JP3659346B2 - Shiba genetic transformation method - Google Patents

Description

１０ｍｌの懸濁培養物中の細胞を、５０ｍｌのポリプロピレンチューブ（Ｂｅｃｔｏｎ Ｄｉｃｋｉｎｓｏｎ Ｌａｂｗａｒｅ、ＵＳＡ）中において、２０分間２５００ｒｐｍで遠心分離することによって収集し、そして１０ｍｌの液体感染培地（表１のＬｑＭＳＡＳ）中で、穏やかにボルテックスすることによって再懸濁した。
【００３４】
アセトシリンゴンを、１００ｍｇ／ｍｌ濃度で、ジメチルスルホキシド中に適切な量の粉末を溶解することによって調製し、そして暗所で４℃にて保存した。これを、必要とされる場合に、適切な最終濃度まで滅菌培地に添加した。ＭＳＣＧ４培地において生長したカルスを、Ａｇｒｏｂａｃｔｅｒｉｕｍ細胞懸濁液中に１分間浸漬した。滅菌濾紙上での脱水後、このカルスを、４ｍｇ／ｌ ２，４−Ｄを有する共存培養培地（ＭＳＡＳ）において、暗所で１５日間２６±１℃にて培養した。共存培養後、このカルスを、界面活性剤（０．０２％プルロニック（ｐｌｕｒｏｎｉｃ）Ｆ６８）を補充した滅菌二重蒸留水中で、この洗浄溶液が透明になるまでボルテックスすることによって徹底的に洗浄し、そして最終的に、１０００ｍｇ／ｌカルベニシリン（ｃａｌｂｅｎｉｃｉｌｌｉｎ）および０．０２％界面活性剤を含有する滅菌二重蒸留水中で洗浄した。洗浄後、このカルスの半分を、Ｓｃｈｅｎｋら（１９９８）によって記載された方法によるＧＵＳ活性アッセイのために使用した。
【００３５】
いくつかの因子を、形質転換効率を最適化するために試験した。（１）浸潤についての異なるカルス型の比較のために、１型、２型、および３型のカルスを、６日間、２，４−Ｄを含まないＭＳＡＳ培地において共存培養した；（２）共存培養期間の比較のために、カルスを、３、６、９、１２、１５、１８、２１、２４、および２７日間、共存培養した；（３）培地成分の試験のために、異なる量の２，４−Ｄ（０、１、２、４、および８ｍｇ／ｌ）、ＣａＣｌ₂（０、１１、１１０、２２０、および４４０ｍｇ／ｌ）、およびアセトシリンゴン（０、１０、５０、１００、および２００ｍｇ／ｌ）を、ＭＳＡＳ培地に添加した。
【００３６】
（ｐＧＰＴＶ−ＨＢ共存培養）
シバカルスのＡ．ｔｕｍｅｆａｃｉｅｎｓ感染についての最適化条件は、ドナーとしての３型カルスの使用、９日間の共存培養期間、培地からの２，４−ＤおよびＣａＣｌ₂の排除、ならびに１００ｍｇ／ｌアセトシリンゴンの使用を含んでいた。実験に使用された、すべての培地およびそれらの組成を、表１に要約する。ｐＧＰＴＶ−ＨＢ共存培養後、カルスを、濾紙を載せたＭＳＣＧ培地に移し、そして暗所で２〜４週間培養した。次いで、このカルスを、苗条誘導のために、濾紙を載せたＭＳＳＩ培地に移した。種々の濃度（１２５、２５０、および５００ｍｇ／ｌ）のカルベニシリンを、苗条誘導培地に添加した。誘導された苗条を、引き続いて、４週間の発根および選択のために、ＭＳＲＳ培地に移した。５ｍｇ／ｌビアラフォスおよび１０ｍｇ／ｌハイグロマイシンを、ビアラフォス抵抗性小植物およびハイグロマイシン抵抗性小植物の選択のために使用した。選択培地上で発根した植物を、抗生物質およびビアラフォスを有さないＭＳＰＧ培地に移し、そしてさらに生長させた。次いで、完全に生長した植物を土壌を含むポットに移し、そして３０℃、８０％相対湿度、および冷白色蛍光管によって提供される３０μｍｏｌ ｍ^-2ｓ^-1の強度を有するイラディアンスでの１８時間光周期に設定された環境制御グロースチャンバにおいて生長させた。根が十分に発生した時点で、ポットを温室に移した。
【００３７】
（トランスジェニックシバ植物の分析）
推定トランスジェニックシバ植物が、除草剤に対して抵抗性であるか否かを試験するために、この推定トランスジェニックシバ植物に、５ｇ／ｌの除草剤溶液（１ｇ／ｌビアラフォス、Ｍｅｉｊｉ Ｓｅｉｋａ、Ｊａｐａｎ）を噴霧した。さらに、除草剤抵抗性遺伝子が、植物ゲノム中に組み込まれているか否かを研究するために、ＲｏｇｅｒおよびＢｅｎｄｉｃｈ（１９８５）の方法に従って、ゲノムＤＮＡを、トランスジェニック植物の葉から単離した。ＨｉｎｄＩＩＩまたはＢａｍＨＩで消化した２０μｇのゲノムＤＮＡを、０．８％アガロースゲルにおいて分離し、標準的な方法（Ｓｏｕｔｈｅｒｎ １９７５）によってナイロンメンブレン（Ｈｙｂｏｎｄ−Ｎ⁺）にブロットし、そして³²Ｐ標識化したｈｐｔ遺伝子配列またはｂａｒ遺伝子配列で探索した。これらのプローブを、ランダムプライミング法（Ｓａｍｂｒｏｏｋら、１９８９）によって調製した。
【００３８】
（結果）
（Ａｇｒｏｂａｃｔｅｒｉｕｍ感染に適切なカルス形態）
種々の組合せの２，４−ＤおよびＢＡを含むＭＳ培地における成熟種子由来１型カルスの培養は、１〜４型のカルスの混合物を生じた（表２）。
【００３９】
表２は、異なる組合せの生長調節物質を含む培地における、シバ１型カルスの異なる生長を示す。１０ｍｇの１型カルスを、２，４−ＤおよびＢＡの種々の組合せ、ならびに２ｇ／ｌゲルライトを有するＭＳ培地において、暗所で、５週間２８℃にて生長させた。各値は、１０個のカルスから算出された平均を示す。カルスの形態を、以下によって評価した；１＝やや白色〜黄色、緻密および非破砕性、２＝白色、緻密および破砕性、３＝黄色、緻密および非常に破砕性、４＝半透明、軟性および水っぽい。
【００４０】
【表２】

結果として生じた４つの型のカルスを、形態学的に特徴付けた。１型カルスは、やや白色〜黄色または薄緑色であり、緻密かつ非破砕性の構造を有する。複数の苗条原基が、いくつかの１型カルスの表面に観察された（図１Ａ）。２型カルスは白色であり、緻密かつ破砕性の構造を有する（図１Ｂ）。３型カルスは黄色であり、そして緻密かつ非常に破砕性の構造を有した（図１Ｃ）。４型カルスは、軟性の水っぽい外観を有して半透明であるという点で、独特である（図１Ｄ）。１〜３型のカルスは再生可能であったが、４型カルスは再生不可能であった。１型および２型カルスの最良の生長および発生率（それぞれ、６７．４±９．１ｍｇ、５８％、および３２．３±５．２ｍｇ、２８％）および３型カルスの最良の生長および発生率（３３．７±１１．３ｍｇ、５９％）は、それぞれ、１および４ｍｇ／ｌの２，４−Ｄと組合せて０．０１ｍｇ／ｌ ＢＡを含むＭＳ培地において得られた（表２）。再生不可能な４型カルスの発生率は、２，４−Ｄと組合せて０．０１ｍｇ／ｌ ＢＡを添加することによって、１／１．８倍〜１／３．４倍に減少した。従って、１または４ｍｇ／ｌの２，４−Ｄと組合せて０．０１ｍｇ／ｌのＢＡを、それぞれ、１〜２型または３型のカルスのさらなる生長のために使用した。２型および３型のカルスの継代培養は、１型カルス継代培養での出現率と類似した出現率で、１型、２型、３型、および４型カルスの混合物を生じた。しかし、４型カルスの継代培養は、４型カルスのみを生じた。興味深いことに、１〜３型のカルスは、ホルモンの異なる組合せを使用した場合に、形態学的なバリエーションを示した。さらに、継代培養する前に３日間、カルスを明所で継代培養した場合、緑色カルスが出現した。この緑色カルスからは、苗条誘導培地において、小さな白子植物が再生した（図２Ｃ）。４型カルスの発生を抑制することによって、そして継代培養工程毎に緑色カルスを選択することによって、カルス系統からの緑色植物の再生可能性は、２年間にわたって、いかなる有害な影響も伴なわずに維持され得る（図２Ｃ）。
【００４１】
カルスの形態と苗条の再生可能性との間の密接な関係は、種々の植物種において実証されてきた（ＡｒｍｓｔｒｏｎｇおよびＧｒｅｅｎ １９８５；ＫｅおよびＬｅｅ １９９６；ＬｕｏおよびＪｉａ １９９８）。ナガハグサ（Ｋｅｎｔｕｃｋｙ ｂｌｕｅｇｒａｓｓ）（Ｐｏａ ｐｒａｔｅｎｓｉｓ Ｌ．）では、子葉鞘および胚から誘導されるカルスは、形態および破砕性に基づいて４つの型に分類され得る（ＫｅおよびＬｅｅ １９９６）。ナガハグサの４型カルスは、柔らかく、構造を有さず、そして半透明であり、そこから苗条が全く再生されなかった。この結果は、シバで得た結果と類似している。ナガハグサにおける４型カルスの誘導は、オーキシンによってのみ制御され得、一方、シバの誘導は、サイトカイニンレベルを減少させることによって制御することができた。Ａｓｔｒａｎｇａｌｕｓ ａｄｓｕｒｇｅｎｓでは、胚軸由来のカルスがまた４つの型に分類されたが（ＬｕｏおよびＪｉａ １９９８）、４つのカルスの型のいずれもが、８ヶ月間継代培養した後でさえ、形態の変化を示さなかった。他方、４型を除き、１〜３型のシバカルス型の各々の継代培養は、４つのカルス型を生じた。シバ、ナガハグサ、およびＡｓｔｒａｎｇａｌｕｓ ａｄｓｕｒｇｅｎｓ間におけるこれらの差異は、異なる種間の差示的再生可能性を反映する。
【００４２】
（形質転換効率に影響を及ぼす因子）
（カルス型の効果）
ＭＳＣＧ１培地上で生長した１型および２型のカルス、ならびにＭＳＣＧ４培地上で生長した３型カルスを、Ａ．ｔｕｍｅｆａｃｉｅｎｓ ＥＨＡ１０１（ｐＩＧ１２１Ｈｍ）と、６日間、２，４−Ｄを含まない共存培養培地で共存培養し、そしてＧＵＳ発現の測定を使用して、異なるカルス型と形質転換効率との間の関係を検査した（表３、図２Ａ）。
【００４３】
表３は、異なるカルス型の形質転換効率を示す。３つのカルス型を、２，４−Ｄを含まないＭＳＡＳ培地（表１を参照のこと）上で、６日間暗所で、２６℃で共存培養した。３つの複製を測定し平均した。
【００４４】
【表３】

ＧＵＳ発現は、１型および３型カルス上で検出されたが、２型カルス上では青斑は検出されなかった。３型カルスは、１ｇのカルス新鮮重量あたり７２１±２０１個の青斑という最も高いＧＵＳ発現頻度を生じた。これは、１型カルスにおいて観察されたものよりも３４倍高かった。この結果は、他のいずれのカルスの型よりも、３型カルスがより容易に形質転換されることを示す。
【００４５】
（２，４−Ｄ濃度の効果）
カルスの単位新鮮重量あたりの青斑の数によって決定された各カルス型の形質転換頻度はまた、表２に示される共存培養培地における２，４−Ｄ濃度によって影響を受けた。共存培養培地における２，４−Ｄの効果を、ＭＳＣＧ４培地上で生長したカルス上の青斑を計数することによって検査した（図３）。共存培養の９日後、青斑は、２，４−Ｄを含まない共存培養培地上でのカルスの新鮮重量１ｇあたり７９３±１４５個であった。しかし、青斑の数は、２，４−Ｄ濃度が増加するにつれ減少した（図３）。この観察は、２，４−Ｄが、Ａｇｒｏｂａｃｔｅｒｉｕｍ媒介形質転換効率に対して負の効果を有することを示唆する。これは、他の植物において観察されたものとは対照的である。Ａｇｒｏｂａｃｔｅｒｉｕｍ媒介形質転換における活性な細胞分裂についての正の役割は、種々の植物種において議論されてきた。アマ（ＭｃＨｕｇｈｅｎら、１９８９）およびナス（Ｃｌａｕｄｉａら、２０００）において、Ａｇｒｏｂａｃｔｅｒｉｕｍ感染の前の２，４−Ｄを含む再生培地での外植片の予備培養は、効率的な遺伝子移入にとって必須であった。傷を受けた外植片における活性な細胞分裂は、Ｔ−ＤＮＡフラグメントの植物ゲノムＤＮＡへの組み込みを大きく改善した（ＭｃＨｕｇｈｅｎら、１９８９；Ｍｕｔｈｕｋｕｍａｒら、１９９６；Ｃｌａｕｄｉａら、２０００）。２，４−Ｄを含まない共存培養培地が、シバカルスの細胞分裂を活性化させ得ること、またはそれ自体の遺伝子移入を促進し得ることは、正確な分子機構は解明されていないが、可能である。
【００４６】
（共存培養期間）
２〜３日の共存培養の期間が、イネ科の形質転換において一般的に使用されてており（Ｈｉｅｉら、１９９４；Ｒａｓｈｉｄら、１９９６；Ｄｏｎｇら、１９９６；Ｉｓｈｉｄａら、１９９６、Ｃｈｅｎｇら、１９９７）、一方、５〜７日までの延長した共存培養の期間が、ｌｉｌｉｕｍ ｕｓｉｔａｔｉｓｓｉｍｕｍ、シトレンジ（ｃｉｔｒａｎｇｅ）、およびアガパンサス（ａｇａｐａｎｔｈｕｓ）においてＡｇｒｏｂａｃｔｅｒｉｕｍ媒介形質転換効率を増大させることが示されている（Ｃｅｒｖｅｒａら、１９９８；Ｓｕｚｕｋｉら、２００１）。共存培養期間の延長により、細菌細胞の膨大な増殖が生じ、そして通常再生頻度は減少する（Ｃｅｒｖｅｒａら、１９９８）。細菌細胞の過剰増殖が６日間の共存培養期間に観察されたが、ボルテックスによる完全な洗浄により、カルスを細菌の汚染から保護することが可能であり、そして形質転換効率は減少しなかった（データ示さず）。
【００４７】
（共存培養培地におけるＣａＣｌ₂濃度）
Ａ．ｔｕｍｅｆａｃｉｅｎｓ媒介形質転換頻度に対するＣａＣｌ₂の効果を調査した。カルシウムを含まない共存培養培地は、ＧＵＳ発現青斑の数を有意に増大させた（図４）。青斑は、ＣａＣｌ₂を含まない共存培養培地上で、１ｇの新鮮カルス重量あたり１３６８個であったが、ＣａＣｌ₂濃度が増大するにつれ減少した。しかし、カルスの生長は、カルシウムを含まない共存培養培地において、カルシウムを含む培地より遅かった。したがって、カルスは、共存培養後にカルシウムを含有するカルス生長培地に迅速に移されなければならない。同様のカルシウム効果が、Ｈｅｖｅａ ｂｒａｓｕｌｉｅｎｓｉｓで観察された（Ｍｏｎｔｏｒｏら、２０００）。
【００４８】
カルシウムは、病原感染に対する植物応答において重要な役割を演じ、そして最近の生理学研究において議論されている（Ｄｉｅｒｋ １９９８）。このカルシウム媒介植物防御機構はまた、Ａｇｒｏｂａｃｔｅｒｉａｌ細胞により感染させたときに、シバカルスにおいて誘発され得る。これは、シバカルスがカルシウムを含まない共存培養培地でより容易に感染される理由を説明し得る。
【００４９】
（アセトシリンゴン濃度の効果）
フェノール化合物であるアセトシリンゴンは、ＡｇｒｏｂａｃｔｅｒｉｕｍのＴｉプラスミドにおけるｖｉｒ遺伝子発現を活性化する誘導物質として周知である（Ｓｔａｃｈｅｌら、１９８５）。アセトシリンゴンは、ジャポニカイネおよびインディカイネ（Ｈｉｅｉら、１９９４；Ｒａｓｈｉｄら、１９９６）、ファラエノフォシス（ｐｈａｒａｅｎｏｐｓｉｓ）、ランおよびアガパンサス（Ｓｕｚｕｋｉら、２００１）のような単子葉植物において、Ａｇｒｏｂａｃｔｅｒｉｕｍ媒介形質転換に重要であることが示されている。アセトシリンゴンは、キュウリ、ブロッコリ、ダイズ、およびシトレンジのような双子葉植物における形質転換効率もまた大いに増大する（Ｎｉｓｈｉｂａｙａｓｈｉら、１９９６；Ｃｅｒｖｅｒａら、１９９８；Ｓａｎｔａｒｅｍら、１９９８；Ｈｅｎｚｉら、２０００）。
【００５０】
アセトシリンゴンを含まない共存培養培地を使用した場合、形質転換効率が非常に低く、１ｇのカルス新鮮重量あたり８６．３±２４．０個の青斑であった。５０ｍｇ／リットルのアセトシリンゴンをその共存培養培地に添加すると、青斑の数が有意に９倍増大した（図５）。しかし、１００ｍｇ／リットル濃度よりも高い濃度は、形質転換に対して負の効果を有していた。特に、２００ｍｇ／ｌのアセトシリンゴン含有培地上での共存培養したカルスは、共存培養後にカルス培養培地上で生長しなかった。
【００５１】
（ｂａｒ遺伝子でのシバの形質転換）
シバ植物のための最適な形質転換系を評価するために、除草剤抵抗性遺伝子（ｂａｒ）を含有するｐＧＰＴＶ−ＨＢ形質転換ベクターでシバ植物を遺伝的に形質転換するために、確立されたプロトコルを適用した。このプロトコルの主要な構成要素は、３型カルスおよび９日間の共存培養期間の使用であった。この共存培養培地は、２，４−ＤおよびＣａＣｌ₂を含まなかったが、５０ｍｇ／リットルのアセトシリンゴンを含んだ。その共存培養カルスを、カルス生長培地で生長させ、そしてハイグロマイシンおよびビアラフォス選択なしで苗条誘導に供した。時折、Ａｇｒｏｂａｃｔｅｒｉａｌ細胞混入を、１２５ｍｇ／リットルのカルベニシリン含有培地上で観察したので、２５０ｍｇ／リットルのカルベニシリンを苗条誘導培地において使用した。
【００５２】
選択を、１０ｍｇ／リットルのハイグロマイシンまたは５ｍｇ／リットルのビアラフォスを含有する発根培地上でのみ行った。発根した苗条は、４週間後に現れた。この発根した苗条を、再度、新鮮選択培地上で２〜４週間継代培養した場合、最初の選択培地上で発根したエスケープ（ｅｓｃａｐｅ）が最終的に枯死し、一方、推定の真のトランスジェニック植物が発根しそして活発に伸長した。抵抗性植物の頻度は、約２０．５％であった（表４）。
【００５３】
表４は、Ａ．ｔｕｍｅｆａｃｉｅｎｓ感染カルスから再生した苗条の生存率を示す。ＥＨＡ１０１（ｐＧＰＴＶ−ＨＢ）感染カルスから再生した苗条を、５ｍｇ／リットルのビアラフォス（Ｂ）および１０ｍｇ／リットルのハイグロマイシン（Ｈ）を含有するＭＳＲＳ培地（表１を参照のこと）上で４週間培養した。生存率は、産生した抵抗性植物／播種した苗条の数に、１００をかけたものを表す。
【００５４】
【表４】

（トランスジェニックシバ植物に対する除草剤抵抗性アッセイ）
トランスジェニック植物および非トランスジェニック植物を、土壌中に確立した後、それらに、５ｇ／リットル除草剤溶液（Ｍｅｉｊｉ Ｓｅｉｋａ、Ｊａｐａｎ）を２週間毎日適用した。除草剤溶液を２週間散布した後、トランスジェニック植物は、ビアラフォス塗布を生存し、そして成熟まで成長した。しかし、コントロール植物は、成長を止め、そして最終的に枯死した（図３）。この結果は、ｂａｒ遺伝子が、トランスジェニック植物において正常に発現されることを示す。
【００５５】
（ＤＮＡゲルブロット分析）
トランスジェニックシバ植物の除草剤抵抗性がその植物ゲノムに組み込まれたｂａｒ遺伝子に由来することを確認するために、ビアラフォス抵抗性植物を、ＤＮＡゲルブロット分析によって分析した。ゲノムＤＮＡをトランスジェニック植物から単離し、ＨｉｎｄＩＩＩで消化し、そしてｂａｒまたはｈｐｔ特異的プローブとハイブリダイズさせた（図６Ａ）。同時に、コントロール植物からのゲノムＤＮＡをまた、同一のやり方で分析した。コントロールサンプルは、いかなるバンドを示さなかった（図７Ｂ、レーンｃ；図７Ｃ、レーンｃ）。一方、トランスジェニック植物からのサンプルは、ｂａｒおよびｈｐｔ特異的プローブと特異的にハイブリダイズするバンドを示した（図７Ｂ、レーン１；図７Ｃ、レーン１）。ｐＧＰＴＶ−ＨＢのＴ−ＤＮＡフラグメントは、単一のＨｉｎｄＩＩＩ部位を有するので（図７Ａ）、ハイブリダイズしたバンドの数は、トランスジェニックシバ植物に組み込まれた遺伝子コピーのコピー数を反映していた。検出されたバンドは、ｐＧＰＴＶ−ＨＢのマップ（図７Ａ）から予想されたような、ｂａｒまたはｈｐｔ特異的プローブについてのそれぞれ１．６ｋｂまたは１．８ｋｂよりも大きいフラグメントを表した。これは、組み込まれた遺伝子のコピー数が２であったことを示す（図７Ｂ、レーン１；図７Ｃ、レーン１）。ＢａｍＨＩおよびｂａｒプローブでのゲノムＤＮＡサザンブロットはまた、同じ結果を与えた。これは、コピー数が、２であることを示す（図７Ｂ、レーン２）。これらの結果は、２つの安定な組み込み事象が存在したことを示し、明らかに、このトランスジェニック植物において観察された除草剤抵抗性が、組み込まれたｂａｒ遺伝子によって決定されたことを実証する。
【００５６】
本発明におけるシバのＡ．ｔｕｍｅｆａｃｉｅｎｓ媒介形質転換系は、シバならびに密接に関連する芝草種の遺伝子操作を迅速化させる。本発明は、シバの遺伝子形質転換のための最適化された培地組成および培養条件を提供し、これは、目的の遺伝子が、生物的および非生物的ストレスに対しての抵抗性を改良するため、および生長率を調節するために、シバに導入されるべき場合に特に有益である。
【００５７】
（参考文献）
【表５】

（要約）
本発明は、シバ植物（Ｚｏｙｓｉａ ｊａｐｏｎｉｃａ Ｓｔｅｕｄ．）に対する効率的な遺伝子形質転換系を提供する。シバ形質転換のための最適化された培地組成および培養条件もまた提供される。このシバについての確実な形質転換系を、カルス生長および植物再生に有意に影響するいくつかの因子を最適化することによって開発した。カルス型および共存培養期間は、形質転換効率に絶対的に影響した。２，４−Ｄ、ＣａＣｌ₂およびアセトシリンゴンの濃度もまた、重要な因子であった。最良の結果は、３型カルスを、２，４−Ｄを含まない共存培養培地上で６日間共存培養した場合に達成された。共存培養の間のカルシウムの除去および５０ｍｇ／ｌのアセトシリンゴンの添加は、その効率を劇的に増強させた。本発明はまた、ビアラフォス抵抗性シバ植物を提供し、これは、ゴルフ場および競技場に使用され、維持費を節約し得る。
【００５８】
【発明の効果】
本発明に従って、Ａ．ｔｕｍｅｆａｃｉｅｎｓ媒介感染によって、シバを目的の遺伝子で遺伝的に形質転換することが現在可能である。
【図面の簡単な説明】
【図１】図１は、カルス生長培地上で生長した、４つの異なる型のカルスを示す写真である。（Ａ）１型カルスは、やや白色〜黄色または薄緑色を有し、そして緻密かつ非破砕性の構造を示す。（Ｂ）２型カルスは、白色であり、そして緻密かつ破砕性の構造を示す。（Ｃ）３型カルスは、黄色であり、そして緻密かつ非常に破砕性の構造を示す。（Ｄ）４型カルスは、半透明の軟性の水っぽい外見を有する。この４つのカルス型は、表２に要約されるような生長調節物質の異なる組み合わせを補充した、カルス生長培地上で得られた。
【図２】図２は、ｐＧＰＴＶ−ＨＢベクター（図７を参照のこと）を保有するＡ．ｔｕｍｅｆａｃｉｅｎｓを感染させたシバカルスにおける、トランスジェニックＧＵＳ発現を示す写真である。（Ａ）シバカルスにおけるトランスジェニックＧＵＳ発現。（Ｂ）Ａ．ｔｕｍｅｆａｃｉｅｎｓ感染後の、濾紙を載せ、そして抗生物質を含まないＭＳＣＧＣＢ培地上での、カルスの急速な生長。（Ｃ）濾紙上を載せ、そして抗生物質を含まない苗条誘導培地上での、苗条誘導。（Ｄ）ビアラフォス含有培地上での、苗条伸長および発根。Ａ．ｔｕｍｅｆａｃｉｅｎｓ感染カルスから誘導された苗条を、５ｍｇ／ｌのビアラフォスを含有する選抜発根培地ＭＳＲＳ上で培養した。
【図３】図３は、形質転換効率に対する２，４−Ｄの効果を示す。異なる濃度の２，４−Ｄを含有するカルス増殖培地上で培養したカルスの、１グラムの新鮮重量あたりの青斑を計数した。
【図４】図４は、形質転換効率に対するＣａＣｌ₂の効果を示す。培地に異なる濃度のＣａＣｌ₂を含有させることを除いて、図３の簡単な説明に記載のように、Ａ．ｔｕｍｅｆａｃｉｅｎｓ感染カルスを培養した。
【図５】図５は、形質転換効率に対するアセトシリンゴンの効果を示す。異なる濃度のアセトシリンゴンを含有させることを除いて、図３の簡単な説明に記載のように、この実験を行った。
【図６】図６は、トランスジェニックシバ植物（右）および親シバ植物（左）を示す写真である。これらのシバ植物に、２週間の間、毎日、除草剤溶液を噴霧した（５ｇ／ｌのビアラフォス）。（Ａ）除草剤溶液を噴霧しなかった植物。（Ｂ）２週間の間、除草剤溶液を毎日噴霧した植物。
【図７】図７は、形質転換ベクターの略図、および植物ゲノム中の除草剤抵抗性（ｂａｒ）遺伝子の存在を確認するためのサザンブロット分析を示す。（Ａ）形質転換ベクターｐＧＰＴＶ−ＨＢのベクター地図。Ｐｎｏｓ、ノパリンシンターゼ遺伝子のプロモーター；Ｈｙｇ^R、ハイグロマイシン耐性遺伝子（ｈｐｔ）；ＮＯＳ、ノパリンシンターゼ遺伝子のターミネーター；Ｕｂｉ−Ｐ、トウモロコシ由来のユビキチンプロモーター；Ｂａｒ、ビアラフォス抵抗性遺伝子；Ｂ、ＢａｍＨＩ；Ｅ、ＥｃｏＲＩ；Ｈ，ＨｉｎｄＩＩＩ；Ｓ、ＳａｃＩ。（Ｂ）および（Ｃ）トランスジェニックシバ植物のサザンブロット分析を示す写真である。ゲノムＤＮＡを、ＨｉｎｄＩＩＩ（レーン１）またはＢａｍＨＩ（レーン２）のいずれかで消化した。メンブレンに、ｂａｒ（Ｂ）プローブまたはｈｐｔ（Ｃ）プローブのいずれかをハイブリダイズさせた。形質転換されていない植物のゲノムＤＮＡを、ネガティブコントロールとして含んだ（レーンｃ）。[0001]
BACKGROUND OF THE INVENTION
(Background of the Invention)
The present invention relates to an efficient method for gene transformation of zysiagrass (Zoysia japonica Steud.) (Also called Korean grass). The present invention may be applicable to other closely related turfgrass species. The present invention also provides transgenic barn plants that are resistant to herbicides, as well as experimental methods and processes for the production of such herbicide-resistant barns.
[0002]
The present invention relates to an efficient Agrobacterium tumefaciens-mediated gene transformation method for Zoysia japonica Steud. (Also known as Korean turf), optimized medium composition and culture conditions that greatly affect this transformation efficiency. And related experimental means for developing such transgenic barn plants with herbicide resistance and such transgenic barn with bialaphos resistance gene (bar).
[0003]
[Prior art]
Shiba is one of the most important turfgrass species and is widely cultivated in Far East Asia including Korea, Japan and eastern China and in temperate regions around the world. The growing area of buckwheat has grown rapidly in the United States and other countries in recent years, mainly due to its extraordinary characteristics such as drought resistance and the ability to recover quickly from traffic damage. To do. In addition, Shiva grows well on thin soils in virtually all climates. Due to these useful traits, Shiva is widely used in golf courses, stadiums, roadsides, residential gardens, and river banks. As the market size grows rapidly, consumers demand new varieties with improved resistance to pathogens, herbicides and various environmental stresses. Until recently, such new traits in turfgrass have been developed, mainly using classical breeding methods. However, an increasing number of laboratories and research institutions are striving to apply molecular biological methods to genetically manipulate turfgrass (Inokuma et al., 1998; Park and Ahn 1998). This is because it is now possible to use these methods to develop or modify useful traits in a predictable manner.
[0004]
Many monocotyledons are not easily infected by Agrobacterium tumefaciens, but efficient A. tumefaciens-mediated transformation systems have recently been successfully developed for several grasses such as rice (Hiei et al., 1994; Rashid et al., 1996), maize (Ishida et al., 1996) and wheat (Cheng et al., 1997). Unfortunately, however, such an A. tumefaciens-mediated transformation method has not yet been established for turfgrass except for C. bentgrass (Yu et al., 2000) (Chai and Sticklen, 1998). In particular, genetic transformation of buckwheat is further hampered by several additional technical problems, for example, the seed germination rate of turfgrass is very slow and the production of renewable callus is difficult (Asano 1989). ).
[0005]
Callus morphology is closely related to plant reproducibility, as has been demonstrated in various plant species (Armstrong and Green 1985; Ke and Lee 1996; Luo and Jia 1998). Recently, the inventors have established an efficient callus induction and plant regeneration system for shiba (Bae et al., 2001), which was filed on July 24, 2001 as Japanese Patent Application No. 2001-223278. did.
[0006]
[Problems to be solved by the invention]
In accordance with the present invention, A. It is now possible to genetically transform a buckwheat with a gene of interest by a tumefaciens-mediated infection.
[0007]
Some potential target traits for genetic transformation of buckwheat include improved surface planting coverage, resistance to traffic damage, rapid recovery after damage, resistance to biological and abiotic stress , Growth rate manipulation (acceleration or delay), as well as shade avoidance. Of particular interest is that the growth rate and / or shade avoidance can be manipulated, resulting in a dramatic reduction in maintenance costs for irrigation and mowing.
[0008]
With the rapid accumulation of molecular biology techniques in recent years and the establishment of efficient plant culture and gene transformation systems, any gene that is important in crop science now has an impact on crop yield and quality and environmental adaptability. It can be easily introduced into any desired plant for the purpose of augmentation. In the present invention, the present inventors provide an efficient Agrobacterium tumefaciens-mediated shiba transformation method and a transgenic shiba plant having herbicide resistance.
[0009]
As used herein, the term “gene transformation” refers to a means for introducing a gene or genetic material into a target higher plant in a predictable manner. This gene or genetic material is stably integrated into the plant genome and is inherited through generations.
[0010]
[Means for Solving the Problems]
(Summary of the Invention)
The present invention relates to a reliable Agrobacterium tumefaciens-mediated transformation method for buckwheat, which can be routinely used for genetic transformation of buckwheat or closely related turfgrass species.
[0011]
The present invention also provides a method of genetically transforming buckwheat (genus Zoysia spp.), Which comprises the following steps: i) inducing and growing calli of shiba on modified MS medium containing various hormones Step; ii) Infecting Agrobacterium cells into the callus of this buckwheat and introducing a bialaphos resistant bar gene; A step of co-culturing callus and Agrobacterium cells of the buckwheat; iv) excluding Agrobacterium cells from the coculture medium; and v) a step of regenerating transgenic buckwheat.
[0012]
In addition, Agrobacterium cells have a pGPT-HB transformation vector and type 3 callus (defined in our previous application number 2001-223278) coexisted in co-culture medium for 5-7 days. Incubate.
[0013]
The present invention also provides a transgenic barn by the above-mentioned method, and this barn is stably transformed with the bar gene under the control of a plant-specific promoter. A particular promoter is the ubiquitin promoter.
[0014]
Important medium components or culture conditions evaluated in the present invention include callus type, co-culture period, 2,4-D (2,4-dichlorophenoxyacetic acid), CaCl ₂ , And acetosyringone. The highest transformation efficiency was obtained when type 3 callus was co-cultured on a medium without 2,4-D for 5-7 days. Furthermore, removal of calcium during co-cultivation and inclusion of 30-70 mg / l acetosyringone greatly enhanced this transformation efficiency.
[0015]
When this optimized transformation protocol was used for barn transformation with the bar gene, up to 20.5% of the shoots plated on selective medium showed herbicide resistance.
[0016]
Moreover, in this invention, the transgenic shiba plant which has herbicide resistance is provided. The transgenic buckwheat could survive even after spraying a 5 g / l herbicide solution daily for two weeks and eventually grew to maturity. Control plants, on the other hand, stopped growing and died when treated under the same experimental conditions.
[0017]
Thus, the present invention further provides herbicide-resistant transgenic Shiva callus (deposited under accession number KCTC-10076BP).
[0018]
The present invention relates to a method for gene transformation of shiba (Zoysia spp.), Which comprises the following steps: i) inducing and growing calli of the shiba on a modified MS medium containing various hormones; ii) infecting Agrobacterium cells with the calli of the birch and introducing a bialaphos resistance bar gene; iii) in a co-culture medium containing acetosyringone without 2,4-dichlorophenoxyacetic acid and calcium A method is provided, comprising co-culturing shiba callus and Agrobacterium cells; iv) removing Agrobacterium cells from the co-culture medium; and v) regenerating transgenic shiba.
[0019]
In one embodiment, the Agrobacterium cell has a pGPT-HB transformation vector.
[0020]
In one embodiment, type 3 callus is co-cultured in a co-culture medium for 5-7 days.
[0021]
The present invention also provides a transgenic shiba according to the above method, wherein the bar gene is stably transformed under the control of a plant-specific promoter.
[0022]
In one embodiment, the bar gene is expressed under the control of a ubiquitin promoter.
[0023]
The present invention further provides a transgenic Shiba callus into which a bialaphos resistance bar gene (KCTC-10076BP) has been introduced.
[0024]
The above callus is based on the Budapest Treaty, dated 21 September 2001, Korea Collection for Type Cultures (# 52 Own-Dong, Yuson-ku, Taejon, Seoul, 305-333, Republic).
of Korea) with the registration number KCTC-10076BP.
[0025]
DETAILED DESCRIPTION OF THE INVENTION
(Detailed description of the invention)
Agronomically important plant genetic transformation is a potential method for developing new varieties of plant species with new or improved useful traits. Target traits associated with crop plants include improved or delayed growth rates, increased resistance to biological and abiotic stresses, increased yields, and flexible adaptability to environmental changes. Pre-required for successful genetic transformation of plants is a reliable system for tissue culture and plant regeneration and a robust delivery device of genetic material. With recent advances in plant molecular biology and methodology, any gene of interest can be easily delivered to any plant species of interest. However, some crop plants (especially monocotyledonous plants) are cumbersome to genetic manipulation and are not easy to carry out plant regeneration in ordinary laboratories. Turfgrass is widely used for a variety of purposes (including golf courses, turflands, residential gardens, and recreation parks) and has emerged as a potential commercial target plant for recent plant biotechnological applications. It is up. However, efficient tissue culture and gene transformation systems have not been developed. As a result, biotechnological genetic manipulation of turfgrass has not progressed much.
[0026]
The present invention provides media composition and culture conditions optimized for efficient gene transformation and plant regeneration from seed-derived calli of zysiagrass. Shiva, a subtype of warm turfgrass, is widely distributed and cultivated in temperate zones including Far East Asia. In addition, growing areas have been rapidly expanding in recent years due to several advantages, such as resistance to drought damage and traffic damage. In the present invention, the most efficient transformation was achieved when type 3 callus was co-cultured for 6 days in a co-culture medium without 2,4-D but having 50 mg / l acetosyringone. . Calcium, known to improve transformation efficiency in other plants, has a negative effect. Plant tissue culture and regeneration experiments are routinely performed in well-established plant molecular laboratories and are well known in the art.
[0027]
The present invention also relates to herbicide-resistant buckwheat plants, which can survive even after spraying a bialaphos solution daily for two weeks. By growing this transgenic buckwheat in golf courses and athletic fields, it becomes easier to selectively remove weeds by simply spraying herbicides, and maintenance costs are dramatically reduced. In addition, it is expected that a small amount of fertilizer will be required to grow this transgenic buckwheat. This is because weeds that consume nutrients are efficiently removed. This transgenic buckwheat did not show any morphological distortion and grew well and was indistinguishable from the parent plant.
[0028]
The present invention can also be applied to other closely related turfgrass species as well as other monocotyledonous species, possibly belonging to the Gramineae family. Experimental procedures for the development of such genetically engineered plants are well known in the art. The present invention provides an efficient A. cerevisiae by using a highly reproducible callus line. It is the first invention for tumefaciens-mediated transformation.
[0029]
【Example】
(Example)
(Preparation of reusable callus)
Approximately 100 seeds of Zoysia japonica Steud. Matured were surface sterilized with 1 ml of 2% sodium hypochlorite with stirring in a 2 ml centrifuge tube for 15 minutes using a tube mixer. The seeds were then washed 3 times with sterile double distilled water. For callus induction, about 100 seeds were treated with 3% sucrose (w / v), 100 mg / l alpha ketoglutarate, 4 mg / l thiamine-HCl, 2 mg / l 2,4-D, 0.2 mg / l Placed in MS (Murasize and Skoog 1962) media Petri dishes with filter paper containing BA and 0.2% gellite (w / v). This medium is added to 1.2 to 1.3 kg / cm. ² The pressure was adjusted to 5.8 with HCl before autoclaving at 121 ° C. for 20 minutes. Callus induction was performed in the culture room at 26 ± 1 ° C. for 3 months in complete darkness. Each Petri dish (90 mm diameter) contained approximately 30 ml of medium and was sealed with parafilm (American National Can, USA). 30 μmol m provided by a fluorescent tube ^-2 s ^-1 After an additional 3 days incubation under continuous white light with an intensity of 5%, only the green tissue was transferred to MS medium containing 1 mg / l 2,4-D and 0.2 mg / l BA, and subsequently Incubated in the dark for further growth. After 5 weeks of culture in the dark, callus from independent seeds with green color was transferred and in the dark in MS medium with filter paper containing a combination of different hormones of 2,4-D and BA Further culture was performed. Four types of callus were finally obtained. After subculture at 4-week intervals, type 1, type 2, and type 4 callus were generated in medium containing 1 mg / l 2,4-D, and type 3 callus was transferred to 4 mg / l 2, It was generated in a 4-D containing medium (FIG. 1). Each Petri dish (90 mm diameter) contained 25 ml of media and was sealed with Micropore Surgical Tape (3M Health Care, USA).
[0030]
(Characteristics of four callus types)
To investigate the morphological changes that occur in subcultures of type 1 callus, type 1 callus was isolated in the dark with 2,4-D (1, 2, 4, and 8 mg / l) and BA (0, 0.01 and 0.1 mg / l) was transferred to MS medium containing various hormone combinations. Four callus types were observed after 5 weeks of subculture in the dark. Each callus type was weighed and the weight percentage was calculated. Type 2, type 3 and type 4 callus were also tested in the same manner as with type 1 callus.
[0031]
(Optimization conditions for Agrobacterium infection)
including pIG121Hm; Tumefaciens disarmed strain EHA101 was used in this experiment. The pIG121Hm T-DNA contained a hygromycin resistance gene (hpt), a kanamycin resistance gene (npt), and an intron-gus gene (uidA). Agrobacterium tumefaciens cells were cultured at 160 rpm in a 100 ml Erlenmeyer flask containing 20 ml LB medium (Table 1) supplemented with 50 mg / l hygromycin, 100 mg / l kanamycin, and 100 mg / l spectinomycin (pGPTV-HB). Grow overnight at 28 ° C. with shaking.
[0032]
Table 1 shows the different media and their composition used in the Shiba transformation system.
[0033]
[Table 1]

Cells in 10 ml suspension culture were collected by centrifugation at 2500 rpm for 20 minutes in 50 ml polypropylene tubes (Becton Dickinson Labware, USA) and in 10 ml liquid infection medium (LqMSAS in Table 1) And resuspended by gentle vortexing.
[0034]
Acetosyringone was prepared by dissolving an appropriate amount of powder in dimethyl sulfoxide at a concentration of 100 mg / ml and stored at 4 ° C. in the dark. This was added to sterile media to the appropriate final concentration when required. Callus grown in MSCG4 medium were immersed for 1 minute in Agrobacterium cell suspension. After dehydration on sterile filter paper, the callus was cultured in a co-culture medium (MSAS) with 4 mg / l 2,4-D for 15 days at 26 ± 1 ° C. in the dark. After co-cultivation, the callus is thoroughly washed by vortexing in sterile double distilled water supplemented with detergent (0.02% pluronic F68) until the wash solution is clear, And finally, it was washed in sterile double distilled water containing 1000 mg / l carbenicillin and 0.02% surfactant. After washing, half of this callus was used for a GUS activity assay by the method described by Schenk et al. (1998).
[0035]
Several factors were tested to optimize transformation efficiency. (1) For comparison of different callus types for invasion, type 1, type 2 and type 3 callus were co-cultured in MSAS medium without 2,4-D for 6 days; (2) coexistence For comparison of culture periods, calli were co-cultured for 3, 6, 9, 12, 15, 18, 21, 24, and 27 days; (3) different amounts of 2 for testing of media components , 4-D (0, 1, 2, 4, and 8 mg / l), CaCl ₂ (0, 11, 110, 220, and 440 mg / l) and acetosyringone (0, 10, 50, 100, and 200 mg / l) were added to MSAS medium.
[0036]
(PGPTV-HB co-culture)
Shiva callus Optimized conditions for Tumefaciens infection include use of type 3 callus as donor, co-culture period of 9 days, 2,4-D and CaCl from the medium ₂ As well as the use of 100 mg / l acetosyringone. All media used in the experiment and their composition are summarized in Table 1. After co-culture with pGPTV-HB, the callus was transferred to MSCG medium with filter paper and cultured in the dark for 2-4 weeks. The callus was then transferred to MSSI medium with filter paper for shoot induction. Various concentrations (125, 250, and 500 mg / l) of carbenicillin were added to the shoot induction medium. Induced shoots were subsequently transferred to MSRS medium for 4 weeks rooting and selection. 5 mg / l bialaphos and 10 mg / l hygromycin were used for the selection of bialaphos resistant plantlets and hygromycin resistant plantlets. Plants rooted on the selection medium were transferred to MSPG medium without antibiotics and bialaphos and further grown. The fully grown plants are then transferred to a pot containing soil and 30 μmol m provided by a cold white fluorescent tube at 30 ° C., 80% relative humidity, and ^-2 s ^-1 Growing in an environmentally controlled growth chamber set at 18 hours photoperiod with irradiance having an intensity of When the roots were fully developed, the pots were transferred to the greenhouse.
[0037]
(Analysis of transgenic shiba plants)
In order to test whether the putative transgenic buckwheat plant is resistant to the herbicide, this putative transgenic buckwheat plant was added to a 5 g / l herbicide solution (1 g / l bialaphos, Meiji Seika, Japan). ) Was sprayed. In addition, genomic DNA was isolated from the leaves of transgenic plants according to the method of Roger and Bendich (1985) to study whether the herbicide resistance gene was integrated into the plant genome. 20 μg of genomic DNA digested with HindIII or BamHI was separated on a 0.8% agarose gel and nylon membrane (Hybond-N) by standard methods (Southern 1975). ⁺ ), And ³² Search was made with P-labeled hpt gene sequence or bar gene sequence. These probes were prepared by the random priming method (Sambrook et al., 1989).
[0038]
(result)
(Callus form suitable for Agrobacterium infection)
Culturing of mature seed-derived type 1 callus in MS medium containing various combinations of 2,4-D and BA resulted in a mixture of type 1-4 callus (Table 2).
[0039]
Table 2 shows the different growth of Shiva type 1 callus in media containing different combinations of growth regulators. 10 mg type 1 callus was grown at 28 ° C. in the dark for 5 weeks in MS medium with various combinations of 2,4-D and BA and 2 g / l gellite. Each value represents an average calculated from 10 calli. The callus morphology was evaluated by: 1 = slightly white to yellow, dense and non-friable, 2 = white, dense and friable, 3 = yellow, dense and very friable, 4 = translucent, soft and It ’s watery.
[0040]
[Table 2]

The resulting four types of callus were morphologically characterized. Type 1 callus is slightly white to yellow or light green, and has a dense and non-crushed structure. Multiple shoot primordia were observed on the surface of several type 1 calluses (FIG. 1A). Type 2 callus is white and has a dense and friable structure (FIG. 1B). Type 3 callus was yellow and had a dense and very friable structure (FIG. 1C). Type 4 callus is unique in that it has a soft, watery appearance and is translucent (FIG. 1D). The 1-3 type callus was reproducible, but the 4 type callus was not reproducible. Best growth and incidence of type 1 and type 2 callus (67.4 ± 9.1 mg, 58% and 32.3 ± 5.2 mg, 28%, respectively) and type 3 callus best growth and incidence (33.7 ± 11.3 mg, 59%) were obtained in MS medium containing 0.01 mg / l BA in combination with 1 and 4 mg / l 2,4-D, respectively (Table 2). The incidence of non-renewable type 4 callus was reduced from 1 / 1.8 times to 1 / 3.4 times by adding 0.01 mg / l BA in combination with 2,4-D. Therefore, 0.01 mg / l BA in combination with 1 or 4 mg / l 2,4-D was used for further growth of type 1-2 or type 3 callus, respectively. Type 2 and type 3 callus subcultures resulted in a mixture of type 1, type 2, type 3 and type 4 callus with an appearance rate similar to that of type 1 callus subculture. However, subculture of type 4 callus yielded only type 4 callus. Interestingly, types 1-3 callus showed morphological variation when using different combinations of hormones. Furthermore, when the callus was subcultured in the light for 3 days before subculture, green callus appeared. From this green callus, a small white baby plant was regenerated in the shoot induction medium (FIG. 2C). By suppressing the development of type 4 callus and by selecting green callus for each subculture step, the reproducibility of green plants from the callus line has been without any detrimental effects over a period of two years. (FIG. 2C).
[0041]
A close relationship between callus morphology and shoot reproducibility has been demonstrated in various plant species (Armstrong and Green 1985; Ke and Lee 1996; Luo and Jia 1998). In Kentucky bluegrass (Poa privacyis L.), callus derived from coleoptiles and embryos can be classified into four types based on morphology and friability (Ke and Lee 1996). Nagahagusa type 4 callus was soft, unstructured and translucent, from which no shoots were regenerated. This result is similar to that obtained with Shiva. Induction of type 4 callus in Nagahagusa could only be controlled by auxin, whereas induction of shiba could be controlled by decreasing cytokinin levels. In Astragalus adsurgens, callus derived from hypocotyl was also classified into four types (Luo and Jia 1998), but all four callus types changed their morphology even after 8 months of subculture. Did not show. On the other hand, except for type 4, each subculture of types 1 to 3 gave rise to 4 types of callus. These differences among buckwheat, long-tailed and Astragalus adsurgens reflect the differential reproducibility between different species.
[0042]
(Factors affecting transformation efficiency)
(Callus-type effect)
Type 1 and type 2 callus grown on MSCG1 medium and type 3 callus grown on MSCG4 medium co-cultured with Tumefaciens EHA101 (pIG121Hm) for 6 days in 2,4-D-free co-culture medium and using GUS expression measurements to examine the relationship between different callus types and transformation efficiency (Table 3, FIG. 2A).
[0043]
Table 3 shows the transformation efficiency of different callus types. Three callus types were co-cultured at 26 ° C. in the dark for 6 days on MSAS medium without 2,4-D (see Table 1). Three replicates were measured and averaged.
[0044]
[Table 3]

GUS expression was detected on type 1 and type 3 callus, but no blue spots were detected on type 2 callus. Type 3 callus produced the highest GUS expression frequency of 721 ± 201 blue spots per gram callus fresh weight. This was 34 times higher than that observed in type 1 callus. This result indicates that type 3 callus is more easily transformed than any other type of callus.
[0045]
(Effect of 2,4-D concentration)
The transformation frequency of each callus type, determined by the number of blue spots per unit fresh weight of callus, was also affected by the 2,4-D concentration in the co-culture medium shown in Table 2. The effect of 2,4-D in the co-culture medium was examined by counting the blue spots on the callus grown on MSCG4 medium (FIG. 3). Nine days after co-cultivation, blue spots were 793 ± 145 per gram fresh weight of callus on co-culture medium without 2,4-D. However, the number of blue spots decreased as the 2,4-D concentration increased (FIG. 3). This observation suggests that 2,4-D has a negative effect on Agrobacterium-mediated transformation efficiency. This is in contrast to that observed in other plants. The positive role for active cell division in Agrobacterium-mediated transformation has been discussed in various plant species. In flax (McHughhen et al., 1989) and eggplant (Claudia et al., 2000), preculture of explants in regeneration medium containing 2,4-D prior to Agrobacterium infection is essential for efficient gene transfer. It was. Active cell division in wounded explants greatly improved the integration of T-DNA fragments into plant genomic DNA (McHughhen et al., 1989; Muthukumar et al., 1996; Claudia et al., 2000). It is possible that the co-culture medium without 2,4-D could activate Shiva callus cell division or promote its own gene transfer, although the exact molecular mechanism has not been elucidated. is there.
[0046]
(Co-culture period)
A period of 2-3 days co-culture has been commonly used in gramineous transformation (Hiei et al., 1994; Rashid et al., 1996; Dong et al., 1996; Ishida et al., 1996, Cheng et al., 1997 On the other hand, extended periods of co-culture up to 5-7 days have been shown to increase Agrobacterium-mediated transformation efficiency in lilium usitatissimum, citrange, and agapanthus (Cervera et al., 1998; Suzuki et al., 2001). Extension of the co-cultivation period results in enormous proliferation of bacterial cells and usually reduces the frequency of regeneration (Cervera et al., 1998). Although bacterial cell overgrowth was observed during the 6 day co-culture period, thorough washing with vortexing could protect the callus from bacterial contamination and did not reduce transformation efficiency (data Not shown).
[0047]
(CaCl in co-culture medium ₂ concentration)
A. CaCl for tumefaciens-mediated transformation frequency ₂ The effect of was investigated. Co-culture medium without calcium significantly increased the number of GUS-expressing blue spots (FIG. 4). Blue spots are CaCl ₂ Was 1368 per 1 g of fresh callus weight on a coculture medium not containing CaCl. ₂ It decreased as the concentration increased. However, callus growth was slower in the co-culture medium without calcium than in the medium with calcium. Therefore, callus must be quickly transferred to a callus growth medium containing calcium after co-cultivation. Similar calcium effects were observed with Hevea brasiliensis (Montoro et al., 2000).
[0048]
Calcium plays an important role in plant responses to pathogenic infections and has been discussed in recent physiology studies (Dierk 1998). This calcium-mediated plant defense mechanism can also be induced in Shiva callus when infected by Agrobacterium cells. This may explain why Shiva callus is more easily infected with co-culture medium without calcium.
[0049]
(Effect of acetosyringone concentration)
The phenolic compound acetosyringone is well known as an inducer that activates vir gene expression in the Agrobacterium Ti plasmid (Stachel et al., 1985). Acetosyringone is an Agrobacterium-mediated trait in monocotyledonous plants such as Japonica rice and Indica rice (Hiei et al., 1994; Rashid et al., 1996), Phalaenopsis, Orchid and Agapanthus (Suzuki et al., 2001). It has been shown to be important for conversion. Acetosyringone also greatly increases transformation efficiency in dicotyledonous plants such as cucumbers, broccoli, soybeans, and citrange (Nishibayashi et al., 1996; Cervera et al., 1998; Santarem et al., 1998; Henzi et al., 2000).
[0050]
When a co-culture medium without acetosyringone was used, the transformation efficiency was very low, 86.3 ± 24.0 blue spots per 1 g of callus fresh weight. When 50 mg / liter acetosyringone was added to the co-culture medium, the number of blue spots increased significantly 9-fold (FIG. 5). However, concentrations higher than 100 mg / liter had a negative effect on transformation. In particular, callus co-cultured on a medium containing 200 mg / l acetosyringone did not grow on the callus culture medium after co-culture.
[0051]
(Transformation of Shiba with bar gene)
Established protocol for genetically transforming buckwheat plants with the pGPTV-HB transformation vector containing the herbicide resistance gene (bar) to evaluate the optimal transformation system for the buckwheat plants Applied. The main components of this protocol were the use of type 3 callus and a co-culture period of 9 days. This co-culture medium contains 2,4-D and CaCl ₂ But 50 mg / liter acetosyringone. The co-cultured callus was grown on callus growth medium and subjected to shoot induction without hygromycin and bialaphos selection. Occasionally, Agrobacterium cell contamination was observed on 125 mg / liter carbenicillin-containing medium, so 250 mg / liter carbenicillin was used in the shoot induction medium.
[0052]
Selection was performed only on rooting media containing 10 mg / liter hygromycin or 5 mg / liter bialaphos. Rooted shoots appeared after 4 weeks. When the rooted shoots are subcultured again on fresh selection medium for 2-4 weeks, the escape roots rooted on the first selection medium will eventually die, while the estimated true Transgenic plants rooted and actively expanded. The frequency of resistant plants was about 20.5% (Table 4).
[0053]
Table 4 shows A.I. The survival rate of the shoot regenerated from the callus infected with tumefaciens is shown. Shoots regenerated from callus infected with EHA101 (pGPTV-HB) were cultured for 4 weeks on MSRS medium (see Table 1) containing 5 mg / liter bialaphos (B) and 10 mg / liter hygromycin (H). did. Viability represents the number of resistant plants produced / number of sown shoots multiplied by 100.
[0054]
[Table 4]

(Herbicide resistance assay for transgenic shiba plants)
After transgenic and non-transgenic plants were established in the soil, they were applied daily with a 5 g / liter herbicide solution (Meiji Seika, Japan) for two weeks. After two weeks of spraying with the herbicide solution, the transgenic plants survived the bialaphos application and grew to maturity. However, the control plants stopped growing and eventually died (FIG. 3). This result indicates that the bar gene is normally expressed in transgenic plants.
[0055]
(DNA gel blot analysis)
In order to confirm that the herbicide resistance of transgenic buckwheat plants was derived from the bar gene integrated into its plant genome, bialaphos resistant plants were analyzed by DNA gel blot analysis. Genomic DNA was isolated from transgenic plants, digested with HindIII, and hybridized with a bar or hpt specific probe (FIG. 6A). At the same time, genomic DNA from control plants was also analyzed in the same manner. The control sample did not show any band (FIG. 7B, lane c; FIG. 7C, lane c). On the other hand, the sample from the transgenic plant showed a band that hybridizes specifically with the bar and hpt specific probes (FIG. 7B, lane 1; FIG. 7C, lane 1). Since the T-DNA fragment of pGPTV-HB has a single HindIII site (FIG. 7A), the number of hybridized bands reflected the copy number of the gene copy integrated into the transgenic barn plant. The detected bands represented fragments larger than 1.6 kb or 1.8 kb for bar or hpt specific probes, as expected from the pGPTV-HB map (FIG. 7A). This indicates that the copy number of the incorporated gene was 2 (FIG. 7B, lane 1; FIG. 7C, lane 1). Genomic DNA Southern blots with BamHI and bar probes also gave the same results. This indicates that the copy number is 2 (FIG. 7B, lane 2). These results indicate that there were two stable integration events, clearly demonstrating that the herbicide resistance observed in this transgenic plant was determined by the integrated bar gene.
[0056]
Shiba A. in the present invention. The tumefaciens-mediated transformation system speeds up the genetic manipulation of barn as well as closely related turfgrass species. The present invention provides optimized media composition and culture conditions for genetic transformation of buckwheat, since the gene of interest improves resistance to biological and abiotic stresses , And especially when it should be introduced into the shiba to adjust the growth rate.
[0057]
(References)
[Table 5]

(wrap up)
The present invention provides an efficient gene transformation system for Zoysia japonica Steud. Optimized media composition and culture conditions for shiba transformation are also provided. A reliable transformation system for this barn was developed by optimizing several factors that significantly affect callus growth and plant regeneration. Callus type and co-cultivation period absolutely affected transformation efficiency. 2,4-D, CaCl ₂ And the concentration of acetosyringone was also an important factor. The best results were achieved when type 3 callus were co-cultured for 6 days on co-culture medium without 2,4-D. Removal of calcium during co-culture and addition of 50 mg / l acetosyringone dramatically enhanced its efficiency. The present invention also provides a bialaphos resistant shiba plant that can be used in golf courses and stadiums to save on maintenance costs.
[0058]
【The invention's effect】
In accordance with the present invention, A. It is now possible to genetically transform a buckwheat with a gene of interest by a tumefaciens-mediated infection.
[Brief description of the drawings]
FIG. 1 is a photograph showing four different types of callus grown on callus growth medium. (A) Type 1 callus has a slightly white to yellow or light green color and exhibits a dense and non-crushed structure. (B) Type 2 callus is white and exhibits a dense and friable structure. (C) Type 3 callus is yellow and exhibits a dense and very friable structure. (D) Type 4 callus has a translucent soft watery appearance. The four callus types were obtained on callus growth medium supplemented with different combinations of growth regulators as summarized in Table 2.
FIG. 2 shows A.1 carrying pGPTV-HB vector (see FIG. 7). It is a photograph which shows transgenic GUS expression in Shiba callus infected with tumefaciens. (A) Transgenic GUS expression in Shiva callus. (B) A. Rapid growth of callus on MSCGCB medium loaded with filter paper and free of antibiotics after Tumefaciens infection. (C) Induction of shoots on shoot induction medium placed on filter paper and free of antibiotics. (D) Shoot growth and rooting on a bialaphos-containing medium. A. Shoots derived from Tumefaciens-infected calli were cultured on selective rooting medium MSRS containing 5 mg / l bialaphos.
FIG. 3 shows the effect of 2,4-D on transformation efficiency. Blue spots per gram fresh weight of callus cultured on callus growth medium containing different concentrations of 2,4-D were counted.
FIG. 4 shows CaCl versus transformation efficiency. ₂ The effect of Different concentrations of CaCl in the medium ₂ As described in the brief description of FIG. Tumefaciens-infected calli were cultured.
FIG. 5 shows the effect of acetosyringone on transformation efficiency. This experiment was performed as described in the brief description of FIG. 3, except that different concentrations of acetosyringone were included.
FIG. 6 is a photograph showing a transgenic shiba plant (right) and a parent shiba plant (left). These buckwheat plants were sprayed with herbicide solution daily (5 g / l bialaphos) for 2 weeks. (A) Plants that were not sprayed with the herbicide solution. (B) Plant sprayed daily with herbicide solution for 2 weeks.
FIG. 7 shows a schematic of the transformation vector and Southern blot analysis to confirm the presence of the herbicide resistance (bar) gene in the plant genome. (A) Vector map of transformation vector pGPTV-HB. Pnos, promoter of nopaline synthase gene; Hyg ^R Hygromycin resistance gene (hpt); NOS, nopaline synthase gene terminator; Ubi-P, corn-derived ubiquitin promoter; Bar, bialaphos resistance gene; B, BamHI; E, EcoRI; H, HindIII; S, SacI . (B) and (C) Southern blot analysis of transgenic shiba plants. Genomic DNA was digested with either HindIII (lane 1) or BamHI (lane 2). Either the bar (B) probe or the hpt (C) probe was hybridized to the membrane. Genomic DNA from untransformed plants was included as a negative control (lane c).

Claims (3)

A method for genetic transformation of Shiba (genus Zoysia), comprising the following steps:
i) 3% sucrose (w / v), 100 mg / l alpha ketoglutaric acid, 4 mg / l thiamine-HCl, 2 mg / l 2,4-D, 0.2 mg / l BA, and 0.2% gellite (w / v) inducing and growing callus obtained from the seeds of the buckwheat on MS medium containing v) ;
ii) infecting Agrobacterium cells into the birch callus, and introducing a DNA coding sequence of bialaphos resistant bar into the birch callus ;
iii) co-culturing shiba callus and Agrobacterium cells in a co-culture medium containing acetosyringone without both 2,4-dichlorophenoxyacetic acid and calcium;
step to remove the Agrobacterium cells from iv) the coculture medium; and v) comprising the step of reproducing the said callus into transgenic Sheba method. The bialaphos resistance bar gene DNA coding sequence with being stably transformed transgenic turf produced I by the method of claim 1. The transgenic shiba according to claim 2 , transformed with a corn-derived ubiquitin promoter , wherein the promoter is operably linked to the DNA coding sequence of the bialaphos resistant bar gene .

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