Influence of bacterial density during preculture on Agrobact

ORIGINAL PAPER

In?uence of bacterial density during preculture

on Agrobacterium -mediated transformation of tomato

Nan Gao ?Weishou Shen ?Yu Cao ?Yanhua Su ?Weiming Shi

Received:7December 2008/Accepted:8July 2009/Published online:25July 2009óSpringer Science+Business Media B.V.2009

Abstract An improved bacterial preculture protocol for Agrobacterium -mediated genetic transformation was developed for an economic tomato cultivar (Solanum lyco-persicum L.cv.Zhongshu No.4).Frequencies of transient gene expression and stable transformation were in?uenced by the density of Agrobacterium preculture and not the density of Agrobacterium used for infection.The improved protocol presented in this study depends on the use of an overnight-grown Agrobacterium preculture density of OD 600nm =1.0,diluted 1/10th with Luria-Bertani (LB)liquid medium,and grown for an additional 4h.Cultures are collected and resuspended in a liquid cocultivation medium-I,adjusted to OD 600nm =aadc476f011ca300a6c390f3ing this modi?ed Agro-bacterium preparation,transient b -glucuronidase expression was higher than 90%,and transformation ef?ciency reached 44.7%.This improved transformation is simple,repeatable,does not require a feeder layer,and most notably,the trans-formation frequency is stable and highly ef?cient.Keywords Density of bacterial cells ánptII á

Solanum lycopersicum L.áTransient b -glucuronidase (GUS)expression

Introduction

Tomato (Solanum lycopersicum L.)is an economically important crop.However,abiotic stresses,including salinity,heat,drought,and nutrient de?ciencies (such as phosphate and nitrate)often constrain fruit productivity (Raghothama 2000;Abel et al.2002;Zhu 2002;Bhatnagar-Mathur et al.2008).Developing transgenic plants is an effective approach for improving abiotic stress tolerance (Park et al.2005;Bhatnagar-Mathur et al.2008).Estab-lishment of an ef?cient transformation system is essential.The ?rst successful transformation of tomato was reported by Horsch et al.(1985)using Agrobacterium -mediated transformation.To date,various factors affecting the ef?-ciency of Agrobacterium -mediated transformation have been investigated.These include cocultivation temperature (Dillen et al.1997),type (Frary and Earle 1996;Ellul et al.2003;Park et al.2003;Wu et al.2006)and developmental status of explants (Chyi and Phillips 1987;Hamza and Chupeau 1993;Tabaeizadeh et al.1999;Dan et al.2006;Sun et al.2006),use of feeder-layer cells (van Roekel et al.1993;Qiu et al.2007),addition of phenolic compounds

(Lipp Joa

?o and Brown 1993;Cortina and Culia ′n ˇez-Macia `2004;Sun et al.2006;Wu et al.2006),vector constructs (van Roekel et al.1993;Qiu et al.2007),Agrobacterium strains (van Roekel et al.1993;Wroblewski et al.2005),and concentration (Ellul et al.2003;Dan et al.2006;Qiu et al.2007;Wu et al.2006)and composition of the medium (Hamza and Chupeau 1993;Frary and Earle 1996;Ling et al.1998;Vidya et al.2000;Krasnyanski et al.2001;Pozueta-Romero et al.2001;Park et al.2003;Cortina and

Culia

′n ˇez-Macia `2004;Wu et al.2006).Current transfor-mation ef?ciencies ranges from 6to 40%(Hamza and Chupeau 1993;Frary and Earle 1996;Ling et al.1998;Vidya et al.2000;Krasnyanski et al.2001;Park et al.2003;

N.Gao áY.Cao áY.Su áW.Shi (&)

State Key Laboratory of Soil and Sustainable Agriculture,Institute of Soil Science,Chinese Academy of Sciences,210008Nanjing,China e-mail:wmshi@aadc476f011ca300a6c390f3

N.Gao áY.Cao

Graduate School of the Chinese Academy of Sciences,100039Beijing,China

W.Shen

College of Chemistry and Environmental Science,Nanjing Normal University,210097Nanjing,China

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Plant Cell Tiss Organ Cult (2009)98:321–330DOI 10.1007/s11240-009-9566-2

Sun et al.2006).In spite of these successes,most protocols rely on the use of either cumbersome feeder layers from petunia,tobacco,or tomato,complex media formulations, or time-consuming successive subcultures.Hence,opti-mizing the frequency of transformation of tomato is highly desirable.

Agrobacterium tumefaciens has been widely used to transform numerous plant species,including tomato.The ability of Agrobacterium to transform plants is under highly regulated genetic control,especially for the vir gene (Hansen et al.1994;Gelvin2003;Yuan et al.2007).Over the years,optimal conditions for Agrobacterium vir gene induction have been extensively investigated(Joubert et al. 1995;Hwang and Gelvin2004;Yuan et al.2007).In addition,bacterial concentrations used for in?ltration have also been optimized(Pozueta-Romero et al.2001;Wu et al. 2006;Qiu et al.2007).For tomato transformation,Agro-bacterium concentrations usually range between0.01and 1.0at OD600nm(Lipp Joa?o and Brown1993;Frary and Earle1996;Ling et al.1998;Krasnyanski et al.2001;Po-zueta-Romero et al.2001;Park et al.2003;Dan et al.2006; Wu et al.2006;Qiu et al.2007),and overnight-grown Agrobacterium cultures are usually diluted prior to cocul-tivation(Krasnyanski et al.2001;Dan et al.2006;Wu et al. 2006).McCormick et al.(1986)incubated explants in a 1:20dilution of Agrobacterium C58C1(Rif r)culture;while, Lipp Joa?o and Brown(1993)incubated explants in a1:10 dilution,grown for about4h with Agrobacterium C58C1 (Rif r)culture.On the other hand,Frary and Earle(1996) diluted an overnight-grown Agrobacterium strain LB4404 culture one to twofold to reach OD600nm=0.4to obtain an optimal infection concentration;whereas,Raj et al.(2005)?oated precultured cotyledons in a1:15dilution of Agro-bacterium strain LB4404.Sun et al.(2006)used a1:20–40 dilution of Agrobacterium C58C1(Rif r)culture;while,Qiu et al.(2007)diluted an overnight-grown culture of Agro-bacterium strain EHA101to OD600nm=0.2.In our pilot experiment on tomato transformation,we found that when using cotyledons as explants,transformation frequencies varied greatly,even with the same Agrobacterium strain at the sa …… 此处隐藏：36406字，全部文档内容请下载后查看。喜欢就下载吧 ……