CN102010877B - Agrobacterium rhizogenes K599-mediated chrysanthemum transgenic method - Google Patents

Abstract

The invention provides an agrobacterium rhizogenes K599-mediated chrysanthemum transgenic method with high chrysanthemum regeneration efficiency, which is characterized in that living plant materials are directly used as an acceptor infected by agrobacterium rhizogenes K599 to induce adventitious roots (the wound of a chrysanthemum leaf or the axilla of the leaf is infected by agrobacterium rhizogenes K599 bacterial liquid), the adventitious roots induce callus to perform differentiation culture to obtain a complete plant, and the beneficial effects are mainly reflected in that: the appearance of false positive adventitious roots can be overcome; meanwhile, the aim of effectively inhibiting and killing agrobacterium can be achieved by intercepting the area close to the growth point at the top end of the adventitious root for subculture by utilizing the characteristic that the area of the growth point at the top end is sterile in the adventitious root propagation process and combining the sterilization of the cefamycin, and the subsequent processes of adventitious root induced healing and differentiation do not need to use antibiotics for sterilization, so that the transgenic regeneration efficiency is improved.

Description

A kind of chrysanthemum transgenic method of Agrobacterium rhizogenes K599 mediation

(1) technical field

The present invention relates to a kind of chrysanthemum transgenic method of Agrobacterium rhizogenes K599 mediation.

(2) background technology

Chrysanthemum (Dendranthema morifolium) belongs to the plant of composite family Chrysanthemum, originates in China, is one of China's ten great tradition famous flowers and the world's four big cut-flowers; Also be the famous-object flowers that integrate Cultural Significance and economic worth; And have edible and pharmaceutical use (Dai Silan etc., the progress of Chrysanthemum systematics and chrysanthemum origin, Beijing Forestry University's journal; 2002,24 (5/6): 230-234).Strain shape, flower type and pattern are the important factors that influences flower characteristics such as chrysanthemum; And plant transgenic technology plays an important role in the flower characteristic in improvement, and Shao Hanshuan etc., Petty etc., Zheng etc. successively report and utilize transgenic technology to change genes such as lef, phyA, Phy B-1 over to chrysanthemum, makes form and the florescence etc. of chrysanthemum that change (Shao Hanshuan etc. take place; The research of the clone of Arabidopis thaliana LFY cDNA and conversion chrysanthemum; Botany Gazette, 1999,41 (3): 268-271; Petty et al, Modifying chrysanthemum (Dendranthema grandiflorum) growth habit genetic manipulation, Acta Hort, 2000,508:319-321; Zheng et al; Modification of plant architecture inchrysanthemum by ecotopic expression of the tabacco phytochrome B1 gene; J Amer Soc Hort Sci, 2001,126 (1): 19-26).For plant type, be used for potted plant or ground by the chrysanthemum of purposes, often need that plant is downgraded, branchiness is strong, the characteristics such as number is many of blossoming.At present; Change the strain shape of ornamental plant; Gene commonly used is rol gene (Zuker et al, rolC-transgenic carnation with improved horticultural traits:quantitative and qualitative analysis of greenhouse-grown plants, J Am SocHort Sci from Agrobacterium rhizogenes Ri plasmid; 2001,126:13-18).Kiyokawa etc. have changed Agrobacterium rhizogenes rolA, B, C gene in the Begonia tuberhybrida over to; The result observes plant dwarfing, late blooming; The equal crumple of leaf and petal (Kiyokawa et al, Genetic transformation of Begonia tuberhybrida by Ri rolgenes, Plant Cell Reports; 1996,15 (8): 606-609).Handa etc. import the rol gene in the agueweed grass rough gentian (prairie gentian); Except observing transfer-gen plant dwarfing, leaf crumple; Also observe the cup-shaped variation of corolla (Handa et al, Genetic transformationof Eustoma grandifiorum with rol gene, Acta Hort; 1995,392:209-218).The method that usefulness particle gun such as Zuker and agrobacterium tumefaciens combine imports the rol gene in the oeillet, and the rol gene has reduced the generation of oeillet axillalry bud, strengthens the ability of root of hair.Dolgov etc. import the chrysanthemum kind with the rolC gene and ' among the White Snowdon ', obtain two and transform system, wherein have a system to compare with contrast; Show as and grow thickly, downgrade; And leaf divides (Dolgov et al, Agrobacterialtransformation of chrysanthemum, Acta Hort more; 1997,447:329-334).In addition, (Fu Rongzhao and Liu Min obtain chrysanthemum transfer-gen plant, plant physiology journal, 1998,24 (1): 72-76 through agrobacterium tumefaciens-mediated transformation to have research report to utilize Agrobacterium transgenic method to change the goal gene of difference in functionality over to chrysanthemum; Wang Guanlin etc., the aphid resistance research of GNA gene transformation chrysanthemum and transfer-gen plant, Acta Genetica Sinica, 2004,31 (12): 1434-1438; Big waves etc., the heterogenous expression of AtDREB1A gene in chrysanthemum have improved the patience that plant is coerced arid and salt marsh, Chinese science (C collects), 2006,36 (3): 223-231; Big waves etc., the regeneration of ground-cover chrysanthemum Fall Color somatic embryo approach, the winter resistance of genetic transformation and transfer-gen plant detects, Scientia Agricultura Sinica, 2006,39 (7): 1443-1450; Sun Lei etc. utilize the double T-DNA carrier system to obtain non selecting sign transgene chrysanthemum, gardening journal; 2008,35 (5): 727-734, Jiang Dan etc.; Arabidopis thaliana florescence gene FT transforms cut-flower chrysanthemum ' refreshing horse ', gardening journal, 2010; 37 (3): 423-430), but the regeneration efficiency of chrysanthemum is on the low side in the above-mentioned report.

(3) summary of the invention

The object of the invention provides the chrysanthemum transgenic method of the high Agrobacterium rhizogenes K599 mediation of a kind of chrysanthemum regeneration efficiency.

The technical scheme that the present invention adopts is:

A kind of chrysanthemum transgenic method of Agrobacterium rhizogenes K599 mediation, said method comprises:

(1) Agrobacterium rhizogenes K599 bacterium liquid is infected chrysanthemum aseptic seedling blade or axil, induce the formation transgenic adventitious roots; Infecting the formation transgenic adventitious roots can carry out according to ordinary method; Concrete grammar can be following: with high 5～8cm chrysanthemum tissue culture sterile seedling plant; The above blade in middle part marks with scalper or cuts long 0.5～1cm wound with Dissecting scissors, draws 15～20 μ L Agrobacterium bacterium drops with microsyringe and is added on blade wound or axil place, perhaps draws 5～10 μ L Agrobacterium bacterium liquid with the asepsis injector of 5mL capacity; Syringe needle is inserted blade or axil place, again bacterium liquid is injected.

(2) after transgenic adventitious roots grows into more than the 2cm; The transgenic adventitious roots of clip 0.4～0.8cm length from the top; Changing subculture medium over to breeds; Whenever change new subculture medium at a distance from the transgenic adventitious roots top of 15～20 days clip length 0.4～0.8cm; According to said method carry out succeeding transfer culture and change inducing culture after 2～3 generations again over to and carry out inducing culture, change the callus that induces over to division culture medium and carry out differentiation culture, change the seedling that differentiates over to whole plant that root media forms the band root;

Said subculture medium is for adding the MS substratum of 500mg/L cephamycin;

Said inducing culture is for adding the MS substratum of 0.5mg/L 6-benzyl aminopurine+1mg/L naphthylacetic acid;

Said division culture medium is for adding the MS substratum of 2mg/L 6-benzyl aminopurine;

Said root media is for adding the MS substratum of 0.5mg/L growth hormone;

Above-mentioned cultivation is all carried out in culturing bottle with cover;

When the whole plant that (3) differentiates grows to 5～6cm, open cultivate bottle cap and practice seedling 3d after,

Be transplanted to peat again: rice chaff ash: in 3: 1: 1 the soil of perlite volume ratio, growth under field conditions (factors) obtains the transgenic chrysanthemum.

Agrobacterium rhizogenes K599 can be wild-type Agrobacterium rhizogenes K599 in the said step (1); Also can be for having the Agrobacterium rhizogenes K599 of goal gene, the said Agrobacterium rhizogenes K599 that has goal gene imports wild-type Agrobacterium rhizogenes K599 by the plasmid that contains goal gene and obtains.Said goal gene can be the gene that this area routine is used to improve chrysanthemum form, florescence or strain shape.

Have many reports to point out, containing on the substratum of plant hormone, the blade and the stem that exsomatize self can produce not genetically modified adventive root (Lin Ya etc. in culturing process; Influence the factor of Chinese rose callus induction and differentiation; Molecular Plant Breeding, 2006,4 (2): 223-227; Nolan et al; Auxinup-ulates MtSERK1 expression in both Medicago truncatula root-forming andembryogenic cultures; Plant Physiol; 2003,133 (1): 218-230), the contriver also observes this situation in the isolated culture experiment of carrying out chrysanthemum blade and stem; And the object that utilizes chrysanthemum blade and axil under the live body growth conditions to infect as Agrobacterium; Detect the adventive root that obtains through PCR and be transgenic adventitious roots; Therefore; Utilize living materials directly as the acceptor of Agrobacterium inducing adventitious root, can effectively overcome the appearance of false positive transgenic adventitious roots.In addition, in Agrobacterium transgenic process, must use microbiotic to suppress until killing Agrobacterium after explant and Agrobacterium are cultivated altogether, normally used microbiotic is that concentration is the cephamycin of 250～1000mg/L.Yepes etc. observe cephamycin to the toxic effect of the explant regeneration of chrysanthemum; Differentiation (the Yepes etal that can suppress plant; Agrobacterium tumefaciens versus biolistic-mediated transformation of thechrysanthemum cvs.Polaris and Golden Polaris with nucleocapsid proteingenes of three Tospovirus species; International Symposium on Cut Flowers inthe Tropics species; Acta Hort, 1999,482:209-218).Utilize the aseptic characteristics in apical growing point zone in the adventive root reproductive process among the present invention; Carry out succeeding transfer culture 2～3 times through intercepting adventive root top near the vegetative point zone; Be used in combination the cephamycin sterilization, can suppress well and kill Agrobacterium rhizogenes, therefore; In subsequently root induction callus and atomization, need not re-use microbiotic (cephamycin) sterilization, thereby effectively improve the transgenic regeneration efficiency.

Though had report to utilize agrobacterium-mediated transformation and particle bombardment to obtain the transgenic chrysanthemum at present, its transformation efficiency is still not high.The present invention utilizes Agrobacterium rhizogenes K599 that the blade of chrysanthemum cutting and uncut axil are carried out live body to infect and take root, and its frequency of taking root reaches 88% and 60% respectively, and the callus induction rate of adventive root and callus regeneration rate reach 75% and 63.3% respectively.And the transfer-gen plant of acquisition has realized deriving from the efficiently expressing of external source rolC of K599Ri plasmid, and this provides a good experimental system for the transfer of further carrying out chrysanthemum breeding wheat for semidwarfness and target gene.

The receptor-inducible adventive root that the present invention utilizes the live plant material directly to infect as Agrobacterium rhizogenes K599, its beneficial effect is mainly reflected in: can overcome the appearance of false positive adventive root; Simultaneously; Utilize the aseptic characteristics in apical growing point zone in the adventive root reproductive process; Carry out succeeding transfer culture through intercepting adventive root top near the vegetative point zone, be used in combination the cephamycin sterilization, can reach effective inhibition and kill the purpose of Agrobacterium; In subsequently root induction callus and atomization, need not re-use the microbiotic sterilization, improve the transgenic regeneration efficiency.The present invention utilizes Agrobacterium rhizogenes K599 that the chrysanthemum live body is carried out inducing of adventive root and realizes the regeneration of adventive root, for the breeding wheat for semidwarfness of chrysanthemum and the transfer of target gene provide good experimental technique.

(4) description of drawings

Fig. 1 infects the regeneration plant that forms transgenic adventitious roots and transgenic adventitious roots for Agrobacterium rhizogenes K599 to the chrysanthemum live body: A: the aseptic seedling blade is infected by the K599 live body takes root; B: the aseptic seedling axil is infected by the K599 live body takes root; C: the transgenic adventitious roots tissue culture forms callus; D: transgenic adventitious roots tissue culture regeneration of plantlet; E: the transfer-gen plant of transplant survival.

Fig. 2 carries out the pcr amplification result for transgenic hairly root and regenerated plant; M:1 Kb ladderDNA molecular weight marker; 1～3: transgenic hair shape adventive root; 4: the non-transgenic root; 5～7: transgenic regenerated plant; 8: the non-transgenic plant; 9: Agrobacterium rhizogenes K599Ri plasmid.

Fig. 3 is the expression analysis of rolC gene in chrysanthemum wild-type and transfer-gen plant; WT: non-transgenic chrysanthemum; T01, T02 and T03: transgenic chrysanthemum.

Fig. 4 is the adventive root that utilizes the chrysanthemum commentaries on classics gfp gene of Agrobacterium rhizogenes K599 mediation acquisition.

Fig. 5 is the regeneration plant that utilizes the chrysanthemum commentaries on classics gfp gene of Agrobacterium rhizogenes K599 mediation acquisition.

(5) embodiment

Below in conjunction with specific embodiment the present invention is described further, but protection scope of the present invention is not limited in this:

Embodiment 1:

Materials and methods:

1 agrobacterium strains and cultivation thereof

Experiment uses bacterial classification to be wild-type Agrobacterium rhizogenes K599 (available from the National Collection ofPlant Pathogenic Bacteria in U.K.).With the bacterial classification of-80 ℃ of preservations on the solid medium of LB+Str (Streptomycin sulphate) 50mg/L, rule, 28 ℃ of incubated overnight; Picking list bacterium colony in the liquid culture of LB+Str (Streptomycin sulphate) 50mg/L, 230～250r/min, 28 ℃ of incubated overnight, eugonic bacterium liquid is as further experiment.

2 chrysanthemum materials

With the acceptor material of chrysanthemum (little purple chrysanthemum is available from the flowers market, Hangzhou) tissue culture sterile seedling as the infection induced hair of Agrobacterium rhizogenes K599 shape adventive root.

Inducing of 3 maos of shape adventive root

The eugonic Agrobacterium rhizogenes K599 of incubated overnight is resuspended in the LB substratum light modulation density (OD with after twice of the LB substratum eccentric cleaning ₆₀₀) to about 0.5.With high 5～8cm chrysanthemum tissue culture sterile seedling plant; The above blade in middle part marks with scalper or cuts long 0.5～1cm wound with Dissecting scissors; Draw 15～20 μ L Agrobacterium bacterium drops with microsyringe and be added on blade wound or axil place; Perhaps draw 5～10 μ L Agrobacterium bacterium liquid, syringe needle is inserted blade or axil place, again bacterium liquid is annotated the people with the asepsis injector of 5mL capacity.The chrysanthemum plant continues to cultivate subsequently.

The cultivation of 4 maos of shape adventive root and regeneration

After adventive root grows into more than the 2cm; The about 0.5cm of clip adventive root from the top; Change MS (0)+500mg/L Cef (cephamycin) (being that the MS substratum adds 500mg/L Cef) over to and go up breeding; Whenever the top at a distance from about 15～20 days clip adventive root 0.5cm changes on the new substratum; Change evoked callus on the MS+6-BA 0.5mg/L+NAA 1mg/L inducing culture that does not contain cephamycin again over to after cultivating for 2～3 generations, behind the 30d callus that induces changed on the MS+6-BA 2mg/L and break up, change the seedling that differentiates over to form the band root on the MS+IAA 0.5mg/L substratum whole plant at last.The whole plant that differentiates grows to about 5cm, opens and cultivates bottle cap white silk seedling 3d, is transplanted to contain peat again: rice chaff ash: in the basin alms bowl of perlite (volume ratio 3: 1: 1), and growth under field conditions (factors).

The extraction of 5 Agrobacterium K599 plasmids, hair shape adventive root and regeneration plant DNA

The extraction of Agrobacterium K599 DNA in the same way the method in Taihe county etc. (to Taihe county etc., the detoxifcation of wild-type Agrobacterium rhizogenes K599, heredity, 2001,23 (4): 336-340).The method of extracting hair shape adventive root and regeneration plant DNA is: a small amount of hairly root of clip or blade are put into 2mL Eppendorf centrifuge tube, smash to pieces with glass stick after adding the liquid nitrogen quick-frozen, add 750 μ L DNA extract (100mmol/L Tris-HCl; 20mmol/L EDTA; 500mmol/L NaCl 1.5%SDS), adds isopyknic chloroform: ethanol behind 60 ℃ of water-bath 1h: primary isoamyl alcohol (volume ratio is 80: 16: 4) mixed solution; Room temperature leaves standstill 30min; Put upside down back and forth during this time for several times, centrifugal 10min under 4 ℃, 11000r/min gets upper phase and uses isopyknic isopropanol precipitating; Clean 2 times with 70% ethanol centrifugal 5min under 4 ℃, 6000r/min again, be dissolved in the aseptic ddH of 20 μ L after the drying at room temperature ₂O store in-20 ℃ subsequent use.

The PCR of 6 transgenic hair shape adventive root and regeneration plant identifies

RolC gene order (the Furner et al that delivers according to Furner etc.; An Agrobacteriumtransformation in the evolution of the genus Nicotiana, Nature, 1986; 319:422-427), design and synthesize the PCR primer of amplification rolC.The rolC primer is: rolC-P1:5 '-CTCCTGACATCAAACTCGTC-3 '; P2:5 '-TGCTTCGAGTTATGGGTACA-3 '.The pcr amplification parameter is following: behind 94 ℃ of 5min of initial sex change, then carry out 35 circulations, each circulation comprises 94 ℃ of sex change 45s, and 56 ℃ of annealing 45s and 72 ℃ of extension 90s extend 10min at 72 ℃ at last.Amplified production adopts 1.0% agarose gel electrophoresis, 120V 1h, EtBr (ethidium bromide) dyeing, and (Bio/Rad company) carries out Taking Pictures recording and analysis with gel imaging system.

7 regeneration plant qRT-PCR analyze

Extract test kit (TaKaRa company) with RNA and extract transfer-gen plant and contrast non-transgenic plant leaf mRNA, carry out the RT-PCR amplification with SYBR Green Realtime PCR Master Mix (TaKaRa company).According to chrysanthemum actin gene order (GenBank registration number AB205087) design primer, actin-P1:5 '-ACAACTGGCATTGTGTTGGA-3 ' and actin-P2:5 '-CAGGACATCTGAAACGCTCA-3 ', amplification is as confidential reference items.According to rolC gene order (GenBank registration number X03432) design primer; RolC-qP1:5 '-AAGAAAGTGCGGCGAAGTAA--3 ' and rolC-qP2:5 '-ATCTCAGGGTCAAGGACGTG-3 '; Carry out the quantitative analysis of rolC gene, increase with the MX3000P of Strategene company type quantitative real time PCR Instrument.The quantitative fluorescent PCR condition is initial 95 ℃ of 2min, and 95 ℃ of 30s, 59 ℃ of 30s, 72 ℃ of 20s carry out 40 circulations subsequently, finish the back 4 ℃ of preservations.Calculate rolC gene relative expression quantity (Schmittgen and Livak with reference to the method for Schmittgen and Livak; Analyzing real-time PCR data by thecomparative C (T) method; Nat Protoc, 2008,3 (6): 1101-1108).

The result:

Inducing of 1 mao of shape adventive root

Agrobacterium rhizogenes K599 infects the wound and the axil place of chrysanthemum plant leaf; About 10d, form macroscopic hairly root (Figure 1A; B); K599 infects the blade and the unscathed axil of cutting and induces the frequency of root to be respectively 88% (44/50) and 60% (30/50), the frequency of taking root blade＞axil.And the phenomenon of taking root does not all appear in the wound and the axillalry bud place that contrast the blade that does not drip K599 bacterium liquid.

The cultivation of 2 maos of shape adventive root and plant regeneration

The long 0.5cm of the clip left and right sides adventive root from the top; Changing upward ability fast breeding of MS+500mg/L Cef (cephamycin) over to; 15～20d can grow into more than the 2cm, from about the adventive root 0.5cm of top clip fast breeding, changes on the new substratum, and being commissioned to train through 2～3 can reach effective bacteria-eliminating efficacy after supporting; Change evoked callus on the MS+6-BA 0.5mg/L+NAA 1mg/L inducing culture that does not contain cephamycin again over to; The incision that after cultivating for 1 week on the inducing culture, can see root is expanded projection and is formed yellowish green callus, and the callus structure is tight, and inductivity is 75% (60/70).Behind the 30d callus changed on the MS+6-BA 2mg/L and break up, differentiate green seedling, change at last and form the whole plant with root on the MS+IAA0.5mg/L substratum, the callus differentiation frequency is 63.3% (19/30).Transplant survival behind the regeneration plant process white silk seedling, and can normally bloom (Fig. 1 C, D, E).The regeneration plant form shows as and stunts, has characteristics such as hairly root.

The pcr amplification of rolC gene detects in 3 maos of shape adventive root and the regeneration plant

Adventive root that extraction induces and regenerated plant DNA; Utilization is according to the PCR primer of rolC gene order design; From the hairly root of inducing formation and regeneration plant DNA, all increase DNA fragment specific about the 770bp of expectation, this fragment is identical with the specific fragment size that from the Ri DNA of Agrobacterium rhizogenes K599, amplifies; But not conversion root and non-conversion chrysanthemum leaf DNA do not amplify any band (Fig. 2).The Ri T-DNA that shows wild-type Agrobacterium rhizogenes K599 has been incorporated in the inductive hairly root genome, and is transfer-gen plant by adventive root regenerated plant.

The qRT-PCR of 4 regeneration plant rolC genes analyzes

Contrast as confidential reference items with the aclin gene, quantitative fluorescent PCR (qRT-PCR) analysis shows that the rolC gene has been realized efficiently expressing, and in the non-transgenic plant (WT) of wild-type, does not detect expression signal (Fig. 3) in transfer-gen plant (T01, T02, T03).

Embodiment 2:

(1) (the pBIN-35S-GFP construction process is seen document: Xu Jiming to Taihe county, contains structure and the efficiently expressing in the petunia transgenic adventitious roots of gfp plant transgene expression vector will to have the plasmid pBIN-35S-GFP of GFP plasmagene gfp with freeze-thaw method; Heredity, 2008,30 (8): 1069-1074) import Agrobacterium rhizogenes K599; Promptly in 50 μ L K599 competent cells, add about 0.1 μ g plasmid purification pBIN-35S-GFP; Place 10min on ice behind the mixing, place liquid nitrogen flash freezer 5min, use 28 ℃ of water-bath heat shock 5min immediately; Add 500 μ L LB liquid nutrient mediums, at 28 ℃ of slow shaking culture 2h.Get 100 μ L bacterium liquid separate application on LB+Km (kantlex) 50mg/L+Str (Streptomycin sulphate) 50mg/L solid medium, cultivate about 48h screening resistance bacterium colony for 28 ℃, obtain to have the Agrobacterium rhizogenes K599 of plasmid pBIN-35S-GFP.

(2) with the acceptor material of chrysanthemum tissue culture sterile seedling as the infection induced hair of Agrobacterium rhizogenes K599/pBIN-35S-GFP shape adventive root.The eugonic Agrobacterium rhizogenes K599/pBIN-35S-GFP of incubated overnight is resuspended in the LB substratum light modulation density (OD with after twice of the LB substratum eccentric cleaning ₆₀₀) to about 0.5.With high 5～8cm chrysanthemum tissue culture sterile seedling plant; The above blade in middle part marks with scalper or cuts long 0.5～1cm wound with Dissecting scissors; Draw 15～20 μ L Agrobacterium bacterium drops with microsyringe and be added on blade wound or axil place; Perhaps draw 5～10 μ L Agrobacterium bacterium liquid, syringe needle is inserted blade or axil place, again bacterium liquid is annotated the people with the asepsis injector of 5mL capacity.The chrysanthemum plant continues to cultivate subsequently.About 10d, form macroscopic hairly root.Obtaining chrysanthemum changes the adventive root of gfp gene; With ZEISS microscope (model: Axio imager); (spectral filter FITC) carries out fluoroscopic examination to the adventive root that induces under blue excitation light; Adventive root sends intensive green fluorescence (Fig. 4) under fluorescent microscope, show that the gfp gene is efficiently expressed.

(3) the long 0.5cm of the clip left and right sides adventive root from the top; Changing upward propagation of MS (0)+500mg/L Cef (cephamycin) over to; 15～20d can grow into more than the 2cm; From about the adventive root 0.5cm of top clip fast breeding, change on the new substratum, being commissioned to train through 2～3 reaches effective bacteria-eliminating efficacy after supporting, and changes evoked callus on the MS+6-BA 0.5mg/L+NAA 1mg/L inducing culture that does not contain cephamycin again over to; The incision that after cultivating for 1 week on the inducing culture, can see root is expanded projection and is formed yellowish green callus, and the callus structure is tight.Behind the 30d callus changed on the MS+6-BA 2mg/L and break up, differentiate green seedling, change at last and form whole plant (Fig. 5) on the MS+IAA 0.5mg/L substratum with root.Transplant survival behind the regeneration plant process white silk seedling, and can normally bloom.

Claims (2)

1. the chrysanthemum transgenic method of Agrobacterium rhizogenes K599 mediation, said method comprises:

(1) Agrobacterium rhizogenes K599 bacterium liquid is infected chrysanthemum aseptic seedling blade or axil, induce the formation transgenic adventitious roots;

(2) after transgenic adventitious roots grows into more than the 2cm; The transgenic adventitious roots of clip 0.4～0.8cm length from the top; Changing subculture medium over to breeds; Whenever change new subculture medium at a distance from the transgenic adventitious roots top of 15～20 days clip length 0.4～0.8cm; According to said method going down to posterity to cultivate changes inducing culture again over to after 2～3 generations and carries out inducing culture, changes the callus that induces over to division culture medium and carries out differentiation culture, changes the seedling that differentiates over to whole plant that root media forms the band root;

Said subculture medium is for adding the MS substratum of 500mg/L cephamycin;

Said inducing culture is for adding the MS substratum of 0.5mg/L 6-benzyl aminopurine+1mg/L naphthylacetic acid;

Said division culture medium is for adding the MS substratum of 2mg/L 6-benzyl aminopurine;

Said root media adds the MS substratum of 0.5mg/L growth hormone;

Above-mentioned cultivation is all carried out in culturing bottle with cover;

When the whole plant that (3) differentiates grows to 5～6cm, open cultivate bottle cap and practice seedling 3d after, be transplanted to peat again: rice chaff ash: in 3: 1: 1 the soil of perlite volume ratio, growth under field conditions (factors) obtains the transgenic chrysanthemum.

2. the method for claim 1 is characterized in that: Agrobacterium rhizogenes K599 is the Agrobacterium rhizogenes K599 that has goal gene in the said step (1), imports wild-type Agrobacterium rhizogenes K599 by the plasmid that contains goal gene and obtains.

Images