農(nóng)桿菌介導(dǎo)法

1、農(nóng)桿菌介導(dǎo)轉(zhuǎn)化法農(nóng)桿菌介導(dǎo)轉(zhuǎn)化法 農(nóng)桿菌是普遍存在于土壤中的一種革蘭氏農(nóng)桿菌是普遍存在于土壤中的一種革蘭氏陰性陰性細(xì)菌，細(xì)菌，它能在自然條件下趨化性地感染大多數(shù)雙子葉植物的受傷它能在自然條件下趨化性地感染大多數(shù)雙子葉植物的受傷部位，并誘導(dǎo)產(chǎn)生部位，并誘導(dǎo)產(chǎn)生冠癭瘤或發(fā)狀根冠癭瘤或發(fā)狀根。農(nóng)桿菌農(nóng)桿菌感染柳樹感染柳樹產(chǎn)生產(chǎn)生冠癭瘤冠癭瘤原理：原理： 根癌農(nóng)桿菌和發(fā)根農(nóng)桿菌細(xì)胞中分別含有根癌農(nóng)桿菌和發(fā)根農(nóng)桿菌細(xì)胞中分別含有TiTi質(zhì)粒和質(zhì)粒和RiRi質(zhì)粒質(zhì)粒，其上有一段，其上有一段T-DNAT-DNA，農(nóng)桿菌通過侵染植物傷口，農(nóng)桿菌通過侵染植物傷口進(jìn)入細(xì)胞后，可將進(jìn)入細(xì)胞后，可將T-DNAT-DN

2、A插入到植物基因組中。插入到植物基因組中。 因此，農(nóng)桿菌是一種因此，農(nóng)桿菌是一種天然天然的植物遺傳轉(zhuǎn)化體系。人的植物遺傳轉(zhuǎn)化體系。人們將們將目的基因目的基因插入到經(jīng)過改造的插入到經(jīng)過改造的T-DNAT-DNA區(qū)，借助農(nóng)桿菌的區(qū)，借助農(nóng)桿菌的感染實(shí)現(xiàn)感染實(shí)現(xiàn)外源基因外源基因向植物細(xì)胞的轉(zhuǎn)移與整合，然后通過向植物細(xì)胞的轉(zhuǎn)移與整合，然后通過細(xì)胞和組織培養(yǎng)技術(shù)，再生出轉(zhuǎn)基因植株。細(xì)胞和組織培養(yǎng)技術(shù)，再生出轉(zhuǎn)基因植株。 農(nóng)桿菌介導(dǎo)法起初只被用于雙子葉植物中，近年來，農(nóng)桿菌介導(dǎo)法起初只被用于雙子葉植物中，近年來，農(nóng)桿菌介導(dǎo)轉(zhuǎn)化在一些單子葉植物（尤其是水稻）中也農(nóng)桿菌介導(dǎo)轉(zhuǎn)化在一些單子葉植物（尤其是水稻）中

3、也得到了廣泛應(yīng)用。得到了廣泛應(yīng)用。Story冠癭瘤病冠癭瘤?。弘p子葉植物經(jīng)常發(fā)生，因腫瘤著生地面在近地面的根莖交界處，形似帽狀而得名。1907年， Smith & Townsent 農(nóng)桿菌誘發(fā)冠癭瘤病。1947年， Braun et al. 證實(shí)倆者的關(guān)系，但發(fā)現(xiàn)有的菌株不 致病。提出了假說：tumour-inducing principle,TIP.腫瘤 誘導(dǎo)因子 。60s , 腫瘤組織中含高濃度的氨基酸（octopine , nopaline） 總稱冠癭堿（opine) 。 Petit et al.證實(shí)腫瘤組織 合成的冠癭堿取決于菌株，而且菌株能專一地利用 冠癭堿作為菌株生存的唯一

4、的碳源和氮源。(證實(shí)了TIP)1974年，Zaenen et al, Schell, Van Larebeke et al. 從致瘤農(nóng)桿菌中分離出一類巨大的質(zhì)粒 (tumor inducing plasmid)，稱為Ti質(zhì)粒。 Ti=TIP1977年，Chilton et al.分子雜交技術(shù)證實(shí)腫瘤細(xì)胞中存 在外源的DNA ,與Ti質(zhì)粒的DNA有同源性，是整 合到了植物染色體的農(nóng)桿菌質(zhì)粒DNA片段， T- DNA (transferred DNA),其內(nèi)有致瘤和冠癭 堿合成酶等基因。1981年，Ooms et al.發(fā)現(xiàn)Ti質(zhì)粒上有致瘤區(qū)(virulence region)，Vir區(qū)。Ti質(zhì)粒

5、是根癌農(nóng)桿菌細(xì)胞核外存在的一種環(huán)狀雙鏈DNA分子，長度約200kb，平均周長54.175 .4 um，分子質(zhì)量為（90150）106 Da。在溫度低于30的 條件下，Ti質(zhì)?？煞€(wěn)定地存在于根癌農(nóng)桿菌細(xì)胞內(nèi)。Ti質(zhì)粒Ti質(zhì)粒除上述上述誘導(dǎo)受侵染的植物組織產(chǎn)生冠癭瘤 外，還具有以下幾種重要功能：賦予根癌農(nóng)桿菌附著于植物細(xì)胞的能力；賦予根癌農(nóng)桿菌分解代謝冠癭堿的能力；根癌農(nóng)桿菌的寄主植物范圍；決定所誘導(dǎo)的冠癭形態(tài)和冠癭堿的成分；參與寄主細(xì)胞合成植物激素吲哚乙酸和一些細(xì)胞分裂素 的代謝活.1) Ti質(zhì)粒的結(jié)構(gòu)來自于不同野生型根癌農(nóng)桿菌的Ti質(zhì)?？筛鶕?jù)其產(chǎn)生的冠癭堿類型分為三類：章魚堿(octopine

6、)類 胭脂堿(nopaline)類 農(nóng)桿堿（agropine） 類。Ti質(zhì)粒攜帶著既能分解又能合成這些化合物的酶類和相應(yīng)基因，然而冠癭堿合成基因卻不能在根癌農(nóng)桿菌中表達(dá)，它們只有進(jìn)入植物細(xì)胞后才能表達(dá)，Ti質(zhì)粒上的冠癭堿分解基因產(chǎn)物卻能分解冠癭堿，為宿主細(xì)胞提供能源、氮源和碳源。長度：160-250 kb6大功能區(qū)：1)致癌區(qū)，這個(gè)區(qū)主要合成植物 生長素和細(xì)胞分裂素；2)冠癭堿合成區(qū)；3)冠癭堿分解區(qū)；4)Ti質(zhì)粒接合轉(zhuǎn)移區(qū)(tra)；5)毒性區(qū)（Vir）；6)DNA復(fù)制區(qū)（Rep）。在致癌區(qū)和冠癭堿合成區(qū)的兩側(cè)存在著一個(gè)24 bp直接重復(fù)序列，由這三部分所構(gòu)成的DNA區(qū)域叫做T-DNA，插入

7、植物染色體中的Ti質(zhì)粒片段只有T-DNA。由于T-DNA插入植物細(xì)胞染色體中的位置不相同的，因此植物染色體上可能并沒有可供T-DNA插入的專一性DNA序列。T-DNA脂堿型根癌農(nóng)桿菌Ti質(zhì)粒中T-DNA的左右兩側(cè)是一段24bp的重復(fù)序列,構(gòu)成T-DNA的邊界序列(border sequence),分別稱為左邊界(left border,LB)和右邊界(right border,RB).在某些章魚堿型根癌農(nóng)根癌農(nóng)桿菌Ti質(zhì)粒中T-DNA是以兩個(gè)分開的獨(dú)立片段形式存在，即T-DNA左邊區(qū)段和T-DNA右邊區(qū)段。研究表明，插入在T-DNA邊界序列之間的任何DNA都可被轉(zhuǎn)到植物染色體中。因此Ti質(zhì)?？?/p>

8、用做外源目的基因的載體。T-DNA 區(qū)域中的這些基因只有在T-DNA插入到植物基因組后才能激活表達(dá).植物生長素和細(xì)胞分裂素,可調(diào)節(jié)植物細(xì)胞的生長和發(fā)育,它們的過量表達(dá)刺激植物細(xì)胞大量快速增長而形成冠癭。冠癭堿也是在細(xì)胞內(nèi)合并成分泌出來的,構(gòu)成根癌農(nóng)桿菌生長所須的碳源和氮源.Vir區(qū)（Vir-region），即毒性區(qū)，其長度約為35kb。它們控制根癌農(nóng)桿菌附著于植物細(xì)胞和Ti質(zhì)粒進(jìn)入細(xì)胞有關(guān)部位，與感染后冠癭形成有關(guān)。Vir區(qū)位于T-DNA區(qū)左側(cè)，包含義個(gè)毒性遺傳點(diǎn)（virA、vir、virC、 vir、vir和virG）。vir基因的控制著的轉(zhuǎn)移。virvirvirC virG virvirA

9、Vir區(qū)區(qū)植物細(xì)胞受傷后，細(xì)胞壁破裂，分泌物中含有高濃度的創(chuàng)傷誘導(dǎo)分子（酚類化合物）：如乙酰丁酮（acetosyringone，AS）和-羥基酰丁香酮（-hydroxacetosyringone，OH-AS）。根癌農(nóng)桿菌對(duì)這一類物質(zhì)具有趨化性，在植物細(xì)胞表面附著后，受這些創(chuàng)傷誘導(dǎo)分子的刺激，Ti質(zhì)粒vir區(qū)毒性基因被激活和表達(dá)。目前已經(jīng)發(fā)現(xiàn)9種信號(hào)因子，均為水溶性酚類化合物。其中乙酰丁香酮（acetosyringone，AS）和羥基乙酰丁香酮（OH-AS）的作用較強(qiáng)，兒茶酚、原兒茶酚、沒食子酸、焦性沒食子酸、二羥基苯甲酸、香草酚和對(duì)羥基苯酚處理農(nóng)桿菌時(shí)也對(duì)Vir區(qū)的基因表達(dá)起促進(jìn)作用。雙子葉植

10、物在在農(nóng)桿菌侵染時(shí)可以形成大量的信號(hào)因子，而使T- DNA可以成功的轉(zhuǎn)入；而單子葉植物需要加入外源酚類物質(zhì)，才能激活Vir區(qū)的基因，達(dá)到轉(zhuǎn)基因的目的。最先激活表達(dá)的是virA基因，它編碼感受蛋白，位于細(xì)菌細(xì)胞膜的疏水區(qū)，可接受環(huán)境中的信號(hào)分子。在virA蛋白的激活下，virG基因表達(dá)，virG蛋白經(jīng)磷酸化由非活性態(tài)變?yōu)榛罨癄顟B(tài)，進(jìn)而激活vir區(qū)其他基因表達(dá)。virA基因virG基因其中virD基因產(chǎn)物virD1蛋白是一種DNA松弛酶，它可使DNA從超螺旋型轉(zhuǎn)變?yōu)樗沙谛蜖顟B(tài)；而virD2蛋白則能切割已呈松弛態(tài)的T-DNA，2個(gè)邊界產(chǎn)生缺口，使單鏈T-DNA得以釋放。VirE基因所表達(dá)的virE2

11、蛋白是單鏈T-DNA結(jié)合蛋白?？墒筎-DNA形成1個(gè)細(xì)長的核酸蛋白復(fù)合物（T-復(fù)合體），以此保護(hù)T-DNA不被包內(nèi)外的核酸酶降解。最先激活表達(dá)的是virA基因，它編碼感受蛋白，位于細(xì)菌細(xì)胞膜的疏水區(qū)，可接受環(huán)境中的信號(hào)分子。在virA蛋白的激活下，virG基因表達(dá)，virG蛋白經(jīng)磷酸化由非活性態(tài)變?yōu)榛罨癄顟B(tài)，進(jìn)而激活vir區(qū)其他基因表達(dá)。其中virD基因產(chǎn)物virD1蛋白是一種DNA松弛酶，它可使DNA從超螺旋型轉(zhuǎn)變?yōu)樗沙谛蜖顟B(tài)；而virD2蛋白則能切割已呈松弛態(tài)的T-DNA，2個(gè)邊界產(chǎn)生缺口，使單鏈T-DNA得以釋放。VirE基因所表達(dá)的virE2蛋白是單鏈T-DNA結(jié)合蛋白?？墒筎-DNA

12、形成1個(gè)細(xì)長的核酸蛋白復(fù)合物（T-復(fù)合體），以此保護(hù)T-DNA不被包內(nèi)外的核酸酶降解。T-復(fù)合體依次穿過根癌農(nóng)桿菌和植物細(xì)胞膜及細(xì)胞壁。并進(jìn)入植物細(xì)胞核，最終整合進(jìn)入植物核基因組。T-DNA的轉(zhuǎn)移機(jī)理比較復(fù)雜，依賴于T-DNA區(qū)和vir區(qū)共同參與，涉及多個(gè)基因表達(dá)及一系列蛋白質(zhì)和核酸的相互作用。Table1 Summary of vir Gene Products Locus Size(kb) ORFsa Proteins Size(kDa) Locationb FunctionvirA2.0190Mplant signal sensor, protein kinaseVirG1.0130Ct

13、ranscriptional activatorVirD4.5416,47,21,75C/M?T-DNA border endonuclease (VirD1 and VirD2); pilot protein?nuclear localization?(VirD2)VirC2.0226,23C?processing of T-DNA(VirC1)VirE2.027, 60.5C/M?single-strand DNA-binding protein (VirE2) virB9.51126,12,11,87,23,32,5.5,25,32,48,38MT-DNA transfer appara

14、tus?T-DNA加工和轉(zhuǎn)移加工和轉(zhuǎn)移 Vir基因表達(dá)調(diào)控的問題，知道基因表達(dá)調(diào)控的問題，知道VirA和和VirG基因誘導(dǎo)其它基因誘導(dǎo)其它Vir基基因的表達(dá)，當(dāng)因的表達(dá)，當(dāng)VirD操縱元的被誘導(dǎo)表達(dá)后，其中的操縱元的被誘導(dǎo)表達(dá)后，其中的VirD1和和VirD2蛋白質(zhì)具有核酸內(nèi)切酶的活性蛋白質(zhì)具有核酸內(nèi)切酶的活性(Yanofsky MF. 1986)，能夠在能夠在T-DN A邊界重復(fù)序列的特異位點(diǎn)切開邊界重復(fù)序列的特異位點(diǎn)切開T-DNA單鏈，因此單鏈，因此T-DNA往往以單鏈形式進(jìn)入植物細(xì)胞。但是在植物細(xì)胞中也發(fā)往往以單鏈形式進(jìn)入植物細(xì)胞。但是在植物細(xì)胞中也發(fā)現(xiàn)雙鏈現(xiàn)雙鏈T-DNA分子。分子。切

15、刻位點(diǎn)切刻位點(diǎn)(nick site)可作為可作為DNA從從5向向3合成的起始位合成的起始位點(diǎn)，新的點(diǎn)，新的DNA鏈合成后，鏈合成后，T-DNA單鏈即被替換單鏈即被替換(displacement)釋放出來釋放出來(圖圖2)。當(dāng)然這種缺刻也可通。當(dāng)然這種缺刻也可通過重組系統(tǒng)把過重組系統(tǒng)把T-DNA雙鏈從雙鏈從Ti質(zhì)粒上解離下來。質(zhì)粒上解離下來。缺失研究也表明：右邊界重復(fù)序列缺失后，T-DNA不能轉(zhuǎn)移，而左邊界重復(fù)序列的缺失會(huì)稍稍降低T-DNA轉(zhuǎn)移頻率(Timmerman B1988)，這進(jìn)一步說明T-DNA單鏈?zhǔn)窃趶挠疫吔绲阶筮吔?向3替換合成中釋放出來的。右邊界的缺失，使得右邊界的缺失，使得DN

16、A合成不能起始，因而合成不能起始，因而T-DNA不不能釋放，所以能釋放，所以T-DNA不能轉(zhuǎn)移到植物細(xì)胞。而左邊界的缺不能轉(zhuǎn)移到植物細(xì)胞。而左邊界的缺失，僅會(huì)影響失，僅會(huì)影響DNA合成過程的終止，可能會(huì)降低導(dǎo)致合成過程的終止，可能會(huì)降低導(dǎo)致T-DNA單鏈釋放，而不會(huì)明顯影響單鏈釋放，而不會(huì)明顯影響T-DNA單鏈的形成。單鏈的形成。冠癭也是在冠癭堿胞內(nèi)合并成分泌出來的,被冠癭堿分解基因分解成根癌農(nóng)桿菌生長所須的碳源和氮源。冠癭堿分解區(qū)Agrobacterium-mediated transformation Advantages:1. Cleaner inserts2. Higher co-ex

17、pression3. More flexible in tissue types(in planta transformation) almost any type of tissues or cells 4. Mostly widely used system ,both monocot and dicot5. Easy in manipulation ,Disadvantages 1. Only good for nuclear transformation2. Limited in host range,genotype-dependent3. Detrimental to some t

18、issuesIntroduction to AgrobacteriumMoloecular mechanisms of T-DNA transfer into the plant genome Factors influencing the T-DNA delivery Regeneration and selection of transgenic plantsTypesA.tumefaciens : the causative agent of plant disease crowng gall.A.rhizogenes : provokes hair roots. Both crown

19、gall and root hair are neoplastic diseases (plant tumors), both gram-negative soil bacterium. Based on the opines produced in the tumors,Ti plasmids are divided in four groups,while the Ri plasmids fall in three groups.Ti plasmidsu Octopine: pTiAch5,pTiAg57,or pTi19955 Nopaline: pTiC58,pTi1D135,pTi2

20、23 Leucinnopine: pTi542,pTiAT4D,L-succinamopine: pTiEU6,pTiAT181 Ri plasmidsu Mannopine: pRi8196,pRiTR7 Agropine: pRi1855,pRi15834,pRiA4 Cucumopine: pRi1659 Strains: Agrobacterium Strains and the origins of Agrobacterium strains(see attached table 1 and 2)refernce plasmid1:238-253(1978)Examples:C58

21、(C58 chromosome,pTi C58)ABI (C58 chromosome,pTiMp90RK,disarmed TiC58)GV3850 (C58 chromosome,pTiGV3850,disarmed TiC58)A281 (C58 chromosome,pTiBo542)EHA101 (C58 chromosome,pTiEHA101,disarmed pTiBo542 EHA105 (C58 chromosome,pTiEHA105,disardisarmed pTiBo542)T37 (T37 chromosome,pTi37)A208 (C58 chromosome

22、,Pti37)Ach5 (Ach5 chromosome,pTiAch5)LBA4404 (Ach5 chromosome,TiAL4404,disarmed pTiAch5) Strains: Agrobacterium Strains and the origins of Agrobacterium strains(see attached table 1 and 2)refernce plasmid1:238-253(1978)Examples:C58 (C58 chromosome,pTi C58)ABI (C58 chromosome,pTiMp90RK,disarmed TiC58

23、)GV3850 (C58 chromosome,pTiGV3850,disarmed TiC58)A281 (C58 chromosome,pTiBo542)EHA101 (C58 chromosome,pTiEHA101,disarmed pTiBo542 EHA105 (C58 chromosome,pTiEHA105,disardisarmed pTiBo542)T37 (T37 chromosome,pTi37)A208 (C58 chromosome,Pti37)Ach5 (Ach5 chromosome,pTiAch5)LBA4404 (Ach5 chromosome,TiAL44

24、04,disarmed pTiAch5)Molecular mechanisms of T-DNA transfer into plant genome Steps in T-DNA delivery1) host perception2) pre-infection attachment 3) induction and initiation of virulence genes 4) synthesis and assembly of the promiscuous pilus 5) processing and delivery of the T-DNA 6) T-DNA integra

25、tion1)host perception: chemical signals produced naturally by plant roots serve chemoattractants such as sucrose,glucose and fructose as well as amino acids like valine and arginine are good chemoattractants.plant isofolavomoids such as formoneton and coumestrol initiating the transcription of chrom

26、osomal genes of Agrobacterium involved in facilitating root colonization.most of the Vir genes except for genes of the virb and vire operons are required for Agrobacterium motility.2).pre-infection attachment a) Agrobacterium must attach to its host cells before the T-DNA is processed and readied fo

27、r transfer,following chemotaxis to the host plant .b) attachment is independent of Ti or Ri plasmid.c) A.tumefaciens adherence to plant cells is saturable .d) The attachment sites are thought to be specific receptors (2,000 such receptors on carrot suspension culture cells)where only live bacterium

28、binds.e) Each plant cell has enough attachment sites for several bacterlum,but plant cells are transformed by only one or a few bacterium .No loss of competence of plant cell causes by the firsrt agrobacterum infection.f) Chromosomal genes involved in attachment chva,chcb,exoc:synthesis of a cyclic

29、-1,2-glucan, implicated in plant cell binding. cel:cellulose synthesizing genes.agrobacterium cells forms large aggregates microscopically, seen as clumps tetheted by cellulose fibrils. pscA:exopolisaccharide systhesis and secretion leading to the systhesis of cellulose fibrils. att: root colonizati

30、on3) induction and initiation of virulence genes a)virA: autophosphorilating protein respond to the environmental signals by transferring a phosphate group to VirG protein.The induction of vir genes expression is sensitive to temperature(nopaline strain(t37,c58)octopine strains(ach5,lba4404)(see fig

31、ure)b)agrobacterium growth phase the best agrobacterium growth phase is mid to late log phase.c)Vir gene induction(agrobacterium induction medium) presence of phenolic compounds(5-200 umol); low temperature(15-25with a pick at 19); acidic medium(ph5.1-5.7); presence of monosaccharides(d-galaturinic

32、acid at 100u m,d-glucuronic acid 10mm,d-glucose10mm)phosphate starvation(from absence to0.1mm phosphate);often used media for bacterium growth and induction:mg/l or ab minimal(see the formula of mg/l and ab)d)mutant VirA or VirG (constitutive expresslon of VirA or VirG )highly increased expression o

33、f other vir gene, such as VirBAs, pH, as well as temperature dependency E)Multiple copy of VirG,VirD and VirB operons super-binary vector(see map) 2)Inoculation and co-culture conditionsa)enhancing attachmentssurfacts:pluronic acid,silwet(l77), soap et alb) Agrobacterium cell densityc)inoculation an

34、d co-culture time periodd)Temperatur 20 to 27e) inoculation and co-culture mediumf)feeder layers tobacco suspension cells g) vacuum infiltration microwoundimgb)Agrobacterium cell density in general, higher density increases T-DNA delivery ,but causes plant cell death .Depending on the explant type a

35、nd species. The Agrobacterium cell densities can be arranged from 10 7to 3x10 9. OD660 1=3x10 9 cells/mlexample: wheat immature embryos 0.51.0 rice embryogenic callus 1.01.5 corn immature embryos 1.0 soybean 0.30.6 cotton 0.30.6c)inoculation and co-culture time period depending on species, explants,

36、the oprimal inoculation time period varies.Examples: Tobacco leaf: second10min cotton hypocit: 13min peanut leaf: 10min soybean cotyledon mode : 1030min rice ECcs and IEs: 5min corn IEs: 5min wheat IEs and ECs: 60min Wheat suspension cells: 15minCo-culture time varies from 2 to 3 days, usually 3days

37、e)inoculation and co-culture medium in general, inoculation and co-culture medium are the same.Since Agrobactereium vir induction requires minimal salts, try lower salts such as 1/5x or 1/10 x; inoculation and co-culture medium should contain induction chemicals such as AS and sugar; auxins such as

38、2,4-D and picloram enhance T-DNA delivery ,and cytokinins such as BA and TDZ inhibit the T-DNA delivery.3)explants and pretreatment of explantsa)types of explants dicot species: more explants available for infection ,such as leaf, cotyledon, stems, seed explants, meristem, mature or immature embryio

39、s, embtyogenic callus et al. monocot species: less explants for infection .the main explants are immature embryos or the callus derived from. most regenerable explants are usually used for transformation; Avoid the explants are sensitive to Agrobacterium infection, such as microspores and protoplast

40、 cells transformation competence varies among explants intre/intra apecies.Example:intra-species: rice preculture immature embryos and embryogenic callus inter-species: embryogenic callus between rice,corn and wheat.b)physiology conditions of the explants wounding vs no wounding almost all dicot exp

41、lants requires wounding ,while monocot explants dont need wounding before during inocuationsonication-mediated transformation bombardment before infection freshly prepared explants usually more competent than the precultured explants. Including auxins such as 2,4-d and picloram in the preculture med

42、ium enhances T-DNA delivery. Avoiding cytokinins, especially TDZ in the pre-culture medium. DNA-demethylation:5-azacytidine. c)pretreatments: Desiccation:air dry of embryogenic callus of sugarcane. Osmotic treatment:corn freshly isolated immature embryos. Cold vs warm treatments:sometimes,it works!

43、4)efficiency of T-DNA delivery and plant cell recoveryOnce sufficient T-DNA delivery is achieved with an explant, the conditions favor the plant cell recovery and subsequenrly cell eivision and development are crucial for generating stable transgebic plants. An efficient T-DNA delivery system is prerequisite for an efficient stable transformation ayatem in most of the circumstances. Agrobacterium is always toxic to plant cells. a)strategies for enhancing the plant cell recovery i