CN105177042B - A kind of method that plant polygenic inheritance converts - Google Patents

Abstract

The invention discloses a kind of method that plant polygenic inheritance converts, belong to plant biotechnology field.Its content includes the foundation of the embryo callus subculture system of term single gene type high-quality height Agrobacterium infection rate；Agrobacterium containing different binary vectors infects above-mentioned wound healing system, and obtains the transgenic positive plant containing multiple different exogenous genes by dual anti-screening.It is mainly used in solving plant and is difficult to genetic transformation and a difficult problem for multiple characters comprehensive improvement.Experimental data shows, the method using the present invention can be successfully obtained the positive transgenic plant with term single gene type background in 45 months, genetic transformation efficiency reaches as high as 60%, it is capable of converting 200 genophores for each person every year, produces the work efficiency of the positive transgenic plant containing multiple different external source genes of interest independent more than 3000.

Description

A kind of method that plant polygenic inheritance converts

Technical field

The invention belongs to plant biotechnology field, convert more particularly to a kind of plant polygenic inheritance Method, particularly relate to a kind of plant polygenic inheritance convert method.

Background technology

With switchgrass and Miscanthus, the energy grass as representative belongs to the perennial tall and big draft energy-source plant of C4, rich in wood Matter cellulose series biomass, yield is big, strong adaptability and be not take up ploughing, and meets Biomass Energy Development " no Strive grain with the people, do not strive with grain ground " fundamental state policy.No matter current main energy sources grass product kind is in plantation and farmland Management aspect, or in breeding and genetic improvement field, all have accumulated substantial amounts of data and rich experience. Meanwhile, the strong adaptability of energy grass, geographic range is wide.Additionally, switchgrass and the full-length genome of Miscanthus Sequence measures complete the most substantially, and the redundant gene resource that it contains urgently is developed.But in the face of the sequence of magnanimity How information, screen acquisition by high-throughout genetic conversion system and have the genetic resources of potential using value, Remaining the huge challenge currently faced, therefore to receive the world each in the genetic improvement research of energy grass The extensive concern of state's researcher.Monocotyledonous grasses is belonged to, as this section yet with energy grass Majority of plant is the same, and the genetic transformation efficiency of energy grass is low and poor stability.

Compared with other energy grass, switchgrass is important energy-source plant, and the grass family being also traditional is herded Grass, therefore the genetic transformation starting of switchgrass is relatively early, research experience relative abundance.The current willow set up The outer implant of branch millet genetic transformation system mainly uses children's fringe, immature caryopsis or the embryo of mature seed induction Property callus.Compared with the above two, switchgrass mature seed abundance, when drawing materials not by development of plants Phase and the impact of growing environment, and the genetic conversion system that the callus using mature seed to induce is set up, Its efficiency is generally higher than immaturity caryopsis and children's fringe system.It addition, the transgenic switchgrass plant being previously reported by Major part uses hygromycin selection to obtain, and minority is produced by bialaphos screening, but uses hygromycin simultaneously The polygenes transformation technology of screening dual anti-with bialaphos is not yet set up.Current monoclonal antibody screening can proceed to Ramulus Salicis Babylonicae The number of most exogenous genes of millet is 3, therefore if able to set up switchgrass dual anti-screening system, then At least the number that proceeds to of exogenous gene can be doubled such that it is able to realize important agronomy multiple to switchgrass The comprehensive regulation of shape and MOLECULE DESIGN.As most of list plants, switchgrass is difficult to carry out genetic transformation. The efficiency of most of switchgrass genetic conversion system of report is less than 10% at present.Though there being the something lost of only a few switchgrass Biography transformation system efficiency is more than 10%, but its repeatability and stability are strong, and the genetic background of material source mixes Miscellaneous.This is primarily due to switchgrass is the most affine plant of selfing, and each seed all represents a different base Because of type, therefore use the callus of seed mixture, callus quality certainly will be caused very different, not only Affect the stability of genetic transformation efficiency, and the transgenic plant obtained has different genetic backgrounds, latent Affecting its subsequent analysis and application.

Up to now, existing 28 countries have planted different types of transgenic on the soil of 1.8 hundred million hectares Crop, area when transgenic plant was planted first than 1996 adds 100 times.Nearly 20 years of past Numerous studies and comprehensive analytical data confirm, transgenic technology makes the consumption of chemical pesticide decrease 37%, crop Yield adds 22%, and peasant's profit adds 68%, and therefore grain is pacified by research and development and the use of transgenic technology Entirely, sustainable development and environment/climate change are made that contribution.But, the commercialization of transgenic plant is ground Send out firstly the need of producing thousands of independent transfer-gen plants, then choose above-mentioned colony carries out a thousand li One or the screening operation that Not one like can be found in ten thousand.It addition, in order to meet the purpose commercially produced, need plant Multiple various traits carry out comprehensive improvement and MOLECULE DESIGN, therefore to reach transgenic switchgrass plant business Metaplasia is produced and the purpose of application, needs the efficient polygenic inheritance transformation system of development badly multiple important to switchgrass Economical character carries out comprehensive improvement, thus directive breeding goes out dedicated fiber biomass resource new varieties, for fiber Biomass liquid fuel produces and provides abundance stable and can the quality raw materials of Efficient Conversion.

Summary of the invention:

It is an object of the invention to provide a kind of plant polygenic inheritance method for transformation, it is provided that a kind of efficiently polygenes Genetic conversion system, for genetic improvement and the MOLECULE DESIGN of plant important economical trait.

The present invention is achieved by the following technical solution:

A kind of method that plant polygenic inheritance converts, its content includes:

The foundation of the embryo callus subculture system of term single gene type high-quality height Agrobacterium infection rate；

Agrobacterium containing different binary vectors infects above-mentioned wound healing system, and is obtained containing many by dual anti-screening The transgenic plant positive plant of individual different exogenous gene.

Further, the surface of described term single gene type high-quality embryo callus subculture system is faint yellow, structure Loosening and have gentle gloss, its method set up includes: the term single gene type wound healing of plant maturation seed System sets up, and high-quality embryo callus subculture system is obtained, finally by visual method screening and wound healing system regeneration rate assessment method The regeneration rate of all term single genes type high-quality embryo callus subculture system obtained is all higher than 90%.

Further, described the plant callus of efficiency rating is infected from above-mentioned screening institute for Agrobacterium Obtain the high-quality embryo callus subculture system of multiple term single gene type, wherein, from each wound healing system all carefully Born of the same parents are respectively provided with identical genetic background, concrete grammar be will containing pANIC6A binary vector Agrobacterium bacterium solution with Term single gene type high-quality embryo callus subculture system hatches altogether, selects to infect the efficiency wound healing system more than 30% and is The embryo callus subculture system of term single gene type high-quality height Agrobacterium infection rate, then carries out subculture expanding propagation, and is used for The foundation of follow-up plant efficient genetic transforming method.

On the basis of technique scheme, described agrobacterium strains is EHA105 or AGL1.

Further, the described binary vector for plant efficient polygenic inheritance conversion contains hygromycin respectively With selected gene, in addition to containing above-mentioned riddled basins, it is suitable for the plasmid that the present invention uses Possibly together with one or more different genes of interest.

The present invention can be used for process LAN or RNAi in plant and suppresses any gene.Genes of interest can be resistance to removing Grass agent gene, cell wall controlling gene, tillering number determine that gene, stem stalk thickness control gene, base of blooming Cause or stress resistance gene.

Heretofore described plant is the unifacial leaf the most affine plant of grass family selfing, including switchgrass and Miscanthus Equal energy source grass and graminous pasture plant.

Heretofore described resistance screening reagent is hygromycin and double propylamine phosphorus.

A kind of plant polygenic inheritance convert method, it specifically include plant maturation seed callus induction, The acquisition of the embryo callus system of term single gene type high-quality height Agrobacterium infection rate and agriculture bacillus mediated Polygenic inheritance step of converting.

Described plant maturation seed callus induction, sterilizes the mature seed of plant and is inoculated in after cleaning On callus inducing medium, incubator temperature 25 ± 2 DEG C, light culture 6-7 week, it is thus achieved that callus.

The acquisition of the embryo callus system of described term single gene type high-quality height Agrobacterium infection rate, institute The callus obtained uses visual method to screen, and selects the most faint yellow, short texture and has gentle The embryo callus of gloss be high-quality embryo callus, the high-quality embryo callus that will obtain Separate half callus to be inoculated on callus regeneration culture medium culturing base, group training room temperature 25 ± 2 DEG C, Light application time 16h every day light/8h is dark, cultivates 4-5 week, it is thus achieved that regeneration bud, and carries out the assessment of regeneration efficiency, Then the regeneration rate wound healing system more than 90% is selected to carry out subculture, incubator temperature 25 ± 2 DEG C, light culture 3-4 In individual week, it is subsequently used for Agrobacterium and infects the assessment of efficiency, finally select Agrobacterium to infect efficiency healing more than 30% Wound system is the embryo callus system of term single gene type high-quality height Agrobacterium infection rate, then carries out expanding propagation, Incubator temperature 25 ± 2 DEG C, light culture, every 3-4 week subculture is once.

Described agriculture bacillus mediated polygenic inheritance converts, the term single gene type high-quality of described expanding propagation Callus in the embryo callus system of high Agrobacterium infection rate is divided into fritter, is subsequently placed in threeway and is dried Evacuation in ware, uses the Agrobacterium bacterium solution containing different binary vectors to described switchgrass term single gene type High-quality embryo callus is hatched altogether, then removes the Agrobacterium bacterium solution of residual, 25 ± 2 DEG C, dark In co-culture 3 days, the callus after co-culturing is transferred in resistant calli screening culture medium MSD3HB Screening, incubator temperature 25 ± 2 DEG C, light culture, every 3 weeks transfer once, until kanamycin-resistant callus tissue group Knit and grow, the resistant calli of acquisition is transferred in resistant calli screening culture medium MSK2HB, group Training room temperature 25 ± 2 DEG C, light application time 16h every day light/8h is dark, and every 4 weeks transfer once, until resistance Bud grows；The resistance regeneration bud obtained is forwarded on root media MSRHB, group training room temperature 25 ± 2 DEG C, Light application time 16h every day light/8h is dark；The regrowth obtained conventionally is transplanted in soil, 2 Zhou Houzai Raw Seedling survives.

Further, described resistant calli screening culture medium MSD3HB (pH=5.8) is MS minimal medium Add 30g/L sucrose, 3mg/L2,4-D, 30mg/L hygromycin, 2.4mg/L double propylamine phosphorus, 400mg/L head Plug oxime sodium and 7.5g/L agar, autoclaving 15min.

Further, described resistant buds regeneration culture medium MSK2HB (pH=5.8) is that MS minimal medium adds 30g/L sucrose, 2mg/L kinetins, 10mg/L hygromycin, 1.2mg/L double propylamine phosphorus, 400mg/L cephalo Plug oxime sodium and 7.5g/L agar, autoclaving 15min.

Further, described resistant buds root media MSRHB (pH=5.8) is that 1/2MS minimal medium adds The double propylamine phosphorus of 15g/L sucrose, 10mg/L hygromycin, 1.2mg/L, 400mg/L Cefotaxime sodium and 7.5g/L Agar, autoclaving 15min.

The genetic transformation of heretofore described all term single gene type high-quality height Agrobacterium infection rate wound healing system Efficiency is all higher than 30%, and the highest transformation efficiency can reach 60%, it is possible to once proceed at least 6 differences Exogenous gene, produce the independent positive transgenic plant containing multiple exogenous genes more than 3000.

Gene clone, vector construction and Agrobacterium in the present invention are infected and standard method all can be used to realize.

Central characteristics and the inventive concept of the present invention include:

1) by screening and the foundation of term single gene type high-quality embryo callus subculture system, Agrobacterium is infected quick by enrichment The callus of sense, thus a large amount of Agrobacteriums overcoming previously used seed mixture wound healing system to be caused are infected The interference to Agrobacterium-mediated genetic transformation of the insensitive tissue.

2) the dual anti-screening system of method, resistant calli and resistant buds is infected by simple efficient Agrobacterium System, significantly improves the quantity of Genetic Transformation in Higher Plants efficiency and transgenic.

Present invention beneficial effect compared with prior art:

1) screening is rapidly: in the present invention, high-quality embryonic callus induction is in plant maturation seed, its source Abundant, it is possible to filtered out high-quality embryo callus subculture system with high throughput by visual method, the screening cycle is 3-4 Month.

2) outer implant is sufficient: the high-quality embryo callus subculture system screening acquisition in the present invention can be held by subculture expanding propagation Continuation of insurance is deposited, and is not affected period by development of plants, is not only able to ensure the outer implant material for genetic transformation Lasting sufficient supplies, and it greatly shortens the cycle converted.

3) genotype is homogeneous: each high-quality embryo callus subculture system of use all results from a corresponding kind Son, the healing cell in the most each wound healing system has identical genotype, thus turns base produced by ensureing Because plant has the follow-up research and development of identical genetic background, beneficially transgenic plant.

4) transformation efficiency is high: the present invention is by first to term single gene type high-quality height Agrobacterium infection rate wound healing System screens, and the term single gene type high-quality height Agrobacterium infection rate wound healing system filtered out carries out follow-up something lost Passing and convert, the genetic transformation efficiency producing positive transgenic plant is more than 30%, and what wherein conversion ratio was the highest can Reach 60%, and infect the transplanting of transgenic plant greenhouse from the Agrobacterium of callus and only need 4-5 month, It is thus possible to realize converting for each person every year 200 genophores, produce more than 3000 containing outside multiple differences The independent positive transgenic plant of source gene.

5) can transformed gene quantity many: the method for the present invention uses hygromycin and double dual anti-screenings of propylamine phosphorus first, Doubling of number gene can be realized rapidly, it is achieved outside at least 6 differences in the quantity of existing gene transformation The disposable conversion of source gene, therefore the present invention is possible not only to the multiple important characters to plant and realizes comprehensively changing Good, it is also possible to multiple key genes of a certain specific metabolic pathway to be carried out molecular regulation simultaneously, thus obtains Previously it is difficult to the new character that obtains and new quality.

Accompanying drawing explanation

Outside the term single gene type high-quality embryo callus subculture system of switchgrass mature seed induction in Fig. 1: embodiment 1 Morphological characteristic.

Fig. 2: embodiment 1 and 2 infects what efficiency evaluation and polygenic inheritance converted for switchgrass Agrobacterium Binary vector T-DNA sketch.A.pANIC6A binary vector contains red fluorescent protein gene (RFP), available The Agrobacterium observing assessment switchgrass term single gene type high-quality embryo callus subculture system in microscope infects efficiency； B.pANDA-PvCOMTRi binary vector contains hygromycin gene (hph)；C.pANIC6E-OsmiR156OE Binary vector contains double propylamine phosphorus resistant gene (bar) of herbicide；B. difference is carried respectively with C. binary vector Plant resistance to environment stress screening-gene, can be used for the foundation of switchgrass polygenic inheritance transformation system；

Switchgrass genetic transformation process agriculture bacillus mediated in Fig. 3: embodiment 2.A. switchgrass maturation kind it is derived from The term single gene type high-quality height Agrobacterium of son infects efficiency embryo callus；Healing after B. Agrobacterium is infected Injured tissue co-cultures；C. resistant calli is grown in hygromycin and double propylamine phosphorus screening culture medium；D. tide Mycin and the resistant buds of double propylamine phosphorus screening；E. the resistance Seedling of growth on root media；F. greenhouse-grown Transgenic switchgrass plant；G. the GUS dyeing of positive transgenic switchgrass plant leaf blade and stem section is identified.

The PCR of the transgenic switchgrass plant containing multiple exogenous genes in Fig. 4: embodiment 2 identifies.A:PCR Identify the existence of hph, COMT-RNAi and nptII gene in double transgenic switchgrass plant converted；B: In the double transgenic switchgrass plant converted of A:PCR qualification, bar, gus-plus and OSmiR156 gene deposits ?；M:DNA Marker；The switchgrass plant of 1-10: double conversions；The wild type willow of Ck-: non-transgenic Branch millet plant；CK+:pANDA-PvCOMTRi and pANIC6E-OsmiR156OE binary vector.

Detailed description of the invention:

It is established as reality below with what switchgrass term single gene type high-quality height Agrobacterium infected efficiency embryo callus subculture system Executing example to be described principle and the feature of the present invention, example is served only for explaining the present invention, is not intended to Limit the scope of invention.Material, reagent, binary vector and Agrobacterium etc. used in following embodiment, as Without specified otherwise, all can be bought by commercial sources from company, described MS minimal medium is purchased from PhytoTechnology Laboratories (article No.: M519).

Embodiment 1: switchgrass term single gene type high-quality height Agrobacterium infects the foundation of efficiency embryo callus subculture system, It comprises the following steps:

1), after being sterilized 2 hours with 3% (W/V) calcium hypochlorite by the mature seed of switchgrass, aseptic steaming is used Distilled water cleans 3 times (each 5 minutes), is subsequently placed in 4 DEG C of refrigerator overnight, is continuing with 5% (W/V) next day Calcium hypochlorite is sterilized 1.5 hours, then uses sterile distilled water to clean 3 times (each 5 minutes), is inoculated in On callus inducing medium (MSD5), and each seed is numbered, incubator temperature 25 ± 2 DEG C, Light culture 6-7 week, it is thus achieved that callus.

2) callus step (1) obtained uses visual method to screen, according to callus Form and soft or hard degree are classified, and select the most faint yellow, short texture and have gentle gloss Embryo callus (Fig. 1), carries out the screening of subsequent step；

3) high-quality embryo callus step (2) obtained (is numbered and corresponding seed numbering Unanimously) separating half callus to be inoculated in MSK2 culture medium, group training room temperature 25 ± 2 DEG C, during illumination Between every day 16h light/8h dark, cultivate 4-5 week, it is thus achieved that regeneration bud (numbering is numbered consistent with corresponding seed), And carry out the assessment of regeneration efficiency, final select regeneration rate embryo callus subculture system more than 90% to carry out subsequent step Screening；

4) high-quality height regeneration rate (> 90% that step (3) is obtained) embryo callus system carry out Expanding propagation, each independent wound healing system expanding propagation 10 dish, incubator temperature 25 ± 2 DEG C, light culture, every 3-4 week Subculture is once；

5) line of the Agrobacterium tumefaciems AGL1 containing pANIC6A binary vector (Fig. 2 A) that will preserve at-80 DEG C It is inoculated in LB to add on the solid medium of 50mg/L rifampicin and 50mg/L kanamycin, overnight trains for 28 DEG C Supporting, then picking list colony inoculation adds the liquid training of 50mg/L rifampicin and 50mg/L kanamycin in LB Support in base, 28 DEG C of shaking table (200rpm) incubated overnight to OD₆₀₀Reach 0.6, then add acetosyringone extremely Final concentration of 200 μm ol/L, continue to cultivate 2h to OD₆₀₀Reach 0.8-1.0,3500rpm, 20 DEG C to be centrifuged 15min collects Agrobacterium bacterial sediment, and uses the resuspended thalline of MSD3 fluid medium, adjusts OD₆₀₀To 0.3.

6) use the Agrobacterium bacterium solution of preparation in step (5) single to the switchgrass obtained in step (4) 10min is hatched altogether in genotype high-quality embryo callus subculture system, and wherein callus hatches front use prior to Agrobacterium The tweezers of sterilizing are uniformly divided into the fritter of 1-2cm, are subsequently placed in evacuation 10 minutes in threeway drying basin, connect And hatch altogether 10 minutes, then use aseptic filter paper to remove the Agrobacterium bacterium solution of residual, 25 ± 2 DEG C, dark In co-culture 3 days.

7) to the callus after co-culturing 3 days in step (6), red by fluorescence microscopy Microscopic observation The presence or absence statistics of fluorescin (RFP) signal infects efficiency, and the efficiency of selection wound healing system more than 30% is list One genotype high-quality height Agrobacterium infects efficiency embryo callus subculture system, then carries out subculture expanding propagation, and after being used for The foundation of continuous switchgrass high-efficiency genetic transforming method.

Described switchgrass mature seed is low ground type switchgrass kind Alamo, and primordial seed is picked up from for 2012 The Ardmore of Oklahoma, United States, and obtained for wound healing by outbreeding in Qingdao in 2013 A large amount of F1 generation seeds of tissue induction.

Described seed callus inducing culture MSD5 (pH=5.8) is that MS minimal medium adds 30g/L Sucrose, 5mg/L2,4-D and 7.5g/L agar, autoclaving 15min.

Described callus regeneration culture medium MSK2 (pH=5.8) is that MS minimal medium adds 30g/L sugarcane Sugar, 2mg/L kinetins and 7.5g/L agar, autoclaving 15min.

The numbering of described all switchgrass high-quality embryo wound healing system is all numbered consistent with the seed initially originated, In each the embryo callus subculture system hence set up, all cells is all from, from same seed, having identical base Because of type background.

Described Agrobacterium is Agrobacterium tumefaciems AGL1, and carries pANIC6A binary vector, this carrier T-DNA carries red fluorescent protein gene (RFP) (Fig. 2 A), can be used for fluorescence microscopy Microscopic observation by agriculture The callus that bacillus is successfully infected.

Described Agrobacterium is infected method and includes hatching altogether and evacuation of callus and Agrobacterium, it is possible to aobvious Write raising Agrobacterium infects efficiency.

Described Agrobacterium infects efficiency according to the callus lines number occurring RFP signal after co-culturing: invade Callus lines number before dye calculates and obtains, all switchgrass term single gene type high-quality embryos screened The Agrobacterium of wound healing system is infected efficiency and is all higher than 30%, and the highest infects efficiency more than 80%.

Described switchgrass term single gene type high-quality embryo callus subculture system is except training by conventional callus subculture Support outside preserving, it is also possible to number corresponding regrowth with this wound healing system in greenhouse by transplanting, by this plant Tiller carry out asexual propagation and preserve 10-12, and corresponding term single gene type high-quality embryo callus subculture Tissue then can be re-established by children's fringe callus induction.

Embodiment 2: the efficient acquisition of the transgenic switchgrass plant containing multiple exogenous genes, its main feature Including following operating procedure:

1) pANDA-PvCOMTRi and pANIC6E-OsmiR156OE pair is contained respectively by preserve at-80 DEG C Unit carrier (Fig. 2 B and C) Agrobacterium tumefaciems AGL1 streak inoculation in LB add 50mg/L rifampicin and On the solid medium of 50mg/L kanamycin, 28 DEG C of incubated overnight, then picking list colony inoculation adds in LB Add in the fluid medium of 50mg/L rifampicin and 50mg/L kanamycin, 28 DEG C of shaking table (200rpm) mistakes Night cultivates to OD₆₀₀Reach 0.6, then add acetosyringone and cultivate to final concentration of 200 μm ol/L, continuation 2h to OD₆₀₀Reach 0.8-1.0,3500rpm, 20 DEG C of centrifugal 15min and collect Agrobacterium bacterial sediment, and make With the resuspended thalline of MSD3 fluid medium, adjust OD₆₀₀To 0.6, then containing above-mentioned preparation respectively The bodies such as the Agrobacterium tumefaciems AGL1 bacterium solution of pANDA-PvCOMTRi and pANIC6E-OsmiR156OE binary vector Long-pending mixing.

2) use the Agrobacterium bacterium solution prepared in step (1) to the switchgrass term single gene described in example 1 Type high-quality height Agrobacterium is infected efficiency embryo callus subculture system (Fig. 3 A) and hatches 10min, wherein wound healing altogether Organize and hatch, prior to Agrobacterium, the fritter that the tweezers of front sterilizing are uniformly divided into 1-2cm, be subsequently placed in threeway dry Evacuation 10 minutes in dry ware, hatch 10 minutes the most altogether, then use aseptic filter paper to remove the agriculture of residual Bacillus bacterium solution, co-cultures 3 days (Fig. 3 B) by 25 ± 2 DEG C in dark.

3) callus after co-culturing in step (2) is transferred and is carried out in solid screening culture medium MSD3HB Screening, incubator temperature 25 ± 2 DEG C, light culture, every 3 week switchings are once, long until resistant calli Go out, the about 1.5-2.0 month (Fig. 3 C).

4) resistant calli obtained in step (2) is transferred on regeneration culture medium MSK2HB, group training Room temperature 25 ± 2 DEG C, light application time 16h every day light/8h is dark, and every 4 weeks transfer once, until resistant buds Grow, the about 1.5-2.0 month (Fig. 3 D).

4) the resistance regeneration bud obtained in step (3) is forwarded on root media MSRHB, group training room Temperature 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. 3 E).

5) being conventionally transplanted in soil by the regrowth obtained in step (4), after 2 weeks, regrowth becomes Live, and in 28 ± 2 DEG C of greenhouse, dark according to 16h light every day time/8h, light intensity is healthy in 390lE/m2/S1 Growth, identifies (Fig. 3 F and G) for positive transgenic switchgrass plant.

Described Agrobacterium tumefaciems type is in addition to AGL1, it is also possible to use EHA105.

Described Agrobacterium tumefaciems AGL1 carries respectively pANDA-PvCOMTRi and PANIC6E-OsmiR156OE binary vector, wherein, comprises tide in the T-DNA of pANDA-PvCOMTRi carrier Neomycin phosphotransferase gene (hph), kalamycin resistance gene (nptII) and in being used for suppressing switchgrass The Gateway cassette (Fig. 2 B) of source COMT gene expression；PANIC6E-OsmiR156OE carrier T-DNA comprises anti-herbicide gene (bar), beta-glucuronic acid glycosidase genes (gus-plus) and is used for The Gateway cassette (Fig. 2 C) of external source OsmiR156 overexpression.

Described liquid MSD3 culture medium (pH=5.8) infected for Agrobacterium is that MS minimal medium adds Add 30g/L sucrose, 3mg/L2,4-D, autoclaving 15min.

Described resistant calli screening culture medium MSD3HB (pH=5.8) is that MS minimal medium adds 30g/L sucrose, 3mg/L2,4-D, 30mg/L hygromycin, 2.4mg/L double propylamine phosphorus, 400mg/L cephalo plug Oxime sodium and 7.5g/L agar, autoclaving 15min.

Described resistant buds regeneration culture medium MSK2HB (pH=5.8) is that MS minimal medium adds 30g/L sugarcane Sugar, the double propylamine phosphorus of 2mg/L kinetins, 10mg/L hygromycin, 1.2mg/L, 400mg/L Cefotaxime sodium and 7.5g/L agar, autoclaving 15min.

Described resistant buds root media MSRHB (pH=5.8) is that 1/2MS minimal medium adds 15g/L sugarcane Sugar, the double propylamine phosphorus of 10mg/L hygromycin, 1.2mg/L, 400mg/L Cefotaxime sodium and 7.5g/L agar, Autoclaving 15min.

The soil of described transgenic switchgrass plantlet of transplant is turfy soil and Vermiculitum (1:1), is spending after transplanting Use preservative film to cover tissue cultured seedling above basin, use pallet, topped up with water in pallet bottom flowerpot, throw off after 1 week Preservative film, tissue cultured seedling survival rate is 100%.

Embodiment 3: the Molecular Identification of the transgenic switchgrass positive plant containing multiple exogenous genes, it is main Feature includes following operating procedure:

Greenhouse-grown transgenic switchgrass plant after 1 month, takes the blade of top 8-9cm, and is cut into 3-4cm Segment, use 2 × CTAB method (Zhang Xiaoxiang etc., the improvement CTAB of a kind of Rapid Isolation of Wheat genomic DNA Method, China agronomy circular, 2012,28:46-49) extract DNA, and carry out exogenous gene/sequence (hph, NptII, COMT-RNAi, bar, gus-plus and OsmiR156) PCR amplification, identify positive transgenic Switchgrass plant (Fig. 4).

Described for exogenous gene/sequence (hph, NPTII, COMT-RNAi, bar, gus-plus and OsmiR156) the PCR primer sequence detected is respectively as follows:

Hph3:AAGGAATCGGTCAATACACTACATGG

Hph4:AAGACCAATGCGGAGCATATACG

NptIIF:CGTCCTTTGCTCGGAAGAGTATGAA

NptIIR:GACGCAGAAGGCAATGTCATACCAC

COMT-RNAiF:AACAGTTCCTGATTAACCACAAACC

COMT-RNAiR:GCCAGAAGTTCTTTTTCCAGTACC

BarF:AGTCGACCGTGTACGTCTCC

BarR:GAAGTCCAGCTGCCAGAAAC

Gus-plusF:CACGGTGCCGGCCTATCTGA

Gus-plusR:GCTTGCGACCACTTTGCCTTCCT

OsmiR156F:CACCACAGTTTAATTTATTTCTTGG

OsmiR156R:CTAGGCAGAAAATTTAACAGGAG

The amplification condition of PCR is: 95 DEG C, 2min；Then carrying 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 polygenic inheritance transformation efficiency by PCR detection comprise exogenous gene/sequence (hph, nptII, COMT-RNAi, bar, gus-plus and OsmiR156) plant number: Agrobacterium preinfective wound healing group Knit block number and calculate acquisition.

Use in example 1 switchgrass of the part term single gene type high-quality embryo callus subculture system detection screening acquisition Polygenic inheritance transformation efficiency is more than 30%, and wherein in example 1, Agrobacterium infects the polygenes of the highest wound healing system Genetic transformation efficiency is 60%, it is possible to converts at least 6 exogenous genes in switchgrass simultaneously, for each person every year may be used Convert 200 genophores, produce the transgenic willow containing multiple different external source genes of interest more than 3000 Branch millet plant.

Technical solution of the present invention is applicable not only to switchgrass, applies also for the self-incompatible grass family such as Miscanthus The polygenic inheritance transformation system of energy grass and herbage is set up.

It should be noted last that, above example is merely to illustrate technical scheme and unrestricted, Although the present invention has been described in detail by above-described embodiment, the most any amendment to the present invention program or Equivalent, all without departing from the spirit and scope of technical solution of the present invention.

Claims (7)

1. the method that a plant polygenic inheritance converts, it is characterised in that its content includes:

The foundation of the embryo callus subculture system of term single gene type high-quality height Agrobacterium infection rate；

Agrobacterium containing different binary vectors infects above-mentioned wound healing system, and is obtained containing many by dual anti-screening The transgenic plant positive plant of individual different exogenous gene；

The surface of described term single gene type high-quality embryo callus subculture system is faint yellow, short texture and has temperature The gloss of profit, its method set up includes: the term single gene type wound healing system of plant maturation seed sets up, high-quality Amount embryo callus subculture system is obtained by visual method screening and wound healing system regeneration rate assessment method, the final all lists obtained The regeneration rate of one genotype high-quality embryo callus subculture system is all higher than 90%；

The plant callus infecting efficiency rating for Agrobacterium is obtained multiple single base from above-mentioned screening Because of the high-quality embryo callus subculture system of type, wherein, it is respectively provided with identical from all cells in each wound healing system Genetic background, it is will be containing pANIC6A binary vector Agrobacterium bacterium that Agrobacterium infects efficiency rating concrete grammar Liquid is hatched altogether with term single gene type high-quality embryo callus subculture system, selects the wound healing system infecting efficiency more than 30% It is the embryo callus subculture system of term single gene type high-quality height Agrobacterium infection rate, then carries out subculture expanding propagation, and Foundation for follow-up polygenes high-efficiency genetic transforming method.

The method that a kind of plant polygenic inheritance the most according to claim 1 converts, it is characterised in that described Agrobacterium strains is EHA105 or AGL1.

The method that a kind of plant polygenic inheritance the most according to claim 1 converts, it is characterised in that described Binary vector contains hygromycin and selected gene respectively, and the plasmid that the applicable present invention uses contains There is one or more different genes of interest.

The method that a kind of plant polygenic inheritance the most according to claim 3 converts, it is characterised in that purpose base Because of be Bar gene, cell wall controlling gene, tillering number determine gene, stem stalk thickness control gene, Floral genes or stress resistance gene.

The method that a kind of plant polygenic inheritance the most according to claim 1 converts, it is characterised in that described Plant is the unifacial leaf the most affine plant of grass family selfing.

The method that a kind of plant polygenic inheritance the most according to claim 1 converts, it is characterised in that described double The reagent of resistance screening is hygromycin and double propylamine phosphorus.

The method that a kind of plant polygenic inheritance the most according to claim 1 converts, it is characterised in that its tool Body step includes the induction of plant maturation seed callus, term single gene type high-quality height Agrobacterium infection rate The acquisition of embryo callus system and agriculture bacillus mediated polygenic inheritance step of converting；

Described plant maturation seed callus induction, sterilizes the mature seed of plant and is inoculated in after cleaning On callus inducing medium, incubator temperature 25 ± 2 DEG C, light culture 6-7 week, it is thus achieved that callus；

Described seed callus inducing culture MSD5 be MS minimal medium add 30g/L sucrose, 5mg/L2,4-D and 7.5g/L agar, autoclaving 15min, pH=5.8；

The acquisition of the embryo callus system of described term single gene type high-quality height Agrobacterium infection rate: institute The callus obtained uses visual method to screen, and selects the most faint yellow, short texture and has gentle The embryo callus of gloss be high-quality embryo callus, the high-quality embryo callus that will obtain Separate half callus to be inoculated in MSK2 culture medium, group training room temperature 25 ± 2 DEG C, light application time every day 16h light/8h is dark, cultivates 4-5 week, it is thus achieved that regeneration bud, and carries out the assessment of regeneration efficiency, then selects The raw rate wound healing system more than 90% carries out subculture, incubator temperature 25 ± 2 DEG C, and light culture 3-4 is all, then Infect the assessment of efficiency for Agrobacterium, finally select Agrobacterium to infect the efficiency wound healing system more than 30% and be list The embryo callus system of one genotype high-quality height Agrobacterium infection rate, then carries out expanding propagation, incubator temperature Spending 25 ± 2 DEG C, light culture, every 3-4 week subculture is once；

Described agriculture bacillus mediated polygenic inheritance converts, the term single gene type high-quality of described expanding propagation Callus in the embryo callus system of high Agrobacterium infection rate is divided into fritter, is subsequently placed in threeway and is dried Evacuation in ware, uses the Agrobacterium bacterium solution containing different binary vectors to described term single gene type high-quality The embryo callus of high Agrobacterium infection rate is hatched altogether, then removes the Agrobacterium bacterium solution of residual, and 25 ± 2 DEG C, co-culturing 3 days in dark, the callus after co-culturing is transferred in resistant calli screening training Supporting base MSD3HB to screen, incubator temperature 25 ± 2 DEG C, light culture, every 3 weeks transfer once, directly Grow to resistant calli, the resistant calli of acquisition is transferred to resistant calli screening culture medium On MSK2HB, group training room temperature 25 ± 2 DEG C, light application time 16h every day light/8h is dark, every 4 week switchings one It is secondary, until resistant buds grows；The resistance regeneration bud obtained is forwarded on root media MSRHB, group training room Temperature 25 ± 2 DEG C, light application time 16h every day light/8h is dark；The regrowth obtained conventionally is transplanted to In soil, after 2 weeks, regrowth survives；

Described resistant calli screening culture medium MSD3HB be MS minimal medium add 30g/L sucrose, The double propylamine phosphorus of 3mg/L2,4-D, 30mg/L hygromycin, 2.4mg/L, 400mg/L Cefotaxime sodium and 7.5g/L Agar, autoclaving 15min, pH=5.8；

Described resistant buds regeneration culture medium MSK2HB is that MS minimal medium adds 30g/L sucrose, 2mg/L The double propylamine phosphorus of kinetins, 10mg/L hygromycin, 1.2mg/L, 400mg/L Cefotaxime sodium and 7.5g/L fine jade Fat, autoclaving 15min, pH=5.8；

Described resistant buds root media MSRHB be 1/2MS minimal medium add 15g/L sucrose, The double propylamine phosphorus of 10mg/L hygromycin, 1.2mg/L, 400mg/L Cefotaxime sodium and 7.5g/L agar, high pressure Sterilizing 15min, pH=5.8.