carrageenin.
2, the conversion process of activation of Agrobacterium and embryo callus: the single colony inoculation of Agrobacterium EHA105 that will contain goal gene shakes cultivation in activation medium; Through centrifugal recovery thalline; Be resuspended in again and continue the concussion cultivation in the gene induced substratum of equal-volume vir; To induce Agrobacterium plasmid vir expression of gene, this bacterium liquid is the Agrobacterium engineering bacteria liquid that transforms usefulness; The sugarcane embryo callus is changed in the Agrobacterium bacterium liquid, be inoculated in after the immersion on the common culture medium and cultivate altogether down, be inoculated on the division culture medium then in dark condition.Said activation medium is to be basic medium with YEP, and adds corresponding microbiotic Kan 50 μ g/mL, Str50 μ g/mL, Rif 50 μ g/mL.The gene induced substratum of said vir is to be basic medium with the MS substratum; Wherein macroelement is revised as 1/5 of former content, and adds 1mg/L2.4-D, 10mmol/L fructose, 10mmol/L glucose, 30g/L sucrose, 100-150 μ mol/L Syringylethanone.Said altogether culture medium is to be basic medium with the MS substratum; Wherein macroelement is revised as 1/5 of former content, and adds 1mg/L2.4-D, 10mmol/L fructose, 10mmol/L glucose, 30g/L sucrose, 100-150 μ mol/L Syringylethanone, carrageenin 20g/L.Said division culture medium is to be basic medium with the MS substratum, and adds 1mg/L6-BA, 0.2mg/LKT, 30g/L sucrose, 8g/L carrageenin.
3, the differentiation regeneration and the screening process of embryo callus: the embryo callus that will be inoculated on the division culture medium places 1500lux; Being cultured to embryoid under the 14h/d illumination condition forms; Then embryoid is changed on the substratum of sprouting, continue to place 1500lux, the 14h/d illumination condition is cultivated down and is produced budlet; Then budlet is transferred on the seedling substratum; 1500lux is cultured under the 14h/d illumination condition and grows up to seedling, and the seedling that will grow up to 0.5cm-3cm is transferred on the resistance substratum in 1500lux; The 14h/d illumination condition is cultivated down and was obtained resistant plant in 18-25 days, the resistant plant individual plant is incubated at the strong plantlets and rootage that carries out resistant plant on the strengthening seedling and rooting substratum by numbering for single bottle cultivated 18-25 days.The said substratum of sprouting is to be basic medium with the MS substratum, and adds 0.2mg/L6-BA, 30g/L sucrose, 8g/L carrageenin.Said one-tenth seedling substratum is to be basic medium with the MS substratum, and adds 30g/L sucrose, 8g/L carrageenin.Said resistance substratum is to be basic medium with the MS substratum, and adds 5mg/LIBA, 250ul/LPPT, 30g/L sucrose, 8g/L carrageenin.Said strengthening seedling and rooting substratum is to be basic medium with the MS substratum, and adds 5mg/LIBA, 250ul/LPPT, 30g/L sucrose, 8g/L carrageenin, 50-100ml/L Sucus Cocois.
4, transform plantlet DNA/RNA trace extraction and PCR and RT-PCR qualification process: by the blade of the resistant plant of numbering clip individual plant list flask culture on the strengthening seedling and rooting substratum; Shred and be placed on freezing 3-8 second in the liquid nitrogen; Grind, extract DNA, carry out PCR then and detect with the CTAB method.Use the same method through the plant of PCR test positive and to extract RNA and carry out RT-PCR and detect.At last PCR and RT-PCR being detected the plant of all being positive takes out to clean from substratum and is transplanted to booth after refining seedling.
The present invention carries out the experiment of genetic transformation to sugarcane, and it is following that institute obtains data list:
Sugarcane changing effect data
| The gene title | Callus usage quantity (individual) | Transformation time | Screening resistant plant quantity | Detect positive plant quantity | Screening efficiency | Transformation efficiency | Transform consuming time |
| 1900GUS | 10=200 of 20 ware * | 09.1.17- 09.5.13 | 44 strains | 40 strains | 90.9％ | 20.0％ | 116 days |
| 1900SST1 | 25 ware * 10=250 are little | 09.8.1- 09.12.15 | 50 strains | 46 strains | 92.0％ | 18.4％ | 112 days |
| 1900SST2 | 10=300 of 30 ware * | 09.8.3- 09.12.25 | 80 strains | 70 strains | 87.5％ | 23.3％ | 115 days |
| 1900SST3 | 10=300 of 30 ware * | 09.9.4- 09.1.10 | 82 strains | 75 strains | 91.5％ | 25.0％ | 125 days |
* annotate: screening efficiency=detection positive plant quantity/screening resistant plant quantity
Transformation efficiency=detection positive plant quantity/use callus quantity
From above data declaration, the present invention brings up to the genetic transformation efficiency of sugarcane agrobacterium-mediated transformation about 20% in sugarcane genetically modified breeding field, has precedence over previous 3% greatly; Screening efficiency has reached 90%, and transformation time also shortens to about 4 months from original half a year.
The present invention with the spire tissue at sugarcane apical growing point place as the explant induction embryo callus; The acceptor material that infects as Agrobacterium, through first differentiation culture, after carry out the PPT screening of high density; After obtaining resistant plant; Adopt DNA, the RNA trace extractive technique of innovation, extract the DNA/RNA of resistance seedling, carry out PCR, RT-PCR detection then; Plant through PCR, RT-PCR test positive is transplanted to booth again, treats to carry out after plant grows up the detection of other molecule and Physiology and biochemistry again.Compared with present technology, the present invention can increase substantially transformation efficiency, shortens the time that transforms, and only needs just can obtain in 4 months the transfer-gen plant of PCR, RT-PCR test positive, has also reduced the conversion cost.
Embodiment
Below in conjunction with embodiment the present invention is elaborated.
The present invention includes the process of inducing of sugarcane embryo callus; The activation of Agrobacterium reaches the conversion process to the sugarcane embryo callus; Transform the differentiation regeneration and the screening process of back embryo callus, transform plantlet DNA, RNA trace extraction and PCR and RT-PCR qualification process.
Following embodiment is that the plant expression vector PCBI1900 that utilizes this laboratory to build carries out genetic transformation to the sugarcane embryo callus.
Specific operation process is following:
1, embryo callus induces
With good cultivation of sugar cane kind (ROC22) is the transformation receptor material, selects the healthy and strong plant of field growing, gets its tail and slightly partly successively peels off outside Lao Ye; Get its spire tissue apart from about the about 10cm of apical growing point as explant, earlier with behind 75% the alcohol immersion 30s, again with 0.1% mercuric chloride processing 12min; The back with aseptic filter paper suck dry moisture, is carefully peelled off outermost one deck leaf tissue with aseptic washing 3～4 times again; Then its crosscut is become the thick thin slice of 0.2～0.5mm and be inoculated on the inducing culture; Cultivated 20-30 days down in 25 ℃ of dark conditions, can grow embryo callus, can directly be used for transforming.
2, the preparation of Agrobacterium engineering bacteria liquid
To contain through the Agrobacterium that PCR identifies on the corresponding antibiotic YEP flat board and rule, picking list colony inoculation in activation medium, in 28 ℃, 200rpm shaking culture to logarithmic phase (about 24h); Enlarged culturing contains in the identical antibiotic YEP substratum to 100mL in the 150mL triangular flask again, and being cultured to OD is (10h) about 0.3-1.5, and bacterium liquid is transferred in the centrifuge tube; 4 ℃, the centrifugal 5min of 5000rpm abandons supernatant; Blot raffinate, the resuspended thalline of the gene induced substratum of vir with equal-volume (100mL) places 28 ℃ again; 200rpm shaking culture 1-3h, to induce Agrobacterium Vir expression of gene, this bacterium liquid is the Agrobacterium engineering bacteria liquid that transforms usefulness.
3, Agrobacterium bacterium liquid is contaminated the sugarcane embryo callus and is cultivated altogether
Place ready Agrobacterium engineering bacteria liquid to soak about 30-40min the sugarcane embryo callus, during stir embryo callus gently with aseptic nipper.Afterwards, with aseptic strainer elimination bacterium liquid, then embryo callus is transferred on the filter paper; In Bechtop, dry; Until dry and comfortable, the no moisture film of material surface, the fritter that is cut into 0.3-0.5cm to the material structure piece again is transferred on the common culture medium, and dark is cultivated 3-4d altogether about 22 ℃.Cultivate the back altogether once with the sterilized water washing embryo callus that contains Car200mg/L; Again with aseptic washing 2-3 time; Wash once with the MS liquid nutrient medium more at last; Place on the aseptic filter paper Bechtop blow the material that dries in the air dry and comfortable after, embryo callus is transferred on the division culture medium that contains Car200mg/L, 28 ℃ of light are cultivated down.
4, the embryo callus seedling differentiation after the During Agrobacterium
The embryo callus that is inoculated in division culture medium is placed 28 ℃, and 1500lux, 14h/d illumination condition cultivate down can differentiate embryoid two weeks; Again embryoid is transferred on the substratum of sprouting; Continue to place 1500lux, the 14h/d illumination condition is cultivated to produce budlet down, after two weeks budlet is transferred on the seedling substratum; In 1500lux, the 14h/d illumination condition is cultivated about three weeks down and is made it grow up to seedling.
5, sugarcane transforms the screening of plantlet
The seedling of selecting 0.5cm-3cm is transferred on the resistance substratum in 1500lux; The 14h/d illumination condition was cultivated down about three weeks, single bottle of resistance plantlet individual plant is incubated at the strong plantlets and rootage that carries out resistant plant on the strengthening seedling and rooting substratum by numbering cultivates and can grow up to the good resistant plant of root system development 3 weeks.
6, the trace of the total DNA of resistant plant extracts
By numbering with the resistant plant blade 10-30mg of sterile scissors clip individual plant list flask culture on the M4 substratum; Shred and directly put into the 2.0mL centrifuge tube; Centrifuge tube is placed the freezing several seconds of liquid nitrogen, leaf tissue is smashed to pieces, adopt CTAB method trace to extract the DNA of resistant plant with glass stick.
7, the PCR of resistant plant detects
With the total DNA of plant is template; Insert a regional gene design primer with plant expression vector pCBI 1900, do over against photograph, be negative contrast with the DNA of unconverted sugarcane with the pCBI1900 DNA; The PCR product carries out 1% agarose gel electrophoresis analysis and photograph; Reclaim the PCR product, and be connected on the pMD18-T carrier, check order and analyze the homology of this gene order on its sequence and the pCBI1900 plant expression vector.
8, the RNA of resistant plant extracts
, shred and directly put into 2.0mL centrifuge tube (DEPC processing) with the plant leaf 10-30mg of sterile scissors clip by numbering, place liquid nitrogen to smash to pieces with glass stick after the freezing several seconds immediately, adopt the Trizo1 method to extract RNA through the PCR test positive.
9, the RT-PCR of resistant plant detects
RNA with the PCR positive plant is that template is carried out the RT-PCR detection; Primer detects identical with resistant plant PCR; Do over against photograph with pCBI 1900 DNAs, be negative contrast with the DNA of unconverted sugarcane, the RT-PCR product carries out 1% agarose gel electrophoresis analysis and photograph.
10, the transplanting of transformed plant
To be the male plantlet of transplant to warmhouse booth through PCR and RT-PCR detection.
Claims (1)
1. an efficient sugarcane genetically modified method fast is characterized in that: comprise the activation of inducing process, Agrobacterium of sugarcane embryo callus and the conversion process of embryo callus, differentiation regeneration and screening process, conversion plantlet DNA/RNA trace extraction and the PCR and the RT-PCR qualification process of embryo callus;
1), the sugarcane embryo callus induce process: the spire with the apical growing point place of good cultivation of sugar cane kind is organized as explant; Explant is thinly sliced after sterilization, aseptic water washing, aseptic filter paper blot its surperficial moisture and is inoculated on the inducing culture, under the lucifuge condition, is cultured to and induces embryo callus; Said inducing culture is to be basic medium with the MS substratum, and adds 1-2mg/L 2,4-D, 30g/L sucrose, 8g/L carrageenin;
2), the conversion process of activation of Agrobacterium and embryo callus: the single colony inoculation of Agrobacterium EHA105 that will contain goal gene shakes cultivation in activation medium; Through centrifugal recovery thalline; Be resuspended in again and continue the concussion cultivation in the gene induced substratum of equal-volume vir; To induce Agrobacterium plasmid vir expression of gene, this bacterium liquid is the Agrobacterium engineering bacteria liquid that transforms usefulness; The sugarcane embryo callus is changed in the Agrobacterium bacterium liquid, be inoculated in after the immersion on the common culture medium and cultivate altogether down, be inoculated on the division culture medium then in dark condition; Said activation medium is to be basic medium with YEP, and adds corresponding microbiotic Kan 50 μ g/mL, Str50 μ g/mL, Rif 50 μ g/mL; The gene induced substratum of said vir is to be basic medium with the MS substratum; Wherein macroelement is revised as 1/5 of former content, and adds 1mg/L2.4-D, 10mmol/L fructose, 10mmol/L glucose, 30g/L sucrose, 100-150 μ mol/L Syringylethanone; Said altogether culture medium is to be basic medium with the MS substratum; Wherein macroelement is revised as 1/5 of former content, and adds 1mg/L2.4-D, 10mmol/L fructose, 10mmol/L glucose, 30g/L sucrose, 100-150 μ mol/L Syringylethanone, carrageenin 20g/L; Said division culture medium is to be basic medium with the MS substratum, and adds 1mg/L6-BA, 0.2mg/LKT, 30g/L sucrose, 8g/L carrageenin;
3), the differentiation regeneration and the screening process of embryo callus: the embryo callus that will be inoculated on the division culture medium places 1500lux; Being cultured to embryoid under the 14h/d illumination condition forms; Then embryoid is changed on the substratum of sprouting, continue to place 1500lux, the 14h/d illumination condition is cultivated down and is produced budlet; Then budlet is transferred on the seedling substratum; 1500lux is cultured under the 14h/d illumination condition and grows up to seedling, and the seedling that will grow up to 0.5cm-3cm is transferred on the resistance substratum in 1500lux; The 14h/d illumination condition is cultivated down and was obtained resistant plant in 18-25 days, the resistant plant individual plant is incubated at the strong plantlets and rootage that carries out resistant plant on the strengthening seedling and rooting substratum by numbering for single bottle cultivated 18-25 days; The said substratum of sprouting is to be basic medium with the MS substratum, and adds 0.2mg/L6-BA, 30g/L sucrose, 8g/L carrageenin; Said one-tenth seedling substratum is to be basic medium with the MS substratum, and adds 30g/L sucrose, 8g/L carrageenin; Said resistance substratum is to be basic medium with the MS substratum, and adds 5mg/LIBA, 250ul/LPPT, 30g/L sucrose, 8g/L carrageenin; Said strengthening seedling and rooting substratum is to be basic medium with the MS substratum, and adds 5mg/LIBA, 250ul/LPPT, 30g/L sucrose, 8g/L carrageenin, 50-100ml/L Sucus Cocois;
4), transform plantlet DNA/RNA trace extraction and PCR and RT-PCR qualification process: by the blade of the resistant plant of numbering clip individual plant list flask culture on the strengthening seedling and rooting substratum; Shred and be placed on freezing 3-8 second in the liquid nitrogen; Grind, extract DNA, carry out PCR then and detect with the CTAB method; Use the same method through the plant of PCR test positive and to extract RNA and carry out RT-PCR and detect; At last PCR and RT-PCR being detected the plant of all being positive takes out to clean from substratum and is transplanted to booth after refining seedling.
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| US9447421B2 (en) | 2010-06-24 | 2016-09-20 | Syngenta Participations Ag | Methods for Agrobacterium-mediated transformation of sugar cane |
| WO2013090137A1 (en) | 2011-12-15 | 2013-06-20 | Syngenta Participations Ag | Compositions and methods for increased expression in sugar cane |
| CN102559748B (en) * | 2012-03-17 | 2012-12-19 | 福建农林大学 | Simple and convenient agrobacterium tumefaciens-mediated sugarcane transgene method |
| CN102911929B (en) * | 2012-10-16 | 2013-08-07 | 福建农林大学 | Method for preparing leaf receptor material for sugarcane transgene transient expression |
| BR102014023930A2 (en) | 2014-09-26 | 2016-05-17 | Ctc Ct De Tecnologia Canavieira S A | method for transforming a plant cell or plant tissue using agrobacterium, transgenic plant, transgenic cell or transgenic tissue, culture medium, and using a method for transforming a plant cell or plant tissue |
| CN107517855A (en) * | 2017-10-19 | 2017-12-29 | 广西大学 | The method for obtaining sugarcane health seedling |
| CN114058640A (en) * | 2021-12-01 | 2022-02-18 | 云南省农业科学院甘蔗研究所 | Efficient agrobacterium-mediated sugarcane genetic transformation method |
| CN115747255B (en) * | 2022-10-21 | 2023-07-25 | 中国热带农业科学院热带生物技术研究所 | Efficient sugarcane transgenic method |
| CN116491421A (en) * | 2023-05-26 | 2023-07-28 | 广西大学 | Gene gun-mediated sugarcane embryo genetic transformation method |
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|---|---|---|---|---|
| CN1644026A (en) * | 2005-01-19 | 2005-07-27 | 中国热带农业科学院热带生物技术研究所 | Culturing method for sugarcane germ plasm |
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| CN1644026A (en) * | 2005-01-19 | 2005-07-27 | 中国热带农业科学院热带生物技术研究所 | Culturing method for sugarcane germ plasm |
Non-Patent Citations (2)
| Title |
|---|
| 于兰等.农杆菌介导的BT基因导入甘蔗的研究.《广西农学报》.2007,第22卷(第6期),全文. * |
| 罗敬萍.农杆菌介导甘蔗基因转化技术的优化.《热带作物学报》.2003,第24卷(第4期),全文. * |
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