JP5932832B2 - 植物細胞の適格性を改善する方法 - Google Patents
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Description
本出願は、2010年12月17日出願の米国仮出願第61/424,136号の優先権の利益を主張する。上記の出願の開示は全て参照として本明細書に組み込まれる。
実施例1
この実施例は、滅菌蒸留水(SDW)にPEGを含有する組成物と接触させた乾燥ダイズ胚の増強された形質転換を示す。ダイズcv.A3525乾燥胚を、米国特許出願公開第2008/0280361号に記載された方法に従って切除し、SDWに溶解した5、10、20、または50%PEG−4000の様々な量と1時間接触させた。胚をSDWで5〜6回すすぎ、米国特許出願公開第2009/0138985号に記載された方法に従って形質転換した。胚を、元のOriRiまたはOriV複製を有し、アグロバクテリウムのABIまたはAB30株に含まれた2T形質転換ベクターで形質転換した。胚を、スペクチノマイシン選択培地で再生させた。表4に示されているように、5、10、および20%PEG組成物と接触させた胚は、INO培地またはSDW単独と接触させた胚と比較して、一般に、増強された形質転換頻度および単一コピー事象の増強された頻度を示した。50%PEGと接触させた胚も、SDWと接触させた胚(TF=2.3%)と比較して改善されたTF(7%)を示した。50%PEGでの実験は、AB30/OriRi作成物により実施した。ダイズのスペクチノマイシン選択培地の組成については表5を参照すること。
実施例2
この実施例は、INOまたはINO培地中にPEGを含有する組成物と接触させた乾燥ダイズ胚の増強された形質転換を示す。ダイズcv.A3525乾燥胚を、米国特許出願公開第2008/0280361号に記載された方法に従って切除し、INOに溶解した20%PEG−4000と1時間接触させた。胚を無添加INOで5〜6回すすぎ、米国特許出願公開第2009/0138985号に記載された方法に従って形質転換した。胚を、元のOriRi複製を有し、アグロバクテリウムのAB30株に含まれた2T形質転換ベクターで形質転換した。胚を、スペクチノマイシン選択培地で再生させた。表6に示されているように、20%PEG組成物と接触させた胚は、一般に、INO培地単独と接触させた胚と比較して、2つの異なる実験において増強された形質転換頻度を示した。INO培地の組成については表7を参照すること。
実施例3
この実施例は、BGM単独と比較した、マメ発芽培地(BGM−表8)にPEGを含有する組成物と接触させた乾燥ダイズ胚の増強された形質転換を示す。ダイズcv.A3525乾燥胚を、米国特許出願公開第2008/0280361号に記載された方法に従って切除し、BGMに溶解した10%または20%PEG−4000と1時間接触させた。胚をBGMで5〜6回すすぎ、米国特許出願公開第2009/0138985号に記載された方法に従って形質転換した。胚を、元のOriV複製を有し、アグロバクテリウムのAB30株に含まれた2T形質転換ベクターで形質転換した。胚を、スペクチノマイシン選択培地で再生させた。表9に示されているように、10%および20%PEG組成物と接触させた胚は、BGM単独と接触させた胚と比較して増強された形質転換頻度を示した。
実施例4
この実施例は、PEGと比較したTFに対する他の浸透性化合物の効果を示す。様々なPEG種による処理のTF増強も比較する。ダイズcv.A3555乾燥胚を、米国特許出願公開第2008/0280361号に記載された方法に従って切除し、SDWに溶解したPEG−4000、PEG−6000、PEG−8000、マンニトール、ソルビトール、またはグリセロールの様々な量と1時間接触させた。次に胚をSDWで5〜6回すすぎ、米国特許出願公開第2009/0138985号に記載された方法に従って形質転換した。胚を、元のOriRi複製を有し、アグロバクテリウムのAB30株に含まれた2T形質転換ベクターで形質転換した。胚を、スペクチノマイシン選択培地で再生させた。表10に示されているように、10%および20%PEG組成物と接触させた胚は、他の浸透性化合物または水単独で処理した胚と比較して、増強された形質転換頻度を示した。
実施例5
この実施例は、PEGが乾燥胚外植片の水和率の調節に有用であることを示す。ダイズcv.A3525乾燥胚を、米国特許出願公開第2008/0280361号に記載された方法に従って切除した。次に外植片を、INOに溶解した20%PEG4000またはINO単独のいずれかと接触させた。水分取り込み率は、処理の際に定期的な間隔で外植片の試料を取り、それらを重量測定に基づいた破壊オーブン試験に付し、そこで外植片を約100℃のオーブンでおよそ2日間乾燥した。水分率は、これらの重量損失により決定した。表11および12にそれぞれ示されているように、第1の研究は、1、4、および24時間の水和率を検査し、一方、第2の研究は、0、15、30、45、および60分間の水和率の率を検査した。両方の研究は、20%PEG4000と接触させた乾燥ダイズ胚外植片による水分取り込み率が低減されていることを示す。により水和を調節する
実施例6
この実施例は、水中のPEGと接触させた乾燥切除胚外植片が、水単独と接触させたものと比較して少ない出液水を放出することを示す。ダイズcv.A3555乾燥胚を、米国特許出願公開第2008/0280361号に記載された方法に従って切除し、SDW中20%PEG−4000またはSDW単独と1時間接触させた。それぞれの培地中の外植片を、オービタルシェイカーに75RPMで入れ、室温でインキュベートした。各群の培地の試料をインキュベーションの0、15、30、45、および60分後に取り出し、光学密度(300nmでの吸光度、適切な培地による空試験)および伝導率(マイクロジーメンス)を測定して出液水を分析した。表13に示されているように、出液水の光学密度は、20%PEG処理の試料と比較して水対照試料においてはるかに高かった。伝導率についても同じことが当てはまった。注:星印(*)で示された読み取り値は、使用した分光光度計が最大範囲の3.295を有したので、試料の1/10希釈で実施し、次に10を掛けた。
実施例7
この実施例は、アグロバクテリウム仲介形質転換の前に接触させたときの、水和ダイズ胚の形質転換頻度に対するPEG処理の効果を示す。ダイズcv.A3525ダイズ胚を、米国特許出願公開第2008/0280361号に記載された方法に従って切除し、SDW中のPEG−4000の多様な量と1時間接触させた。胚をSDWで5〜6回すすぎ、米国特許第7,402,734号に記載された方法に従って形質転換した。胚を、元のOriRi複製を有し、アグロバクテリウムのAB30株に含まれた2T形質転換ベクターで形質転換した。胚を、スペクチノマイシン選択培地で再生させた。表14に示されているように、PEG処理は、既に水和されたダイズ成熟胚のTFの増強に有用であるとは思われなかった。PEG処理は乾燥胚および下記のトウモロコシ未熟胚で例示されているように未熟胚に有用でありうる。
実施例8
この実施例は、形質転換頻度に対する、同時培養の際のPEG組成物の効果を示す。ダイズcv.A3555乾燥胚を、米国特許出願公開第2008/0280361号に記載された方法に従って切除し、SDWに溶解した20%PEG−4000と1時間接触させた。胚をSDWで5〜6回すすぎ、米国特許出願公開第2009/0138985号に記載された方法に従って形質転換した。胚を、元のOriRi複製を有し、アグロバクテリウムのAB30株に含まれた2T形質転換ベクターで形質転換した。次に形質転換された細胞を、1、5、10、または20%PEG4000を含有するINO培地で同時培養した。同時培養培地は1ppmのTDZも含有した。胚を、スペクチノマイシン選択培地で再生させた。表15に示されているように、PEGが同時培養培地に含まれている処理は、PEGが同時培養の前に使用された処理と比較して低いTFを生じた。このことは、形質転換の前のPEG処理が細胞の細菌仲介形質転換の適格性を改善するが、同時培養の際のPEGはTFを低減すると思われることを示唆している。
実施例9
この実施例は、PEGおよび異なる量(%)のPEGをカルス生産の改善のために使用できることを示す。トウモロコシの雌穂からトウモロコシ胚を、本開示の他の部分に記載されているように手作業で単離し、集めた。胚を、1mLのLynx2304または異なる量のPEG8000(Sigma P−2139)を含有する1mLのLynx2304培地のいずれかに、6℃で4日間暗黒に保存した。それぞれの処理では4回の反復を実施した。続いて胚をカルス誘導培地Lynx1074で約2週間培養した。Lynx1074および2304培地の組成については表17を参照すること。表16に示されているように、対照処理(保存なし)では、単離の後にLynx2304培地で直接培養したとき、100%の胚がカルスを生産した。1%または5%のPEG8000に保存されたとき、僅かな率の胚がカルスを生産した。PEGなしの培地に保存されたとき、カルスを生産した胚はなかった(処理2)。10または20%のPEG8000に保存された胚の100%がカルスを生産した(処理5および6)。
実施例10
実施例11
実施例12
実施例13
実施例15
実施例16
実施例17
実施例18
実施例19
実施例20
この実施例は、滅菌水にPEGを含有する組成物と接触させた乾燥ワタ胚の増強された形質転換を示す。ワタcv.DP393−0053種を、10%クロロックス漂白剤で10分間無菌化した。次にこれらを種乾燥機で一晩乾燥して、平均内部水分量の3.4%を達成した。最初にディスク型粉砕機のGP140型(Modern Process Equipment Corp.,Chicago,II)で種を加工し、次にClipper Eclipse 324(Clipper Separation Technologies;A.T.Ferrell Company,Bluffton,Ind)のような自動化篩い分け空気流分離装置で加工して、種皮、微粉および他の破片のような種の不要な部分の大部分を除去することにより、乾燥胚を乾燥種から切除した。保持した種材料を沸騰が止まるまで(約30秒間)液体窒素に浸け、次に、米国特許出願公開第2008/0280361号に記載されているように、追加的にスクリーンして、子葉、種皮および他の破片のような不要な材料から望ましい胚外植片材料を単離する前に、ディスク型粉砕機で再び加工した。乾燥胚を、滅菌水に溶解した20%PEG−4000と1時間接触させた。胚をRO水で4分間すすぎ、次に、米国特許第8,044,260号に記載された方法に従ってアグロバクテリウム仲介形質転換に付した。胚を、スペクチノマイシン選択培地で再生させた。表28に示されているように、20%PEG組成物と接触させた胚は、一般に、INO培地(PEGなし)と接触させた胚と比較して増強された形質転換頻度を示した。ワタのスペクチノマイシン選択培地の組成については表29を参照すること。
Claims (19)
- 植物細胞を形質転換の前に有効量のポリエチレングリコール含有組成物と接触させることを含む、前記植物細胞の細菌仲介形質転換の適格性を改善する方法。
- 前記ポリエチレングリコールの前記有効量が1〜25容量%である、請求項1に記載の方法。
- 前記ポリエチレングリコールの前記有効量が20容量%である、請求項2に記載の方法。
- 前記ポリエチレングリコールの分子量が200〜10000である、請求項1に記載の方法。
- 前記ポリエチレングリコールの分子量が4000〜8000である、請求項4に記載の方法。
- 有効量の少なくとも1つの植物成長調整剤が前記ポリエチレングリコール含有組成物と共に提供される、請求項1に記載の方法。
- 前記植物成長調整剤が、オーキシン、サイトカイニン、またはこれらの組み合わせを含む、請求項6に記載の方法。
- 前記オーキシンが、IAA、2,4−D、NAA、IBA、ジカンバ、またはこれらの組み合わせからなる群より選択される、請求項7に記載の方法。
- 前記オーキシンの量が0.001mg/L〜30mg/Lである、請求項7に記載の方法。
- 前記サイトカイニンが、BAP、ゼアチン、カイネチン、TDZ、またはこれらの組み合わせからなる群より選択される、請求項7に記載の方法。
- 前記サイトカイニンの量が0.001mg/L〜30mg/Lである、請求項7に記載の方法。
- 前記ポリエチレン含有組成物が前記植物細胞と30分〜10日間接触させられる、請求項1に記載の方法。
- 前記ポリエチレン含有組成物が前記植物細胞と3日〜7日間接触させられる、請求項12に記載の方法。
- 前記ポリエチレン含有組成物が前記植物細胞と30分〜300分間接触させられる、請求項12に記載の方法。
- 前記植物細胞を全植物に再生させることを更に含む、請求項1に記載の方法。
- 前記植物細胞が、種、葉、茎、根、未熟胚、成熟胚、カルス、小胞子、分裂組織、子葉、胚軸、上胚軸、中胚軸、子葉鞘、根生、幼芽、または生殖組織の細胞を含む、請求項1に記載の方法。
- 前記植物細胞が、トウモロコシ、ダイズ、ワタ、カノーラ、サトウキビ、タマネギ、メロン、サトウダイコン、またはコムギの細胞を含む、請求項1に記載の方法。
- 前記細菌仲介形質転換が、アグロバクテリウム(Agrobacterium)仲介、リゾビウム(Rhizobium)仲介、シノリゾビウム(Sinorhizobium)仲介、メソリゾビウム(Mesorhizobium)仲介、およびブラディリゾビウム(Bradyrhizobium)仲介形質転換である、請求項1に記載の方法。
- 前記ポリエチレングリコールと接触させた前記植物細胞を、形質転換の前に非PEG含有組成物においてすすぐステップを更に含む、請求項1に記載の方法。
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