NZ625401B2 - Method for improved transformation using agrobacterium - Google Patents
Method for improved transformation using agrobacterium Download PDFInfo
- Publication number
- NZ625401B2 NZ625401B2 NZ625401A NZ62540112A NZ625401B2 NZ 625401 B2 NZ625401 B2 NZ 625401B2 NZ 625401 A NZ625401 A NZ 625401A NZ 62540112 A NZ62540112 A NZ 62540112A NZ 625401 B2 NZ625401 B2 NZ 625401B2
- Authority
- NZ
- New Zealand
- Prior art keywords
- surfactant
- cells
- plant
- transformation
- plant cell
- Prior art date
Links
- 230000001131 transforming Effects 0.000 title claims abstract description 117
- 241000589158 Agrobacterium Species 0.000 title claims abstract description 48
- 239000004094 surface-active agent Substances 0.000 claims abstract description 90
- 239000007788 liquid Substances 0.000 claims abstract description 36
- 210000001161 Embryo, Mammalian Anatomy 0.000 claims abstract description 18
- ZQTYRTSKQFQYPQ-UHFFFAOYSA-N trisiloxane Chemical compound [SiH3]O[SiH2]O[SiH3] ZQTYRTSKQFQYPQ-UHFFFAOYSA-N 0.000 claims abstract description 15
- 241000196324 Embryophyta Species 0.000 claims description 165
- 239000002609 media Substances 0.000 claims description 93
- 210000002257 embryonic structures Anatomy 0.000 claims description 86
- 240000008042 Zea mays Species 0.000 claims description 50
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 47
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 claims description 40
- 235000009973 maize Nutrition 0.000 claims description 40
- 239000007787 solid Substances 0.000 claims description 9
- 239000001963 growth media Substances 0.000 claims description 7
- 239000002736 nonionic surfactant Substances 0.000 claims description 5
- 239000002671 adjuvant Substances 0.000 claims description 4
- 230000000240 adjuvant Effects 0.000 claims description 4
- 239000002280 amphoteric surfactant Substances 0.000 claims description 3
- 239000003945 anionic surfactant Substances 0.000 claims 2
- 210000004027 cells Anatomy 0.000 description 130
- 210000001519 tissues Anatomy 0.000 description 37
- 229920003013 deoxyribonucleic acid Polymers 0.000 description 30
- LINPVWIEWJTEEJ-UHFFFAOYSA-N methyl 2-chloro-9-hydroxyfluorene-9-carboxylate Chemical compound C1=C(Cl)C=C2C(C(=O)OC)(O)C3=CC=CC=C3C2=C1 LINPVWIEWJTEEJ-UHFFFAOYSA-N 0.000 description 28
- 239000000203 mixture Substances 0.000 description 25
- 206010020649 Hyperkeratosis Diseases 0.000 description 24
- 238000002474 experimental method Methods 0.000 description 23
- 238000011069 regeneration method Methods 0.000 description 22
- 239000003550 marker Substances 0.000 description 20
- 238000000034 method Methods 0.000 description 20
- 108091005946 yellow fluorescent protein Proteins 0.000 description 17
- 230000008929 regeneration Effects 0.000 description 16
- 241000625014 Vir Species 0.000 description 15
- 230000014509 gene expression Effects 0.000 description 14
- 239000011780 sodium chloride Substances 0.000 description 13
- 239000000126 substance Substances 0.000 description 13
- OJOBTAOGJIWAGB-UHFFFAOYSA-N Acetosyringone Chemical compound COC1=CC(C(C)=O)=CC(OC)=C1O OJOBTAOGJIWAGB-UHFFFAOYSA-N 0.000 description 12
- 241000502171 Distylium racemosum Species 0.000 description 12
- 229940029983 VITAMINS Drugs 0.000 description 12
- 229940021016 Vitamin IV solution additives Drugs 0.000 description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 12
- 230000012010 growth Effects 0.000 description 12
- 150000003839 salts Chemical class 0.000 description 12
- 239000011782 vitamin Substances 0.000 description 12
- 235000013343 vitamin Nutrition 0.000 description 12
- 229930003231 vitamins Natural products 0.000 description 12
- SQGYOTSLMSWVJD-UHFFFAOYSA-N Silver nitrate Chemical compound [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 description 11
- 230000002363 herbicidal Effects 0.000 description 11
- 239000004009 herbicide Substances 0.000 description 11
- 239000002253 acid Substances 0.000 description 10
- 201000009910 diseases by infectious agent Diseases 0.000 description 10
- 239000012499 inoculation medium Substances 0.000 description 10
- CDAISMWEOUEBRE-UHFFFAOYSA-N inositol Chemical compound OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 10
- 150000002500 ions Chemical class 0.000 description 10
- KWYUFKZDYYNOTN-UHFFFAOYSA-M potassium hydroxide Inorganic materials [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 10
- 230000000284 resting Effects 0.000 description 10
- 239000000725 suspension Substances 0.000 description 10
- CZMRCDWAGMRECN-UGDNZRGBSA-N D-sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 9
- CZMRCDWAGMRECN-GDQSFJPYSA-N Sucrose Natural products O([C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](CO)O1)[C@@]1(CO)[C@H](O)[C@@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-GDQSFJPYSA-N 0.000 description 9
- 238000004519 manufacturing process Methods 0.000 description 9
- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- 239000005720 sucrose Substances 0.000 description 9
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 8
- 229960002429 Proline Drugs 0.000 description 8
- 230000003115 biocidal Effects 0.000 description 8
- 230000002708 enhancing Effects 0.000 description 8
- 230000002068 genetic Effects 0.000 description 8
- 235000018102 proteins Nutrition 0.000 description 8
- 102000004169 proteins and genes Human genes 0.000 description 8
- 108090000623 proteins and genes Proteins 0.000 description 8
- 241000589155 Agrobacterium tumefaciens Species 0.000 description 7
- 239000003242 anti bacterial agent Substances 0.000 description 7
- 235000005822 corn Nutrition 0.000 description 7
- 235000005824 corn Nutrition 0.000 description 7
- 230000002255 enzymatic Effects 0.000 description 7
- 230000002401 inhibitory effect Effects 0.000 description 7
- 229940064005 Antibiotic throat preparations Drugs 0.000 description 6
- 229940083879 Antibiotics FOR TREATMENT OF HEMORRHOIDS AND ANAL FISSURES FOR TOPICAL USE Drugs 0.000 description 6
- 229940042052 Antibiotics for systemic use Drugs 0.000 description 6
- 229940042786 Antitubercular Antibiotics Drugs 0.000 description 6
- 241001058146 Erium Species 0.000 description 6
- 229940093922 Gynecological Antibiotics Drugs 0.000 description 6
- 229940024982 Topical Antifungal Antibiotics Drugs 0.000 description 6
- 238000009632 agar plate Methods 0.000 description 6
- 239000005018 casein Substances 0.000 description 6
- 235000021240 caseins Nutrition 0.000 description 6
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- 239000003112 inhibitor Substances 0.000 description 6
- 230000003993 interaction Effects 0.000 description 6
- 229940079866 intestinal antibiotics Drugs 0.000 description 6
- 239000000463 material Substances 0.000 description 6
- 229940005935 ophthalmologic Antibiotics Drugs 0.000 description 6
- 101700082413 tant Proteins 0.000 description 6
- 230000035897 transcription Effects 0.000 description 6
- GOCUAJYOYBLQRH-UHFFFAOYSA-N 2-[4-[3-chloro-5-(trifluoromethyl)pyridin-2-yl]oxyphenoxy]propanoic acid Chemical compound C1=CC(OC(C)C(O)=O)=CC=C1OC1=NC=C(C(F)(F)F)C=C1Cl GOCUAJYOYBLQRH-UHFFFAOYSA-N 0.000 description 5
- 241000894006 Bacteria Species 0.000 description 5
- FPPNZSSZRUTDAP-UWFZAAFLSA-N Carbenicillin Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)C(C(O)=O)C1=CC=CC=C1 FPPNZSSZRUTDAP-UWFZAAFLSA-N 0.000 description 5
- 229960003669 Carbenicillin Drugs 0.000 description 5
- 241000238631 Hexapoda Species 0.000 description 5
- 240000007594 Oryza sativa Species 0.000 description 5
- 235000007164 Oryza sativa Nutrition 0.000 description 5
- 230000001580 bacterial Effects 0.000 description 5
- 238000010367 cloning Methods 0.000 description 5
- 230000018109 developmental process Effects 0.000 description 5
- 239000000413 hydrolysate Substances 0.000 description 5
- 230000000977 initiatory Effects 0.000 description 5
- 239000003921 oil Substances 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 235000009566 rice Nutrition 0.000 description 5
- 241000894007 species Species 0.000 description 5
- KWIUHFFTVRNATP-UHFFFAOYSA-N Betaine Natural products C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 description 4
- 210000002421 Cell Wall Anatomy 0.000 description 4
- IWEDIXLBFLAXBO-UHFFFAOYSA-N Dicamba Chemical compound COC1=C(Cl)C=CC(Cl)=C1C(O)=O IWEDIXLBFLAXBO-UHFFFAOYSA-N 0.000 description 4
- 229920002459 Intron Polymers 0.000 description 4
- 241000209510 Liliopsida Species 0.000 description 4
- SUKJFIGYRHOWBL-UHFFFAOYSA-N Sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- 229920001577 copolymer Polymers 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 238000005755 formation reaction Methods 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 4
- 238000002744 homologous recombination Methods 0.000 description 4
- 238000003898 horticulture Methods 0.000 description 4
- 238000002955 isolation Methods 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000006011 modification reaction Methods 0.000 description 4
- 235000019198 oils Nutrition 0.000 description 4
- 229920001296 polysiloxane Polymers 0.000 description 4
- 238000004114 suspension culture Methods 0.000 description 4
- 241000701489 Cauliflower mosaic virus Species 0.000 description 3
- 210000000349 Chromosomes Anatomy 0.000 description 3
- 229920001405 Coding region Polymers 0.000 description 3
- 229920000742 Cotton Polymers 0.000 description 3
- 239000005504 Dicamba Substances 0.000 description 3
- 102000007698 EC 1.1.1.1 Human genes 0.000 description 3
- 108010021809 EC 1.1.1.1 Proteins 0.000 description 3
- 240000008529 Triticum aestivum Species 0.000 description 3
- 241000607479 Yersinia pestis Species 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 239000006285 cell suspension Substances 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 239000000306 component Substances 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 230000023753 dehiscence Effects 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 230000001939 inductive effect Effects 0.000 description 3
- 230000000749 insecticidal Effects 0.000 description 3
- 230000000670 limiting Effects 0.000 description 3
- 210000000056 organs Anatomy 0.000 description 3
- -1 polysiloxane Polymers 0.000 description 3
- 230000001105 regulatory Effects 0.000 description 3
- 230000000392 somatic Effects 0.000 description 3
- 230000001629 suppression Effects 0.000 description 3
- 235000021307 wheat Nutrition 0.000 description 3
- LWTDZKXXJRRKDG-KXBFYZLASA-N (-)-Phaseollin Natural products C1OC2=CC(O)=CC=C2[C@H]2[C@@H]1C1=CC=C3OC(C)(C)C=CC3=C1O2 LWTDZKXXJRRKDG-KXBFYZLASA-N 0.000 description 2
- GINJFDRNADDBIN-FXQIFTODSA-M (2S)-2-[[(2S)-2-[[(2S)-2-azaniumyl-4-[methyl(oxido)phosphoryl]butanoyl]amino]propanoyl]amino]propanoate Chemical compound [O-]C(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H]([NH3+])CCP(C)([O-])=O GINJFDRNADDBIN-FXQIFTODSA-M 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N 1-[(1S,2R,3R,4S,5R,6R)-3-carbamimidamido-6-{[(2R,3R,4R,5S)-3-{[(2S,3S,4S,5R,6S)-4,5-dihydroxy-6-(hydroxymethyl)-3-(methylamino)oxan-2-yl]oxy}-4-formyl-4-hydroxy-5-methyloxolan-2-yl]oxy}-2,4,5-trihydroxycyclohexyl]guanidine Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- UNFWWIHTNXNPBV-WXKVUWSESA-N Actinospectacin Chemical compound O([C@@H]1[C@@H](NC)[C@@H](O)[C@H]([C@@H]([C@H]1O1)O)NC)[C@]2(O)[C@H]1O[C@H](C)CC2=O UNFWWIHTNXNPBV-WXKVUWSESA-N 0.000 description 2
- 102000007469 Actins Human genes 0.000 description 2
- 108010085238 Actins Proteins 0.000 description 2
- 241000766754 Agra Species 0.000 description 2
- 241000589156 Agrobacterium rhizogenes Species 0.000 description 2
- 241000219430 Betula pendula Species 0.000 description 2
- 235000006008 Brassica napus var napus Nutrition 0.000 description 2
- 240000000385 Brassica napus var. napus Species 0.000 description 2
- 235000006618 Brassica rapa subsp oleifera Nutrition 0.000 description 2
- 235000004977 Brassica sinapistrum Nutrition 0.000 description 2
- IAKHMKGGTNLKSZ-INIZCTEOSA-N Colchicine Chemical compound C1([C@@H](NC(C)=O)CC2)=CC(=O)C(OC)=CC=C1C1=C2C=C(OC)C(OC)=C1OC IAKHMKGGTNLKSZ-INIZCTEOSA-N 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N D-Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 108010000700 EC 2.2.1.6 Proteins 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 229920002148 Gellan gum Polymers 0.000 description 2
- IAJOBQBIJHVGMQ-UHFFFAOYSA-N Glufosinate Chemical compound CP(O)(=O)CCC(N)C(O)=O IAJOBQBIJHVGMQ-UHFFFAOYSA-N 0.000 description 2
- 240000007842 Glycine max Species 0.000 description 2
- 235000010469 Glycine max Nutrition 0.000 description 2
- 239000005562 Glyphosate Substances 0.000 description 2
- XDDAORKBJWWYJS-UHFFFAOYSA-N Glyphosate Chemical compound OC(=O)CNCP(O)(O)=O XDDAORKBJWWYJS-UHFFFAOYSA-N 0.000 description 2
- 241000219146 Gossypium Species 0.000 description 2
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 2
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 2
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 2
- 240000004658 Medicago sativa Species 0.000 description 2
- 108020004999 Messenger RNA Proteins 0.000 description 2
- WHNWPMSKXPGLAX-UHFFFAOYSA-N N-Vinylpyrrolidone Chemical compound C=CN1CCCC1=O WHNWPMSKXPGLAX-UHFFFAOYSA-N 0.000 description 2
- NWBJYWHLCVSVIJ-UHFFFAOYSA-N N-benzyladenine Chemical compound N=1C=NC=2NC=NC=2C=1NCC1=CC=CC=C1 NWBJYWHLCVSVIJ-UHFFFAOYSA-N 0.000 description 2
- JQXXHWHPUNPDRT-ZNQWNCHJSA-N O([C@](C1=O)(C)O/C=C/[C@@H]([C@H]([C@@H](OC(C)=O)[C@H](C)[C@H](O)[C@H](C)[C@@H](O)[C@@H](C)\C=C\C=C(C)/C(=O)Nc2c(O)c3c(O)c4C)C)OC)c4c1c3c(O)c2C=NN1CCN(C)CC1 Chemical compound O([C@](C1=O)(C)O/C=C/[C@@H]([C@H]([C@@H](OC(C)=O)[C@H](C)[C@H](O)[C@H](C)[C@@H](O)[C@@H](C)\C=C\C=C(C)/C(=O)Nc2c(O)c3c(O)c4C)C)OC)c4c1c3c(O)c2C=NN1CCN(C)CC1 JQXXHWHPUNPDRT-ZNQWNCHJSA-N 0.000 description 2
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 2
- 229940081190 Rifampin Drugs 0.000 description 2
- 239000005708 Sodium hypochlorite Substances 0.000 description 2
- 240000003768 Solanum lycopersicum Species 0.000 description 2
- 240000001016 Solanum tuberosum Species 0.000 description 2
- 235000002595 Solanum tuberosum Nutrition 0.000 description 2
- 229940090121 Sulfonylureas for blood glucose lowering Drugs 0.000 description 2
- YWBFPKPWMSWWEA-UHFFFAOYSA-O TRIAZOLOPYRIMIDINE Chemical compound BrC1=CC=CC(C=2N=C3N=CN[N+]3=C(NCC=3C=CN=CC=3)C=2)=C1 YWBFPKPWMSWWEA-UHFFFAOYSA-O 0.000 description 2
- 229920000401 Three prime untranslated region Polymers 0.000 description 2
- 108090000848 Ubiquitin Proteins 0.000 description 2
- 102400000757 Ubiquitin Human genes 0.000 description 2
- 235000017585 alfalfa Nutrition 0.000 description 2
- 235000017587 alfalfa Nutrition 0.000 description 2
- UHOVQNZJYSORNB-UHFFFAOYSA-N benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 2
- 239000007844 bleaching agent Substances 0.000 description 2
- 235000008984 brauner Senf Nutrition 0.000 description 2
- 230000001488 breeding Effects 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 235000013339 cereals Nutrition 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000013080 embryo development ending in birth or egg hatching Effects 0.000 description 2
- 230000013144 embryo development ending in seed dormancy Effects 0.000 description 2
- 239000003623 enhancer Substances 0.000 description 2
- 239000000216 gellan gum Substances 0.000 description 2
- 235000010492 gellan gum Nutrition 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 102000005396 glutamine synthetase family Human genes 0.000 description 2
- 108020002326 glutamine synthetase family Proteins 0.000 description 2
- 229940097068 glyphosate Drugs 0.000 description 2
- 239000005090 green fluorescent protein Substances 0.000 description 2
- 239000010903 husk Substances 0.000 description 2
- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 description 2
- 230000002147 killing Effects 0.000 description 2
- 238000009630 liquid culture Methods 0.000 description 2
- 230000001404 mediated Effects 0.000 description 2
- 229920002106 messenger RNA Polymers 0.000 description 2
- 229910001507 metal halide Inorganic materials 0.000 description 2
- 150000005309 metal halides Chemical class 0.000 description 2
- QYDYPVFESGNLHU-KHPPLWFESA-N methyl oleate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC QYDYPVFESGNLHU-KHPPLWFESA-N 0.000 description 2
- 230000003287 optical Effects 0.000 description 2
- 230000036961 partial Effects 0.000 description 2
- 230000008506 pathogenesis Effects 0.000 description 2
- 125000000951 phenoxy group Chemical group [H]C1=C([H])C([H])=C(O*)C([H])=C1[H] 0.000 description 2
- 230000006308 pollination Effects 0.000 description 2
- 229920001888 polyacrylic acid Polymers 0.000 description 2
- 229920000136 polysorbate Polymers 0.000 description 2
- 235000012015 potatoes Nutrition 0.000 description 2
- 229960001225 rifampicin Drugs 0.000 description 2
- KEAYESYHFKHZAL-UHFFFAOYSA-N sodium Chemical compound [Na] KEAYESYHFKHZAL-UHFFFAOYSA-N 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
- 235000012424 soybean oil Nutrition 0.000 description 2
- 239000003549 soybean oil Substances 0.000 description 2
- 229960000268 spectinomycin Drugs 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 description 2
- 230000017613 viral reproduction Effects 0.000 description 2
- 230000001018 virulence Effects 0.000 description 2
- 230000000007 visual effect Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- DNIAPMSPPWPWGF-GSVOUGTGSA-N (-)-propylene glycol Chemical compound C[C@@H](O)CO DNIAPMSPPWPWGF-GSVOUGTGSA-N 0.000 description 1
- OYIKARCXOQLFHF-UHFFFAOYSA-N (5-cyclopropyl-1,2-oxazol-4-yl)-[2-methylsulfonyl-4-(trifluoromethyl)phenyl]methanone Chemical compound CS(=O)(=O)C1=CC(C(F)(F)F)=CC=C1C(=O)C1=C(C2CC2)ON=C1 OYIKARCXOQLFHF-UHFFFAOYSA-N 0.000 description 1
- 229920000160 (ribonucleotides)n+m Polymers 0.000 description 1
- CAAMSDWKXXPUJR-UHFFFAOYSA-N 1,5-dihydro-4H-imidazol-4-one Chemical class O=C1CNC=N1 CAAMSDWKXXPUJR-UHFFFAOYSA-N 0.000 description 1
- VJYIFXVZLXQVHO-UHFFFAOYSA-N 1-(2-chlorophenyl)sulfonyl-3-(4-methoxy-6-methyl-1,3,5-triazin-2-yl)urea Chemical compound COC1=NC(C)=NC(NC(=O)NS(=O)(=O)C=2C(=CC=CC=2)Cl)=N1 VJYIFXVZLXQVHO-UHFFFAOYSA-N 0.000 description 1
- PRPINYUDVPFIRX-UHFFFAOYSA-N 1-Naphthaleneacetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CC=CC2=C1 PRPINYUDVPFIRX-UHFFFAOYSA-N 0.000 description 1
- ROOPEIGRBDVOKP-UHFFFAOYSA-N 2,3-di(butan-2-yl)phenol Chemical compound CCC(C)C1=CC=CC(O)=C1C(C)CC ROOPEIGRBDVOKP-UHFFFAOYSA-N 0.000 description 1
- XBVFJKRTALSVOX-UHFFFAOYSA-N 2,3-di(butan-2-yl)phenol;2-methyloxirane;oxirane Chemical compound C1CO1.CC1CO1.CCC(C)C1=CC=CC(O)=C1C(C)CC XBVFJKRTALSVOX-UHFFFAOYSA-N 0.000 description 1
- 229940100228 Acetyl Coenzyme A Drugs 0.000 description 1
- ZSLZBFCDCINBPY-ZSJPKINUSA-N Acetyl-CoA Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCSC(=O)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 ZSLZBFCDCINBPY-ZSJPKINUSA-N 0.000 description 1
- UDMBCSSLTHHNCD-KQYNXXCUSA-N Adenosine monophosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O UDMBCSSLTHHNCD-KQYNXXCUSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 240000005781 Arachis hypogaea Species 0.000 description 1
- 241000193388 Bacillus thuringiensis Species 0.000 description 1
- 229940097012 Bacillus thuringiensis Drugs 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- 229960001561 Bleomycin Drugs 0.000 description 1
- 108010006654 Bleomycin Proteins 0.000 description 1
- 239000005489 Bromoxynil Substances 0.000 description 1
- UPMXNNIRAGDFEH-UHFFFAOYSA-N Bromoxynil Chemical compound OC1=C(Br)C=C(C#N)C=C1Br UPMXNNIRAGDFEH-UHFFFAOYSA-N 0.000 description 1
- 229940041514 Candida albicans extract Drugs 0.000 description 1
- 229940089639 Cornsilk Drugs 0.000 description 1
- NDUPDOJHUQKPAG-UHFFFAOYSA-N Dalapon Chemical compound CC(Cl)(Cl)C(O)=O NDUPDOJHUQKPAG-UHFFFAOYSA-N 0.000 description 1
- 102000016680 Dioxygenases Human genes 0.000 description 1
- 108010028143 Dioxygenases Proteins 0.000 description 1
- 108010066133 EC 1.5.1.11 Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 108090000331 Firefly luciferases Proteins 0.000 description 1
- 102100004985 GUSB Human genes 0.000 description 1
- 108010060309 Glucuronidase Proteins 0.000 description 1
- 239000005561 Glufosinate Substances 0.000 description 1
- 108010088778 Glycine max beta-conglycinin protein Proteins 0.000 description 1
- 240000002024 Gossypium herbaceum Species 0.000 description 1
- 235000004341 Gossypium herbaceum Nutrition 0.000 description 1
- 101710011863 HPD Proteins 0.000 description 1
- 240000006669 Helianthus annuus Species 0.000 description 1
- 235000003222 Helianthus annuus Nutrition 0.000 description 1
- 240000005979 Hordeum vulgare Species 0.000 description 1
- 235000007340 Hordeum vulgare Nutrition 0.000 description 1
- 229940088597 Hormone Drugs 0.000 description 1
- 241000282619 Hylobates lar Species 0.000 description 1
- 108010060231 Insect Proteins Proteins 0.000 description 1
- 239000005571 Isoxaflutole Substances 0.000 description 1
- 241000229754 Iva xanthiifolia Species 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N Kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 description 1
- 229920001732 Lignosulfonate Polymers 0.000 description 1
- 239000005578 Mesotrione Substances 0.000 description 1
- KPUREKXXPHOJQT-UHFFFAOYSA-N Mesotrione Chemical compound [O-][N+](=O)C1=CC(S(=O)(=O)C)=CC=C1C(=O)C1C(=O)CCCC1=O KPUREKXXPHOJQT-UHFFFAOYSA-N 0.000 description 1
- 230000036740 Metabolism Effects 0.000 description 1
- 240000008962 Nicotiana tabacum Species 0.000 description 1
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 1
- ILUJQPXNXACGAN-UHFFFAOYSA-N O-Anisic acid Chemical compound COC1=CC=CC=C1C(O)=O ILUJQPXNXACGAN-UHFFFAOYSA-N 0.000 description 1
- 101710010904 PCBD2 Proteins 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 229920002065 Pluronic® P 105 Polymers 0.000 description 1
- 241001506308 Potato virus T Species 0.000 description 1
- 229960004063 Propylene glycol Drugs 0.000 description 1
- ABOOPXYCKNFDNJ-UHFFFAOYSA-N Quizalofop Chemical compound C1=CC(OC(C)C(O)=O)=CC=C1OC1=CN=C(C=C(Cl)C=C2)C2=N1 ABOOPXYCKNFDNJ-UHFFFAOYSA-N 0.000 description 1
- 230000025458 RNA interference Effects 0.000 description 1
- 229920000970 Repeated sequence (DNA) Polymers 0.000 description 1
- YAHZABJORDUQGO-NQXXGFSBSA-N Ribulose-1,5-bisphosphate Chemical compound OP(=O)(O)OC[C@@H](O)[C@@H](O)C(=O)COP(O)(O)=O YAHZABJORDUQGO-NQXXGFSBSA-N 0.000 description 1
- 240000000111 Saccharum officinarum Species 0.000 description 1
- 235000007201 Saccharum officinarum Nutrition 0.000 description 1
- 240000003829 Sorghum propinquum Species 0.000 description 1
- 235000011684 Sorghum saccharatum Nutrition 0.000 description 1
- 229960005322 Streptomycin Drugs 0.000 description 1
- LSNNMFCWUKXFEE-UHFFFAOYSA-L Sulphite Chemical compound [O-]S([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-L 0.000 description 1
- 101700062054 TGL3 Proteins 0.000 description 1
- 229960002180 Tetracycline Drugs 0.000 description 1
- OFVLGDICTFRJMM-WESIUVDSSA-N Tetracycline Chemical compound C1=CC=C2[C@](O)(C)[C@H]3C[C@H]4[C@H](N(C)C)C(O)=C(C(N)=O)C(=O)[C@@]4(O)C(O)=C3C(=O)C2=C1O OFVLGDICTFRJMM-WESIUVDSSA-N 0.000 description 1
- 239000004098 Tetracycline Substances 0.000 description 1
- 235000012545 Vaccinium macrocarpon Nutrition 0.000 description 1
- 244000291414 Vaccinium oxycoccus Species 0.000 description 1
- 235000002118 Vaccinium oxycoccus Nutrition 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 235000007244 Zea mays Nutrition 0.000 description 1
- 229930000028 abscisic acids Natural products 0.000 description 1
- JLIDBLDQVAYHNE-OAHLLOKOSA-N abscisin II Chemical compound OC(=O)C=C(C)C=C[C@@]1(O)C(C)=CC(=O)CC1(C)C JLIDBLDQVAYHNE-OAHLLOKOSA-N 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 125000000129 anionic group Chemical group 0.000 description 1
- 239000002363 auxin Substances 0.000 description 1
- 238000004166 bioassay Methods 0.000 description 1
- OYVAGSVQBOHSSS-UAPAGMARSA-O bleomycin A2 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC=C(N=1)C=1SC=C(N=1)C(=O)NCCC[S+](C)C)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C OYVAGSVQBOHSSS-UAPAGMARSA-O 0.000 description 1
- 230000032823 cell division Effects 0.000 description 1
- 230000002759 chromosomal Effects 0.000 description 1
- 229960001338 colchicine Drugs 0.000 description 1
- 230000000295 complement Effects 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 230000000875 corresponding Effects 0.000 description 1
- 235000004634 cranberry Nutrition 0.000 description 1
- 244000038559 crop plants Species 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000032459 dedifferentiation Effects 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- MOTZDAYCYVMXPC-UHFFFAOYSA-N dodecyl hydrogen sulfate Chemical compound CCCCCCCCCCCCOS(O)(=O)=O MOTZDAYCYVMXPC-UHFFFAOYSA-N 0.000 description 1
- 229940043264 dodecyl sulfate Drugs 0.000 description 1
- 230000000408 embryogenic Effects 0.000 description 1
- 229910001651 emery Inorganic materials 0.000 description 1
- 241001233957 eudicotyledons Species 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 235000012055 fruits and vegetables Nutrition 0.000 description 1
- 101710010393 ftt-2 Proteins 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 239000003349 gelling agent Substances 0.000 description 1
- 210000004602 germ cell Anatomy 0.000 description 1
- 101710010895 glpV Proteins 0.000 description 1
- 239000003862 glucocorticoid Substances 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 230000002209 hydrophobic Effects 0.000 description 1
- 239000003617 indole-3-acetic acid Substances 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 229940088649 isoxaflutole Drugs 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 101700041898 lcrF Proteins 0.000 description 1
- 101700066242 lip3 Proteins 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 108010083942 mannopine synthase Proteins 0.000 description 1
- 239000012533 medium component Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000035786 metabolism Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 229960000485 methotrexate Drugs 0.000 description 1
- 229940073769 methyl oleate Drugs 0.000 description 1
- 230000002906 microbiologic Effects 0.000 description 1
- 239000011859 microparticle Substances 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 230000014075 nitrogen utilization Effects 0.000 description 1
- 108010058731 nopaline synthase Proteins 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- IAYPIBMASNFSPL-UHFFFAOYSA-N oxane Chemical compound C1CO1 IAYPIBMASNFSPL-UHFFFAOYSA-N 0.000 description 1
- 235000020232 peanut Nutrition 0.000 description 1
- 230000000149 penetrating Effects 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000003375 plant hormone Substances 0.000 description 1
- 230000027086 plasmid maintenance Effects 0.000 description 1
- 230000001402 polyadenylating Effects 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229920002102 polyvinyl toluene Polymers 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000000644 propagated Effects 0.000 description 1
- 235000013772 propylene glycol Nutrition 0.000 description 1
- 210000001938 protoplasts Anatomy 0.000 description 1
- 230000022532 regulation of transcription, DNA-dependent Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000010187 selection method Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 235000019333 sodium laurylsulphate Nutrition 0.000 description 1
- 239000011877 solvent mixture Substances 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 235000019364 tetracycline Nutrition 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 101700008042 virB Proteins 0.000 description 1
- 101710032620 virC Proteins 0.000 description 1
- 101710032619 virD Proteins 0.000 description 1
- 101700084103 virF Proteins 0.000 description 1
- 230000003612 virological Effects 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
- 239000001231 zea mays silk Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
Abstract
Disclosed is a method for plant cell transformation comprising exposing immature embryo plant cells to Agrobacterium cells in a liquid medium containing a non-ionic trisiloxane surfactant, the surfactant having a concentration of 0.001 weight percentage to 0.08 weight percentage in the liquid medium, and wherein the non-ionic trisiloxane surfactant is not a trisiloxane alkoxylate. , and wherein the non-ionic trisiloxane surfactant is not a trisiloxane alkoxylate.
Description
METHOD FOR IMPROVED TRANSFORMATION USING AGROBACTERIUM
CROSS REFERENCE TO D APPLICATIONS
This application claims the benefit of US. Provisional Patent Application Serial
No. 61/576,138 filed December 15, 2011.
BACKGROUND
Plant transformation generally encompasses the methodologies required and utilized
for the introduction of a plant—expressible foreign gene into plant cells, such that fertile progeny
plants may be obtained which stably maintain and express the foreign gene. Numerous members
of the monocotyledonous and ledonous classifications have been transformed. Transgenic
agronomic crops, as well as fruits and vegetables, are of commercial interest. Such crops e
but are not limited to maize, rice, soybeans, canola, er, alfalfa, m, wheat, cotton,
peanuts, tomatoes, potatoes, and the like.
Several techniques are known for introducing foreign genetic material into plant cells,
and for obtaining plants that stably maintain and express the introduced gene. Such techniques
include acceleration of genetic material coated onto microparticles directly into cells (cg, US.
Patent No. 050 and US. Patent No. 5,141,131). Other transformation technology includes
WHISKERSTM technology (see, e.g., US. Patent No. 5,302,523 and US. Patent No. 5,464,765).
oporation technology has also been used to transform plants. See, e.g, WO 87/06614,
US. Patent No. 5,472,869, US. Patent No. 5,384,253, WO 92/09696, and WO 93/21335.
Additionally, fusion of plant lasts with liposomes containing the DNA to be delivered,
direct ion of the DNA, as well as other possible methods, may be employed.
Once the inserted DNA has been integrated into the plant genome, it is usually
relatively stable throughout uent generations. The transformed cells grow inside the
plants in the usual manner. They can form germ cells and transmit the transformed trait(s) to
progeny plants. Such plants can be grown in the normal manner and may be crossed with plants
that have the same transformed hereditary factors or other hereditary factors. The ing
hybrid individuals have the corresponding ypic properties, for example, the ability to
control the feeding of plant pest insects.
1001685931
A number of alternative techniques can also be used for inserting DNA into
a host
plant cell. Those techniques include, but are not limited to, transformation with T—DNA
delivered by Agrobacterium tumefaciens or Agrobacz‘erium rhizogenes as the transformation
agent. Plants may be ormed using Agrobacterium technology, as described, for example,
in US. Patent No. 5,177,010, US. Patent No. 5,104,310, European Patent Application No.
013 1624B 1, European Patent Application No. 120516, European Patent Application No.
159418B1, European Patent Application No. , US. Patent No. 5,149,645, US. Patent No.
,469,976, US. Patent No. 5,464,763, U.S. Patent No. 4,940,838, US. Patent No. 4,693,976,
European Patent Application No. , European Patent Application No. , European
Patent Application No. , European Patent Application No. 604662, an Patent
Application No. 627752, European Patent Application No. 0267159, European Patent
Application No. 0292435, US. Patent No. 019, US. Patent No. 5,463,174, US. Patent No.
785, US. Patent No. 5,004,863, and US. Patent No. 5,159,135. The use ofT-DNA—
ning vectors for the transformation of plant cells has been intensively researched and
sufficiently described in European Patent Application 120516; An et al., (1985, EMBO J. 4:277—
284); Fraley et al., (1986, Crit. Rev. Plant Sci. 4: 1~46), and Lee and Gelvin (2008, Plant Physiol.
146:325—332), and is well established in the field.
A critical first step in the transformation of plant cells by Agrobacterium
spp. is close
contact, binding, or nce ofthe bacterial cells to the cells ofthe host plant to be
transformed. After cell—cell binding, the biology of T—DNA transfer from Agrobacterium to
plant cells is known. See, e.g., Gelvin, 2003, Microbiol. Molec. Biol. Rev. 67:16—37; and
Gelvin, 2009, Plant Physiol. 150: 1665—1676. At minimum, at least a T—DNA right border repeat,
but often both the right border repeat and the left border repeat of the Ti
or Ri plasmid will be
joined as the flanking region of the genes desired to be inserted into the plant cell. The left and
right T—DNA border repeats are crucial cis—acting sequences required for T—DNA transfer.
Various trans—acting components are encoded within the total Agrobacterium
genome. Primary
amongst these are the ns encoded by the vir genes, which are normally found as a series of
operons on the Ti or Ri plasmids. s Ti and Ri plasmids differ somewhat in the
complement of vir genes, with, for example, virF not always being present. Proteins encoded by
vir genes perform many different functions, ing recognition and signaling of plant
cell/bacteria interaction, induction of vir gene ription, formation of a Type IV secretion
1001685931
channel, recognition ofT—DNA border repeats, formation of T-strands, transfer of T-strands to
the plant cell, import of the T—strands into the plant cell s, and integration of T-strands into
the plant nuclear chromosome, to name but a few. See, e.g, Tzfira and Citovsky, 2006, Curr.
Opin. Biotechnol. -154.
IfAgrobacterium strains are used for transformation, the DNA to be inserted into the
plant cell can be cloned into special plasmids, for example, either into an intermediate (shuttle)
vector or into a binary vector. Intermediate vectors are not capable of independent replication in
cterium cells, but can be manipulated and replicated in common Escherichia coli
molecular cloning strains. It is common that such intermediate s comprise sequences,
framed by the right and left T—DNA border repeat regions, that
may include a selectable marker
gene functional for the selection of transformed plant cells, a cloning linker, cloning polylinker,
or other sequence which can function as an introduction site for genes destined for plant cell
transformation. Cloning and lation of genes desired to be transferred to plants can thus
be easily performed by standard methodologies in E. coli, using the shuttle vector as a cloning
vector. The finally manipulated shuttle vector can subsequently be introduced into
Agrobacterium plant transformation s for further work. The intermediate vector can be
transferred into Agrobacterium by means of a helper plasmid (via bacterial conjugation), by
oporation, by chemically mediated direct DNA transformation, or by other known
methodologies. Shuttle s can be integrated into the Ti or Ri plasmid or derivatives thereof
by homologous recombination owing to sequences that are homologous between the Ti or Ri
plasmid, or derivatives thereof, and the intermediate plasmid. This homologous recombination
(i.e. plasmid integration) event thereby provides a means of stably maintaining the altered shuttle
vector in Agrobacterium, with an origin of replication and other plasmid maintenance functions
provided by the Ti or Ri d portion of the co~integrant d. The Ti or Ri plasmid also
comprises the vir regions comprising vir genes necessary for the transfer of the T—DNA. It is
common that the plasmid carrying the vir region is a mutated Ti or Ri plasmid r plasmid)
from which the T-DNA region, including the right and left T—DNA border repeats, have been
deleted. Such pTi-derived plasmids, having onal vir genes and g all or substantially
all of the T—region and associated ts are descriptively referred to herein as helper
plasmids.
1001685931
The superbinary system is a specialized example of the shuttle vector/homologous
recombination system (reviewed by Komari er al., 2006, I_n: Methods in Molecular Biology (K.
Wang, ed.) No. 343: Agmbacterium ols, pp.15-4l; and Komori et al., 2007, Plant Physiol.
145:1155-1160). Strain 4(pSB 1) harbors two independently-replicating plasmids,
pAL4404 and pSB l. pAL4404 is a smid-derived helper plasmid which contains an intact
set of vir genes (from Ti plasmid pTiACHS), but which has no T—DNA region (and thus no T—
DNA left and right border repeat sequences). Plasmid pSBl supplies an additional partial set of
vir genes derived from pTiB0542; this partial Vir gene set includes the virB operon and the virC
, as well as genes virG and virD] . One example of a shuttle vector used in the
superbinary system is pSBl l, which ns a cloning polylinker that serves as an introduction
site for genes destined for plant cell transformation, flanked by Right and Left T—DNA border
repeat regions. Shuttle vector pSBll is not capable of independent replication in
Agrobacterium, but is stably maintained as a co—integrant plasmid when integrated into pSBl by
means of homologous recombination between common sequences present on pSBl and p88] 1.
Thus, the fully modified T—DNA region introduced into LBA4404(pSB 1) on a modified pSB l 1
vector is productively acted upon and erred into plant cells by Vir ns d from
two different Agrobacterium Ti plasmid sources (pTiACHS and pTiB0542). The Agrobacterium
tumefaciens host strain ed with the superbinary system is LBA4404(pSBl). The
superbinary system has proven to be particularly useful in transformation of monocot plant
species. See Hiei et al., (1994) Plant J. 62271—282; and lshida et al., (1996) Nat. Biotechnol.
—750.
In addition to the vir genes ed by Agrobacterium Ti ds, other,
chromosomally-borne virulence controlling genes (termed chv genes) are known to control
certain aspects of the interactions ofAgrobacterium cells and plant cells, and thus affect the
overall plant transformation frequency (Pan et (11., 1995, Molec. Microbiol. 17:259—269). Several
of the chromosomally-borne genes required for Virulence and attachment are grouped together in
a chromosomal locus spanning 29 kilobases (Matthysse et al., 2000, Biochim. Biophys. Acta
1490:208-212).
In on to numerous technologies for transforming plants, the type of tissue which
is contacted with the foreign genes may vary as well. Such tissue may include, but is not limited
to, embryogenic tissue, callus tissue types I and II, hypocotyl, and meristem. Almost all plant
1001685931
tissues may be ormed during dedifferentiation using appropriate techniques within the skill
of an artisan. One skilled in the field of plant transformation will understand that multiple
methodologies are available for the production of transformed plants, and that they may be
modified and specialized to accommodate ical differences between various host plant
species. Plant ts (for example, pieces of leaf, ts of stalk, meristems, roots, but also
protoplasts or sion-cultivated cells) can ageously be cultivated with Agrobacterium
tumefaciens or Agmbacz‘erium rhizogenes for the transfer of the DNA into the plant cell.
Callus cultures Plant tissue cultures may advantageously be cultivated with
Agrobacterium tumefaciens or Agrobacterz'um rhizogenes for the transfer of the DNA into the
plant cell, and are generally initiated from sterile pieces of a whole plant that may consist of
pieces of organs, such as leaves or roots, or maybe specific cell types, such as pollen or
endosperm. Many features of the explant are known to affect the efficiency of culture initiation.
It is thought that any plant tissue can be used as an explant, if the correct conditions are found.
lly, younger, more rapidly growing tissue (or tissue at an early stage of development) is
most ive. Explants cultured on the appropriate medium can give rise to an unorganized,
g, and dividing mass of cells (callus). In culture, callus can be maintained more or less
indefinitely, provided that it is subcultured on to fresh medium periodically. During callus
formation, there is some degree of de-differentiation, both in morphology (a callus is usually
composed of unspecialized parenchyma cells) and metabolism.
Callus cultures are extremely important in plant biotechnology. Manipulation of the
plant hormone ratios in the culture medium can lead to the development of shoots, roots, or
somatic embryos from which whole plants can uently be produced (regeneration). Callus
cultures can also be used to initiate cell suspensions, which are used in a variety of ways in plant
transformation studies.
Cell suspension cultures Callus cultures, y speaking, fall into one oftwo
categories: compact or friable. In compact callus, the cells are y aggregated, whereas in
friable callus, the cells are only loosely associated with each other and the callus becomes soft
and breaks apart easily. Friable callus provides the um to form cell-suspension cultures.
ts from some plant species or particular cell types tend not to form friable callus, making
it difficult to initiate cell suspension. The friability of the callus can sometimes be improved by
manipulating the medium components, by repeated subculturing, or by ing it on semi-solid
1001685931
medium m with a low tration of gelling agent). When friable callus is placed into
a liquid medium and then agitated, single cells and/or small clumps of cells
are released into the
medium. Under the correct conditions, these released cells ue to
grow and divide,
eventually ing a cell-suspension culture. Cell suspensions can be ined relatively
simply as batch cultures in conical flasks and are propagated by repeated subculturing into fresh
medium. After subculture, the cells divide and the biomass of the culture increases in
characteristic fashion. Cell suspension cultures may advantageously be cultivated with
Agrobacterium tumefaciens or Agrobacterium rhizogenes for the transfer of the DNA into the
plant cell.
Shoot tip and meristem culture The tips of shoots (which contain the shoot apical
meristem) can be cultured in vitro, producing clumps of shoots from either axillary or
adventitious buds and may advantageously be cultivated with Agrobaclerium lumefociens
Agrobacterium rhizogenes for the transfer of the DNA into the plant cell. Shoot meristem
cultures are used for cereal regeneration (seedlings can be used as donor material).
Embryo culture Embryos can be used as explants to generate callus cultures or
somatic s. Both immature and mature embryos can be used as explants. Immature,
embryo—derived genic callus is a tissue used in monocotyledon plant regeneration and
may advantageously be cultivated with Agrobacterium lumefaciens for the transfer of the DNA
into the plant cell. Immature embryos are an intact tissue that is e of cell division to give
rise to callus cells that can differentiate to produce tissues and
organs of a whole plant.
Immature embryos can be obtained from the fertilized ears of a mature maize plant, for e,
from plants pollinated using the methods fer et al. (1982, Growing maize/or
purposes. In: Maize for Biological Research. W. F. an, Ed. UNIVERSITY PRESS,
University ofNorth , Grand Forks, ND). Exemplary methods for isolating immature
embryos from maize are described by Green and Phillips (Crop Sci. 15:417-421 (1976)).
Immature embryos are preferably isolated from the developing ear using aseptic methods and
held in sterile medium until use. The use ofAgrobacterium in transformation of immature
embryos is disclosed by Sidorov & Duncan, (2009, Methods in Molecular Biology: Transgenic
Maize, vol.526 Chapter 4, M. Paul Scott (Ed.)) and in US. Patent No. 840.
Microspore culture Haploid tissue can be cultured in vitro by using pollen or anthers
as an explant and may advantageously be cultivated with Agrobacterium tumefaciens for the
1001685931
transfer of the DNA into the plant cell. Both callus and embryos can be produced from pollen.
Two approaches can be taken to e cultures in vitro from haploid tissue. In the first,
anthers (somatic tissue that surrounds and contains the pollen) are cultured on solid medium.
Pollen-derived embryos are subsequently produced via dehiscence of the mature anthers. The
dehiscence of the anther s both on its isolation at the t stage and on the correct
culture conditions. In some s, the reliance on natural dehiscence can be circumvented by
cutting the wall of the . In the second method, anthers are cultured in liquid medium, and
pollen released from the anthers can be d to form embryos. Immature pollen can also be
extracted from developing anthers and cultured directly.
Many of the cereals (rice, wheat, barley, and maize) require medium supplemented
with plant growth tors for pollen or anther culture. Regeneration from microspore explants
can be obtained by direct embryogenesis, or via a callus stage and subsequent embryogenesis.
Haploid tissue cultures can also be initiated from the female gametophyte (the ovule).
In some cases, this is a more efficient method than using pollen or anthers.
Plants ed from haploid cultures may not be haploid. This can be a
consequence
of chromosome doubling during the culture period. Chromosome doubling (which
may be
induced by treatment with chemicals such as colchicine)
may be an advantage, as in many cases
haploid plants are not the desired outcome of regeneration from haploid tissues. Such plants are
often referred to as di—haploids, because they contain two copies of the same haploid
genome.
Following transformation of any of the aforementioned plant materials by cultivation
with Agrobaclerium tumefaciens for the transfer of the DNA into the plant cell, whole plants
may then be regenerated from the infected plant material following ent in suitable growth
conditions and culture medium, which may contain antibiotics or herbicides for selection of the
transformed plant cells. The plants so obtained can then be tested for the
presence of the inserted
DNA.
Cell transformation (including plant cell transformation)
may involve the construction
of an expression vector which will on in a ular cell. Such a vector may comprise
DNA that includes a gene under control of, or ively linked to, a regulatory element (for
example, a promoter). The expression vector may n one or more such operably-linked
gene/regulatory t combinations. The (s) may be in the form of a plasmid and can
be used alone or in combination with other plasmids to provide transformed cells using
1001685931
transformation methods as described herein to incorporate transgene(s) into the genetic material
of a plant cell.
Plant cell expression vectors may include at least one genetic marker, operably linked
to a regulatory element (a promoter, for example) that allows transformed cells ning the
marker to be either recovered by negative selection (22a, inhibiting growth of cells that do not
contain the able marker gene) or by ve selection (226., screening for the t
d by the genetic marker). Many selectable marker
genes suitable for plant transformation
are well known in the transformation arts and include, for example, genes that code for
enzymes
that metabolically detoxify a selective chemical agent which
may be an antibiotic or an
herbicide, or genes that encode an altered target which may be insensitive to the inhibitor. A few
positive selection methods are also known in the art. The individually ed selectable
marker gene may accordingly permit the selection oftransformed cells while the growth of cells
that do not contain the inserted DNA can be suppressed by the selective compound. The
preference for a particular selectable marker gene is at the discretion of the artisan, but any of the
following able markers may be used, as well as any other gene not listed herein which
could function as a selectable . Examples of selectable markers include, but
are not
d, to ance or tolerance to Kanamycin, G418, Hygromycin, Bleomycin, Methotrexate,
Phosphinothricin (Bialaphos), Glyphosate, Imidazolinones, Sulfonylureas and
Triazolopyrimidine herbicides, such as Chlorosulfuron, Bromoxynil, and DALAPON.
In addition to a selectable marker, it may be desirable to use a reporter gene. In some
instances a reporter gene may be used without a selectable marker. Reporter genes are genes
which typically do not provide a growth age to the ent organism or tissue. The
reporter gene typically encodes for a protein which provides for some phenotypic change or
enzymatic property. le reporter genes include, but are not limited to, those that encode
beta—glucuronidase (GUS), firefly luciferase, or scent proteins such as green fluorescent
protein (GFP) or yellow fluorescent protein (YFP, essentially as disclosed in US. Patent No.
7,951,923).
Regardless of transformation technique utilized, the foreign gene can be incorporated
into a gene transfer vector adapted to express the foreign
gene in the plant cell by including in
the vector a plant promoter. In addition to plant promoters, promoters from a variety of sources
can be used efficiently in plant cells to express foreign genes. For example, promoters of
1001685931
bacterial origin, such as the octopine synthase promoter, the nopaline synthase promoter, the
mannopine synthase promoter; promoters of Viral origin, such as the 358 and 198 promoters of
cauliflower mosaic Virus (CaMV), a promoter from sugarcane bacilliforrn Virus, and the like may
be used. Plant-derived promoters include, but are not limited to ribulose-l,6-bisphosphate
(RUBP) carboxylase small t (ssu), beta-conglycinin promoter, phaseolin promoter, ADH
(alcohol dehydrogenase) er, hock promoters, ADF (actin depolymerization factor)
promoter, and tissue specific promoters. Promoters may also contain certain enhancer sequence
elements that may improve the transcription ncy. l enhancers include, but are not
limited to, alcohol dehydrogenase l (ADHl) intron 1 and ADHl-intron 6. Constitutive
promoters may be used. Constitutive promoters direct continuous gene expression in nearly all
cells types and at nearly all times (Lag. actin, ubiquitin, CaMV 358). Tissue specific promoters
are responsible for gene expression in specific cell or tissue types, such as the leaves or seeds
Examples of other promoters that may be used include those that are active during a certain stage
of the plant's development, as well as active in c plant tissues and organs Examples of
such promoters include, but are not limited to, promoters that are root specific, pollen—specific,
embryo specific, corn silk c, cotton fiber specific, seed erm specific, and phloem
specific.
Under certain circumstances, it may be desirable to use an inducible promoter. An
inducible promoter is responsible for expression of genes in se to a c signal, such
as: physical stimulus (ag. heat shock genes); light (e. g. Ribulose—bis—phosphate 1,5
carboxylase); hormone (e. g. glucocorticoid); antibiotic (e.g. Tetracycline); metabolites; and
stress (eg. drought). Other desirable transcription and translation elements that function in
plants also may be used, such as, for e, 5' untranslated leader sequences, and 3' RNA
transcription ation and poly—adenylate addition signal sequences. Any suitable plant—
specific gene transfer vector known to the art may be used.
enic crops containing insect resistance (IR) traits are prevalent in corn and
cotton plants hout North America, and usage of these traits is expanding globally.
Commercial transgenic crops combining IR and herbicide tolerance (HT) traits have been
developed by multiple seed companies. These include combinations of IR traits conferred by
Bacillus thuringiensis (B.t.) insecticidal proteins and HT traits such as tolerance to Acetolactate
Synthase (ALS) inhibitors such as Sulfonylureas, olinones, Triazolopyrimidine,
1001685931
Sulfonanilides, and the like, Glutamine Synthetase (GS) inhibitors such as Bialaphos,
Glufosinate, and the like, 4-HydroxyPhenyleruvate Dioxygenase (HPPD) inhibitors such as
Mesotrione, Isoxaflutole, and the like, S—EnoleruvylShikimatePhosphate Synthase (EPSPS)
inhibitors such as Glyphosate and the like, and Acetyl—Coenzyme A ylase (ACCase)
inhibitors such as Haloxyfop, Quizalofop, op, and the like. Other examples are known in
which transgenically provided proteins provide plant tolerance to ide chemical classes
such as phenoxy acids herbicides and loxyacetates auxin herbicides (see
A2), or phenoxy acids ides and aryloxyphenoxypropionates herbicides (see WO
2005/l07437Al). The ability to control multiple pest problems through 1R traits is a valuable
cial product concept, and the convenience of this product concept is enhanced if insect
control traits and weed control traits are combined in the same plant. Further, improved value
may be obtained via single plant combinations of [R traits conferred by a B.t. insecticidal protein
with one or more additional HT traits such as those mentioned above, plus one or more
additional input traits (e. g. other insect resistance conferred by B.t.—derived or other insecticidal
proteins, insect ance conferred by mechanisms such as RNAi and the like, disease
resistance, stress tolerance, improved nitrogen utilization, and the like), or output traits (e. g. high
oils content, healthy oil ccmposition, nutritional improvement, and the like). Such combinations
may be obtained either through conventional breeding (e.g. breeding stack) or jointly as a novel
transformation event involving the simultaneous introduction of le genes (eg lar
stack). Benefits include the y to manage insect pests and improved weed control in a crop
plant that provides secondary benefits to the producer and/or the consumer. Thus, the methods
of this disclosure can be used to provide transformed plants with combinations of traits that
comprise a te agronomic package of improved crop quality with the ability to flexibly and
cost effectively control any number of mic issues.
SUMMARY
Methods for plant cell transformation are described. These s include exposing
the plant cells to Agrobacterium cells in a liquid medium containing a surfactant. The
Agrobacterium cells can be scraped from a solid medium or grown in a liquid growth medium
prior to being suspended in the liquid medium containing the surfactant. The concentration of
surfactant can be in the range of 0.001 weight percent to 0.08 weight t. The surfactant can
1001685931
be a non-ionic trisiloxane surfactant and more than one surfactant can be used. The plant cells
can be maize cells. The plant cells can be exposed to continuous light after exposure to the
Agrobacz‘erium cells.
DESCRIPTION OF DRAWINGS
Figure l is a bar graph showing the enhancement of maize immature embryo
transformation when the surfactant THRU® S 233 is added to the Infection Medium
used to create a suspension ofAgmbacterium cells (harboring plasmid pEPSlOS3) prior to co—
cultivation.
Figure 2 is a bar graph showing the enhancement of maize re embryo
transformation when the surfactant BREAK~THRU® S 233 is added to the Infection Medium
used to create a suspension ofAgrobacz‘erium cells prior to co-cultivation. The Plasmids used for
each ment shown in Figure 2 include: Experiment 1 = pEPS1053; GOI = IPT, selectable
marker = aad]. Experiment 2 = pEPS 1038; GOI=GF14, able marker = aadl. Experiment 3
and Experiment 4 = pEPSlOZ7; no GOI, selectable marker = aad].
DETAILED PTION
Methods to increase transformation ncy in plants when using cterium
are described. The methods include exposing plant cells to Agrobacterium cells in a liquid
medium containing a surfactant. Some methods include exposing the plant cells to continuous
light after exposure to the Agrobaclerium cells. es ofplants useful with these methods
include maize plants and immature maize embryos.
s ofAgrobacterz‘um differ from one another in their ability to transform plant
cells of various species. Regardless of the particular combination ofAgrobacterium strain/host
plant considered, Agrobacterium acts through attachment to the host cell during transformation.
See McCullen and Binns, 2006, Ann. Rev. Cell. Dev. Biol. 22:101w127; and Citovsky et al.,
2007, Cell. Microbiol. 929—20. For this reason, methods that enhance binding ofAgmbacterium
cells to plant cells, such as those disclosed herein using surfactants, may e increases in
transformation efficiency. Enhancing the binding ofAgrobacterium cells to plant cells is
different for ent species and tissue types as different plant species, and further, different
tissues of a plant of a single species, can differ in chemical and biochemical composition of their
1001685931
cell walls. Further, such ences may also vary during different developmental stages of a
single plant tissue.
Additionally different genera and species of bacteria, and indeed, different strains of a
bacterial species, often differ in chemical and biochemical composition of their cell walls, and
these differences can change during the bacterial growth cycle. Increases in plant ormation
efficiencies by the methods disclosed herein thus may result from the ability of surfactants to
se hydrophobic repulsive interactions between crerz’um cell walls and plant cell
walls, and thus allow intimate ell interactions to occur.
One may therefore utilize the chemical differences between different tant
agents to promote cell—cell ctions between cells ofdifferent Agrobacterium strains (and
different growth phases of such cells) and cells and tissues of different host plants during various
phases of e of the plant tissues so that enhanced transformation efficiencies may be
observed.
Surfactants belong to several chemical classes, and one skilled in the field ofplant
transformation will understand that different chemical classes of surfactants
may be used to
enhance plant transformation efficiency with different plant hosts. Examples of surfactants from
these al classes useful with the s disclosed herein include adjuvants, non-ionic
surfactants, anionic tants, oil based surfactants, amphoteric surfactants, and polymeric
surfactants. An example of a preferred surfactant useful with the methods described herein is a
non—ionic trisiloxane surfactant such as BREAK—THRU® S233 from Evonik Industries (Essen,
Germany). Examples of further preferred surfactants useful with the methods described herein
include trisiloxane alkoxylates, ethoxylated soybean oils, l ethoxylate C—13s, C12-C14—
alkyldimethyl betaines, and di—sec—butylphenol ethylene oxide—propylene oxide block co—
polymers. Table 1 presents an non-limiting list of surfactants of various chemical type that
be used to practice the methods described herein.
Table l. Surfactants grouings, cial names and chemicalalction/class.
EVONIK
BREAK-THRU® Polyether—modified
INDUSTRIES AG
Adjuvant S 240 polysiloxane
(Essen, Germany)
BREAK—THRU® Organo-modified EVONIK
S 243 polysiloxane INDUSTRIES AG
1001685931
anew
SILWET® 618 Trisiloxane alkoxylate
ELLIOTT
Blend of organosilicone &
HI~WETT® ' ~ CHEMICALS LTD.
other organic fluids.
(Aukland, NZ)
DOW CORNING
SYLGARD® 309 Non—ionic silicone surfactant
(Midland, MI)
HENKEL
AGRIMUL® PG 2069 Alkylpolyglucoside CORPORATION
(Berkeley, CA)
MONSON
Alcohol ethoxylate C~l3
TRYCOL® 5941 IES, INC
(9 mole EO*)
(Leominster, MA)
Alcohol ethoxylate (3-13 STEPAN COMPANY
Non-ionic MAKON® TD—6
(6 mole E0) (Northtield, IL)
tant
AICOhOl Ethoxylate C _ 13
MAKON® TD~12 STEPAN COMPANY
(12 mole EO)
l ethoxylate C~13
TRYCOL® 5993A MONSON
(3 mole EO) COMPANIES, INC
AGRILIANCE LLC
Non—ionic surfactant and
PREFERENCE® . . (Inver Grove Heights,
anti—foamlng agent
. BASF
PLURONIC® P105 gi‘ififggli‘gp:51);1:36 CORPORATION
p y
nnati, OH)
A ' ' HUNTSMAN
3:31 fionate yS d' 1m alk I benzene
NANSA® HS 90/s CORPORATION
S gig;“ a p
(The Woodlands, TX)
SLS (no trade name) SIGMA—ALDRICH
Sodlum lauryl sulfate.
(St. Louis, MO)
EMERY®/EMGARD® HENKEL
Methyloleate/surfactants
ATION
Ethoxylated soybean oil
AGNIQUE® SBO—IO BASF
Oil-based (10 mole POE**) CORPORATION
di-sec-butylphenol ne DOW
UPTAKE® oxide—propylene oxide block AGROSCIENCES
co-polymer (Indianapolis, IN)
LOVELAND
LI-700® soy-oil derived, nic
PRODUCTS INC.
penetrating surfactant
(Greele , CO)
1001685931
X® (C-12) Amine oxide STEPAN COMPANY
AKZONOBEL
100% Alkoxylated fatty
ADSEE® AB 650 ' ’ SURFACE
Amphoteric 2:21;: + wetting agent + CHEMISTRY LLC
surfactant (Chicago, IL)
BREAK-THRU® G Fatty acid amido alkyl EVONIK
850 e RIES AG
GERONOL® CF/AS Betaines, C12—I4- RHODIA INC
alkyldimethyl (Cranberry, NJ)
ORKLA INDIA PVT"
BORRESPERSE® NA Lignosulfonate LTD
(Vashi, India)
MORWET® D425 Alkylnaphthalene sulfonate MONSON
condensate COMPANIES, INC
ATLOXTM 4913 . . CRODA WEST
Non Ionic comb polymer_
Chino Hills, CA
METASPERSETM Modified e acryllc" .
- 3
500L CRODA WEST
polymer
P I . INTERNATIONAL
0 ymeric
AGRIMER® AL 1 O Alkylated Vinylpyrrolidone LTY
copolymers PRODUCTS
, NJ)
CEVOL® 205 . CELANESE LTD.
Polyvmyl alcohol
(Dallas, TX)
. AKZONOBEL
ALCOSPERSE® 725 E
pHgdgzlihgt’gany dy y
CHEMISTRY LLC
1,2—propanediol, Alcohol
TACTICTM LOVELAND
ethoxylate, silicone polycther
PRODUCTS INC.
copolymer
*EO : moles of ethylene oxide reacted with a particular hydrophobe
**POE : moles ofpropylene oxide d with a particular hydrophobe
The methods disclosed herein utilize the transformation—enhancing properties
surfactants to dramatically increase transformation efficiency in plants such
as immature maize
embryos by Agrobacz‘erium (e.g., Agrobacterium tumefaciens). The surfactants used with the
methods described herein are selected, as suggested above, based
upon the ability to promote
cell-cell interactions that will enhance transformation efficiency. The concentration of
tant
in the liquid medium can be 0.001 weight percent to 0.08 weight
percent, 0.001 weight percent to
0.07 weight percent, 0.001 weight percent to 0.06 weight
percent, 0.001 weight percent to 0.05
weight percent, 0.001 weight percent to 0.04 weight percent, 0.001 weight percent to 0.035
1001685931
weight percent, 0.001 weight t to 0.03 weight percent, 0.001 weight percent to 0.025
weight percent, 0.001 weight t to 0.02 weight percent, 0.001 weight percent to 0.015
weight percent, 0.001 weight percent to 0.01 weight t, or 0.005 weight percent to 0.01
weight percent.
One or more additional surfactants can also be used with the methods described
herein. As indicated, the transformation efficiency is ent
on a variety of factors ing
plant species and —type and Agrobacterium strain. Given the variety of interactions
involved, a system of two or more surfactants can provide enhanced transformation efficiency.
The additional surfactants used in a system of two or
more surfactants can be ed, for
example, from Table 1.
The methods described herein are broadly applicable to a variety of plant species
varieties including monocotyledons and dicotyledons. Crops ofinterest include but are not
limited to maize, rice, soybeans, canola, sunflower, alfalfa, sorghum, wheat,
cotton, s,
tomatoes, potatoes, and the like. The methods herein can be used with cells at s stages of
pment, e.g., immature embryos. Thus, the methods described herein can be used to
transform maize re embryos. The size ofimmature embryos used in the
methods
described herein can vary. For example, immature embryos can be greater than
or equal to 1.5
mm and less than or equal to 2.5 mm in length.
The external environment the cells are ined in after
exposure to Agrobaclerium
according to the methods described herein can be controlled. For example, temperature, pH, and
other components of growth medium the cells are placed
upon after transformation according to
the methods described herein can be varied and are generally well known
to those of skill in the
art. One ofthose variables is exposure to light. The methods bed herein
can include
exposing the plant cells to common 18 hour light/6 hour dark protocols or alternatively to
continuous light after exposure to the Agrobacterium cells. For example, cells treated according
to the methods described herein can be exposed to 24-hour white fluorescent light conditions
weeks after treatment, e.g., until the regeneration and plantlet isolation
stages of plant
preparation.
An additional method includes preparing a liquid medium containing
a surfactant,
suspending Agrobacterium cells in the liquid , and exposing plant cells to the
Agrobacterium cells in the liquid medium containing the surfactant. The Agrobacterium cells
1001685931
can be scraped from a solid medium prior to being suspended in the liquid medium containing
tant. Additionally, the Agrobacterz'um cells can be grown in a liquid growth medium prior
to being ded in the liquid medium containing a surfactant.
ols and s for transforming plants using Agrobacterium are well known
to those of skill in the art of molecular biology. Any types of methods known for the
use of
Agrobacterium in orming plants can be used with the methods described . The
examples below provide embodiments of methods demonstrating the effectiveness of the
methods described herein, but are not ed to be limitations on the
scope of the claims.
All patents, patent applications, provisional applications, and publications referred to
or cited herein are incorporated by reference in their entirety to the extent they
are not
inconsistent with the explicit teachings of this specification.
EXAMPLES
The following examples illustrate procedures for practicing the claims. The examples
and embodiments described herein are for illustrative
purposes only and various modifications or
changes in light thereof will be suggested to persons skilled in the art and are to be included
within the spirit and scope ofthe claims. All percentages are by weight and all solvent mixture
proportions are by volume unless otherwise noted. All temperatures are in degrees Celsius.
EXAMPLE 1. Agrobacterz‘um transformation for generation of superbinary vectors.
The Agrobacterium superbinary system is conveniently used for transformation of
monocot plant hosts. Methodologies for constructing and validating superbinary vectors are well
sed and incorporated herein by reference (Operating Manual for Plasmid pSBl, Version
3.1, available from Japan Tobacco, Inc., Tokyo, Japan). Standard molecular biological and
microbiological methods were used to generate superbinary plasmids. Verification/validation of
the structure of the inary plasmid was done using methodologies
as ted in the
Operating Manual for Plasmid pSBl.
cterium strains harboring various superbinary plasmids were used in this
work. All these plasmids ned, as the selectable marker/herbicide tolerance
gene, the
coding ce (CBS) for the AADl protein (US. Patent 7,838,733), whose expression was
under the transcriptional control of a rice actinl promoter and associated intron 1, essentially as
1001685931
disclosed in U.S. Patent No. 5,641,876 and GENBANKTM Accession No. EU155408.1.
Termination of transcription and polyadenylation of the aad] mRNAs were determined by a
maize lipase 3'UTR, essentially as disclosed as bases 921 to 1277 of GENBANKTM Accession
No. gb]L359l3.1[MZELIPASE and in U.S. Patent No. 7,179,902. In on, the inary
plasmids harbored a gene whose expression was not expected to affect transformation frequency.
In particular, in plasmid pEP81083, a CDS encoding a YFP protein (essentially as disclosed in
US Patent No. 7,951,923) (transcription of which was controlled by a maize ubiquitin 1 promoter
with associated intron 1; U.S. Patent No. 5,510,474), and whose mRNAs were terminated by a
maize Per5 3'UTR (U.S. Patent No. 6,384,207» was advantageously used as a visual marker to
monitor transformation and determine relative transformation efficiencies. Other superbinary
plasmids used to ify the methods disclosed here (plasmids pEPSlOl3, l8,
pEPS 1028, pEPSlOB6, pEPSlO38, pEP81059, pEPSlO64, pEP81066, pEPS1068, pEPS6004,
and pEPS6008) harbored a CDS encoding a Dow AgroSciences etary protein, expression
of which was controlled by the same transcription/termination elements as were used for the YFP
CDS.
Expression onFP was used to measure the efficiency oftransformation in some
experiments. ormation efficiency percentages were calculated as the number of calli that
displayed sion of YFP, divided by the number oftreated embryos, times 100. YFP
expression was ed by visual observation using either an Olympus SZX12 us
America Inc; Center Valley, PA) or a Leica M165FC (Leica Microsystems Inc.; Buffalo Grove,
IL) fluorescent microscope, with YFP filters covering the ranges for excitation at 5 14 nm and
emission measured at 527 run.
In other experiments that employed Agmbacterz’um strains ing superbinary
ds lacking the YFP gene, transformation efficiencies were calculated following
® analysis (Life Technologies; Carlsbad, CA) of progeny plants ed from
embryos that were selected by means of resistance to Haloxyfop. The TAQMAN® components
used were specific for the aad] coding region. Transformation efficiencies were calculated from
the number of TAQMAN®~positive events determined, divided by the number of treated
embryos, times 100. An "event" for these purposes was considered to be an embryo that
ed one or more TAQMAN®-verified plant(s). An individual embryo was considered to
be one event regardless of how many plants it may have produced.
1001685931
EXAMPLE 2. Transformation of maize by Agrobacterz'um strains (Transformation
Protocol 1).
The basic work flow is summarized as s. Embryos are extracted from
immature ears of corn at the developmental stage at which the
young embryos are about 1.4 to
1.9 mm in length. When different transformation ions are to be compared, approximately
equal numbers of embryos isolated from a single ear are divided amongst all the treatments. The
embryos are incubated with a suspension containing cterium cells and surfactant (or not,
for comparison), then are moved to solid—medium plates and co—cultivation is allowed for 3
to 5
days. The d embryos are transferred onto a medium containing antibiotics (for the
suppression and killing of the Agrobacterz'um cells) and compounds for the selective isolation of
genetically transformed corn tissues and plants. The corn tissue (usually, but not limited to,
) is grown on ion medium until plants are regenerated. These plants are tested to
confirm their genetic transformation and those having a desired modification
are grown to
maturity for seed tion.
Immature Embryo Production Seeds from a B 104 inbred were planted into on—
pots ning SUNSHINE CUSTOM BLEND 160 (SUN GRO HORTICULTURE; Bellevue,
WA). The plants were grown in a greenhouse using a combination of high pressure sodium and
metal halide lamps with a 16:8 hour Light:Dark photoperiod. To obtain immature embryos for
transformation, controlled sib—pollinations were performed. Immature embryos were isolated at
to 13 days post—pollination when embryos were approximately 1.4 to 1.9
mm in size. Maize
ears were surface sterilized after ng the husks and silks by immersing in 50% commercial
bleach (CLOROX®, 5.25% sodium hypochlorite) with TWEEN®—20 (1
or 2 drops per 500 mL)
for 10 minutes and triple—rinsed with sterile water.
Immature embryos were aseptically isolated directly into a micro centrifuge tube
containing 2 mL of Infection Medium with suspended Agrobacterz‘um cells, and surfactant as
appropriate. The embryos were incubated with the suspension bacterium cells,
ning surfactant (or not, for control experiments), for 5-30 minutes.
A suspension ofAgrobacterium cells containing a superbinary vector
was prepared
by first growing the cells as a lawn for 4 days at 25°, or 3 days at 28°, on solid agar plates
containing YEP (gm/L: Yeast Extract, 5; Peptone, 10; NaCl, 5; agar, 15) with 50 mg/L
5931
Spectinomycin; 10 mg/L Rifampicin; and 50 mg/L omycin. (In some experiments, the
Agrobacterium cells were grown on solid LB medium (SIGMA ALDRICH; St. Louis, MO) 20
gm/L, with antibiotics as above.) This e was ed from a single colony isolate
established under the same conditions. One or two loopfuls of cells
were d from the lawn,
then uniformly resuspended (by gently pipetting
up and down) in Infection Medium (IflVI) to an
optical density at 600 nm (OD600) of 0.35 to 0.45. Infection Medium contains: 4.33 gm/L MS
salts; IX ISU Modified MS Vitamins; 68.4 gm/L e; 36 gm/L glucose; 700 mg/L
L-proline;
3.3 mg/L Dicamba—KOH; and 100 pM acetosyringone (prepared in DMSO);
at pH 5.2.
Depending upon the experiment, an appropriate amount of surfactant solution (e.
g. BREAK—
THRU® S 233 at 0.01% final concentration) was added to the ion Medium
after
suspending the cells.
The Agrobacterium and embryo solution was incubated for 5 to 30 minutes
at room
temperature, and then the embryos were transferred to Co—cultivation Medium, which contained
4.33 gm/L MS salts; 1X ISU Modified MS Vitamins; 30 gm/L
e; 700 mg/L L—proline; 100
mg/L myo-inositol; 3.3 mg/L Dicamba—KOH; 100 mg/L Casein Enzymatic Hydrolysate; 15 mg/L
AgNO3; 100 uM acetosyringone; and 3 gm/L GELZANTM; at pH 5.8. Co-cultivation incubation
was for 3 to 4 days at 250 under 24~hour white fluorescent light (approximately 50 pEm"2s'I).
Resting and Selection After co—cultivation, the embryos (36 embryos/plate)
were
carefully transferred to fresh non—selection Resting Medium containing 4.33 gm/L MS salts; 1X
ISU Modified MS Vitamins; 30 gm/L
sucrose; 700 mg/L L—proline; 3.3 mg/L Dicamba in KOH;
100 mg/L myo—inositol; 100 mg/L Casein Enzymatic Hydrolysate; 15 mg/L
AgNO3; 0.5 gm/L
MES; 250 mg/L Carbenicillin; and 2.3 gm/L GELZANTM; at pH 5.8. Incubation was continued
for 7 days at 280 in 24~hour white fluorescent light (approximately 50 ‘l).
Following the 7 day resting period, the embryos were transferred to Selection
Medium. For selection of maize tissues transformed with a superbinary plasmid containing
plant expressible aad] able marker gene, the embryos (36/plate) were first transferred to
Selection Medium I, which comprised Resting Medium (above) containing 100 nM
R-Haloxyfop
acid (0.0362 mg/L). The embryos were incubated for 1 week (28°; continuous light), and then
transferred to ion Medium 11 which comprised g Medium with 500 nM
R-Haloxyfop
acid (0.1810 mg/L), on which they were incubated under continuous light for
an additional 7
1001685931
days. At this time they were moved to fresh Selection Medium 11 and incubation was continued
as above for an additional week.
Those skilled in the art of maize transformation will understand that other methods
selection of transformed plants are available when other plant expressible selectable
marker
genes (e.g. herbicide tolerance genes) are used.
Pre-regeneration Following the selection process, cultures were transferred to Pre—
regeneration Medium containing 4.33 gm/L MS salts; 1X ISU Modified MS Vitamins; 45 gm/L
sucrose; 350 mg/L L—proline; 100 mg/L myO—inositol; 50 mg/L Casein Enzymatic Hydrolysate;
1.0 mg/L AgNO3; 0.25 gm/L MES; 05 mg/L aleneacetic acid in
NaOH; 2.5 mg/L ic
acid in ethanol; 1 mg/L 6—benzylaminopurine; 250 mg/L Carbenicillin; 2.5 gm/L GELZANTM;
and 500 nM R—Haloxyfop acid; at pH 5.8. Incubation was ued for 7 days at 280 under
continuous white fluorescent light as above.
Regeneration and plantlet ion For regeneration, the cultures were transferred to
Regeneration Medium I containing 4.33 gm/L MS salts; 1X ISU Modified MS Vitamins; 60
gm/L e; 100 mg/L myo—inositol; 125 mg/L Carbenicillin; 2.5 gm/L GELZANTM; and 500
nM R—Haloxyfop acid; at pH 5.8 and plantlets were d to
generate and grow at 280 under
continuous white fluorescent light for
up to 3 weeks.
When ets reached a suitable growth stage, they
were excised with a forceps and
scalpel and transferred to Regeneration Medium 11 containing 4.33 gm/L MS salts; IX ISU
Modified MS Vitamins; 30 gm/L sucrose; 100 mg/L myo—inositol; 3.0 gm/L
TM; at pH
.8; and incubated at 280 under continuous white fluorescent light as above to allow for further
growth and development of the shoot and roots.
Seed production Plants were transplanted into METRO-MIX 360 soilless growing
medium (SUN GRO HORTICULTURE; BELLEVUE, WA) and hardened—off in
a growth room.
Plants were then transplanted into SUNSHINE CUSTOM BLEND 160 soil mixture
and grown
to flowering in the ouse. Controlled pollinations for seed production were conducted.
EXAMPLE 3. ormation of maize by Agrobacterium strains formation
Protocol 2).
The basic work flow is summarized as follows. Embryos
are extracted from
immature ears of corn at the developmental stage at which the
young embryos are about 1.8 to
1001685931
2.4 mm in length. When different transformation conditions
are to be compared, approximately
equal numbers of embryos isolated from a single ear are divided amongst all the treatments. The
embryos are incubated with a suspension containing Agrobacterium cells and surfactant (or not,
for comparison), then are moved to solid-medium plates and co-cultivation is d for
1 to 4
days. The d embryos are erred onto a medium containing antibiotics (for the
suppression and killing of the Agrobacterz'um cells) and compounds for the selective ion of
genetically transformed corn tissues and plants. The corn tissue ly, but not limited to,
callus) is grown on selection medium until plants are regenerated. These plants are tested to
confirm their genetic transformation and those having a desired modification
are grown to
maturity for seed production.
re Embryo Production Seeds from maize inbred line B104 (an Iowa State
variety commercially released in the early 1980’s) were d into 4—gallon—pots ning
SUNSHINE CUSTOM BLEND 160 (SUN GRO HORTICULTURE; Bellevue, WA). The
plants were grown in a greenhouse using a combination of high pressure sodium and metal
halide lamps with a 16:8 hour Dark photoperiod. To obtain immature embryos for
transformation, controlled sib-pollinations were performed. Immature embryos were isolated at
to 13 days post—pollination when embryos were approximately 1.8 to 2.4
mm in size. Maize
ears were surface sterilized after removing the husks and silks by immersing in 50% cial
bleach (CLOROX®, 6.15% sodium hypochlorite) with TWEEN®—20 (1
or 2 drops per 500 mL)
for 10 minutes and triple—rinsed with sterile water.
Alternatively, maize ears can be surface sterilized by thorough spraying with a freshly
prepared solution of 70% ethanol until the ear is completely soaked. Prior to use, the ear is
allowed to air dry for half an hour in a sterile transfer hood to allow the ethanol solution
tely evaporate.
Immature embryos were aseptically isolated directly into a micro centrifuge tube
containing 2 mL of ation Medium with suspended Agrobacterium cells, and surfactant as
appropriate. The embryos were incubated with the suspension ofAgrobacterium cells,
containing surfactant (or not, for control experiments), for 5-30 minutes.
A suspension bacterium cells containing a superbinary vector
was prepared
by first growing the cells in 125 mL (in 500 mL baffled flask) of LB medium (SIGMA
ALDRICH; St. Louis, MO) 20 gm/L, containing 50 mg/L Spectinomycin; 10 mg/L Rifampicin;
1001685931
and 50 mg/L Streptomycin with shaking (250
rpm in the dark) at 26° for 6 hr. This culture was
established by 1:5 dilution of a 25 mL overnight culture (grown in the
same medium) into the
fresh medium. Cells were pelleted by fugation for 15 min at 3500
rpm at 4°, then
uniformly resuspended (by gently pipetting up and down) in Inoculation Medium (InM) to an
optical density of approximately 1.0 at 600 nm (OD600). Inoculation Medium contains: 2.2 gm/L
MS salts (Frame el al. (2011, Genetic TransZormaz‘z‘on Using Maize Immature Zygptz'c
Embryos.
I_n Plant Embryo Culture Methods and ols: s in Molecular Biology. T. A. Thorpe
and E. C. Yeung, (Eds), Springer Science and ss Media, LLC.
pp 327—341); 1X ISU
Modified MS Vitamins (Frame et at, 2011 supra); 68.4 gm/L
sucrose; 36 gm/L glucose; 115
mg/L L—proline; 100 mg/L ositol; and 200 uM acetosyringone (prepared in DMSO); at pH
.4. Depending upon the ment, an appropriate amount of surfactant solution
(e. g.
BREAK—THRU® S 233 at 0.01% final concentration) was added to the Inoculation Medium
after suspending the cells.
The Agrobacterium and embryo solution was ted for 5 to 15 s
at room
temperature, and then the embryos were transferred to Co—cultivation Medium, which contained
4.33 gm/L MS salts; 1X ISU Modified MS Vitamins; 30 gm/L
sucrose; 700 mg/L L—proline; 3.3
mg/L Dicamba in KOH (3,6—dichloro~o—anisic acid or 3,6—dichloro—2—methoxybenzoic acid); 100
mg/L myo—inositol; 100 mg/L Casein Enzymatic Hydrolysate; 15 mg/L AgN03; 100 uM
acetosyringone in DMSO; and 3 gm/L GELZANTM (SIGMA—ALDRICH); at pH 5.8. Co—
cultivation incubation was for 3 to 4 days at 25° under continuous white fluorescent
light
(approximately 50 pEm'Zs‘l).
Resting and Selection After co—cultivation, the embryos (36 s/plate) were
carefully erred to non—selection Resting Medium containing 4.33 gm/L MS salts; 1X ISU
Modified MS Vitamins; 30 gm/L sucrose; 700 mg/L L—proline; 3.3 mg/L Dicamba in
KOH; 100
mg/L myo—inositol; 100 mg/L Casein Enzymatic Hydrolysate; 15 mg/L AgN03; 0.5 gm/L MES
(2—(N—morpholino)ethanesulfonic acid monohydrate (PHYTOTECHNOLOGIES LABR.;
Lenexa, KS); 250 mg/L icillin; and 2.3 gm/L TM; at pH 5.8. Incubation was
continued for 7 days at 28° in continuous white fluorescent light conditions
as above.
Following the 7 day resting period, the embryos were transferred to Selection
Medium. For selection of maize tissues transformed with a superbinary plasmid containing
plant expressible aad] selectable marker gene, the embryos (18 embryos/plate) were first
5931
transferred to Selection Medium I which ted of the Resting Medium (above), and
containing 100 nM R-Haloxyfop acid (0.0362 mg/L). The embryos were incubated for 1 week,
and then erred (12 embryos/plate) to Selection Medium 11, which
consisted of Resting
Medium ), and with 500 nM R-Haloxyfop acid (0.1810 mg/L),
on which they were
ted for an additional 2 weeks. Transformed isolates were obtained over the course of
approximately 4 to 6 weeks at 28° under 24-hour white fluorescent light conditions
(approximately 50 uEm‘Zs‘l). Recovered isolates were transferred to fresh Pre—Regeneration
medium for initiation of regeneration and timber analysis.
Those skilled in the art of maize ormation will understand that
other methods of
ion of transformed plants are available when other plant expressible
selectable marker
genes (e.g. herbicide tolerance genes) are used.
Pre—regeneration Following the selection process, cultures exposed to the 24—hour
light regime were transferred (6 to 8 calli/plate) to Pre—regeneration Medium containing 4.33
gm/L MS salts; 1X ISU Modified MS Vitamins; 45 gm/L sucrose; 350 mg/L
L-proline; 100
mg/L myo—inositol; 50 mg/L Casein Enzymatic ysate; 1.0 mg/L AgNOg; 0.25 gm/L
MES;
0.5 mg/L naphthaleneacetic acid in NaOH; 2.5 mg/L abscisic acid
in ethanol; 1 mg/L 6-
benzylaminopurine; 250 mg/L Carbenicillin; 2.5 gm/L GELZANTM; and 500 nM xyfop
acid; at pH 5.8. Incubation was continued for 7 to 14 days at 28° continuous white fluorescent
light (approximately 50 uEm"2s'1).
Regeneration and plantlet isolation For regeneration, the es were transferred
(up to 12 calli per PHYTATRAYTM (PHYTOTECHNOLOGIES LABR.)) to a primary
Regeneration Medium containing 4.33 gm/L MS salts; 1X ISU Modified MS Vitamins; 60
gm/L
sucrose; 100 mg/L myo—inositol; 125 mg/L Carbenicillin; 3.5 gm/L GELLAN GUM G434
TECHNOLOGIES LABR.); and 500 nM R—Haloxyfop acid; at pH 5.8 and
plantlets
were allowed to te and grow for up to 3 weeks.
When plantlets reached 3 to 5 cm in length, they
were transferred (6 plants per
PHYTATRAYTM) to Plant Growth Medium containing 4.33 gm/L MS salts; 1X ISU Modified
MS Vitamins; 30 gm/L sucrose; 100 mg/L myo-inositol; 3.5 gm/L
GELLAN GUM G434; and
0.5 mg/L indoleacetic acid in NaOH; at pH5.8, and incubated
at 25° under 16-hour white
fluorescent light conditions (approximately 50 uEm‘Zs’l) to allow for further
growth and
development ofthe shoot and roots.
1001685931
Seed production Plants were transplanted into METRO-MIX 360 soilless
g medium
(SUN GRO HORTICULTURE; UE, WA) and hardened-off in
a growth room. Plants
were then transplanted into SUNSHINE CUSTOM BLEND 160 soil
mixture and grown to
flowering in the greenhouse. Controlled pollinations for seed production
were conducted.
EXAMPLE 4. Transformation efficiencies using Agrobacz‘erium cells
grown in
liquid medium.
Agrobacterium superbinary strain LBA4404(pEPS
1083) was used to transform maize
immature embryos by the method disclosed in Example 2
(Transformation Protocol 1).
Comparisons were made of the transformation efficiencies obtained when the A
grobacterium
cells were scraped from YEP
agar plates and resuspended in Infection Medium (HM), versus
experiments done at the same time using cterium cells
grown in liquid medium LB,
harvested by centrifugation and resuspended in 11M.
, Comparative ormation efficiencies
were determined at various stages of the
s by ng the s of yellow fluorescent
spots (YFP+) on treated tissue pieces one to five weeks after initiation of the
transformation
experiments. Table 2 summarizes the results obtained.
Table 2. Comparison of transformation efficiencies using Agrobacterium
inocula prepared from cells scraped from LBA4404(pEPSlO83}
agarplates or harvested after grthh in liquid culture.
Experiment Stage of Agra. N0. of N0. of
Number YFP Count Growth Embryos Treated YFP+ Calli (%)
Plate 108 40/108 (37)
Selection
1 Liquid 103 103/103 (100)
Medium I Plate 108 66/108 (61)
Liquid 36 36/36 (100)
Plate 76 0/76 (0)
2 Pre—Regeneration Liquid 83 32/83 (39)
Medium
L Plate 107
L 0/107 (0)
Liquid—1 100 18/100(18) 1
Plate 71
4 0/71 (0)
3 Pre-Regeneration L Liquid 4 72 1; 26/72 (36)
Medium 4
Plate
1. ‘1 91 0/91 (0)
Liquid 78 7/78 (9)
, Plate 73
_1 4/73 (5)
4 Riga???“ '—
Liquid 97
L 51/97 (53)
Plate 3 69 1_ 3/69 (4)
1001685931
Liquid 59 36/59 (61)
The results summarized in Table 2 demonstrate that infection of maize embryos
using Agrobacterz'um cells freshly harvested from liquid culture provides significantly higher
transformation efficiencies than is obtained using cells scraped from
agar plates.
E 5. Improvement oftransformation efficiencies by addition of surfactant
to Transformation Protocol 1.
Agra/bacterium superbinary strain LBA4404(pDAB108652) was used to transform
maize re embryos by the methods disclosed in Example 2. Plasmid pDABlO8652
contains the YFP coding region, whose expression was driven by the ZmUbil
promoter, and also
harbors the am]! herbicide tolerance coding region under expression control ofthe rice actinl
promoter. Comparisons were made of the transformation efficiencies obtained when the
Agrobaclerium cells were suspended in HM g surfactant, versus experiments done at the
same time with lfiVI containing added surfactant BREAK—THRU® S 233 at various
concentrations. ormation efficiencies were calculated by counting calli with fluorescent
s (each callus arising from a single embryo) after 4 weeks of Haloxyfop selection. At this
time, the cent sectors were large and therefore the tissues represented stably transformed
sectors. The results summarized in Table 3 demonstrate that use of surfactant increases
transformation efficiencies, and that there is a sensitivity of the enhancing effect
on the
concentration of tant used.
Table 3. Effect of s concentrations of surfactant BREAK~THRU® S 233
transformation efficiencies.
Experiment Surfactant No. Embryos
Plasmid Transformation
Number Concentration Treated Efficiency (%)
1 pDAB108652 0% 245 2.86%
:i _] 0.005% 126 l— 14.29%
0.01% 129 8.53%
2 —i_pDAB108652_'_ _J
0% _[ _]
272 0.74%
[_ 0.02% 135
l_ f 6.67%
|_ 0.04% 140 0%
cterz'um inary strain LBA4404(pEPS 1083) was used to transform maize
immature embryos by the method disclosed in Example 2. Comparisons
were made ofthe
5931
transformation efficiencies obtained when the Agrobacterium cells were suspended in IflVI
lacking surfactant, versus experiments done at the same time in the presence of added surfactant
in the HM. Comparative ormation efficiencies were determined at various stages of the
process by counting the s of yellow fluorescent spots (YFP+) on treated tissue pieces one
to five weeks after initiation of the transformation experiments. Table 4 summarizes the results
obtained.
In some experiments, the Agrobacterium cells were washed with IflVl (with, or
without, surfactant) before the cocultivation step by suspension and gentle centrifugation
("wash" in Table 4). Further, in ment 5 (Table 4) 200 uM acetosyringone, (rather than
100 pM as is specified in Example 2) was used to induce vir
gene expression, and the
Agrobacterz'um cells were grown on a plate of LB medium with appropriate antibiotics, rather
than YEP medium.
Table 4. Enhancement of transformation efficiency by Agrobaclerium through the use of a
surfactant. Surfactants BREAK-THRU® S 233, PREFERENCE® or TM were added to
the Infection Medium (working concentration 0.01%) used to create a suspension of
Agmbacterium cells prior to co—cultivation.
No. of No. of % of
EXP StagCeOolifFP Treatment Embryos Embryos s
d YFP+ YFP+
No surfactant i 50 28 56
l Resting Medium S 233* 48 33 69
PREFERENCE® i 48 29 60
Selection No surfactant 60 10 17
Medium 1 S 233 60 39 65
No surfactant 64 20 31
Selection S 233 + lfM wash** 6O 44 73
Medium 1 HM + S 233 wash 66 24 36
S 233 + S 233 wash 72 55 76
No surfactant 36 0 0
Selection 8 233 + HM wash
4 _|_ 36 4 T_ 11
Medium 1 HM + S 233 wash 36 8 22
S 233 + S 233 wash 36 19 53
[_ No. of No. of % of
EXP Stafifo‘l’lfanP Treatment Embryos Calli Embryos
Treated YFP+ YFP+
1001685931
Medium II S 233 60 29/46 48
n--—3 Medium 11 S 233 193 15/78
No surfactant 132
Pre~Regeneration
S 233 110
Medium
TACTICTM 114
* S 233 is THRU® S 233
** IfM is Infection Medium used
to suspend and/or wash the Agrobacterium cells.
*** The Agrobacterz’um cells
were grown on an LB medium plate with antibiotics and vir
gene sion was induced with 200 uM acetosyringone.
The experiments summarized in Table 4 y show that the
presence of surfactant
BREAK—THRU® S 233 in the Infection Medium used to re—suspend the Agrobacterium cells
scraped from solid medium plates dramatically increases the transformation efficiencies of
re embryos. Further, tant TACTICTM has a positive but less dramatic effect on
enhancing transformation efficiency.
In a r ification ofthe methods of this disclosure, immature maize
embryos were transformed with cells ofAgrobacterium strain LBA4404(pEPSlO83) by the
methods of Example 2. Transformation efficiencies were monitored by the
appearance of YFP+
spots or sectors on developing calli from immature embryos. The left side of Figure 1 shows
five experiments (Experiments 1 to 5) using Agrobaclerium cells scraped from solid
agar plates,
and the right side of Figure 1 shows results from three experiments (Experiments 6 to 9) in
which the Agrobacterium cells were harvested from liquid-grown cultures. In combined
Experiments 1 through 5, transformation efficiencies were sed in s from all nine of
the ears harvested (100%) and the transformation ency increases were statistically
significant (Fisher's Exact p<—0.05) in embryos from six of the nine ears (67%). In combined
Experiments 6 through 9 d grown Agrobacreria), embryos from all eight of the ears
harvested (100%) showed a statistically significant increase in transformation efficiency. Thus,
it is clear from the results summarized in Figure 1 that addition of BREAK-THRU® S 233 to the
ion Medium dramatically increases transformation efficiencies of maize immature
embryos, in some cases resulting in transformation efficiencies of over 90%.
In another illustration of the methods of this disclosure, immature maize embryos
were transformed with cells ofAgrobacterium strain LBA4404 harboring various plasmids (all
1001685931
of which contained the aad] able marker gene) by the methods of Example 2. As before,
the experimental treatments compared transformation efficiencies with, or without, the use of
0.01% surfactant BREAK—THRU® S 233. The embryos were regenerated and taken all the way
through Haloxyfop selection to plant production. Thus, the data were collected at a substantially
later stage than that summarized in Figure 1. Transformation efficiency percentages were
ated by dividing the number of embryos that produced a transgenic plant ("an event") by
the number of treated immature s, times 100. For this purpose, an embryo was counted
as a single event even if it produced multiple transgenic plants. The results of three experiments
using cz‘erz'um cells scraped from agar plates are shown in Figure 2 (Experiments 1, 2 and
3). In addition, Figure 2 shows the results of an ment (Experiment 4) in which the
Agrobacterium cells were grown in liquid medium, harvested by centrifugation, and resuspended
in [M (with, or without, BREAK—THRU® S 233). Paired bars in Figure 2 show the responses of
embryos from individual ears.
It is clear from the data in Figure 2 that addition ofthe surfactant BREAK—THRU® S
233 results in a dramatic se in Agrobacterium—mediated transformation efficiencies of
maize immature embryos, regardless of previous growth configuration of the cterium
cells and regardless ofthe gene composition ofthe orming plasmid. In combined
Experiments 1, 2, and 3, transformation efficiencies were increased in embryos from 23 of the 26
ears harvested (88%) and the transformation efficiency increases were statistically significant
(Fisher's Exact p<—0.05) in embryos from 12 of the 26 ears (46%). In Experiment 4 (liquid
growth Agmbacleria) embryos from 10 of the 12 cars harvested (83%) showed an increase in
transformation efficiency, and the increase was statistically cant in one of the 12 ears
(8%).
EXAMPLE 6. ison of transformation—enhancing action of surfactants of
various chemical classes.
Table 1 provides a non—limiting list of surfactants of several chemical classes.
Transformation experiments of immature embryos were conducted using Transformation
Protocol 2 as provided in Example 3. Agrobacterium cells harboring s plasmids were
suspended in Inoculation Medium (InM) containing BREAK—THRU® S 233 or various other
surfactants (all at a concentration of 0.01%), and transformation rates (measured by a ®
assay of the curd] gene were compared 7 to 10 weeks after initiation of the experiment.
1001685931
ormation efficiency percentages were calculated by dividing the number of embryos that
ed a transgenic plant ("an event") by the number of treated re embryos, times 100.
For this purpose, an embryo was counted as a single event even if it produced multiple
transgenic plants. Table 5 presents the transformation efficiencies obtained.
Table 5. ison of transformation efficiencies obtained with surfactants of various
chemical classes. BREAK—THRU® S 233 and other surfactants were used at a concentration of
0.01%.
No' Of
Experiment . Transformation
Plasmld Surfactant
4 Embryos
ncy ( A1). 0
Treated
BREAK-THRU® s 233 756 25
pBPS 1036
SILWET® H8429 498 4.1
BREAK—THRU® S 233 648 16.4
pEPS6008
AGNIQUE® 880-10 648 14.5
BREAK-THRU® s 233 648 15
pEPS 1066
SILWET® H8429 648 5
BREAK—THRU® s 233 647 14.4
ADSEE® AB—650 660 1.9
BREAK~THRU® s 233 534 23
pEPS6004
TRYCOL® 5941 540 15
BREAK-THRU® s 233 648 20
6 pEPS 1013
METASPERSE® SOOL 648 9
BREAK—THRU® s 233 390 16.3
7 pEPSlO64
UPTAKE® 324 5.6
BREAK—THRU® s 233 432 9
8 pEPs1018
ALCOSPERSE® 725 432 0
BREAK—THRU® s 233 427 23
9 pEPSlO38
GERENOL® CF/AS 30 432 27
BREAK—THRU® s 233 468 29
E13810”
BREAK-THRU® s 233 408
11 —_pEPSlozg
BREAK-THRU®SZ33_428
12 pEPSlO64
1001685931
* On
e 4 ears were used in a given experiment. Embryos ted from a single ear were
split between the two treatments in each ment.
The results summarized in Table 5 demonstrate that the use of BREAK—THRU® S
233, when included in the Inoculation Medium used to re—suspend the Agrobacrerz'um inoculum
cells grown in and harvested from liquid medium, provided transformation ncies that
were
superior to those obtained with the majority of the other surfactants tested. In three experiments
(Experiment 2, Experiment 9, and Experiment 11), the transformation efficiencies observed were
nearly the same n the two surfactants.
EXAMPLE 7. ormation results from ent operators.
One d in the art of maize transformation will understand that plant
transformation methodologies often require considerable expertise that is acquired
over months
or years of experimentation. Transformation efficiencies may vary over a wide
range due to
inconsistencies in the ways in which procedures are practiced by different
operators. Thus, it is
advantageous to provide maize transformation procedures that improve predictability in
transformation efficiencies obtained by different operators at different times. ormations of
maize immature embryos were performed over a period of several months using the s of
Example 3 (Agrobacterium strain LBA4404 harboring various plasmids) and containing
BREAK—THRU® S 233 in the Inoculation Medium. Transformation efficiencies were estimated
from counts of Haloxyfop—tolerant callus tissues obtained. Table 6 summarizes the results
obtained.
Table 6. Transformation efficiencies obtained by multiple operators using methods that
incorporate surfactant in the Inoculation Medium.
Operator No. of . Estimated Standard
Ears Used Trans Eff. % 7" Deviation
1001685931
0-18
-_-——
*From manual event counts — not all events were verified by Taqman® analysis. Escape rate
for Haloxyfop selection is approximately 5%.
**Average ormation Efficiency for all operators.
The results summarized in Table 6. show that the transformation protocol disclosed in
Example 3, when practiced with the inclusion ofBREAK—THRU® S 233 in the Inoculation
Medium, provides a robust and predictable methodology that reduces operator—to operator
variation in transformation efficiency. Further, the improved predictability of the methods
allows a more accurate determination of the size of the experiment (6.g. s of embryos
that must be treated) to obtain a desired e (e.g. numbers of transformed events obtained).
The t invention is not limited in scope by the embodiments disclosed herein
which are intended as illustrations ofa few aspects ofthe invention and any embodiments which
are functionally equivalent are within the scope of this invention. Various modifications of the
methods in addition to those shown and bed herein will become apparent to those skilled in
the art and are intended to fall within the scope of the appended claims. Further, while only
certain representative combinations of the method steps disclosed herein are specifically
discussed in the embodiments above, other combinations of the method steps will become
nt to those skilled in the art and also are intended to fall within the scope of the appended
claims. Thus a combination of steps may be explicitly mentioned herein; however, other
combinations of steps are included, even though not explicitly stated. Where a value is explicitly
recited, it is to be understood that values which are about the same quantity or amount as the
recited value are also within the scope of the ion. Where a range of values is recited, each
intervening integer value, and each fraction thereof, between the recited upper and lower limits
of that range is also specifically disclosed, along with each sub-range between such . The
term “comprising” and variations f as used herein is used synonymously with the term
“including” and variations thereof and are open, non-limiting terms. As used , the terms
“modify” or “alter”, or any forms thereof, mean to , alter, replace, delete, substitute,
remove, vary, or transform.
1148
THE
Claims (15)
- l. A method for plant cell transformation comprising ng immature embryo plant cells to Agrobacterium cells in a liquid medium containing a non—ionic trisiloxane surfactant, the surfactant having a concentration of 0.001 weight t to 0.08 weight percent in the liquid medium, and wherein the non—ionic trisiloxane surfactant is not a trisiloxane alkoxylate.
- 2. The method for plant cell transformation of claim 1, r comprising an additional surfactant.
- 3. The method for plant cell transformation of claim 2, n the additional surfactant is an adjuvant, a non—ionic surfactant, an anionic surfactant, an oil based surfactant, an amphoteric surfactant, or a polymeric surfactant.
- 4. The method for plant cell transformation of any one of claims 1 to 3, wherein the plant cells are maize cells.
- 5. The method for plant cell transformation of any one ofclaims 1 to 4, wherein the immature embryos are greater than or equal to 1.5 mm and less than or equal to 2.5 mm in length.
- 6. The method for plant cell transformation of any one ofclaims l to 5, wherein the plant cells are exposed to continuous light after exposure to the Agrobaclerium cells.
- 7. A method for plant cell ormation comprising: ing a liquid medium containing a non—ionic trisiloxane surfactant, wherein the non— ionic trisiloxane surfactant is not a trisiloxane alkoxylate and wherein the non—ionic surfactant has a concentration of 0.001 weight percent to 0.08 weight percent in the liquid medium; suspending Agrobacterz‘um cells in the liquid medium; and exposing immature embryo plant cells to the Agrobacterium cells in the liquid medium containing the surfactant. l 001 684940
- 8. The method for plant cell transformation of claim 7, wherein the Agrobacterium cells are d from a solid medium prior to being suspended in the liquid medium containing a surfactant.
- 9. The method for plant cell transformation of claim 7, wherein the Agrobacterz‘um cells are grown in a liquid growth medium prior to being suspended in the liquid medium containing a surfactant.
- 10. The method for plant cell ormation of any one of claims 7 to 9, further comprising an onal surfactant.
- 1 l. The method for plant cell transformation of claim 10, wherein the additional surfactant is an adjuvant, a non—ionic surfactant, an anionic surfactant, an oil based surfactant, an amphoteric surfactant, or a polymeric surfactant.
- 12. The method for plant cell transformation of any one of claims 7 to l 1, wherein the plant cells are maize cells.
- 13. The method for plant cell transformation of any one of claims 7 to l 1, wherein the immature embryos are 1.5 to 2.5 mm in length.
- 14. The method for plant cell transformation of any one of claims 7 to 13, wherein the plant cells are exposed to uous light after re to the Agrobacterium cells.
- 15. The method for plant cell transformation ofclaim l or 7, substantially as hereinbefore described. ;FLIZQIXZ LIZQIXZ Ln Experiment
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201161576138P | 2011-12-15 | 2011-12-15 | |
US61/576,138 | 2011-12-15 | ||
PCT/US2012/069769 WO2013090734A1 (en) | 2011-12-15 | 2012-12-14 | Method for improved transformation using agrobacterium |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ625401A NZ625401A (en) | 2017-02-24 |
NZ625401B2 true NZ625401B2 (en) | 2017-05-25 |
Family
ID=
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20130157369A1 (en) | Method for improved transformation using agrobacterium | |
US7186889B2 (en) | Method for genetic transformation of woody trees | |
JP2018503392A (en) | Method for performing site-specific modification in complete plants by gene transient expression | |
MX2007002083A (en) | Methods for plant regeneration, transformation and production of insect resistant transgenic okra. | |
CA2505499C (en) | Methods of plant regeneration and transformation | |
Soliman et al. | Efficient transformation and regeneration of fig (Ficus carica L.) via somatic embryogenesis | |
Ozias-Akins et al. | Progress in the development of tissue culture and transformation methods applicable to the production of transgenic peanut | |
JP4463456B2 (en) | High-efficiency Agrobacterium-mediated transformation of cotton using petiole grafts | |
CN101186910B (en) | Transgene method for peanut | |
US20040237133A1 (en) | Method for transformation of mono-and di-cotyledonous plants using meristematic tissue and nodal callus from dicotyledonous plants | |
US11946057B2 (en) | Pre-conditioning treatments to improve plant transformation | |
NZ625401B2 (en) | Method for improved transformation using agrobacterium | |
Beyaz et al. | Explant position effect on gene transformation to flax (Linum usitatissimum L.) via Agrobacterium tumefaciens | |
Mangena | A simplified in-planta genetic transformation in soybean | |
Petolino et al. | Plant cell culture: a critical tool for agricultural biotechnology | |
US20230235344A1 (en) | Plant cell treatments to improve plant transformation | |
Do et al. | Agrobacterium-mediated transformation of Nang Thom Cho Dao, an indica rice variety | |
CH694207A5 (en) | A method for producing transformed coffee plants and transgenic coffee plants. | |
Sumithra et al. | Effect of wounding methods on regeneration and transformation in Gossypium herbaceum and Gossypium hirsutum cotton genotypes. | |
WO2024065009A1 (en) | Methods of plant manipulation | |
CN115605082A (en) | Transformation method | |
Zheng et al. | Genetic transformation of Allium cepa mediated by Agrobacterium tumefaciens | |
Smith et al. | Tissue culture for crop improvement. | |
EP1092778A1 (en) | Agrobacterium-based transformation method for Beta vulgaris | |
Pannetier et al. | Genetic engineering and the improvement of rice and cotton |