CN105296529A - Genetic transformation method for panicum virgatum L. - Google Patents
Genetic transformation method for panicum virgatum L. Download PDFInfo
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Abstract
The invention discloses a genetic transformation method for panicum virgatum L., and belongs to the technical field of plant bioengineering. The method comprises disinfection of mature panicum virgatum L. seeds, induction and propagation expanding of type II high-quality embryonic callus, kanamycin screening to obtain kanamycin-resistant callus tissue after agrobacterium infection, kanamycin-resistant callus tissue regeneration and rooting, and finally acquisition of positive transgenic panicum virgatum L. plants. The method is mainly used for solving the problems of large-scale screening and commercialized production of transgenic panicum virgatum L.. Experimental data show that by the adoption of the method, low-cost kanamycin can be used as a transgenic plant resistance screening reagent, the positive transgenic panicum virgatum L. plants are successfully obtained in 4-5 months, genetic transformation efficiency reaches up to 60%, 100 genetic vectors can be transformed by each person every year, and the working efficiency of producing more than 2,000 independent positive transgenic panicum virgatum L. plants is achieved.
Description
Technical field
The invention belongs to plant biotechnology field, relate to a kind of method of Genetic Transformation in Higher Plants particularly, particularly relate to a kind of method of switchgrass genetic transformation.
Background technology
Fibrous biomass resource is raw material the abundantest on the earth, but due to the shortage of some gordian techniquies, the current fibrous biomass resource of Energy production that can be used as is less than 1%.Genetic engineering breeding can overcome the gene barrier between species fast, thus it is degeneration-resistant and can the novel energy grass resource of Efficient Conversion to cultivate high yield at short notice.Therefore engineered strategy is adopted, excavate and identify the key gene resource that can determine energy grass biomass, quality and resistance, and obtain corresponding genetic improvement plant, contribute to the bottleneck problem solving resource shakiness and the bad existed in fibrous biomass development and utilization process.
Switchgrass (
panicumvirgatuml.) be C4 perennial (10-12) tall and big herbaceous plant, main as herbage and energy grass, be important fibrous biomass resource.Along with the development of high throughput sequencing technologies, the full-length genome of switchgrass completes order-checking.But in the face of the sequence information of magnanimity, how to obtain the genetic resources with potential using value by high-throughout genetic conversion system screening, remain current a faced huge challenge, therefore the genetic improvement research of switchgrass receives the extensive concern of countries in the world researcher.As most of list plant, switchgrass is difficult to carry out genetic transformation by agriculture bacillus mediated method.The efficiency of most of switchgrass genetic conversion system of current report is lower than 10%.In addition, previously the transgenosis switchgrass plant major part of report used hygromycin selection to obtain, and minority is produced by bialaphos screening, but the switchgrass genetic conversion system using kantlex to carry out screening not yet is set up.
As most of transgenic plant are commercially produced, the acquisition of excellent transgenosis switchgrass plant needs to carry out a thousand li from thousands of independently transgenic plant population and chooses one or the screening operation that Not one like can be found in ten thousand.Therefore setting up that the efficient and switchgrass of low cost heredity turns is primary goal and the core content of the commercialization research and development being transgenosis energy-source plant, is also the basis of switchgrass Main Agronomic Characters genetic improvement and molecular designing.Kantlex is cheap, commercially available price is at 2-3 unit/g, and the average commercially available price of Totomycin and bialaphos is respectively 1500 yuan/g and 18000 yuan/g, if the kalamycin resistance screening system of graminous pasture and energy grass therefore can be set up, greatly will reduce switchgrass transgenic plant production cost, there are huge commercial applications potentiality and wide market outlook.And the foundation of this system contributes to the directive breeding accelerating dedicated fiber biomass resource new variety, produce for fibrous biomass liquid fuel and provide sufficient stable and can the high-quality low cost production raw material of Efficient Conversion.
Summary of the invention
The object of this invention is to provide the genetic conversion system that a kind of switchgrass high efficiency, low cost is provided, for genetic improvement and the molecular designing of graminous pasture and energy-source plant important economical trait.
The present invention is achieved by the following technical solution:
A method for switchgrass genetic transformation, its content comprises:
Use the Agrobacterium containing binary vector to infect switchgrass typeII type high quality embryo callus, and obtain positive transgenic switchgrass plant by kalamycin resistance screening system.
Further, described switchgrass typeII type high quality embryonic callus induction is from the seed of switchgrass maturation, the surface of this callus is faint yellow, short texture and has gentle gloss, its method set up comprises: the sterilization of switchgrass mature seed, the induction of callus, the acquisition of typeII type high quality embryo callus and expand numerous.
On the basis of technique scheme, described agrobacterium strains is
eHA105or
aGL1.
Further, the described binary vector for switchgrass genetic transformation contain kalamycin resistance gene (
nptII), except containing except above-mentioned riddled basins, be applicable to the plasmid of the present invention's use also containing one or more different goal gene.
The present invention is used in process LAN or RNAi in plant and suppresses any gene.Goal gene can be that stress resistance gene, Bar gene, cell walls regulatory gene and biomass size determine gene etc.
The ecotype of the switchgrass described in the present invention is low ground type and highland type.
Resistance screening reagent described in the present invention is kantlex.
A kind of method of switchgrass genetic transformation, it specifically comprises the sterilization of switchgrass mature seed, the induction of typeII type high quality embryo callus and expand numerous, kantlex screening obtain Agrobacterium infect after kanamycin-resistant callus tissue and kanamycin-resistant callus tissue regeneration and take root and finally obtain positive transgenic switchgrass plant step.
Described switchgrass mature seed sterilization, mature seed 3%(W/V by switchgrass) after Losantin sterilizes 2 hours, sterile distilled water is used to clean 3 times (each 5 minutes), then 4 DEG C of refrigerator overnight are placed in, continuing next day to use 5%(W/V) Losantin sterilizes 1.5 hours, then uses sterile distilled water to clean 3 times (each 5 minutes).
The acquisition of described typeII type high quality embryo callus, is inoculated in the switchgrass mature seed after sterilization cleaning on callus inducing medium (MSD5), incubator temperature 25 ± 2 DEG C, in light culture 6-7 week, obtains callus.Visual method is adopted to screen obtained callus, select wherein faint yellow, short texture and the embryo callus with gentle gloss is typeII type high quality embryo callus, the typeII embryo callus of acquisition being carried out subculture expands numerous, incubator temperature 25 ± 2 DEG C, light culture, every 3-4 week subculture once.
The Agrobacterium of described callus is infected, and typeII type high quality embryo callus numerous for described expansion is divided into fritter, is then placed in threeway drying basin and vacuumizes, use containing carry kalamycin resistance gene (
nptII) the Agrobacterium bacterium liquid of binary vector described switchgrass typeII type high quality embryo callus is hatched altogether, then residual Agrobacterium bacterium liquid is removed, 25 ± 2 DEG C, Dual culture 3 days in dark, callus after Dual culture is transferred in resistant calli screening culture medium MSD3K and screen, incubator temperature 25 ± 2 DEG C, light culture, every 3 week switchings once, until resistant calli grows, the resistant calli of acquisition is transferred on resistant calli screening culture medium MSK2K, group training chambers temp 25 ± 2 DEG C, light application time 16h every day light/8h is dark, every 4 week switchings once, until resistant buds grows, the resistance regeneration bud obtained is forwarded on root media MSR, group training chambers temp 25 ± 2 DEG C, and light application time 16h every day light/8h is dark, the regrowth obtained conventionally is transplanted in soil, and after 2 weeks, regrowth survives.
Further, described resistant calli screening culture medium MS3DK (pH=5.8) is MS minimum medium interpolation 30g/L sucrose, 3mg/L2,4-D, 150mg/L kantlex, 400mg/L Cefotaxime sodium and 7.5g/L agar, autoclaving 15min.
Further, described resistant buds regeneration culture medium MSK2K (pH=5.8) is MS minimum medium interpolation 30g/L sucrose, 2mg/L kinetin, 50mg/L kantlex, 400mg/L Cefotaxime sodium and 7.5g/L agar, autoclaving 15min.
Further, described resistant buds root media MSR (pH=5.8) is 1/2MS minimum medium interpolation 15g/L sucrose, 400mg/L Cefotaxime sodium and 7.5g/L agar, autoclaving 15min.
The average genetic transformation efficiency of the Agrobacterium infection rate callus system described in the present invention is 60%, infecting from Agrobacterium the cycle that switchgrass callus transplants to transgenic plant greenhouse is 4-5 month, at least 100 genes can be transformed for each person every year, produce and be greater than 2000 independently positive transgenic plant.
Gene clone in the present invention, vector construction and Agrobacterium are infected and standard method all can be used to realize.
Central characteristics of the present invention is switchgrass genetic conversion system combining efficient and the large advantage of low cost two; and in switchgrass, successfully set up kalamycin resistance screening system first, thus guarantee positive transgenic plant the commercially producing for herbage and energy grass obtaining sufficient amount.
The present invention's beneficial effect compared with prior art:
1) transformation efficiency is high: the present invention produces the genetic transformation efficiency of positive transgenic switchgrass plant up to 60%, infect the transplanting of transgenic plant greenhouse from the Agrobacterium of callus and only need 4-5 month, thus can realize transforming at least 100 genophores for each person every year, produce the working efficiency being greater than 2000 independently positive transgenic plant.
2) with low cost: plant resistance to environment stress selective agent is the important factor determining transgenic plant production cost, method of the present invention uses cheap kantlex as the resistance screening reagent of transgenosis switchgrass plant first, be 1/100 of Totomycin and the bialaphos use cost generally used in current switchgrass genetic transformation, thus greatly reduce the cost that transgenic plant commercially produce.
3) restricted little: in the present invention, high quality embryonic callus induction is in plant maturation seed, its abundance, the typeII type high quality embryo callus of sufficient amount can be provided, and the high quality embryo callus obtained expands numerous lasting preservation by subculture, not by the restriction in development of plants period, the lasting sufficient supplies of the explant material for genetic transformation can be ensured.
In table 1 switchgrass genetic transformation, the use cost of different plant resistance to environment stress selective agent compares
Accompanying drawing explanation
Fig. 1: the typeII type high quality embryo callus of switchgrass mature seed induction in embodiment 1.
Fig. 2: switchgrass genetic transformation flow process agriculture bacillus mediated in embodiment 2.A. in embodiment 2 for the binary vector T-DNA sketch of switchgrass genetic transformation.PMDC100 binary vector contain kalamycin resistance gene (
nptII), can be used for the foundation of switchgrass genetic conversion system; B. Agrobacterium infect after callus Dual culture; C. the resistant calli of kantlex screening; D. the resistant buds of kantlex screening; The resistant buds (resistance seedling) of E. taking root; F. the transgenosis switchgrass plant of greenhouse-grown.
Fig. 3: the PCR qualification of transgenosis switchgrass plant in embodiment 3.M:DNAMarker; 1-13: the switchgrass plant of conversion; The negative control (without DNA profiling) of Ck-:PCR system; WT: not genetically modified wild-type switchgrass plant; CK+:pMDC100 binary vector plasmid.
embodiment:
Be described principle of the present invention and feature with the embodiment that is established as of low ground type switchgrass genetic conversion system below, example, only for explaining the present invention, is not intended to limit scope of invention.Material used in following embodiment, reagent, binary vector and Agrobacterium etc., if no special instructions, all can be bought from company by commercial sources, described MS minimum medium is purchased from PhytoTechnologyLaboratories (article No.: M519).
embodiment 1:the acquisition of switchgrass typeII type high quality embryo callus and expand numerous, it comprises the following steps:
1) by the mature seed 3%(W/V of switchgrass) after Losantin sterilizes 2 hours, sterile distilled water is used to clean 3 times (each 5 minutes), then 4 DEG C of refrigerator overnight are placed in, continuing next day to use 5%(W/V) Losantin sterilizes 1.5 hours, and then use sterile distilled water to clean 3 times (each 5 minutes), be inoculated on callus inducing medium (MSD5), and each seed is numbered, incubator temperature 25 ± 2 DEG C, in light culture 6-7 week, obtains callus.
2) callus that step (1) obtained adopts visual method to select wherein faint yellow, short texture and has typeII type high quality embryo callus (Fig. 1) of gentle gloss, be inoculated on MSD5 substratum, carry out preserving and expand numerous, incubator temperature 25 ± 2 DEG C, light culture, every 3-4 week subculture once.
Described switchgrass mature seed is low ground type switchgrass kind Alamo, and primordial seed picks up from the Ardmore of Oklahoma, United States for 2012, and is obtained in 2013 a large amount of F1 generation seeds being used for callus induction by outbreeding in Qingdao.
Described seed callus inducing culture MSD5(pH=5.8) be MS minimum medium interpolation 30g/L sucrose, 5mg/L2,4-D and 7.5g/L agar, autoclaving 15min.
embodiment 2:the acquisition of transgenosis switchgrass plant, its principal feature comprises following operation steps:
1) agrobacterium tumefaciens containing pMDC100 binary vector (Fig. 2 A) will preserved at-80 DEG C
aGL1streak inoculation is added on the solid medium of 50mg/L Rifampin and 50mg/L kantlex in LB, 28 DEG C of incubated overnight, then picking list colony inoculation adds in the liquid nutrient medium of 50mg/L Rifampin and 50mg/L kantlex in LB, and 28 DEG C of shaking table (200rpm) incubated overnight are to OD
600reach 0.6, then adding Syringylethanone to final concentration is 200 μm of ol/L, continues to cultivate 2h to OD
600reach 0.8-1.0,3500rpm, 20 DEG C of centrifugal 15min collect Agrobacterium bacterial sediment, and use the resuspended thalline of MS3D liquid nutrient medium, adjustment OD
600to 0.3.
2) the Agrobacterium bacterium liquid of preparation in step (1) is used to hatch 10min altogether to the switchgrass typeII type high quality embryo callus described in example 1, wherein callus is evenly divided into the fritter of 1-2cm prior to the tweezers that Agrobacterium hatches front sterilizing, then be placed in threeway drying basin and vacuumize 10 minutes, then 10 minutes are hatched altogether, then aseptic filter paper is used to remove residual Agrobacterium bacterium liquid, 25 ± 2 DEG C, Dual culture 3 days (Fig. 2 B) in dark.
3) callus after Dual culture in step (2) transferred in solid screening culture medium MSD3K screen, incubator temperature 25 ± 2 DEG C, light culture, every 3 week switchings once, until resistant calli grows, about 1.5-2.0 month (Fig. 2 C).
4) be transferred on regeneration culture medium MSK2K by the resistant calli of acquisition in step (3), group training chambers temp 25 ± 2 DEG C, light application time 16h every day light/8h is dark, and every 4 weeks transfer once, until resistant buds grows, and about 1.5-2.0 month (Fig. 2 D).
5) be forwarded on root media MSR by the resistance regeneration bud obtained in step (4), group training chambers temp 25 ± 2 DEG C, light application time 16h every day light/8h is dark, and the root system development of about 1 month regeneration bud is perfect (Fig. 2 E).
6) regrowth obtained in step (5) is conventionally transplanted in soil, regrowth survives after 2 weeks, and in greenhouse 28 ± 2 DEG C, dark according to 16h light/8h every day time, light intensity is healthy growth in 390lE m2 S1, for positive transgenic switchgrass plant qualification (Fig. 2 F).
Described agrobacterium tumefaciens type except
aGL1outward, can also use
eHA105.
Described agrobacterium tumefaciens
aGL1in carry pMDC100 binary vector, comprise in the T-DNA of this carrier kalamycin resistance gene (
nptII) (Fig. 2 A).
The described liquid MS3D substratum (pH=5.8) infected for Agrobacterium is that MS minimum medium adds 30g/L sucrose, 3mg/L2,4-D, autoclaving 15min.
Described resistant calli screening culture medium MS3DK (pH=5.8) is MS minimum medium interpolation 30g/L sucrose, 3mg/L2,4-D, 150mg/L kantlex, 400mg/L Cefotaxime sodium and 7.5g/L agar, autoclaving 15min.
Described resistant buds regeneration culture medium MSK2K (pH=5.8) is MS minimum medium interpolation 30g/L sucrose, 2mg/L kinetin, 50mg/L kantlex, 400mg/L Cefotaxime sodium and 7.5g/L agar, autoclaving 15min.
Described resistant buds root media MSR (pH=5.8) is 1/2MS minimum medium interpolation 15g/L sucrose, 400mg/L Cefotaxime sodium and 7.5g/L agar, autoclaving 15min.
The soil of described transgenosis switchgrass plantlet of transplant is turfy soil and vermiculite (1:1), and above flowerpot, use preservative film to cover tissue cultured seedling after transplanting, use pallet bottom flowerpot, topped up with water in pallet, throw off preservative film after 1 week, tissue cultured seedling surviving rate is 100%.
embodiment 3:the Molecular Identification of the transgenosis switchgrass plant obtained by kalamycin resistance screening, its principal feature comprises following operation steps:
The transgenosis switchgrass plant of greenhouse-grown after 1 month, get the blade of top 8-9cm, and be cut into the segment of 3-4cm, adopt 2 × CTAB method (Zhang Xiaoxiang etc., an improvement CTAB method for Rapid Isolation of Wheat genomic dna, Chinese agronomy circular, 2012,28:46-49) extract DNA, and carry out foreign gene (
nptII) pcr amplification, qualification positive transgenic switchgrass plant (Fig. 3).
Described for foreign gene (
nptII) the PCR primer sequence that detects is:
nptIIF：CGTCCTTTGCTCGGAAGAGTATGAA
nptIIR：GACGCAGAAGGCAATGTCATACCAC
The amplification condition of PCR is: 95 DEG C, 2min; Then carry out 30 circulations, the condition of each circulation is.94 DEG C, 30s; 55 DEG C, 30s; 72 DEG C, 30s; Last 72 DEG C, 10min.
Described switchgrass genetic transformation efficiency by PCR detect comprise foreign gene (
nptII) plant number: the preinfective callus lines number of Agrobacterium calculates and obtains.
Described agriculture bacillus mediated switchgrass genetic transformation efficiency is 60%, can transform 100 genophores for each person every year, produces and is greater than 2000 independently positive transgenic switchgrass plant.
It should be noted last that, above embodiment is only unrestricted for illustration of technical scheme of the present invention, although above-described embodiment is to invention has been detailed description, therefore any amendment to the present invention program or equivalent replacement, all do not depart from the spirit and scope of technical solution of the present invention.
Claims (9)
1. a method for switchgrass genetic transformation, is characterized in that its content comprises:
Use the Agrobacterium containing binary vector to infect switchgrass typeII type high quality embryo callus, and obtain positive transgenic switchgrass plant by kalamycin resistance screening system.
2. the method for a kind of switchgrass genetic transformation according to claim 1, it is characterized in that the surface of described typeII type high quality embryo callus subculture system is faint yellow, short texture and has gentle gloss, its method set up comprises: the sterilization of switchgrass mature seed and callus induction, the acquisition of typeII type high quality embryo callus and expand numerous.
3. the method for a kind of switchgrass genetic transformation according to claim 1, is characterized in that the described switchgrass callus for Agrobacterium-mediated genetic transformation system is from above-mentioned obtained typeII type high quality embryo callus.
4. the method for a kind of switchgrass genetic transformation according to claim 1, is characterized in that described agrobacterium strains is
eHA105or
aGL1.
5. the method for a kind of switchgrass genetic transformation according to claim 1, is characterized in that described binary vector contains kalamycin resistance gene, and the plasmid being applicable to the present invention's use contains one or more different goal gene.
6. the method for a kind of switchgrass genetic transformation according to claim 5, is characterized in that goal gene is stress resistance gene, Bar gene, cell walls regulatory gene and biomass size controlling gene etc.
7. the method for a kind of switchgrass genetic transformation according to claim 1, is characterized in that described switchgrass is for highland type and low ground type.
8. the method for a kind of switchgrass genetic transformation according to claim 1, is characterized in that the reagent of described resistance screening is kantlex.
9. the method for a kind of switchgrass genetic transformation according to claim 1, it is characterized in that its concrete steps comprise the sterilization of switchgrass mature seed, the induction of typeII type high quality embryo callus and expand numerous, kantlex screening obtain Agrobacterium infect after kanamycin-resistant callus tissue and kanamycin-resistant callus tissue regeneration and take root and finally obtain positive transgenic switchgrass plant step;
Described switchgrass mature seed callus induction, being inoculated on callus inducing medium after the mature seed of switchgrass sterilization cleaning, incubator temperature 25 ± 2 DEG C, in light culture 6-7 week, obtains callus;
Described seed callus inducing culture MSD5 is that MS minimum medium adds 30g/L sucrose, 5mg/L2,4-D and 7.5g/L agar, autoclaving 15min, pH=5.8;
The acquisition of described typeII type high quality embryo callus system: adopt visual method to screen obtained callus, select wherein faint yellow, short texture and the embryo callus with gentle gloss is typeII type high quality embryo callus, the high quality embryo callus of acquisition is inoculated on MSD5 substratum, carry out expansion numerous, incubator temperature 25 ± 2 DEG C, light culture, every 3-4 week subculture is once;
Described agriculture bacillus mediated switchgrass genetic transformation, typeII type high quality embryo callus numerous for described expansion is divided into fritter, then be placed in threeway drying basin to vacuumize, the Agrobacterium bacterium liquid containing binary vector is used to hatch altogether described typeII type high quality embryo callus, then residual Agrobacterium bacterium liquid is removed, 25 ± 2 DEG C, Dual culture 3 days in dark, callus after Dual culture is transferred in resistant calli screening culture medium MSD3K and screen, incubator temperature 25 ± 2 DEG C, light culture, every 3 week switchings once, until resistant calli grows, the resistant calli of acquisition is transferred on resistant calli screening culture medium MSK2K, group training chambers temp 25 ± 2 DEG C, light application time 16h every day light/8h is dark, every 4 week switchings once, until resistant buds grows, the resistance regeneration bud obtained is forwarded on root media MSR, group training chambers temp 25 ± 2 DEG C, and light application time 16h every day light/8h is dark, the regrowth obtained conventionally is transplanted in soil, and after 2 weeks, regrowth survives,
Described resistant calli screening culture medium MS3DK is that MS minimum medium adds 30g/L sucrose, 3mg/L2,4-D, 150mg/L kantlex, 400mg/L Cefotaxime sodium and 7.5g/L agar, autoclaving 15min, pH=5.8;
Described resistant buds regeneration culture medium MSK2K is that MS minimum medium adds 30g/L sucrose, 2mg/L kinetin, 50mg/L kantlex, 400mg/L Cefotaxime sodium and 7.5g/L agar, autoclaving 15min, pH=5.8;
Described resistant buds root media MSR is that 1/2MS minimum medium adds 15g/L sucrose, 400mg/L Cefotaxime sodium and 7.5g/L agar, autoclaving 15min, pH=5.8.
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| CN115710590A (en) * | 2022-11-29 | 2023-02-24 | 中国科学院青岛生物能源与过程研究所 | Wild barley agrobacterium-mediated callus infection method |
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| CN116751791A (en) * | 2023-07-21 | 2023-09-15 | 中国科学院青岛生物能源与过程研究所 | Application of PvPSK3 gene in improving genetic transformation efficiency of gramineous plants |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110122332A (en) * | 2019-06-06 | 2019-08-16 | 西北农林科技大学 | A kind of method of broom corn millet spire in vitro culture and plant regeneration |
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| CN115710590B (en) * | 2022-11-29 | 2024-02-02 | 中国科学院青岛生物能源与过程研究所 | Agrobacterium tumefaciens mediated callus infection method |
| CN116042894A (en) * | 2022-12-05 | 2023-05-02 | 中国科学院青岛生物能源与过程研究所 | Application of PvCKX4 and miR156-SPL-CKX4 molecular module in switchgrass trait improvement |
| CN116042894B (en) * | 2022-12-05 | 2023-06-16 | 中国科学院青岛生物能源与过程研究所 | Application of PvCKX4 and miR156-SPL-CKX4 molecular module in switchgrass trait improvement |
| CN116751791A (en) * | 2023-07-21 | 2023-09-15 | 中国科学院青岛生物能源与过程研究所 | Application of PvPSK3 gene in improving genetic transformation efficiency of gramineous plants |
| CN116751791B (en) * | 2023-07-21 | 2024-02-02 | 中国科学院青岛生物能源与过程研究所 | Application of PvPSK3 gene in improving genetic transformation efficiency of gramineous plants |
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