JP4424784B2 - Methods for improving the efficiency of gene transfer into plant cells - Google Patents
Methods for improving the efficiency of gene transfer into plant cells Download PDFInfo
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- JP4424784B2 JP4424784B2 JP15802599A JP15802599A JP4424784B2 JP 4424784 B2 JP4424784 B2 JP 4424784B2 JP 15802599 A JP15802599 A JP 15802599A JP 15802599 A JP15802599 A JP 15802599A JP 4424784 B2 JP4424784 B2 JP 4424784B2
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- C—CHEMISTRY; METALLURGY
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
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- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
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Description
【０００１】
【発明の属する技術分野】
本発明は、植物細胞への遺伝子導入の効率を向上させる方法に関する。
【０００２】
【従来の技術】
アグロバクテリウムによる形質転換法は、一般的に、効率が高い、導入される遺伝子のコピー数が少ない、T-DNAという特定の領域を断片化させることなく導入できる、短期間の培養により形質転換体を得ることができるため培養変異が少ないなど、多くの優れた特徴を持っている。このため、さまざまな植物種で最も有用な形質転換の手段として広く用いられている。
【０００３】
このように、アグロバクテリウム法は非常に優れた植物の形質転換方法であるが、形質転換の成否ならびに効率は、植物種、遺伝子型ならびに用いる植物組織に依存して大きく異なるのが実状である（Potrykus et al. 1998(参考文献(33))）。すなわち、形質転換に成功していない植物種があるほか、ごく一部の品種のみ形質転換が可能な植物種も多い。また、利用可能な組織が限定されており大量の材料を取り扱うことができない植物種もある。遺伝子組換えにより実用的な品種を作出するには、多数の形質転換植物を作出した上で、目的とする形質を持った系統を選抜する必要がある。しかしながら、この目的に即し多数の形質転換体を容易に得ることができる作物の種類は、現状では一部に限定されている。したがって、このような問題点を解決することができる改良手法の開発が強く望まれている。
【０００４】
アグロバクテリウムを介する形質転換方法自体は、植物種により供試材料や培養に用いる培地の組成などを異にするものの、材料となる組織にアグロバクテリウムの懸濁液を接触させ、共存培養の後に形質転換細胞の選抜を行い、形質転換植物を作出するという操作ではほぼ共通している。材料となる植物組織には対しては、通常、必要に応じ滅菌処理を行うがそれ以外に特別な処理を施すことなくアグロバクテリウムの感染が行われる（Rogers et al. 1988(参考文献(34)), Visser 1991(参考文献(38)), McCormick 1991(参考文献(29)), Lindsey et al. 1991(参考文献(28))）。従って、形質転換系の改良は、アグロバクテリウムの菌系、ベクター構成、培地組成、選抜マーカー遺伝子やプロモーターの種類、供試組織の種類などを中心に研究が行われてきた。
【０００５】
これに対し、アグロバクテリウムを接種する前の植物組織を、遺伝子導入が生じやすい生理的状態に変換するという考え方に基づく研究は、ほとんど行われていない。何らかの簡便な処理により、そのような生理的状態に変換することができればたいへん利用価値が高く、遺伝子導入効率の向上に加え、従来困難であった植物種や遺伝子型の形質転換を可能にする顕著な効果も期待される。これまでの植物組織への前処理に関する研究例としては、パーティクルガン(Bidney et al., 1992(参考文献(5)))および超音波(Trick et al., 1997(参考文献(37)))処理が上げられる。どちらも物理的に組織を付傷することでバクテリアの植物組織内への侵入を促し、感染対象となる植物細胞を増加させることを目的としている。しかしながら、これは従来より広く行われているリーフディスク法(Horsch et al., 1985(参考文献(17)))を発展させたものに過ぎず、新規な考え方に基づく処理法ではない。なお、効果の程度や汎用性は明らかでなく、一般的な手法として用いられていないのが現状である。
【０００６】
【発明が解決しようとする課題】
従って、本発明の目的は、従来のアグロバクテリウム法による遺伝子導入方法よりも高い効率で組織を付傷することなく簡便に遺伝子導入を行うことができる、植物細胞への遺伝子導入の効率を向上させる方法を提供することである。
【０００７】
【課題を解決するための手段】
本願発明者らは、鋭意研究の結果、アグロバクテリウム属細菌を用いた遺伝子導入方法において、遺伝子導入に供する植物細胞又は植物組織を遠心処理することにより、遺伝子導入効率を有意に向上させることができることを見出し本発明を完成した。
【０００８】
すなわち、本発明は、植物細胞又は植物組織を遠心処理することを伴う、アグロバクテリウム属細菌を介して行われる植物細胞への遺伝子導入の効率を向上させる方法を提供する。
【０００９】
【発明の実施の形態】
本発明の方法では、アグロバクテリウム属細菌を介した遺伝子導入方法において、遺伝子を導入する植物細胞又は植物組織を遠心処理することを伴う。植物細胞又は植物組織は、遠心処理した後、通常の重力下でアグロバクテリウム属細菌と接触させてもよいし、遠心処理しながらアグロバクテリウム属細菌と接触させてもよい。好ましくは、植物細胞又は植物組織を遠心処理した後、通常の重力下でアグロバクテリウム属細菌と接触させる方法である。
【００１０】
遠心処理条件は、用いる植物の種類等に応じて適宜選択されるが、通常、１００Ｇ〜２５万Ｇ、好ましくは５００Ｇ〜２０万Ｇ、さらに好ましくは１０００Ｇ〜１５万Ｇ程度の遠心加速度範囲で行われる。また、遠心処理の時間は、遠心加速度及び用いる植物の種類等に応じて適宜選択されるが、通常１秒間以上行うことが好ましい。なお、遠心時間の上限は特にないが、通常、１０分間程度で目的を達成することができる。なお、遠心処理時間は、遠心加速度が大きい場合には極く短い時間、例えば１秒以下でも遺伝子導入効率を有意に向上させることができる。一方、遠心加速度が小さい場合には、遠心処理を長く行うことにより遺伝子導入効率を有意に向上させることができる。特に好ましい遠心処理条件は、５００Ｇ〜２０万Ｇ、特には１０００Ｇ〜１５００００Ｇで１秒間〜２時間程度の場合が多いが、その植物細胞又は植物組織にとっての適切な遠心処理条件は、ルーチンな実験により容易に設定することができる。
【００１１】
本発明の方法は、アグロバクテリウム属細菌と接触させる植物細胞又は植物組織として遠心処理したものを用いる、又は遠心処理を行いながらアグロバクテリウム属細菌と接触させることを特徴とするものであり、アグロバクテリウム属細菌を用いた遺伝子導入あるいは形質転換方法自体としては、周知の方法をそのまま適用することができる。
【００１２】
アグロバクテリウム属細菌を用いた植物への遺伝子導入あるいは形質転換方法自体は、この分野において周知であり、広く用いられている。
【００１３】
土壌細菌アグロバクテリウム（Agrobacterium tumefaciens）が多くの双子葉植物に根頭癌腫病（crown gall disease）を引き起こすことは古くから知られており、1970年代には、Tiプラスミドが病原性に関与すること、さらにTiプラスミドの一部であるT-DNAが植物ゲノムに組み込まれることが発見された。その後このT-DNAには癌腫の誘発に必要なホルモン（サイトカイニンとオーキシン）の合成に関与する遺伝子が存在し、細菌遺伝子でありながら植物中で発現することが明らかにされた。T-DNAの切り出しと植物への伝達にはTiプラスミド上のヴィルレンス領域（vir領域）に存在する遺伝子群が必要であり、またT-DNAが切り出されるためにはT-DNAの両端に存在するボーダー配列が必要である。他のアグロバクテリウム属細菌であるAgrobacterium rhizogenesもRiプラスミドによる同様なシステムを有している（図３及び図４）。
【００１４】
アグロバクテリウムの感染によってT-DNAが植物ゲノムに組み込まれるので、T-DNA上に所望の遺伝子を挿入するとこの遺伝子も植物ゲノムに組み込まれることが期待された。しかしながら、Tiプラスミドは190kb以上と巨大であるため、標準的な遺伝子工学手法ではプラスミド上のT-DNA上に遺伝子を挿入することは困難であった。そのため、T-DNA上に外来遺伝子を挿入するための方法が開発された。
【００１５】
まず、腫瘍性のTiプラスミドのT-DNAからホルモン合成遺伝子が除去されたディスアーム型の菌系（disarmed strains）であるLBA4404(Hoekema et al., 1983(参考文献(12)))、C58C1(pGV3850) (Zambryski et al., 1983(参考文献(40)))、GV3Ti11SE(Fraley et al., 1985(参考文献(9)))などが作製された（図３）。これらを用いることにより、所望の遺伝子をアグロバクテリウムのTiプラスミドのT-DNA中に、あるいは所望の遺伝子を有するT-DNAをアグロバクテリウムに導入する2種類の方法が開発された。このうちの一つは、遺伝子操作が容易で所望の遺伝子の挿入が可能であり、大腸菌で複製ができる中間ベクターを、アグロバクテリウムのディスアーム型TiプラスミドのT-DNA領域中に、三系交雑法（triparental mating）(Ditta et al., 1980(参考文献(8)))を介して相同組換えにより導入する方法であり、中間ベクター法と呼ばれる(Fraley et al., 1985(参考文献(9)); Fraley et al., 1983(参考文献(10)); Zambryski et al., 1983(参考文献(40))、特開昭59-140885号（EP116718）)。もう一つは、バイナリーベクター（binary vector）法とよばれるもので（図３）、T-DNAの植物への組み込みにvir領域が必要であるが、機能するために同じプラスミド上に存在する必要はないという結果(Hoekema et al., 1983)に基づいている。このvir領域にはvirA、virB、virC、virD、virE及びvirGが存在し、（植物バイオテクノロジー事典（エンタプライズ株式会社発行（1989）））、vir領域とはこのvirA、virB、virC、virD、virE及びvirGの全てを含むものをいう。したがって、バイナリーベクターは、T-DNAをアグロバクテリウムと大腸菌の両方で複製可能な小さなプラスミドに組み込んだものであり、これをディスアーム型Tiプラスミドを有するアグロバクテリウムに導入して用いる。アグロバクテリウムへのバイナリーベクターの導入には、エレクトロポレーション法や三系交雑法などの方法により行うことができる。バイナリーベクターには、pBIN19(Bevan, 1984(参考文献(4)))、pBI121(Jefferson, 1987(参考文献(19)))、pGA482(An et al., 1988(参考文献(2))、特開昭60-70080号（EP120516）)などがあり、これらをもとに数多くの新たなバイナリーベクターが構築され、形質転換に用いられている。また、Ri プラスミドのシステムにおいても、同様なベクターが構築され形質転換に用いられている。
【００１６】
アグロバクテリウムA281(Watson et al., 1975(参考文献(39)))は、強病原性（super-virulent）の菌系であり、その宿主範囲は広く、形質転換効率も他の菌系より高い(Hood et al.,1987(参考文献(13)); Komari, 1989(参考文献(21)))。この特性は、A281が有するTiプラスミドのpTiBo542によるものである(Hood et al., 1984(参考文献(16)); Jin et al., 1987(参考文献(20)); Komari et al., 1986(参考文献(24)))。
【００１７】
pTiBo542を用いて、これまでに2つの新しいシステムが開発されている。一つはpTiBo542のディスアーム型のTiプラスミドを有する菌系EHA101(Hood et al., 1986)およびEHA105(Hood et al., 1993)を用いたものであり、これらを上述のバイナリーベクターシステムに適用することにより、形質転換能力の高いシステムとして種々の植物の形質転換に利用されている。もう一つは、スーパーバイナリーベクター（'super-binary' vector）(Hiei et al., 1994(参考文献(11)); Ishida et al., 1996(参考文献(18)); Komari et al., 1999(参考文献(26))、WO94/00977号、WO95/06722号)システムである（図４）。このシステムは、vir領域（virA、virB、virC、virD、virE及びvirG（以下、これらをぞれぞれ「vir断片領域」ということもある。））を持つディスアーム型のTiプラスミドおよびT-DNAを有するプラスミドからなることから、バイナリーベクターシステムの一種である。しかしながら、T-DNAを有する側のプラスミド、即ちバイナリーベクターにvir断片領域のうち、少なくとも一つのvir断片領域を実質的に取除いたvir領域の断片（このうち好ましくは少なくともvirB又はvirGを含む断片、さらに好ましくはvirB及びvirGを含む断片）を組み込んだ(Komari, 1990a(参考文献(22)))スーパーバイナリーベクターを用いる点で異なる。なお、スーパーバイナリーベクターを有するアグロバクテリウムに、所望の遺伝子を組み込んだT-DNA領域を導入するには、三系交雑法を介した相同組換えが容易な手法として利用できる(Komari et al., 1996(参考文献(25)))。このスーパーバイナリーベクターシステムは、上述の種々のベクターシステムと比べて、多くの植物種で非常に高い形質転換効率をもたらすことが明らかとなっている(Hiei et al., 1994(参考文献(11)); Ishida et al., 1996(参考文献(18)); Komari, 1990b(参考文献(23)); Li et al., 1996(参考文献(27)); Saito et al., 1992(参考文献(35)))。
【００１８】
本発明の方法においては、宿主となるアグロバクテリウム属細菌としては、特に限定されないが、Agrobacterium tumefaciens （例えば上述のAgrobacterium tumefaciens LBA4404(Hoekema et al., 1983(参考文献(12)))およびEHA101(Hood et al., 1986(参考文献(15))）を好ましく用いることができる。
【００１９】
本発明の方法によれば、アグロバクテリウム属細菌における病原性（vir）領域の遺伝子群の発現に基づく遺伝子導入系であれば、特に限定されることなく有意な効果を得ることができる。したがって、上述の中間ベクター、バイナリーベクター、強病原性のバイナリーベクター、スーパーバイナリーベクターなどいずれのベクターシステムに対しても用いることができ、本発明による効果を得ることができる。これらのベクター類を改変した異なるベクターシステムを用いた場合においても同様である（例えば、アグロバクテリウム属細菌のvir領域の一部または全部を切り出し付加的にプラスミド中に組み込む、vir領域の一部または全部を切り出し新たなプラスミドの一部としてアグロバクテリウムに導入するなど）。また、当然ではあるが本発明の方法によれば、野生型のアグロバクテリウム属細菌においても、植物へ野生型のT-DNA領域の導入効率を高め、事実上感染効率を向上することができる。
【００２０】
植物に導入しようとする所望の遺伝子は、上記プラスミドのT-DNA領域中の制限酵素部位に常法により組み込むことができ、当該プラスミドに同時に若しくは別途組込んだカナマイシン、パロモマイシン等の薬剤に対する耐性を有する遺伝子等の適当な選択マーカーに基づいて選択することができる。大型で多数の制限部位を持つものは、通常のサブクローニングの手法では所望のDNAをT-DNA領域内に導入することが必ずしも容易でないことがある。このような場合には、三系交雑法により、アグロバクテリウム属細菌の細胞内での相同組換えを利用することで目的のDNAを導入することができる。
【００２１】
また、プラスミドをAgrobacterium tumefaciens等のアグロバクテリウム属細菌に導入する操作は従来法により行うことができ、例としては、上記した三系交雑法やエレクトロポレーション法、エレクトロインジェクション法、PEGなどの化学的な処理による方法などが含まれる。
【００２２】
植物に導入しようとする遺伝子は、従来の技術と同様に基本的にはT-DNAの左右境界配列の間に配置されるものである。しかし、プラスミドが環状であるため、境界配列の数は１つでもよく、複数の遺伝子を異なる部位に配置しようとする場合には、境界配列が3個以上あってもよい。また、アグロバクテリウム属細菌中で、TiまたはRiプラスミド上に配置されてもよく、または他のプラスミド上に配置されてもよい。さらには、複数の種類のプラスミド上に配置されてもよい。
【００２３】
アグロバクテリウム属細菌を介して遺伝子導入を行う方法は、植物細胞又は植物組織をアグロバクテリウム属細菌と単に接触させることにより行うことができる。例えば、１０6 〜１０11細胞／ｍｌ程度の細胞濃度のアグロバクテリウム属細菌懸濁液を調製し、この懸濁液中に植物細胞又は植物組織を３〜１０分間程度浸漬後、固体培地上で数日間共存培養することにより行うことができる。
【００２４】
遺伝子導入に供される細胞又は組織は、何ら限定されるものではなく、葉、根、茎、実、その他いずれの部位であってもよいし、カルスのような脱分化したものでも脱分化していない胚等であってもよい。また、植物の種類も何ら限定されないが、被子植物が好ましく、被子植物ならば双子葉植物でも単子葉植物でもよい。
【００２５】
下記実施例において具体的に示されるように、本発明の方法によれば、従来のアグロバクテリウム法に比較して、遺伝子導入の効率が有意に向上する。
【００２６】
【実施例】
以下、本発明を実施例に基づきより具体的に説明する。もっとも、本発明は下記実施例に限定されるものではない。
【００２７】
実施例１
（1）アグロバクテリウムの菌系およびプラスミド
アグロバクテリウムおよびそのべクターには、LBA4404（pBI121）(pBI121は米国クローンテック社より市販、(Jefferson RA 1987(参考文献(19)))、LBA4404（pIG121Hm）(Hiei, Y. et al., 1994(参考文献(11))、LBA4404(pTOK233)(Hiei et al., 1994（参考文献(11)）)およびLBA4404(pSB133）（図２）を用いた。
【００２８】
なお、pSB133の構築は、以下のように行った。pGA482(An G et al., 1985(参考文献(3)))を制限酵素Sal Iで消化して得た6.2 kbのＤＮＡ断片を、pSB11(Komari et al., 1996(参考文献(25))をSalIで消化して得られる5.1 kbpのＤＮＡ断片と結合してプラスミドを作製した。次いで、このプラスミドを制限酵素EcoRI、BglIIで消化して8.6 kbのＤＮＡ断片を得た。このＤＮＡ断片を平滑化処理し、BglIIリンカー(TaKaRa社製)を挿入してプラスミドpSB27を得た。このpSB27を制限酵素HindIIIで消化し、pIG221(Ohta S et al., 1990(参考文献(32))をHind IIIで消化することで得られる3.1 kbの35Sプロモーター及びイントロン介在GUS遺伝子を含む断片を挿入してpSB33を得た。pSB33を大腸菌LE392株に導入した後、Triparental mating法(Ditta G et al., 1980(参考文献(8))により、pSB1(Komari et al., 1996(参考文献(25)))を有するアグロバクテリウムLBA4404株に導入した。pSB133はアグロバクテリウム内でpSB1とpSB33の間の相同組換えにより得られた。pBI121のT-DNA領域には、ノパリン合成酵素遺伝子（nos）のプロモーターにより制御されるカナマイシン耐性遺伝子（nptII）、カリフラワーモザイクウィルス（CaMV）の35Sプロモーターにより制御されるGUS遺伝子を有する。pIG121Hm及びpTOK233のT-DNA領域には、nosプロモーターにより制御されるnptII遺伝子、35Sプロモーターにより制御されるhpt遺伝子、35Sプロモーターにヒマのカタラーゼ遺伝子のイントロンが介在するGUS遺伝子を有する。また、pSB133のT-DNA領域には、nosプロモーターにより制御されるnptII遺伝子、CaMVの35Sプロモーターに制御されヒマのカタラーゼ遺伝子のイントロンが介在するGUS遺伝子を有する(図２)。なお、pSB133及びpTOK233は形質転換能力が高いスーパーバイナリーベクター(Komari, T. et al., 1999(参考文献(26)))である。
【００２９】
（2）供試品種および組織
供試品種として、日本稲品種のコシヒカリおよび月の光を用いた。開花後8〜14日目の未熟種子の頴を除去し、70％エタノールで数秒、ツイーン20を含む1％次亜塩素酸ナトリウム水溶液で15分間滅菌処理を行った。滅菌水で数回洗浄後、長さ1.5〜2mmの未熟胚を摘出し供試組織とした。
【００３０】
（3）遠心処理
イネ未熟胚を滅菌水入りのチュ−ブの中に入れ、微量高速遠心機、大型高速遠心機もしくは超高速遠心機を用いて、760G〜150,000Gの遠心処理を行った。遠心処理終了後、未熟胚にアグロバクテリウムを接種した。
【００３１】
（4）接種および共存培養
未熟胚への接種および共存培養の方法は、 Hiei et al. (1994)（参考文献(11)）によった。すなわち、遠心処理後、チューブ内部の滅菌水を除き、アグロバクテリウムの懸濁液を加え、5〜30秒間ボルテックスミキサーにより攪拌した。 バクテリア懸濁液の調製は、ＡＢ培地（Chilton, M-D et al., 1974(参考文献(6))）上で３〜１０日間培養したアグロバクテリウムのコロニーを白金耳でかきとり、修正ＡＡ培地（ＡＡ主要無機塩類、ＡＡアミノ酸及びＡＡビタミン類（Toriyama K. et al., 1985(参考文献(36))、ＭＳ微量塩類（Murashige, T et al., 1962(参考文献(30))、1.0 g/l カザミノ酸、100 μＭアセトシリンゴン、0.2 M ショ糖、0.2 M グルコース）に懸濁することにより行った。約5分間室温で静置した後、共存培養用の培地に置床した。共存培養用の培地としては、2N6-AS培地(Hiei et al. 1994(参考文献(11)))の無機塩類をR2培地(Ohira et al. 1973(参考文献(31)))の組成に変更して用いた。ただし、主要無機塩類（KNO3, KH2PO4, CaCl22H2O, MgSO47H2O）については1/2の濃度で培地に添加した。なお、接種菌密度は1×108 〜1×109 cfu/ml に調整した。共存培養は3〜13日間行い、一部の未熟胚についてX-Glucを処理することによるGUS発現を調査した（Hiei et a1.1994）（参考文献(11)）。すなわち、共存培養処理直後、組織を0.1% Triton X-100 を含む0.1 M リン酸緩衝液(pH6.8) に浸漬し、３７℃で１時間静置した。リン酸緩衝液でアグロバクテリウムを除去した後、1.0 mM ５−ブロモ−４−クロロ−３−インドリル−β−Ｄ−グルクロン酸(X-gluc)および20% メタノールを含むリン酸緩衝液を添加した。３７℃で２４時間処理した後、青色の呈色を示す組織を顕微鏡下で観察した。
【００３２】
（5）形質転換細胞の選抜
共存培養後、未熟胚およびカルスを250mg/l カルベニシリンおよび250mg/l セフォタキシムを含み、200mg/lパロモマイシンまたは10〜30mg/lハイグロマイシンを含む1次選抜培地に移植し、30℃明条件下で1〜2週間培養した。1次選抜培地には、 Hiei et al. (1994)（参考文献(11)）による2N6K培地に30g/lのD-ソルビトールを添加した培地を用いた（K培地）。また、Hiei et al. (1994)(参考文献(11))による2N6培地（Ｎ6 の無機塩およびビタミン類（Chu C. C. 1978 (参考文献(7))）、1 g/l カザミノ酸、2 mg/l ２，４−Ｄ）の(NH4)2SO4を232 mg/lとしAA培地(Toriyama et al., 1985(参考文献(36)))のアミノ酸類を添加した培地についても試験に供した（N培地）。
【００３３】
1次選抜培地上に形成されたカルスを、250mg/lセフォタキシムおよび250mg/l カルベニシリンを含み、200mg/lパロモマイシンもしくは80mg/lハイグロマイシンを含む2次選抜培地上に移植し、30℃明条件下で1〜2週間の培養を行った。2次選抜培地には、Hiei et al. (1994)（参考文献(11)） によるN6-7培地 の(NH4)2SO4を232 mg/lとしAA培地(Toriyama et al., 1985(参考文献(36)))のアミノ酸類を添加した培地を使用した。なお、パロモマイシンを合有する上記の1次および2次選抜培地には、培地固化剤に8g/lアガロースを使用した。耐性カルスの出現率は、2次選抜後に調査した。
【００３４】
（6）形質転換体の再分化
未熟胚の胚盤部位から得られた選抜薬剤耐性のカルスを、250mg/lカルベニシリンおよび250mg/lセフォタキシムを含み、100mg/lパロモマイシンまたは50mg/lハイグロマイシンを含む再分化培地N6S3培地(Hiei et al. 1994（参考文献(11)））上に置床した。
【００３５】
（7）再分化個体におけるGUS発現の調査
25℃明条件下で4〜5週間の再分化培養を行なって得られた各薬剤耐性の再分化植物の葉片を、上記のようにX-Glucを処理することにより、GUS発現を調査した（Hiei et a1.1994（参考文献(11)））。再分化個体は500倍のHyponex水溶液中に移植し、25℃明条件下で約2週間育苗した後、温室内のポットヘ移植した。
【００３６】
(8) 結果
(i)遠心処理効果の検討
微量高速遠心機、大型高速遠心機および超高速遠心機を用いてイネの未熟胚への遠心処理効果を調べた結果、10KGから100KGの範囲の処理で遺伝子の導入効率が高まった(表1, 2, 3, 6)。処理時間については10分間の処理で明らかな効果が認められた(表4, 5)。また、コシヒカリと月の光の品種間でのGUSの一過性発現頻度に違いは認められなかった。なお、遠心処理は遺伝子導入効率の向上だけでなくカルス誘導を促進する効果が認められたことから、ほかの植物種を含めて、培養におけるカルスの誘導および増殖に有用であることが示唆された。
【００３７】
表６の結果から超高速遠心機を用いた250KGの60分処理では、月の光未熟胚からのカルス誘導が全く認められなかった。しかし、110KGの60分処理ではカルス誘導が確認され、GUS発現も高率で認められた。 同様にコシヒカリについても超高速遠心機を用いた250 KG・60分処理では、未熟胚からのカルス誘導が認められなかった。以上の結果から、イネ未熟胚における遠心処理の効果の範囲は5KG〜200KGと考えられ、処理方法の簡便性を考慮すると微量高速遠心機および大型高速遠心機を使用する場合には、20KG,40KG処理が適当と考えられる。さらに表9, 10, 11の結果から、形質転換能力が高いとされるスーパーバイナリーベクターを有するLBA4404(pSB133)のみならず、通常のバイナリーベクターであるLBA4404 (pIG121Hm)でも、20KG・60分の遠心処理により未熟胚を用いた形質転換が可能であることが明らかとなった。
【００３８】
(ii) 遠心処理と共存培養期間の検討
表-7, 8の結果から共存培養期間が３日より6, 13日がトランジェントアッセイで高いGUS発現効率を示した。共存培養期間が9日についても別の実験で高いGUS発現が認められた。現在、共存培養期間が異なる各種未熟胚を一次選抜培地上(10ppmハイグロマイシン, 200ppmパロモマイシン)で培養しているが、9,13日共存の区では、3,6日の共存区と比較して薬剤耐性カルスの出現率が低い傾向にある。
【００３９】
(iii) 遠心処理による形質転換効率の調査
現在、上記により作出したGUS陽性の形質転換体(表4,5)をそれぞれ順化し、栽培を継続している。一部分の系統については、採種を終了し稔性調査を行った。その結果、遠心処理した形質転換体は無処理の形質転換体(コシヒカリ、月の光)と比較し、形態および稔性に差は認められなかった。
【００４０】
Hiei et al. (1994（参考文献(11)））は、イネのカルスを材料として比較的高い効率で形質転換が行うことができることを報告している。また、Aldemita RR et al. 1996(参考文献(1))は、イネの未熟胚を用いた形質転換例を報告している。これらの形質転換手法をより効率よく安定して実施するために、上述した遠心処理法は非常に有効である。特に、未熟胚は栽培環境に左右されやすく形質転換に好適な未熟胚材料を常時得ることは容易ではないが、遠心処理を施すことにより安定した高い形質転換効率を維持することが可能である。Hiei et al. (1994（参考文献(11)））は、形質転換能力の高いベクターであるスーパーバイナリーベクターがイネの形質転換効率を向上させることを示した。また、Aldemita et al., 1996(参考文献(1))によれば、スーパーバイナリーベクターのLBA4404(pTOK233)を用いた試験においてのみ、形質転換体を得ている。本研究における遠心処理法は、通常のバイナリーベクターを用いた場合においても、スーパーバイナリーベクターに匹敵するか、それ以上の遺伝子導入効率を得ることができる。また、スーパーバイナリーベクターと遠心処理法を併用することにより、より一層効率を向上させることが可能である。さらに、遠心処理法を用いることにより、これまで全く形質転換体を得ることができなかった品種においても形質転換体を得ることができるものと推察される。
【００４１】
【表１】
表１ 各種遠心処理と共存培養後のGUS発現結果 (供試菌系：LBA4404/pSB133)
( )内は胚盤におけるGUS発現領域の面積 -:なし,+:小, ++:中, +++:大
【００４２】
【表２】
表２ コシヒカリ未熟胚からのパロモマイシン耐性カルスの出現率 (供試菌系：LBA4404/pSB133)
遠心処理時間：10分、共存培養期間：3〜5日
【００４３】
【表３】
表3月の光未熟胚からのパロモマイシン耐性カルスの出現率(供試菌系：LBA4404/pSB133)
遠心処理時間：10分、共存培養期間：3〜5日
【００４４】
【表４】
表4遠心処理時間と共存培養後のGUS発現結果（品種：コシヒカリ）
胚盤領域におけるGUS発現領域の面積 +:小, ++:中, +++:大
【００４５】
【表５】
表-5 遠心処理時間とパロモマイシン耐性カルスの出現率（品種：コシヒカリ）
耐性カルスの出現した未熟胚数／供試未熟胚数
【００４６】
【表６】
表6 遠心処理強度と共存培養後のGUS発現(品種：月の光)
1）微量高速遠心機 2)大型高速遠心機 3)超高速遠心機
胚盤部に占めるGUS発現領域の割合 -:なし,±:＜1/8, +:1/8-1/4, ++:＞1/4
【００４７】
【表７】
表7 遠心処理および共存培養期間と共存培養後のGUS発現(品種:月の光)
それぞれの回転数に対し、60分間の遠心処理
胚盤部に占めるGUS発現領域の割合 -:なし,±:＜1/8, +:1/8-1/4, ++:＞1/4
【００４８】
【表８】
表8 遠心処理および共存培養期間と共存培養後のGUS発現(品種:コシヒカリ)
それぞれの回転数に対し、60分間の遠心処理
胚盤部に占めるGUS発現領域の割合 -:なし,±:＜1/8, +:1/8-1/4, ++:＞1/4
【００４９】
【表９】
表9 LBA4404(pBI121)による形質転換結果（品種：月の光）
【００５０】
【表１０】
表10 LBA4404(pIG121Hm)による形質転換結果（品種：月の光）
【００５１】
【表１１】
表11 LBA4404(pBI121)による形質転換結果（品種：コシヒカリ）
【００５２】
【表１２】
表12 LBA4404(pSB133)による形質転換結果（品種：コシヒカリ）
【００５３】
実施例 2
大きさ約1.2 mmのトウモロコシ未熟胚（品種A188、農林水産省生物資源研究所より入手）を無菌的に取り出し、LS-inf液体培地で一回洗浄した。遠心管に未熟胚と100 μＭのアセトシリンゴンを含むLS-inf培地2.0 mlに約1 x 109 cfu/mlの濃度で、Agrobacterium tumefaciens LBA4404(pSB131)（Ishida et al. 1996(参考文献(18))）を懸濁した液を加え、40,000G, 4℃で30分間遠心処理した。対照の未熟胚は、前記と同様の細菌懸濁液中で30分間、室温で静置した。処理後、緩やかに攪拌した後、胚軸面が培地に接するようにLS-AS培地に置床した。また、遠心処理後の未熟胚への接種は、以下の通り行った。無菌的に取り出した未熟胚をLS-inf液体培地で一回洗浄した後、同液体培地を含む遠心管に移し、20 KGまたは40 KGで4℃、1時間の遠心処理を行った。対照は液体培地中で1時間、室温で静置した。処理後、液体培地を除き、約1 x 109 cfu/mlの濃度でLBA4404(pSB131)を懸濁した液を加え、緩やかに攪拌した。5分間室温で静置した後、胚軸面が培地に接するように10 μＭ AgNO3を含むLS-AS培地に置床した。25℃、暗黒下で3日間共存培養した後、一部の未熟胚を採取し、実施例１と同様にX-glucによりGUS遺伝子のトランジェントな発現を調査した。なお、上記の培地および培養法は、Ishida, Y.et al. 1996(参考文献(18))に記載の方法に従った。
【００５４】
LBA4404(pSB131)を接種したA188未熟胚でのGUS遺伝子のトランジェントな発現を表１３に示す。いずれの未熟胚もGUS遺伝子の発現を示したが、対照の未熟胚に比べ、遠心処理を行った未熟胚では、より広い範囲での発現を示すものが多く確認された。遠心処理による遺伝子導入部位の増大は、アグロバクテリウム菌とともに遠心処理を行った場合、遠心処理後アグロバクテリウム菌を接種した場合の両方で認められた。また、遠心強度及び処理時間を変えた場合でも対照に比べより広い範囲でのGUS遺伝子の発現が認められた。
【００５５】
以上の結果から、遠心処理した未熟胚を選抜培地で培養すれば、対照に比べより高い効率で、形質転換植物の得られる可能性が示された。また、従来のアグロバクテリウム法では形質転換できなかったA188以外のトウモロコシ品種（Ishida et al. 1996(参考文献(18))）についても遠心処理することにより形質転換植物の得られる可能性が示唆された。
【００５６】
【表１３】
表１３ A188未熟胚でのGUS遺伝子のトランジェントな発現
試験1はアグロバクテリウム菌共存下で遠心処理を行った。試験2は遠心処理後、アグロバクテリウム菌の接種を行った。
【００５７】
【発明の効果】
本発明により、従来のアグロバクテリウム法による遺伝子導入方法よりも高い効率で、組織を付傷することなく簡便に遺伝子導入を行うことができる、植物細胞への遺伝子導入の効率を向上させる方法が提供された。本発明の方法は、単子葉植物に対しても双子葉植物に対しても適用可能である。
【００５８】
参考文献
(1) Aldemita RR, Hodges TK (1996) Agrobacterium tumefaciens-mediated transformation of japonica and indica rice varieties. Planta 199: 612-617
(2) An, G., Evert, P.R., Mitra, A. and Ha, S.B. (1988) Binary vectors. In Gelvin, S.B. and Schilperoort, R.A. (eds.), Plant Molecular Biology Manual A3. Kluwer Academic Press, Dordrecht, pp. 1-19.
(3) An, G., Watson, BD., Stachel, S., Gordon, MP. & Nester, EW., (1985) New cloning vehicles for transformation of higher plants. EMBO J., 4:277-288.
(4) Bevan, M. (1984) Binary Agrobacterium vectors for plant transformation. Nucleic Acids Res., 12, 8711-8721.
(5) Bidney, D., Scelonge, C., Martich, J., Burrus, M., Sims, L., and Huffmanm G. (1992) Microprojectile bombardment of plant tissues increases transformation frequency by Agrobacterium tumefaciens. Plant Mol. Biol., 18, 301-313.
(6) Chilton, M-D., Currier, TC. Farrand, SK. Bendich, AJ. Gordon, MP. & Nester EW. (1974) Agrobacterium tumefaciens DNA and PS8 bacteriophage DNA not detected in crown gall tumers . Proc. Natl. Acad. Sci. USA, 71:3672-3676
(7) Chu, C. C., (1978) Proc. Symp. Plant Tissue Culture, Science Press Peking, pp.43-50
(8) Ditta, G., Stanfield, S., Corbin, D. and Helinski, D.R. (1980) Broad host range DNA cloning system for Gram-negative bacteria: Construction of gene bank of Rhizobium meliloti. Proc. Natl. Acad. Sci. USA, 77, 7347-7351.
(9) Fraley, R.T., Rogers, S.G., Horsch, R.B., Eicholtz, D.A. and Flick, J.S. (1985) The SEV system: a new disarmed Ti plasmid vector for plant transformation. Bio/technology, 3, 629-635.
(10) Fraley, R.T., Rogers, S.G., Horsch, R.B., Sanders, P.R., Flick, J.S., Adams, S.P., Bittner, M.L., Brand, L.A., Fink, C.L., Fry, J.S., Galluppi, G.R., Goldberg, S.B., Hoffmann, N.L. and Woo, S.C. (1983) Expression of bacterial genes in plant cells. Proc Natl Acad Sci USA, 80, 4803-4807.
(11) Hiei, Y., Ohta, S., Komari, T. and Kumashiro, T. (1994) Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. The Plant Journal, 6, 271-282.
(12) Hoekema, A., Hirsch, P.R., Hooykaas, P.J.J. and Schilperoort, R.A. (1983) A binary plant vector strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature, 303, 179-180.
(13) Hood, E.E., Fraley, R.T. and Chilton, M.-D. (1987) Virulence of Agrobacterium tumefaciens strain A281 on legumes. Plant Physiol, 83, 529-534.
(14) Hood, E.E., Gelvin, S.B., Melchers, L.S. and Hoekema, A. (1993) New Agrobacterium helper plasmids for gene transfer to plants. Transgenic Res., 2, 208-218.
(15) Hood, E.E., Helmer, G.L., Fraley, R.T. and Chilton, M.-D. (1986) The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T-DNA. J. Bacteriol., 168, 1291-1301.
(16) Hood, E.E., Jen, G., Kayes, L., Kramer, J., Fraley, R.T. and Chilton, M.-D. (1984) Restriction endonuclease map of pTiBo542, a potential Ti-plasmid vector for genetic engineering of plants. Bio/technology, 2, 702-709.
(17) Horsch, R. B., Fry, J. E., Hoffmann, N. L., Eichholtz, D., Rpgers, S. G. and Fraley, R. T.(1985) A simple and general method for transferring genes into plants. Science 227, 1229-1231.
(18) Ishida, Y., Saito, H., Ohta, S., Hiei, Y., Komari, T. and Kumashiro, T. (1996) High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Nature Biotechnol, 14, 745-750.
(19) Jefferson, R.A. (1987) Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol. Biol. Rep., 5, 387-405.
(20) Jin, S., Komari, T., Gordon, M.P. and Nester, E.W. (1987) Genes responsible for the supervirulence phenotype of Agrobacterium tumefaciens A281. J. Bacteriol., 169, 4417-4425.
(21) Komari, T. (1989) Transformation of callus cultures of nine plant species mediated by Agrobacterium. Plant Sci., 60, 223-229.
(22) Komari, T. (1990a) Genetic characterization of double-flowered tobacco plant obtained in a transformation experiment. Theor. Appl. Genet., 80, 167-171.
(23) Komari, T. (1990b) Transformation of cultured cells of Chenopodium quinoa by binary vectors that carry a fragment of DNA from the virulence region of pTiBo542. Plant Cell Reports, 9, 303-306.
(24) Komari, T., Halperin, W. and Nester, E.W. (1986) Physical and functional map of supervirulent Agrobacterium tumefaciens tumor-inducing plasmid pTiBo542. J. Bacteriol., 166, 88-94.
(25) Komari, T., Hiei, Y., Saito, Y., Murai, N. and Kumashiro, T. (1996) Vectors carrying two separate T-DNAs for co-transformation of higher plants mediated by Agrobacterium tumefaciens and segregation of transformants free from selection markers. Plant J, 10, 165-174.
(26) Komari, T. and Kubo, T. (1999) Methods of Genetic Transformation: Agrobacterium tumefaciens. In Vasil, I.K. (ed.) Molecular improvement of cereal crops. Kluwer Academic Publishers, Dordrecht, pp. 43-82.
(27) Li, H.-Q., Sautter, C., Potrykus, I. and Puonti-Kaerlas, J. (1996) Genetic transformation of cassava (Manihot esculenta Crantz). Nature Biotechnol., 14, 736-740.
(28) Lindsey, K., Gallois, P. and Eady, C. (1991) Regeneration and transformation of sugarbeet by Agrobacterium tumefaciens. Plant Tissue Culture Manual B7:1-13. Kluwer Academic Publishers.
(29) McCormick, S. (1991) Transformation of tomato with Agrobacterium tumefaciens. Plant Tissue Culture Manual B6:1-9. Kluwer Academic Publishers.
(30) Murashige, T. and Skoog, F. (1962) Physiol. Plant 15:473-497.
(31) Ohira, K., Ojima, K., Fujiwara, A. (1973) Studies on the nutrition of rice cell culture I. A simple, defined medium for rapid growth in suspension culture. Plant Cell Physol., 14:1113-1121.
(32) Ohta, S., Mita, S., Hattori, T., Namamura, K. (1990) Construction and expression in tobacco of aβ-glucuronidase (GUS) reporter gene containing an intron within the coding sequence. Plant Cell Physiol. 31: 805-813.
(33) Potrykus, I., Bilang, R., Futterer, J., Sautter, C. and Schrott, M. (1998) Agricultural Biotecnology, NY:Mercel Dekker Inc. pp. 119-159.
(34) Rogers, S.G., Horsch, R.B. and Fraley, R. T. (1988) Gene transfer in plants: Production of transformed plants using Ti plasmid vectors. Method for Plant Molecular Biology, CA: Academic Press Inc. pp.423-436.
(35) Saito, Y., Komari, T., Masuta, C., Hayashi, Y., Kumashiro, T. and Takanami, Y. (1992) Cucumber mosaic virus-tolerant transgenic tomato plants expressing a satellite RNA. Theor. Appl. Genet., 83, 679-683.
(36) Toriyama, K. and Hinata, K. (1985) Plant Sci. 41:179-183
(37) Trick, H.N. and Finer, J.J. (1997) SAAT: sonication-assisted Agrobacterium-mediated transformation. Transgenic Research 6:329-336.
(38) Visser, R.G.F. (1991) Regeneration and transformation of potato by Agrobacterium tumefaciens. Plant Tissue Culture Manual B5:1-9. Kluwer Academic Publishers.
(39) Watson, B., Currier, T.C., Gordon, M.P., Chilton, M.-D. and Nester, E.W. (1975) Plasmid required for virulence of Agrobacterium tumefaciens. J Bacteriol, 123, 255-264.
(40) Zambryski, P., Joos, H., Genetello, C., Leemans, J., Van Montagu, M. and Schell, J. (1983) Ti plasmid vector for the introduction of DNA into plant cells without alteration of their normal regeneration capacity. EMBO J, 2, 2143-2150.
【図面の簡単な説明】
【図１】本発明の方法に好ましく用いることができるスーパーバイナリーベクターの例であるpTOK233の構築方法を示す図である。
【図２】本発明の方法に好ましく用いることができるスーパーバイナリーベクターの例であるpSB133の遺伝子地図を示す図である。
【図３】アグロバクテリウム属細菌の主要な２種類のベクターシステムである中間ベクターシステムとバイナリーベクターシステムの構築過程を示す模式図である。
【図４】アグロバクテリウム ツメファシエンスの強病原性菌株A281に由来する２種類のバイナリーベクターシステムを示す模式図である。
【符号の説明】
virB Agrobacterium tumefaciens A281に含まれるＴｉプラスミドpTiBo542のヴィルレンス領域中のvirB遺伝子
virC Agrobacterium tumefaciens A281に含まれるＴｉプラスミドpTiBo542のヴィルレンス領域中のvirC遺伝子
virG Agrobacterium tumefaciens A281に含まれるＴｉプラスミドpTiBo542のヴィルレンス領域中のvirG遺伝子
BL アグロバクテリウム属細菌のＴ−ＤＮＡの左ボーダー配列
BR アグロバクテリウム属細菌のＴ−ＤＮＡの右ボーダー配列
TC テトラサイクリン抵抗性遺伝子
SP スペクチノマイシン抵抗性遺伝子
IG イントロンＧＵＳ遺伝子
HPT ハイグロマイシン抵抗性遺伝子
K 制限酵素ＫｐｎＩ部位
H 制限酵素ＨｉｎｄIII 部位
Ampr アンピシリン耐性遺伝子
BAR bar遺伝子
Pnos ノパリン合成酵素遺伝子のプロモーター
Tnos ノパリン合成酵素遺伝子のターミネーター
P35S CaMV 35Sプロモーター
COS, cos ラムダファージのCOS部位
ORI, ori ColE1の複製開始点
NPT,NPTII カナマイシン抵抗性遺伝子
Vir アグロバクテリウム属細菌のTiプラスミドの全vir領域
S Vir 強病原性アグロバクテリウム属細菌のTiプラスミドpTiBo542の全vir領域
s vir* TiプラスミドpTiBo542のvir領域の一部を含む断片[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for improving the efficiency of gene transfer into plant cells.
[0002]
[Prior art]
Agrobacterium transformation methods are generally highly efficient, have a low copy number of the gene to be introduced, can be introduced without fragmenting a specific region called T-DNA, and are transformed by short-term culture. Since the body can be obtained, it has many excellent features such as fewer culture variations. For this reason, it is widely used as the most useful means of transformation in various plant species.
[0003]
As described above, the Agrobacterium method is a very excellent method for transforming plants, but the success or failure of transformation and the efficiency vary greatly depending on the plant species, genotype and plant tissue used. (Potrykus et al. 1998 (reference (33))). In other words, there are plant species that have not been successfully transformed, and there are many plant species that can be transformed with only a few varieties. In addition, there are plant species that are limited in available tissues and cannot handle large amounts of materials. In order to produce a practical variety by genetic recombination, it is necessary to produce a large number of transformed plants and then select a line having the desired trait. However, the types of crops that can easily obtain a large number of transformants for this purpose are limited to a part at present. Therefore, development of an improved technique that can solve such problems is strongly desired.
[0004]
Although the Agrobacterium-mediated transformation method itself varies depending on the plant species, the composition of the test material and the culture medium used for the culture, etc., the Agrobacterium suspension is brought into contact with the tissue to be used as a material for co-culture. The operation of selecting a transformed cell later to produce a transformed plant is almost common. The plant tissue that is the material is usually sterilized as necessary, but is infected with Agrobacterium without any other special treatment (Rogers et al. 1988 (references (34 )), Visser 1991 (reference (38)), McCormick 1991 (reference (29)), Lindsey et al. 1991 (reference (28))). Therefore, improvement of the transformation system has been carried out mainly on the Agrobacterium strain, vector composition, medium composition, selection marker gene and promoter type, test tissue type, and the like.
[0005]
On the other hand, there has been little research based on the idea of converting plant tissue before inoculation with Agrobacterium into a physiological state where gene transfer is likely to occur. If it can be converted into such a physiological state by some simple treatment, it is very useful, and in addition to improving gene transfer efficiency, it is possible to transform plant species and genotypes that have been difficult in the past. The effect is also expected. Examples of previous research on plant tissue pretreatment include particle gun (Bidney et al., 1992 (reference (5))) and ultrasound (Trick et al., 1997 (reference (37))) Processing is raised. Both are intended to increase the number of plant cells to be infected by physically injuring the tissue to promote the entry of bacteria into the plant tissue. However, this is only a development of the leaf disk method (Horsch et al., 1985 (reference (17))), which has been widely used, and is not a processing method based on a new concept. In addition, the degree of effect and versatility are not clear, and it is not used as a general method at present.
[0006]
[Problems to be solved by the invention]
Therefore, the object of the present invention is to improve the efficiency of gene introduction into plant cells, which can perform gene introduction more easily and without damaging the tissue at a higher efficiency than conventional gene introduction methods by the Agrobacterium method. Is to provide a way to make it happen.
[0007]
[Means for Solving the Problems]
As a result of intensive studies, the inventors of the present invention can significantly improve gene transfer efficiency by centrifuging plant cells or plant tissues to be used for gene transfer in gene transfer methods using Agrobacterium. The present invention has been completed.
[0008]
That is, the present invention provides a method for improving the efficiency of gene introduction into plant cells carried out via Agrobacterium, which involves centrifugation of plant cells or plant tissues.
[0009]
DETAILED DESCRIPTION OF THE INVENTION
The method of the present invention involves centrifugation of a plant cell or a plant tissue into which a gene is introduced in a gene introduction method via Agrobacterium. The plant cell or plant tissue may be centrifuged and then contacted with Agrobacterium under normal gravity, or may be contacted with Agrobacterium while being centrifuged. The method is preferably a method in which plant cells or plant tissues are centrifuged and then contacted with Agrobacterium under normal gravity.
[0010]
Centrifugal treatment conditions are appropriately selected according to the type of plant used, etc., but are usually performed in a centrifugal acceleration range of about 100G to 250,000G, preferably 500G to 200,000G, more preferably about 1000G to 150,000G. Is called. In addition, the time for the centrifugal treatment is appropriately selected according to the centrifugal acceleration, the type of plant to be used, and the like, but it is usually preferable to carry out for 1 second or longer. The upper limit of the centrifugation time is not particularly limited, but the object can usually be achieved in about 10 minutes. In addition, when the centrifugal acceleration is large, the gene introduction efficiency can be significantly improved even if the centrifugation time is extremely short, for example, 1 second or less. On the other hand, when the centrifugal acceleration is small, gene introduction efficiency can be significantly improved by performing the centrifugation process for a long time. Particularly preferred centrifugation conditions are 500 G to 200,000 G, particularly 1000 G to 150,000 G, often for about 1 second to 2 hours, but appropriate centrifugation conditions for the plant cells or plant tissues are determined by routine experiments. It can be set easily.
[0011]
The method of the present invention is characterized in that a plant cell or plant tissue to be contacted with an Agrobacterium is centrifuged or contacted with an Agrobacterium while performing the centrifugation, As a gene introduction or transformation method using Agrobacterium, known methods can be applied as they are.
[0012]
Methods for gene introduction or transformation into plants using Agrobacterium are well known and widely used in this field.
[0013]
The soil bacterium Agrobacterium tumefaciens has long been known to cause crown gall disease in many dicotyledonous plants. In the 1970s, the Ti plasmid was involved in pathogenicity. Furthermore, it was discovered that T-DNA, which is part of the Ti plasmid, was integrated into the plant genome. Later, it was revealed that this T-DNA contains genes involved in the synthesis of hormones (cytokinin and auxin) necessary for tumor induction and is expressed in plants despite being a bacterial gene. For excision of T-DNA and transmission to plants, a group of genes present in the virulence region (vir region) on the Ti plasmid is required, and in order for T-DNA to be excised, it exists at both ends of T-DNA. A border array is required. Another Agrobacterium genus Agrobacterium rhizogenes has a similar system using the Ri plasmid (FIGS. 3 and 4).
[0014]
Since T-DNA was integrated into the plant genome by infection with Agrobacterium, it was expected that when a desired gene was inserted into T-DNA, this gene was also integrated into the plant genome. However, since the Ti plasmid is as large as 190 kb or more, it has been difficult to insert a gene on T-DNA on the plasmid using standard genetic engineering techniques. Therefore, a method for inserting foreign genes on T-DNA has been developed.
[0015]
First, LBA4404 (Hoekema et al., 1983 (reference (12))), C58C1 (disarmed strains) in which hormone synthesis genes have been removed from T-DNA of the neoplastic Ti plasmid, pGV3850) (Zambryski et al., 1983 (reference (40))), GV3Ti11SE (Fraley et al., 1985 (reference (9))) and the like were prepared (FIG. 3). By using these, two methods have been developed for introducing a desired gene into T-DNA of an Agrobacterium Ti plasmid or T-DNA having a desired gene into Agrobacterium. One of them is an easy-to-operate gene that allows insertion of a desired gene. An intermediate vector that can be replicated in Escherichia coli is incorporated into the T-DNA region of an Agrobacterium disarm-type Ti plasmid. This is a method of introduction by homologous recombination via triparental mating (Ditta et al., 1980 (reference (8))), and is called the intermediate vector method (Fraley et al., 1985 (reference ( 9)); Fraley et al., 1983 (reference (10)); Zambryski et al., 1983 (reference (40)), JP-A-59-140885 (EP116718)). The other is called the binary vector method (Figure 3), which requires the vir region for T-DNA integration into plants, but must be on the same plasmid to function. Based on the result (Hoekema et al., 1983). There are virA, virB, virC, virD, virE and virG in this vir region (Plant Biotechnology Encyclopedia (issued by Enterprise Co., Ltd. (1989))), and the vir region is this virA, virB, virC, virD, Includes all virE and virG. Therefore, the binary vector is one in which T-DNA is incorporated into a small plasmid that can be replicated in both Agrobacterium and E. coli, and this is introduced into Agrobacterium having a disarm type Ti plasmid. The introduction of the binary vector into Agrobacterium can be performed by a method such as electroporation method or three-way hybridization method. Binary vectors include pBIN19 (Bevan, 1984 (reference (4))), pBI121 (Jefferson, 1987 (reference (19))), pGA482 (An et al., 1988 (reference (2))), Kaisho 60-70080 (EP120516)), and many new binary vectors have been constructed and used for transformation. In the Ri plasmid system, a similar vector has been constructed and used for transformation.
[0016]
Agrobacterium A281 (Watson et al., 1975 (reference (39))) is a super-virulent strain with a broad host range and higher transformation efficiency than other strains. High (Hood et al., 1987 (reference (13)); Komari, 1989 (reference (21))). This property is due to the Ti plasmid pTiBo542 of A281 (Hood et al., 1984 (reference (16)); Jin et al., 1987 (reference (20)); Komari et al., 1986. (Reference (24))).
[0017]
Two new systems have been developed so far using pTiBo542. One was using the strains EHA101 (Hood et al., 1986) and EHA105 (Hood et al., 1993) with the disarmed Ti plasmid of pTiBo542, which were applied to the binary vector system described above. By doing so, it is utilized for transformation of various plants as a system with high transformation ability. The other is the 'super-binary' vector (Hiei et al., 1994 (11); Ishida et al., 1996 (18)); Komari et al., 1999 (reference (26)), WO94 / 00977, WO95 / 06722) system (FIG. 4). This system consists of disarmed Ti plasmid and T- with vir regions (virA, virB, virC, virD, virE and virG (hereinafter sometimes referred to as “vir fragment regions”)). Since it consists of a plasmid with DNA, it is a kind of binary vector system. However, the plasmid on the side having T-DNA, that is, a fragment of the vir region obtained by substantially removing at least one vir fragment region of the vir fragment region from the binary vector (of these, preferably a fragment containing at least virB or virG And more preferably, a fragment containing virB and virG) (Komari, 1990a (reference (22))). In order to introduce a T-DNA region into which a desired gene has been incorporated into Agrobacterium having a super binary vector, homologous recombination via a three-line hybridization method can be used (Komari et al. , 1996 (reference (25))). This super binary vector system has been shown to provide very high transformation efficiencies in many plant species compared to the various vector systems described above (Hiei et al., 1994 (11)). Ishida et al., 1996 (reference (18)); Komari, 1990b (reference (23)); Li et al., 1996 (reference (27)); Saito et al., 1992 (reference) (35))).
[0018]
In the method of the present invention, the host genus Agrobacterium is not particularly limited, but Agrobacterium tumefaciens (for example, the above-mentioned Agrobacterium tumefaciens LBA4404 (Hoekema et al., 1983 (reference (12))) and EHA101 ( Hood et al., 1986 (reference document (15))) can be preferably used.
[0019]
According to the method of the present invention, a significant effect can be obtained without particular limitation as long as it is a gene transfer system based on the expression of a gene group of a pathogenicity (vir) region in an Agrobacterium. Therefore, the present invention can be used for any of the vector systems such as the intermediate vectors, binary vectors, strong pathogenic binary vectors, and super binary vectors described above, and the effects of the present invention can be obtained. The same applies to the case of using different vector systems obtained by modifying these vectors (for example, a part of the vir region in which part or all of the vir region of Agrobacterium is excised and additionally incorporated into the plasmid) Or cut out the whole and introduce it into Agrobacterium as a part of a new plasmid). In addition, of course, according to the method of the present invention, in the wild type Agrobacterium, the efficiency of introducing the wild type T-DNA region into the plant can be increased, and the infection efficiency can be effectively improved. .
[0020]
A desired gene to be introduced into a plant can be incorporated into a restriction enzyme site in the T-DNA region of the plasmid by a conventional method, and has resistance to drugs such as kanamycin and paromomycin incorporated into the plasmid simultaneously or separately. Selection can be based on an appropriate selection marker such as a gene possessed. A large one having many restriction sites may not always be easy to introduce a desired DNA into a T-DNA region by a normal subcloning technique. In such a case, the target DNA can be introduced by utilizing homologous recombination in the cells of the genus Agrobacterium by a three-line hybridization method.
[0021]
In addition, the operation of introducing a plasmid into an Agrobacterium genus such as Agrobacterium tumefaciens can be carried out by conventional methods. Examples include the three-line hybridization method, electroporation method, electroinjection method, PEG, and other chemical methods. The method by typical processing is included.
[0022]
The gene to be introduced into the plant is basically arranged between the left and right border sequences of T-DNA, as in the prior art. However, since the plasmid is circular, the number of boundary sequences may be one, and when a plurality of genes are to be arranged at different sites, there may be three or more boundary sequences. They may also be placed on Ti or Ri plasmids in Agrobacterium or on other plasmids. Furthermore, it may be arranged on a plurality of types of plasmids.
[0023]
The method of introducing a gene via an Agrobacterium can be performed by simply contacting a plant cell or plant tissue with an Agrobacterium. For example, 106 -1011Prepare a suspension of Agrobacterium at a cell concentration of about cells / ml, soak plant cells or plant tissue in this suspension for about 3 to 10 minutes, and co-culture on a solid medium for several days Can be performed.
[0024]
The cells or tissues to be used for gene transfer are not limited in any way, and may be any part of leaves, roots, stems, berries, or other parts, and even those that have been dedifferentiated such as callus will be dedifferentiated. It may be an embryo that is not. Moreover, the kind of plant is not limited at all, but angiosperm is preferable. As long as it is angiosperm, it may be a dicotyledonous plant or a monocotyledonous plant.
[0025]
As specifically shown in the following examples, according to the method of the present invention, the efficiency of gene transfer is significantly improved as compared with the conventional Agrobacterium method.
[0026]
【Example】
Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.
[0027]
Example 1
(1) Agrobacterium strain and plasmid
Agrobacterium and its vectors include LBA4404 (pBI121) (pBI121 is commercially available from Clontech, USA (Jefferson RA 1987 (reference (19))), LBA4404 (pIG121Hm) (Hiei, Y. et al., 1994 (reference (11)), LBA4404 (pTOK233) (Hiei et al., 1994 (reference (11))) and LBA4404 (pSB133) (FIG. 2) were used.
[0028]
PSB133 was constructed as follows. A 6.2 kb DNA fragment obtained by digesting pGA482 (An G et al., 1985 (reference (3))) with the restriction enzyme Sal I was converted into pSB11 (Komari et al., 1996 (reference (25))). Was ligated with a 5.1 kbp DNA fragment obtained by digestion with SalI to prepare a plasmid, which was then digested with restriction enzymes EcoRI and BglII to obtain a 8.6 kb DNA fragment. And a BglII linker (manufactured by TaKaRa) was inserted to obtain plasmid pSB27, which was digested with restriction enzyme HindIII, and pIG221 (Ohta S et al., 1990 (reference (32)) was converted to HindIII. A fragment containing a 3.1 kb 35S promoter and an intron-mediated GUS gene obtained by digestion with E. coli was inserted to obtain pSB33, which was introduced into E. coli strain LE392, followed by the Triparental mating method (Ditta G et al., 1980). (Reference (8)) was introduced into Agrobacterium strain LBA4404 having pSB1 (Komari et al., 1996 (Reference (25))). It was obtained by homologous recombination between pSB1 and pSB33 in C. The pBI121 T-DNA region contains a kanamycin resistance gene (nptII), cauliflower mosaic virus (nopaline synthase gene (nos) promoter). The GUS gene is controlled by the 35S promoter of CaMV) The T-DNA region of pIG121Hm and pTOK233 contains the nptII gene controlled by the nos promoter, the hpt gene controlled by the 35S promoter, and the castor catalase gene of the 35S promoter. In addition, the T-DNA region of pSB133 contains the nptII gene controlled by the nos promoter and the GUS gene controlled by the CaMV 35S promoter and the intron of the castor catalase gene. (Fig. 2) Note that pSB133 and pTOK233 are super binary vectors (Kom ari, T. et al., 1999 (reference (26))).
[0029]
(2) Test variety and organization
Japanese rice varieties Koshihikari and Tsukihikari were used as test varieties. The buds of immature seeds on the 8th to 14th days after flowering were removed, and sterilization was performed with 70% ethanol for several seconds and with 1% sodium hypochlorite aqueous solution containing Tween 20 for 15 minutes. After washing several times with sterilized water, immature embryos having a length of 1.5 to 2 mm were extracted and used as test tissues.
[0030]
(3) Centrifugal processing
Rice immature embryos were placed in a tube containing sterilized water, and centrifuged at 760G to 150,000G using a micro high speed centrifuge, a large high speed centrifuge, or an ultra high speed centrifuge. After completion of centrifugation, immature embryos were inoculated with Agrobacterium.
[0031]
(4) Inoculation and co-culture
The method of inoculation to immature embryos and co-culture was according to Hiei et al. (1994) (reference (11)). That is, after centrifuging, the sterilized water inside the tube was removed, an Agrobacterium suspension was added, and the mixture was stirred with a vortex mixer for 5 to 30 seconds. The bacterial suspension was prepared by scraping Agrobacterium colonies cultured on an AB medium (Chilton, MD et al., 1974 (reference (6))) for 3-10 days with a platinum loop, and using a modified AA medium ( AA major inorganic salts, AA amino acids and AA vitamins (Toriyama K. et al., 1985 (reference (36)), MS trace salts (Murashige, T et al., 1962 (reference (30))), 1.0 g / l casamino acid, 100 μM acetosyringone, 0.2 M sucrose, 0.2 M glucose) The mixture was allowed to stand at room temperature for about 5 minutes and then placed on a medium for co-cultivation. As a culture medium, the inorganic salts of 2N6-AS medium (Hiei et al. 1994 (reference (11))) were changed to the composition of R2 medium (Ohira et al. 1973 (reference (31))). However, major inorganic salts (KNO)Three, KH2POFour, CaCl22H2O, MgSOFour7H2O) was added to the medium at a concentration of 1/2. The inoculum density is 1 x 108 ~ 1 × 109 Adjusted to cfu / ml. Co-cultivation was performed for 3 to 13 days, and GUS expression by treating X-Gluc for some immature embryos was investigated (Hiei et a1.1994) (reference (11)). That is, immediately after the co-culture treatment, the tissue was immersed in a 0.1 M phosphate buffer solution (pH 6.8) containing 0.1% Triton X-100 and allowed to stand at 37 ° C. for 1 hour. After removing Agrobacterium with phosphate buffer, add phosphate buffer containing 1.0 mM 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid (X-gluc) and 20% methanol did. After treatment at 37 ° C. for 24 hours, a structure showing a blue color was observed under a microscope.
[0032]
(5) Selection of transformed cells
After co-cultivation, immature embryos and callus were transferred to a primary selection medium containing 250 mg / l carbenicillin and 250 mg / l cefotaxime, 200 mg / l paromomycin, or 10-30 mg / l hygromycin, and 1 Cultured for ~ 2 weeks. As the primary selection medium, a medium in which 30 g / l D-sorbitol was added to 2N6K medium according to Hiei et al. (1994) (reference (11)) was used (K medium). In addition, 2N6 medium (N6 inorganic salts and vitamins (Chu CC 1978 (reference (7))), 1 g / l casamino acid, 2 mg / l by Hiei et al. (1994) (reference (11)) l 2,4-D) (NHFour)2SOFourA medium supplemented with amino acids of AA medium (Toriyama et al., 1985 (reference (36))) at 232 mg / l was also subjected to the test (N medium).
[0033]
Callus formed on the primary selection medium was transplanted onto the secondary selection medium containing 250 mg / l cefotaxime and 250 mg / l carbenicillin and containing 200 mg / l paromomycin or 80 mg / l hygromycin, and light conditions at 30 ° C Incubated for 1-2 weeks. The secondary selection medium was N6-7 medium (NH) according to Hiei et al. (1994) (reference (11)).Four)2SOFourA medium supplemented with amino acids of AA medium (Toriyama et al., 1985 (reference (36))) was used. In addition, 8 g / l agarose was used as a medium solidifying agent in the above primary and secondary selection media combined with paromomycin. The appearance rate of resistant callus was investigated after the second selection.
[0034]
(6) Redifferentiation of transformants
Selective drug-resistant callus obtained from the scutellum site of immature embryos is redifferentiation medium N6S3 medium (Hiei et al. 1994 (reference (11))).
[0035]
(7) Investigation of GUS expression in redifferentiated individuals
GUS expression was investigated by treating X-Gluc as described above on leaf pieces of each drug-resistant redifferentiated plant obtained by performing redifferentiation culture for 4 to 5 weeks under light conditions at 25 ° C ( Hiei et a1.1994 (reference (11))). The redifferentiated individuals were transplanted in a 500-fold Hyponex aqueous solution, grown for about 2 weeks under light conditions at 25 ° C., and then transplanted to a pot in a greenhouse.
[0036]
(8) Result
(i) Examination of centrifugal treatment effect
As a result of examining the effect of centrifugal treatment on immature embryos of rice using a micro high-speed centrifuge, a large-scale high-speed centrifuge, and an ultra-high speed centrifuge, gene introduction efficiency was increased by treatments ranging from 10KG to 100KG (Table 1, 2, 3, 6). As for the treatment time, a clear effect was observed after 10 minutes treatment (Tables 4 and 5). In addition, there was no difference in the transient frequency of GUS between Koshihikari and Tsukihikari varieties. In addition, the effect of centrifuging not only improved the gene transfer efficiency but also promoted callus induction, suggesting that it is useful for the induction and growth of callus in culture, including other plant species. .
[0037]
From the results shown in Table 6, callus induction from lunar light immature embryos was not observed at all in 250 KG for 60 minutes using an ultrahigh speed centrifuge. However, callus induction was confirmed by treatment with 110KG for 60 minutes, and GUS expression was also observed at a high rate. Similarly, calligraphy from immature embryos was not observed in Koshihikari after treatment with 250 KG for 60 minutes using an ultra-high speed centrifuge. From the above results, the range of effects of centrifugation in immature rice embryos is considered to be 5KG to 200KG. Considering the simplicity of the processing method, 20KG, 40KG are required when using a micro high speed centrifuge and a large high speed centrifuge. Processing is considered appropriate. Furthermore, from the results of Tables 9, 10, and 11, not only LBA4404 (pSB133) having a super binary vector that is considered to have high transformation ability, but also LBA4404 (pIG121Hm), which is a normal binary vector, is centrifuged at 20 KG for 60 minutes. It became clear that transformation using immature embryos was possible by the treatment.
[0038]
(ii) Examination of centrifugation and co-culture period
From the results of Tables 7 and 8, the GUS expression efficiency was higher in the transient assay in the coculture period from 3 days to 6 and 13 days. High GUS expression was observed in another experiment even when the co-cultivation period was 9 days. Currently, various immature embryos with different co-cultivation periods are cultured on the primary selection medium (10 ppm hygromycin, 200 ppm paromomycin). The incidence of drug-resistant callus tends to be low.
[0039]
(iii) Investigation of transformation efficiency by centrifugation
Currently, GUS-positive transformants (Tables 4 and 5) produced as described above are acclimatized, and cultivation is continued. For some strains, seeding was completed and fertility studies were conducted. As a result, the transformed transformant showed no difference in morphology and fertility compared to the untreated transformant (Koshihikari, Tsukihikari).
[0040]
Hiei et al. (1994 (reference (11))) reported that transformation can be performed with relatively high efficiency using rice callus. Aldemita RR et al. 1996 (reference (1)) reports a transformation example using immature rice embryos. In order to carry out these transformation techniques more efficiently and stably, the above-described centrifugation method is very effective. In particular, immature embryos are easily influenced by the cultivation environment, and it is not easy to always obtain immature embryo materials suitable for transformation, but stable high transformation efficiency can be maintained by performing centrifugation treatment. Hiei et al. (1994 (reference (11))) showed that a super-binary vector, a vector with high transformation ability, improved the transformation efficiency of rice. In addition, according to Aldemita et al., 1996 (reference (1)), transformants were obtained only in tests using the super binary vector LBA4404 (pTOK233). The centrifugation method in this study can obtain a gene transfer efficiency comparable to or higher than that of a super binary vector even when a normal binary vector is used. Further, the efficiency can be further improved by using a super binary vector and a centrifugal treatment method in combination. Furthermore, it is presumed that by using the centrifugation method, a transformant can be obtained even in cultivars for which a transformant could not be obtained at all.
[0041]
[Table 1]
Table 1. Results of GUS expression after various centrifugation treatments and co-culture (Test strain: LBA4404 / pSB133)
(): GUS expression area in the scutellum-: None, +: Small, ++: Medium, +++: Large
[0042]
[Table 2]
Table 2 Appearance rate of paromomycin-resistant callus from immature embryos of Koshihikari (test strain: LBA4404 / pSB133)
Centrifugation time: 10 minutes, co-cultivation period: 3-5 days
[0043]
[Table 3]
Table 3. Appearance rate of paromomycin-resistant callus from photo immature embryos in March (test strain: LBA4404 / pSB133)
Centrifugation time: 10 minutes, co-cultivation period: 3-5 days
[0044]
[Table 4]
Table 4 Centrifugation time and GUS expression results after co-cultivation (variety: Koshihikari)
Area of GUS expression region in scutellum region +: Small, ++: Medium, +++: Large
[0045]
[Table 5]
Table-5 Centrifugation time and appearance rate of paromomycin-resistant callus (variety: Koshihikari)
Number of immature embryos with resistant callus / number of test immature embryos
[0046]
[Table 6]
Table 6 Centrifugal treatment strength and GUS expression after co-culture (Cultivar: Moonlight)
1) Micro high speed centrifuge 2) Large high speed centrifuge 3) Ultra high speed centrifuge
Proportion of GUS expression area in the scutellum-: None, ±: <1/8, +: 1-8-1 / 4, ++:> 1/4
[0047]
[Table 7]
Table 7 Centrifugal treatment and co-culture period and GUS expression after co-culture (Cultivar: Moonlight)
Centrifugation for 60 minutes for each rotation speed
Proportion of GUS expression area in the scutellum-: None, ±: <1/8, +: 1-8-1 / 4, ++:> 1/4
[0048]
[Table 8]
Table 8 Centrifugal treatment and co-culture period and GUS expression after co-culture (Cultivar: Koshihikari)
Centrifugation for 60 minutes for each rotation speed
Proportion of GUS expression area in the scutellum-: None, ±: <1/8, +: 1-8-1 / 4, ++:> 1/4
[0049]
[Table 9]
Table 9 Results of transformation with LBA4404 (pBI121) (variety: moonlight)
[0050]
[Table 10]
Table 10 Results of transformation with LBA4404 (pIG121Hm) (variety: moonlight)
[0051]
[Table 11]
Table 11 Transformation results with LBA4404 (pBI121) (variety: Koshihikari)
[0052]
[Table 12]
Table 12 Results of transformation with LBA4404 (pSB133) (variety: Koshihikari)
[0053]
Example 2
A maize immature embryo (variety A188, obtained from the National Institute of Bioresources, Ministry of Agriculture, Forestry and Fisheries) of about 1.2 mm in size was aseptically removed and washed once with LS-inf liquid medium. Approximately 1 x 10 in 2.0 ml of LS-inf medium containing immature embryos and 100 μM acetosyringone in a centrifuge tube9 A suspension of Agrobacterium tumefaciens LBA4404 (pSB131) (Ishida et al. 1996 (reference (18))) at a concentration of cfu / ml was added and centrifuged at 40,000 G, 4 ° C. for 30 minutes. Control immature embryos were allowed to stand at room temperature for 30 minutes in the same bacterial suspension as described above. After the treatment, the mixture was gently stirred and then placed on the LS-AS medium so that the hypocotyl surface was in contact with the medium. Inoculation of immature embryos after centrifugation was performed as follows. The immature embryos aseptically removed were washed once with LS-inf liquid medium, transferred to a centrifuge tube containing the same liquid medium, and centrifuged at 20 KG or 40 KG at 4 ° C. for 1 hour. The control was left in liquid medium for 1 hour at room temperature. After processing, except liquid medium, about 1 x 109 A solution in which LBA4404 (pSB131) was suspended at a concentration of cfu / ml was added and gently stirred. After standing at room temperature for 5 minutes, 10 μM AgNO so that the hypocotyl surface is in contact with the mediumThreeAnd placed on LS-AS medium containing. After co-culturing for 3 days in the dark at 25 ° C., some immature embryos were collected, and the transient expression of the GUS gene was examined by X-gluc as in Example 1. The medium and culture method described above were according to the method described in Ishida, Y. et al. 1996 (reference document (18)).
[0054]
Table 13 shows the transient expression of the GUS gene in A188 immature embryos inoculated with LBA4404 (pSB131). Although all immature embryos showed GUS gene expression, many immature embryos that had been subjected to centrifugation were confirmed to show expression in a wider range than the control immature embryos. The increase in the gene introduction site by centrifugation was observed both when Agrobacterium was centrifuged and when Agrobacterium was inoculated after centrifugation. In addition, even when the centrifugal strength and the treatment time were changed, expression of the GUS gene was observed in a wider range than the control.
[0055]
From the above results, it was shown that when cultured immature embryos were cultured in a selective medium, transformed plants could be obtained with higher efficiency than the control. It is also suggested that maize varieties other than A188 that could not be transformed by the conventional Agrobacterium method (Ishida et al. 1996 (reference (18))) could be obtained by transforming. It was done.
[0056]
[Table 13]
Table 13 Transient expression of GUS gene in A188 immature embryos
In Test 1, centrifugation was performed in the presence of Agrobacterium. Test 2 was inoculated with Agrobacterium after centrifugation.
[0057]
【The invention's effect】
According to the present invention, there is provided a method for improving the efficiency of gene introduction into plant cells, which can perform gene introduction more easily than a conventional Agrobacterium method and without damaging the tissue. sponsored. The method of the present invention is applicable to both monocotyledonous and dicotyledonous plants.
[0058]
References
(1) Aldemita RR, Hodges TK (1996) Agrobacterium tumefaciens-mediated transformation of japonica and indica rice varieties.Plana 199: 612-617
(2) An, G., Evert, PR, Mitra, A. and Ha, SB (1988) Binary vectors.In Gelvin, SB and Schilperoort, RA (eds.), Plant Molecular Biology Manual A3. Kluwer Academic Press, Dordrecht , pp. 1-19.
(3) An, G., Watson, BD., Stachel, S., Gordon, MP. & Nester, EW., (1985) New cloning vehicles for transformation of higher plants.EMBO J., 4: 277-288.
(4) Bevan, M. (1984) Binary Agrobacterium vectors for plant transformation.Nucleic Acids Res., 12, 8711-8721.
(5) Bidney, D., Scelonge, C., Martich, J., Burrus, M., Sims, L., and Huffmanm G. (1992) Microprojectile bombardment of plant tissues increases transformation frequency by Agrobacterium tumefaciens. Biol., 18, 301-313.
(6) Chilton, MD., Currier, TC. Farrand, SK. Bendich, AJ. Gordon, MP. & Nester EW. (1974) Agrobacterium tumefaciens DNA and PS8 bacteriophage DNA not detected in crown gall tumers. Proc. Natl. Acad Sci. USA, 71: 3672-3676
(7) Chu, C. C., (1978) Proc. Symp. Plant Tissue Culture, Science Press Peking, pp. 43-50
(8) Ditta, G., Stanfield, S., Corbin, D. and Helinski, DR (1980) Broad host range DNA cloning system for Gram-negative bacteria: Construction of gene bank of Rhizobium meliloti. Proc. Natl. Acad. Sci. USA, 77, 7347-7351.
(9) Fraley, R.T., Rogers, S.G., Horsch, R.B., Eicholtz, D.A. and Flick, J.S. (1985) The SEV system: a new disarmed Ti plasmid vector for plant transformation.Bio/technology, 3, 629-635.
(10) Fraley, RT, Rogers, SG, Horsch, RB, Sanders, PR, Flick, JS, Adams, SP, Bittner, ML, Brand, LA, Fink, CL, Fry, JS, Galluppi, GR, Goldberg, SB , Hoffmann, NL and Woo, SC (1983) Expression of bacterial genes in plant cells.Proc Natl Acad Sci USA, 80, 4803-4807.
(11) Hiei, Y., Ohta, S., Komari, T. and Kumashiro, T. (1994) Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. The Plant Journal, 6, 271-282.
(12) Hoekema, A., Hirsch, PR, Hooykaas, PJJ and Schilperoort, RA (1983) A binary plant vector strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Ti-plasmid.Nature, 303, 179 -180.
(13) Hood, E.E., Fraley, R.T. and Chilton, M.-D. (1987) Virulence of Agrobacterium tumefaciens strain A281 on legumes.Plant Physiol, 83, 529-534.
(14) Hood, E.E., Gelvin, S.B., Melchers, L.S. and Hoekema, A. (1993) New Agrobacterium helper plasmids for gene transfer to plants.Transgenic Res., 2, 208-218.
(15) Hood, EE, Helmer, GL, Fraley, RT and Chilton, M.-D. (1986) The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T-DNA.J. Bacteriol., 168 , 1291-1301.
(16) Hood, EE, Jen, G., Kayes, L., Kramer, J., Fraley, RT and Chilton, M.-D. (1984) Restriction endonuclease map of pTiBo542, a potential Ti-plasmid vector for genetic engineering of plants.Bio/technology, 2, 702-709.
(17) Horsch, R. B., Fry, J. E., Hoffmann, N. L., Eichholtz, D., Rpgers, S. G. and Fraley, R. T. (1985) A simple and general method for transferring genes into plants.Science 227, 1229-1231.
(18) Ishida, Y., Saito, H., Ohta, S., Hiei, Y., Komari, T. and Kumashiro, T. (1996) High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens Nature Biotechnol, 14, 745-750.
(19) Jefferson, R.A. (1987) Assaying chimeric genes in plants: the GUS gene fusion system.Plant Mol. Biol. Rep., 5, 387-405.
(20) Jin, S., Komari, T., Gordon, M.P. and Nester, E.W. (1987) Genes responsible for the supervirulence phenotype of Agrobacterium tumefaciens A281. J. Bacteriol., 169, 4417-4425.
(21) Komari, T. (1989) Transformation of callus cultures of nine plant species mediated by Agrobacterium.Plant Sci., 60, 223-229.
(22) Komari, T. (1990a) Genetic characterization of double-flowered tobacco plant obtained in a transformation experiment.Theor. Appl. Genet., 80, 167-171.
(23) Komari, T. (1990b) Transformation of cultured cells of Chenopodium quinoa by binary vectors that carry a fragment of DNA from the virulence region of pTiBo542.Plant Cell Reports, 9, 303-306.
(24) Komari, T., Halperin, W. and Nester, E.W. (1986) Physical and functional map of supervirulent Agrobacterium tumefaciens tumor-inducing plasmid pTiBo542. J. Bacteriol., 166, 88-94.
(25) Komari, T., Hiei, Y., Saito, Y., Murai, N. and Kumashiro, T. (1996) Vectors carrying two separate T-DNAs for co-transformation of higher plants mediated by Agrobacterium tumefaciens and segregation of transformants free from selection markers.Plant J, 10, 165-174.
(26) Komari, T. and Kubo, T. (1999) Methods of Genetic Transformation: Agrobacterium tumefaciens.In Vasil, I.K. (ed.) Molecular improvement of cereal crops.Kluwer Academic Publishers, Dordrecht, pp. 43-82.
(27) Li, H.-Q., Sautter, C., Potrykus, I. and Puonti-Kaerlas, J. (1996) Genetic transformation of cassava (Manihot esculenta Crantz). Nature Biotechnol., 14, 736-740.
(28) Lindsey, K., Gallois, P. and Eady, C. (1991) Regeneration and transformation of sugarbeet by Agrobacterium tumefaciens.Plant Tissue Culture Manual B7: 1-13.Kluwer Academic Publishers.
(29) McCormick, S. (1991) Transformation of tomato with Agrobacterium tumefaciens.Plant Issue Culture Manual B6: 1-9.Kluwer Academic Publishers.
(30) Murashige, T. and Skoog, F. (1962) Physiol. Plant 15: 473-497.
(31) Ohira, K., Ojima, K., Fujiwara, A. (1973) Studies on the nutrition of rice cell culture I. A simple, defined medium for rapid growth in suspension culture.Plant Cell Physol., 14: 1113 -1121.
(32) Ohta, S., Mita, S., Hattori, T., Namamura, K. (1990) Construction and expression in tobacco of aβ-glucuronidase (GUS) reporter gene containing an intron within the coding sequence.Plant Cell Physiol 31: 805-813.
(33) Potrykus, I., Bilang, R., Futterer, J., Sautter, C. and Schrott, M. (1998) Agricultural Biotecnology, NY: Mercel Dekker Inc. pp. 119-159.
(34) Rogers, S.G., Horsch, R.B. and Fraley, R.T. (1988) Gene transfer in plants: Production of transformed plants using Ti plasmid vectors.Method for Plant Molecular Biology, CA: Academic Press Inc. pp.423-436.
(35) Saito, Y., Komari, T., Masuta, C., Hayashi, Y., Kumashiro, T. and Takanami, Y. (1992) Cucumber mosaic virus-tolerant transgenic tomato plants expressing a satellite RNA. Appl. Genet., 83, 679-683.
(36) Toriyama, K. and Hinata, K. (1985) Plant Sci. 41: 179-183
(37) Trick, H.N. and Finer, J.J. (1997) SAAT: sonication-assisted Agrobacterium-mediated transformation.Transgenic Research 6: 329-336.
(38) Visser, R.G.F. (1991) Regeneration and transformation of potato by Agrobacterium tumefaciens.Plant Tissue Culture Manual B5: 1-9.Kluwer Academic Publishers.
(39) Watson, B., Currier, T.C., Gordon, M.P., Chilton, M.-D. and Nester, E.W. (1975) Plasmid required for virulence of Agrobacterium tumefaciens. J Bacteriol, 123, 255-264.
(40) Zambryski, P., Joos, H., Genetello, C., Leemans, J., Van Montagu, M. and Schell, J. (1983) Ti plasmid vector for the introduction of DNA into plant cells without alteration of their normal regeneration capacity.EMBO J, 2, 2143-2150.
[Brief description of the drawings]
FIG. 1 is a diagram showing a construction method of pTOK233, which is an example of a super binary vector that can be preferably used in the method of the present invention.
FIG. 2 is a diagram showing a gene map of pSB133, which is an example of a super binary vector that can be preferably used in the method of the present invention.
FIG. 3 is a schematic diagram showing the construction process of an intermediate vector system and a binary vector system, which are two main types of vector systems of the genus Agrobacterium.
FIG. 4 is a schematic diagram showing two types of binary vector systems derived from the strongly pathogenic strain A281 of Agrobacterium tumefaciens.
[Explanation of symbols]
virB gene in the virulence region of Ti plasmid pTiBo542 contained in virB Agrobacterium tumefaciens A281
virC gene in the virulence region of Ti plasmid pTiBo542 contained in Agrobacterium tumefaciens A281
virG gene in the virulence region of Ti plasmid pTiBo542 contained in virG Agrobacterium tumefaciens A281
BL Left border sequence of T-DNA of Agrobacterium
BR Right border sequence of T-DNA of Agrobacterium
TC tetracycline resistance gene
SP spectinomycin resistance gene
IG intron GUS gene
HPT hygromycin resistance gene
K restriction enzyme KpnI site
H restriction enzyme HindIII site
Ampr ampicillin resistance gene
BAR bar gene
Pnos promoter of nopaline synthase gene
Tnos nopaline synthase gene terminator
P35S CaMV 35S promoter
COS, cos COS site of lambda phage
ORI, ori ColE1 replication start point
NPT, NPTII kanamycin resistance gene
The entire vir region of Ti plasmid of Vir Agrobacterium
S Vir The entire vir region of Ti plasmid pTiBo542 of the strongly pathogenic Agrobacterium
s vir * Ti fragment containing part of the vir region of pTiBo542
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