TR201812130T4 - Method for developing sugar beet plants resistant to herbicides. - Google Patents
Method for developing sugar beet plants resistant to herbicides. Download PDFInfo
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- TR201812130T4 TR201812130T4 TR2018/12130T TR201812130T TR201812130T4 TR 201812130 T4 TR201812130 T4 TR 201812130T4 TR 2018/12130 T TR2018/12130 T TR 2018/12130T TR 201812130 T TR201812130 T TR 201812130T TR 201812130 T4 TR201812130 T4 TR 201812130T4
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- protoplasts
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Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Herbisitlere karşı dirençli bir şeker pancarı bitkisi üretilmesi için bir yöntem olup, bir şeker pancarı bitkisinden izole edilen stomatal koruyucu hücrelerinden protoplastların elde edilmesi, bir ALS herbisitini söz konusu hücreler için ölümcül olan bir konsantrasyonda içeren bir bileşimin hücrelere uygulanması ve sağ kalan hücrelerden şeker pancarı bitkilerinin rejenere edilmesi adımlarını içermektedir.A method of producing a herbicide-resistant sugar beet plant, comprising obtaining protoplasts from stomatal protective cells isolated from a sugar beet plant, applying a composition comprising an ALS herbicide to a cells lethal to said cells and regenerating the sugar beet plants from the surviving cells to regenerate the sugar beet plants. steps.
Description
TARIFNAME HERBISITLERE KARSI DIRENÇLI SEKER PANCARI BITKILERINI GELISTIRMEK IÇIN YÖNTEM Bulusun Alani Mevcut bulus, herbisitlere, örnegin asetohidroksiasit sentaz enzimi (ALS) inhibitörlerine karsi dirençli olan seker pancari bitkilerini üretmek için bir yöntemle ilgilidir. DESCRIPTION DEVELOPING SUGAR BEET PLANTS THAT ARE RESISTANT TO HERBICIDES METHOD FOR Field of Invention The present invention relates to herbicides, such as acetohydroxyacid synthase enzyme (ALS) inhibitors. relates to a method for producing sugar beet plants that are resistant to
Mevcut bulus ayrica, bu yöntemle elde edilen bitkilerle ilgilidir. The present invention also relates to plants obtained by this method.
Seker pancari, iliman ve subtropik bölgelerde önemli bir tarimsal mahsuldür. Sugar beet is an important agricultural crop in temperate and subtropical regions.
Modern tarimda, yabani ot çogalmasini yönetmek için herbisitler yaygin olarak kullanilmaktadir. In modern agriculture, herbicides are widely used to manage weed overgrowth. is used.
ALS inhibitörleri gibi herbisitlere karsi dirençli olan seker pancari bitkilerinin gelistirilmesi transgenik yaklasimlar kullanilarak yapilabilmektedir. Development of sugar beet plants resistant to herbicides such as ALS inhibitors can be done using transgenic approaches.
Aslinda, bir herbisite karsi direnç kazandiran bir geni tasiyan yabanci DNA'nin uygulanmasi, seker pancari dahil olmak üzere çesitli tarla mahsullerinde basarili bir sekilde gerçeklestirilmistir. In fact, foreign DNA carrying a gene that confers resistance to an herbicide is Its application has been successful in a variety of field crops, including sugar beet. was carried out in the following way.
WO 95/10178 sayili belge, Bialaphos herbisitine karsi transgenik olarak uyarilmis direnci açiklamaktadir. Direnoi kodlayan gen, seker pancarindan alinan koruyucu hücrelerin protoplastlarina uygulanmis, ardindan bu protoplastlar seker pancari bitkileri olarak rejenere edilmistir. Bu bitkiler kimyasal fosfinotrisine ve bunun türevi glufosinata karsi da dirençlidir. WO 95/10178 transgenicly induced against Bialaphos herbicide explains the resistance. Gene encoding resistance, protective from sugar beet applied to the protoplasts of the cells, then these protoplasts were applied to sugar beet plants. as regenerated. These plants have the chemical phosphinothricin and its derivative glufosinata. is also resistant.
Transforme edilmemis bitkiler Bialaphos'a karsi direnç kazanmamistir. Untransformed plants did not acquire resistance to Bialaphos.
En iyi durumda, 28 adet transforme edilmis kallus 83000 protoplasttan rejenere edilmistir; en kötü durumda, 1 kallus 190 000 protoplasttan rejenere edilmistir. At best, 28 transformed calluses regenerated from 83000 protoplasts. has been made; In the worst case, 1 callus was regenerated from 190 000 protoplasts.
Akabinde, bu kalluslarin bazilari somatik embriyogenez gösterebilmektedir ve seker pancari fidelerini %1 verimlilikle ve oldukça avantajli bir özellik olarak görülen sekilde Bununla birlikte, yaygin olarak embriyo gibi seker pancari organlarindan ve/veya yaprak disklerinden elde edilen eksplantlardan alinan kalluslarin daha dogrudan transformasyonuna dayali olan bu yaklasim alanda yaygin olarak kullanilmamaktadir. Subsequently, some of these calli may show somatic embryogenesis and sugar beet seedlings with 1% productivity, which is seen as a very advantageous feature. However, it is commonly derived from sugar beet organs such as embryos and/or leaves. More direct analysis of calli from explants obtained from This approach, which is based on transformation, is not widely used in the field.
Seker pancari mahsulünde oldugu gibi, DNA vektörlerine ve/veya yabanci genlerin yerlestirilmesine dayali olmayan, herbisitlere karsi dirençli bitkilerin gelistirilmesi için önemli bir ihtiyaç halen bulunmaktadir. As in the sugar beet crop, DNA vectors and/or foreign genes for the development of herbicide-resistant plants that are not based on There is still an important need.
Diger yandan, ALS inhibitörleri gibi herbisitlere karsi dirençli seker pancari bitkisinin gelistirilmesi teorik olarak, dogal olusumlu bir direnç geni içerecek olan bir seker pancari bitkisi ile klasik yetistiricilikle yapilabilmektedir. Ancak bu yaklasim zaman alicidir ve basvuru sahibinin bilgisine göre, dogal olusumlu ALS inhibitörüne karsi dirençli bitkiler belgelenmis olmasina ragmen, en azindan çesitli yabani ot türü dahil olmak üzere seker pancari disindaki türler için basarili sonuçlanmamistir. Özellikle bir ikili mutantin (yani ayni ALS geninde iki mutasyona sahip bir bitkinin) dogada olusmasi pek mümkün degildir. On the other hand, resistance to herbicides, such as ALS inhibitors, is found in sugar beet plants. a sugar beet, whose development would theoretically contain a naturally occurring resistance gene. It can be done with classical cultivation with the plant. However, this approach is time consuming and to the applicant's knowledge, plants resistant to the naturally occurring ALS inhibitor sugar, including at least a variety of weed species not successful for species other than beet. Specifically, a double mutant (i.e. a plant with two mutations in the same ALS gene) is unlikely to occur in nature is not.
WO 98/0252? sayili patent basvurusu, sülfonilüre herbisitleri gibi bazi ALS inhibitörlerine karsi dirençli olan seker pancari bitkisinin üretimi için bir yöntemi açiklamakta olup, bu yöntem B. vulgari's eksplantlarindan elde edilen kalluslarin sülfonilüreye tabi tutulmasi ve bu herbisitin varliginda büyüyebilen birkaç spontan mutanttan bitkilerin rejenere edilmesi adimlarini içermektedir. WO 98/0252? Some ALS, such as sulfonylurea herbicides, A method for the production of sugar beet plants resistant to inhibitors This method explains that calli obtained from B. vulgari's explants exposure to a sulfonylurea and several spontaneous plants that can grow in the presence of this herbicide. It includes the steps of regenerating plants from the mutant.
Bu yöntem, ALS geninde bir mutasyona sahip bir bitki vermis olup, burada kodlanmis ALS enzimindeki 188. pozisyondaki (Arabidopsi's thaliana ALS enzimindeki 197. pozisyona karsilik gelmektedir) prolin bir serinle ikame edilmistir. Ancak bu mutant ticari olarak kullanilmamaktadir çünkü tercih edilen modern sülfonilüreler olan ALS herbisitleri (örn. foramsülfuron) ise islemler gerekli doz oraninda tarla çalismalarinda bir miktar fitotoksisite sergilemektedir. istisna B. vui'garfs eksplantlarindan elde edilen kalluslarin foramsülfurona tabi tutulmasi ve dolayisiyla foramsülfuron dahil olmak üzere birkaç ALS inhibitörüne karsi dirençli seker pancari bitkisi ile sonuçlanmasidir. This method yielded a plant with a mutation in the ALS gene, where it was encoded position 188 in the ALS enzyme (Arabidopsi's thaliana 197 in the ALS enzyme). corresponds to the position) proline is substituted by a serine. However, this mutant are not used as modern sulfonylureas, ALS herbicides. (e.g. foramsulfuron), on the other hand, the operations are carried out at the required dose rate in some amount in field studies. exhibits phytotoxicity. the exception is foramsulfuron exposure of calli from B. vui'garfs explants and thus resistant to several ALS inhibitors, including foramsulfuron resulting in a sugar beet plant.
Bu yöntem, ALS geninde bir mutasyona sahip bir bitki vermis olup, burada kodlanmis ALS enzimindeki 569. pozisyondaki (Arabidopsi's iha/fena ALS enzimindeki 574. pozisyona karsilik gelmektedir) triptofan bir Iösinle ikame edilmistir. This method yielded a plant with a mutation in the ALS gene, where it was encoded At position 569 in the ALS enzyme (574th position in Arabidopsi's iha/fena ALS enzyme). corresponds to the position) tryptophan is substituted by an Iosine.
Bu homozigot 569/569 mutantinin tarla çalismalari, foramsülfurona, iyodosülfurona (bir baska ALS inhibitörü) ve farkli ALS inhibitörlerinin karisimlarina karsi iyi direnç göstermistir. Field studies of this homozygous 569/569 mutant have shown that foramsulfuron, iodosulfuron (a good resistance to other ALS inhibitors) and mixtures of different ALS inhibitors has shown.
Bu yayinlanan yöntemlerin her ikisi de embriyolar gibi bagimsiz eksplantlardan gelen kalluslarin izole edilmesine iliskin oldukça yerlesik adimlardan faydalanmaktadir. Ancak bu, zaman alici bir yaklasim olup, (i) fazla sayida taze embriyonun izole edilmesini, (ii) bunlarin agar ile katilastirilmis kültür ortaminda yinelemeli kültürünü ve (iii) morfolojik seçme yaklasimlarini kullanarak rejenere edilebilir kalluslarin seçilmesini kapsama ktadir. Both of these published methods come from individual explants such as embryos. utilizes well-established steps to isolate calluses. However This is a time-consuming approach, requiring (i) isolating large numbers of fresh embryos, (ii) their replicative culture in agar-solidified culture medium and (iii) morphological selection of regenerable calluses using selection approaches. is covered.
Seker pancarina genetik özellikler aktarilmasinin diger stratejileri gelistirilmis olup, bunlar baslangiç malzemesi olarak (stomatal koruyucu hücre protoplastlarindan farklidir olan) mesofil protoplastlarina dayanmaktadir (Krens ve ark., 1990, Theor. appl. Genet., cilt 79, syf. 390-396). biyoteknoloji uygulamalarini açiklamaktadir. Yedi farkli in vitro kültür teknigi açiklanmaktadir. Bunlarin arasinda, ya transformasyon amacina yönelik stomatal koruyucu hücrelerini ya da solmus hipokotil ekSpIantlarindan gelen kirilgan kallusu köken alan protoplastlarin kültürü yer almakta olup, ikincisi çok daha verimlidir. Ancak protoplastlarin kullanildigi bu yaklasim büyük zorluklarla iliskilendirilmistir. deneyi gerçeklestirmek için 500000 stomatal koruyucu hücre protoplastinin kültürünü kullanmistir. Transformasyon verimliligi %2'den fazla olmustur. Diger taraftan, bitkilerin in vitro kültürünün stomatal bozulma ile iliskili oldugu bildirilmis olup, ilgili hücrelerde sorunlara ve dolayisiyla bir mutasyonel olayi içeren bir yöntemin gerçeklestirilmesi için gereken miktarlar gibi çok yüksek miktarda stomatal koruyucu hücre protoplastlarinin üretimine isaret etmektedir. Other strategies for transferring genetic traits to sugar beet have been developed. these as starting material (different from stomatal guard cell protoplasts is based on mesophyll protoplasts (Krens et al., 1990, Theor. appl. Genet., vol 79, p. 390-396). explains biotechnology applications. Seven different in vitro culture techniques is explained. Among them, stomatal for the purpose of either transformation guard cells or fragile callus from wilted hypocotyl appendages. The culture of the protoplasts originating from the origin takes place, and the latter is much more productive. However This approach, using protoplasts, has been associated with great difficulties. culture of 500000 stomatal guard cell protoplasty to perform the experiment. has used. The transformation efficiency was more than 2%. On the other hand, plants It has been reported that in vitro culture is associated with stomatal deterioration, and for realizing a method involving problems and hence a mutational event. of very high amounts of stomatal guard cell protoplasts, such as the required amounts. refers to its production.
Bulusun Özeti Genis bir yönde mevcut bulus, bir herbisite karsi dirençli olan bir mutant seker pancari bitkisi üretilmesi için, asagidaki adimlari içeren bir yöntemi açiklamaktadir: - bir seker pancari bitkisinden izole edilen stomatal koruyucu hücrelerinden . bu protoplastlarin in vitro kültürüne, bu herbisiti in vitro kültürlenmis hücrelerin uygulanmasi ve . bu in vitro kültürlenmis hücrelerin sag kalan hücrelerinden seker pancari bitkilerinin rejenere edilmesi, . muhtemelen, bu herbisitin hedefledigi peptidi/peptitleri kodlayan gende bir mutasyona sahip olan rejenere edilmis seker pancari bitkilerinin seçilmesi, burada bu stomatal koruyucu hücre protoplastlari bir seker pancari bitkisi olarak rejenere olma kapasitelerine göre önceden seçilmekte olup, burada önceden seçilmis protoplastlarin %10”dan fazla olan (büyüyen protoplastlarin sayisi:kültüre koyulan toplam protoplast sayisi) bölünme ve canli kallus olarak rejenere olma olasiligi vardir, burada söz konusu protoplastlardan elde edilen kalluslarin %10*dan fazla olan (filiz veren kallus sayisi:toplam kallus sayisi) filiz gelistirme kapasitesi vardir ve burada bu herbisit bu protoplastlarin 20 000 OOO'dan fazlasina uygulanmaktadir. Summary of the Invention The present invention in a broad aspect is a mutant sugar beet that is resistant to an herbicide. describes a method for producing the plant, which includes the following steps: - from stomatal guard cells isolated from a sugar beet plant . to the in vitro culture of these protoplasts, this herbicide to the in vitro cultured cells implementation and . sugar beet from the surviving cells of these in vitro cultured cells regeneration of plants, . possibly a gene encoding the peptide(s) targeted by this herbicide selection of regenerated sugar beet plants that have the mutation, here these stomatal guard cell protoplasts are used as a sugar beet plant. are preselected according to their capacity to regenerate, where more than 10% of protoplasts (number of growing protoplasts: cultured total number of protoplasts) are likely to divide and regenerate as viable callus, wherein more than 10%* of calli obtained from said protoplasts (sprout number of callus giving: total number of callus) has the capacity to develop sprouts and here this the herbicide is applied to more than 20 000 OOO of these protoplasts.
Mevcut yöntemde kullanilan herbisit ALS genini hedef almayan bir herbisit olabilmektedir. The herbicide used in the present method is an herbicide that does not target the ALS gene. can happen.
Tercih edilen herbisitler (ALS'yi hedef almayan) asagidakilerden olusan gruptan seçilmektedir: - 4-HPPD inhibitörleri (mesela mezotrion, izoksaflutol, pirasülfotol, benzobisiklon, benzofenap, pirazolinat, pirazoksifen, tembotrion, topramezon, sulkotrion ve sulkotrion), karotenoid biyosentez inhitörleri (mesela flurtamon, fluridon, flurokloridon, beflubutamid, norflurazon, pikolinafen ve diflufenikan); - EPSP sentaz inhibitörleri (mesela glifosat veya glifosat-trimesyum); fotosistem ll inhibitörleri (mesela Fenil-karbamatlar, örnegin fenmedifam veya desmedifam, Piridazinonlar (örnegin kloridazon = pirazon), Triazinler (örnegin siyanazini remtal, eglinazin-etil, proglinazin-etil, ametrin, atrazin, desmetrin, dimetametrin, prometon, prometrin, propazin, simazin, simetrin, terbumeton, terbütilazin, terbütrin, metoprotin), Triazinonlar (örnegin metamitron, metribuzin, hekzazinon), Urasiller (bromasil, lenasil, terbasil), Üreler (dimefuron, izoproturon, linuron, monolinuron, etidimuron, metabenztiyazuron, tebutiuron, diuron, fenuron, neburon, siduron, izouron, klorobromuron, klorotoluron, kloroksuron, fluometuron, metobromuron, metoksuron, tiyazafluron, monuron, sikluron, monolinuron) veya amikarbazon, solan, propanil, bentazon, bromoksinil. ioksinil, bromofenoksim, piridat, piridafol; fotosistem I inhibitörleri (mesela dikuat veya parakua): hücre bölünmesi inhibitörleri (mesela karbetamid, klorprofam, profam, naproanilid, difenamid, napropamid, butenaklor, metazaklor, dietatiI-etil, asetoklor, alaklor, butaklor, propaklor Monsanto, propizoklor, dimetaklor, dimetenamid, metolaklor, pretilaklor, S-metolaklor, petoksamid, tenilklor, anilofos, kafenstrol, indanofan, bromobutid, piperofos, flufenaset, mefenaset, fentrazamid): mikrotübül düzeneginin inhibitörleri (mesela propizamid = pronamid, tebutam, klortal-dimetil = DCPA, flukloralin, pendimetalin, butralin, benefin=benfluralin, etalfluralin, orizalin, trifluralin, prodiamin, dinitramin, butamifos, ditiyopir, tiyazopir); protoporfirinojen oksidaz inhibitörleri (mesela Difenileterler (asiflorfen-sodyum, bifenoks, etoksifen-etil, klornitrofen, floroglikofen-etil, oksiflorfen, klometoksifen, flordifen, fomesafen, Iaktofen, nitrofen, aklonifen), N-fenilftalimidler (sinidon-etil, flumiklorak-pentil, flumioksazin), Oksadiazoller (oksadiargil, oksadiazon) Oksazolidinedionlar, (pentoksazon), Fenilpirazoller (fluazolat, piraflufen-etil), Pirimidindionlar (saflufenasil, benzfendizon, butafenasil), Tiyadiazoller (tidiazimin, flutiaset-metil), Triazolinonler (azafenidin, karfentrazon-etil sülfentrazon), piraklonil, profluazol, flufenpir-etil ); . Asetil CoA karboksilaz inhibitörleri (mesela Ariloksifenoksipropiyonatlar (mesela klodinafop-proparjil, sihalofop-bütil, diklofop-metil, fenoksaprop-P-etil, fluazifop-P- bütil, haloksifop-etotil, haloksifop-metil, haloksifop-P-metil, propakuizafop, kuizalofop-P-etil veya kuizalofop-P-tefuril), Sikloheksandionlar (mesela alloksidim, bütroksidim, kletodim, sikloksidim, profoksdim, setoksidim, tepraloksidim veya tralkoksidim), Fenilpirazolin (pinoksaden)); - hücre çeperi sentez inhibitörleri (mesela indaziflam, izoksaben, klortiamid, diklobenil, kuinklorak, flupoksam); . glutamin sent(et)az inhibitörleri (glufosinat-amonyum veya bialafos=bilanafos) ve . sentetik oksin (mesela TBA, dikamba, kloramben, benazolin-etil 4, diklorprop-P, mekoprop-P, 2,4,5-T (weedar) 2,4-D (Weedar), 2,4-DB (Bütirol), diklorprop, MCPB, mekoprop, MCPA-tiyoetil, klomeprop, Agrokson 4, aminopiralid, klopiralid, fluroksipir, halauksifen-metil, pikloram, triklopir, kuinklorak, kuinmerak), daha tercihen 4-HPPD inhibitörleri, karotenoid biyosentez inhibitörleri, EPSP sentaz inhibitörleri, fotosistem ll inhibitörleri, fotosistem I inhibitörleri, mikrotübül düzenegi inhibitörleri, protoporfirinojen oksidaz inhibitörleri ve sentetik oksinden olusan gruptan seçilmektedir. Preferred herbicides (not targeting ALS) are from the group consisting of: is selected: - 4-HPPD inhibitors (eg mesotrione, isoxaflutol, pyrasulfotol, benzobicyclone, benzofenap, pyrazolinate, pyrazoxifen, tembotrione, topramezone, sulcotrione and sulcotrione), carotenoid biosynthesis inhibitors (eg flurtamon, fluridone, flurochloridone, beflubutamide, norflurazone, picolinafen and diflufenican); - EPSP synthase inhibitors (eg glyphosate or glyphosate-trimecium); photosystem II inhibitors (eg Phenyl-carbamates, eg phenmedipham or desmedifam, Pyridazinones (eg chloridazone = pyrazone), Triazines (eg cyanazine remtal, eglinazine-ethyl, proglinazine-ethyl, ametrine, atrazine, desmethrin, dimetametrin, prometone, promethrin, propazine, simazine, symmetryn, terbumetone, terbutylazine, terbuthrin, metoprotin), Triazinones (for example, metamitron, metribuzin, hexazinone), Uracils (bromacil, lenasil, terbacil), Ureas (dimefuron, isoproturon, linuron, monolinuron, etidymuron, metabenzthiazuron, tebutiuron, diuron, fenuron, neburon, siduron, isouron, chlorobromuron, chlorotoluron, chloroxuron, fluometuron, methobromuron, methoxuron, thiazafluron, monuron, cycluron, monolinuron) or amicarbazone, solan, propanil, bentazone, bromoxynil. ioxynil, bromofenoxime, pyridate; pyridafol; photosystem I inhibitors (eg diquat or paraquaa): cell division inhibitors (eg carbetamide, chlorpropham, propham, naproanilide, diphenamide, napropamide, butenachlor, metazachlor, dietatili-ethyl, acetochlor, alachlor, butachlor, propachlor Monsanto, propisochlor, dimethachlor, dimethenamide, metolachlor, pretilachlor, S-metolachlor, petoxamide, tenylchlor, anilofos, cafenstrol, indanofan, bromobutide, piperofos, flufenacet, mefenacet, phentrazamide): inhibitors of microtubule assembly (eg propizamide = pronamide, tebutam, chlortal-dimethyl = DCPA, fluchloralin, pendimethalin, butraline, benefin = benfluralin, etalfluralin, oryzalin, trifluralin, prodiamin, dinitramine, butamifos, dithiopyr, thiazopyr); protoporphyrinogen oxidase inhibitors (eg Diphenylethers (aciflorfen-sodium, bifenox, ethoxyphene-ethyl, chlornitrophen, fluoroglycofen-ethyl, oxyflorfen, clomethoxyphene, flordifen, fomesafen, Iaktofen, nitrofen, aclonifen), N-phenylphthalimides (sinidon-ethyl, flumiclorac-pentyl, flumioxazine), Oxadiazoles (oxadiargil, oxadiazon) Oxazolidinediones, (pentoxazone), Phenylpyrazoles (fluazolate, piraflufen-ethyl), Pyrimidinediones (saflufenasil, benzfendizone, butafenacil), Thiadiazoles (thidiazimine, fluthiacet-methyl), Triazolinones (azafenidine, carfentrazone-ethyl sulfentrazone), pyraclonil, profluazole, flufenpyr-ethyl); . Acetyl CoA carboxylase inhibitors (eg Aryloxyphenoxypropionates (eg. clodinafop-propargile, cihalofop-butyl, diclofop-methyl, fenoxaprop-P-ethyl, fluazifop-P- butyl, haloxyfop-ethothyl, haloxyfop-methyl, haloxyfop-P-methyl, propaquizafop, quizalofop-P-ethyl or quizalofop-P-tefuryl), Cyclohexandiones (eg alloxidim, butroxydim, cletodime, cycloxidim, profoxdim, cetoxydim, tepraloxidim or tralkoxydim), Phenylpyrazoline (pinoxadene)); - cell wall synthesis inhibitors (eg indaziflam, isoxaben, chlortiamide, diclobenil, quinclorac, flupoxam); . glutamine synthase(et)ase inhibitors (glufosinate-ammonium or bialafos=bilanafos) and . synthetic auxin (eg TBA, dicamba, chloramben, benazoline-ethyl 4, dichlorprop-P, mecoprop-P, 2,4,5-T (weedar) 2,4-D (Weedar), 2,4-DB (Butyrole), dichlorprop, MCPB, mecoprop, MCPA-thioethyl, clomeprop, Agroxon 4, aminopyralid, clopyralid, fluroxypyr, halauxifen-methyl, picloram, triclopyr, quinclorac, quinmerak), more preferably 4-HPPD inhibitors, carotenoid biosynthesis inhibitors, EPSP synthase inhibitors, photosystem II inhibitors, photosystem I inhibitors, microtubule assembly inhibitors, protoporphyrinogen oxidase inhibitors and synthetic auxin. is selected.
Son derece tercih edilen bir baska herbisit ise bir ALS inbihitörüdür. Another highly preferred herbicide is an ALS inhibitor.
Bu nedenle, mevcut bulus asetohidroksiasit sentaz enziminin (ALS) bir veya daha fazla inhibitörüne karsi dirençli olan bir mutant seker pancari bitkisinin üretilmesi için, asagidaki adimlari içeren bir yöntemle ilgilidir: . bir seker pancari bitkisinden izole edilen stomatal koruyucu hücrelerinden . söz konusu protoplastlarin in vitro kültürüne, bir veya daha fazla ALS inhibitörünü in vitro kültürlenmis hücreler için (bunlarin %99'undan daha fazlasi için (tercihen olan (yine de bazi mutantlarin kaçmasina izin veren) bir konsantrasyonda içeren bir bilesim uygulanmasi ve . söz konusu in vitro kültürlenmi's hücrelerin sag kalan hücrelerinden seker pancari bitkilerinin rejenere edilmesi, burada söz konusu stomatal koruyucu hücre protoplastlari bir seker pancari bitkisi olarak rejenere olma kapasitelerine göre önceden seçilmekte olup, burada önceden seçilmis protoplastlarin %10'dan fazla olan (büyüyen protoplastlarin sayisizkültüre koyulan toplam protoplast sayisi) bölünme ve canli kallus olarak rejenere olma olasiligi vardir, burada söz konusu protoplastlardan elde edilen kalluslarin %10'dan fazla olan (filiz veren kallus sayisiztoplam kallus sayisi) filiz gelistirme kapasitesi vardir ve burada söz konusu ALS inhibitörleri söz konusu protoplastlarin 20 000 000'undan fazlasina ve hatta 50 000 000'undan fazlasina uygulanmaktadir. Therefore, the present invention contains one or more of the acetohydroxyacid synthase enzyme (ALS) enzymes. To produce a mutant sugar beet plant that is resistant to its inhibitor, It relates to a method with the following steps: . from stomatal guard cells isolated from a sugar beet plant. . in vitro culture of said protoplasts, one or more ALS inhibitors for cells cultured in vitro (more than 99% of these (preferably (which still allows some mutants to escape) applying a composition and . sugar beet from the surviving cells of said in vitro cultured cells regenerating plants, wherein said stomatal guard cell protoplasts according to their regenerative capacity as a sugar beet plant. preselected, where more than 10% of preselected protoplasts (number of growing protoplasts, total number of protoplasts cultured) there is a possibility of dividing and regenerating as living callus, here it is More than 10% of calli from protoplasts (sprouting callus) innumerable total number of calluses) has the capacity to develop sprouts and here it is ALS inhibitors affect more than 20 000 000 of these protoplasts and even It is applied to more than 50 000 000 of them.
Tercihen bu yöntem, farkli genotiplerdeki seker pancari bitkilerinden gelen stomatal koruyucu hücrelerinin izole edilmesi ve her genotip için, söz konusu protoplastlar kültüre koyuldugunda söz konusu protoplastlarin büyüyen oraninin ölçülmesi alt adimlarini içermektedir. Preferably, this method uses stomata from sugar beet plants of different genotypes. isolating guard cells and, for each genotype, the protoplasts in question were cultured. sub-steps of measuring the growing rate of protoplasts in question when placed contains.
Tercihen bu yöntem ayrica, avantajli bir biçimde ALS geninde bir mutasyonu tespit etmek için ve/veya ALS geninde bir veya daha fazla, tercihen bir, iki veya daha fazla mutasyonu olan rejenere edilmis seker pancari bitkilerini seçmek için, sag kalan in vitro kültürlenmi's hücrelerden gelen rejenere bitkilerin genomunun sekanslanmasi adimini içermektedir. Preferably, this method also advantageously detects a mutation in the ALS gene. and/or one or more, preferably one, two or more, in the ALS gene surviving in vitro to select regenerated sugar beet plants with the mutation the step of sequencing the genome of regenerated plants from cultured cells contains.
Arjinin 372, Serin 648 ve Glisin 649`dan olusan gruptan seçilen amino asitleri kodlayan pozisyonlarda ALS geninde bir veya birkaç mutasyonu olan seker pancari mevcut bulusun yöntemi ile elde edilmektedir ve/veya seçilmektedir. 188. pozisyondaki Prolinde ve 569. pozisyondaki Triptofanda bir mutasyonu olan bir baska tercih edilen seker pancari bitkisi mevcut bulusun yöntemi ile elde edilmektedir ve/veya seçilmektedir. Encodes amino acids selected from the group consisting of Arginine 372, Serine 648 and Glycine 649. sugar beet with one or more mutations in the ALS gene at positions obtained and/or selected by the method of the invention. Proline in position 188 and another preferred sugar having a mutation in Tryptophan at position 569 The beet plant is obtained and/or selected by the method of the present invention.
Tercihen, iki veya daha fazla mutasyon olan seker pancari bir allelde iki mutasyonu sahip olup, kodlanmis peptidin ALS inhibitörlerine karsi (özellikle birkaç ALS inhibitörü içeren bilesimlere karsi) direnç için sinerji olusturan iki mutasyonu barindirdigi anlamina gelmektedir. En Çok tercih edilen seker pancarinin bir örnegi bir allelde 188. pozisyondaki Prolinde ve 569. pozisyondaki Triptofanda bir mutasyonu olan (ve muhtemelen ikinci allelde ayni mutasyonlari olan; alternatif olarak, ikinci allel farkli mutasyonlari barindirmaktadir) seker pancaridir. Preferably, sugar beet with two or more mutations has two mutations in an allele. against ALS inhibitors of the encoded peptide (especially several ALS inhibitors). This means that it contains two mutations that create synergies for resistance (against compounds containing is coming. An example of the most preferred sugar beet is 188 in one allele. with a mutation in Proline at position 569 and Tryptophan at position 569 (and possibly having the same mutations in the second allele; alternatively, the second allele is different contains mutations) is sugar beet.
Alternatif olarak, iki mutasyona sahip olan seker pancarinin her allelde bir mutasyonu Mevcut bulusun yöntemi, Prolin 1881 kodlayan pozisyonlarda ALS geninde bir olarak mutasyona ugramistir), Fenilalanin 573, Serin 648 ve Glisin 649”u kodlayan pozisyonlarda ALS geninde bir veya daha fazla mutasyona sahip bir seker pancari bitkisinin rejenere edilmesine olanak vermektedir. Tercih edilen seker pancarinin bir örnegi, 188. pozisyondaki Prolinde bir mutasyona ve ayni allelde bir baska mutasyona (muhtemelen triptofan 569 veya Trp569Gly degil) sahip bir seker pancaridir. Alternatively, sugar beet with two mutations has one mutation in each allele. The method of the present invention uses a gene in the ALS gene at positions coding for Proline 1881. mutated as phenylalanine), encoding Phenylalanine 573, Serine 648, and Glycine 649. a sugar beet with one or more mutations in the ALS gene at positions It allows the plant to be regenerated. One of the preferred sugar beet for example, a mutation in Proline at position 188 and another mutation in the same allele. (probably not tryptophan 569 or Trp569Gly).
Mevcut bulusun yöntemi, Triptofan 569'u (tercihen Lösin olarak mutasyona ugramistir) kodlayan pozisyonda ALS geninde bir mutasyona ve Glisin 112, Alanin 113, Metiyonin pozisyonlarda ALS geninde bir veya daha fazla mutasyona sahip bir seker pancari bitkisinin rejenere edilmesine olanak vermektedir. The method of the present invention uses Tryptophan 569 (preferably mutated to Leucine) a mutation in the ALS gene at the coding position and Glycine 112, Alanine 113, Methionine a sugar beet with one or more mutations in the ALS gene at positions It allows the plant to be regenerated.
Tercih edilen seker pancarinin bir örnegi, 569. pozisyondaki triptofanda bir mutasyona ve ayni allelde bir baska mutasyona (muhtemelen Prolin 188 veya Pro188Ser degil) sahip bir seker pancaridir. An example of the preferred sugar beet has a mutation in tryptophan at position 569. and another mutation in the same allele (probably not Proline 188 or Pro188Ser) It is a sugar beet.
Mevcut bulusun yöntemi, ALS geninde bir veya daha fazla mutasyona sahip bir seker pancari bitkisinin rejenere edilmesine olanak vermekte olup, burada söz konusu bir Triptofan 569, Serin 648 ve Glisin 649'dan olusan gruptan seçilmektedir, burada söz konusu Alanin 113 Valin veya Treonin olarak mutasyona ugramistir, burada söz konusu Prolin 188 Treonin, Arjinin, Lösin, Glutamin veya Alanin olarak mutasyona ugramistir, burada söz konusu Alanin 198 Valin olarak mutasyona ugramistir, burada söz konusu Aspartat 371 Glutamat olarak mutasyona ugramistir, burada söz konusu Arjinin 372 Histidin olarak mutasyona ugramistir, burada söz konusu Triptofan 569 Glisin olarak mutasyona ugramistir, burada söz konusu Serin 648 Treonin olarak mutasyona ugramistir ve burada söz konusu Glisin 649 Aspartat olarak mutasyona ugramistir. The method of the present invention is a sugar with one or more mutations in the ALS gene. allows the beet plant to be regenerated, wherein said a It is selected from the group consisting of Tryptophan 569, Serine 648 and Glycine 649, where mention the subject Alanine 113 has mutated to Valine or Threonine, where Proline 188 is mutated into Threonine, Arginine, Leucine, Glutamine or Alanine, wherein said Alanine is mutated to 198 Valine, wherein in question Aspartate 371 is mutated to Glutamate, where Arginine 372 is It is mutated to histidine, where the Tryptophan 569 is called Glycine. mutated, wherein said Serine 648 mutates as Threonine and here said Glycine 649 is mutated to Aspartate.
Tercihen, bu spesifik mutasyonlardan ikisine veya daha fazlasina sahip seker pancari bir allelde iki mutasyona sahip olup, kodlanmis peptidin ALS inhibitörlerine karsi (özellikle birkaç ALS inhibitörü içeren bilesimlere karsi) direnç için sinerji olusturan iki mutasyonu barindirdigi anlamina gelmektedir. Preferably, sugar beet with two or more of these specific mutations having two mutations in one allele, the encoded peptide against ALS inhibitors Two synergies for resistance (especially against compositions containing several ALS inhibitors) It means that it contains the mutation.
Mevcut bulusun yöntemi, ALS geninde Prolin 188'i kodlayan pozisyonda bir mutasyona ve ALS geninde Triptofan 5691u kodlayan pozisyonda bir mutasyona sahip bir seker pancari bitkisinin rejenere edilmesine olanak vermektedir. The method of the present invention involves a mutation at the position encoding Proline 188 in the ALS gene. and a sugar with a mutation at the position encoding Tryptophan 5691u in the ALS gene It allows the beet plant to be regenerated.
Avantajli bir biçimde, bu yöntem bir veya daha fazla ALS inhibitörünün bilesimin (bir veya daha fazla ALS inhibitörü içerir) in vitro kültürlenmis hücrelerin en az %99'u (tercihen en az %99.9'u ve hatta %99.99'u) için ölümcül oldugu konsantrasyonunun çikarimda bulunulmasi ön adimini içermektedir. Advantageously, this method allows the composition of one or more ALS inhibitors (a or more ALS inhibitors) at least 99% of cells cultured in vitro (preferably at least 99.9% or even 99.99%) of the concentration at which it is fatal for includes the preliminary step of making inferences.
In vitro kültürlenmis stomatal koruyucu hücre protoplastlarina eklenecek olan bilesimde bulunan tercih edilen ALS inhibitörü (söz konusu bilesim ilaveten, tercihen sülfonilüre ALS inhibitörü, mesela tienkarbazon-metilden baska bir siniftan olan bir baska ALS inhibitörü içermektedir) tercihen 10'9 mol/l ila (daha tercihen) 10"6 mol/l arasi bir konsantrasyonda olan foramsülfurondur. Composition to be added to stomatal guard cell protoplasts cultured in vitro The preferred ALS inhibitor available (the compound in addition, preferably a sulfonylurea Another ALS inhibitor, eg from a class other than thiencarbazone-methyl inhibitor) preferably from 10'9 mol/l to (more preferably) 10'6 mol/l concentration of foramsulfuron.
In vitro kültürlenmis stomatal koruyucu hücre prot0plastlarina eklenecek olan bilesimde bulunan bir baska uygun ALS inhibitörü etoksisülfürondur (muhtemelen bu bilesim ayrica diger ALS inhibitörlerini içermektedir). Composition to be added to stomatal guard cell protoplasts cultured in vitro Another suitable ALS inhibitor available is ethoxysulfuron (probably this composition) also includes other ALS inhibitors).
Mevcut bulusun ilgili bir yönü, mevcut bulusun yöntemi ile elde edilebilen mutasyona ugramis bir seker pancari bitkisidir, mesela triptofan 569 ve/veya prolin 188lde bir mutasyona sahip bir seker pancaridir. A related aspect of the present invention is the mutation achievable by the method of the present invention. ugramis is a sugar beet plant, eg tryptophan 569 and/or proline 188l It is a sugar beet with a mutation.
Mevcut bulusun bir baska ilgili yönü, SEKANS TANIM No:3 (veya SEKANS TANIM No:4) ve/veya SEKANS TANIM No:5 (veya SEKANS TANIM NO:6)”yi içeren mutasyona ugramis bir seker pancari bitkisidir. saklanan ürüne karsilik gelmektedir. Another relevant aspect of the present invention is SEQ ID NO:3 (or SEQ ID NO. No:4) and/or SEQ ID No:5 (or SEQ ID NO:6)” It is a mutated sugar beet plant. corresponds to the stored product.
Mevcut bulus ayrica, bulusa ait mutasyona ugramis bitkiden elde edilen doku veya bitki parçasi (örnegin stomatal koruyucu hücreleri veya yaprak seritleri) veya tohumlarin yani sira bunlarin bir baska genetik özelligin takdimi için kullanimlari ile ilgilidir. The present invention also includes tissue or plant derived from the mutated plant of the invention. part (for example, stomatal guard cells or leaf strips) or seeds, i.e. next is their use for the presentation of another genetic trait.
Mevcut bulus ayrica, mevcut bulus kapsaminda gelistirilen (iyi biçimde rejenere olan) stomatal koruyucu hücre protoplastlarini ALS inhibitörlerine direnç disinda bir baska genetik özelligin takdimi için kullanimi ile ilgilidir. The present invention also includes (well regenerated) stomatal guard cell protoplasts other than resistance to ALS inhibitors relates to its use for the presentation of genetic traits.
Bulusun Detayli Açiklamasi Bulus sahipleri, bir stomatal koruyucu hücre protoplastindan bir seker pancari bitkisinin rejenerasyonunun çok zor olmasina ve düsük düzeyde olusmasina ve alanda olagan sekilde kullanildigi gibi eksplantlardan elde edilen kalluslardan dogrudan rejenerasyona göre daha uzun ve daha karmasik prosedürler gerektirmesine ragmen, seker pancarinin stomatal koruyucu hücresinden gelen protoplastlarin ALS inhibitörleri gibi herbisitlere karsi direnci tetiklemek için iyi bir baslangiç maddesini temsil ettigini kesfetmislerdir. Detailed Description of the Invention The inventors describe a sugar beet plant from a stomatal guard cell protoplasty. regeneration is very difficult and occurs at a low level and is not common in the field. Direct regeneration from calluses from explants as used Although it requires longer and more complex procedures than protoplasts from stomatal guard cell to herbicides such as ALS inhibitors They discovered that it represents a good starting material for inducing resistance against it.
Aslinda, eksplantlardan gelen kallus (kalluslar) farklilasmamis (veya genel hale getirilmis) hücrelerin bir kitlesi olup, bunlar, uygun kültür kosullarinda farklilasacak (veya yeniden farklilasacak) ve tamamen fonksiyonel bir seker pancari bitkisini rejenere edecektir. Bu yöntemde, tohumlar büyük miktarlarda toplanmakta, ardindan kolayca sterilize edilmekte eksplantlar bu sekilde büyük miktarlarda elde edilmektedir. Bu yöntem, isin büyük bir kisminin steril bir ortamin zorluklari olmadan gerçeklestirilmesine olanak sagladigindan elverislidir. In fact, callus(s) from explants are undifferentiated (or generalized). bred) cells that will differentiate (or will differentiate again) and regenerate a fully functional sugar beet plant. will. In this method, seeds are collected in large quantities, then easily Sterilized explants are thus obtained in large quantities. This The method allows most of the work to be done without the rigors of a sterile environment. It is convenient because it provides the opportunity.
Diger taraftan, stomatal koruyucu hücreleri bitkide iyi sekilde tanimlanmis bir düzene sahiptir ve bunlarin bitki dokusundan izolasyonu tek tek hücrelere, ardindan bir islemden sonra tek tek protoplastlara yol açmaktadir. On the other hand, stomatal guard cells are arranged in a well-defined order in the plant. and their isolation from plant tissue individual cells, then a gives rise to individual protoplasts after processing.
Mevcut bulusun yönteminde, fideler in vitro olarak büyütülmeli ve steril kosullarda muhafaza edilmelidir, ardindan stomatal koruyucu hücreleri bu küçük fidelerden izole edilmektedir ve steril kosullar muhafaza edilirken protoplastlar elde edilmektedir, ardindan bu tek tek protoplastlar tekrar hücre çeperlerini üretmeleri, ilk önce mikro- kalluslar olarak, ardindan bir seker pancari bitkisi olarak büyümeleri için uyarilmaktadir. In the method of the present invention, the seedlings must be grown in vitro and kept under sterile conditions. must be maintained, then isolate the stomatal guard cells from these small seedlings. and protoplasts are obtained while maintaining sterile conditions, These individual protoplasts then regenerate their cell walls, first micro- as calli, then stimulated to grow as a sugar beet plant.
Seker pancarinin stomatal koruyucu hücre protoplastlarinin çok hassas olduklari, bunun da bunlarin uygulama kapsamini azalttigi bulunmustur: örnegin bir mutajenin eklenmesinin, istenen genetik özelligin ortaya çikmasini desteklemek yerine çogu durumda protoplastlar için ölümcül oldugu bulunmustur. The stomatal guard cell protoplasts of sugar beet are very sensitive. they have also been found to reduce the scope of application: for example, a mutagen rather than supporting the emergence of the desired genetic trait, In this case, it was found to be fatal for protoplasts.
Bir baska deyisle, mutasyonla istenen genetik özelligi kazanmis olan bir seker pancari bitkisini gelistirmek için baslangiç maddesi olarak stomatal koruyucu hücre protoplastlarinin kullanilmasinda basarili olmasi tamamen beklenmedik bir durumdur. In other words, a sugar beet that has acquired the desired genetic trait by mutation. stomatal guard cell as starting material for plant growth Its success in using protoplasts is completely unexpected.
Ayrica, bulus sahipleri stomatal koruyucu hücre protoplastlarinin bölünme kapasitesinin seker pancari genotiplerine oldukça bagli oldugunu bulmustur: genotiplerin çogunlugundan gelen stomatal koruyucu hücre protoplastlari çok düsük (neredeyse sifir) bölünme (büyüme) kapasitesi sergilerken, bazi spesifik genotiplerden gelen protoplastlarin in vitro büyümek için oldukça büyük bir kapasitesi vardir. In addition, the inventors have shown that the dividing capacity of stomatal guard cell protoplasts found that it was highly dependent on sugar beet genotypes: stomatal guard cell protoplasts from the majority are very low (almost zero) exhibit dividing (growth) capacity, while some specific genotypes Protoplasts have a fairly large capacity to grow in vitro.
Bulus sahipleri, bu stomatal koruyucu hücre protoplastlarinin oldukça büyük miktarlarinin oldugu kati ortam üzerindeki (polimer içeren ortam, mesela aljinat veya agaroz benzerini içeren ortam) kültürün, ardindan kültürlenmis maddenin (normalde rejenere edilmis kallus formundadir) ALS inhibitörleri gibi herbisitlere maruz birakilmasinin, eklenen mutajenler olmadan spontan mutasyonlarin çok nadir oldugu bilinmesine ve yöntemin bir veya daha fazla ALS inhibitörüne karsi istenen direnci sergileyen halihazirda var olan bir mutant bitkinin seçilmesinde bir popülasyon içerisindeki genetik çesitlilikten faydalanamamasina ragmen bu herbisit molekülüne karsi dirençli olan bazi mutasyona ugramis hücrelerin seçilmesi ile sonuçlandigini saptamistir. The inventors believe that these stomatal guard cell protoplasts are quite large. on solid media (medium containing polymer, eg alginate or medium containing the agarose analogue), followed by the cultured substance (normally in the form of regenerated callus) exposed to herbicides such as ALS inhibitors be left, spontaneous mutations without added mutagens are very rare. known and desired resistance of the method to one or more ALS inhibitors. a population in selecting an already existing mutant plant that exhibits Although it cannot benefit from the genetic diversity in its results in the selection of some mutated cells that are resistant to determined.
ALS inhibisyonu gibi herbisitlere karsi direnç kazanmis olan mutasyona ugramis bu hücrelerin bazilari ayrica canli seker pancari bitkileri olarak rejenere olabilmistir. This mutated species that has acquired resistance to herbicides such as ALS inhibition some of the cells were also able to regenerate as live sugar beet plants.
Protoplastlarin pratikte kullanilmasi çok zor olmasina ragmen, mevcut bulus yine de herbisite karsi direnç kazanmis olan çok hizli sekilde stabil olan mutantlar vermistir. Although protoplasts are very difficult to use in practice, the present invention still yielded very rapidly stable mutants that acquired resistance to the herbicide.
Mevcut bulusun yöntemi, muhtemelen bu gende en az bir mutasyona ve/veya diger genlerdeki diger mutasyonlara ek olarak herbisitin, örnegin bir ALS inhibitörünün hedefi olan enzimde bir veya birkaç mutasyon (mesela ALS geninde bir mutasyon, burada ALS geni, Arabidopsis thaliana ALS”nin 574. pozisyonuna karsilik gelen ALS proteininin 569. pozisyonunda triptofandan farkli bir amino asit içeren bir ALS proteinini kodlamaktadir) dahil olmak üzere bir veya birkaç mutasyona sahip olan herbisitlere karsi dirençli (ALS inhibitörüne dirençli) seker pancari bitkilerinin üretilmesine olanak vermektedir. The method of the present invention is likely to cause at least one mutation in this gene and/or other target of herbicide, for example an ALS inhibitor, in addition to other mutations in genes one or more mutations in the enzyme (for example, a mutation in the ALS gene, where ALS The gene is located at position 569 of the ALS protein, which corresponds to position 574 of Arabidopsis thaliana ALS. encodes an ALS protein containing an amino acid different from tryptophan at position ) resistant to herbicides (ALS) with one or more mutations, including It allows the production of sugar beet plants (resistant to the inhibitor).
Bu nedenle mevcut bulusun bir yönü, bir veya daha fazla ALS inhibitörüne karsi dirençli olan seker pancari bitkisinin (Beta vulgari's; dolayisiyla ayrica Beta vulgari's mari'tima ssp) üretilmesi için, asagidaki adimlari içeren bir yöntemdir: . (ALS inhibitörlerine karsi hassas) bir seker pancari bitkisinden izole edilen stomatal koruyucu hücrelerinin (bunlarin protoplastlarinin) elde edilmesi; . bu in vitro kültürlenmis hücrelere, bir veya daha fazla ALS inhibitörü içeren bir bilesimin bu in vitro kültürlenmis hücreler için ölümcül olan bir konsantrasyonda uygulanmasi ((yabani tip) hücrelerin en az %99'u ALS inhibitörü tarafindan öldürülmektedir, ancak bazi mutantlar islemden kaçabilmektedir) ve sag kalan hücrelerden seker pancari bitkilerinin rejenere edilmesi. Therefore, one aspect of the present invention is resistance to one or more ALS inhibitors. sugar beet plant (Beta vulgari's; therefore also Beta vulgari's mari'tima ssp) is a method that includes the following steps: . isolated from a sugar beet plant (sensitive to ALS inhibitors) obtaining stomatal guard cells (their protoplasts); . These in vitro cultured cells are given a drug containing one or more ALS inhibitors. of the compound at a concentration that is lethal to these in vitro cultured cells. administration (at least 99% of (wild-type) cells by ALS inhibitor killed, but some mutants can escape the process) and regeneration of sugar beet plants from surviving cells.
Bu yöntem, (örnegin WO 95/10178 sayili belgede açiklanan) bir transgenin takdimine bir alternatif olarak görülebilir. Ancak endojen genlerin mutasyonuna dayali olan bir yöntem transgenik yaklasima göre daha zahmetlidir. Aslinda, bir transgenik (mutasyona ugramis) genin takdimi çok daha hizli, esnek ve öngörülebilirdir. This method allows for the introduction of a transgene (disclosed, for example, in WO 95/10178). can be seen as an alternative. However, a mutation based on endogenous genes The method is more demanding than the transgenic approach. In fact, a transgenic (mutated ugramis) gene delivery is much more rapid, flexible, and predictable.
Transgen ile uyarilmis direncin olmamasi, ALS inhibitörlerine karsi kazanilmis direncin, bir (yabanci) DNA'nin, örnegin ALS inhibitörlerinin toksisitesini lokal olarak azaltan bir protein (örn. ALS inhibitörlerini parçalayan veya intraselüler konsantrasyonunu azaltan bir enzim) veya ihhibitörlerin varliginda bile önemli aktivite ve fonksiyonelligi koruyan bir ALS mutant enzimi gibi ALS inhibitörlerine karsi dirençli bir ALS proteini gibi dogrudan ALS inhibitörlerine karsi direnç saglayan bir proteini kodlayan bir (yabanci) DNA'nin, stomatal koruyucu hücre protoplastlarina (veya bundan elde edilen hücrelere) in vitro insersiyonundan dogrudan kaynaklanmadigini belirtmektedir. Absence of transgene-induced resistance, acquired resistance to ALS inhibitors, a substance that locally reduces the toxicity of a (foreign) DNA, eg ALS inhibitors protein (e.g. that breaks down ALS inhibitors or reduces their intracellular concentration) an enzyme) or an enzyme that maintains significant activity and functionality even in the presence of inhibitors. directly as an ALS protein that is resistant to ALS inhibitors, such as the ALS mutant enzyme. A (foreign) DNA encoding a protein that confers resistance to ALS inhibitors, into stomatal guard cell protoplasts (or cells derived from it) states that it is not caused directly by in vitro insertion.
Ancak bulusun yöntemi ile elde edilen bitki ayrica bu soyda bir baska genetik özelligi toplamak için bir transgenik bitki ile melezlenebilir. Mevcut bulusla elde edilen bitki (veya bunun bir parçasi) ayrica (mevcut bulusun yönteminde elde edilen genetik özellikten farkli olan) bir baska genetik özelligi takdim etmek için müteakip bir transgenik yöntemde kullanilabilir. However, the plant obtained by the method of the invention also has another genetic feature in this strain. It can be crossed with a transgenic plant for collection. The plant (or part of it) also (from the genetic trait obtained in the method of the present invention) a subsequent transgenic trait to introduce another genetic trait can be used in the method.
Muhtemelen, bu yöntem ayrica izole edilmis stomatal koruyucu hücre protoplastlarinin kültürüne bir mutajen ajani eklenmesi adimini içermektedir. Presumably, this method may also be used to detect isolated stomatal guard cell protoplasts. includes the step of adding a mutagen agent to the culture.
Uygun mutajen ajanlari fiziksel (mesela UV veya X-isini) maruziyeti veya kimyasal hatta %2.5,te) maruziyettir. Ancak, protoplast viyabilitesinin mesela %0.2,den fazla EMS ile islem gibi alisilagelmis sekilde gerçeklestirilen bazi mutajenez islemlerinden zararli bir biçimde etkilendigi gösterilmistir. Suitable mutagen agents include physical (eg UV or X-ray) exposure or chemical even at 2.5%) exposure. However, more than 0.2% of protoplast viability, for example, EMS Harmful from some mutagenesis processes, such as treatment with has been shown to be affected.
Alternatif olarak, bu yöntem bu nedenle bir mutajen ajan eklenmesi adimini içermemektedir. Alternatively, this method therefore avoids the step of adding a mutagenic agent. does not include.
Mevcut bulus baglaminda, bir herbisit tercihen, belirli bir dozda uygulandiginda yabani ot kontrolü için kullanilan herhangi bir molekülü belirtmektedir. In the context of the present invention, an herbicide is preferably wild when applied at a given dose. refers to any molecule used for weed control.
Mevcut yöntemde (bu herbisite karsi dirençli seker pancari bitkilerini gelistirmek için) kullanilan tercih edilen herbisitlerin bir peptitde spesifik bir bilinen aktivitesi vardir (böylece ilgili gendeki tek bir mutasyon bu herbisite karsi direnç kazandirabilmektedir). In the current method (to develop sugar beet plants resistant to this herbicide) preferred herbicides used have a specific known activity in a peptide (so that a single mutation in the relevant gene can confer resistance to this herbicide).
Bir baska deyisle, tercih edilen herbisitler (çogunlukla spesifik biçimde bir bitki enziminin aktivitesini inhibe eden) bir peptit hedefi için spesifiktir ve/veya (hedef gendeki bir mutasyon için dirençli seker pancarini taramak için bir pozisyonda olmasi amaciyla) bunlarin hedef peptidi bilinmektedir. In other words, the preferred herbicides (often specifically the expression of a plant enzyme) specific for a peptide target (which inhibits its activity) and/or (a in order to be in a position to screen for resistant sugar beet for mutation) their target peptide is known.
Mevcut bulus baglaminda, bir ALS inhibitörü ALS geninin fonksiyonunu inhibe eden herhangi bir molekülü ifade etmektedir. In the context of the present invention, an ALS inhibitor inhibits the function of the ALS gene. means any molecule.
Tercihen, mevcut bulusta kullanilan ALS inhibitörleri ALS disindaki diger (seker pancari) enzimlerini (büyük ölçüde) inhibe etmemektedir. Preferably, the ALS inhibitors used in the present invention are other than ALS (sugar beet). It does not inhibit enzymes (to a large extent).
Avantajli bir biçimde, ALS inhibitörleri yabani tip ALS enziminin fonksiyonunun fazlasinin) inhibe edildigi, ancak ilgili olmayan enzimlerin fonksiyonunun büyük ölçüde etkilenmedigi bir konsantrasyonda mevcut bulusta seçilmekte ve kullanilmaktadir. Advantageously, ALS inhibitors are the function of wild-type ALS enzyme. The function of enzymes that are inhibited, but not related, is largely is selected and used in the present invention at a concentration that is not affected.
(Bulusu gerçeklestirmek için) uygun ALS inhibitörleri sülfonilüre herbisitleri, sülfonilaminokarboniltriazolinon herbisitleri, imidazolinon herbisitleri, triazolopirimidin herbisitleri ve pirimidinil(tiyo)benzoat herbisitlerinden olusan gruptan seçilmektedir. Suitable ALS inhibitors (to carry out the invention) are sulfonylurea herbicides, sulfonylaminocarbonyltriazolinone herbicides, imidazolinone herbicides, triazolopyrimidine and pyrimidinyl(thio)benzoate herbicides.
Avantajli bir biçimde, herbisit bilesimi en az bir sülfonilüre herbisiti ve en az bir triazolopirimidin içermektedir. Advantageously, the herbicide composition includes at least one sulfonylurea herbicide and at least one Contains triazolopyrimidine.
(Bulusu gerçeklestirmek için) tercih edilen ALS inhibitörleri foramsülfuron, amidosülfuron, tienkarbazon-metil, etoksisülfuron ve bunlarin karisimidir (özellikle tienkarbazon-metil ve ya foramsülfuron ya da amidosülfuron içeren bir bilesim); ancak, rejenere edilen seker pancari mutanti avantajli bir biçimde birkaç ALS inhibitörüne karsi dirençlidir. The preferred ALS inhibitors (to carry out the invention) are foramsulfuron, amidosulfuron, thiencarbazone-methyl, ethoxysulfuron and mixtures thereof (especially a composition containing thiencarbazone-methyl and either foramsulfuron or amidosulfuron); however, The regenerated sugar beet mutant advantageously resists several ALS inhibitors. is resistant.
(ALS inhibitörlerinin karisimlari dahil) diger ALS inhibitörleri kullanilabilir ve teknikte uzman kisi hangi mutasyonun belirli bir ALS inhibitörüne karsi güçlü direnç sagladigini bilir (örn. 569. pozisyonda triptofanin mutasyonunun foramsülfuron direnci ile iliskili oldugu bilinmektedir); dolayisiyla, mevcut yöntemin esnekligi ve verimliligi göz önünde bulunduruldugunda, teknikte uzman kisi birkaç mutasyon ve dolayisiyla ALS inhibitörlerine (örn. ALS inhibitörlerinin karisimlarina) karsi daha genis direnç kazanilmis olan seker pancari bitkilerini tasarlayabilir. Other ALS inhibitors (including mixtures of ALS inhibitors) can be used and the art The expert will know which mutation confers strong resistance to a particular ALS inhibitor. (e.g., mutation of tryptophan at position 569 is associated with foramsulfuron resistance. known to be); Therefore, considering the flexibility and efficiency of the current method, available, the skilled artisan can identify several mutations and hence ALS. greater resistance to inhibitors (eg, mixtures of ALS inhibitors) be able to design sugar beet plants.
Daha tercihen, bu yöntem stomatal koruyucu hücre protoplastlarinin tamamen fonksiyonel bir seker pancari bitkisine rejenere olma kapasitesi açisindan bir seker pancari bitki genotip (hatti) seçilmesi ön adimini içermektedir ve/veya mevcut bulusun yöntemi iyi rejenere olan seker pancari genotiplerinden (hatlarindan) izole edilen stomatal koruyucu hücre protoplastlari üzerinde gerçeklestirilmektedir. More preferably, this method completely removes stomatal guard cell protoplasts. sugar in terms of its regenerative capacity to a functional sugar beet plant beet plant genotype (line) selection includes the preliminary step and/or isolated from sugar beet genotypes (lines) that regenerated well carried out on stomatal guard cell protoplasts.
(Stomatal koruyucu hücre protoplastlarinin bir seker pancari bitkisi olarak rejenere olma yetenegi açisindan seker pancari bitki genotiplerinin seçilmesine iliskin) uygun bir ön adim, (farkli genotip geçmislerinden gelen) en az 10 farkli seker pancari bitki genotipinin, tercihen en az 15 farkli genotipin ve hatta en az 30 farkli genotipin, stomatal koruyucu hücre protoplastlarinin bir seker pancari bitkisi olarak rejenere olma kapasitesi açisindan (çok daha tercihen in vitro büyüme ve/veya kallus olusturma kapasiteleri açisindan) karsilastirilmasini (verim veya parazitik enfeksiyonlara karsi direnç gibi olasi avantajli özelliklerinden bagimsiz olarak) ve mevcut bulusun yöntemini gerçeklestirmek için iyi rejenere olan bir genotipin (hattin) seçilmesini kapsamaktadir. (Regeneration of stomatal guard cell protoplasts as a sugar beet plant a suitable preliminary study (on the selection of sugar beet plant genotypes) in terms of step, at least 10 different sugar beet plants (from different genotype histories) genotype, preferably at least 15 different genotypes and even at least 30 different genotypes, stomatal capacity of guard cell protoplasts to regenerate as a sugar beet plant (much more preferably in vitro growth and/or callus-forming capacities) in terms of potential (such as yield or resistance to parasitic infections) independently of its advantageous properties) and to realize the method of the present invention It involves selecting a genotype (line) that regenerates well for
Mevcut bulus baglaminda, iyi rejenere olan stomatal koruyucu hücre protoplastlari, (büyüme:toplam) bölünme ve/veya büyüme ve/veya canli seker pancari kallusu olarak rejenere olma (uygun kültür ortaminda ve mevcut bulusun yönteminde uygulanacak toksik molekül/herbisit gibi ekzojen seleksiyon basinci olmadan büyütüldügünde) olasiligi olan protoplastlari belirtmektedir. In the context of the present invention, stomatal guard cell protoplasts that regenerate well, (growth:total) as division and/or growth and/or live sugar beet callus regeneration (to be applied in the appropriate culture medium and method of the present invention) when grown without exogenous selection pressure such as toxic molecule/herbicide) indicates possible protoplasts.
Kallus (kalluslar) farklilasmamis hücrelerin bir kitlesini ifade etmektedir. Teknikte kalluslar, embriyo gibi eksplantlardan ya da yaprak veya kotiledon kökenli parenkimden elde edilen eksplantlardan elde edilebilmektedir. Ancak, mevcut bulus baglaminda, kalluslar (iyi rejenere olan) stomatal koruyucu hücre protoplastlarinin büyümesinin sonucudun Bu iyi rejenere olan protoplastlarla elde edilen kalluslarin %10'dan fazla (filiz veren kallus sayisi : toplam kallus sayisi; filizztoplam), tercihen %20'den fazla (filiz:toplam) ve hatta %30'dan fazla (filiz:toplam) filiz gelistirme kapasitesi vardir. Callus (calli) refers to a mass of undifferentiated cells. in technique calli, from explants such as embryos or parenchyma of leaf or cotyledon origin obtained from explants. However, in the context of the present invention, calli (well regenerating) stomatal guard cell protoplasts you were the result More than 10% of the calli obtained from these well regenerating protoplasts (sprouting number of calli: total number of callus; sproutstotal), preferably more than 20% (sprouts:total) and even more than 30% (sprout:total) sprout development capacity.
Tercihen, iyi rejenere olan seker pancari stomatal koruyucu hücre protoplastlari, fazla) olan canli seker pancari bitkisi olarak rejenere olma kapasitesine sahip protoplastlari ifade etmektedir. Preferably, well regenerating sugar beet stomatal guard cell protoplasts, It has the capacity to regenerate as a live sugar beet plant. stands for protoplasts.
Bu yöntemde herbisiti (örn. bir veya daha fazla ALS inhibitörü) içeren bilesim bu (iyi rejenere olan) seker pancari stomatal hücre protoplastlarinin 20 000 OOO'dan daha fazlasinin in vitro kültürüne uygulanmaktadir. In this method, the composition containing the herbicide (eg, one or more ALS inhibitors) is this (well More than 20 000 OOO of regenerating sugar beet stomatal cell protoplasts applied to in vitro culture of excess.
Tercihen, (bir veya daha fazla) ALS inhibitörünü içeren bilesim bu (iyi rejenere olan) seker pancari stomatal koruyucu hücre protoplastlarinin 50 000 OOO'undan daha fazlasinin in vitro kültürüne uygulanmaktadir. stomatal koruyucu hücre protoplastlari, polimer içeren ortam (mesela aijinat veya agaroz içeren ortam) üzerinde büyütülmüstür. Preferably, this is the composition containing (one or more) ALS inhibitors (well regenerated) More than 50 000 OOO of sugar beet stomatal guard cell protoplasts applied to in vitro culture of excess. stomatal guard cell protoplasts, polymer-containing medium (eg aginate or grown on agarose-containing medium).
Muhtemelen, bu (iyi rejenere olan) seker pancari stomatal koruyucu hücre protoplastlari, (bir veya daha fazla) ALS inhibitörünü (mesela foramsülfuron ve muhtemelen tienkarbazon-metil) içeren bilesimin uygulanmasindan önce polimer (aljinat) içeren ortamda en az yaklasik 1 hafta (tercihen yaklasik 3 hafta ve/veya 4 haftadan az) büyütülmektedir. Presumably, this (well regenerating) sugar beet stomatal guard cell protoplasts, (one or more) ALS inhibitors (eg foramsulfuron and polymer prior to application of the composition containing possibly thiencarbazone-methyl) (alginate) at least about 1 week (preferably about 3 weeks and/or 4 weeks) less than a week) is enlarged.
Tercihen, bu yöntem ayrica mutasyona ugramis stomatal koruyucu hücresinin büyümesinin ve yabani tip stomatal koruyucu hücresinin (ve/veya saf ve/veya henüz herbisitle islem görmemis) ALS inhibitörü içermeyen bir ortamda büyümesinin karsilastirilmasi ve muhtemelen ilgili yabani tip hücrenin en az %75 büyümesini, tercihen en az %90 büyümesini koruyan mutantlarin seçilmesi adimini içermektedir. Preferably, this method also allows the mutated stomatal guard cell to be growth and wild-type stomatal guard cell (and/or naive and/or not yet herbicide-free) growth in an ALS inhibitor-free medium. comparison and possibly at least 75% growth of the wild-type cell of interest, preferably, selecting mutants that retain at least 90% growth.
Tercihen veya ilaveten, bu yöntem ayrica mutasyona ugramis hücreden rejenere edilmis seker pancarinin büyümesinin ve/veya veriminin ve yabani tip (ve/veya saf vei'veya henüz ALS inhibitörü ile islem görmemis) seker pancarinin sera testlerinde ve herhangi bir ALS inhibitörü olmadan agronomik kosullarda büyümesinin karsilastirilmasi ve muhtemelen yabani tip seker pancarinin en az %75 büyümesini ve/veya verimini, tercihen en az %90 büyümesini ve/veya verimini koruyan ALS inhibitörüne dirençli mutant(lar) olan seker pancari bitkilerinin seçilmesi adimini içermektedir. Preferably or additionally, this method also regenerates from the mutated cell. growth and/or yield of cultivated sugar beet and wild-type (and/or pure vei'or not yet treated with ALS inhibitor) in the sera tests of sugar beet and Comparison of growth under agronomic conditions without any ALS inhibitor. and possibly at least 75% growth and/or yield of wild-type sugar beet, resistant to ALS inhibitor, preferably maintaining at least 90% growth and/or yield includes the step of selecting sugar beet plants that are mutant(s).
Tercihen, bu yöntem ayrica sag kalan protoplastlardan rejenere edilmis bitkilerin sekanslanmasi ve/veya herbisite (örn. bir veya daha fazla ALS inhibitörüne) karsi dirençle iliskili olan (olabilen) bir veya birkaç mutasyonun tanimlanmasi adimini içermektedir. Preferably, this method is also used for plants regenerated from surviving protoplasts. sequencing and/or against herbicides (eg, one or more ALS inhibitors) the step of identifying one or more mutations that are (could be) associated with resistance. contains.
Mevcut bulus baglaminda, “mutasyon” terimi tercihen, elde edilen bitkinin ALS inhibitörleri gibi herbisitlere karsi bir direnç kazanmis olacagi sekilde ilgili amino asitte bir degisikligine neden olan, herbisitin (örn. ALS proteininin) hedefledigi peptidi kodlayan nükleotid sekansindaki bir (tek bir) degisikligi ifade etmektedir. In the context of the present invention, the term "mutation" preferably means ALS from the resulting plant. in the relevant amino acid so that it has acquired a resistance to herbicides such as inhibitors encoding the peptide targeted by the herbicide (eg, the ALS protein) that causes a change in It refers to a (single) change in the nucleotide sequence.
Bir baska deyisle, mevcut bulus baglaminda, “mutasyon” tercihen herbisite (ALS inhibitörüne) karsi bir dirence olanak veren “nokta mutasyonuna” esdeger olarak anlasilmaktadir. Bu dogrultuda, “birkaç mutasyon” tercihen, mevcut bulusta bir (yigin) çoklu nokta mutasyonunu ifade etmekte olup, her bir nokta mutasyonu bir herbisite (örn. bir ALS inhibitörüne ve/veya ALS inhibitörü olmayan bir herbisite) bir direnç saglamak üzere kodlanmis amino asitte bir degisiklige neden olmaktadir. Bu nedenle, tercihen, mevcut bulus baglaminda “mutasyon”, nükleotid sekansinda kodlanmis proteini modifiye etmeyen bir degisikligi (mesela kodlama üçlüsünün üçüncü amino asidindeki bir degisikligi), herbisit (mesela ALS inhibitörü) direnci ile iliskili olmayan amino asitlerdeki bir degisikligi ve nükleotid sekansindaki çoklu eszamanli degisiklikleri içermemektedir. In other words, in the context of the present invention, "mutation" is preferably herbicide (ALS) equivalent to a "point mutation" that allows a resistance against is understood. In this regard, "several mutations" are preferably one (stack) in the present invention. refers to multiple point mutations, each point mutation being a herbicide (eg. confer resistance to an ALS inhibitor and/or a non-ALS inhibitor herbicide) It causes a change in the coded amino acid. Therefore, preferably "mutation" in the context of the present invention is the protein encoded in the nucleotide sequence a non-modifying change (for example, in the third amino acid of the coding triplet) a change), amino acid not associated with herbicide (eg ALS inhibitor) resistance a change in acids and multiple simultaneous changes in the nucleotide sequence does not include.
Avantajli bir biçimde, (bir veya daha fazla) ALS inhibitörüne karsi dirençle iliskili mutasyonlarin (tercihen ALS genindeki bir veya iki mutasyonun) tanimlanmasi adimi bu mutasyonu kapsayan oligonükleotid primerlerinin gelistirilmesi ile baglantilidir. Advantageously associated with resistance to (one or more) ALS inhibitors. this is the step of identifying mutations (preferably one or two in the ALS gene). associated with the development of oligonucleotide primers that contain mutation.
Avantajli bir biçimde, bu ALS genindeki mutasyonlari tanimlama adimini, yabani tip ve mutasyona ugramis ALS genlerinin kodladigi proteinin (in vitro) enzimatik aktivite ölçümleri takip etmektedir. Advantageously, the step of identifying mutations in this ALS gene, wild-type and enzymatic activity of the protein (in vitro) encoded by mutated ALS genes following the measurements.
Tercihen, yabani tip ALS enziminin ve mutasyona ugramis ALS enziminin bu enzimatik ölçümleri bir veya daha fazla ALS inhibitörü varliginda (bir inhibisyon egrisini elde etmek için bir veya birkaç konsantrasyonda) gerçeklestirilmektedir. Preferably, this enzymatic expression of the wild-type ALS enzyme and the mutated ALS enzyme measurements in the presence of one or more ALS inhibitors (to obtain an inhibition curve) for one or more concentrations) is carried out.
Muhtemelen, yabani tip enzimin ve mutasyona ugramis enzimin bu enzimatik ölçümü (ayrica) ALS inhibitörü olmadan gerçeklestirilmektedir (mutasyona ugramis enzimin enzimatik aktivitesini karsilastirmak için; tercihen ALS inhibitörü olmadan, mutasyona ugramis enzim yabani tip enzimin aktivitesinin en az %50'sini, daha tercihen en az korumaktadir). Presumably, this enzymatic measurement of wild-type enzyme and mutated enzyme (also) without ALS inhibitor to compare its enzymatic activity; mutate, preferably without ALS inhibitor ugramis enzyme has at least 50% of the activity of the wild-type enzyme, more preferably at least protects).
Tercihen, mevcut bulusun yöntemi farkli konsantrasyonlarda bir veya daha fazla ALS inhibitörü içeren bilesimlerin karsilastirilmasi ve ALS inhibitörünün ve/veya bu bilesim içerisinde özel bir formülasyondaki ALS inhibitörünün seker pancari bitkisinden izole edilen stomatal koruyucu hücre protoplastinin (mesela en az bir hafta süreyle aljinat üzerinde büyütülen stomatal koruyucu hücre protoplastlarinin) in vitro kültürü için ölümcül oldugu konsantrasyonun çikarimda bulunulmasini adimini içermektedir. Örnegin, (bir veya daha fazla) ALS inhibitörünün seker pancari bitkisinden izole edilen stomatal koruyucu hücre protoplasti için ölümcül oldugu konsantrasyonun çikarimda bulunulmasi adimi, yabani tip seker pancari bitkisinden (ve/veya saf ve/veya henüz ALS inhibitörü ile islem görmemis) izole edilen stomatal koruyucu hücre protoplastlarinin in vitro kültüründe gerçeklestirilmektedir. Preferably, the method of the present invention contains one or more ALS at different concentrations. Comparison of the compositions containing the inhibitor and the comparison of the ALS inhibitor and/or this composition isolated from sugar beet plant of ALS inhibitor in a special formulation. Stomatal guard cell protoplasty (for example, alginate for at least one week) for in vitro culture of stomatal guard cell protoplasts grown on It includes the step of inferring the concentration at which it is lethal. For example, (one or more) ALS inhibitors isolated from sugar beet plant Inferring the concentration at which it is fatal for stomatal guard cell protoplasty The step of finding is from wild-type sugar beet plants (and/or pure and/or not yet ALS inhibitor of the isolated stomatal guard cell protoplasts. performed in in vitro culture.
Mevcut bulus baglaminda, (bir veya daha fazla) ALS inhibitörü içeren bilesimin ölümcül konsantrasyonu, kültürlenmis hücrelerin en az %99'unu, tercihen en az %99.9,unu, daha tercihen en az %99.99tunu öldürmek için yeterli olan (yine de bazi mutantlarin bu islemden kaçmasina olanak veren) bir konsantrasyonu ifade etmektedir. In the context of the present invention, the lethality of the composition containing (one or more) ALS inhibitors concentration of at least 99%, preferably at least 99.9% of the cultured cells, more preferably sufficient to kill at least 99.99% (although some mutants denotes a concentration (which allows one to escape the process).
Alternatif olarak veya ilaveten, bu (bir veya daha fazla) ALS inhibitörünün seker pancari bitkisinden izole edilen stomatal koruyucu hücre protoplastlarinin in vitro kültürü için ölümcül olan konsantrasyonun çikarimda bulunulmasi adimi (ayrica) mutasyona ugramis stomatal koruyucu hücrelerin in vitro kültüründe (ALS geninde bir mutasyon kazanmis olan ve ALS inhibitörlerine karsi dirençli olan hücrelerde) gerçeklestirilmektedir. Alternatively or additionally, this (one or more) ALS inhibitors for in vitro culture of stomatal guard cell protoplasts isolated from the step of inferring the concentration that is lethal (also) mutates ugramis stomatal guard cells (a mutation in the ALS gene) in cells that have acquired and are resistant to ALS inhibitors) is carried out.
ALS inhibitörünün ölümcül konsantrasyonunun (bu ALS inhibitörünü içeren bir bilesimde) saf ve mutasyona ugramis hücreler üzerinde karsilastirmasi avantajli bir biçimde bir oran olarak (veya test edilen ALS inhibitörü basina bir oran olmak üzere birkaç oran olarak) ifade edilmistir. The lethal concentration of the ALS inhibitor (that contains an ALS inhibitor) composition) on naïve and mutated cells. as a ratio (or a ratio per ALS inhibitor tested) expressed in several ratios).
Tercihen, bir ALS inhibitörü için, saf hücrelerde ölümcül konsantrasyon mutasyona ugramamis hücrelere göre 50 kat daha düsüktür, daha tercihen saf hücrelerde ölümcül konsantrasyon mutasyona ugramis hücrelere göre 200 kat daha düsüktür, yine daha tercihen saf hücrelerde ölümcül konsantrasyon mutasyona ugramis hücrelere göre 1000 kat daha düsüktür. Preferably, for an ALS inhibitor, the lethal concentration in naïve cells is mutated. 50 times lower than in intact cells, more preferably lethal in naïve cells concentration is 200 times lower than in mutated cells, again preferably 1000 lethal concentration in naïve cells than in mutated cells times lower.
(Mevcut bulusun yönteminde kullanilan) herbisit, foramsülfuron gibi en az bir ALS inhibitörü içeren (inhibitörlerin) bir karisim olabilir. At least one ALS such as herbicide (used in the method of the present invention), foramsulfuron It may be a mixture of inhibitor(s).
Muhtemelen, bulusun yönteminde kullanilan ALS inhibitörü sülfonilüre (örn. foramsülfuron) ve iyodosülfuron, amidosülfuron ve tienkarbazon-metilden olusan gruptan seçilen bir baska ALS inhibitörü gibi ALS inhibitörlerinin bir karisimidir. Presumably, the ALS inhibitor used in the method of the invention is sulfonylurea (eg. foramsulfuron) and iodosulfuron, amidosulfuron and thiencarbazone-methyl It is a mixture of ALS inhibitors such as another ALS inhibitor selected from the group.
Tercihen, bulusun yönteminde kullanilan ALS inhibitörü, foramsülfurondur (veya bunu içermektedir), örnegin aljinat içeren bir ortamda protoplastlarin bir haftalik (veya üç haftalik) in vitro kültürüne (daha tercihen bu kültürlenmis protoplastlardan rejenere konsantrasyonda hücrelerin in vitro kültürü sirasinda muhafaza edilen foramsülfurondur (veya bunu içermektedir). Preferably, the ALS inhibitor used in the method of the invention is foramsulfuron (or contains, for example, protoplasts in a medium containing alginate for one week (or three weekly) to in vitro culture (more preferably regenerated from these cultured protoplasts) is the foramsulfuron that is retained during in vitro culture of cells at a concentration (or includes it).
Mevcut bulusun ilgili bir yönü, (örnegin bu yöntem bir veya daha fazla herbisit (ALS inhibitörü degil) kullanimini veya bir veya daha fazla ALS inhibitörünün kullanimini içerdiginde ya da bir ALS inhibitörünün ve bir ALS inhibitörü olmayan bir herbisitin kullanimini içerdiginde) bu yöntemle elde edilebilen mutasyona ugramis bir seker pancari bitkisidir. A related aspect of the present invention (for example, this method is using one or more herbicides (ALS) inhibitor) or use of one or more ALS inhibitors or containing an ALS inhibitor and an herbicide that is not an ALS inhibitor. a mutated sugar obtainable by this method beet plant.
Fenilalanin 573, Serin 648 ve Glisin 649'den olusan gruptan seçilen amino asitleri kodlayan pozisyonlarda ALS geninde bir veya birkaç mutasyona sahip (mevcut bulusun yöntemi ile elde edilebilen) bir seker pancaridir. Amino acids selected from the group consisting of Phenylalanine 573, Serine 648 and Glycine 649 have one or more mutations in the ALS gene at coding positions It is a sugar beet that can be obtained by the method of
Tercih edilen (mevcut bulusun yöntemi ile elde edilebilen) seker pancari Alanin 113 (örn. Valin veya Treonin olarak mutasyona ugramistir), Treonin, Arjinin, Lösin, Glutamin veya Alanin olarak mutasyona ugramis olan Prolin 188, Alanin 196 (örn. Valin olarak mutasyona ugramistir), Aspartat 371 (örn. Glutamat olarak mutasyona ugramistir), Arjinin 372 (örn. Histidin olarak mutasyona ugramistir), Glisin olarak mutasyona ugramis Triptofan 569, Serin 648 (örn. Treonin olarak mutasyona ugramistir) ve Glisin 649'dan (örn. ASpartat olarak mutasyona ugramistir) olusan gruptan seçilen amino asitleri kodlayan pozisyonlarda ALS geninde bir veya birkaç mutasyona sahiptir. Preferred sugar beet Alanine 113 (obtainable by the method of the present invention) (e.g. mutated to Valine or Threonine), Threonine, Arginine, Leucine, Glutamine or Proline 188 mutated to Alanine, Alanine 196 (e.g. as Valine mutated), Aspartate 371 (e.g. mutated to Glutamate), Arginine 372 (eg mutated to Histidine) mutated to Glycine ugramis Tryptophan 569, Serine 648 (e.g. mutated to Threonine) and Glycine Amino selected from the group consisting of 649 (eg, mutated to Aspartate) have one or more mutations in the ALS gene at positions encoding acids.
Mevcut bulusun ilgili bir yönü, ALS geninde, kodlanmis ALS enziminde 569. pozisyondaki (Arabidopsis thaliana ALS enziminde 574. pozisyona karsilik gelmektedir) triptofanin bir baska amino asitle (örnegin lösin) sübstitüe edildigi bir mutasyonu ve muhtemelen bir baska (bir veya birkaç) mutasyonu, tercihen ALS geninde bir baska (bir veya birkaç) mutasyonu, örnegin ALS geninde bir baska amino asit sübstitüsyonuna neden olan bir mutasyonu içeren mutasyona ugramis bir seker pancaridir (veya seker pancarindan izole edilmis bir mutasyona ugramis stomatal koruyucu hücre gibi bir mutasyona ugramis seker pancari bitki hücresidir). A related aspect of the present invention is in the ALS gene, encoded by the ALS enzyme 569. (corresponding to position 574 in the Arabidopsis thaliana ALS enzyme) a mutation in which tryptophan is substituted with another amino acid (for example, leucine), and possibly another (one or more) mutations, preferably another (one or more) in the ALS gene or several) mutations to another amino acid substitution, for example, in the ALS gene is a mutated sugar beet (or sugar) containing a mutation that causes such as a mutated stomatal guard cell isolated from beetroot mutated sugar beet plant cell).
Bir baska tercih edilen seker pancari bitkisi 569. pozisyonda Triptofanin Lösin olarak bir 648 ve Glisin 649'dan olusan gruptan seçilen amino asitleri kodlayan pozisyonlarda ALS geninde bir veya birkaç mutasyona sahiptir. Another preferred sugar beet plant is at position 569 as Tryptophan Leucine. ALS at positions encoding amino acids selected from the group consisting of 648 and Glycine 649 have one or more mutations in the gene.
Tercih edilen (mevcut bulusun yöntemi ile elde edilebilen) bir seker pancari 569. pozisyonda Triptofanin Lösin olarak bir mutasyonuna ve Alanin 113 (örn. Valin veya Treonin olarak mutasyona ugramistir), Treonin, Arjinin, Lösin, Glutamin veya Alanin olarak mutasyona ugramis olan Prolin 188, Alanin 196 (örn. Valin olarak mutasyona ugramistir), Aspartat 371 (örn. Glutamat olarak mutasyona ugramistir), Arjinin 372 (örn. A preferred sugar beet (obtainable by the method of the present invention) is 569. At position Tryptophan has a mutation as Leucine and Alanine 113 (e.g. Valine or mutated to Threonine), Threonine, Arginine, Leucine, Glutamine or Alanine Proline 188 mutated to Alanine 196 (e.g. mutated to Valine) mutated to Glutamate), Aspartate 371 (e.g. mutated to Glutamate), Arginine 372 (e.g. mutated to Glutamate).
Histidin olarak mutasyona ugramistir), Serin 648 (örn. Treonin olarak mutasyona ugramistir) ve Glisin 649'dan (örn. Aspartat olarak mutasyona ugramistir) olusan gruptan seçilen amino asitleri kodlayan pozisyonlarda ALS geninde bir veya birkaç mutasyona sahiptir. mutated to histidine), Serin 648 (e.g. mutated to Threonine) mutated to Aspartate) and Glycine 649 (e.g. mutated to Aspartate) one or more of the ALS gene at positions encoding amino acids selected from the group has mutation.
Bu mutasyona ugramis seker pancari bitkisi sülfonilüre (öm. foramsülfuron) gibi bir veya birkaç ALS inhibitörüne ve avantajli bir biçimde tercihen iyodosülfuron, amidosülfuron ve tienkarbazon-metilden olusan gruptan seçilen diger ALS inhibitörlerine karsi dirençlidir. This mutated sugar beet plant contains a substance such as a sulfonylurea (e.g. foramsulfuron) or several ALS inhibitors and advantageously preferably iodosulfuron, amidosulfuron and It is resistant to other ALS inhibitors selected from the group consisting of thiencarbazone-methyl.
Mevcut bulusun ilgili bir yönü, ALS geninde, kodlanmis ALS enziminde 188. pozisyondaki prolinin (Arabi'dopsi's thali'ana ALS enziminde 197. pozisyona karsilik gelmektedir) bir baska amino asitle (mesela serin) sübstitüe edildigi bir mutasyonu içeren bir mutasyona ugramis seker pancari bitkisidir (veya seker pancarindan izole edilen mutasyona ugramis bir stomatal koruyucu hücre gibi bir mutasyona ugramis seker pancari bitki hücresidir). A related aspect of the present invention is in the ALS gene, encoded by the ALS enzyme 188. proline at position 197 (corresponding to position 197 in Arabi'dopsi's thali'ana ALS enzyme) a mutation in which it is substituted with another amino acid (e.g. serine) is a mutated sugar beet plant (or isolated from sugar beet) containing mutated, such as a mutated stomatal guard cell sugar beet plant cell).
Alternatif olarak, (mevcut bulusun yöntemi ile elde edilebilen) tercih edilen bir seker pancari bitkisi 188. pozisyonda Prolinin Serin olarak bir mutasyonuna ve Glisin 112, olusan gruptan seçilen amino asitleri kodlayan pozisyonlarda ALS geninde bir veya birkaç mutasyona sahiptir. Alternatively, a preferred sugar (obtainable by the method of the present invention) The beet plant has a mutation at position 188 of Proline as Serin and Glycine 112, one or more in the ALS gene at positions encoding amino acids selected from the group consisting of It has several mutations.
Tercih edilen bir seker pancari bitkisi (mevcut bulusun yöntemi ile elde edilebilir) ALS geninde 188. pozisyonda Prolinin Serin olarak bir mutasyonuna ve ALS geninde Alanin 113'ün (örn. Valin veya Treonin olarak mutasyona ugramistir), Aspartat 371 (örn. A preferred sugar beet plant (obtainable by the method of the present invention) ALS a mutation of Proline at position 188 in the Serine gene and Alanine in the ALS gene. 113 (e.g. mutated to Valine or Threonine), Aspartate 371 (e.g. threonine).
Glutamat olarak mutasyona ugramistir), Arjinin 372 (örn. Histidin olarak mutasyona ugramistir), Glisin olarak mutasyona ugramis Triptofan 569, Serin 648 (örn. Treonin olarak mutasyona ugramistir) ve Glisin 649iu (örn. Aspartat olarak mutasyona ugramistir) kodlayan pozisyonlarda bir veya birkaç mutasyona sahiptir. mutates to Glutamate), Arginine 372 (e.g. mutates to Histidine) mutated to Glycine, Tryptophan 569, Serine 648 (e.g. Threonine) mutated as Aspartate) and Glycine 649iu (e.g. mutated as Aspartate) ugram) has one or more mutations at the coding positions.
Mevcut bulusun bir baska ilgili yönü, ALS enziminde 569. pozisyonda triptofanin bir mutasyonunu ve ALS enziminde 188. pozisyondaki profilin bir mutasyonunun yani sira muhtemelen bir baska (bir veya birkaç) mutasyonu, tercihen ALS geninde bir baska (bir veya birkaç) mutasyonu içeren mutasyona ugramis bir seker pancari bitkidir (veya seker pancarindan izole edilen mutasyona ugramis bir stomatal koruyucu hücre gibi mutasyona ugramis bir seker pancari bitki hücresidir). Another relevant aspect of the present invention is that tryptophan at position 569 in the ALS enzyme is a mutation and a mutation of the profile at position 188 in the ALS enzyme, as well as possibly another (one or more) mutations, preferably another (one or more) in the ALS gene a mutated sugar beet plant (or sugar beet) containing one or more mutations as a mutated stomatal guard cell isolated from beetroot is a mutated sugar beet plant cell).
Tercihen, bu mutasyona ugramis seker pancari bitkisinin ALS geni (bunun bir alleli) SEKANS TANIM No:3 veya SEKANS TANIM NO:5”e karsilik gelmektedir. Preferably, the ALS gene of this mutated sugar beet plant (an allele of it) It corresponds to SEQ ID NO:3 or SEQ ID NO:5.
Avantajli bir biçimde, mevcut bulusun mutasyona ugramis seker pancari bitkisi SEKANS TANIM No:3 (bir allelde) ve SEKANS TANIM NO:5'i (ikinci allelde) içermektedir. Advantageously, the mutated sugar beet plant of the present invention SEQ ID NO:3 (on one allele) and SEQ ID NO:5 (on the second allele) contains.
Muhtemelen, mevcut bulusun mutasyona ugramis seker pancari bitkisi SEKANS TANIM NO:3'ü (bir allelde) ve ya SEKANS TANIM No:1 ya da SEKANS TANIM NO:7'yi (ikinci allelde) içermektedir. Presumably, the mutated sugar beet plant of the present invention SEQUENCE DESCRIPTION NO:3 (on one allele) or either SEQ ID NO:1 or SEQ ID NO:7 (on the second allele) in the allele).
Mevcut bulusun bir baska ilgili yönü, bir veya daha fazla mutasyonu kapsayan bir nükleotid fragmanidir (en az 20 veya en az 25 ardisik nükleotidden, ancak 200'den az ardisik nükleotidden, tercihen 50'den az ardisik nükleotidden 0lusmaktadir); muhtemelen bu fragman, bir primer veya prob olarak kullanima yöneliktir (örnegin nükleotidik olmayan bir parça ile ya da radyoaktivite kullanilarak ya da seker pancarinin ALS genine yabanci olan bir nükleik asit sekansi ile etiketlenmis bir probla ayrica etiketlenmis bir nükleotid prob içermektedir). Another relevant aspect of the present invention is a method involving one or more mutations. is a nucleotide fragment (of at least 20 or at least 25 consecutive nucleotides, but less than 200 consecutive nucleotides, preferably less than 50 consecutive nucleotides); possibly this fragment is intended for use as a primer or probe (e.g. with a non-nucleotidic moiety or using radioactivity or A probe labeled with a nucleic acid sequence foreign to the ALS gene also contains a labeled nucleotide probe).
Mevcut açiklamanin bir baska ilgili yönü, toksik moleküle (herbisit) karsi direnci olan seker pancari bitkilerinin marker destekli seçimine yönelik mutasyonu kapsayan bu nükleotid fragmaninin kullanimidir. Örnekler Karsilastirmali örnek ALS herbisitinin yabani tip seker pancari eksplantlari olan kalluslara eklenmesi sonrasinda mutasyona ugramis seker pancari alanda basarili bir sekilde üretildiginden elde edilen seker pancari genotipini (hattini) seçmis ve bunlarin stomatal koruyucu hücrelerinden protoplastlari izole etmistir. Another relevant aspect of the present disclosure is resistance to toxic molecule (herbicide). involving mutation for marker-assisted selection of sugar beet plants. use of the nucleotide fragment. Examples comparative example Addition of ALS herbicide to calli with wild-type sugar beet explants Since the mutated sugar beet was successfully produced in the field after selected the sugar beet genotype (line) obtained and their stomatal protective isolated protoplasts from cells.
Bu protoplastlarin birkaç milyonu (deneysel olarak ortalama yaklasik 2 ila yaklasik 5 milyon ve 11 milyona kadar; toplamda ALS herbisiti yaklasik 150 milyon protoplasta uygulanmistir) WO 95/10178 sayili belgedeki gibi izole edilmis, aljinat içeren kültür ortamina yerlestirilmis ve 10'9 ila 10'6 mol/l foramsülfuron içeren MS kültür ortami ile islemden geçirilmistir. Several million of these protoplasts (experimentally average about 2 to about 5 million and up to 11 million; In total, ALS herbicide is applied to approximately 150 million protoplasts. isolated, alginate-containing culture as in WO 95/10178 with MS culture medium containing 10'9 to 10'6 mol/l foramsulfuron has not been processed.
Bulus sahipleri daha sonra, WO 95/10178 sayili belgede açiklanan protokolü izleyerek seker pancarini rejenere etmis ve yalnizca birkaç kallusun ALS inhibitörü karsisinda sag kaldigini gözlemlemistir. Ancak, bir istisna olarak, bu rejenere edilmis kalluslarin hiçbiri seker pancari bitkisi olarak gelisememistir. Rejenere edilen tek seker pancari bitkisi ALS geninde (foramsülfuron hedef enzimini kodlayan) mutasyon göstermemistir. The inventors then follow the protocol described in WO 95/10178. It regenerated the sugar beet and only a few calluses survived against the ALS inhibitor. observed it. However, as an exception, none of these regenerated calli It has not developed as a sugar beet plant. The only regenerated sugar beet plant ALS gene (encoding foramsulfuron target enzyme) did not show mutations.
Bu nedenle, parental hattinin, (bir eksplant kökenli) kalluslarin bir herbisite dogrudan maruz birakilmasi temelinde, bu herbisite karsi mutasyondan kaynakli direnç kazandigi gösterilmis olan bu seker pancari hatti, bu yöntem stomatal koruyucu hücre protoplastlarini kapsadigi zaman ayni amaç için faydali olmamistir. Örnek 1 Iyi rejenere olan protoplastlar için seker pancari genotiplerinin (hatlarinin) seçilmesi Bulus sahipleri daha sonra, stomatal koruyucu hücre protoplastlarindan rejenerasyon kapasiteleri bakimindan birkaç seker pancari bitki genotipini karsilastirmistir. Therefore, the parental line of calli (from an explant) can be directly applied to an herbicide. mutation-induced resistance to this herbicide based on exposure to this sugar beet line shown, this method stomatal guard cell it was not useful for the same purpose when it covered the protoplasts. Example 1 Determination of sugar beet genotypes for well regenerating protoplasts. selection of (lines) The inventors then regenerate from stomatal guard cell protoplasts. compared several sugar beet plant genotypes in terms of capacity.
Bulus sahipleri, yaklasik %001 (ve hatta daha az) rejenere olma kapasitesine sahip genotipler ve %0.1 'den fazla rejenere olma kapasitesine sahip birkaç genotip bulmustur. Inventors have the capacity to regenerate approximately 001% (or even less). genotypes and found several genotypes with greater than 0.1% regenerative capacity.
Bulus sahipleri ayrica, protoplastlarin büyümesi (bunlarin büyüme ve in vitro bölünme kapasiteleri), filiz olusturmak üzere kalluslarin büyüme kapasitesi ve bir bitkiyi rejenere etmek üzere büyüyen kalluslarin orani arasinda bir ayrim olusturmustur. The inventors also noted that the growth of protoplasts (their growth and division in vitro) capacities), the growth capacity of calluses to form shoots, and the ability to regenerate a plant. A distinction has been made between the proportion of calluses growing to
Tablo 1: bazi seker pancari genotiplerinin karsilastirilmasi Genotip protoplastlarlgram Büyüyen Filiz Elde edilen hücrelerin olusturma bitkilerin yüzdesi yüzdesi yüzdesi Bir bütün olarak alindiginda stomatal koruyucu hücre protoplastinin tam bir seker pancari bitkisi olarak rejenere olma kapasitesini yansitan degerler “Rel1” hücre hatti için daha yüksek olmasina ragmen, bu hücre hattinin mevcut bulusun yürütülmesi için yeter ölçüde faydali olmadigi düsünülmüstür. Table 1: Comparison of some sugar beet genotypes Genotype Protoplasts Obtained from Growing Sprout forming cells of plants percent percent percent Taken as a whole, stomatal guard cell protoplasty is a complete sugar Values reflecting its capacity to regenerate as a beet plant are for the “Rel1” cell line. Although higher, this cell line is sufficient to carry out the present invention. It is not considered to be beneficial to any extent.
Bulus sahipleri ayrica "büyüyen hücre yüzdesi” parametresinin mevcut bulusu yürütmek için diger parametrelerden çok daha önemli oldugu sonucuna varmistir. Inventors can also use the "percentage of growing cells" parameter to carry out the current invention. concluded that it is much more important than other parameters for
Bulus sahipleri in vitro büyüyebilen stomatal koruyucu hücre protoplastlarinin Örnek 2 Protoplastlarin herbisit islemi Bulus sahipleri karsilastirmali Örnek'teki ile ayni yaklasimi uygulamis, ancak iyi büyüyen stomatal koruyucu hücre protoplastlarini temel almistir (örnegin Örnek 1'de büyüyen stomatal koruyucu hücre protoplastlarinin yüksek bir oranina sahip diger seker pancari bitkileri de kullanilabilir). The inventors of stomatal guard cell protoplasts that can grow in vitro Example 2 Herbicide treatment of protoplasts The inventors applied the same approach as in the Comparative Example, but with good based on growing stomatal guard cell protoplasts (for example, in Example 1 other sugar with a high proportion of growing stomatal guard cell protoplasts beet plants can also be used).
Toplamda, yaklasik 68 milyon iyi büyüyen stomatal koruyucu hücre protoplasti 10'6M'ye kadar foramsülfuron içeren bir ALS herbisit bilesimi ile islemden geçirilmistir. In total, approximately 68 million well-growing stomatal guard cell protoplasty reaches 10'6M. treated with an ALS herbicide composition containing as much as foramsulfuron.
Bulus sahipleri 46 kallus elde etmistir. The inventors obtained 46 calluses.
Birkaç rejenere edilmis bitki hedef gen olan ALS geninde bir mutasyon göstermektedir: her durumda 569. pozisyonda triptofana yönelik kodonda bir mutasyon (W569L; Arabio'opsis thaiiana'da 574. pozisyondaki triptofana karsilik gelmektedir). Bu mutantin ALS genlerindeki iki alIeI SEKANS TANlM No:3 ve SEKANS TANlM No:7 ile kodlanmaktadir. Diger büyüyen kalluslar sekanslanmistir ve ALS geninde mutasyonlara sahiptir (diger pozisyonlarda mutasyonlar da dahil) ancak bitki olarak rejenere olamamistir. Several regenerated plants show a mutation in the target gene, the ALS gene: in each case a mutation in the codon for tryptophan at position 569 (W569L; It corresponds to tryptophan at position 574 in Arabio'opsis thaiana). This mutant Two genes in ALS genes with SEQ ID NO: 3 and SEQ ID NO: 7 is coded. Other growing calli have been sequenced and are susceptible to mutations in the ALS gene. (including mutations at other positions) but regenerates as a plant could not.
Bulus sahipleri böylece mevcut bulusun yönteminin, özellikle bu yöntem yabanci DNA kullanimini kapsamadigi ve/veya genetik elemanlari kodlayan DNA vektörlerinin takdiminin ALS inhibitörlerine karsi direnç kazandirdiginin zaten bilindigi ve yalnizca birkaç ay içinde pozitif sonuçlar verdigi için bir herbisite karsi dirence neden olan gelistirilmis mutasyonlari olan bitkiler gelistirmek için çok faydali oldugu sonucuna varmistir. The inventors thus consider that the method of the present invention, in particular this method DNA vectors encoding genetic elements and/or presentation is already known to confer resistance to ALS inhibitors, and only that cause resistance to an herbicide because it produces positive results within a few months. concluded that it is very useful for developing plants with enhanced mutations. has arrived.
Bulus sahipleri daha sonra bu yöntemi tekrarlamis ve mutasyonlarin sayisini artirmak için protoplastlara bir mutajen (% uygulamistir. Örnek 3 Seker pancarlarinin ALS inhibitör islemi Bulus sahipleri. mutasyona ugramis SEKANS TANIM NO:3'e (bu mutasyon için heterozigot) ve yabani tip (saf) seker pancari ticari türüne sahip rejenere seker pancarlarinin davranisini karsilastirmistir. The inventors then repeated this method and increased the number of mutations. applied a mutagen (%) to protoplasts. Example 3 ALS inhibitory treatment of sugar beets Invention owners. mutated to SEQ ID NO:3 (for this mutation heterozygous) and wild type (pure) sugar beet commercial type regenerated sugar compared the behavior of beets.
(Heterozigot) mutasyona ugramis tür, etkisini güçlendirmek için herbisit bir organik bilesikle (25 g/sa kolza tohumu metil ester) birlestirildiginde dahi Foramsülfurona (12.5 g/sa; 3 uygulamaya kadar) iyi direnç göstermistir. The (heterozygous) mutated strain is a herbicide organic to potentiate its effect. Foramsulfuron (12.5), even when combined with the compound (25 g/h rapeseed methyl ester) g/h; up to 3 applications) showed good resistance.
Beklendigi gibi, yabani tip (saf) bitki birinci uygulamadan sonra dahi Foramsülfurona karsi çok hassas olmustur. As expected, the wild-type (pure) herb showed Foramsulfuron even after the first application. has been very sensitive.
Ayni deney amidosülfuron (15 g Isa) kullanilarak gerçeklestirilmis ve mutasyona ugramis bitkilerde ayni direnç seviyesini vermistir. The same experiment was performed using amidosulfuron (15 g Isa) and mutated It gave the same resistance level in the affected plants.
Diger taraftan, yabani tip (saf) bitkiler özellikle organik bilesikle birlestirildiginde ve/veya birkaç amidosülfuron uygulamasindan sonra amidosülfurona karsi çok hassas olmustur. On the other hand, wild type (pure) plants especially when combined with organic compound and/or became very sensitive to amidosulfuron after several administrations of amidosulfuron.
Ayni deney iyodosülfuron (3.5 g/sa) kullanilarak gerçeklestirilmis ve iyodosülfuron eklendiginde mutasyona ugramis bitkilerde iyi bir direnç seviyesi göstermistir, ancak bu direnç iyodosülfuron organik bilesikle birlikte uygulandiginda azalmistir. The same experiment was carried out using iodosulfuron (3.5 g/h) and iodosulfuron showed a good level of resistance in mutated plants when added The resistance was reduced when iodosulfuron was administered with the organic compound.
Beklendigi gibi, yabani tip (saf) bitki bir uygulamadan sonra dahi ve organik bilesik olmadan iyodosülfurona karsi çok hassas olmustur. As expected, even after one application of wild-type (pure) plant and organic compound It has been very sensitive to iodosulfuron without
Ayni deney 7.5 g/sa tienkarbazon-metil kullanilarak gerçeklestirilmis ve mutasyona ugramis bitkilerde iyodosülfuron için olanla yaklasik ayni direnç seviyesini vermistir. The same experiment was carried out using 7.5 g/h thiencarbazone-methyl and the mutation It gave approximately the same level of resistance as for iodosulfuron in cultivated plants.
Yabani tip (saf) bitki test edilen konsantrasyonlarda ve organik bilesik eklemesine bakilmaksizin tienkarbazon-metile karsi çok hassas olmustur. The wild-type (pure) plant was allowed at the tested concentrations and the addition of organic compounds. was very sensitive to thiencarbazone-methyl regardless of
Bulus sahipleri, yabani tiple karsilastirma yoluyla, SEKANS TANlM NO:3'ü (Budapeste Anlasmasi NCIMB 42051 kapsaminda saklanmistir) içeren mutasyona ugramis seker pancari bitkisinin foramsülfurona karsi en iyi direnci sundugu sonucuna varmistir. The inventors obtained SEQ ID NO: 3 (Budapest) by comparison with wild type. mutated sugar containing (reserved under NCIMB 42051) concluded that the beet plant offered the best resistance to foramsulfuron.
Bulus sahipleri ayrica, bu (heterozigot) mutasyona ugramis bitkinin ayrica diger kimyasal siniflara ait olan inhibitörlere karsi direnç dahil olmak üzere diger ALS inhibitörlerine karsi (kismen olsa da) biraz direnç kazanmis oldugu sonucuna varmistir. Örnek 4 ALS geninde diger mutasyonlara sahip seker pancarlarinin ALS inhibitör Bulus sahipleri daha sonra SEKANS TANlM No:3 ve SEKANS TANIM NO:5'i (iki farkli allelde) içeren mutasyona ugramis bir seker pancari bitkisi gelistirmistir. Ortaya çikan bu ikili mutant Budapeste Anlasmasi kapsaminda NClMB 42050 altinda saklanmistir. Hem SEKANS TANlM No:3 hem de SEKANS TANlM NO:5'i içeren bir bitki örnegin tek mutant NCIMB 42051'e uygulanan bir müteakip mutajenez adimini içeren birkaç teknige dayali olarak üretilebilmektedir. The inventors also noted that this (heterozygous) mutated plant also other ALS, including resistance to inhibitors belonging to chemical classes concluded that it had acquired some (albeit partial) resistance to its inhibitors. Example 4 ALS inhibitory activity of sugar beets with other mutations in the ALS gene. The inventors then issued SEQ ID NO: 3 and SEQ ID NO: 5 (two different developed a mutated sugar beet plant containing This is what turns out The double mutant is stored under NClMB 42050 under the Budapest Agreement. both For example, a plant containing SEQ ID NO:3 and SEQ ID NO:5 several techniques including a subsequent mutagenesis step applied to mutant NCIMB 42051. can be produced based on
Bulus sahipleri daha sonra bu ikili mutant bitkinin (amino asit 569'da bir allelde bir mutasyon ve amino asit 188'de diger allelde bir mutasyon) direncini tekli mutant (569. pozisyonda bir mutasyon) seker pancari ile karsilastirmistir. The inventors later developed this double mutant plant (an allele in amino acid 569). mutation and a mutation in the other allele at amino acid 188) resistance to a single mutant (569. a mutation at position) with sugar beet.
Ikili mutant bitki hatti en azindan, Örnek 3'teki direnç özelliklerinin tümünü korumustur ve ayrica organik bilesiklerle bilesim haline getirildiginde bile tienkarbazon-metile karsi ve amidosülfuron islemlerine karsi iyi bir direnç (tarla uygulamasi ile uyumlu) kazanmistir. The double mutant plant line retained at least all of the resistance traits in Example 3 and also against thiencarbazone-methyl even when compounded with organic compounds and good resistance to amidosulfuron treatments (compatible with field application) has won.
Bu nedenle, bu ikili mutant bitki tekli mutant bitkisine (ALS geninde 569. pozisyonda) atfedilen dirençle kiyaslandiginda birkaç ALS inhibitörüne karsi gelistirilmis, sinerjik direnç göstermektedir. Örnek 5 Sera çalismalari: Dogrudan karsilastirmada farkli seker pancarlarinin ALS inhibitör islemi Mevcut bulusa göre (yukarida Örnek 4'te açiklandigi gibi, “Hat A”) SEKANS TANIM No:3 ve SEKANS TANIM NO:5'i (iki farkli allelde) içeren mutasyona ugramis seker pancari bitkileri, kodlanmis ALS enzimindeki 569. pozisyonunda triptofanin bir lösinle sübstitüe edildigi seker pancari bitkileri (“Hat B"), kodlanmis ALS enzimindeki 188. pozisyondaki prolinin bir serinle sübstitüe edildigi WO 98/02527”de açiklanan seker pancari bitkileri (“Hat C”) ve 569 ve 188 pozisyonlarda bir mutasyona sahip olmayan geleneksel tür (yabani tip) seker pancari bitkileri (“Hat WT") ile dogrudan karsilastirmada farkli ALS inhibitörleri ile islemden geçirilmistir. Therefore, this double mutant plant is referred to as a single mutant plant (at position 569 in the ALS gene). Developed, synergistic, against several ALS inhibitors compared with the implied resistance shows resistance. Example 5 Greenhouse studies: ALS of different sugar beets in direct comparison inhibitor process SEQUENCE DESCRIPTION According to the present invention (“Line A” as described in Example 4 above) Mutated sugar containing No:3 and SEQ ID NO:5 (in two different alleles) beet plants, tryptophan at position 569 in the encoded ALS enzyme with a leucine sugar beet plants (“Line B”) to which it is substituted, encoded in the ALS enzyme 188. sugar as described in WO 98/02527, in which proline at position 1 is substituted by a serine beet plants (“Line C”) and do not have a mutation at positions 569 and 188 directly with traditional type (wild type) sugar beet plants (“Hat WT”) treated with different ALS inhibitors for comparison.
Bahsedilmis olan dört farkli seker pancari bitkisinin tohumlarinin birkaç grubu serada ayri ayri dikilmistir ve monographie "Entwicklungsstadien mono- und dikotyler Pflanzen", 2nd edition, 2001, ed. Uwe Meier, Biologische Bundesanstalt für Land und Forstwirtschaft kaynagina göre Beta vulgari's L. ssp. Vulgaris (örn. 4 yaprak (ikinci çift) katlanmamis) için BBCH 14 evresine kadar büyütülmüstür. Akabinde, seker pancari bitkilerinin elde edilen ayri gruplarinin her biri tek tek Tablo 2'de belirtilen miktarlarda (g/sa) bir ALS inhibitörü (ALS-in) ile islemden geçirilmistir. Several batches of seeds of the four different sugar beet plants mentioned were in the greenhouse. individually sewn and monographie "Entwicklungsstadien mono- and dicots Pflanzen", 2nd edition, 2001, ed. Uwe Meier, Biologische Bundesanstalt für Land und According to Forstwirtschaft source Beta vulgari's L. ssp. Vulgaris (eg 4 leaves (second pair) unfolded) was magnified to stage BBCH 14. Subsequently, sugar beet Each of the separate groups of plants obtained individually in the amounts indicated in Table 2. (g/h) was treated with an ALS inhibitor (ALS-in).
Ilgili ALS inhibitörünün uygulanmasindan 14 gün sonra, her seker pancari bitkisi için hasar (yani fitotoksisite) %0 (yani hasar yok, fitotoksisite yok) ila %100 (yani bitkiler tamamen ölmüstür) skalasinda puanlanmistir. puanlama Tablo 2'de gösterilmistir. 14 days after application of the respective ALS inhibitor, for each sugar beet plant damage (ie phytotoxicity) 0% (ie no damage, no phytotoxicity) to 100% (ie plants completely dead) scale. scoring is shown in Table 2.
Foramsülfu ron Iyodosülfuron-metil-Na Amidosülfuron Tienkarbazon-metil Bisbyribac-Na Metosulam ALS-in g/sa Hat A Bitkilerin her grubu için ortalama ilaveten, her seker pancari bitkisi için tipik erken fenotip tienkarbazon-metil ve foramsülfuron içeren bir karisimla islemden geçirildikten sonra incelenmistir. Her Hat için temsili bir erken fenotip Sekil 1'de gösterilmistir (Sekil 1). foramsulfuron Iodosulfuron-methyl-Na Amidosulfuron Thiencarbazone-methyl Bisbyribac-Na my method ALS-in g/h Line A Average for each group of plants In addition, the typical early phenotype for each sugar beet plant is thiencarbazone-methyl and It was studied after treatment with a mixture containing foramsulfuron. Each Line A representative early phenotype is shown in Figure 1 (Figure 1).
Sekil 1 ayrica, mevcut bulusa göre seker pancari bitkilerinin (“Hat A") gelistirilmis ALS inhibitör direnci gösterdigini ortaya koymaktadir, yani diger erken fenotiplere kiyasla üstün büyüme ve daha az fitotoksisite etkileri gözlemlenmistir. Örnek 6 Tarla çalismalari: Dogrudan karsilastirmada farkli seker pancarlarinin ALS inhibitör islemi Tablo 3: ALS inhibitörlerinin seker pancari bitkisine etkisi. Degerler, ölçülen hasarin ortalama yüzdesini temsil etmektedir. Figure 1 also shows the improved ALS of sugar beet plants (“Line A”) according to the present invention. It demonstrates inhibitory resistance, that is, compared to other early phenotypes. Superior growth and less phytotoxic effects were observed. Example 6 Fieldwork: Different sugar beets in direct comparison ALS inhibitor process Table 3: Effect of ALS inhibitors on sugar beet plant. The values are the measured damage represents the average percentage.
ISLEMDEN GEÇIRILMEMIS 2Sg/HA (Foramsülfuron) (Tienkarbazon) (iyodosülfuron) (mesosülfuron) HOESTAR 30g/Ha (amidosülfuron) (etoksisülfuron) RAPTOR 40 g/Ha (imazamoks) TACCO SOg/Ha (metosulam) NOMINEE 50g/Ha (bispyribac) MOTlVELL 60 g/Ha (nikosülfuron) CROPPER SX 8 g/Ha (metosülfuron) 574 ve 574 197 homo LEXUS 50 DF10g/Ha 70 (flupirsülfuron) ATTRIBUT 70 g/Ha 91 (propoksikarabazon) SlMPLlClTY 509/Ha (pirksisulam) 97 PRlMUS 10 nga (florasulam) 99 POlNTER SX 309/Ha (tribenuron) 98 CATO 13 g/Ha (rimsülfuron) 68 MONITOR 80 WG 109/Ha 93 (sülfosülfuron) DEBUT YX115 g/Ha 0 (triflusülfuron) EVEREST 40g/Ha (flukarbazon) 93 HARMONY 7.5 nga (tiensülfuron) 98 574 ve 574 197 homo Bulus sahipleri, ticari bilesimleri yabani otlarin yok edilmesine olanak veren dozda test etmistir. NOT PROCESSED 2Sg/HA (Foramsulfuron) (Tiencarbazone) (iodosulfuron) (mesosulfuron) HOESTAR 30g/Ha (amidosulfuron) (ethoxysulfuron) RAPTOR 40 g/Ha (imazamox) TACCO SOg/Ha (metosulam) NOMINEE 50g/Ha (bispyribac) MOTIVELL 60 g/Ha (nicosulfuron) CROPPER SX 8 g/Ha (methosulfuron) 574 and 574 197 homos LEXUS 50 DF10g/Ha 70 (flupirsulfuron) ATTRIBUT 70 g/Ha 91 (propoxycarbazone) SlMPLlClTY 509/Ha (pirxisulam) 97 PRlMUS 10 nga (florasulam) 99 POLNTER SX 309/Ha (tribenuron) 98 CATO 13 g/Ha (rimsulfuron) 68 MONITOR 80 WG 109/Ha 93 (sulfosulfuron) DEBUT YX115 g/Ha 0 (triflusulfuron) EVEREST 40g/Ha (flucarbazone) 93 HARMONY 7.5 nga (thiensulfuron) 98 574 and 574 197 homos The inventors test commercial compositions at a dose that allows weed eradication. has done.
Hassas kontrol (yani ALS geninde mutasyonu olmayan seker pancari) biri hariç tüm herbisitler tarafindan öldürülmüstür. Bulus sahipleri kontrol (islem görmemis) bitkisine az hasar ölçmüs olup, bu bazi durumlarda %35'e ve hatta %40'a varmaktadir. Bu Diger taraftan, 569. pozisyonda (574) heterozigot olan seker pancari bitkisi birkaç herbisit bilesimine karsi kismen dirençli hale gelmistir. Her iki allelde 569 mutasyonunu (574) içeren ve dolayisiyla (569/569) homozigot olan bitki daha da artirilmis bir dirence sahiptir: yalnizca 7 herbisit bilesimi orta derecede toksiktir (%5'ten %35'e kadar). Precision control (ie sugar beet without mutation in the ALS gene) all but one killed by herbicides. The inventors have little access to the control (untreated) plant. damage has been measured, which in some cases reaches 35% or even 40%. This On the other hand, the sugar beet plant heterozygous at position 569 (574) has several It has become partially resistant to herbicide composition. 569 mutation in both alleles The plant containing (574) and thus (569/569) homozygous has a further increased resistance. has: only 7 herbicide compositions are moderately toxic (from 5% to 35%).
ALS geninin bir allelinde 569. (574) pozisyonda mutasyonu ve ALS geninin ikinci allelinde 188. (197) pozisyonda bir mutasyonu içeren bir seker pancari bitkisi de gelistirilmis direnç kazanmistir, çünkü 9 herbisit bilesimi orta derecede toksiktir ve yalnizca 3'ü oldukça toksiktir. Sasirtici bir biçimde, ALS inhibitörüne karsi güçlü direnç (569) saglayan bir mutasyonun kayboldugu ve yalnizca zayif direnç (188) saglayan bir mutasyonun eklendigi böyle bir bitki bu tarla testinin 3 farkli kosulunda homozigot (5691569) bitkiye göre daha da iyi direnç saglamaktadir. Mutation at position 569 (574) in one allele of the ALS gene and a second mutation of the ALS gene A sugar beet plant also contains a mutation at position 188 (197) in its allele. It has gained enhanced resistance because the 9 herbicide compositions are moderately toxic and only 3 are highly toxic. Surprisingly, strong resistance to ALS inhibitor (569), a mutation that confers only weak resistance (188) is lost and such a plant to which the mutation has been added is homozygous in 3 different conditions of this field test. (5691569) provides even better resistance than the plant.
Bilgisayar Çiktisi (Elektronik Form olarak Orijinal) (Bu belge uluslararasi basvuru belgesinin bir parçasi degildir ve uluslararasi basvuru belgesi olarak sayilmamaktadir) 0-1 Saklanan Mikroorganizma(lar) veya Diger Biyolojik Madde ile ilgili Form PCT/ROM 34 (SAFE) Bildirimleri (PCT Yönetmeligi mükerrer M. 13) 0-1-1 Sunlarla hazirlanmistir PCT Çevrimiçi Basvuru Versiyon 35.000.235 MTIFOP 1-3-1 1-3-2 1-3-3 1-3-4 Uluslararasi Basvuru No. dosya referansi Asagidaki bildirimler, saklanan mikroorganizma(lar) veya tarifnamede asagidaki yerlerde anilan diger biyolojik maddeler ile Paragraf numarasi Saklanan ürünün tanimlamasi Saklayan kurumun adi Saklayan kurumun adresi Saklama tarihi Erisim Numarasi Ek Bildirimler Bildirimlerin Yapildigi Tanimlanan BPSESSOO1OPC NCIMBNCIMB Ltd. Computer Printout (Original in Electronic Form) (This document is for international reference It is not part of the document and is used as an international application document. not counted) 0-1 Contained Microorganism(s) or Related to Other Biological Substance Form PCT/ROM 34 (SAFE) Notices (PCT Regulation repeated M. 13) 0-1-1 Prepared with PCT Online Application Version 35,000,235 MTIFOP 1-3-1 1-3-2 1-3-3 1-3-4 International Application No. file reference The following notifications are stored microorganism(s) or in the following places in the description with other biological substances mentioned paragraph number Description of the stored product Name of the holding institution The address of the depositing institution retention date Access Number Additional Notices Identified That Notifications Are Made BPSESSOO1OPC NCIMBNCIMB Ltd.
Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB21 9YA, Birlesik Krallik Tüm tanimlamalar 2-3-1 2-3-2 2-3-3 2-3-4 SADECE KABUL OFISI KULLANIMI IÇINDIR 0-4 Bu form, uluslararasi Asagidaki bildirimler, saklanan mikroorganizma(lar) tarifnamede asagidaki yerlerde anilan diger biyolojik maddeler ile Paragraf numarasi Saklanan ürünün tanimlamasi Saklayan kurumun adi Saklayan kurumun adresi Saklama tarihi Erisim Numarasi Ek Bildirimler NCIMBNCIMB Ltd. Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB21 9YA, United Kingdom All descriptions 2-3-1 2-3-2 2-3-3 2-3-4 FOR ACCEPTANCE OFFICE USE ONLY 0-4 This form is international The following notifications are stored microorganism(s) in the following places in the description with other biological substances mentioned paragraph number Description of the stored product Name of the holding institution The address of the depositing institution retention date Access Number Additional Notices NCIMBNCIMB Ltd.
Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB21 9YA, Birlesik Krallik Bildirimlerin Yapildigi Tanimlanan Tüm tanimlamalar alinmistir: (evet veya hayir) basvuru ile birlikte 0-4- Yetkili memur lsabelle 1 Aoustin YALNIZCA ULUSLARARASI OFISIN KULLANIMI IÇINDIR 0-5 Bu form, uluslararasi Ofis tarafindan alinmistir: 0-5-1 Yetkili memur SEKANS LISTESI <110> SES <130> SESSOO1O <160> 8 <170> Patentln version 3.5 <210>1 <211> 1995 <212> DNA <213> Beta vulgaris <220> <221> CDS <222>(1yr1995) <223> 4D6834 WT all <400> 1 <210>2 <211>664 <212> PRT <213> Beta vulgaris <400> 2 (3111 1.311 371:- (3111 <210> 3 <211> 1995 <212> DNA <213> Beta vulgaris <220> <221› misc_feature <222> (1)..(1994) A511. <220> <221> CDS <222>(1y41995) <400> 3 <210>4 <211> 664 <212> PRT <213> Beta vulgaris <400> 4 Met Aia Ala Thr Phe Thr Asn Pro Thr ?ne Ser Pro Ser Ser Thr Pro Leu Thr Lya Thr Leu Lys Ser Gln Ser Ser Ile Ser Ser Thr Leu Pro <210> 5 <211> 1995 <212> DNA <213> Beta vulgaris <220> <221 misc_feature <222> (1)..(1994) <223> Pro Mutant <220> <221CDS <222>(1y41995) <400> 5 13519 <210>6 <211>664 <212> PRT <213> Beta vulgaris <400> 6 Arg Vai Ser Asn Vai Ala Asp Leu Azg Ala Ala Iie 610 615 Asp Thr Pro Giy Pro Tyr Lou Lou Asp Va] Ile Val 625 630 His Val Leu Pro Met :Je Pro Ser Gly Ala Gly Phe 645 650 Thr Glu Gly Asp Gly Arg Thr Ser <210> 7 <211> 1995 <212> DNA <213> Beta vulgaris <220> <221> misc_feature <222> (1)..(1994) <223> 4D6834 al2 WT <220> <221> CDS <222> (1)..(1995) <400> 7Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB21 9YA, United Kingdom All Identified Definitions Where Notifications Are Made taken: (Yes or no) application with 0-4- Authorized officer Isabelle 1 Austin FOR INTERNATIONAL OFFICE USE ONLY 0-5 This form has been received by the International Office: 0-5-1 Authorized officer SEQUENCE LIST <110> SOUND <130> SESSOO1O <160> 8 <170> Patented version 3.5 <210>1 <211> 1995 <212> DNA <213> Beta vulgaris <220> <221> CDS <222>(1yr1995) <223> 4D6834 WT all <400> 1 <210>2 <211>664 <212> PRT <213> Beta vulgaris <400> 2 (3111) 1,311 371:- (3111) <210> 3 <211> 1995 <212> DNA <213> Beta vulgaris <220> <221› misc_feature <222> (1)..(1994) A511. <220> <221> CDS <222>(1y41995) <400> 3 <210>4 <211> 664 <212> PRT <213> Beta vulgaris <400> 4 Met Aia Ala Thr Phe Thr Asn Pro Thr ?ne Ser Pro Ser Ser Thr Pro Leu Thr Lya Thr Leu Lys Ser Gln Ser Ser Ile Ser Ser Thr Leu Pro <210> 5 <211> 1995 <212> DNA <213> Beta vulgaris <220> <221 misc_feature <222> (1)..(1994) <223> Pro Mutant <220> <221CDS <222>(1y41995) <400> 5 13519 <210>6 <211>664 <212> PRT <213> Beta vulgaris <400> 6 Arg Vai Ser Asn Vai Ala Asp Leu Azg Ala Ala Iie 610 615 Asp Thr Pro Wear Pro Tyr Lou Lou Asp Va] With Val 625 630 His Val Leu Pro Met :Je Pro Ser Gly Ala Gly Phe 645 650 Thr Glu Gly Asp Gly Arg Thr Ser <210> 7 <211> 1995 <212> DNA <213> Beta vulgaris <220> <221> misc_feature <222> (1)..(1994) <223> 4D6834 al2 WT <220> <221> CDS <222> (1)..(1995) <400> 7
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