抗除草剂的草类物种的开发 The development of herbicide-resistant grass species

技术领域 Technical Field

[0001] 本文所揭示的本发明大致涉及对选择性除草剂具有抗性的草类和开发所述草类的方法。 [0001] The present invention disclosed herein relates generally to a method grasses and the grasses develop selective herbicide resistance.

背景技术 Background

[0002] 海雀稗（Seashore paspalum/Paspalum vaginatum)是一种温季草坪草,一般适合于沙丘环境。 [0002] seashore paspalum (Seashore paspalum / Paspalum vaginatum) is a warm season grass, generally suitable for dune environment. 海雀稗的有利性质包括其对盐、水浸和干旱的耐受性。 Seashore paspalum advantageous properties, including their salts, flooding and drought tolerance. 这些特征使得雀稗属（paspalum)成为用于任何或所有这些环境问题都会成为问题的比赛场地的优质草坪草候选者。 These features make paspalum (paspalum) become candidates for high-quality turfgrass any or all of these environmental issues will be a problem of venue. 举例来说，高尔夫球场设计师推荐海雀稗用于盐或水质会影响草坪草生长和维护的热带或亚热带沿海地区的新球场。 For example, a golf course designer seashore paspalum is recommended for salt or water quality will affect the new stadium turf grass growth and maintenance of tropical or subtropical coastal areas. 另外，许多现有的高尔夫球场已用雀稗属替代狗牙根(bermudagrass/Cynodon dactylon)。 In addition, many of the existing golf courses have been used paspalum alternative bermudagrass (bermudagrass / Cynodon dactylon). 与狗牙根相比，雀稗属需要的氮气更少并且对用含盐或劣质水冲洗的耐受性更好，这会降低管理成本并改善冲冼灵活性。 Compared to bermudagrass, nitrogen paspalum and require less water to wash with saline or poor better tolerated, which will reduce administrative costs and improve flexibility wash.

[0003] 用雀稗属替代狗牙根的主要局限性是狗牙根的重新建植。 [0003] The paspalum alternative Bermudagrass main limitation is re-planting bermudagrass. 狗牙根具有高竞争性并且一旦建植就难以去除。 Bermudagrass has a highly competitive and it is difficult to remove once planting. 狗牙根和其它似杂草的草类会大大降低雀稗属草坪的审美价值和质量。 Bermudagrass and other grass-like weeds will greatly reduce the paspalum turf aesthetic value and quality. 因此，需要控制或限制雀稗属繁殖区中的狗牙根或似杂草的草类生长。 Therefore, the need to control or limit the paspalum breeding areas in bermudagrass or grasses grow like weeds. 为控制雀稗属繁殖草坪草区中的似杂草的草类的生长，需要开发对选择性除草剂具有抗性的雀稗属草坪草。 To control paspalum turfgrass breeding area growing like weeds grasses, we need to develop paspalum selective herbicides in the case of lawn grass. 过去开发抗除草剂的草坪草的方法包括使用基因工程方法。 Turfgrass method developed in the past include the use of herbicide-resistant genetic engineering methods. 然而，由于政府关于使用基因改造植物的条例和限制，因此通过基因工程方法制造的植物可能难以商业化。 However, due to government regulations and restrictions on the use of genetically modified plants and therefore is produced by genetic engineering of plants may be difficult to commercialize. 因此， 本发明的实施例包括开发对除草剂具有非转基因抗性的草坪草栽培品种，以及具有转基因抗性的栽培品种。 Thus, embodiments of the present invention include the development of non-transgenic herbicide-resistant cultivars of turfgrass, and transgenic resistant cultivars.

发明内容 DISCLOSURE

[0004] 本发明的实施例涉及一种选择和培养的来自黍亚科群组的抗乙酰辅酶A羧化酶抑制剂除草剂的植物，或其组织、种子或子代。 [0004] Embodiments of the present invention relates to a method to select and train a group from Panicodae anti-acetyl coenzyme A carboxylase inhibitor herbicide plant, or a tissue, seed or progeny. 在一些实施例中，从已经历选择方法的抗除草剂的未分化细胞再生抗乙酰辅酶A羧化酶抑制剂除草剂的植物，其中所述选择方法包括：从黍亚科群组的植物提供未分化细胞的愈合组织，使所述愈合组织与足以延缓生长或杀死愈合组织的量的至少一种除草剂接触，基于所述除草剂的不同作用选择至少一种抗性细胞，和从所述至少一种抗性细胞再生所述品种的能生存的整个植物。 In some embodiments, has experienced from herbicide-resistant selection method undifferentiated cell regeneration anti-acetyl coenzyme A carboxylase inhibitor herbicide plant, wherein the selection method comprising: providing a plant Panicodae group undifferentiated cells of the healing tissue, so that the healing tissue and retard growth or kill an amount sufficient to heal tissue contacting at least one herbicide, select at least one resistant cell based on the different roles of the herbicide, and from the said at least one entire plant resistant cell regeneration of the species can survive. 在一些实施例中，所述植物是非转基因植物。 In some embodiments, the plant is non-transgenic plants.

[0005] 在本发明的一些实施例中，抗乙酰辅酶A羧化酶抑制剂除草剂的植物是黍族(Paniceae)的成员。 [0005] In some embodiments of the present invention, the anti-acetyl coenzyme A carboxylase inhibitor herbicide plant is millet family (Paniceae) members. 在一些实施例中，抗乙酰辅酶A羧化酶抑制剂除草剂的植物是一种选自以下的群组的植物：地毯草属（Axonopus)(地毯草（carpetgrass))、马唐属（Digiteria) (马唐草（crabgrass))、稗属（Echinochloa)、黍属（Panicum)、雀稗属（Paspalum)(百喜草(Bahiagrass)) (Pennisetum) (Setaria)禾口1¾卩十胃属(Stenotaphrum) (圣奥古斯丁草（St. Augustine grass))。 In some embodiments, the anti-acetyl coenzyme A carboxylase inhibitor herbicide plant is a plant selected from the following group: carpet grass genus (Axonopus) (carpet grass (carpetgrass)), crabgrass (Digiteria ) (crabgrass (crabgrass)), Echinochloa (Echinochloa), Panicum (Panicum), paspalum (Paspalum) (bahia grass (Bahiagrass)) (Pennisetum) (Setaria) Hekou 1¾ stomach Jie ten genera (Stenotaphrum ) (St. Augustine grass (St. Augustine grass)). 在一些实施例中，抗乙酰辅酶A羧化酶抑制剂除草剂的植物是一种选自以下的群组的植物：海雀稗（seashore paspalum/P. vaginatum)、剪股颖属草（bent grass)、高羊茅草（tall fescue grass)、结缕草（Zoysiagrass)、狗牙根（狗牙根属（Cynodon spp))、草地早熟禾（Kentucky Bluegrass)、得克萨斯早熟7^ (Texas Bluegrass) > H^Jft (Perennial ryegrass) > Sf 41 ^ (buffalograss/Buchloe dactyloides)、百足草(假俭草(Eremochloa ophiuroides))和圣奥古斯丁草（偏序钝叶胃(Stenotaphrum secundatum)) > WMM- ( WMiM-M )禾口15¾胃(Bahiagrass/Paspalum notatum)。 In some embodiments, the anti-acetyl coenzyme A carboxylase inhibitor herbicide plant is a plant selected from the following groups: seashore paspalum (. Seashore paspalum / P vaginatum), Agrostis grass (bent grass), tall fescue (tall fescue grass), Zoysia (Zoysiagrass), bermudagrass (Cynodon dactylon (Cynodon spp)), Bluegrass (Kentucky Bluegrass), Texas precocious 7 ^ (Texas Bluegrass)> H ^ Jft (Perennial ryegrass)> Sf 41 ^ (buffalograss / Buchloe dactyloides), centipede grass (centipedegrass (Eremochloa ophiuroides)) and St. Augustine grass (partial order Stenotaphrum stomach (Stenotaphrum secundatum))> WMM- (WMiM-M) Hekou 15¾ stomach (Bahiagrass / Paspalum notatum).

[0006] 在本发明的一些实施例中，抗乙酰辅酶A羧化酶抑制剂除草剂的植物对乙酰辅酶A羧化酶（ACCase)抑制剂具有抗性。 [0006] In some embodiments of the present invention, the anti-acetyl coenzyme A carboxylase inhibitor herbicide plant acetyl coenzyme A carboxylase (ACCase) inhibitor resistance. 在一些实施例中，抗乙酰辅酶A羧化酶抑制剂除草剂的植物对环己二酮除草剂、丙酸芳氧基苯氧酯除草剂、苯基吡唑啉除草剂或其混合物具有抗性。 In some embodiments, the anti-acetyl coenzyme A carboxylase inhibitor herbicide plants cyclohexanedione herbicide, an aryloxy phenoxy propionic acid herbicide ester, phenyl-pyrazoline herbicides, or mixtures thereof having anti- sex. 在一些实施例中，抗乙酰辅酶A羧化酶抑制剂除草剂的植物对至少一种选自以下的群组的除草剂具有抗性：禾草灭（alloxydim)、丁苯草酮（butroxydim)、 氯丙氧定(cloproxydim)、环苯草酮(profoxydim)、稀禾定(sethoxydim)、环苯草酮(clefoxydim)、克草同（clethodim)、环杀草（cycloxydim)、得杀草（tepraloxydim)、苯草酮（tralkoxydim)、炔禾灵（chloraizfop)、炔草酸（clodinafop)、氯伏草（clofop)、赛伏草(cyhalofop)、禾草灵（diclofop)、恶唑禾草灵（fenoxaprop)、噻唑禾草灵(fenthiaprop) > 精吡氟禾草灵（fluazaf op-butyl)、吡氟禾草灵（fluazifop)、合氯氟（haloxyfop)、异恶草醚（isoxapyrifop)、恶唑酰草胺（metamifop)、普拔草（propaquizafop)、快伏草(quizalofop)、三氟丙酸(trifop)和唑啉草酯(pinoxaden)。 In some embodiments, the anti-acetyl coenzyme A carboxylase inhibitor herbicide plant for at least one selected from the group of herbicides: alloxydim (alloxydim), styrene-butadiene oxadiazon (butroxydim) , chlorpromazine oxygen set (cloproxydim), benzene ring oxadiazon (profoxydim), sethoxydim (sethoxydim), benzene ring oxadiazon (clefoxydim), g grass with (clethodim), ring herbicide (cycloxydim), you have to kill grass ( tepraloxydim), benzene oxadiazon (tralkoxydim), alkyne Wo Ling (chloraizfop), clodinafop (clodinafop), chlorine-volt grass (clofop), game-volt grass (cyhalofop), diclofop (diclofop), fenoxaprop ( fenoxaprop), thiazole diclofop (fenthiaprop)> fine fluazifop (fluazaf op-butyl), fluazifop (fluazifop), co-chlorofluorocarbons (haloxyfop), different evil grass ether (isoxapyrifop), oxazole acid alachlor (metamifop), general weeding (propaquizafop), fast-volt grass (quizalofop), trifluoroacetic acid (trifop) and pinoxaden (pinoxaden).

[0007] 在本发明的一些实施例中，抗乙酰辅酶A羧化酶抑制剂除草剂的植物的除草剂抗性是通过乙酰辅酶A羧化酶基因的至少一个选自以下的群组的氨基酸位置的突变来赋予： 1756、1781、1999、2027、2041、2078、2099和2096。 [0007] In some embodiments of the invention, the herbicide-resistant anti-acetyl coenzyme A carboxylase inhibitor herbicide plant by acetyl coenzyme A carboxylase gene of at least one amino acid selected from the following group mutation position to impart: 1756,1781,1999,2027,2041,2078,2099 and 2096. 在一些实施例中，除草剂抗性是通过氨基酸位置1781的异亮氨酸突变成亮氨酸来赋予。 In some embodiments, the herbicide resistance by amino acid at position 1781 of isoleucine mutation leucine to impart.

[0008] 本发明的实施例还涉及一种如任一上述段落中所述的抗乙酰辅酶A羧化酶抑制剂除草剂的植物的子代。 [0008] Embodiments of the present invention also relates to a method as claimed in any one of the above paragraph, the progeny of anti-acetyl coenzyme A carboxylase inhibitor herbicide plants. 在一些实施例中，所述子代是抗乙酰辅酶A羧化酶抑制剂除草剂的植物亲代有性繁殖的结果。 In some embodiments, the progeny is an anti-acetyl coenzyme A carboxylase inhibitor herbicide plant parental result of sexual reproduction. 在一些实施例中，所述子代是抗乙酰辅酶A羧化酶抑制剂除草剂的植物亲代无性繁殖的结果。 In some embodiments, the progeny is an anti-acetyl coenzyme A carboxylase inhibitor herbicide plant parental result of asexual reproduction.

[0009] 本发明的实施例还涉及一种如任一上述段落中所述的抗乙酰辅酶A羧化酶抑制剂除草剂的植物的种子或其子代。 [0009] Embodiments of the present invention also relates to a method as claimed in any one of the above paragraphs of the anti-acetyl coenzyme A carboxylase inhibitor herbicide plant seeds or progeny thereof.

[0010] 本发明的实施例涉及包含如任一上述段落中所述的抗乙酰辅酶A羧化酶抑制剂除草剂的植物或其子代或种子的草地。 [0010] Embodiments of the present invention relates to compositions comprising as any of the above paragraphs of the anti-acetyl coenzyme A carboxylase inhibitor herbicide plant or its progeny or seed grass. 本发明的实施例还涉及一种包含如任一上述段落中所述的抗乙酰辅酶A羧化酶抑制剂除草剂的植物或其子代或种子的草坪草苗圃地。 Embodiments of the present invention further relates to an as claimed in any one of the above paragraphs of the anti-acetyl coenzyme A carboxylase inhibitor herbicide plant or its progeny or seed turfgrass nursery. 在本发明的实施例中，提供一种包含如任一上述段落中所述的抗乙酰辅酶A羧化酶抑制剂除草剂的植物或其子代或种子的商业草坪、高尔夫球场或牧场。 In an embodiment of the present invention, there is provided commercial lawn plant or its progeny or seeds comprising as claimed in any one of the above paragraphs of the anti-acetyl coenzyme A carboxylase inhibitor herbicide, golf course or pasture.

[0011] 本发明的实施例还涉及一种从黍亚科群组中鉴别抗除草剂植物的方法，其包括： 从黍亚科群组的植物提供未分化细胞的愈合组织，使所述愈合组织与足以延缓生长或杀死愈合组织的量的至少一种除草剂接触，基于所述除草剂的不同作用选择至少一种抗性细胞，和从所述至少一种抗性细胞再生所述品种的能生存的整个植物，其中所述再生植物对至少一种除草剂具有抗性。 Example [0011] The present invention also relates to a method of identifying the group from Panicodae herbicide-resistant plants, comprising: providing a callus of undifferentiated cells from plants Panicodae group, so the healing Organization and retard growth or kill sufficient amount of callus at least one herbicide exposure, based on the different roles of the herbicide resistant cell selecting at least one, and from the at least one resistance cell regeneration of the species viable whole plant, wherein at least one of the regenerated plants resistant to herbicide. 在一些实施例中，所述方法进一步包括将至少一种抗性细胞扩增为多种未分化细胞。 In some embodiments, the method further comprises the amplification of at least one resistant cell undifferentiated cells. 在一些实施例中，从非转基因植物提供未分化细胞的愈合组织。 In some embodiments, the non-transgenic plants provide callus of undifferentiated cells.

7[0012] 在本发明的一些实施例中，方法中所提供的植物是一种选自黍族的植物。 7 [0012] In some embodiments of the present invention, the method provided in the plant is a plant family selected from millet. 在一些实施例中，所述植物是一种选自以下的群组的植物：地毯草属（地毯草）、马唐属（马唐草）、稗属、黍属、雀稗属（百喜草）、狼尾草属、狗尾草属和钝叶草属（圣奥古斯丁草）。 In some embodiments, the plant is a plant selected from the following group: carpet grass genus (grass carpet), crabgrass (Digitaria grass), barnyard grass, Panicum, Paspalum (Bahiagrass ), Pennisetum, Setaria genus and species of the genus (St. Augustine grass). 在一些实施例中，所述植物是一种选自以下的群组的植物：海雀稗、剪股颖属草（剪股颖属(Agrostis spp))、高羊茅草（tall fescue)、结缕草、狗牙根（狗牙根属）、草地早熟禾、得克萨斯早熟禾、黑麦草、野牛草、百足草（假俭草）和圣奥古斯丁草（偏序钝叶草）、地毯草(地毯草属）和百喜草。 In some embodiments, the plant is a plant selected from the following groups: seashore paspalum, Agrostis grass (Agrostis (Agrostis spp)), tall fescue (tall fescue), Junction thread grass, bermudagrass (Cynodon spp.), Bluegrass, Texas bluegrass, ryegrass, buffalo grass, centipede grass (centipedegrass) and St. Augustine grass (partial order Stenotaphrum), carpet grass (genus carpet ) and bahia grass.

[0013] 在本发明的一些实施例中，方法中所使用的至少一种除草剂是乙酰辅酶A羧化酶(ACCase)抑制剂。 [0013] In some embodiments of the present invention, the method used in at least one herbicide is acetyl coenzyme A carboxylase (ACCase) inhibitor. 在一些实施例中，至少一种除草剂是选自以下的群组：禾草灭、丁苯草酮、氯丙氧定、环苯草酮、稀禾定、环苯草酮、克草同、环杀草、得杀草、苯草酮、炔禾灵、炔草酸、氯伏草、赛伏草、禾草灵、恶唑禾草灵、噻唑禾草灵、精吡氟禾草灵、吡氟禾草灵、合氯氟、 异恶草醚、恶唑酰草胺、普拔草、快伏草、三氟丙酸及唑啉草酯。 In some embodiments, the at least one herbicide selected from the following group: alloxydim, styrene-butadiene oxadiazon, chlorpromazine given oxygen, benzene ring oxadiazon, sethoxydim, grass one benzene ring, g grass with ring herbicide, have to kill grass, grass ketone benzene, alkyne Wo Ling, clodinafop, chlorine-volt grass, grass race volts, diclofop, fenoxaprop, thiazole diclofop, fluazifop fine, fluazifop, co chlorofluorocarbons, different evil grass ether, oxazole acid alachlor, general weeding, fast-volt grass, trifluoroacetic acid and pinoxaden.

[0014] 在本发明的一些实施例中，植物的除草剂抗性是通过乙酰辅酶A羧化酶基因的选自以下的群组的至少一个氨基酸位置的突变来赋予：1756、1781、1999、2027、2041、2078、 2099和2096。 [0014] In some embodiments of the invention, the herbicide-resistant plant by acetyl coenzyme A carboxylase gene selected from the group of at least one mutation at amino acid position to impart: 1756,1781,1999, 2027,2041,2078, 2099 and 2096. 在一些实施例中，除草剂抗性是通过氨基酸位置1781的异亮氨酸突变成亮氨酸来赋予。 In some embodiments, the herbicide resistance by amino acid at position 1781 of isoleucine mutation leucine to impart.

[0015] 本发明的实施例还涉及一种抗除草剂植物的可再生细胞的组织培养，其是通过如上述段落中所述的方法来进行。 Example [0015] The present invention also relates to a herbicide-resistant plant tissue culture of regenerable cells, which is obtained by the method as described in the above paragraph to.

[0016] 在本发明的实施例中，一种控制抗除草剂植物附近的杂草的方法，其中所述抗除草剂植物是通过上述段落中所述的方法来鉴别，所述方法包括：使至少一种除草剂与所述杂草和抗除草剂植物接触，其中所述至少一种除草剂是以足以抑制同一物种的非选择植物生长或足以抑制杂草生长的速率与杂草和植物接触。 [0016] In an embodiment of the present invention, a method of controlling weeds in herbicide-resistant plant nearby, where the herbicide-resistant plants are authenticated through the method described in the preceding paragraphs, the method comprising: in contact with said at least one weed herbicide and herbicide-resistant plant, wherein the at least one herbicide is sufficient to suppress non-selected plant of the same species or sufficient to inhibit the growth rate of the growth of weeds and weed plants in contact with . 在一些实施例中，抗除草剂植物对乙酰辅酶A羧化酶（ACCase)抑制剂具有抗性。 In some embodiments, the herbicide-resistant plant acetyl coenzyme A carboxylase (ACCase) inhibitor resistance. 在一些实施例中，方法包括使除草剂直接与抗除草剂植物接触。 In some embodiments, the method comprises direct contact with the herbicide herbicide-resistant plants. 在一些实施例中，方法包括使除草剂与抗除草剂植物所在的生长培养基接触。 In some embodiments, the method comprises a growth medium where the herbicide and herbicide-resistant plant contact.

[0017] 在一些实施例中，抗除草剂植物对环己二酮除草剂、丙酸芳氧基苯氧酯除草剂、苯基吡唑啉除草剂或其混合物具有抗性。 [0017] In some embodiments, herbicide-resistant plants cyclohexanedione herbicide, an aryloxy phenoxy propionic acid herbicide ester, phenyl-pyrazolone having herbicide resistance, or mixtures thereof. 在一些实施例中，抗除草剂植物是非转基因植物。 In some embodiments, herbicide-resistant plants are non-transgenic plants.

[0018] 在本发明的一些实施例中，植物的除草剂抗性是通过乙酰辅酶A羧化酶基因的至少一个选自以下的群组的氨基酸位置的突变来赋予：1756、1781、1999、2027、2041、2078、 2099和2096。 [0018] In some embodiments of the invention, the herbicide-resistant plant by acetyl coenzyme A carboxylase gene mutation in at least one amino acid selected from the group of the following locations to impart: 1756,1781,1999, 2027,2041,2078, 2099 and 2096. 在一些实施例中，除草剂抗性是通过乙酰辅酶A羧化酶基因的氨基酸位置1781的异亮氨酸突变成亮氨酸来赋予。 In some embodiments, the herbicide resistance by amino acid position acetyl coenzyme A carboxylase gene of 1781 isoleucine mutation leucine to impart.

[0019] 在一些实施例中，方法中所使用的至少一种除草剂是选自以下的群组：禾草灭、丁苯草酮、氯丙氧定、环苯草酮、稀禾定、环苯草酮、克草同、环杀草、得杀草、苯草酮、炔禾灵、 炔草酸、氯伏草、赛伏草、禾草灵、恶唑禾草灵、噻唑禾草灵、精吡氟禾草灵、吡氟禾草灵、合氯氟、异恶草醚、恶唑酰草胺、普拔草、快伏草、三氟丙酸及唑啉草酯。 [0019] In some embodiments, the at least one herbicide used in the method is selected from the following group: alloxydim, butylbenzene oxadiazon, chloropropyl given oxygen, oxadiazon benzene ring, sethoxydim, oxadiazon benzene ring, g grass with ring herbicide, have to kill grass, grass ketone benzene, alkyne Wo Ling, clodinafop, chlorine-volt grass, grass race volts, diclofop, fenoxaprop, thiazole diclofop , fine fluazifop, fluazifop, co chlorofluorocarbons, different evil grass ether, oxazole acid alachlor, general weeding, fast-volt grass, trifluoroacetic acid and pinoxaden.

[0020] 本发明的实施例涉及一种以ATCC寄存编号_寄存的海雀稗特异性DNA标 [0020] Embodiments of the present invention relates to a deposit of ATCC Accession number _ seashore paspalum specific DNA standard

记，或其能够鉴别抗除草剂的草类栽培品种的片段。 Remember, or be able to identify herbicide-resistant grass cultivars fragments. 在一些实施例中，海雀稗特异性DNA标记包含SEQ ID NO ：5或其片段。 In some embodiments, the seashore paspalum specific DNA markers comprising SEQ ID NO: 5 or a fragment thereof.

[0021] 本发明的实施例还涉及一种鉴别抗除草剂植物的方法，其包括：获得植物的遗传 Example [0021] The present invention also relates to a method to identify herbicide-resistant plants, which include: access to genetic plants

8学样品，和测定所述样品中存在或不存在乙酰辅酶A羧化酶基因的位置1781的突变，其中存在位置1781的突变指示植物具有除草剂抗性。 8 school sample, and the sample was measured in the presence or location of acetyl coenzyme A carboxylase gene mutations 1781 does not exist, where there is a mutation 1781 position indicator plant herbicide resistance. 还涵盖乙酰辅酶A羧化酶的位置1781的标记在鉴别抗除草剂植物的方法中的用途。 Also covers acetyl coenzyme A carboxylase 1781 marks the location of use in identifying herbicide-resistant plants method.

[0022] 本发明的实施例涉及一种标记辅助育种的方法，其包括以下步骤：鉴别所关注用于育种和选择的特征，其中所述特征与乙酰辅酶A羧化酶基因有关；提供带有能够赋予植物对乙酰辅酶A羧化酶抑制剂除草剂的抗性的乙酰辅酶A羧化酶序列变异体的第一植物， 其中所述植物进一步包含所关注之特征；用第二植物培育所述第一植物；鉴别育种步骤的子代为具有所述乙酰辅酶A羧化酶序列变异体；和基于所述鉴别步骤选择可能具有所关注的特征的子代。 [0022] Embodiments of the present invention relates to a method for marker-assisted breeding, which comprises the steps of: identifying a feature of interest for breeding and selection, wherein the feature and acetyl coenzyme A carboxylase genes; provided with The first plant capable of conferring resistance to acetyl coenzyme A carboxylase inhibitor herbicide acetyl coenzyme A carboxylase sequence variants, wherein the plant further comprises the characteristic of interest; with the second plant breeding The first plant; discriminating breeding child took the step having the acetyl coenzyme A carboxylase sequence variants; and based on the identification step of selecting features of interest may have offspring. 在一些实施例中，所述特征选自：性状或基因。 In some embodiments, the feature is selected from: a trait or gene. 在一些实施例中，所述性状是至少一种选自由以下组成的群组的性状：除草剂耐受性、疾病抗性、害虫昆虫抗性、脂肪酸改变、蛋白质或碳水化合物代谢、生长速率增加、胁迫耐受性增强、优先成熟、感官性质增强、形态特征改变、不能繁殖、其它农业性状、工业用途的性状或改善消费者吸引力的性状。 In some embodiments, the trait is a trait of at least one selected from the group consisting of: herbicide tolerance, disease resistance, insect pest resistance, alter the fatty acid, protein or carbohydrate metabolism, increased growth rate , enhanced stress tolerance, priority mature, enhanced organoleptic properties, morphological changes can not reproduce, other agronomic traits, traits for industrial uses or improve consumer appeal traits.

[0023] 在本发明的一些实施例中，方法中所包括的乙酰辅酶A羧化酶序列变异体包括以下至少一个位置的变化:1756、1783、1999、2027、2041、2078、2099和2096。 [0023] In some embodiments of the present invention, a method included in the acetyl coenzyme A carboxylase sequence variants include changes in at least one location: 1756,1783,1999,2027,2041,2078,2099 and 2096. 在一些实施例中，植物抗性所针对的除草剂是至少一种选自以下的群组的除草剂：禾草灭、丁苯草酮、氯丙氧定、环苯草酮、稀禾定、环苯草酮、克草同、环杀草、得杀草、苯草酮、炔禾灵、炔草酸、氯伏草、赛伏草、禾草灵、恶唑禾草灵、噻唑禾草灵、精吡氟禾草灵、吡氟禾草灵、合氯氟、异恶草醚、恶唑酰草胺、普拔草、快伏草、三氟丙酸及唑啉草酯. In some embodiments, the plant resistance against the herbicide is at least one selected from the group of herbicides: alloxydim, styrene-butadiene oxadiazon, chlorpromazine given oxygen, benzene ring oxadiazon, sethoxydim , benzene ring oxadiazon, g grass with ring herbicide, have to kill grass, grass phenyl ketone, alkyne Wo Ling, clodinafop, chlorine-volt grass, grass race volts, diclofop, fenoxaprop, thiazole grass Ling, Jing fluazifop, fluazifop, co chlorofluorocarbons, different evil grass ether, oxazole acid alachlor, general weeding, fast-volt grass, trifluoroacetic acid and pinoxaden.

[0024] 在一些实施例中，方法中所包括的鉴别步骤包括选自以下的过程：序列变异体的分子检测、乙酰辅酶A羧化酶抑制剂抗性的观察和通过应用乙酰辅酶A羧化酶抑制剂进行选择。 [0024] In some embodiments, the authentication step method included include those selected from the following processes: molecular detection of sequence variants observed acetyl coenzyme A carboxylase inhibitor resistance and through the application of acetyl coenzyme A carboxylase inhibitor selected.

[0025] 本发明的实施例涉及一种用包含具有从ATCC寄存编号_的序列衍生的 [0025] Embodiments of the present invention relates to a storage number _ comprises a sequence derived from ATCC

至少250个碱基的DNA区段转化的转基因植物，和其子代植物。 The transgenic plant DNA segments at least 250 bases of transformed plants and their progeny. 在一些实施例中，所述子代植物选自：回交子代、杂交、克隆子代和同胞交配子代。 In some embodiments, the progeny plant is selected from: backcross progeny, hybrids, clone progeny and sib-mating progeny. 在一些实施例中，所述DNA区段包含从SEQ ID NO ：5衍生的至少个250碱基。 In some embodiments, the DNA segment comprises from SEQ ID NO: 5 derived from at least a 250 bases.

[0026] 本发明的实施例还涉及一种含有包含从ATCC寄存编号_的序列衍生的 [0026] Embodiments of the present invention also relates to a method comprising comprising Accession number _ sequence derived from ATCC

至少250个碱基的DNA区段的转化细胞。 At least 250 bp DNA segment of transformed cells. 在一些实施例中，所述DNA区段包含从SEQ ID NO： 5衍生的至少个250碱基。 In some embodiments, the DNA segment comprises from SEQ ID NO: 5 derived from at least a 250 bases.

[0027] 在本发明的实施例中，提供一种鉴别乙酰辅酶A羧化酶基因的位置1781的突变的方法，所述方法包括从细胞获得遗传学样品，通过在扩增步骤中使用SV384F引物和SV348R 引物来选择性扩增DNA片段，和对所述DNA片段测序以确定存在或不存在乙酰辅酶A羧化酶基因的位置1781的突变，其中DNA片段中存在突变指示细胞中存在位置1781的突变。 [0027] In an embodiment of the present invention, there is provided a position acetyl coenzyme A carboxylase gene to identify mutations of 1781, the method comprises obtaining a genetic sample from a cell by using SV384F primers in the amplification step and SV348R primers to selectively amplify DNA fragments, and sequencing of the DNA fragments to determine the presence or location of acetyl coenzyme A carboxylase gene mutation does not exist 1781, which indicates the presence of mutant DNA fragments present in the cell location in 1781 mutation.

附图说明 Brief Description

[0028] 所属领域的技术人员应了解下文所述的图示仅用于说明性目的。 [0028] Those skilled in the art will appreciate shown below for illustrative purposes only. 所述图示无论如何也不打算限制本发明教示的范围。 The illustration in no way intended to limit the scope of the present teachings.

[0029] 图1是植物的脂肪酸生物合成路径的图。 [0029] Figure 1 is a plant fatty acid biosynthesis pathway FIG.

[0030] 图2是用于如本文所揭示的选择非转基因抗除草剂植物的除草剂选择方案的一实施例的说明。 [0030] FIG. 2 is used as disclosed herein select a non-transgenic herbicide resistant plants herbicide options for a description of the embodiments. [0031] 图3是说明海雀稗的稀禾定剂量-反应曲线的图。 [0031] FIG. 3 is said Ariake paspalum sethoxydim dose - response curve of FIG.

[0032] 图4是生长于含有稀禾定的愈合组织诱发培养基上的海雀稗的抗稀禾定愈合组织的照片。 [0032] FIG. 4 is grown containing sethoxydim callus induced on medium seashore paspalum anti sethoxydim callus photos.

[0033] 图5是一系列色谱图，其说明如本文所揭示而选择的抗除草剂海雀稗植物的乙酰辅酶A羧化酶基因的位置1781的氨基酸突变。 [0033] FIG. 5 is a series of chromatograms, which illustrates the position of acetyl coenzyme A carboxylase gene as disclosed herein and selected herbicide-resistant plants of seashore paspalum 1781 amino acid mutations.

[0034] 图6是说明在处理后7天（DAT)对照植物和如本文所揭示而选择的抗除草剂植物对kgment™稀禾定的反应的照片。 [0034] FIG. 6 is seven days after treatment (DAT) control plants and as disclosed herein and selected herbicide-resistant plants kgment ™ sethoxydim reaction of photos.

[0035] 图7是说明在处理后7天（DAT) kgment™稀禾定对对照植物和如本文所揭示而选择的抗除草剂植物的损伤的图。 [0035] FIG. 7 is in seven days after treatment (DAT) kgment ™ sethoxydim to control plants and as described in Fig selected herbicide-resistant plants revealed damage.

[0036] 图8是说明在处理后14天（DAT)对照植物和如本文所揭示而选择的抗除草剂植物对kgment™稀禾定的反应的照片。 [0036] FIG. 8 is within 14 days after treatment (DAT) control plants and as disclosed herein and selected herbicide-resistant plants kgment ™ sethoxydim reaction of photos.

[0037] 图9是说明在处理后9天（DAT) kgment™稀禾定对对照植物和如本文所揭示而选择的抗除草剂植物的损伤的图。 [0037] FIG. 9 is illustrated in FIG. 9 days after treatment (DAT) kgment ™ sethoxydim to control plants and as disclosed herein and selected herbicide-resistant plant of injury.

[0038] 图10是说明在处理后21天（DAT)对照植物和如本文所揭示而选择的抗除草剂植物对kgment™稀禾定的反应的照片。 [0038] FIG. 10 is 21 days after treatment (DAT) control plants and as disclosed herein and selected herbicide-resistant plants kgment ™ sethoxydim reaction of photos.

[0039] 图11是说明在处理后21天（DAT) kgment™稀禾定对对照植物和抗除草剂植物的损伤的图。 [0039] FIG. 11 is in 21 days (DAT) kgment ™ sethoxydim damage control herbicide-resistant plants and reprocessing plants Fig.

[0040] 图12是说明在处理后42天（DAT)在用kgment™稀禾定处理后对照植物和如本文所揭示而选择的抗除草剂植物的平均干重的图。 [0040] FIG. 12 is in 42 days (DAT) after treatment with sethoxydim kgment ™ treated control plants and the average dry as disclosed herein and the choice of herbicide-resistant plant of heavy Fig.

[0041] 图13是说明在处理后21天（DAT)Poast™稀禾定对对照植物和如本文所揭示而选择的抗除草剂植物的损伤的图。 [0041] FIG. 13 is illustrated in FIG. 21 days after treatment (DAT) Poast ™ sethoxydim to control plants and as disclosed herein and selected herbicide-resistant plant of injury.

[0042] 图14是说明在处理后21天（DAT)Fusilade II™精吡氟禾草灵除草剂对对照植物和如本文所揭示而选择的抗除草剂植物的损伤的图。 [0042] FIG. 14 is 21 days after treatment (DAT) Fusilade II ™ fine fluazifop and herbicides to control plants as described in Fig selected herbicide-resistant plants revealed damage.

[0043] 图15是说明在处理后21天（DAT)Acclaim Extra™ II精恶唑禾草灵(fenoxaprop-p-butyl)除草剂对对照植物和抗除草剂植物的损伤的图。 [0043] FIG. 15 is an explanatory (DAT) Acclaim Extra ™ II fenoxaprop (fenoxaprop-p-butyl) herbicide damage control plants and herbicide-resistant plant of FIG. 21 days after treatment.

[0044] 图16是由植物居间分生组织获得的愈合组织产生的一实施例的说明。 [0044] FIG. 16 is intervening callus from plants obtained by meristem produced an embodiment.

具体实施方式 DETAILED DESCRIPTION

[0045] 对选择性除草剂的抗性可提供控制各种草坪草物种中的似杂草的草类的高效方法。 [0045] The selective herbicides efficient way to control a variety of grasses in turf grass species like weeds available. 已提出用于开发抗除草剂植物的基因工程方法，然而由于政府关于使用基因改造植物的条例和限制，因此这些方法可能难以商业化。 Has been proposed for the development of anti-herbicide plant genetic engineering methods, but because government regulations and restrictions on the use of genetically modified plants, so these can be difficult to commercialize. 相比之下，目前通过非转基因方法得到的具有除草剂抗性的植物的环境释放不服从严格的政府条例。 In contrast, the present method is obtained by non-transgenic plant environments with the release of herbicide resistance disobedience strict government regulations. 因此，本发明的实施例涉及筛选和选择抗除草剂的草坪草植物的方法，包括不通过转基因而有效的方法。 Thus, embodiments of the present invention relates to the screening and selection of herbicide-resistant turf grass plant, comprising not transgenic and effective way.

[0046] ^X [0046] ^ X

[0047] 除非另作说明，否则所属相关领域的技术人员应根据常规用途来了解术语。 [0047] Unless otherwise specified, belong to the relevant skill in the art should be based on a general purpose to understand terms.

[0048] 如本文中所使用，术语“外植体”是指包括分生组织的植物组织。 [0048] As used herein, the term "explant" is meant to include plant tissue meristems. 其也可指包括(但不限于）一个或一个以上胚、子叶、下胚轴、叶基、中胚轴、胚芽、原生质体和胚轴的植物组织。 It may also be meant to include (but are not limited to) one or more embryos, cotyledons, hypocotyls, leaf base, the hypocotyl, germ, protoplasts and plant tissue Axes.

[0049] 如本文中所使用，术语“愈合组织”是指可生长或维持在培养基中以产生遗传一致细胞的未分化的植物细胞块。 [0049] As used herein, the term "callus" refers to the growth or maintenance of undifferentiated plant cell block in a medium to produce the genetic identity of cells.

[0050] 如本文中所使用，术语“抗除草剂”或“除草剂耐受”（包括其任何变化形式）是指植物从与足以引起同一物种的非抗性植物的生长延缓或死亡的量的除草剂接触中恢复、 在接触后存活和/或茁壮成长。 [0050] As used herein, the term "herbicide-tolerant" or "herbicide tolerance" (including any variations thereof) refers to an amount sufficient to cause the growth of a plant from a non-resistant plant of the same species or delaying death The herbicide exposure recover after exposure to survive and / or thrive. 足以引起非抗性植物的生长或死亡的除草剂的量通常在约2μΜ到约100 μ M除草剂浓度的范围内。 An amount sufficient to cause growth of non-herbicide-resistant plant or death is usually in the range of from about 2μΜ to about 100 μ M concentration of the herbicide. 在一些实施例中，除草剂的足够量在约5μ M到约50 μ M除草剂浓度、约8 μ M到约30 μ M除草剂浓度或约10 μ M到约25 μ M除草剂浓度的范围内。 In some embodiments, a sufficient amount of the herbicide of about 5μ M to about 50 μ M of herbicide concentration, from about 8 μ M to about 30 μ M of herbicide concentration, or from about 10 μ M to about 25 μ M of herbicide concentration range. 或者，足以引起非抗性植物的生长或死亡的除草剂的量在每公顷约25克活性成分（g ai ha-1)到约6500g ai ha—1除草剂施用的范围内。 Alternatively, an amount sufficient to cause the growth of non-herbicide-resistant plants per hectare or death in about 25 grams of active ingredient (g ai ha-1) from about 6500g ai ha-1 range to the herbicide. 在一些实施例中，除草剂的足够量在约50g ai ha—1到约5000g ai ha"1除草剂施用、约75g ai ha"1到约2500g ai ha"1除草剂施用、约IOOg ai ha4 到约1500g ai ha—1 除草剂施用、或约250g ai ha4 到约IOOOg ai ha-1 除草剂施用的范围内。 In some embodiments, a sufficient amount of the herbicide of about 50g ai ha-1 to about 5000g ai ha "1 herbicide, about 75g ai ha" 1 to about 2500g ai ha "1 herbicide, about IOOg ai ha4 to about 1500g ai ha-1 herbicide, or about 250g ai ha4 to about IOOOg ai ha-1 within the scope of application of the herbicide.

[0051] 如本文中所使用，术语“标记辅助选择”是指通过检测与所需性状有关的一个或一个以上标记来选择植物的所需性状的过程。 [0051] As used herein, the term "marker assisted selection" refers to one or more related tag to select the desired traits in plants with desired traits through the detection process. 所述标记可为表型标记，例如除草剂或抗生素抗性。 The mark may be a phenotypic markers, such as herbicide or antibiotic resistance. 而且，所述标记可为分子标记，例如一种或一种以上多态现象（如下文所述）、DNA或RNA酶、或容易检测的其它序列。 Furthermore, the label may be a molecular marker, e.g., one or more polymorphism (as described below), DNA or RNA enzymes, or other sequences easily detectable.

[0052] 对于个别植物为“外源性”的多核苷酸是通过除有性杂交外的任何方法引入植物中的多核苷酸。 [0052] For individual plants as "exogenous" polynucleotide is a polynucleotide introduced into a plant by any method other than sexual crossing outside. 下文描述可用来完成的方法的实例，并且包括转化、基因枪法、电穿孔等。 The method described below to complete examples available, and include transformation, particle bombardment, electroporation and the like. 所述含有外源性核酸的植物是称作Rtl (用于在活体外从转化细胞再生的植物）产生转基因植物。 The plant containing the exogenous nucleic acid is referred Rtl (for plants regenerated from transformed cells in vitro) to produce transgenic plants. R。 R. 还可指无论是转基因还是非转基因的任何其它再生植物。 Also refers to whether transgenic or non-transgenic any other regeneration plant genes.

[0053] 如本文中所使用，术语“转基因”描述含有人工改造的基因组的非天然存在的植物，其中所述植物在其基因组中包括外源性核酸分子，所述分子可来源于相同物种或不同物种，包括非植物物种。 [0053] As used herein, the term "transgenic" describes a non-naturally occurring plant containing artificial modification of the genome, wherein the plant comprises an exogenous nucleic acid molecule in its genome, the molecule can be derived from the same species or different species, including non-plant species. 外源性核酸分子可为基因调控元件，诸如启动子、增强子或其它调控元件，或可含有可与天然或异源基因调控元件有关的编码序列。 Exogenous nucleic acid molecule may be a gene regulatory elements, such as promoters, enhancers or other regulatory elements, or may contain native or heterologous gene regulatory element associated coding sequence. 通过有性杂交或通过自体受精产生的转基因植物是所述植物的子代。 Transgenic plants by sexual crossing or produced by self-fertilization is the progeny of the plant.

[0054] 如本文中所使用，“多态现象”意思是在一个或一个以上个体的群体中核酸序列的一个或一个以上基因座存在一个或一个以上变化。 [0054] As used herein, "polymorphism" means one or more individuals in the population of a nucleic acid sequence of one or more loci presence of one or more changes. 所述变化可包含（但不限于）一个或一个以上碱基变化、一个或一个以上核苷酸插入或一个或一个以上核苷酸缺失。 The changes may include (but are not limited to) one or more base changes, one or more nucleotide insertion or deletion of one or more nucleotides. 多态现象包括单核苷酸多态现象（SNP)、简单序列重复（SSR)、插入缺失（indel)(插入和缺失）、限制片段长度多态现象、单倍体和标记SNP。 Polymorphism includes single nucleotide polymorphism (SNP), a simple sequence repeat (SSR), insertion deletion (indel) (insertions and deletions), restriction fragment length polymorphisms, haplotypes and tag SNP. 另外，多态现象可包括遗传标记、基因、DNA衍生序列、RNA衍生序列、启动子、基因的5 '未翻译区、基因的3 '未翻译区、微小RNA (microRNA)、 siRNA、定量性状基因座（quantitative trait locus，QTL)、卫星标记、转基因、mRNA、ds mRNA、转录概况或甲基化模式。 In addition, the polymorphism may include genetic markers, genes, DNA-derived sequences, RNA-derived sequence, a promoter, gene 5 'untranslated region, 3 genes' untranslated region, micro RNA (microRNA), siRNA, quantitative trait locus Block (quantitative trait locus, QTL), marker, genetically, mRNA, ds mRNA, transcriptional profiles or methylation patterns. 多态现象可由核酸复制的随机处理、通过突变诱发、由移动基因组元件、由拷贝数变化和在减数分裂过程期间引起，诸如不等交换、基因组加倍和染色体破坏和融合。 Polymorphism may be a random process of DNA replication, through mutation induction, the mobile genomic elements, caused by the copy number changes and during the process of meiosis, such as unequal exchange, genome duplication and chromosome damage and integration. 变化可常见于群体中或可较低频率地存在于群体中，前者在普通植物育种方面具有较大效用且后者可能与稀少但重要的表型变化相关。 Changes may be common in the population or less frequently exist in the population, the former has a larger utility plant breeding in general and the latter may be associated with rare but important phenotypic changes.

[0055] 如本文中所使用，“标记”是指多态核酸序列或核酸特征。 [0055] As used herein, "labeled" refers to a polymorphic nucleic acid sequence or nucleic acid feature. 在更广泛的方面，“标记” 可为可用于区别有机体之间的可遗传差异的可检测特征。 In a broader context, "tag" may be genetic differences can be used to distinguish between organisms detectable characteristics. 所述特征的实例可包括遗传标记、蛋白质组成、蛋白质水平、油组成、油水平、碳水化合物组成、碳水化合物水平、脂肪酸组成、脂肪酸水平、氨基酸组成、氨基酸水平、生物聚合物、药物、淀粉组成、淀粉水平、可发酵淀粉、发酵产率、发酵效率、能量产率、次要化合物、代谢物、形态特征和农业学特征。 Examples of characteristics may include genetic markers, protein composition, protein levels, oil composition, oil levels, carbohydrate composition, carbohydrate levels, fatty acid composition, fatty acid levels, amino acid composition, amino acid levels, biopolymers, pharmaceuticals, starch composition , starch levels, fermentable starch, fermentation yield, fermentation efficiency, energy yield, secondary compounds, metabolites, morphological characteristics and agriculture.

[0056] 如本文所使用，“标记分析”指的是一种使用特定方法检测特定基因座的多态性的方法，例如测量至少一种表型（例如种子颜色、花朵颜色或其他可目测的性状）、限制性片段长度多态性（RFLP)、单碱基延伸、电泳、序列比对、等位基因特异性寡核苷酸杂交（ASO)、 随机扩增多态DNA(RAPD)、基于微阵列的技术和核酸定序技术等。 [0056] As used herein, "labeled analyte" refers to a method for detecting a particular locus using a particular method polymorphism methods, e.g., measuring at least one phenotype (e.g., seed color, flower color, or other visual observation Characters), restriction fragment length polymorphism (RFLP), single base extension, electrophoresis, sequence alignment, allele specific oligonucleotide hybridization (ASO), random amplified polymorphic DNA (RAPD), based on technology and nucleic acid sequencing microarray technology.

[0057] 如本文所使用，“基因型”指的是表型的遗传组成，且此可使用标记物间接表征或通过核酸定序直接表征。 [0057] As used herein, "genotype" refers to a heritable phenotype of the composition, and this can be indirectly characterized using markers or directly characterized by nucleic acid sequencing. 适合标记物包括表型特征、代谢特征、遗传标记或一些其他类型的标记物。 Suitable markers include phenotypic characteristics, metabolic characteristics, genetic markers, or some other type of marker. 基因型可构成至少一种遗传标记物基因座的等位基因或至少一种单倍体窗的单倍体。 Genotype may constitute at least one genetic marker loci or at least one haplotype allele haplotype window. 在一些实施例中，基因型可表示单个基因座，且在其他实施例中，可表示全基因组的基因座。 In some embodiments, the genotype may represent a single locus, and in other embodiments, may represent a locus of the whole genome. 在一些实施例中，基因型可以反映部分染色体、整个染色体、部分基因组和整个基因组的序列。 In some embodiments, the genotype can reflect part of the chromosome, the entire chromosomal sequence, and a portion of the genome of the entire genome.

[0058] 如本文所使用，“数量性状基因座（QTL) ”指的是数量上控制到一定程度的基因座， 其可表示一般连续分布的形状。 [0058] As used herein, "quantitative trait locus (QTL)" refers to a certain degree of control on the number of loci, which may represent the general shape of a continuous distribution.

[0059] 如本文所使用，“核酸序列片段”指的是聚核苷酸的一部分核苷酸序列或多肽的一部分胺基酸序列。 [0059] As used herein, "nucleic acid sequence fragment" refers to a part of the amino acid sequence of a portion of the nucleotide sequence of a polynucleotide or polypeptide. 核苷酸序列的片段可以编码保留天然或相应全长蛋白质的生物活性的蛋白质片段。 The nucleotide sequences may encode fragments retained biological activity of native or corresponding full-length protein protein fragments. 核苷酸序列的片段范围为至少约20个核苷酸、约50个核苷酸、约100个核苷酸、 约250个核苷酸，且至多基因的全长核苷酸序列或编码本文所揭示蛋白质的序列。 Range nucleotide sequence fragment is at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, about 250 nucleotides, and up to the full-length nucleotide sequence or gene encoding this article sequence of the protein disclosed.

[0060] 供筛选的适合植物 [0060] for screening for plants

[0061] 本发明的实施例针对由除草剂抗性细胞（所述细胞已经历除草剂选择过程）再生的黍亚科（Panicodae)除草剂抗性植物，以及其识别方法。 [0061] Embodiments of the present invention are directed by the herbicide-resistant cells (the cell selection process has undergone herbicide) reproduced Panicodae (Panicodae) herbicide-resistant plant, as well as its identification method. 所述植物可以从以下各物的群组中选出：柳叶箬族(Isachneae tribe)、Neurachneae 族、Arundinellaeae 族和黍族（Paniceae tribe)。 The plant may be selected from the group of the following material: bamboo willow family (Isachneae tribe), Neurachneae family, Arundinellaeae family and millet family (Paniceae tribe). 在一些实施例中，所述植物可为选自表A或表B中所提供的清单的属的任何成员。 In some embodiments, the plant can be any member of the genus A or Table B is selected from the list provided in Table. 适用于本发明的例示性（非详尽）植物清单包括黍族的成员，例如地毯草(Carpetgrass)、马唐草、百喜草、圣奥古斯丁草(St. Augustine grass)和栗(millet)，包括狐尾草（Foxtail)(谷子（Setaria italical))、珍珠粟（Pearl，Pennisetam glaucum) 禾口泰(Proso,Panicum miliaceum ；通常禾尔为“常见，，泰、高梁泰(broom corn millet)、赤泰(hog millet)或白黍子(white millet)。 Useful in the present invention is shown (non-exhaustive) plant list includes members of the millet family, such as carpet grass (Carpetgrass), crabgrass, bahia grass, St. Augustine grass (St. Augustine grass) and chestnut (millet), including foxtail grass (Foxtail) (millet (Setaria italical)), pearl millet (Pearl, Pennisetam glaucum) Hekou Thai (Proso, Panicum miliaceum; usually Wo Seoul as "common ,, Thailand, sorghum Thai (broom corn millet), red Thai ( hog millet) or white millet (white millet).

[0062] 在一些实施例中，所述植物为具有商业应用价值的草坪草种类，例如高尔夫球场、 田径场、商业绿化、商业或家庭草坪、和牧场。 [0062] In some embodiments, the plant is turfgrass species have commercial applications, such as golf courses, athletic fields, green business, commercial or home lawns, and pastures. 例示性草坪草种类包括（但不限于）海雀稗、百喜草、狗牙根（狗牙根属)、blue gramma grass、野牛草、地毯草（地毯草属）、百足草（假俭草）、克育草（kikuyugrass)、垂穗草（sideoats grama)、圣奥古斯丁草（偏序钝叶草）、结缕草、一年生早熟禾(annual bluegrass)、一年生黑麦草（annual ryegrass)、加拿大早熟禾(Canada bluegrass)、细叶型羊茅（chewings fescue)、细弱剪股颖（colonial bentgrass) ^1¾'¾¾¾!¾!!¾ (creeping bentgrass)、^水草(crested wheatgrass)、扁(fairway wheatgrass)、硬羊茅(hard fescue)、草地早熟禾(Kentucky bluegrass)、得克萨斯早熟禾、果园草（orchard grass)、黑麦草、红牛毛草（red fescue)、红顶草（redtop)、 粗莲苗系草(rough bluegrass)、羊茅(sheep fescue)、无芒雀麦(smooth bromegrass)、 高羊茅草、猫尾草（Timothygrass)、绒毛剪股颖(velvet bentgrass)、碱茅(weeping alkaligrass)、纳托尔氏脸茅(western wheatgrass)等。 Exemplary turfgrass species include (but are not limited to) the seashore paspalum, bahia grass, bermudagrass (Cynodon dactylon), blue gramma grass, buffalo grass, carpet grass (carpet genus), Momotari grass (centipedegrass), g and grass (kikuyugrass), vertical spike grass (sideoats grama), St. Augustine grass (partial order Stenotaphrum), Zoysia grass, annual bluegrass (annual bluegrass), annual ryegrass (annual ryegrass), Canada bluegrass ( Canada bluegrass), Zoysia type fescue (chewings fescue), thin bentgrass (colonial bentgrass) ^ 1¾'¾¾¾! ¾ !! ¾ (creeping bentgrass), ^ plants (crested wheatgrass), flat (fairway wheatgrass), hard Fescue (hard fescue), Kentucky bluegrass (Kentucky bluegrass), Texas bluegrass, orchard grass (orchard grass), ryegrass, Red Bull teasel (red fescue), red top grass (redtop), Lin Miao-based rough grass ( rough bluegrass), fescue (sheep fescue), smooth bromegrass (smooth bromegrass), tall fescue, timothy grass (Timothygrass), hair bentgrass (velvet bentgrass), Puccinellia (weeping alkaligrass), Nuttall Mao's face (western wheatgrass) and so on. [0063] 表A.黍亚科属成员（根据族编组） [0063] Table A. Panicodae are members (according to family grouping)

[0064] [0064]

[0065] 表B.黍亚科的黍族的属成员 It is a member of [0065] Table B. Panicodae millet family

[0066] [0066]

[0068] 在本发明实施例中，待经受本发明方法的植物可以是天然存在的植物、栽培的非转殖基因植物或已通过基因方式修饰的植物，例如转殖基因植物。 [0068] In the embodiment of the invention, the method of the present invention is to be subjected to a plant may be naturally occurring plant, cultivation of non-genetically modified plants colonization or modified by genetic means plants, such as gene transfer plant colonization.

[0069] 愈合组织来源[0070] 可以从植物产生可以活体外培养的愈合组织或大量未分化细胞的任何部分采集外植体选择。 [0069] healing tissue source [0070] can produce callus can live cultured or any part of a large number of undifferentiated cells collected explant select from plants. 举例而言，可以从植物的夹层分生组织、未成熟花序或叶片分生组织获得外植体选择。 For example, from the mezzanine plant meristems, immature inflorescence meristems obtained or blade explant selection. 在一些实施例中，可以从植物种子，或其片段或区段获得外植体选择。 In some embodiments, the explant selection can be obtained from plant seeds, or a fragment or segment.

[0071] 在外植体收集之前，可以对源组织或种子进行灭菌步骤，从而避免活体外微生物污染。 [0071] before explant collection, you can organize or seed source for sterilization step, so as to avoid vitro microbial contamination. 灭菌可以包括在例如约10V/V%至100V/V%溶液等漂白溶液中冲洗、在例如约50v/ Sterilization may include, for example, washing bleaching solution approximately 10V / V% to 100V / V% solution, and the like, for example, from about 50v /

至95V/V%溶液等纯溶液（例如乙醇溶液）中冲洗、和/或在灭菌去离子水中冲洗。 To 95V / V% solution, such as pure solutions (e.g. ethanol) in the rinsing, and / or in sterile deionized water rinse. 灭菌步骤可以在不会使植物材料致死的任何温度下进行，优选为约20°C至约42°C。 Sterilization step may be in the plant material does not make any lethal temperature, preferably about 20 ° C to about 42 ° C.

[0072] 可以使用多种年龄的干燥外植体（在低湿气条件下从种子切下的外植体）或经干燥的湿外植体（在水合/吸入后从种子切下且随后脱水和储存的外植体）。 [0072] You can use a variety of ages and dried explants (under low moisture conditions in explants from seed cut) or dried wet explants (after hydration / dehydration inhalation and subsequently cut from the seed and Storage explants). 在一些实施例中，外植体相对“年轻”，因为其在使用之前从种子移除不到一天，例如约1至M小时，例如约2、3、5、7、10、12、15、20或23小时。 In some embodiments, explants relatively "young", because it removed less than a day, for example, about 1 to M-hour from the seeds before use, for example, about 2,3,5,7,10,12,15, 20 or 23 hours. 在一些实施例中，视用于保持外植体活力的储存条件而定，外植体可能储存了较长时间，包括数天、数周、数月或甚至数年。 In some embodiments, depending on the explants for maintaining the vitality of the storage conditions, the explants may be stored for a long time, including the number of days, weeks, months or even years. 所属领域的技术人员可以理解，可以使储存时间最优化，从而可以获得有效愈合组织形成。 Those skilled in the art will appreciate, can optimize storage time, can be obtained efficiently callus formation.

[0073] 在一些实施例中，干燥种子或外植体可以首先例如通过吸入例如水或灭菌液体等液体注液、再干燥且随后用于产生愈合组织。 [0073] In some embodiments, the dried seeds or the explants e.g. by inhalation may be first sterile liquid such as water or liquid such as liquid injection, and then dried and subsequently used to produce callus.

[0074] 外植体可以从水合种子、干燥耐储存种子、干燥的水合外植体的部分再水合（其中“水合”和“再水合”定义为种子内部湿气百分比的可测量变化）、或“注液”的种子采出； 即种子已开始发芽但已适当放在停滞未决有利条件下来完成发芽过程。 [0074] Explants from hydrated seeds, dried storable seed, partially rehydrated dried hydrated explant (where "hydrated" and "rehydration" is defined as the percentage of moisture inside the seed measurable changes), or seed "pouring" of recovery; that seed has begun to sprout but has been stalled pending favorable conditions for an appropriate place to complete the germination process down. 所属领域的技术人员将能够使用各种水合方法且使诱发愈合组织之前的培育时间的长度最优化。 Those skilled in the art will be able to use various hydration methods and that the length of incubation time before the induced callus optimization. 所得新颖外植体可储存且可发芽和/或用于在提供了适当条件时诱发愈合组织形成。 The resulting novel explants stored and can germinate and / or for providing the appropriate conditions to induce the formation of callus. 因此，新干燥可储存分生组织外植体可以称作人工种子。 Therefore, the new store dried meristem explants can be called artificial seed.

[0075] 在适当植物培养基中培养外植体选择以促进愈合组织形成。 [0075] explants cultured in appropriate plant selection medium to promote healing tissue. 举例来说，植物培养基可为MS/B5培养基（村重(Murashige)和斯库格(Skoog). 1962.植物生理学(Physiol Plant) 15 :473-497 ；甘博格（Gamborg)等人，1968.实验细胞研究（Exp Cell Res) 50 ： 151-158，其各以全文引用的方式并入本文中），所述培养基补充有植物生长素和包括胺基酸、碳水化合物和盐等养料。 For example, a plant culture medium for MS / B5 medium (village weight (Murashige) and Skoog (Skoog) 1962. Plant Physiology (Physiol Plant) 15:. 473-497; Gan Borg (Gamborg) et al. 1968. Experimental Cell Research (Exp Cell Res) 50:. 151-158, which all manner entirety herein incorporated by reference), supplemented with auxin and including amino acids, carbohydrates and salts nourishment. 已知当适当补充时支持植物组织生长和发育的多种组织培养基，包括由外植体选择形成愈合组织。 Supports a variety of tissue culture plant tissue growth and development of the known replenished when appropriate, including the explants formed callus selection. 所述组织培养基可以作为商业制剂购买或由所属领域的技术人员常规制备和改质。 The tissue culture media can be purchased or by those skilled in the art conventionally prepared and modified as a commercial preparation. 所述培养基的实例包括（但不限于）村重和斯库格(1962.植物生理学15 =473-497)；楚(Chu)等人(1975.中国科学(Scientia Sinica) 18 ： 659-668)；林斯麦尔（Linsmaier)和斯库格(Skoog) (1965.植物生理学（Physiol Plant) 18 :100-127)；内宫有里(Uchimiya)和村重(Murashige) (1962.植物生理学(Plant Physiol) 15 ：73)；甘博格等人(1968.实验细胞研究50 :151-158)；顿坎(Duncan)等人(1985.植物（Planta) 165 :322-332)；劳埃德(Lloyd)和麦肯(McCown) (1981. ProcAnt Plant Propagator' s Soc 30:421-427)；尼特克(Nitsch)和尼特克(Nitsch) (1969.科学（Science) 163 :85-87)；以及斯肯克(Schenk)和希尔德布兰德(Hildebrandt) (1972. Can J Bot 50 ： 199-204)所述的培养基；前述文献各自以全文引用的方式并入本文中。 Examples of the medium include (but are not limited to) the village of weight and Skoog (1962. Plant Physiology 15 = 473-497); Chu (Chu), et al (1975. Chinese science (Scientia Sinica) 18: 659-668 ); Springs Maier (Linsmaier) and Skoog (Skoog) (1965. Plant Physiology (Physiol Plant) 18: 100-127); the inner palace Yuri (Uchimiya) village weight (Murashige) (1962. Plant Physiology (Plant Physiol) 15: 73); Gan Borg et al. (1968. Experimental Cell Research 50: 151-158); Dayton Hom (Duncan) et al. (1985. Plant (Planta) 165: 322-332); Lloyd Germany (Lloyd) and McCann (McCown) (1981. ProcAnt Plant Propagator 's Soc 30: 421-427); Nite Ke (Nitsch) and Nite Ke (Nitsch) (1969. Science (Science) 163: 85- 87); and (1972. Can J Bot 50 Si Kenke (Schenk) and Hildebrand (Hildebrandt): Medium 199-204) said; their entirety by the foregoing documents are incorporated by reference herein. 所属领域的技术人员可作相应补充以制造出这些培养基的衍生物所属领域的技术人员了解， 用于转型和再生的培养基和例如养料和生长调节剂等培养基补充通常针对特定目标作物或相关变种最优化。 Those skilled in the art can make the appropriate culture medium supplemented with manufacturing these derivatives of ordinary skill in the art will appreciate, for the transformation and regeneration of the medium and regulating agents such as nutrients and growth medium was supplemented with usual crop specific target or Related variant optimization. 组织培养基可以补充有碳水化合物，例如（但不限于）葡萄糖、蔗糖、 Tissue culture media can be supplemented with carbohydrates, such as (but not limited to) glucose, sucrose,

15麦芽糖、甘露糖、果糖、乳糖、半乳糖和/或右旋糖，或一定比率的碳水化合物。 15 maltose, mannose, fructose, lactose, galactose, and / or dextrose, or a certain percentage of carbohydrates. 试剂市场有售且可以购自多个供应商（参看例如西格马化工公司（Sigma Chemical Co.)，圣路易斯(¾. Louis)，密苏里州(Mo.)；和植物科技实验室(PhytoTechnology Laboratory),肖尼(Shawnee Mission)，堪萨斯州（Kans.))。 Reagents are commercially available and can be purchased from multiple vendors (see, for example Sigma Chemical Company (Sigma Chemical Co.), St. Louis (¾ Louis), Missouri (Mo.);. And Plant Technology Laboratory (PhytoTechnology Laboratory) Shawnee (Shawnee Mission), Kansas (Kans.)). 可以包括其他适合植物生长素，包括（但不限于） 麦草畏（dicamba)2，4-二氯苯氧基乙酸（〃 2，4_D")等。愈合组织诱发形成可以视外植体选择而定，且可以根据所属领域的技术人员熟知的方案选择和最优化。 It may include other suitable auxin, including (but not limited to) dicamba (dicamba) 2,4- dichlorophenoxy acetic acid (〃 2,4_D "), etc. can be subject to induce the formation of callus explant selection may be and can be selected and optimized in accordance with the skilled person familiar with the plan.

[0076] 愈合组织形成的评估 [0076] the formation of healing tissue assessment

[0077] 在出现第一传代培养之前评估各基因型产生愈合组织的能力。 [0077] In the first subculture appear before evaluating different genotypes generated ability to heal tissue. 可以通过观测每个产生愈合组织的基因型的外植体数目来测定最多产细胞系。 It can be determined by observing the number of genotypes of each generation to heal tissue explant most productive cell lines. 在约30天之后，相关数值量表可应用于愈合组织。 In about 30 days after the relevant value scale can be used to heal tissue. 举例而言，视外植体产生的愈合组织的量而定，数值量表可以由1至5 的额定值组成。 For example, depending on the amount of callus explant may be generated, numerical rating scale from 1 to 5 may be made of the composition. 例示性额定值5可以表示外植体产生大量愈合组织，而额定值1指定为可见愈合组织产量极低的外植体。 Exemplary Rating 5 can represent a large amount of explants callus tissue, and is designated as a rated output of low visibility callus explant. 在定额后，移除每一个愈合组织且传代培养。 After the quotas are removed and each callus subculture. 每一个外植体产生的愈合组织可以识别为个别细胞系。 Each callus explant produced can be identified as individual cell lines. 举例来说，可每两周或三周进行每一个愈合组织的传代培养。 For example, it can be performed every two or three weeks a callus subculture.

[0078] 对除草剂的剂量反应的评估 [0078] evaluation of the dose-response herbicides

[0079] 通过将每一个待测试基因型的愈合组织放在一系列诱发培养板上来评定用于筛选抗性愈合组织的适当除草剂浓度，所述培养板具有不同浓度的除草剂。 [0079] By each genotype to be tested in a series of induced callus culture plates to assess the proper concentration of the herbicide-resistant calli for screening, the culture plates with different concentrations of herbicide. 剂量反应分析中测试的除草剂浓度范围优选为预测致死剂量的O至15倍，更优选为预测致死剂量的2至10 倍，且通常为预测致死剂量的约3至5倍。 Herbicide concentration in the range of the dose-response analysis of the test is preferably O to 15 times the lethal dose of prediction, the predicted lethal dose is more preferably 2 to 10 times, and usually lethal dose prediction about 3-5 times. 如剂量反应分析所测定，打算用于筛选抗性愈合组织的除草剂浓度可以比无对照愈合组织生长的最小剂量大30-50%。 Measured as dose-response analysis, intended for screening tissue concentration of herbicide resistant healing can heal the smallest dose tissue growth 30-50% compared with no controls.

[0080] 除草剂抗性细胞的选择 [0080] selection of herbicide-resistant cells

[0081] 为了选择除草剂抗性细胞，如剂量反应分析所测定，可以将成熟愈合组织放在含有适当除草剂浓度的愈合组织培养基中。 [0081] In order to select herbicide resistant cells, as measured by dose response analysis can be mature callus on the callus culture medium containing the appropriate concentration of the herbicide. 在筛选过程期间，可以将愈合组织传代培养至新鲜培养板中。 During the screening process, it can heal tissue subcultured to fresh culture plates. 在识别抗性愈合组织之后，可以将其传代培养至诱发培养基上以供额外生长， 这足以支持再生。 After identifying resistant calli can be subcultured onto a medium for additional growth induced, which is sufficient to support regeneration.

[0082] 除草剂抗性细胞再生成完整植物 [0082] Herbicide-resistant cells to regenerate a whole plant

[0083] 自植物培养基移除愈合组织，且接种于适当再生培养基上。 [0083] removed from plant callus culture and seeded on a suitable regeneration medium. 已知多种组织培养基在适当补充时支持植物组织生长、发育和再生。 Known to support a variety of tissue culture media supplemented when appropriate plant tissue growth, development and regeneration. 所述组织培养基可以作为商业制剂购买或由所属领域的技术人员常规制备和改质。 The tissue culture media can be purchased or by those skilled in the art conventionally prepared and modified as a commercial preparation. 所述培养基的实例包括（但不限于）上文所列出的培养基。 Examples of the medium include (but are not limited to) the media listed above. 作为非限制性实例，可以通过将每一个抗性细胞系的愈合组织放在培养基上再生海雀稗，所述培养基由补充有1.2%ig L—1 CuSOjP 1. 125!^/!/1 BAP(6-苯甲基胺基嘌呤） 的MS/B5基础培养基组成。 As a non-limiting example, the healing of tissue by a resistant cell lines on seashore paspalum regeneration medium supplemented with a medium consisting of 1.2% ig L-1 CuSOjP 1. 125! ^ /! / 1 BAP (6- benzyl amino purine) in the MS / B5 basal medium composition. 再生培养基可以视植物组织来源、以及所属领域技术人员已知的适当再生培养基的选择和再生方案而定。 Plant tissue regeneration medium may optionally sources, and selection and regeneration programs known to those skilled in the appropriate regeneration medium may be.

[0084] 再生可在容器中的固体或液体培养基上进行，所述容器例如是皮氏培养皿、烧瓶、 槽或任何其它用于培养的合适腔室。 [0084] In the regeneration vessel can be a solid or a liquid culture medium, e.g., the container is a petri dish, a flask, grooves or any other chamber suitable for cultivation. 容器可视情况被密封（例如用过滤胶带），以便促进再生植物的气体交换。 Optionally sealed container (e.g., by filtration tape), in order to promote plant regeneration gas exchange. 生长室条件可介于约20°C或更低到40°C或更高之间。 Growth chamber conditions can be between about 20 ° C or lower to between 40 ° C or higher. 在一些实施例中，合适的生长温度可在约22°C到37°C、约25°C到35°C或约到32°C的范围内。 In some embodiments, the appropriate growth temperature of about 22 ° C to 37 ° C, about 25 ° C to 35 ° C or about 32 ° C within the range of. 黑暗：光照暴露可在约1小时黑暗：23小时光照到约12小时黑暗或更长：12小时光照或更短的范围内。 Dark: light exposure can range from about one hour of darkness: 23 hours of light to about 12 hours of darkness or longer: the range of 12 hours of light or less. 在一些实施例中，黑暗：光照暴露可在约2小时黑暗：22小时光照到约10小时黑暗：14小时光照、约4小时黑暗：20小时光照到约8小时黑暗：16小时光照的范围内。 In some embodiments, the dark: light exposure can range from about two hours of darkness: 22 hours of light to about 10 hours of darkness: 14 hours of light, about 4 hours of darkness: 20 hours of light to about eight hours of darkness: 16 hours of light in the range of . 黑暗：光照暴露之后可存在约1小时到10小时黑暗、约2小时到8小时黑暗或约4 小时到6小时黑暗中的任一种。 Dark: After light exposure may be present from about 1 hour to 10 hours of darkness, about 2 hours to 8 hours of darkness, or about four hours to six hours in the dark either. 在一些实施例中，黑暗期之后可存在其他黑暗周期：光照暴露之后可存在任何适于再生的组合的黑暗暴露。 In some embodiments, there may be other dark period after the dark period: the presence of darkness after light exposure can regenerate any suitable combination of exposures. 根据所属领域中熟知的方案选择适当的光强度以促进生长。 Select the appropriate light intensity according to relevant programs known in the art to promote growth. 举例来说，为了促进海雀稗的生长和再生，可对生长中的植物提供大约等于通用（GE)冷白光灯泡在66-95 μ E πΓ2 s—1的强度下提供的光强度。 For example, in order to promote seashore paspalum growth and regeneration, growing plants can provide approximately equal Universal (GE) light intensity cool white bulb at 66-95 μ E πΓ2 s-1 intensity provided.

[0085] 再牛棺物的子代 Progeny [0085] and then cattle coffin matter

[0086] 再生植物可无性繁殖。 [0086] regenerated plants can be asexual reproduction. 举例来说，再生植物可自我授粉。 For example, self-pollinate regenerated plants. 在一些实施例中，花粉可从再生植物获得，且与具有第二种所要特性的另一种植物的种子生长植物杂交。 In some embodiments, the pollen can be obtained from regenerated plants, and plant seeds with another having a second characteristic of the growth of plants to be hybridized. 在一些实施例中，花粉可从具有第二种所要特性的植物获得且用于对再生植物授粉。 In some embodiments, the pollen can be obtained from a plant having a second desired characteristics and is used to pollinate regenerated plants. 再生植物的子代可例如是种子或繁殖扦插物，其中再生植物的除草剂抗性是从亲本遗传得到。 The progeny of regenerated plants can, for example, seeds or cuttings propagation, wherein the herbicide-resistant plant regeneration is obtained from the parent inheritance. 此外，再生植物可自我杂交或近亲杂交以产生抗性等位基因的纯合植物品系。 In addition, self-regeneration plant to produce hybrid or inbred resistance allele of homozygous plant lines. 在一些情况下，所述纯合植物可具有比初始选择的杂合植物高的抗性水平。 In some cases, the homozygous plants can have a heterozygous plants higher level of resistance than initially selected.

[0087] 营养繁殖可通过使用草皮、楔形枝、小枝和匍匐茎实现。 [0087] vegetative propagation through the use of turf, wedge-shaped branches, twigs and stolons implementation. 当用于草坪草品种时，所述草的营养繁殖产生的子代通常是纯系的（遗传上相同）。 When used in turf grass species, the offspring of the grass is usually generated by vegetative propagation (genetically identical) pure line. 纯系营养品种产生外观非常均 Nutrition produce pure line varieties look very average

一的草皮。 A turf.

[0088] 某些品种仅由营养方法繁殖；具有这种特征的例示性品种包括观赏植物、小果和树。 [0088] Certain breeds only from vegetative propagation methods; with this feature Exemplary species include ornamental plants, small fruit and trees.

[0089] 除草剂抗件的分子表征 Molecular characterization [0089] Anti-member herbicide

[0090] 植物中导致除草剂抗性的突变可通过从植物组织提取DNA和随后进行PCR扩增来表征。 [0090] plant herbicide resistance mutations caused by extraction of DNA from plant tissue and subsequent PCR amplification characterization. 植物DNA可经由许多DNA提取方法提取，例如CTAB方法（莱斯纳（Lassner)等人，1989.植物分子生物学报道(Plant Mol. Biol. R印.）7 ： 116-128，其以全文引用的方式并入本文中）、SDS-乙酸钾方法（得拉波塔（Deilaporta)等人1983.植物分子生物学报道（Plant Molecular Biology Reporter) 1 ： 19-21，其以全文引用的方式并入本文中）、 叶组织的直接扩增（波托姆（Berthomieu)和梅耶（Meyer) 1991.植物分子生物学（Plant Molecular Biology) 17 :555_557，其以全文引用的方式并入本文中）、烧沸方法(伊可达(Ikeda)等人2001.植物分子生物学报道(Plant Molecular Biology Reporter) 19(1)： 27-32，其以全文引用的方式并入本文中）、碱性处理方法（辛（Xin)等人2003.生物技术(BioTechniques) 34 :820_826，其以全文引用的方式并入本文中）、FTA.®卡片或任何其它有效的用于植物的DNA提取方法。 DNA can be extracted via a number of plant DNA extraction methods, such as CTAB method (Leissner (Lassner) et al., 1989 Plant Molecular Biology reported (Plant Mol Biol R India) 7:.... 116-128, reference in its entirety which is incorporated herein), SDS- potassium acetate method (available Rapota (Deilaporta) et al. 1983. Plant Molecular Biology reported (Plant Molecular Biology Reporter) 1: 19-21, which is incorporated by reference in its entirety direct amplification of this article), leaf tissue (Botuo Mu (Berthomieu) and Meyer (Meyer) 1991. Plant Molecular Biology (Plant Molecular Biology) 17: 555_557, which in its entirety is incorporated by reference herein). boiling method (Yi up (Ikeda), et al. 2001. Plant Molecular Biology reported (Plant Molecular Biology Reporter) 19 (1): 27-32, which is incorporated by reference in its entirety herein), alkali treatment (oct (Xin) et al. 2003. Biotechnology (BioTechniques) 34: 820_826, which is incorporated by reference in its entirety herein), FTA.® card or any other effective method of DNA extraction for plants. 用于启动赋予除草剂抗性的DNA区域的PCR扩增的引物可被设计成匹配最大数量的可能相关物种的保守侧接序列。 Primers for imparting herbicide resistance to start DNA region amplified by PCR can be designed to match the maximum possible number of related species conserved flanking sequences.

[0091]鉴另Ilij乙酰辅Sl A羧化Sfeffl泡丨齐卯余丨抗件相关的突变 [0091] Another Ilij acetyl Kam Fu Sl A carboxylated Sfeffl bubble Shu Qi Mao Yu Shu anti pieces associated mutations

[0092] 可评估通过本文揭示的方法鉴别为对乙酰辅酶A羧化酶抑制剂除草剂具有抗性的植物的乙酰辅酶A羧化酶基因内的基因突变。 [0092] can be assessed by the methods disclosed herein identified as acetyl coenzyme A carboxylase inhibitor herbicide gene mutation acetyl coenzyme A carboxylase resistant genes within the plant. 举例来说，在一些实施例中，基因突变可导致乙酰辅酶A 羧化酶蛋白在残基Gin 1756、He 1781、Trp 1999、Trp 2027、lie 2041、Asp 2078,Cys 2088和/或Gly 2096上的突变。 For example, in some embodiments, gene mutations can lead to acetyl coenzyme A carboxylase protein residues Gin 1756, He 1781, Trp 1999, Trp 2027, lie 2041, Asp 2078, Cys 2088 and / or the Gly 2096 mutations. 在一些实施例中，那些残基上的突变可包括（但不限于）亮氨酸、丙氨酸、缬氨酸、半胱氨酸、天冬氨酸、甘氨酸、精氨酸和谷氨酸。 In some embodiments, the mutated residues that may include (but are not limited to) leucine, alanine, valine, cysteine, aspartic acid, glycine, arginine and glutamic acid . 在一些实施例中，乙酰辅酶A羧化酶蛋白内的氨基酸取代可例如是Gin 1756取代为Glu、He 1781 取代为Leu、He 1781 取代为Ala、He 1781 取代为Val、iTrp 1999 取代为Cys、iTrp 2027 取 In some embodiments, the amino acid acetyl coenzyme A carboxylase protein may be, for example, the substitution of Gin 1756 substitution Glu, He 1781 was substituted with Ala substitution Leu, He 1781, He 1781 replaced by Val, iTrp 1999 replaced by Cys, Take iTrp 2027

17代为Cys、He 2041 取代为Asp、He 2041 取代为Val、Asp 2078 取代为Gly、Asp 2078 取代为Val、Cys 2088取代为Arg和/或Gly 2096取代为Ala等。 17 on behalf of Cys, He 2041 replaced by Asp, He 2041 replaced by Val, Asp 2078 replaced by Gly, Asp 2078 replaced by Val, Cys 2088 unsubstituted substituted with Arg and Ala / Gly 2096 or the like. 在一些实施例中，氨基酸取代可为例如上文所述位置的位置上的两个或两个以上突变的组合，涉及上文所述位置的改变。 In some embodiments, amino acid substitutions can be, for example, the position of the above two or location on a combination of the above mutations, involving changing the position of the above. 同样地，在一些实施例中，可在这些位置和/或所属领域技术人员已知可作为乙酰辅酶A羧化酶与乙酰辅酶A羧化酶抑制剂之间的接触或相互作用的位置的其它位置上进行其它保守取代。 Likewise, in some embodiments, may be in these positions and / or known to persons skilled in the art can be used as contact or other interaction acetyl coenzyme A carboxylase and acetyl coenzyme A carboxylase inhibitors position between perform other conservative substitution position.

[0093] 导致ACC蛋白中氨基酸取代的乙酰辅酶A羧化酶基因中的突变包括表C中所列的突变。 [0093] result in ACC protein amino acid substitution acetyl coenzyme A carboxylase gene mutations include mutations listed in Table C.

[0094] 表C.与乙酰辅酶A羧化酶抑制剂除草剂抗性有关的氨基酸取代的总结 [0094] Table C. and acetyl coenzyme A carboxylase inhibitor herbicide-resistant amino acid substitutions related summary

[0095] [0095]

[0096] 此外，乙酰辅酶A羧化酶除草剂抗性可通过任何所述氨基酸位置上的任何保守取代赋予。 [0096] In addition, acetyl coenzyme A carboxylase herbicide resistance by any Conservative position on any of the amino acid substitution conferred. 表D提供保守取代的列表。 Table D provides a list of conservative substitutions.

[0097] 表D.保守氨基酸取代 [0097] Conservative amino acid substitutions in Table D.

[0098] [0098]

[0099] 评估对除草剂的总体棺物抗件 [0099] The overall assessment of the herbicide versus coffin pieces

[0100] 可通过比较除草剂抗性细胞系与除草剂敏感对照株的除草剂暴露效果来评估总体植物除草剂抗性。 [0100] to assess the overall effect of plant herbicide resistance can be exposed by comparing the herbicide herbicide and herbicide resistant cell lines sensitive control strain. 除草剂暴露可通过用各种施用量（在相关物种的已知致命剂量的0到20倍的范围内）的除草剂处理除草剂抗性植物和除草剂敏感对照株来实现。 Herbicide exposure can be administered by a variety of content (in the range of 0-20 times the related species known lethal dose) of the herbicide-resistant plant herbicides and herbicides sensitive control strain to achieve.

[0101] 除草布丨抗件 [0101] The herbicidal Bushukangjian

[0102] 本发明实施例涉及本文所揭示的在用于在商业应用的植物中产生除草剂抗性的方法和组合物。 Implementation [0102] The present invention disclosed herein relates to methods and compositions for producing herbicide resistance in plants in commercial applications. 在本发明的实施例中，选择且鉴别抗乙酰辅酶A羧化酶抑制剂除草剂的植物。 In an embodiment of the present invention, the selection and identification of anti-acetyl coenzyme A carboxylase inhibitor herbicide plant.

[0103] 已知乙酰辅酶A羧化酶（ACCase)以两种形式存在：真核和原核形式。 [0103] Known acetyl coenzyme A carboxylase (ACCase) exists in two forms: eukaryotic and prokaryotic form. 原核形式由四个亚单位构成，而真核形式为具有独特功能域的单个多肽（哈伍德（Harwood)等人1988. 植物分子生物学（Plant Molecular Biology) 39 ： 101-138，其以全文引用的方式并入本文中）。 Prokaryotic form consists of four subunits, and eukaryotic form of a single polypeptide (Harwood (Harwood) with unique functional domains, et al. 1988. Plant Molecular Biology (Plant Molecular Biology) 39: 101-138, cited in its entirety which is incorporated herein by reference). 乙酰辅酶A在脂质生物合成的第一个关键步骤中被乙酰辅酶A羧化酶羧化形成丙二酰辅酶A。 Acetyl coenzyme A critical first step in lipid biosynthesis is acetyl coenzyme A carboxylase malonyl-CoA carboxylase to form A. 在大多数植物中，乙酰辅酶A羧化酶划分为两种形式（佐佐木（Sasaki)等人1995.植物生理学(Plant Physiology) 108 :445_449，其以全文引用的方式并入本文中）。 In most plants, acetyl coenzyme A carboxylase is divided into two forms (Sasaki (Sasaki), et al. 1995. Plant Physiology (Plant Physiology) 108: 445_449, which in its entirety is incorporated by reference herein). 已知叶绿体为脂质合成的主要场所；但乙酰辅酶A羧化酶也可存在于细胞溶质中。 It is known primarily as a place chloroplast lipid synthesis; however acetyl coenzyme A carboxylase also be present in the cytosol. 大多数植物在叶绿体中具有原核形式，且在细胞溶质中具有真核形式。 Most plants have chloroplasts form of prokaryotic and eukaryotic forms having the cytosol. 四聚原核蛋白由四个独特基因编码，一个位于叶绿体基因组中。 Nucleotide tetramer composed of four distinct gene encoding a chloroplast genome located. 真核形式由大小为约12，OOObp的核基因编码（波德文斯基(Podkowinski)等人1996.美国科学院院报(PNAS)93 :1870_1874，其以全文引用的方式并入本文中）。 Eukaryotic form determined by the size of about 12, OOObp nuclear genes encoding (Pod Stravinsky (Podkowinski) et al. 1996. National Academy of Sciences (PNAS) 93: 1870_1874, which in its entirety is incorporated by reference herein). 草的独特性在于在细胞溶质与叶绿体中均可见真核形式的乙酰辅酶A 羧化酶（前述佐佐木（Sasaki)等人1995)。 Grass is unique in that in the cytosol and the chloroplast are visible eukaryotic form of acetyl coenzyme A carboxylase (the aforementioned Sasaki (Sasaki) et al. 1995). 在草的质体和细胞溶质中，真核形式的乙酰辅酶A羧化酶很类似，编码其之基因也很类似（刚尼奇（Gornicki)等人1994.美国科学院院报（PNAS)91 :6860-6864,其以全文引用的方式并入本文中）。 In the grass plastid and cytosol, the eukaryotic form of acetyl coenzyme A carboxylase is very similar, it is also very similar gene coding (just Nicky (Gornicki) et al. 1994. National Academy of Sciences (PNAS) 91: 6860-6864, which is incorporated in its entirety by reference herein). 然而，尽管质体和细胞溶质中真核形式的乙酰辅酶A羧化酶之间具有同源性，但细胞溶质形式不受抑制乙酰辅酶A羧化酶的除草剂影响（德尔叶(Delye) 2005.植物生理学(Plant Physiology) 137 :794-806,¾ 以全文引用的方式并入本文中）。 However, in spite of homology the plastid and the cytosol between eukaryotic form of acetyl coenzyme A carboxylase, but not cytosolic form of acetyl coenzyme A carboxylase inhibiting herbicides influence (Bender leaves (Delye) 2005 physiology (Plant Physiology) 137: 794-806, ¾ incorporated by reference in its entirety herein by reference).

[0104] 充当乙酰辅酶A羧化酶（ACCase)抑制剂的除草剂可阻断植物的脂质生物合成，从而造成活性生长区（诸如分生组织）的膜被破坏。 [0104] serves as acetyl coenzyme A carboxylase (ACCase) inhibitors block the herbicide plant lipid biosynthesis, resulting in active growth areas (such as meristem) of the film is destroyed. 乙酰辅酶A羧化酶抑制剂例如丙酸芳氧基苯氧酯（APP)化学家族（也称作F0P)和环己二酮（CHD)家族（也称作DIM)。 Acetyl coenzyme A carboxylase inhibitors such as propionic acid aryloxyphenoxyalkyl ester (APP) chemical family (also known as F0P) and cyclohexanedione (CHD) family (also known as DIM).

[0105] 因此，本发明实施例涉及选用于抗乙酰辅酶A羧化酶抑制剂除草剂的植物和其鉴别方法。 [0105] Accordingly, embodiments of the invention selected for anti-acetyl coenzyme A carboxylase inhibitor herbicide plant and its identification method involves. 在一些实施例中，植物抗环己二酮除草剂、丙酸芳氧基苯氧酯除草剂、苯基吡唑啉除草剂或其混合物。 In some embodiments, the plant anti-cyclohexanedione herbicide, an aryloxy phenoxy propionic acid herbicide ester, phenyl-pyrazoline herbicides, or mixtures thereof. 在一些实施例中，植物对至少一种选自表E中所提供的清单的除草剂具有抗性。 In some embodiments, the plant of at least one compound selected from the list in Table E provided in the herbicides.

[0106] 表E.乙酰辅酶A羧化酶抑制剂 [0106] Table E. acetyl coenzyme A carboxylase inhibitors

[0107] [0107]

19 19

[0108] Fusion w/Fluazifop [0108] Fusion w / Fluazifop

[0109] [0109]

Axial Axial

[0110] 除草用环己二酮包括（但不限于）稀禾定乙氧基亚氨基）-丁基]-5-[2-(乙硫基）丙基]-3_羟基-2-环己烯-1-酮，以名称P0AST™购自巴斯夫公司(BASF)(新泽西州帕斯攀尼(Parsippany, NJ)))、克草同（(E， E)-(士）-2-[1-[[(3_氯-2-丙烯基）氧基]亚氨基]丙基]-5-[2-(乙硫基）丙基]-3-羟基-2-环己烯-1-酮；以SELECT™购自雪佛龙化学公司（住友）（Chevron Chemical (Valent))(加利福尼亚州弗雷斯诺(Fresno, Calif.))),氯丙氧定（(E， E)-2-[l-[[(3-氯-2-丙烯基）氧基]亚氨基]丁基]-5-[2-(乙硫基）丙基]-3-羟基-2-环己烯-1-酮；以SELECT0NE™购自雪佛龙化学公司（住友）（Chevron Chemical(Valent))(加利福尼亚州弗雷斯诺(Fresno, Calif.)))和苯草酮(2_[1_(乙氧基亚氨基）丙基]_3_羟基-5-均三甲苯基环己烯-2-烯酮，以GRASP™购自美国陶氏化学公司（Dow Chemical USA) (密歇根州米德兰(Midland, Mich·)))。 [0110] The herbicidal cyclohexanedione including (but not limited to) sethoxydim ethoxy-imino) - butyl] -5- [2- (ethylthio) propyl] -2-hydroxy -3_ hexene-1-one, to name P0AST ™ available from BASF Corporation (BASF) (New Jersey Parsippany (Parsippany, NJ))), g grass with ((E, E) - (disabilities) -2- [ 1 - [[(3_-chloro-2-propenyl) oxy] imino] propyl] -5- [2- (ethylthio) propyl] -3-hydroxy-2-cyclohexene-1 one; to SELECT ™ available from Chevron Chemical Company (Sumitomo) (Chevron Chemical (Valent)) (. California Fresno (Fresno, Calif))), oxygen given chlorpromazine ((E, E) -2 - [l - [[(3- chloro-2-propenyl) oxy] imino] butyl] -5- [2- (ethylthio) propyl] -3-hydroxy-2-cyclohexen - 1-one; to SELECT0NE ™ available from Chevron Chemical Company (Sumitomo) (Chevron Chemical (Valent)) (. California Fresno (Fresno, Calif))) and benzene oxadiazon (2_ [1_ (ethoxymethyl Nokia amino) propyl] _3_ hydroxy-5-mesityl cyclohexene-2-enone to GRASP ™ available from The Dow Chemical Company (Dow Chemical USA) (Midland, Michigan (Midland, Mich ·))). 另外，除草用环己二酮包括(但不限于)环苯草酮、环杀草和得杀草。 In addition, the herbicidal cyclohexanedione include (but are not limited to) oxadiazon benzene ring, the ring and have herbicidal weed control.

[0111] 除草用丙酸芳氧基苯氧酯和/或芳氧基苯氧基丙酸展现针对植物的一般和选择性除草活性。 [0111] The herbicidal acid aryloxyphenoxyalkyl ester and / or aryloxyphenoxypropionic acid exhibit general and selective herbicidal activity against plants. 在这些化合物中，芳氧基可为苯氧基、吡啶基氧基或喹喏啉基。 Among these compounds, the aryloxy group may be phenoxy, pyridyloxy or quinoxaline group. 除草用丙酸芳氧基苯氧酯包括（但不限于）合氯氟G2-[4-[[3-氯-5-(三氟甲基）-2-吡啶基]氧基] 苯氧基]-丙酸），其以VERDICT™购自美国陶氏化学公司（Dow Chemical USA)(密歇根州米德兰（Midland. Mich.)))、禾草灵（（(+)-2-½-(2，4-二氯苯氧基）-苯氧基]丙酸）， 以H0EL0N™购自赫司特劳塞尔公司（Hoechst-Roussel Agri-Vet Company)(新泽西州萨默维尔（Somerville，NJ)))、恶唑禾草灵（（士）(6-氯-2-苯并恶唑基）氧基]苯氧基]丙酸；以WHIP™购自赫司特劳塞尔公司(Hoechst-Roussel Agri-Vet Company)(新泽西州萨默维尔（Somerville，NJ)))、吡氟禾草灵（（士）K4-[[5_(三氟甲基）_2_吡啶基]氧基]苯氧基）丙酸；以FUSILADE™购自ICI美洲公司（ICI Americas)(特拉华州威尔明顿（WilrningtomDel.)))、精吡氟禾草灵（(R)_2-[4-[[5_(三氟甲基）_2_吡啶基]氧基]苯氧基]丙酸；以FUSILADE 2000™购自ICI美洲公司（ICI Americas)(特拉华州威尔明顿（WilmingtomDel·)))、快伏草（（士）K4[ (6-氯-2-喹喏啉基）-氧基]苯氧基]丙酸；以ASSURE™购自杜邦公司(Ε. I. DuPont de Nemours)(特拉华州威尔明顿(Wilmington, Del.)))和炔草酸。 Herbicidal aryloxy phenoxy propionic acid ester include (but are not limited to) co-chlorofluorocarbon G2- [4 - [[3- chloro-5- (trifluoromethyl) -2-pyridinyl] oxy] phenoxy ] - propionic acid) which VERDICT ™ available from The Dow Chemical Company)), diclofop (((+) (Dow Chemical USA) (Michigan Midland (Midland Mich..) - 2-½- (2,4-dichlorophenoxy) - phenoxy] propionic acid) to H0EL0N ™ available from Hoechst Straw Searle company (Hoechst-Roussel Agri-Vet Company) (New Jersey Somerville (Somerville, NJ))), fenoxaprop ((disabilities) (6-chloro-2-benzoxazolyl) oxy] phenoxy] propionic acid; in WHIP ™ available from Hoechst Straw Searle company ( Hoechst-Roussel Agri-Vet Company) (New Jersey Somerville (Somerville, NJ))), fluazifop ((disabilities) K4 - [[5_ (trifluoromethyl) _2_ pyridyl] oxy] phenoxy) propionic acid; to FUSILADE ™, available from ICI Americas, Inc. (. ICI Americas) (Wilmington, Delaware (WilrningtomDel))), fine fluazifop ((R) _2- [4- [[5_ (trifluoromethyl) _2_ pyridyl] oxy] phenoxy] propionic acid; to FUSILADE 2000 ™, available from ICI Americas, Inc. (ICI Americas) (Wilmington, Delaware (WilmingtomDel ·) )), fast-volt grass ((disabilities) K4 [(6- chloro-2-quinoxalin-yl) - oxy] phenoxy] propionic acid; to ASSURE ™ available from DuPont (Ε I. DuPont de Nemours. ) (Wilmington, Delaware (Wilmington, Del.))) and clodinafop.

[0112] 除草用环己二酮或除草用丙酸芳氧基苯氧酯或除草用苯基吡唑啉的类似物 [0112] The herbicidal cyclohexanedione or herbicidal acid aryloxyphenoxyalkyl herbicidal phenyl ester or analogs pyrazoline

[0113] 乙酰辅酶A羧化酶抑制剂中包括结构上与本文所揭示的除草用环己二酮、除草用丙酸芳氧基苯氧酯或除草用苯基吡唑啉有关的除草剂，诸如类似物、代谢物、中间物、前驱物、盐等。 [0113] acetyl coenzyme A carboxylase inhibitors include structure disclosed herein herbicidal cyclohexanedione, herbicidal acid aryloxyphenoxyalkyl herbicidal phenyl ester or related pyrazoline herbicides, such analogs, metabolites, intermediates, precursors, salts and the like.

[0114] 用相关基因转化 [0114] transformed with the relevant genes

[0115] 在本文所揭示的方法中，分离特定DNA片段且克隆到载体中以供转化植物组织或细胞。 [0115] In the method disclosed herein, a specific DNA fragment isolated and cloned into a vector for transformation of plant tissue or cells. 举例来说，从A细胞系（实例）的乙酰辅酶A羧化酶基因分离384个碱基对片段，其中已鉴别到在乙酰辅酶A羧化酶蛋白的位置1781处异亮胺酸突变为亮胺酸(“Ilel781LeU” 或“I1781L”）。 For example, the separation from A cell line (example) acetyl-CoA carboxylase gene A 384 bp fragment, which had been in a position to identify the acetyl coenzyme A carboxylase protein of 1781 iso-leucine mutation bright tryptophan ("Ilel781LeU" or "I1781L"). 鉴别的所述片段可用于转化本文所揭示的植物组织和细胞。 The fragments identified can be used to transform plant tissue and cells disclosed herein.

[0116] 已开发各种方法将基因转移到植物组织中，包括（但不限于）高速显微喷射、显微注射、电穿孔、直接DNA吸收和细菌介导的转化。 [0116] Various methods have been developed to transfer genes into plant tissues, including (but not limited to) high-speed jet microscopy, microinjection, electroporation, direct DNA uptake and bacterial-mediated transformation. 已知介导植物细胞转化的细菌包括根瘤菌科(Rhizobiaceae)的多个种，包括（但不限于）土壤杆菌属（Agrobacterium sp.)、中华根瘤菌属（Sinorhizobium sp.)、中间根瘤菌属（Mesorhizobium sp.)和慢生根瘤菌属(Bradyrhizobium sp.)(例如布鲁斯阿茨（Broothaerts)等人，2005.自然（Nature) 433: 6四-633和美国专利申请公开案2007/0271627，其各自以全文引用的方式并入本文中）。 Known mediated transformation of plant cells comprising a plurality of species of bacteria Rhizobiaceae (Rhizobiaceae), including (but not limited to) Agrobacterium (Agrobacterium sp.), Sinorhizobium (Sinorhizobium sp.), Intermediate Rhizobium (. Mesorhizobium sp) and Mesorhizobium (such as Bruce Artz (Broothaerts) et al., 2005 Nature (Nature) 433 (Bradyrhizobium sp.):. 6 four -633 and U.S. Patent Application Publication 2007/0271627, which each incorporated by reference in its entirety herein). 所述转化的标靶可为未分化的愈合组织、已分化组织、源自特定细胞系的细胞群体等。 The target can be transformed callus undifferentiated, differentiated tissue, cells derived from cell lines and other specific groups. 共培养和后续步骤可在约23°C或低于25°C的温度下，且可在高达约35°C或40°C或高于40°C下，在黑暗条件下或在光（例如光亮的Percival培育箱）下执行2到5天（例如，光期为16小时光/8小时黑暗，光强度彡5 μ Ε,诸如约5-200 μ E或允许正常质体发育的其他照明条件）。 Co-cultivation and the subsequent steps can be at a temperature of about 23 ° C or lower than, 25 ° C, and can be up to about 35 ° C or 40 ° C or above 40 ° C below, in the dark or in the light (for example, execution 2-5 days (for example, a light period of 16 hours light / 8 hours dark shiny Percival incubator), the light intensity 彡 5 μ Ε, such as about 5-200 μ E or allow other normal lighting conditions plastid development ).

[0117] 含有经分离DNA片段的载体可含有多个有利于转化植物细胞或组织且调节结构核酸序列表达的遗传组分。 [0117] vector containing the isolated DNA fragments may contain more than one beneficial transformation of plant cells or tissues and regulate the expression of the genetic component of the structure of nucleic acid sequence.

[0118] 在一些实施例中，载体可含有可选择、可筛选或可评分的标记基因。 [01] In some embodiments, the carrier may contain selectable, screenable or scorable marker gene. 在本文中这些遗传组分也称作功能遗传组分，因为其产生提供鉴别已转化植物的功能的产物或具有农学效用的产物。 In this article these genetic components are also referred to as functional genetic components, because it produces provide identification function has been transformed plant products or with agronomic utility product. 充当选择或筛选手段的DNA可在可再生植物组织中用于产生以下化合物， 其将赋予植物组织对化合物的抗性，否则所述化合物对植物组织有毒。 Acts as a selection or screening means may be used in DNA renewable plant tissue to produce the following compound, which compound will provide the plant tissue resistance to otherwise toxic compounds on the plant tissue. 多个可筛选或可选择标记基因在所述领域中已知且可用于本发明。 A plurality of screening or selectable marker gene in the art and can be used in the present invention are known. 适用作标记的相关基因包括（但不限于）⑶S、绿色萤光蛋白（GFP)、萤光素酶（LUX)等。 Related genes useful as markers include (but are not limited to) ⑶S, green fluorescent protein (GFP), luciferase (LUX) and the like. 已知其他示范性标记且包括β-葡糖醛酸酶（GUQ，其编码各种发色底物的酶（杰佛森（Jefferson)等人1987.生物化学学会学报（Biochem Soc Trans) 15 :7_19 ；杰佛森（Jefferson)等人1987.欧洲分子生物学学会报告（EMBO J)6:3901-3907，其各自以全文引用的方式并入本文中）；R-基因座基因，其编码调节植物组织中花青甙色素（红色）产生的产物（德尔波特（Dellaporta)等人1988. 染色体结构与功能：新概念的影响（Chromosome Structure and Function ：Impact of New Concepts).第18 届斯塔德勒遗传学研讨会(18th Stadler Genetics Symposium) 11 ： 观3-观2，其以全文引用的方式并入本文中）；β-内酰胺酶基因（萨克里菲（Sutcliffe) 等人1978.美国国家科学院院刊（Proc Natl Acad Sci USA) 75 :3737_3741，其以全文引用的方式并入本文中）；已知编码各种发色底物的酶的基因（例如PADAC，发色头孢菌素(cephalosporin))；萤光素酶基因（欧文(Ow)等人1986.科学(Science) 234 :856-859, 其以全文引用的方式并入本文中）；编码可转化发色儿茶酚的儿茶酚双加氧酶的xylE基因(左瓦斯基(Zukowsky)等人1983.美国国家科学院院刊(Proc Natl Acad Sci USA)80 ： 1101-1105，其以全文引用的方式并入本文中）；α-淀粉酶基因（伊卡图Qkatu)等人1990.生物技术（Bio/TechnoDS :241-242，其以全文引用的方式并入本文中）；酪胺酸酶基因（卡茨(Katz)等人1983.普通与应用微生物学杂志(J Gen Microbiol) 129 =2703-2714, 其以全文引用的方式并入本文中），其编码能够将酪胺酸氧化成DOPA与多巴醌的酶，DOPA 和多巴醌又缩合成黑色素；绿色萤光蛋白（艾利欧（Elliot)等人1999.植物细胞报告(Plant Cell R印）18 :707_714，其以全文引用的方式并入本文中）和α半乳糖苷酶。如所属领域中所熟知，可使用植物转化的其他方法，例如米其（Miki)等人（1993.植物分子生• fgLii^：巾白勺力夕去(Methods in Plant Molecular Biology and Biotechnology), 格里克(Glick)与汤普森(Thompson)(编），CRC出版社(CRC Press)，公司(Inc.)：波卡若顿（Boca Raton)，第67-88页，其以全文引用的方式并入本文中）所述，包括使用微粒轰击（例如美国专利第5，914，451号；麦卡比(McCabe)等人1991.生物/技术(Bio/ Technology) 11 :596-598 ；美国专利第5，015，580号；美国专利第5，550，318号；和美国专利第5，538，880号；前述各文献以全文引用的方式并入本文中)。 Other exemplary tags are known and include β- glucuronidase (GUQ, which encodes a variety of chromogenic substrate of the enzyme (Jefferson (Jefferson), et al. 1987. Biochemical Society TECHNOLOGY (Biochem Soc Trans) 15: 7_19; Jefferson (Jefferson), et al. 1987. Report of the European Molecular Biology Society (EMBO J) 6: 3901-3907, which are each incorporated by reference in its entirety herein); R- locus gene, which encodes a regulator Plant tissue anthocyanin pigments (red) generated product (Delporte (Dellaporta) et al. 1988. chromosome structure and function: the impact of the new concept (Chromosome Structure and Function: Impact of New Concepts) 18th Starr. Chandler Genetics Symposium (18th Stadler Genetics Symposium) 11: View of 3- Concept 2, which in its entirety is incorporated by reference herein); β- lactamase gene (Sakelifei (Sutcliffe), et al. 1978. PNAS (Proc Natl Acad Sci USA) 75: 3737_3741, which in its entirety is incorporated by reference herein); a known gene encoding the enzyme substrates hair color (for example PADAC, chromogenic cephalosporin (cephalosporin)); luciferase gene (Owen (Ow), et al. 1986. Science (Science) 234: 856-859, which is incorporated by reference in its entirety herein); color-coding can be converted to catechol catechol dioxygenase of xylE gene (left Kowalski (Zukowsky), et al. 1983. PNAS (Proc Natl Acad Sci USA) 80: 1101-1105, which is incorporated by reference in its entirety herein by reference) ; α- amylase gene (Figure Ica Qkatu), et al. 1990. Biotechnology (Bio / TechnoDS: 241-242, which is incorporated by reference in its entirety herein); tyrosinase gene (Katz (Katz) et al. 1983. Common and Applied Microbiology Journal (J Gen Microbiol) 129 = 2703-2714, which in its entirety is incorporated by reference herein) encoding can be oxidized to DOPA and tyrosine dopaquinone enzyme, DOPA and dopaquinone and shrink melanin synthesis; green fluorescent protein (Yi Liou (Elliot), et al. 1999. Plant Cell Reports (Plant Cell R India) 18: 707_714, which is incorporated by reference in its entirety herein) and . α-galactosidase as well known in the art, other methods may be used for plant transformation, such as Mickey (Miki) et al. (1993. Plant Molecular Biology • fgLii ^: towel white spoon to force evening (Methods in Plant Molecular Biology and Biotechnology), Glick (Glick) and Thompson (Thompson) (series), CRC Press (CRC Press), the Company (Inc.): If Boca Raton (Boca Raton), p. 67-88, which incorporated by reference in its entirety herein), the use of microparticle bombardment comprising (e.g., U.S. Patent No. 5,914,451; McCabe (McCabe), et al. 1991. Bio / Technology (Bio / Technology) 11: 596 -598; U.S. Patent No. 5,015,580; U.S. Pat. No. 5,550,318; and U.S. Patent No. 5,538,880; each of which in the preceding entirety is incorporated by reference herein).

[0119] 转基因植物可通过所述领域中已知的方法和组合物从已转化植物细胞再生。 [0119] The transgenic plant by methods known in the art and compositions from a transformed plant cell regeneration. 举例来说，使用土壤杆菌转化方法形成的转基因植物通常含有插入一个染色体中的单个简单重组DNA序列，且称作转基因事件。 For example, formed using Agrobacterium transformation methods typically contains transgenic plant chromosome insert a single simple recombinant DNA sequence, and called transgenic event. 所述转基因植物可称作杂合有所插入的外源序列。 The transgenic plant can be referred to be heterozygous exogenous sequence inserted. 对于一种转基因的转基因植物纯合子可通过含有单个外源基因序列的独立分离的转基因植物与自身（例如R。植物）有性配对（自身受精）产生R1种子而获得。 For a genetically homozygous transgenic plants by transgenic plants containing a single foreign gene sequence of independent and separate itself (for example R. plant) sexual partners (self-fertilization) produces R1 seed obtained. 所产生的1/4的R1种子纯合有这个转基因。 R1 seed produced 1/4 homozygous have the transgene. R1种子发芽得到可测试接合性的植物，通常使用可区别杂合子与纯合子的SNP测定或热扩增测定（亦即接合性测定）。 R1 seed germination obtain a test engagement of plants, usually distinguishing heterozygous and homozygous SNP assay or hot amplification assay (ie engagement of the assay). 或者，可从数个R1植物产生&子代且对其进行测试，其中所有个体均具有抗性的均质&子代表示纯合R1亲本。 Alternatively, from several R1 plants produce & progeny and to test, in which all individuals are resistant homogeneous & progeny homozygous R1 represents parents.

[0120] 为证实转基因植物中外源DNA或“转基因”的存在，可执行多种测定。 [0120] To confirm the presence of DNA or "transgenic" exogenous transgenic plants, perform a variety of assay. 所述测定包括例如“分子生物”测定，诸如南方墨点法（Southern blotting)和北方墨点法（northern blotting)以及PCR、INVADER™测定；“生物化学”测定，诸如侦测蛋白产物的存在，例如通过免疫方法（ELISA和西方墨点法（western blot))或通过酶促功能；植物部分测定，诸如叶子或根测定；以及通过分析完整再生植物的表型。 The assays include, for example, "molecular biological" measurement, Southern blot (Southern blotting) and Northern blotting (northern blotting) and PCR, INVADER ™ assay such as; "biochemical" measurement, such as to detect the presence of the protein product, for example, by immunoassay (ELISA and western blot (western blot)) or by enzymatic function; plant parts determination, such as leaf or root determination; and by analyzing the phenotype of complete regeneration of plants.

[0121] 针对一种突变进行选择且在植物中证实之后，或将转基因引入植物中之后，突变或转基因可引入与第一个植物通过杂交有性相容的任何植物中，而无需在第二个植物中直接选择突变体或转化第二个植物。 After After [0121] For a confirmed mutation and selection in plants, or introduced into the transgenic plants, or transgenic mutations may be introduced first with any plant sexually compatible plant by hybridization, and without the need for a second a plant or converted directly select a mutant second plant. 因此，如本文所用之术语“子代”可表示源自本发明制备的亲本植物的任一代子代，其中子代包含所要基因型或表型，无论是转基因或非转基因。 Thus, as used herein the term "progeny" is derived from the preparation of the present invention can be expressed in any generation progeny parent plant, which contains the desired offspring genotype or phenotype, whether transgenic or non-transgenic. 视习知用法和/或常规定义而定，“转基因植物”因此可具有任何代。 Depending conventional usage and / or conventional definition set, "transgenic plant" and therefore can have no representative. 对植物进行“杂交”而提供相对于起始植物细胞系具有一或多个所选突变、表型和/或添加的转基因或等位基因的植物细胞系可通过将起始或基础植物与包含突变等位基因、转基因等的供体植物细胞系杂交而将特定序列引入植物细胞系中。 Plants are "hybrid" plant provided with respect to the starting cell line having the selected one or more mutations, the phenotype and / or addition of a transgenic plant cell lines or allele can be prepared by starting or basic plant comprising mutant allele, such as genetically modified plant line hybrid donor cells specific sequence introduced into a plant cell lines. 为达成这个目的，克例如执行以下步骤：(a)种植第一（起始细胞系）和第二（包含所要转基因或等位基因的供体植物细胞系）亲本植物的种子；(b)使第一和第二亲本植物的种子生长成具有花的植物；（c)用第二亲本植物的花粉对 To achieve this goal, grams such as the following steps: (a) planting (starting cell line) and a second parent plant seeds (containing a desired transgene or allele of the donor plant cell line) first; (b) make The first and second seed parent plant flower plants have grown; (c) with a second parent plant pollen

23第一亲本植物的花授粉；和（d)收集具有已受精花的亲本植物上产生的种子。 23 first parent plant pollination; and (d) the collection has been fertilized seeds produced on the parent plant flowers.

[0122]肖編細申·佘·_勿錄性诚 [0122] Xiao Shen · She compiled a fine record of honesty · _ Do

[0123] 去除不适宜杂草可通过用除草剂处理需要排他性生长抗性植物种的区域来完成， 其中已对这些除草剂具有抗性。 [0123] by removing inappropriate weeds treated with a herbicide-resistant plant requires exclusive area growing species to complete, which has been resistant to these herbicides. 因此，本发明实施例涉及在通过本文所揭示方法鉴别的除草剂抗性植物附近控制杂草的方法，包括：使至少一种除草剂与杂草和除草剂抗性植物接触，其中至少一种除草剂用足以抑制相同物种的非选择植物和/或需要抑制的杂草种的生长或使其死亡的速率接触杂草和植物。 Accordingly, the present invention embodiments of the method in the vicinity by the method disclosed herein to identify herbicide-resistant plant involves controlling weeds, comprising: at least one herbicide contact with weeds and herbicide-resistant plant, wherein at least one with a non-selective herbicide sufficient to inhibit plant and / or in need of inhibition of weed species or the same species so that the rate of growth of weeds and dead plants contacts. 非选择植物通常对除草剂无抗性。 Non-selective herbicide generally non-resistant plants.

[0124] 在一些实施例中，除草剂可直接与抗除草剂植物和杂草接触。 [0124] In some embodiments, the herbicide may be in direct contact with the herbicide-resistant plants and weeds. 举例来说，除草剂可直接在抗除草剂植物和杂草上撒播。 For example, the herbicide can be broadcast directly on herbicide-resistant plants and weeds. 或者，除草剂可直接在抗除草剂植物和杂草上喷洒。 Alternatively, the herbicide can be sprayed directly on the herbicide-resistant plants and weeds. 可将除草剂施用于抗除草剂植物和杂草的其他方法包括（但不限于）撒播或喷洒在含有抗除草剂植物和杂草的区域或地块上。 Other methods for applying herbicides herbicide-resistant plants and weeds may include (but are not limited to) broadcast or sprayed on the area or parcel containing the herbicide-resistant plants and weeds.

[0125] 在一些实施例中，可使除草剂接触抗除草剂植物和杂草所处的生长培养基或添加到这个生长培养基中。 [0125] In some embodiments, the contact can herbicide and herbicide-resistant plant or weed growth medium which added to the growth medium. 生长培养基可为（但不限于）土壤、泥煤、污泥、泥浆或砂子。 Growth medium for (but not limited to) the soil, peat, sludge, mud or sand. 在其他实施例中，除草剂可包括在灌溉植物的水中。 In other embodiments, the herbicide may be included in the water for irrigation of plants.

[0126] 通常，足以使非抗性植物或非选择植物生长或死亡的除草剂的量介于约2μ M或2μΜ以下到约ΙΟΟμΜ或ΙΟΟμΜ以上的除草剂浓度。 [0126] Typically, sufficient to select the amount of non-resistant plant or plant growth or death herbicide 2μΜ between about 2μ M or less to about ΙΟΟμΜ ΙΟΟμΜ or more herbicide concentration. 在一些实施例中，足量除草剂介于约5 μ M到约50 μ M除草剂浓度，约8 μ M到约30 μ M除草剂浓度，或约10 μ M到约25 μ M除草剂浓度。 In some embodiments, a sufficient amount of herbicide between about 5 μ M to about 50 μ M herbicide concentration of approximately 8 μ M to about 30 μ M herbicide concentration, or about 10 μ M to about 25 μ M Herbicide concentration. 或者，足以使非抗性植物生长或死亡的除草剂的量介于每公顷约25公克活性成分（g ai ha—1)到约6500g ai ha—1除草剂施用。 Alternatively, an amount sufficient to cause growth or death of a non-resistant plant herbicides is between about 25 grams per hectare of the active ingredient (g ai ha-1) to about 6500g ai ha-1 herbicide. 在一些实施例中，足量除草剂介于约50g ai ha—1到约5000g ai ha—1除草剂施用，约75g ai ha—1到约2500g ai ha—1除草剂施用，约IOOg ai ha-1 到约1500g ai ha-1 除草剂施用，或约250g ai ha-1 到约IOOOg ai ha-1 除草剂施用。 In some embodiments, a sufficient amount of herbicide between about 50g ai ha-1 to about 5000g ai ha-1 herbicide, about 75g ai ha-1 to about 2500g ai ha-1 herbicide, about IOOg ai ha -1 to about 1500g ai ha-1 herbicide, or about 250g ai ha-1 to about IOOOg ai ha-1 herbicide.

[0127] 标记物辅助的选择方法 [0127] marker-assisted selection method

[0128] 标记物辅助的选择（MAQ也称为分子育种或标记物辅助的育种（MAB)，是指通过检测植物中的一种或一种以上标记物来选择植物中所需性状的方法，其中标记物与所需性状有关联。在一些实施例中，用于MAS的标记物为分子标记物。在其它实施例中，标记物为如上所讨论的表型标记物。 [0128] marker-assisted selection (MAQ also called molecular breeding or marker-assisted breeding (MAB), refers to plants by detecting one or more markers to select the method desired traits in plants, wherein the marker associated with the desired trait. In some embodiments, markers for MAS as molecular markers. In other embodiments, the phenotypic marker is a marker as discussed above.

[0129] 在分子育种计划中，可使用遗传标记物等位基因来鉴别在一个标记物基因座、一些基因座或单倍型上含有所需基因型，因此预计将所需基因型连同相关所需表型一起传给子代的植物。 [0129] In the molecular breeding programs may be used to identify the genetic marker allele at one marker locus, some of the locus or haplotype containing the desired genotype on, it is expected that the desired genotype together with the relevant need phenotype together passed to offspring plants. 标记物可用于植物育种中，这是因为一旦这些标记物建立，其将不受环境或上位相互作用的影响。 Markers can be used in plant breeding, because once established these markers, it will not affect the environment or host interactions. 此外，某些类型的标记物适合于高通量检测，能够以成本有效的方式迅速地鉴别。 In addition, certain types of markers suitable for detection of high-throughput, cost-effective manner possible to rapidly discriminating.

[0130] 由于分子标记物中存在等位基因的差异，所以可以通过统计学评价分离群体的基因型和表型来鉴别数量性状基因座（quantitative trait loci，QTL)。 [0130] Since the allelic differences in molecular markers, it is possible to identify quantitative trait loci (quantitative trait loci, QTL) separated by statistical evaluation of genotype and phenotype groups. 定位QTL的方法为所属领域中所熟知，并描述于例如WO 90/04651 ；美国专利第5，492，547号、美国专利第5，981，832号、美国专利第6，455，758号；菲林·加西亚（Flint-Garcia)等人，2003植物生物学年鉴(Ann. Rev. Plant Biol.) 54 :357-374中，前述各参考文献以全文引用的方式并入本文中。 Location QTL approach to art for well known and are described, for example, WO 90/04651; U.S. Patent No. 5,492,547, US Patent No. 5,981,832, US Patent No. 6,455,758; Film Garcia (Flint-Garcia), et al., 2003 Yearbook of Plant Biology (Ann Rev. Plant Biol..) 54: 357-374, the foregoing references are incorporated by reference in its entirety herein. 在这些情况下，通过代替用基因分型进行高成本、时间密集的表型分析，使用标记物推断表型可使育种计划更为经济。 In these cases, the high cost of using genotyping performed by replacing, time-intensive phenotypic analysis using phenotypic markers can be inferred more economical breeding programs. 标记物方法允许在植物成熟之前选择，由此节省时间且有效地利用土地。 Marker allows selection before plant maturation, thereby saving time and efficient use of land. 也可以在种子水平上进行选择，以便种植优选种子（美国专利公开案第2005/000213435号和美国专利公开案第2007/000680611号，前述各案以全文引用的方式并入本文中）。 You can also select the seed level in order to planting seeds preferred (US Patent Publication No. 2005/000213435 and U.S. Patent Publication No. 2007/000680611, the foregoing case in its entirety is incorporated by reference herein). 另外，育种计划可以设计成通过靶向特定基因型，明确地推动具体、 有利的表型的频率（美国专利第6，399，855号，以全文引用的方式并入本文中）。 In addition, breeding programs can be designed by targeting particular genotype, specifically to promote specific, advantageous phenotype frequencies (U.S. Patent No. 6,399,855, incorporated by reference in its entirety herein). 可以连续地监测这些群落的准确度，以确保维持预测能力，从而告知培育决定（美国专利申请案2005/0015827,以全文引用的方式并入本文中）。 Continuously monitoring the accuracy of these communities, in order to ensure the maintenance of the predictive power, thereby informing foster decision (U.S. Patent Application 2005/0015827, incorporated by reference in its entirety herein).

[0131] 因此，本发明的实施例涉及标记物辅助的育种方法，这些方法包括：鉴别用于育种和选择的相关特征，其中此特征与乙酰辅酶A羧化酶基因有关联；提供携带能够赋予植物乙酰辅酶A羧化酶抑制剂除草剂抗性的乙酰辅酶A羧化酶序列变异体的第一植物，其中此植物另外包含此相关特征；将第一植物与第二植物一起培育；鉴别培育步骤中具有此乙酰辅酶A羧化酶序列变异体的子代；和基于鉴别步骤，选择可能具有此相关特征的子代。 [0131] Accordingly, embodiments of the present invention relates to marker-assisted breeding methods, which include: identification of relevant characteristics for breeding and selection, in which this feature and acetyl coenzyme A carboxylase gene are associated; providing portable can give The first plant plant acetyl coenzyme A carboxylase inhibitor herbicide-resistant acetyl-CoA carboxylase A sequence variant, in which the plant additionally includes related features; incubated with the first plant with a second plant; identification nurture This step has progeny acetyl coenzyme A carboxylase sequence variants; and based on the identification step of selecting the relevant characteristics may have this progeny. 相关特征可以为选自以下群组的任一者或多者：除草剂耐性、抗病性、抗虫性、脂肪酸、蛋白质或碳水化合物代谢改变、生长速度提高、胁迫耐性增强、优选成熟、感官性质增强、地貌特征改变、无菌、其它农业性状、工业用途的性状或提高消费者吸引力的性状。 Related features can be selected from the group of any one or more of: herbicide tolerance, disease resistance, insect resistance, fatty acids, protein or carbohydrate metabolism change, grow faster, enhanced stress tolerance, preferably mature, sensual enhance nature, geomorphology change, sterile, other agronomic traits, traits for industrial uses or increase consumer appeal traits.

[0132] 在一些实施例中，可基于核酸来分析遗传多态现象是否存在，从而用于选择繁殖种群中的种子或植物。 [0132] In some embodiments, the nucleic acid can be analyzed based on the existence of genetic polymorphism, which is used to select breeding population of seeds or plants. 分析可用于选择包含遗传标记物或与其相联的基因、QTL、等位基因或基因组区域（单倍型）。 Analysis can be used to select contain genetic markers or genes associated therewith, QTL, alleles, or genomic regions (haplotypes). 举例来说，标记物可以为包括对应于乙酰辅酶A羧化酶蛋白质中选自以下群组的至少一个氨基酸位置的变异的乙酰辅酶A羧化酶序列变异体：Gin 1756、Ile 1781、Trp 1999、Trp 2027、Ile 2041、Asp 2078、Cys 2088 和Gly 2096。 For example, the marker may comprise corresponding to acetyl coenzyme A carboxylase protein selected from the group of at least one amino acid position variation of acetyl coenzyme A carboxylase sequence variant: Gin 1756, Ile 1781, Trp 1999 , Trp 2027, Ile 2041, Asp 2078, Cys 2088 and Gly 2096. 在一些实施例中，变异可以为选自以下群组的至少一者：Glnl756Glu、Ilel781Leu、Ilel781Ala、 Ilel781Val、Trpl999Cys、Trp2027Cys、Ile2041Asp、Ile2041Val、Asp2078Gly、Asp2078Val、 Cys2088Arg和Gly2096Ala。 In some embodiments, the at least one variation can be selected from the following group: Glnl756Glu, Ilel781Leu, Ilel781Ala, Ilel781Val, Trpl999Cys, Trp2027Cys, Ile2041Asp, Ile2041Val, Asp2078Gly, Asp2078Val, Cys2088Arg and Gly2096Ala. 核酸分析方法为所属领域中所知，且包括（但不限于）基于PCR的检测方法（举例来说TaqMan分析）、微阵列方法和核酸定序法。 Nucleic acid analysis method known in the art as relevant, and include (but are not limited to) PCR-based detection methods (for example, TaqMan assays), microarray methods, and nucleic acid sequencing method. 在一些实施例中，通过使用核酸扩增方法，简化DNA、RNA或cDNA的样品中多态现象部位的检测。 In some embodiments, the nucleic acid amplification method by using a simplified test sample DNA, RNA, or cDNA of the polymorphism site. 这些方法特别提高覆盖多态现象部位，或包括此部位和远离或靠近此部位的序列的聚核苷酸的浓度。 These methods are particularly improve coverage polymorphism site, or include this site and the polynucleotide concentration away from or close to this part of the sequence. 这些扩增的分子可易于通过凝胶电泳、荧光检测方法或其它方式检测。 These molecules can be easily amplified by gel electrophoresis, fluorescence detection methods or other means testing. 因此，扩增分析、这些分析中所用的寡核苷酸和通过这些分析产生的相应核酸产物也可用于标记物辅助的育种计划中以通过选择育种来选择具有所需性状的子代。 Therefore, the amplification analyzes that used in these analyzes oligonucleotides and corresponding nucleic acid product was also used marker-assisted breeding programs to be selected by selective breeding progeny having the desired trait.

[0133] 同样，可基于仅仅表型分析，进行基于乙酰辅酶A羧化酶抑制剂除草剂抗性的MAS。 [0133] Similarly, phenotypic analysis may be based only, based acetyl coenzyme A carboxylase inhibitor herbicide resistance MAS. 开始培育植物并选择，或工程改造，使得相关性状与赋予乙酰辅酶A羧化酶抑制剂抗性的等位基因非随机相关（关联）。 Began to cultivate plants and choose, or engineered so that related traits conferring acetyl coenzyme A carboxylase inhibitor-resistant allele non-random correlation (association). 接着此植物可与具有其它所需性状的植物杂交。 Then the plant may have other plants with desired traits hybridization. 推测显示乙酰辅酶A羧化酶抑制剂抗性的植物也携带与抗性标记物关联的性状。 Speculate display acetyl coenzyme A carboxylase inhibitor resistant plants also carry markers associated with the resistance trait. 关联越紧密/ 越高，推测越肯定。 The more closely related / higher, suggesting that the more sure. 因此，乙酰辅酶A羧化酶抑制剂抗性等位基因通过产生例如经受住另外致死量的乙酰辅酶A羧化酶抑制剂的植物，可用作MAS的表型标记物，或者由于简化与抗性等位基因相关联的序列变异体的检测而可以用作分子标记物。 Therefore, acetyl coenzyme A carboxylase inhibitor resistance allele for example by producing lethal withstand additional acetyl coenzyme A carboxylase inhibitors plants, MAS can be used as phenotypic markers, or due to simplification and anti- Detection of allelic sequence variant associated and may be used as molecular markers. 举例来说，可以分析与乙酰辅酶A羧化酶序列差异相关联的除草剂抗性。 For example, you can analyze and acetyl coenzyme A carboxylase sequence differences associated with herbicide resistance. 除草剂抗性性状可包括对选自以下群组的任一种或多种除草剂的抗性：禾草灭、丁苯草酮、氯丙氧定、环苯草酮、稀禾定、环苯草酮、克草同、环杀草、得杀草、苯草酮、炔禾灵、炔草酸、氯伏草、赛伏草、禾草灵、精恶唑禾草灵、噻唑禾草灵、精吡氟禾草灵、吡氟禾草灵、合氯氟、异恶草醚、恶唑酰草胺、普拔草、快伏草、三氟丙酸和唑啉草酯。 Herbicide resistance traits may include any one or more selected from the group of herbicides: alloxydim, styrene-butadiene oxadiazon, chlorpromazine given oxygen, benzene ring oxadiazon, sethoxydim, ring benzene oxadiazon, g grass with ring herbicide, have to kill grass, grass ketone benzene, alkyne Wo Ling, clodinafop, chlorine-volt grass, grass race volts, diclofop, fenoxaprop, thiazole diclofop , fine fluazifop, fluazifop, co chlorofluorocarbons, different evil grass ether, oxazole acid alachlor, general weeding, fast-volt grass, trifluoroacetic acid and pinoxaden. 可如本文中所述，评估通过应用乙酰辅酶A羧化酶抑制剂除草剂和观测对除草剂的抗性进行选择的方法。 It may be as described herein, assessed by use of acetyl coenzyme A carboxylase inhibitor herbicide and resistance to herbicides observation method selection.

[0134] MAS方案在所属领域中众所周知，且使用各种标记作为工具。 [0134] MAS program is well known in the art, and using various markers as a tool. 举例来说，MAS 在美国专利第5，437，697号、美国专利公开案第2005/000204780号、美国专利公开案第2005/000216545号、美国专利公开案第2005/000218305号、美国专利公开案第2006000504538号、美国专利第6，100,030号和麦克尔(Mackill) (2008.生物科学(Phil Trans R Soc B) 363 =557-572)中有所描述，上述各者以引用的方式并入本文中。 For example, MAS in U.S. Patent No. 5,437,697, US Patent Publication No. 2005/000204780, US Patent Publication No. 2005/000216545, US Patent Publication No. 2005/000218305, U.S. Patent Publication No. 2006000504538, US Patent No. 6,100,030 and Michael (Mackill) (2008. Biological Sciences (Phil Trans R Soc B) 363 = 557-572) are described, each of the above are incorporated herein by reference in. 因此，所属领域的技术人员可在MAS方案中使用本发明的抗性表型或序列作为工具来选择与乙酰辅酶A羧化酶抑制剂抗性等位基因相关的性状。 Thus, those skilled in the art as a tool to select and acetyl coenzyme A carboxylase inhibitor resistance allele related traits using resistant phenotype or sequence of the invention in the MAS program.

[0135] 已详细描述本发明，明显的是，在不背离随附权利要求中所定义的本发明范畴下， 修改、变化和等效实施例为可能的。 [0135] The present invention has been described in detail, it is apparent, in the present invention without departing from the scope of the appended claims, as defined, modifications, variations, and equivalent embodiments are possible. 此外，应了解，以非限制性实例形式提供本发明中的所有实例。 In addition, it is understood, non-limiting example in the form of the present invention provides all of the instances.

[0136] 实例 [0136] Examples

[0137] 提供下列非限制性实例以进一步说明本文所公开的本发明实施例所属领域的技术人员应了解随后实例中所公开的技术代表已发现可在实践本发明实施例中发挥良好功用的途径，且因此可被认为构成本发明实践模式的实例。 [0137] The following non-limiting examples to further illustrate the cases of ordinary skill in the art disclosed herein, the present invention is to be understood that the subsequent technical representative examples disclosed embodiment has been found to play a good way function of the present invention may be implemented in practice and thus it may be considered to constitute an instance practice patterns of the present invention. 然而，所属领域的技术人员根据本发明应了解，可在不背离本发明的精神和范畴下，在所公开的特定实施例中作出多种变化， 且仍获得相同或相似的结果。 However, those skilled in the art to be understood according to the present invention can be made without departing from the spirit and scope of the invention, the various changes made in the specific embodiments disclosed, and still get the same or similar results.

[0138]实例 1 [0138] Example 1

[0139] 从植物的居间分生组织中获得的愈合组织产生 [0139] intervening callus obtained from meristem plants produce

[0140] 在图18中说明例示性外植体选择。 [0140] illustrated in FIG. 18 exemplary explant selection. 外植体组织可从含有最上部三片叶片的嫩枝中获得。 Explant tissue can be obtained from the uppermost containing three blades of twigs. 在低于最低叶节点处切割嫩枝，而且可修整各叶片顶部以在杀菌程序期间保持间距。 Cutting shoots below the lowest leaf node, and can be trimmed at the top of each blade during sterilization procedures to maintain the spacing. 将切片放到漂白剂溶液（20%体积/体积）大约10分钟，随后在70%乙醇中放置10 分钟，之后用无菌水冲洗。 The sections were placed in a bleach solution (20% v / v) for about 10 minutes, then placed in 70% ethanol for 10 minutes, then rinsed with sterile water. 去除外侧（较成熟）两片叶片，在茎上留下最新的叶片。 Removing the outer side (more mature) two blades, leaving the latest in stem leaves. 将新叶片在20%漂白剂中杀菌1分钟，在70%乙醇中杀菌1分钟，且随后在无菌水中冲洗。 The new blade sterilized in 20% bleach for 1 minute, sterilized in 70% ethanol for 1 minute, and then rinsed in sterile water. 所述叶片靠近节点的底部为居间分生组织。 The bottom of the blade near the node is intervening meristem. 移除此切片下部5毫米，且将其种植于愈合组织诱导培养基上，所述培养基含有MS基本盐（村重（Murashige)和斯科格（Skoog). 1962.植物生理学（Physiol Plant) 15 :473_497，其以全文引用的方式并入本文中），补充有B5维生素(甘博格O^amborg)等人，1968.实验细胞研究（Exp Cell Res)，50 ： 151-158，其以全文引用的方式并入本文中）、2，4_ 二氯苯氧乙酸（“2，4-0”）、蔗糖，且调节至？ Remove this lower portion 5 mm slices, and be planted in the healing callus induction medium, the medium containing MS basic salts (village weight (Murashige) and Skoog (Skoog). 1962. Plant Physiology (Physiol Plant) 15: 473_497, which in its entirety is incorporated by reference herein), supplemented with vitamin B5 (Gan Borg O ^ amborg) et al., 1968 Experimental Cell Research (Exp Cell Res), 50:. 151-158, which in entirety is incorporated by reference herein), 2,4_-dichlorophenoxyacetic acid ("2,4-0"), sucrose, and adjusted to? 册.5。 .5 Book. 将所种植的外植体在27°C的温度下放到暗处。 The explants were planted into a dark place at a temperature of 27 ° C.

[0141] 表1.愈合组织诱导培养基 [0141] Table 1. callus induction medium

[0142] [0142]

[0143]实例 2 [0143] Example 2

[0144] 从未成熟雀稗属花簇中获得的愈合组织产生 [0144] paspalum immature flower clusters obtained callus generation

[0145] 在花序伸出之前自温室生长的植物获得未成熟花簇，且将其用作产生愈合组织的外植体组织来源。 [0145] Before projecting inflorescence grown plants from the greenhouse to get immature flower clusters and produce it as a source of healing tissue explant tissue. 分离两穗花簇，且用含几滴吐温80 (Tween 80)的10% (体积/体积）漂白剂表面杀菌10分钟，且用无菌水冲洗，之后将其种植于MS培养基上，所述培养基含B5维生素（村重(Murashige)和斯科格(Skoog). 1962.同上文；甘博格(Gamborg)等人，1968. 同上文）和2毫克/升2-4，D。 Two separate clusters of flower spike, and containing a few drops of Tween 80 (Tween 80) 10% (v / v) bleach surface sterilized for 10 minutes, and rinsed with sterile water, after which it was planted on MS medium, the said medium containing vitamin B5 (village weight (Murashige) and Skoog (Skoog) 1962. supra;. Gan Borg (Gamborg) et al., 1968 supra) and 2 mg / L 2-4, D. 获得10种基因型的外植体组织，包括来自格鲁吉亚大学海滨雀禾卑属育禾中计戈丨J (University of Georgia Seashore Paspalum Breeding Program)的8 种实验细胞系、一种所采集的生态型（莫纳克亚（Mauna Kea) (PI 647892))和商业种植的品种“浪花（Seaspray)”。 Get 10 genotypes explants organizations, including the University of Georgia from humble seaside sparrow Wo Wo in the case of infertility total Ge 丨 J (University of Georgia Seashore Paspalum Breeding Program) of eight kinds of experimental cell lines, an ecotype collected ( Variety Mauna Kea (Mauna Kea) (PI 647892)) and the commercial planting of "spray (Seaspray)". 将四种外植体种植于各培养板上，且用耐斯克膜（Nescofilm™) (卡兰研究产品公司(Karlan Research Products Co)；科顿伍德(Cottonwood), AZ)密封培养板。 Adds four explants were planted in each culture plate, and the Gdansk-resistant membrane (Nescofilm ™) (Karan Research Products (Karlan Research Products Co); Branch Cottonwood (Cottonwood), AZ) seal plates. 将外植体在27°C下放到暗处。 The explants were placed in the dark at 27 ° C under. 自这些10种基因型之间产生总共21个细胞系（表2)。 From among these 10 kinds of genotypes generated a total of 21 cell lines (Table 2). 基于基因型和将外植体组织放到诱导培养基上的日期，给与所产生的各愈合组织以细胞系名称。 Based on genotype and tissue explants on induction medium into a date, give each callus cell lines generated with the name.

[0146] 表2.海滨雀稗属的活体外愈合组织产生和选择赋予稀禾定抗性的突变的总结 [0146] Table 2. seashore paspalum in vitro tissue healing and choice gives sethoxydim-resistant mutations summary

[0149] 雀稗属对除草剂的剂量-反应曲线[0150] 使用由作为模型栽培品种的品种『浪花』产生的愈合组织测定培养中雀稗属组织对稀禾定施用量的剂量反应。 [0149] paspalum doses of herbicides - response curve [0150] use the healing tissue as determined by breed, "waves" model cultivar produced culture paspalum organizations sethoxydim application rate of dose response. 通过将来自『浪花』的愈合组织放放到含有2毫克/升2-4，D和8种浓度中1种浓度的稀禾定的MS/B5培养基（村重（Murashige)和斯科格(Skoog). 1962.同上文；甘博格（Gamborg)等人，1968.同上文）上来确定稀禾定浓度对愈合组织生长的作用。 By callus from "spray" is put into containing 2 mg / liter 2-4, D and eight kinds of concentration in one kind of concentration of sethoxydim MS / B5 medium (village weight (Murashige) and Skoog . (Skoog) 1962. supra; Gan Borg (Gamborg) et al., 1968 supra) up to determine sethoxydim concentration on callus growth. 除草剂施用量重复测定6次且包括O μ M、2. 5 μ Μ、5 μ M、7. 5 μ M、10 μ M、 25μΜ、50μΜ及100 μ M稀禾定的浓度。 Determination of the amount of herbicide was repeated 6 times, and including O μ M, 2. 5 μ Μ, 5 μ M, 7. 5 μ M, 10 μ M, 25μΜ, 50μΜ and 100 μ M sethoxydim concentration. 在甲醇中稀释稀禾定，且在高压蒸汽处理的培养基冷却到大约55°C后添加（以防止由于热降解而丧失活性）。 Diluted in methanol sethoxydim, and in the medium autoclaved cooled to about 55 ° C after the addition (to prevent loss of activity due to thermal degradation). 通过在存储前将容器包裹于铝箔中来保护培养基以免光降解。 Before storing the containers by wrapping in aluminum foil to protect from light degradation media.

[0151] 为测量愈合组织生长，称出0. 5克愈合组织，分成9等块且以3X3图案放到各培养板的固体培养基上。 [0151] To measure the healing of tissue growth, weighed 0.5 grams callus, divided into nine blocks, etc. and to 3X3 pattern placed on each plate of solid media. 以完全随机设计将八种稀禾定浓度中各浓度6块重复培养板分布于生长室中的支架上。 In a completely randomized design to eight sethoxydim concentration of each concentration was repeated six plates distributed on growth chamber bracket. 种植后第21天，称量各培养板组织的重量且记录。 The first 21 days after planting, weighed and record the plates organizations. 为传代培养，自各培养板获得0. 5克以进入下一生长期。 A subculture, from each culture plate to obtain 0.5 g to enter the next phase of growth. 此过程持续9周，各培养板得到三个生长测量结果。 This process lasted nine weeks, each plate get three growth measurements. 用各培养板在各测量点（第3周、第6周和第9周）时的重量除以初始重量，获得质量比较增加量。 With each plate at each measurement point (3 weeks, 6 weeks and 9 weeks) weight divided by the initial weight, get quality is to increase the amount. 使用三个连续传代培养物平均的针对各除草剂施用量的愈合组织生长来辨别适用于选择突变体的浓度。 Use three successive subcultures average for each herbicide rate of healing of tissue growth to identify suitable selection concentration mutants. 使用非线性回归将愈合组织回应于稀禾定浓度的生长与负指数式衰减函数拟合（美国SAS 软件研究所（SAS Institute, Inc.) 2008. SAS OnlineDoc ® 9· 2.凯利（Cary)，NC)。 Using non-linear regression to heal tissue in response to the growth in sethoxydim concentration of negative exponential decay function fitting (United States Institute of SAS software (SAS Institute, Inc.) 2008. SAS OnlineDoc ® 9 · 2. Kelly (Cary), NC). 完全抑制愈合组织生长的最低除草剂施用量为7. 5 μ M稀禾定。 The minimum amount of herbicide completely inhibited the growth of the healing tissue is 7. 5 μ M sethoxydim. 为确保功效，选择1. O μ M稀禾定浓度来选择抗性细胞（图3)。 To ensure effectiveness, select 1. O μ M sethoxydim concentration select resistant cells (Fig. 3).

[0152]实例 4 [0152] Example 4

[0153] 选择稀禾定抗性细胞系 [0153] Select sethoxydim-resistant cell lines

[0154] 通过将约6个月大的愈合组织放到含有10 μ M稀禾定的愈合组织诱导培养基（实例1)上来对稀禾定抗性（SR)细胞进行选择。 [0154] By about 6 months into the healing tissue containing 10 μ M sethoxydim callus induction medium (Example 1) up to sethoxydim resistant (SR) cell selection. 使用大培养板（尺寸为245XM5mm)来高效地筛选较多数量的细胞。 The use of large plates (size 245XM5mm) to efficiently screening a large number of cells. 将直径为约4mm的愈合组织放到15X15的格子中，每块培养板得到总共225个愈合组织。 The diameter of about 4mm healing of tissue into the 15X15 grid, each plate to give a total of 225 union organizations. 以3周的时间间隔传代培养愈合组织三次（实例3)，选择期总共9周。 3-week intervals callus subculture three times (Example 3), select a total of nine weeks period. 在容纳补充有10 μ M稀禾定的愈合组织诱导培养基（实例1)的100X15mm皮氏培养皿（petri dish)中传代培养抗性愈合组织1个月，以获得足够的愈合组织。 Accommodating supplemented with 10 μ M sethoxydim callus induction medium (Example 1) 100X15mm petri dish (petri dish) resistance subcultured calli a month, in order to obtain sufficient callus. 此操作提供12周或12周以上的总选择时间。 This action provides more than 12 weeks or 12 weeks of total selection time.

[0155] 总共筛选20，250个愈合组织。 [0155] A total of 20,250 screened a callus. 选择过程产生65个稀禾定抗性（SR)细胞系，表示突变率为每312个愈合组织中有一个抗性品系。 Selection process produces 65 sethoxydim resistant (SR) cell lines, showing mutation rate per 312 union organizations have a resistant strain. 产生SR愈合组织的6个细胞系为莫纳克亚、GA05-025-164、UGA03. 539. 13、UGA05. 025. 181、UGA03. 525. 22 和UGA03. 09E-3。 SR callus generation of six cell lines Mauna Kea, GA05-025-164, UGA03. 539. 13, UGA05. 025. 181, UGA03. 525. 22 and UGA03. 09E-3. 在所有基因型中SR愈合组织的出现率低，且处于0到0.0051的范围内。 In all genotypes SR union organizations appear low, and in the range of 0 to 0.0051. 虽然对于所有基因型来说，回收SR细胞系的概率低，但所回收的SR细胞系的数目不同且处于零到高达每培养板225个愈合组织9个SR细胞系的范围内。 While all genotypes, the probability of recovery of SR cells low, but a different number of recovered cell lines and in SR zero to nine range up within each cell line SR 225 healing tissue culture plate. 对获得抗性愈合组织品系的概率差异的统计分析指示基因型间无显着差异（p = 0. 35)。 There was no significant difference (p = 0. 35) the difference between the probability of acquired resistance to union organization strains of statistical analysis indicated genotypes. 给与抗性愈伤组织以SR名称，自选择培养基中移出，且传代培养以在再生前使组织增加。 SR-resistant callus given name is removed from selective medium, and subcultured to increase prior to regenerate the tissue.

[0156]实例 5 [0156] Example 5

[0157] 稀禾定抗性细胞系再生 [0157] sethoxydim-resistant cell line regeneration

[0158] 试图使所有抗性愈合组织再生。 [0158] trying to make all resistance to heal tissue regeneration. 所用的再生培养基为MS/B5培养基（村重(Murashige)和斯科格(Skoog). 1962.同上文；甘博格(Gamborg)等人，1968.同上文）， The regeneration medium was used MS / B5 medium (village weight (Murashige) and Skoog (Skoog) 1962. supra;. Gan Borg (Gamborg) et al., 1968 supra).

28其补充有1. 24mg/L CuSO4和1. 125mg/L 6-苯甲基氨基嘌呤(BAP)(阿尔皮特(Altpeter) 等人，2005.国际草坪草社会研究期刊international Turfgrass Society Research Journal)，10 :485-489,其以全文引用的方式并入本文中）。 28 supplemented with 1. 24mg / L CuSO4 and 1. 125mg / L 6- benzylamino purine (BAP) (Al Pete (Altpeter) et al., 2005 International Social Research lawn grass journals international Turfgrass Society Research Journal), 10: 485-489, which is incorporated in its entirety by reference herein). 将各稀禾定抗性（SR)细胞系的愈合组织放到五块培养板上的4X4格子中，各愈伤组织的尺寸为约4mm的直径。 The healing Organization sethoxydim-resistant (SR) cell line culture into five 4X4 grid plate, the size of each callus is about 4mm in diameter. 接着将培养板放到生长室中，所述生长室保持25°C及1小时黑夜：23小时白天的光周期，其中由冷光型日光灯提供66-95 μ mol光子HT2S4的光强度。 The plates were then placed in a growth chamber, said growth chamber to maintain 25 ° C and 1 hour dark: 23 hours of light during the day cycle, which provides 66-95 μ mol photon intensity HT2S4 by cold fluorescent lamps. 在30天时间结束时，针对再生评价所有培养板。 At the end of 30 days, renewable for evaluation of all plates. 如果嫩枝出现，就在再生培养基上再传代培养细胞系1个月。 If the shoots appear, subcultured cell lines a month on the regeneration medium.

[0159] 在嫩枝发育后，通过在无生长调节剂下将组织放到MSO培养基（列于下表3中）诱导生根。 [0159] After the shoots development, by the absence of growth regulators will be organized into the rooting medium MSO (listed in Table 3). 当根生长充分（约30天）时，自培养基移出植物，且直接放到花盆中，所述花盆容纳Fafard ®3B(亚加瓦姆镇（Agawam)，马萨诸塞州（MS))混合物与砂的1 ： 1混合物。 When sufficient root growth (about 30 days), the medium was removed from the plant and directly into pots, the pots hold Fafard ®3B (acrylic Ouham town (Agawam), Massachusetts (MS)) mixture Sand with a 1: 1 mixture. 接着将盆栽植物转移到温室中，温室中保持到32°C 10小时白天14小时黑夜的光周期。 Next, the potted plants were transferred to the greenhouse, greenhouse holding to 32 ° C 10 hours of daylight 14 hours dark photoperiod.

[0160] 表3.诱导生根的MSO培养基 [0160] Table 3. rooting medium MSO

[0161] [0161]

[0162] 65个SR细胞系中的2个SR细胞系在再生前死亡，因此剩余63个SR细胞系，使三个细胞系再生：A细胞系、B细胞系和C细胞系。 [0162] 65 cell lines SR 2 SR cells regenerate before death, and therefore the remaining 63 SR cells, regeneration of the three cell lines: A cell lines, B cell lines and C cells. A细胞系和B细胞系源自相同细胞系，所述细胞系来源于2008年1月12日产生的莫纳克亚，而C细胞系源自2008年3月4日产生的实验细胞系UGA03-098E-3。 A cell lines and B cell lines derived from the same cell line, said cell line derived from the January 12, 2008 generated Mauna Kea, and C cell line derived from a March 4, 2008 produced experimental cell lines UGA03 -098E-3. 再生的三种细胞系的愈合组织为致密的且为黄色，而相比来说，大部分细胞系的愈合组织为白色且柔软的。 Healing tissue regeneration in three cell lines was dense and yellow, and contrast, most of the healing tissue cells as white and soft.

[0163]实例 6 [0163] Example 6

[0164] 稀禾定抗性细胞系的分子表征 [0164] Molecular characterization of sethoxydim-resistant cell lines

[0165] 一旦选择SR雀稗属细胞系，就对引起抗性的突变进行表征。 [0165] Once selected SR paspalum cell line, on the right of resistance mutations were characterized. 使用CTAB方法（兰瑟（Lassner)等人，1989.植物分子生物学报道（Plant Mol Biol Report), 7 ： 116-128，其以全文引用的方式并入本文中）从再生植物的愈合组织或叶片组织中提取出DNA。 Using the CTAB method (Lanser (Lassner) et al., Plant Molecular Biology 1989 reported (Plant Mol Biol Report), 7:. 116-128, which is incorporated by reference in its entirety herein by reference) or from callus regenerating plants leaf tissue extracted DNA. 使用乙酰辅酶A羧化酶（ACCase)胺基酸序列（迪徕（D6lye)等人，2005.杂草研究（Weed Research)，45 :323-330,其以全文引用的方式并入本文中）来确定物种间的同源区域。 Use acetyl coenzyme A carboxylase (ACCase) amino acid sequence (Di Lai (D6lye) et al., 2005 Weed (Weed Research), 45:. 323-330, which is incorporated by reference in its entirety herein by reference) to determine regions of homology between species. 使用狗尾草（Setaria viridis)乙酰辅酶A羧化酶的核苷酸序列（基因库AM94805)(迪徕(Delye)等人，2002，植物（Planta)，214 :421_427，其以全文引用的方式并入本文中）来设计扩增海滨雀稗属的同源区域的引物，且改变个别碱基以匹配由基因库的BLAST功能所测定可能的最多数目的草物种。 Use green foxtail (Setaria viridis) acetyl coenzyme A carboxylase nucleotide sequence (gene bank AM94805) (Di Lai (Delye) et al., 2002, Plant (Planta), 214: 421_427, which is incorporated by reference in its entirety in this article) to design amplification primers seashore paspalum homologous regions and changes in individual bases to match the largest possible number of grass species gene pool by the BLAST function measured. 所得引物扩增乙酰辅酶A羧化酶基因的384个碱基对片段，所述片段跨越引起1781位置上Ile向Leu取代的A 向T 转换。 The resulting primer acetyl coenzyme A carboxylase gene of 384 base pair fragment that spans 1781 locations cause Ile, Leu A to T to convert. 引物命名为SV384F(5 ' CGGGGTTCAGTACATTTAT 3'，SEQ ID N0:1)禾口SV348R(5' GATCTTAGGACCACCCAACTG 3'，SEQ ID NO ：2)。 Primers designated SV384F (5 'CGGGGTTCAGTACATTTAT 3', SEQ ID N0: 1) Wo port SV348R (5 'GATCTTAGGACCACCCAACTG 3', SEQ ID NO: 2). 退火温度为53°C，延长时间为30 秒和35个循环。 Annealing temperature of 53 ° C, to extend the time to 30 seconds and 35 cycles. 所研发的对乙酰辅酶A羧化酶基因的2078位置进行定序的引物命名为S VAC2F (5 ' AATTCCTGTTGGTGTCATAGCTGTGGAG 3 '，SEQ ID NO :；3)和SVAClR (5 ' TTCAGATTTATCAACTCTGGGTCAAGCC 3'，SEQ ID NO :4)，且用于扩增此片段的PCR条件与用于扩增1781 的条件相同。 R & D for 2078 position acetyl coenzyme A carboxylase gene sequencing primers were named S VAC2F (5 'AATTCCTGTTGGTGTCATAGCTGTGGAG 3', SEQ ID NO:; 3) and SVAClR (5 'TTCAGATTTATCAACTCTGGGTCAAGCC 3', SEQ ID NO: 4), and PCR conditions used to amplify this fragment with 1781 for the same amplification conditions. SVAC引物扩增跨越乙酰辅酶A羧化酶基因中位置2078的编码区域的520bp 片段。 SVAC primers spanning acetyl coenzyme A carboxylase gene fragment in position 2078 of 520bp coding region.

[0166]实例 7 [0166] Example 7

[0167] 识别稀禾定抗性细胞系且自细胞系再生稀禾定抗性雀稗属 [0167] Recognition sethoxydim-resistant cell lines and cell lines from renewable sethoxydim-resistant paspalum

[0168] 表2总结迄今为止的选择过程，迄今为止，已产生65个稀禾定抗性细胞系。 [0168] Table 2 summarizes the selection process so far, so far, has produced 65 sethoxydim-resistant cell lines. 抗性愈合组织形成的频率为每312个进行完全选择过程的愈合组织1个抗性愈合组织。 Resistant callus formation frequency per 312 callus were completely resistant to the selection process of a healing tissue. 可再生的稀禾定抗性（SR)愈合组织的出现率为每32. 5个抗性愈合组织1个可再生稀禾定抗性愈合组织。 Renewable sethoxydim resistant (SR) callus emergence was 32.5 per resistant calli a renewable sethoxydim-resistant callus. 再生的SR细胞系的出现率为每10，125个通过选择过程的愈合组织1个SR细胞系。 SR regeneration occurrence rate per 10,125 cells a selection process by an SR callus cells.

[0169] 单愈合组织细胞的平均体积测量为1. 3582X 10_5μ L。 [0169] The average volume measurement single callus cells to 1. 3582X 10_5μ L. 此提供每4mm直径的愈合组织块258，000个细胞的近似值。 This provides each block diameter 4mm callus cells 258,000 approximation. 因此，通过选择的20，250个愈合组织含有约52亿个细胞。 Thus, by 20,250 a callus selected contains about 5.2 billion cells. 假定仅单个突变细胞为各SR细胞系的成因，则抗性细胞在本实验中的出现率为每8X IO7 个细胞1个抗性细胞。 Cause assumed only a single mutant cell for the SR cell lines, the resistant cells in this experiment was the emergence of cells per 8X IO7 a resistant cells. 在1781aa位置上获得A向T突变的出现率为1. 74X IO9分之一。 A place to get in 1781aa appear to T mutation rate of 1. 74X IO9 points.

[0170] 迄今为止，四个SR愈合组织（细胞系A、细胞系B、细胞系C和细胞系D)已长成绿色小植物，且两个SR愈合组织（细胞系A和细胞系B)已长成可存活植物。 [0170] To date, four SR callus (cell line A, cell lines B, C cell lines and cell lines D) has grown into a small green plants, and two SR callus (cell lines A and cell line B) It has grown into a viable plant. A细胞系、B细胞系和D细胞系源自相同细胞系莫纳克亚12JAN08，而C细胞系源自2008年3月4日产生的实验细胞系UGA 03-098E-3。 A cell lines, B cell lines and D cell lines derived from the same cell line Mauna Kea 12JAN08, and C cell line derived from a March 4, 2008 produced experimental cell lines UGA 03-098E-3. A细胞系就再生植物来说最多产，产生500个以上的个别植物。 A cell line on regeneration plant is the most productive, generating more than 500 individual plants. B细胞系产生约20个植物。 B cells produce about 20 plants.

[0171] 自65个SR细胞系的63个细胞系中获得ACCase扩增子，且仅有3个细胞系，包括细胞系A(图5)显示位置1781上A转变成T。 [0171] were obtained from 65 cell lines SR 63 cell lines ACCase amplicons, and only three cell lines, including cell lines A (Figure 5) shows the position of 1781 A converted into T. 这些SR细胞系中除位置1781或2078以外的位置上也可能发生突变。 These cell lines SR mutations occur at positions other than the position on the 1781 or 2078. 抗性细胞系对于突变来说是杂合的，因此序列层析谱说明在突变时存在双峰，其中一个峰表示野生型等位基因，且另一峰表示突变等位基因。 Resistant cell line is heterozygous for the mutation, and therefore the presence of the sequence described bimodal chromatogram when mutated, wherein the wild-type allele represents a peak and the other peaks represent mutant allele. 在产生活的植物的两个细胞系中，仅仅细胞系A具有预期的Ile突变成Leu。 Ile plant life in the production of the two cell lines, cell lines A having only the desired mutation into Leu. 下文中给出针对细胞系A所获得的扩增子的遗传序列为SEQ ID NO :5，其中突出和有下划线的密码子指示Ile突变成Leu。 The genetic sequence given below A cell line obtained amplicon of SEQ ID NO: 5, which highlights and underlined mutated codon indicating Ile Leu. 细胞系B在位置1781上具有野生型序列。 B cell lines have the wild-type sequence at position 1781. 因为稀禾定抗性也可由位置2078上Asp突变成Gly所赋予，所以分析细胞系B的DNA是否存在此突变，但没有一个细胞系具有此突变。 Because sethoxydim-resistant but also by the position of 2078 on a Gly Asp mutation conferred, so analysis of DNA cell line B of the existence of this mutation, but none of this mutant cell lines. 细胞系B的稀禾定抗性的性质仍未确定。 Nature sethoxydim-resistant cell line B remains uncertain.

[0172] 500个以上的细胞系A植物已移植到土壤中。 [0172] 500 or more cell lines A plant has been transplanted to soil. 无性繁殖细胞系A的再生植物，以经受除草剂测试，从而在全植物水平上确认西杀草抗性的表达。 Clonal cell lines regenerated plants A to withstand herbicides tested to confirm the expression of the West to kill grasses at the whole plant level.

[0173] SEQ ID NO ：5 [0173] SEQ ID NO: 5

[0174] GGCGATTGGGCCGAAGTCGCATGCTCCCGGCCGCCATGGCGGCCGCGGGAATTCGA [0174] GGCGATTGGGCCGAAGTCGCATGCTCCCGGCCGCCATGGCGGCCGCGGGAATTCGA

[0175] TACCCCTTTTTCAGTACATTTATCTGACTGAAGAAGATTATGCTCGTATTAGCTCTTC [0175] TACCCCTTTTTCAGTACATTTATCTGACTGAAGAAGATTATGCTCGTATTAGCTCTTC

[0176] TGTTATAGCACATAAGCTACAGCTGGACAGCGGTGAAATTAGGTGGATTATTGACT [0176] TGTTATAGCACATAAGCTACAGCTGGACAGCGGTGAAATTAGGTGGATTATTGACT

[0177] CTGTTGTGGGCAAGGAGGATGGGCTTGGTGTTGAGAATTTACATGGAAGTGCTGCT [0177] CTGTTGTGGGCAAGGAGGATGGGCTTGGTGTTGAGAATTTACATGGAAGTGCTGCT

[0178] ATTGCCAGTGCTTATTCTAGGGCATACGAGGAGACATTTACACTTACGTTCGTGACT [0178] ATTGCCAGTGCTTATTCTAGGGCATACGAGGAGACATTTACACTTACGTTCGTGACT

[0179] GGGCGGACTGTAGGAATAGGAGCTTATCTTGCACGACTTGGTATACGGTGCATACA [0179] GGGCGGACTGTAGGAATAGGAGCTTATCTTGCACGACTTGGTATACGGTGCATACA

[0180] GCGTCTTGACCAGCCCATTATTTTAACAGGGTTTTCTGCCCTGAACAAGCTTCTTGG [0181 ] GCGTGAAGTTTACAGCTCCCACATGCAGTTGGGTGGTCCTAAGATCATGGCGACGA[0182] ATGGTGTTGTCCACCTCACTGTTTCAGATGATCTTGAAGGTGTATCCAGTATATTGA [0180] GCGTCTTGACCAGCCCATTATTTTAACAGGGTTTTCTGCCCTGAACAAGCTTCTTGG [0181] GCGTGAAGTTTACAGCTCCCACATGCAGTTGGGTGGTCCTAAGATCATGGCGACGA [0182] ATGGTGTTGTCCACCTCACTGTTTCAGATGATCTTGAAGGTGTATCCAGTATATTGA

[0183] GGTGGCTCAGCTATGTTCCTGCCAACATTGGTGGACCTCTTCCTATTACAAAACCTT [0183] GGTGGCTCAGCTATGTTCCTGCCAACATTGGTGGACCTCTTCCTATTACAAAACCTT

[0184] TGGACCCACCGGACAGACCTGTTGCGTACATCCCTGAGAACACATGCGATCCACGT [0184] TGGACCCACCGGACAGACCTGTTGCGTACATCCCTGAGAACACATGCGATCCACGT

[0185] GCAGCCATCCGTGGTGTAGATGACAGCCAAGGGCAATGGTTGGGTGGTATGTTTGA [0185] GCAGCCATCCGTGGTGTAGATGACAGCCAAGGGCAATGGTTGGGTGGTATGTTTGA

[0186] CAAAGACAGCTTTGTGGAGACATTTGAAGGATGGGCGAAAACAGTTGTCACTGGCA [0186] CAAAGACAGCTTTGTGGAGACATTTGAAGGATGGGCGAAAACAGTTGTCACTGGCA

[0187] GGGCATAGCTTGGAGGAATTCCTGTGGGTGTCATAGCTGTGGAGACACAGAACATG [0187] GGGCATAGCTTGGAGGAATTCCTGTGGGTGTCATAGCTGTGGAGACACAGAACATG

[0188] ATGCAGCTCATCCCTGCTGATCCAGGCCAGCTTGATTCTCATGAGCGATCTGTTCCT [0188] ATGCAGCTCATCCCTGCTGATCCAGGCCAGCTTGATTCTCATGAGCGATCTGTTCCT

[0189] CGGGCTGAACAAGTGTGGTTCCCAGATTCTGCAACCAAGACTGCTCAAGCATTGTT [0189] CGGGCTGAACAAGTGTGGTTCCCAGATTCTGCAACCAAGACTGCTCAAGCATTGTT

[0190] GGACTTCAACCGTGAAGGATTGCCTCTGTTCATCCTTGCTAACTGGAGAGGTTTCTC [0190] GGACTTCAACCGTGAAGGATTGCCTCTGTTCATCCTTGCTAACTGGAGAGGTTTCTC

[0191] TGGTGGACAAAGAGATCTCTTTGAAGGAATTCTTCAGGCTGGGTCAACAATTGTTG [0191] TGGTGGACAAAGAGATCTCTTTGAAGGAATTCTTCAGGCTGGGTCAACAATTGTTG

[0192] AGAACCTTAGGACGTACAATCAACCTGCGTTTGTCTACATTCCTATGGCTGGAGAGC [0192] AGAACCTTAGGACGTACAATCAACCTGCGTTTGTCTACATTCCTATGGCTGGAGAGC

[0193] TGCGTGGAGGAGCTTGGGTTGTGGTTGATAGCAAAATAA [0193] TGCGTGGAGGAGCTTGGGTTGTGGTTGATAGCAAAATAA

[0194] 含有SEQ ID NO ：5的载体于2009年6月寄存于美国模式培养物保藏所（American Type Culture Collection)，美国(USA)弗吉尼亚（Virginia) 20110-2209 马纳萨斯(Manassas)大学大道10801(10801 University Boulevard)，且寄存编号为_。 [0194] comprising SEQ ID NO: 5 carrier in June 2009 deposited at the American Type Culture Collection (American Type Culture Collection), United States of America (USA), Virginia (Virginia) 20110-2209 Manassas (Manassas) University Avenue 10801 (10801 University Boulevard), and Storage numbered _.

[0195]实例 8 [0195] Example 8

[0196] 评估对稀禾定（SEGMENT™除草剂）的全植物抗性 [0196] evaluation of sethoxydim (SEGMENT ™ herbicide) resistance of the whole plant

[0197] 测试从稀禾定抗性细胞系（A细胞系）再生的稀禾定抗性植物在温室中进行的剂量反应实验中在全植物水平上的抗性。 [0197] dose sethoxydim-resistant plant test from sethoxydim-resistant cell lines (A cell line) regeneration reaction in the greenhouse for resistance experiments on whole plant level. 在此实验中，将A细胞系与以下两种除草剂敏感对照株对比：初始亲本株莫纳克亚（PT)，和从组织培养物（TTC)再生的莫纳克亚株。 In this experiment, the A cell lines and two herbicide-sensitive control strain comparison: the initial parent strain Mauna Kea (PT), and culture (TTC) regenerating plants from tissue Mauna Kea. 将植物移植到含有1 ： 1的Fafard ® 3B混合物与砂的混合物的Cone-tainers™(测量为4X1. 4cm, 并逐渐缩减到lcm;斯图维父子公司（Stuewe and Sons he.)，俄勒冈地区科瓦利斯(Corvallis, Oregon))中，并放到温室中的种植台上，处于钠灯下，温室中保持27/32°C白天/黑夜的16小时光周期，持续大约2周，随后施用除草剂处理。 The plants were transplanted to 1 contains: a mixture of Fafard ® 3B mixture with sand. 1 Cone-tainers ™ (measured as 4X1 4cm, and gradually reduced to lcm; Situ Wei & Sons, Inc. (Stuewe and Sons he), Oregon area. Corvallis (Corvallis, Oregon)) in, and placed in the greenhouse cultivation stage, in lower sodium, a greenhouse for 16 h photoperiod 27/32 ° C day / night, for about two weeks, followed by administration herbicide treatment. 使用kgment™除草剂(巴斯夫公司(BASF Corp.)，弗伦翰公园(Florham Park)，新泽西州(NJ))，用每公顷0、50、 100、200、400、800、1600和3200g活性成分的施用量的稀禾定处理三种基因型A细胞系、PT 和TC中的每一种。 Use kgment ™ herbicide (BASF Corporation (BASF Corp.), Florham Park (Florham Park), New Jersey (NJ)), 0,50 per hectare, 100,200,400,800,1600 and 3200g active ingredient The application rate of sethoxydim treatment three genotypes A cell lines, PT, and TC in each. 所有除草剂施用量都是在实验喷洒室中以每公顷1871的喷洒体积应用， 且在干燥之后回到温室梯段上并维持在上文所述的条件下。 All amounts are in experimental herbicide spray chamber to spray volume per hectare 1871 application, and after drying back to the greenhouse bench and maintained under the conditions described above. 在处理后第7、14、21和观天(DAT)，使用0到100的规模（其中0等于无损害且100等于完全死亡）记录作物损害的可见估算值。 After handling the first 14, 21 and Sky (DAT), using scale 0-100 (where 0 equals no damage and 100 equals complete death) records visible crop damage estimates. 在处理后第42天，收获所有植物的地上部分，在50°C的温度下干燥48小时，并称重以确定植物干重。 In the first 42 days after treatment, the aerial parts of the harvest of all plants, at a temperature of 50 ° C for 48 hours and weighed to determine the plant dry weight. 处理是以随机化的完全区组设计安排。 Processing is a randomized complete block design arrangement. 因为有限的植物材料，只有两种TCC复制物是可能的，否则四种复制物用于其它两种基因型（PT和A细胞系）。 Because of the limited plant material, only two replicas TCC is possible, otherwise four duplication was for the other two genotypes (PT and A cell line). 首先使用双因子方差分析来分析数据，随后在除草剂施用量内分析。 First, use two-factor analysis of variance to analyze the data, and then analyze the herbicide rate within. 基因型之间的差异意谓每一种除草剂施用量是使用费雪最小显着差异法（LSD)确定。 Difference means each herbicide rate between genotypes using the Fisher least significant difference (LSD) method to determine.

[0198] 图9说明稀禾定施用量对于三种被测试基因型中的每一种在14DAT的损害等级的影响。 [0198] Figure 9 illustrates sethoxydim application rate for the three genotypes were tested in each of the impact damage 14DAT level. 图11说明稀禾定施用量对于三种被测试基因型中的每一种在21DAT的损害等级的影响。 Figure 11 illustrates sethoxydim application rate for the three levels of impact damage in 21DAT to be tested in each genotype. 双因子方差分析通过除草剂施用量对处理后第7、14、21和观天的损害等级的影响(数据没有显示）指示显着基因型、除草剂施用量和基因型。 Two-factor analysis of variance by affecting the amount of herbicide to the first 14, 21 days after treatment and the concept of damage level (data not shown) indicating a significant genotype, herbicide rate and genotype. A细胞系显示优越的除草剂抗性，即使在每公顷3200g活性成分的最高施用量下（图8，表4)。 A cell lines showed excellent herbicide resistance, even at the highest application rate of active ingredient per hectare 3200g (Figure 8, Table 4). 相比之下，PT和TC在每公顷200g活性成分的施用量下具有30或更大的损害得分，且在等于或大于每公顷800g活性成分的施用量下具有80%或更大的损害得分。 In contrast, PT, and TC has scored 30 or more damage at application rates of active ingredient per hectare 200g, and equal to or greater than the lower application rates of active ingredient per hectare 800g of 80% or greater damage score . 当比较三种基因型中的每一种在每一种除草剂施用量下的平均损害得分时，A细胞系在所有评级日期在大于每公顷IOOg活性成分的所有施用量下具有显着小于PT或TC的损害。 Significantly less than the PT administration at all three genotypes when comparing the amount of the average damage for each in each herbicide rate of scoring, A cell line in all ratings date greater than the active ingredient per hectare IOOg or TC damage. 在3200g ai ha—1或对百足草的最低标示施用量15倍剂量下对细胞系A所观测到的最大损伤得分为7. 5%，百足草为假俭草（Eremochloa ophiuroides (Munro)Hack)，一种对稀禾定天然耐受的草坪草物种。 In 3200g ai ha-1 or Momotari grass 15 times the amount of the minimum labeled dose is administered on the cell line A damage observed maximum score of 7.5%, centipede grass centipedegrass (Eremochloa ophiuroides (Munro) Hack) A native of sethoxydim tolerant turfgrass species.

[0199] 图12呈现在42DAT获取的三种基因型的平均干重。 [0199] FIG. 12 presents three genotypes obtained in 42DAT average dry weight. 两种敏感细胞系PT和TCC的干重回应于稀禾定使用率增加而降低，而CLA的干重即使在大于每公顷1600g活性成分的施用量下仍保持相对稳定。 Two sensitive cell lines PT and TCC's dry weight in response to increased usage of sethoxydim reduced, while CLA dry weight per hectare under even greater than 1600g amount of active ingredient remained relatively stable.

[0200] PT,TC和A细胞系的三种基因型的LD5tl的估算值是每公顷189、276和> 3200g活性成分。 [0200] LD5tl estimates three genotypes of PT, TC and A cell line is 189,276 per hectare and> 3200g active ingredient. 这些数据提供以下证据：细胞系A存在的除草剂抗性水平足够大以有效控制敏感的杂草而无需考虑除草剂损伤。 These data provide the following evidence: the presence of cell lines A herbicide resistance level is large enough to effectively control weeds without regard to sensitive herbicide damage.

[0201] [0201]

〔0202〕 湘室9 [0202] Gordon chamber 9

〔0203〕 进諌沐沛恶«窆)炯澍郐;#浮咏疏办[0204] 起始第二温室实验，以在全植物水平上评估由第二个稀禾定抗性细胞系B细胞系再生的SR植物的稀禾定抗性。 [0203] into 諌 Mu Pei evil «coffin in grave) Kuai Shu Jiong; # floating Yong Shu Office [0204] starting the second greenhouse experiment to evaluate the second sethoxydim-resistant cell line B cells in the whole plant level SR Department of sethoxydim-resistant plant regeneration. 在先前的实验（实例8)中，较高浓度Wkgment™稀禾定除草剂对A细胞系产生极少损伤。 In a previous experiment (Example 8), higher concentrations Wkgment ™ sethoxydim herbicides on cell lines A little damage. 这些损伤的症状更倾向于是表面活性剂的损伤而非稀禾定损伤。 Symptoms of these injuries are more likely to damage a surfactant rather than sethoxydim damage. 因此，选择Poast™除草剂（不含表面活性剂的稀禾定调配物）来表征B细胞系的抗性水平，并将B细胞系的稀禾定抗性水平与A细胞系相比较。 Therefore, the choice Poast ™ herbicide (surfactant-free formulation of sethoxydim) to characterize the resistance level of B cell lines, sethoxydim resistance level cell lines compared with A and B cell lines. 在本实验中，将A细胞系和B 细胞系与以下两种易受除草剂影响的对照组相比较：原始的莫纳克亚亲本细胞系（PT)；和由组织培养再生的莫纳克亚细胞系（TTC)。 In this experiment, the two cell lines A and B cell lines susceptible to herbicide effects compared to the control group: Mauna Kea original parent cell line (PT); and the regeneration of tissue culture Monarch sub-cell line (TTC). 将植物移植到含有1 ： 1 Wi^afard ®;3B混合物与砂的混合物的Cone-tainersTM(测量为4X1. 4cm,并逐渐缩减到Icm ；斯图维父子公司(Stuewe and Sons he.)，俄勒冈地区科瓦利斯（Corvallis，Oregon))中，并放到温室中的种植台上，处于钠灯下，温室中保持27/32°C白天/黑夜的16小时光周期，持续大约2周，随后施用除草剂处理。 The plants were transplanted to contain 1: 1 Wi ^ afard ®; 3B mixture with sand mixture Cone-tainersTM (measured as 4X1 4cm, and gradually reduced to Icm; Situ Wei & Sons, Inc. (Stuewe and Sons he), Oregon. Area Corvallis (Corvallis, Oregon)) in and placed in the greenhouse cultivation stage, in lower sodium, greenhouse holding 27/32 ° C day / night of the 16 hour period, for approximately two weeks, followed by herbicide treatment.

[0205] 使用Poast™除草剂（巴斯夫公司（BASF Corp.)，新泽西州弗洛厄姆帕尔克(Florham Park，NJ))，用0、50、100、200、400、800、1600、3200 和6400g ai ha—1 的稀禾定施用量处理四种基因型（A细胞系、B细胞系、PT和TCC)中的每一者。 [0205] The herbicide Poast ™ (BASF Corporation (BASF Corp.), New Jersey 弗洛厄姆帕 Arcs (Florham Park, NJ)), with 0,50,100,200,400,800,1600,3200 sethoxydim and 6400g ai ha-1 fertilizer processing four genotypes (A cell lines, B cell lines, PT, and TCC) in each. 在实验喷雾室中，所有除草剂施用量都以187L ha—1的喷雾体积施用，干燥后，将植物放回温室中的种植台上，并在上文所述的条件下保持。 In the experiment the spray chamber, all amounts are in herbicide spray volume of 187L ha-1 administration, after drying, the plants were returned to the greenhouse cultivation stage, and maintained at the above conditions. 处理后16、21和28天（DAT)，使用O到100的量表记录下作物损伤的目测估计值，其中0等于无损伤，而100等于完全死亡。 16, 21 and 28 days (DAT) after treatment, the use of O to 100 scale visual estimates of crop damage recorded, where 0 equals no damage, and 100 equals complete death. 本实验为4X9阶乘，即，4种基因型和9种除草剂施用量。 The 4X9 factorial experiment, namely, four genotypes and nine kinds of herbicide rate. 处理是布置成随机的完备型设计。 The deal is arranged complete randomized design. 所用全部四种基因型都是一式四份。 All four genotypes were used in quadruplicate. 首先使用双因素方差分析（SAS，2008)来分析数据，随后在除草剂施用量范围内进行分析。 First, use two-way ANOVA (SAS, 2008) to analyze the data, and then analyzed within herbicide rate ranges. 使用费雪最小显着差异法（LSD)测定在各除草剂施用量下各基因型平均值之间的差异。 Determination of the difference between the averages of each genotype at each herbicide rate using Fisher least significant difference method (LSD).

[0206] 图13说明在21DAT时稀禾定施用量对四种所测试的基因型中每一者的损伤等级的影响。 [0206] FIG. 13 illustrates the effect 21DAT when sethoxydim application rate of the four tested genotypes in each of the damage level. 双因素方差分析指示显着基因型、除草剂施用量以及在16、21和^DAT时除草剂施用量对基因型损伤等级的影响（数据未显示）。 Two-way ANOVA analysis indicated significant genotype influence herbicide rate and herbicide rate at 16, 21 and ^ DAT genotypes damage when levels (data not shown). A细胞系和B细胞系显示出优良的除草剂抗性，甚至在6400g ai ha-1的最高施用量下也是如此（图13)。 A cell lines and B cell lines showed excellent herbicide resistance, even at 6400g ai ha-1 the highest application rate as well (Figure 13). 相比之下，在400g ai ha-1 的施用量下，PT和TCC的损伤评分为27或27以上，而且在1600g ai ha—1的施用量下，损伤评分为80%或80%以上。 In contrast, under 400g ai ha-1 of fertilizer, PT, and TCC of lesion score of 27 or above 27, and in application rate 1600g ai ha-1, and injury score at least 80% or 80%. 当对四种基因型中每一者在每一除草剂施用量下的平均损伤评分进行比较时，在所有等级时期，在高于200g ai ha—1的所有施用量下，A细胞系和B细胞系的损伤都明显少于PT或TCC。 When each of the four genotypes in each herbicide rate of average lesion scores were compared at all times rank at all application rates higher than 200g ai ha-1's, A and B cell lines damage cell lines were significantly less than the PT or TCC. 对于高达6400g ai ha—1的所有施用量，在A细胞系和B 细胞系上所观察到的最大损伤评分也低于20%。 For up to 6400g ai ha-1 all application rate on the A and B cell lines cells observed maximum injury score lower than 20%.

[0207] 对于PT、TC、A细胞系和B细胞系，四种基因型的LD5tl估计值分别为720、782、> 6400、> 6400g ai ha—1。 [0207] For PT, TC, A cell lines and B cell lines, LD5tl estimate of four kinds of genotypes were 720,782,> 6400,> 6400g ai ha-1. 这些数据强有力地证实，A细胞系和B细胞系中存在的除草剂抗性水平能够较好地提供对于易受杂草影响的禾草的有效控制，而且不会涉及除草剂损伤。 These data strongly confirm, A and B cell lines cell lines exist herbicide resistance levels can better provide for the impact of grasses susceptible weeds effectively controlled, and does not involve herbicide damage.

[0208]实例 10 [0208] Example 10

[0209] 稀禾定抗性雀稗属对其它乙酰辅酶A羧化酶抑制剂除草剂的交叉抗药性 [0209] sethoxydim-resistant paspalum other acetyl coenzyme A carboxylase inhibitor herbicide cross resistance

[0210] 稀禾定是称为乙酰辅酶A羧化酶抑制性除草剂类别中的一个成员。 [0210] sethoxydim is known as acetyl coenzyme A carboxylase inhibiting herbicides category one member. 这一家族的除草剂通常分为两组，即环己二酮类（cyclohexanedione，CHD)除草剂，以环己烷环为特征， 常称为“Dims”；和芳氧苯氧丙酸酯类（aryloxyphenoxypropionate，APP)除草剂，常称为lops”。视结构和/或侧链的相似性而定，对稀禾定的抗性可以指示对于乙酰辅酶A羧化酶 This family of herbicides is generally divided into two groups, i.e., cyclohexanedione (cyclohexanedione, CHD) herbicide, characterized cyclohexane ring, often referred to as "Dims"; and aryloxy phenoxypropionic acid ester (aryloxyphenoxypropionate, APP) herbicides, often referred lops ". Depending structure and / or side chain similarity, depending on sethoxydim resistance may indicate the acetyl coenzyme A carboxylase

34抑制剂家族中的一大类除草剂的抗性。 34 inhibitor family of a large class of herbicides. 例如，在具有1781ILE到LEU的突变（最常由稀禾定抗性引起）的数种杂草植物物种中都曾报导过针对CHD和APP除草剂的交叉抗药性（戴尔(Delye), 2005.杂草科学（Weed Science) 53 :728_746，以全文引用的方式并入本文中）。 For example, in several species of weed plants to LEU 1781ILE mutations (most commonly caused by sethoxydim resistance) have been reported in over against CHD and APP herbicides cross-resistance (Dell (Delye), 2005. Weed Science (Weed Science) 53: 728_746, incorporated by reference in its entirety herein). 因此，在一系列温室实验中测定稀禾定抗性A和B细胞系对其它乙酰辅酶A羧化酶抑制性除草剂的抗性。 Therefore, the determination of a series of greenhouse experiments sethoxydim-resistant cell lines A and B to other acetyl coenzyme A carboxylase inhibiting herbicides.

[0211] 在本实验中，将A细胞系和B细胞系与以下两种易受除草剂影响的对照组相比较： 原始的莫纳克亚亲本细胞系（PT)；和由组织培养再生的莫纳克亚细胞系（TTC)。 [0211] In the present experiment, the cell lines A and B cell lines compared with two control groups vulnerable to herbicides on: Mauna Kea original parent cell line (PT); and the regeneration of tissue culture Mauna Kea cell line (TTC). 将植物移植到含有1 ： 1的i^afard ® 混合物与砂的混合物的Cone-tainers™(测量为4 X 14cm， 并逐渐缩减到Icm ；斯图维父子公司，俄勒冈地区科瓦利斯）中，并放到温室中的种植台上， 处于钠灯下，温室中保持27/32°C白天/黑夜的16小时光周期，持续大约2周，随后施用除草剂处理。 The plants were transplanted to contain 1: Cone-tainers ™ i 1 ^ afard ® mixture of sand mixture (measured as 4 X 14cm, and gradually reduced to Icm; Situ Wei & Sons, Inc., Corvallis, Oregon) in By and placed in the greenhouse cultivation stage, in sodium, greenhouse holding 27/32 ° C day / night of the 16 hour period, for approximately two weeks, followed by herbicide treatment.

[0212] 在三个独立的除草剂剂量反应实验中，比较四种基因型A细胞系、B细胞系、 PT、TCC中每一者。 [0212] In three separate herbicide dose-response experiments, compare four kinds of genotype A cell lines, B cell lines, PT, TCC in each. 所测试的除草剂包括精吡氟禾草灵（Fusilade II™)和精恶唑禾草灵(Acclaim Extra™)。 Tested herbicides include fine fluazifop (Fusilade II ™) and fenoxaprop (Acclaim Extra ™). 在各实验中，利用9种适宜的除草剂施用量处理四种基因型中的每一者（一式四份）。 In each experiment, nine kinds of appropriate herbicide rate handle four genotypes in each (four copies). 使用Fusilade II™除草剂（桑吉塔作物保护公司（Syngenta Crop Protection, Inc.)；北卡罗莱纳州格林斯伯勒（Greensboro，NC))，吡氟禾草灵的施用量为0、25、50、100、200、400、800、1600 和3200g ai ha—1 的精吡氟禾草灵施用量。 Use Fusilade II ™ herbicide (Sangeeta crop protection company (Syngenta Crop Protection, Inc.); North Carolina Greensboro (Greensboro, NC)), application rate fluazifop was 0,25 , 50,100,200,400,800,1600 and fine fluazifop application rate of 3200g ai ha-1. 使用Acclaim Extra™除草剂（拜尔环境科学公司（Bayer Environmental Science)，新泽西州蒙特韦尔(Montvale, NJ))，恶唑禾草灵的施用量为0、25、50、100、200、400、800、1600 和3200g ai ha"1的精恶唑禾草灵施用量。在实验喷雾室中，所有除草剂施用量都以187L ha—1的喷雾体积施用，干燥后，将植物放回温室中的种植台上，并在上文所述的条件下保持。处理后21和28天（DAT)，使用0到100的量表记录下作物损伤的目测估计值，其中0等于无损伤，而100 等于完全死亡。本实验为4X9阶乘，S卩，4种基因型和9种除草剂施用量。处理是布置成随机的完备型设计。所用全部四种基因型都是一式四份。首先使用双因素方差分析（SAS， 2008)来分析数据，随后在除草剂施用量范围内进行分析。使用费雪最小显着差异法（LSD) 测定在各除草剂施用量下各基因型平均值之间的差异。 Use Acclaim Extra ™ herbicide (Bayer Environmental Sciences Corporation (Bayer Environmental Science), New Jersey Montreal Vail (Montvale, NJ)), fenoxaprop application rate of 0,25,50,100,200,400 , 800, 1600 and 3200g ai ha "fenoxaprop application rate of 1 in a spray chamber experiments, all amounts are in herbicide spray volume of 187L ha-1 administration, after drying, the plants were returned to the greenhouse the planting stage, and maintained under conditions described above. 21 and 28 days after treatment (DAT), using visual estimates of crop damage at record 0-100 scale, where 0 equals no damage, but 100 equals complete death. This experiment 4X9 factorial, S Jie, four genotypes and nine kinds of herbicide rate. the deal is arranged complete randomized design. The genotypes were quadruplicate with all four First use two-way ANOVA (SAS, 2008) to analyze the data, and then analyzed within herbicide rate range using Fisher least significant difference (LSD) method measured the average between genotype at each herbicide rate differences.

[0213] 图14说明在21DAT时吡氟禾草灵施用量对四种所测试的基因型中每一者的损伤等级的影响。 [0213] FIG. 14 illustrates the influence 21DAT when fluazifop Fertilizer on four genotypes tested each injury level. 双因素方差分析指示显着基因型、除草剂施用量以及在21和^DAT时除草剂施用量对基因型损伤等级的影响（数据未显示）。 Two-way ANOVA analysis indicated significant genotype influence herbicide rate and herbicide rate at 21 and when ^ DAT genotypes damage level (data not shown). 在高于50g ai ha—1的所有施用量下，A 细胞系和B细胞系所显示的损伤不如PT和TCC显着。 In all application rates higher than 50g ai ha-1, and damage cell lines A and B cell lines displayed significantly better than PT and TCC. 对于PT、TC、A细胞系和B细胞系，四种基因型的LD5tl估计值分别为36、37、800和516g ai ha—1。 For PT, TC, A cell lines and B cell lines, LD5tl estimate of four kinds of genotypes were 36,37,800 and 516g ai ha-1. 这些数据强有力地证实了A细胞系和B细胞系都存在针对吡氟禾草灵的交叉抗药性。 These data strongly confirm the A and B cell lines cell lines exist for cross-resistance of fluazifop. 所存在的交叉抗药性水平足以提供针对易受杂草影响的禾草的有效控制，而且不会严重涉及到除草剂损伤。 Level of cross resistance present is sufficient to provide for effective control of susceptible weeds in grasses affected, but not seriously relates to herbicide damage.

[0214] 图15说明在21DAT时恶唑禾草灵施用量对四种所测试的基因型中每一者的损伤等级的影响。 [0214] FIG. 15 illustrates the impact oxazole diclofop application rate of the four tested genotypes in each of the damage grade in 21DAT time. 双因素方差分析指示显着基因型、除草剂施用量以及在21和^DAT时除草剂施用量对基因型损伤等级的影响（数据未显示）。 Two-way ANOVA analysis indicated significant genotype influence herbicide rate and herbicide rate at 21 and when ^ DAT genotypes damage level (data not shown). 在高于50g ai ha—1的所有施用量下，A 细胞系和B细胞系所显示的损伤不如PT和TCC显着。 In all application rates higher than 50g ai ha-1, and damage cell lines A and B cell lines displayed significantly better than PT and TCC. 在本实验中，A细胞系和B细胞系都表现出针对恶唑禾草灵的极高水平的交叉抗药性。 In this experiment, A and B cell lines cell lines exhibited cross resistance against fenoxaprop extremely high level. 在高达1600g ai ha—1的所有恶唑禾草灵施用量下，A细胞系的损伤不超过20%，而甚至在最高施用量3200g ai ha—1下，B细胞系的损伤也不超过20%。 At up to 1600g ai ha-1 of fertilizer all fenoxaprop, damage A cell line does not exceed 20%, and even at the highest application rate of 3200g ai ha-1 under, B cell lines not more than 20 damage %. 对于PT、TC、A细胞系和B细胞系，四种基因型的LD5tl估计值分别为56、22、> 3200和> 3200g ai ha—1。 For PT, TC, A cell lines and B cell lines, LD5tl estimate of four kinds of genotypes were 56,22,> 3200 and> 3200g ai ha-1. 这些数据强有力地证实了A细胞系和B细胞系都存在针对恶唑禾草灵的交叉抗药性。 These data strongly confirm the A and B cell lines cell lines exist for cross-resistance of fenoxaprop. 所存在的交叉抗药性水平能较好地提供针对易受杂草影响的禾草的有效控制，而且不会严重涉及到除草剂损伤。 The existence of cross-resistance level can better provide for effective control of susceptible weed grasses affected, but not seriously relate to herbicide damage.

[0215]实例 11 [0215] Example 11

[0216] 在剪股颖属草中稀禾定抗性细胞系的选择 [0216] Select Agrostis grasses sethoxydim-resistant cell lines

[0217] 为了诱导形成愈合组织，在用力振荡下，于10%漂白剂中对剪股颖属草的种子表面灭菌，持续4小时。 [0217] In order to induce the formation of healing tissue, in force shaking, in 10% bleach for Agrostis grass surface-sterilized seeds for 4 hours. 随后将灭菌过的种子放到表5中所述的愈合组织诱导培养基上（罗(Luo)等人，2003.植物细胞报告(Plant Cell Reports) 22 (9) :645_652，以全文引用的方式并入本文中）。 Then sterilized seeds placed on callus induction medium Table 5 in the (Rom (Luo) et al., 2003 Plant Cell Reports (Plant Cell Reports) 22 (9):. 645_652, in their entirety by reference incorporated herein).

[0218] 表5.用于剪股颖属草的愈合组织诱导培养基 [0218] Table 5 for Agrostis grass callus induction medium

[0219] [0219]

[0220] 一旦从剪股颖属草获得了愈合组织，就如前文所述（实例4)，通过稀禾定选择法筛选这些愈合组织。 [0220] Once the grass Agrostis obtained callus, on previously described (Example 4), screened by healing tissues such as sethoxydim selection. 简单点说，通过将愈合组织放到含有10 μ M稀禾定的愈合组织诱导培养基（表幻上，来选择稀禾定抗性（SR)细胞。使用大培养板（尺寸为245XM5mm)来高效地筛选较多数量的细胞。将直径为约4mm的愈合组织放到15X 15的格子中，得到每块培养板总计225个愈合组织。以3周的时间间隔传代培养愈合组织（实例3)，持续总计9周的选择期。将抗药性愈合组织传代培养到含有补充了10 μ M稀禾定的愈合组织诱导培养基(表幻的100X15mm皮氏培养皿中，培养1个月，以便获得足够的愈合组织。此举将提供总计12周或12周以上的选择时间。 Simply put, by the healing tissue into containing 10 μ M sethoxydim callus induction medium (Table magic to select sethoxydim resistant (SR) cells using large plates (size 245XM5mm) to efficient screening of a large number of cells with a diameter of about 4mm healing of tissue into the grid 15X 15 to give each plate a total of 225 union organization at 3-week intervals subculture callus (Example 3) , a total of nine weeks of continuous selection period would be resistant to the healing of tissue subculture containing supplemented with 10 μ M sethoxydim callus induction medium (Table magic 100X15mm petri dish, and cultured for one month in order to obtain sufficient healing tissue. This will provide more than a total of 12 weeks or 12 weeks of selection time.

[0221]实例 12 [0221] Example 12

[0222] 在剪股颖属草中稀禾定抗性细胞系的再生 [0222] regeneration in Agrostis grasses sethoxydim-resistant cell lines

[0223] 一旦获得了稀禾定抗性愈合组织，就尝试使所有抗药性愈合组织再生。 [0223] Once the sethoxydim-resistant callus, try to make all resistance to the healing tissue regeneration. 所用再生培养基如表6中（罗等人，2003，同上文）所述。 Regeneration media used are shown in Table 6 (Rom et al., 2003, supra) said.

[0224] 表6.用于剪股颖属草的再生培养基 [0224] Table 6 for Agrostis grass regeneration medium

[0225] [0225]

[0226] 可以进行所属领域技术人员已知用于再生稀禾定抗性剪股颖属草愈合组织的任何再生方案。 [0226] may be known to persons skilled in the art for regeneration sethoxydim-resistant grass Agrostis any healing tissue regeneration scheme. 例示性再生方案描述于罗等人0003，同上文）中。 Exemplary regeneration scheme described in Luo et al. 0003, supra) in. 另一例示性再生方案描述于实例5中。 Another exemplary regeneration protocol is described in Example 5.

[0227]实例 13 [0227] Example 13

[0228] 在剪股颖属草中稀禾定抗性细胞系的分子表征 [0228] Molecular characterization of Agrostis grasses sethoxydim-resistant cell lines

[0229] 一旦鉴别出稀禾定抗性（SR)剪股颖属草细胞系，就可以表征引起所述抗药性的突变。 [0229] Once identified sethoxydim resistant (SR) Agrostis grass cell lines, we can characterize the cause of the resistance mutations. 本文中已经描述了用于鉴别乙酰辅酶A羧化酶基因1781位的突变的例示性方案（实例6)。 It has been described herein for identification acetyl coenzyme A carboxylase gene mutation 1781 exemplary program (Example 6). 此外，还可以通过设计引物来扩增包括2027、2041、2078(实例6)和2096位的特定区域，由此分析在剪股颖属草细胞系中乙酰辅酶A羧化酶基因的任何其它位置处的突变（戴尔，2005，同上文）。 In addition, you can also amplify include 2027,2041,2078 (6 instances) and the specific region 2096 by designing primers, whereby analysis Agrostis grass cell lines acetyl coenzyme A carboxylase gene in any other location mutation (Dell, 2005, supra) at. 所属领域技术人员都了解进行序列分析的引物设计和区域扩增方法。 Those skilled in the art are aware of primer design and sequence analysis of the region amplification methods.

[0230]实例 14 [0230] Example 14

[0231] 在剪股颖属草中对稀禾定和乙酰辅酶A羧化酶抑制剂除草剂的抗性的全植物评估 [0231] In Agrostis grass whole plant evaluation of sethoxydim and acetyl coenzyme A carboxylase inhibitor herbicide resistance

[0232] 一旦稀禾定抗性剪股颖属草植物再生，就如本文所述进行稀禾定抗性的全植物评估（实例8和9)。 [0232] Once sethoxydim-resistant Agrostis grass plant regeneration, whole plants evaluate sethoxydim resistant (Examples 8 and 9) as described herein. 此外，也如本文所述进行对其它乙酰辅酶A羧化酶抑制剂除草剂的交叉抗药性评估（实例10)。 In addition, cross-resistance assessment (Example 10) for other acetyl coenzyme A carboxylase inhibitor herbicide as described herein.

[0233]实例 15 [0233] Example 15

[0234] 高羊茅草中愈合组织的诱导 [0234] tall fescue in inducing tissue healing

[0235] 为了诱导形成愈合组织，在50%硫酸中对高羊茅草的种子进行灭菌，持续30分钟，用去离子水和95%乙醇冲洗，并在含有0. 吐温（tween)的100%漂白剂中搅拌30分钟。 [0235] In order to induce the formation of healing tissue in 50% sulfuric acid, tall fescue seeds were sterilized for 30 minutes, deionized water and 95% ethanol, rinsed, and containing 0.1 Tween (tween) 100 % bleach for 30 minutes. 随后，在无菌水中冲洗种子10次，每次4分钟。 Subsequently, the seeds rinsed in sterile water 10 times, each time for 4 minutes. 灭菌后，就将种子放到MS/B5D2培养基上（村重(Murashige)和斯科格（Skoog.) 1962，同上文；甘博格(Gamborg)等人，1968.同上文）以供萌芽。 After sterilization, the seeds will be placed on the MS / B5D2 medium (village weight (Murashige) and Skoog (Skoog) 1962, supra;. Gan Borg (Gamborg) et al., 1968 supra) for bud. 1周后，通过切开种子来使所有萌芽的种子损伤，由此促进愈合组织生长。 After one week, by cutting the seed germination of all the seeds to injury, thereby promoting the healing of tissue growth. 将切开的种子放到愈合组织诱导培养基（如表7中所述）上，以诱导形成愈合组织。 The seeds cut into callus induction medium (Table 7 above) on to induce the formation of callus tissue. 每2 周转移愈合组织，以便繁殖用于接下来的实验。 Transfer every two weeks to heal tissue so that the next test for propagation.

[0236] 表7.用于高羊茅草的愈合组织诱导培养基 Callus induction medium [0236] Table 7 for the tall fescue

[0237] [0237]

[0238]实例 16 [0238] Example 16

[0239] 在高羊茅草稀禾定抗性细胞系的选择 [0239] In the high fescue SETHOXYDIM select resistant cell lines

[0240] 一旦从高羊茅草获得了愈合组织，就如前文所述（实例4)，通过稀禾定选择法筛选这些愈合组织。 [0240] Once the healing of tissue from the tall fescue, on previously described (Example 4), screened by healing tissues such as sethoxydim selection. 简单点说，通过将愈合组织放到含有10 μ M稀禾定的愈合组织诱导培养基（表7)上，来选择稀禾定抗性（SR)细胞。 Simply put, by the healing tissue into containing 10 μ M sethoxydim callus induction medium (Table 7) to select sethoxydim resistant (SR) cells. 使用大培养板（尺寸为245XM5mm)来高效地筛选较多数量的细胞。 The use of large plates (size 245XM5mm) to efficiently screening a large number of cells. 将直径为约4mm的愈合组织放到15X15的格子中，得到每块培养板总计200到250个愈合组织。 The diameter of about 4mm callus into 15X15 grid to give each plate a total of 200-250 healing tissue. 以2周的时间间隔传代培养愈合组织3次（实例3)。 With 2 weeks intervals subcultured calli three times (Example 3). 将抗药性愈合组织传代培养到含有补充了10 μ M稀禾定的愈合组织诱导培养基（表7)的100Χ 15mm皮氏培养皿中，并繁殖至少1个月，以便获得足够的愈合组织。 The resistance to the healing of tissue subculture containing supplemented with 10 μ M sethoxydim callus induction medium (Table 7) 100Χ 15mm petri dishes, and reproduce at least a month in order to obtain sufficient callus.

[0241]实例 17 [0241] Example 17

[0242] 在高羊茅草中稀禾定抗性细胞系中的再生 [0242] In the tall fescue in sethoxydim-resistant cell lines regeneration

[0243] 一旦获得了稀禾定抗性愈合组织，就尝试使所有抗药性愈合组织再生。 [0243] Once the sethoxydim-resistant callus, try to make all resistance to the healing tissue regeneration. 可以使用如表6中（罗等人，2003，同上文）所述的再生培养基。 Regeneration medium can be used as shown in Table 6 (Luo et al., 2003, supra) said. 另一例示性再生方案描述于实例5 中。 Another exemplary regeneration protocol is described in Example 5. 但也可以进行所属领域技术人员已知用于再生稀禾定抗性高羊茅草愈合组织的任何再生方案。 But it can be known to those skilled in any regeneration scheme for renewable sethoxydim-resistant tall fescue tissue healing.

[0244]实例 18 [0244] Example 18

[0245] 在高羊茅草中稀禾定抗性细胞系的分子表征 Molecular characterization [0245] In the tall fescue in sethoxydim-resistant cell lines

[0246] —旦鉴别出稀禾定抗性（SR)高羊茅草细胞系，就可以表征引起所述抗药性的突变。 [0246] - Once identified sethoxydim-resistant (SR) tall fescue cells, it can lead to the characterization of resistance mutations. 本文中已经描述了用于鉴别乙酰辅酶A羧化酶基因1781位的突变的例示性方案（实例6)。 It has been described herein for identification acetyl coenzyme A carboxylase gene mutation 1781 exemplary program (Example 6). 此外，还可以通过设计引物来扩增包括2027、2041、2078(实例6)和2096位的特定区域，由此分析在高羊茅草细胞系中乙酰辅酶A羧化酶基因的任何其它位置处的突变（戴尔，2005，同上文）。 In addition, it can also amplify include (Example 6) and the specific area 2027,2041,2078 2096 by designing primers, which analyze any other position acetyl coenzyme A carboxylase gene in a tall fescue cell lines mutation (Dell, 2005, supra). 所属领域技术人员都了解进行序列分析的引物设计和区域扩增方法。 Those skilled in the art are aware of primer design and sequence analysis of the region amplification methods.

[0247]实例 19 [0247] Example 19

[0248] 在高羊茅草中对稀禾定和乙酰辅酶A羧化酶抑制剂除草剂的抗性的全植物评估 [0248] evaluation of the whole plant sethoxydim and acetyl coenzyme A carboxylase inhibitor herbicide resistance in tall fescue in

[0249] 一旦稀禾定抗性高羊茅草植物再生，就如本文所述进行稀禾定抗性全植物的评估(实例8和9)。 [0249] Once sethoxydim-resistant tall fescue plant regeneration, to carry out a full assessment of sethoxydim-resistant plants (Examples 8 and 9) as described herein. 此外，也如本文所述进行对其它乙酰辅酶A羧化酶抑制剂除草剂的交叉抗药性评估（实例10)。 In addition, cross-resistance assessment (Example 10) for other acetyl coenzyme A carboxylase inhibitor herbicide as described herein.

[0250]实例 20 [0250] Example 20

[0251] 在结缕草中稀禾定抗性细胞系的选择 [0251] Select sethoxydim-resistant cell lines in Zoysiagrasses

[0252] 为了诱导形成愈合组织，在50%硫酸中对结缕草的种子进行灭菌，持续30分钟， 用去离子水和95%乙醇冲洗，并在含有0. 吐温的100%漂白剂中搅拌30分钟。 [0252] In order to induce the formation of healing tissue in 50% sulfuric acid Zoysia grass seeds are sterilized, for 30 minutes, deionized water and 95% ethanol, rinsed, and containing 0.5 Tween 100% bleach The mixture was stirred for 30 minutes. 随后， 在无菌水中冲洗种子10次，每次4分钟。 Subsequently, the seeds rinsed in sterile water 10 times, each time for 4 minutes. 灭菌后，就将种子放到MS/B5D2培养基上（村重(Murashige)和斯科格（Skoog.) 1962，同上文；甘博格(Gamborg)等人，1968.同上文）以供萌芽。 After sterilization, the seeds will be placed on the MS / B5D2 medium (village weight (Murashige) and Skoog (Skoog) 1962, supra;. Gan Borg (Gamborg) et al., 1968 supra) for bud. 1周后，通过切开种子来使所有萌芽的种子损伤，由此促进愈合组织生长。 After one week, by cutting the seed germination of all the seeds to injury, thereby promoting the healing of tissue growth. 将切开的种子放到愈合组织诱导培养基（如表7中所述）上，以诱导形成愈合组织。 The seeds cut into callus induction medium (Table 7 above) on to induce the formation of callus tissue. 每2周转移愈合组织，以便繁殖用于接下来的实验。 Transfer every two weeks to heal tissue so that the next test for propagation.

[0253]实例 21 [0253] Example 21

[0254] 在结缕草中稀禾定抗性细胞系的选择 [0254] Select sethoxydim-resistant cell lines in Zoysiagrasses

[0255] 一旦从结缕草获得了愈合组织，就如前文所述（实例4)，通过稀禾定选择法筛选这些愈合组织。 [0255] Once the healing of tissue from Zoysia, on previously described (Example 4), screened by healing tissues such as sethoxydim selection. 简单点说，通过将愈合组织放到含有10 μ M稀禾定的愈合组织诱导培养基(表7)上，来选择稀禾定抗性（SR)细胞。 Simply put, by the healing tissue into containing 10 μ M sethoxydim callus induction medium (Table 7) to select sethoxydim resistant (SR) cells. 使用大培养板（尺寸来高效地筛选较多数量的细胞。将直径为约4mm的愈合组织放到15X 15的格子中，得到每块培养板总计200到250个愈合组织。以2周的时间间隔传代培养愈合组织3次（实例3)。将抗药性愈合组织传代培养到含有补充了10 μ M稀禾定的愈合组织诱导培养基（表7)的100X15mm 皮氏培养皿中，并繁殖至少1个月，以便获得足够的愈合组织。 Using large plates (size to efficiently filter a large number of cells having a diameter of about 4mm callus 15X 15 into the lattice, each culture plate to give a total of 200 to 250 callus. 2 weeks callus subculture interval three times (Example 3). The resistance to the healing tissue subculture containing supplemented with 10 μ M sethoxydim callus induction medium (Table 7) 100X15mm petri dish and reproduction at least a month, in order to obtain sufficient callus.

[0256]实例 22 [0256] Example 22

[0257] 在结缕草中稀禾定抗性细胞系的再生 [0257] regeneration of Zoysia sethoxydim-resistant cell lines

[0258] 一旦获得了稀禾定抗性愈合组织，就尝试使所有抗药性愈合组织再生。 [0258] Once the sethoxydim-resistant callus, try to make all resistance to the healing tissue regeneration. 可以使用如表6中（罗等人，2003，同上文）所述的再生培养基。 Regeneration medium can be used as shown in Table 6 (Luo et al., 2003, supra) said. 另一例示性再生方案描述于实例5 中。 Another exemplary regeneration protocol is described in Example 5. 但也可以进行所属领域技术人员已知用于再生稀禾定抗性结缕草愈合组织的任何再生方案。 But it can be known to persons skilled in the art for regeneration sethoxydim-resistant Zoysia any healing tissue regeneration scheme.

[0259]实例 23 [0259] Example 23

[0260] 在高羊茅草中稀禾定抗性细胞系的分子表征 Molecular characterization [0260] In the tall fescue in sethoxydim-resistant cell lines

[0261] 一旦鉴别出稀禾定抗性（SR)高羊茅草细胞系，就可以表征引起所述抗药性的突变。 [0261] Once identified sethoxydim resistant (SR) tall fescue cells, it can lead to the characterization of resistance mutations. 本文中已经描述了用于鉴别乙酰辅酶A羧化酶基因1781位的突变的例示性方案（实例6)。 It has been described herein for identification acetyl coenzyme A carboxylase gene mutation 1781 exemplary program (Example 6). 此外，还可以通过设计引物来扩增包括2027、2041、2078(实例6)和2096位的特定区域，由此分析在高羊茅草细胞系中乙酰辅酶A羧化酶基因的任何其它位置处的突变（戴尔，2005，同上文）。 In addition, it can also amplify include (Example 6) and the specific area 2027,2041,2078 2096 by designing primers, which analyze any other position acetyl coenzyme A carboxylase gene in a tall fescue cell lines mutation (Dell, 2005, supra). 所属领域技术人员都了解进行序列分析的引物设计和区域扩增方法。 Those skilled in the art are aware of primer design and sequence analysis of the region amplification methods.

[0262]实例 24 [0262] Example 24

[0263] 在结缕草中对稀禾定和乙酰辅酶A羧化酶抑制剂除草剂的抗性的全植物评估 [0263] In the whole plant Zoysia assessment of sethoxydim and acetyl coenzyme A carboxylase inhibitor herbicide resistance

[0264] 一旦稀禾定抗性结缕草植物再生，就如本文所述进行稀禾定抗性全植物的评估(实例8和9)。 [0264] Once sethoxydim-resistant Zoysia plant regeneration, carried out a full assessment of sethoxydim-resistant plants (Examples 8 and 9) as described herein. 此外，也如本文所述进行对其它乙酰辅酶A羧化酶抑制剂除草剂的交叉抗药性评估（实例10)。 In addition, cross-resistance assessment (Example 10) for other acetyl coenzyme A carboxylase inhibitor herbicide as described herein.

[0265]实例 25 [0265] Example 25

[0266] 通过施用除草剂控制除草剂抗性植物间的杂草种类 [0266] By applying herbicide weed control herbicide-resistant plant species between

[0267] 每周1次用150g ai ha—1稀禾定处理一小块含有狗牙根和稀禾定抗性海滨雀稗的土地，持续3个月的时间。 [0267] 1 week with 150g ai ha-1 sethoxydim treatment containing a small piece of land Bermudagrass and sethoxydim-resistant paspalum, continuing three months. 在3个月的处理期中，观察到，狗牙根慢慢地灭绝，而稀禾定抗性雀稗持续茁壮成长，在这块土地上留下繁殖超过80%的稀禾定抗性雀稗。 In the three-month treatment period was observed, bermudagrass slowly become extinct, while sethoxydim-resistant paspalum continue to thrive in this land leaving more than 80% of the breeding sethoxydim-resistant paspalum.

[0268] 实例洸 [0268] Examples of Guang

[0269] 通过施用组合除草剂控制除草剂抗性植物间的杂草种类 [0269] The administration of a combination of herbicides for weed control herbicide-resistant plant species between

[0270] 每周1次用150g ai ha"1稀禾定和150g ai ha"1恶唑禾草灵处理一小块含有狗牙草和稀禾定抗性海滨雀稗的土地，持续3个月的时间。 [0270] 1 week with 150g ai ha "1 sethoxydim and 150g ai ha" 1 fenoxaprop handle containing a small piece of land in a Dog grass and sethoxydim-resistant paspalum, sustained 3 months. 在3个月的处理期中，观察到，狗牙根慢慢地灭绝，而稀禾定抗性雀稗持续茁壮成长，在这块土地上留下繁殖超过80%的稀禾定抗性雀稗。 In the three-month treatment period was observed, bermudagrass slowly become extinct, while sethoxydim-resistant paspalum continue to thrive in this land leaving more than 80% of the breeding sethoxydim-resistant paspalum.

[0271]实例 27 [0271] Example 27

[0272] 标记辅助的选择：使用除草剂抗性作为标记来鉴别适于选择的性状 [0272] marker-assisted selection: the use of herbicide resistance as a marker to identify adapted to select traits

[0273] 如本文所述（参看实例15-19)培养具有育种目的所需的数种性状的高羊茅草品种，以鉴别所述品种的稀禾定抗性愈合组织细胞系。 [0273] As described herein (see Examples 15-19) were cultured tall fescue varieties with desired traits of several breeding purposes, to identify the species of sethoxydim-resistant callus cell lines. 使这些细胞系再生成一代成熟的植物Ro。 So that in the generation of mature plants to generate these cell lines Ro. 将具有乙酰辅酶A羧化酶I1781L突变（赋予稀禾定抗性）的Rtl植物与缺乏所述数种性状的不同高羊茅草品种杂交。 Rtl plant will have acetyl coenzyme A carboxylase I1781L mutation (given sethoxydim-resistant) with different varieties of tall fescue hybrids lack the several traits. 经过接下来的杂交，某些符合需要的性状显示为非随机地与稀禾定抗性分离。 After hybridization the next, certain traits consistent with the need to display a non-random and sethoxydim resistant isolates. 经过进一步的任选杂交，可定性稀禾定抗性与各相关性状之间的关联。 After further optional hybrid, qualitatively sethoxydim resistance associated with the relevant set of traits. 对于发现与稀禾定抗性有关的每一性状，所述抗性是适用于标记辅助的育种/选择方案的标记。 For each trait discovery and related sethoxydim resistance, said resistance is applied to the tag marker-assisted breeding / selection program.

[0274] 实例沘 [0274] Examples of Bi

[0275] 标记辅助的选择：根据标记表型选择符合需要的相关性状 [0275] marker-assisted selection: Choose meet the needs of the tag phenotypes related traits

[0276] 使用来自实例27的Rtl代或此代植物的子代的稀禾定抗性高羊茅草植物进行标记辅助的育种和选择。 [0276] Examples of use from 27 Rtl-generation or sethoxydim-resistant tall fescue plants of this progeny generation plants were marker-assisted breeding and selection. 将市售的缺乏实例27中所鉴别的相关性状中的一者的高羊茅草品种与来自实例27的稀禾定抗性高羊茅草植物杂交，以形成杂交代。 Commercially available examples of the lack of related traits identified 27 as one of the tall fescue cultivars with resistance from Example sethoxydim 27 tall fescue plant hybridization to form a hybrid generations. 使杂交代的种子萌芽，并用足以杀死或严重延迟非抗药性植物生长的量的稀禾定处理植物。 Generation hybrid seeds sprout, and can kill or severely delayed by the amount of non-resistant plant growth sethoxydim treatment plants. 选择健康的稀禾定抗性植物进行进一步杂交。 Choose a healthy sethoxydim resistant plants for further hybridization. 大量的所述所选植物都携带相关性状。 A large number of the selected plant carries related traits. 使稀禾定抗性植物与市售品种的植物进一步交叉传代，随后用稀禾定处理和选择，产生实质上具有所述市售品种的遗传背景但携带确定与稀禾定抗性相关的符合需要的性状的植株。 So sethoxydim-resistant plant and commercial plant species further cross passages, followed by sethoxydim and selection process, resulting in substantially the genetic background of the commercial cultivars, but determined to carry sethoxydim resistance associated with compliance Characters plants need.

[0277] 实例四 [0277] Four examples

[0278] 标记辅助的选择：根据分子标记选择符合需要的相关性状 [0278] marker-assisted selection: trait markers selected in accordance with the relevant line with the needs of

[0279] 使用来自实例27的Rtl代或此代植物的子代的稀禾定抗性高羊茅草植物进行标记辅助的育种和选择。 [0279] Examples of use from 27 Rtl-generation or sethoxydim-resistant tall fescue plants of this progeny generation plants were marker-assisted breeding and selection. 将市售的缺乏实例27中所鉴别的相关性状中的一者的高羊茅草品种与来自实例27的稀禾定抗性高羊茅草植物杂交，以形成杂交代。 Commercially available examples of the lack of related traits identified 27 as one of the tall fescue cultivars with resistance from Example sethoxydim 27 tall fescue plant hybridization to form a hybrid generations. 使杂交代的种子萌芽，并借助分子方法（例如PCR)针对与I1781L突变相关的SNP的存在筛选来自萌芽植物的样品。 Make hybrid generation of seed germination, and with molecular methods (such as PCR) for the presence of SNP mutations associated with I1781L screening samples from sprouting plants. 例如，可以在扩增分析中使用SV384F和SV384R引物(实例6，SEQ ID NO ：1和2)来检测标记。 For example, you can use SV384F and SV384R primers to amplify the analysis (Example 6, SEQ ID NO: 1 and 2) to detect markers. 杂交植物中分子标记的存在证实了杂交植物也携带与稀禾定抗性有关的符合需要的性状的可能性，如实例27中所论述。 Hybrid plants confirmed the presence of molecular markers also carries the possibility of a hybrid plant traits and sethoxydim resistance related suits your needs, as discussed in Example 27. 选择携带分子标记的植物进行进一步杂交。 Molecular markers to select plants carrying further crossing. 大量的所述所选植物都携带相关性状。 A large number of the selected plant carries related traits. 使具有所述标记的植物与市售品种的植物进一步交叉传代，随后进行进一步分子选择或通过稀禾定处理和选择，产生实质上具有所述市售品种的遗传背景但携带确定与稀禾定抗性相关的符合需要的性状的植株。 Making plants and commercial varieties of plants with the marker further cross passages, followed by a further molecule or by sethoxydim selection process and selection, to produce substantially the genetic background of the commercial cultivars, but carrying determine sethoxydim Resistance to meet the needs associated with plant traits.

[0280] 上文描述的各种方法和技术提供了多种方式来进行本发明。 [0280] various methods and techniques described above provide a variety of ways to carry out the present invention. 此外，所属领域技术人员将认识到来自不同实施例的各种特征的适用性。 In addition, those skilled in the art will recognize the applicability of various features from different embodiments of. 类似地，所属领域技术人员可组合和/或修改上文论述的各种要素、特征和步骤以及各所述要素、特征或步骤的其它已知的等效物，以便执行根据本发明的原理的方法。 Similarly, those skilled in the art may be combined and / or modify the various elements discussed above, features and steps as well as other known equivalents for each of the elements, features, or steps in order to perform according to the principles of the present invention. Methods. 在不同实施例中将特别地包括各种要素、特征和步骤中的一部分，并特别地排除其它的要素、特征和步骤。 Specifically it includes various elements, features, and steps some will be different in the embodiment example, and specifically exclude other elements, features, and steps.

[0281] 尽管已经在某些实施例和实例的内容中揭示了本发明，但所属领域技术人员应了解，本发明的实施例可超出特别揭示的实施例而扩展到其它替代性实施例和/或用途和修改以及其等效物。 [0281] Although the present invention has been disclosed in certain embodiments and examples of the content, but skilled in the art should be appreciated, embodiments of the present invention may be beyond the specifically disclosed embodiments and extended to other alternative embodiments and / or uses and modifications and equivalents thereof.

[0282] 在本发明的实施例中已经揭示了许多变更和替代性要素。 [0282] In an embodiment of the present invention have been disclosed many variations and alternative elements. 所属领域技术人员将显而易见其它变更和替代性要素。 Those skilled in the art will be apparent other modifications and alternative elements.

[0283] 在一些实施例中，术语“一”和“所述”以及在描述本发明的某一特定实施例的上下文（尤其是在以下某些权利要求的上下文）中使用的类似参考物都可解释为涵盖单数和复数形式。 [0283] In some embodiments, the term "one" and "the" and similar reference material in the description of a specific embodiment of the present invention, the context (in particular in the context of some of the following claims) are used interpreted to cover both the singular and plural forms. 本文中有关数字的范围的叙述仅打算用作个别地指示每一单独值在所述范围内的简写方法。 This article described the scope of the relevant figures only intended as individual designation of each individual value within the range of shorthand methods. 除非本文中另作指示，否则各个别值都被并入本说明书中，就如同本文个别地叙述每一值一样。 Unless otherwise indicated herein, each individual value or are incorporated herein as if individually described herein as each value. 除非在本文中另作指示或上下文另外明确地相反指示，否则本文所述的所有方法可以任何次序执行。 Unless otherwise indicated herein or the context clearly dictates otherwise, otherwise, all methods described herein can be performed in any order. 除非另外声明，否则在本文的某些实施例中提供的任何实例和所有实例或示范性术语（例如，“例如”）的使用都仅打算用于更好地说明本发明，并且不形成对本发明的范围的限制。 Unless otherwise provided in any instance in some embodiments herein and all examples, or exemplary terms (eg, "such as") are only intended for the use of better illustrate the present invention and the invention is not formed limited range. 不应将本说明书中的术语解释为指示任何非所主张的要素为实践本发明所必需的。 This description should not be construed as any element of the term non-claimed for the practice of the present invention to be necessary.

[0284] 本文中揭示的替代性要素或实施例的分组不应解释为限制。 Packet alternative elements or embodiments of the [0284] disclosed herein should not be construed as limiting. 各组的成员都可个别地提及和主张，或与所述组的其它成员或者本文所述的其它要素以任何组合形式提及和主张。 Members of the group can be mentioned individually and ideas, or with other elements or other members of the group described herein and claims referred to in any combination. 为便利和/或可专利性起见，一个组的一个或一个以上成员都可包括在一个组中或从一组中缺失。 In order to facilitate and / or patentability purposes, one by one or more members of the group can be included or missing from a group in a group. 当出现任何此类纳入或缺失时，本说明书在本文中应视为含有如此修改的群组，由此满足所附权利要求书中使用的所有马库西群组（Markush group)的书面描述。 When any such inclusion or deletion, the present description herein shall be deemed to contain the group so modified, thereby satisfying the appended claims all used Macuxi groups (Markush group) written description.

[0285] 本文描述了本发明的优选实施例，包括发明者已知的用于进行本发明的最佳模式。 [0285] This paper described a preferred embodiment of the present invention, including known to the inventors for carrying out the best mode of the invention. 所属领域技术人员在阅读了前述描述后，将即刻了解针对这些优选实施例的变更。 Skilled in the art upon reading the foregoing description, it will immediately understand a modified example for these preferred embodiments. 预期所属领域技术人员可在适当时使用所述变更，而且本发明可以不同于本文特别描述的方式实践。 Expected skilled in the art may be used, where appropriate, the change, and the practice of the invention may be practiced otherwise than as specifically described herein. 因此，本发明的许多实施例包括适用法律所允许的针对附加权利要求书中所述的发明主题的所有修改和等效物。 Thus, many embodiments of the present invention include for additional rights under applicable laws permitted in the claims all modifications and equivalents of the inventive subject matter. 此外，除非本文另作指示或上下文另外明确地作相反指示， 否则本发明涵盖上述要素的所有可能的变更的任何组合。 In addition, unless the context indicates otherwise or the context clearly indicates to the contrary, or any combination of these elements to cover all possible variations of the present invention.

[0286] 而且，在本说明书全文引用了多项专利和印刷的出版物。 [0286] In the present specification refers to patents and printed publications. 上述参考文献和印刷的出版物中每一者都是以全文引用的方式个别地并入本文中。 The above cited references and printed publications are based on each individually entirety by reference herein.

[0287] 最后，应了解，本文揭示的本发明的实施例是对本发明原理的说明。 [0287] Finally, it should be appreciated that embodiments of the present invention disclosed herein are the principles of the invention. 可以使用的其它修改都在本发明的范围内。 Other modifications that may be used within the scope of the present invention. 因此，例如（但不限于），可以根据本文的教示而利用本发明的替代性配置。 Thus, for example (but not limited to), according to the teachings herein and the use of an alternative configuration of the present invention. 因此，本发明的实施例不限于所显示和描述者。 Thus, embodiments of the present invention is not limited to those shown and described.