Plant Molecular Biology 20: 1071-1087, 1992. © 1992 KluwerAcademic Publishers. Printed in Belgium.

1071

Multiple copies of virG enhance the transient transformation of celery, carrot and rice tissues by Agrobacterium tumefaciens Chang-Nong Liu 1, Xiu-Qing Li 2'3 and Stanton B. Gelvin 1, Departments of 1Biological Sciences and 2Botany & Plant Pathology, Purdue University, West Lafayette, IN47907, USA (*author for correspondence);3Current address: Department of Agronomy, Purdue University Received 12 May 1992; accepted in revised form 4 August 1992

Key words: Agrobacterium tumefaciens, carrot, celery, gene transformation, GUS expression, rice

Abstract

In an effort to improve the T-DNA-mediated transformation frequency of economically important crops, we investigated the possible enhancement effect of multiple copies of virG genes contained in Agrobacterium tumefaciens strains upon the transient transformation of celery, carrot and rice tissues. Four days after A. tumefaciens infection, we performed histochemical fl-glucuronidase (GUS) assays to determine the frequency of transient transformation of calli from celery and carrot, and explants from rice and celery. Additional copies of octopine- and agropine-type virG genes in A. tumefaciens strains containing an agropine-type Ti-plasmid enhanced the frequency of transient transformation of celery and rice. This enhancement ranged from 25 ~o to five-fold, depending upon the source of the virG gene and the plant tissues inoculated. For both rice and celery, we observed a greater enhancement of transformation using A. tumefaciens strains containing additional copies of an octopine-type virG gene than with strains harboring additional copies of an agropine-type virG gene. Multiple copies of virG genes contained in A. tumefaciens strains harboring a nopaline-type Ti-plasmid had a smaller enhancing effect upon the transformation of celery tissues, and no enhancing effect upon the transformation of rice. In contrast, we obtained a three-fold increase in the transient transformation frequency of carrot calli using an A. tumefaciens strain harboring a nopaline-type Ti-plasmid and additional copies of an octopine-type virG gene. Our results show that multiple copies of virG in A. tumefaciens can greatly enhance the transient transformation frequency of celery, carrot and rice tissues, and that this enhancement is influenced by both the type of Ti-plasmid harbored by A. tumefaciens and by the infected plant species.

Introduction

The genetic transformation of many dicotyledonous plant species using Agrobacterium tumefaciens has become a standard laboratory technique [1, 10, 41]. Celery and carrot are among those species amenable to Agrobacterium-mediated transformation [3, 4, 13]. Rice, as most other agriculturally important monocot cereal crops, is

relatively recalcitrant to infection by A. tumefaciens. To date, the generation of transgenic rice plants by Agrobacterium-mediated transformation has not been reported, although the generation of transgenic tumor callus of a japonica variety of rice was reported by Raineri et al. [32]. Agrobacterium-mediated genetic transformation involves multiple processes, including virgene induction by phenolic compounds and sugars,

1072 T-DNA processing and transfer, the integration of the T-DNA into the plant genome, and its subsequent expression. The combined functions of genes on both the chromosome and the Ti plasmid of A. tumefaciens are required for these processes [reviewed in 12, 24, 33, 44]. VirG protein, the product of the virG gene of the Ti plasmid, functions as a positive transcriptional regulator for the expression of its own and other vir genes after being phosphorylated by VirA protein [17, 23, 35, 43]. The number of copies and the type of virG gene can influence some of the biological properties ofA. tumefaciens. For example, Rogowsky et al. [34] showed that an increase in the copy number of virG could result in a proportional, acetosyringone-independent increase in vir gene expression. We have recently shown that multiple copies of virG in A. tumefaciens allow a high level of vir gene induction by acetosyringone at alkaline pH (C.-N. Liu et aI., manuscript in preparation). Jin etal. [22] showed that a 2.5 kb D N A fragment containing the virG and the 3' end of the virB operon from pTiBo542 is responsible for the supervirulence of the agropine-type strain A. tumefaciens A281. A plasmid containing this fragment conferred a supervirulence phenotype upon A. tumefaciens A348, a nonsupervirulent strain containing the octopine-type Ti-plasmid pTiA6. The virulence of A. tumefaciens A281 could not be increased simply by introducing additional copies of this same fragment into the bacterium. A further increase in virulence could, however, be achieved if the entire virB operon was introduced in addition to the virG gene. In an effort to optimize the conditions for A. tumefaciens infection of rice as well as some dicotyledonous plants, we investigated the possible enhancing effect on transformation conferred upon A. tumefaciens by multiple copies of different types of virG genes. A. tumefaciens strains (containing agropine- or nopaline-type Ti plasmids) with additional copies of either an octopineor an agropine-type virG gene were employed to infect celeriac, carrot, and rice tissues. Binary vectors harboring a cauliflower mosaic virus (CaMV) 35S-uidA construct displaying little or no/?-glucuronidase (GUS) activity in the bacte-

rium were used to monitor the transient transformation of plant tissues. Four days after Agrobacterium infection, histochemical G U S assays were carried out to determine the efficiency of transient transformation. Our results show that introducing extra copies of virG into agropine-type (for rice and celery tissues) or nopaline-type (for carrot tissues) A. tumefaciens strains greatly enhanced the frequency of transient G U S expression in these plants. This report provides a basis for the rapid screening of various combinations of A. tumefaciens strains, infection protocols, and plant tissues to optimize the infection of plants for future stable transformation experiments.

Materials and methods

Plant materials Celery (celeriac or turnip rooted celery, Apium graveolens vat. rapaceum [Mill] Gaud.-Beaup.) and carrot (Daucus carota L.) cultures were induced from hypocotyl explants of celery and carrot in vitro and maintained on agar-solidified MS salts and vitamins [31] containing 1 rag/1 2,4-D, 0.5 mg/1 kinetin, 100 rag/1 inositol, 20 g/1 sucrose, and 2 g/1 glucose in the dark at 25 °C. Celery seedlings were regenerated from these calli on hormone-free MS media. Two rice (Oryza sativa L.) varieties were used in this study. Seeds of IR64, an indica variety, were kindly provided by Dr Steve Linscombe and Dr Norimoto Murai of Louisiana State University, and seeds of the cv. Lemont were a gift of Dr William Park of Texas A&M University. Dehusked mature rice seeds were sterilized in 70~o ethanol for 2 rain and in 50~o Chlorox (v/v, commercial bleach) for 30 rain with shaking. The seeds were transferred to another sterile beaker containing fresh 50 ~o Chlorox for 5 rain. Seeds were washed three times in sterile deionized water, then germinated in Petri plates in the dark at 25 °C for either four (IR64) or five (Lemont) days on a medium consisting of one-quarter strength N6 salts and vitamins [6], 1.0~o sucrose, 0.6~o agar, and lacking plant hormones.

1073 Chemicals and enzymes Ampicillin, carbenicillin, kanamycin, and morpholineethanesulfonic acid (MES) were purchased from Sigma Chemical Co. (St. Louis, MO). Acetosyringone (3', 5'-dimethoxy-4'-hydroxyacetophenone) was purchased from Aldrich Chemical Co. (Milwaukee, WI). X-gluc for GUS assays was from Jersey Lab and Glove Supply (Livingston, NJ). Restriction endonucleases and T4 DNA ligase were purchased from New England Biolabs (Beverly, MA).

Bacterial strains and growth conditions Escherichia coli and Agrobacterium tumefaciens strains used in this study are listed in Table 1. The construction of the binary vectors pCNL1, pCNL28, pCNL29, pCNL30, pCNL35, and pCNL65 is described in Results (Fig. 1). A. tumefaciens strains were grown at 28 °C in YEP medium or AB minimal medium containing 1.0~o sucrose [27]. E. coli strains were grown at 37 °C in LB medium [28]. The concentrations of antibiotics (in #g/ml) used in the growth media were c arbenicillin or ampicillin (100), kanamycin (100), and rifampin (10). A triparental mating procedure [9], using the plasmid pRK2013 [ 11 ], was followed to mobilize binary vectors from E. coli to A. tumefaciens. DNA cloning and transformation orE. coli DH5e with plasmid DNA were performed according to Maniatis etal. [28]. A. tumefaciens EHA105 (At542) was generated from A. tumefaciens EHA101 (kindly provided by Dr Elizabeth Hood, Utah State University) [15] by deletion of the kanamycin resistance gene. pCNL30, pCNL29 and pCNL35 are binary vectors lacking (pCNL30) or containing one copy of either an octopine-type virG gene (from pTiA6; pCNL29) or an agropine-type virG gene (from pTiBo542; pCNL35), respectively. These binary vectors were separately introduced into agropine-type (EHA105 and A281) and nopaline-type (At503 and A208) A. tumefaciens strains, generating At650, At651, At652, At653, At654, At655,

At695, At658, At659, At694, At660, and At661 (Table 1). Disarmed A. tumefaciens strains containing (At651, At652, At654 and At655) or lacking (At650 and At653) additional virG genes on binary vectors were used for celery and carrot transformation. Tumorigenic strains containing (At658, At659, At660, and At661) or lacking (At695 and At694) additional virG genes on binary vectors were used for rice transformation. A. tumefaciens At696 (lacking a Ti plasmid) and A. tumefaciens At643 and At705 (lacking an uidA gene) served as negative controls for GUS expression. A. tumefaciens At699 and At700 are derivatives of A. tumefaciens At650 and At695, respectively, containing an intron in the uidA coding region (see Results). Because the uidA fusion gene in these two strains contains an intron, GUS activity can be expressed only in plant cells after completion of the T-DNA transfer process. A. tumefaciens At487 is a derivative of A. tumefaciens LBA4404 containing the binary vector pBI121 [19].

Preinduction of A. tumefaciens All A. tumefaciens cultures used for transformation were preinduced by acetosyringone before cocultivation with plant tissues. A fresh bacterial colony was picked and grown overnight at 28 ° C in YEP medium containing the appropriate antibiotics. An aliquot of these cells was transferred into AB-sucrose minimal medium and grown to an optical density of 100 Klett units (2 x 109 cells/ ml). These cells were centrifuged at 4 °C for 10 min and washed once with sterile water. The cells were suspended in two volumes of preinduction medium (1.0~o glucose, 1 x AB salts, 20 mM MES, 2 mM sodium phosphate pH 5.6) containing 100#M acetosyringone and incubated at 24 °C for 20 h.

Cocultivation of plant tissues with A. tumefaciens The concentrations of the different A. tumefaciens strains were adjusted to an A60 o of 1 (2 x 109 cells/

1074

A

mas 5' 35S 5'

XhOl/x B /

RE]

RB

p

_~t]/ --k an

~"

LB'~

'~l ~?~4~ )

4~

/H

pCNL1 ~'~P 152 Kb OCSJ ~

x0o,

xo+, _.-r °~+NL~# ,~--

Multiple copies of virG enhance the transient transformation of celery, carrot and rice tissues by Agrobacterium tumefaciens.

In an effort to improve the T-DNA-mediated transformation frequency of economically important crops, we investigated the possible enhancement effect o...
3MB Sizes 0 Downloads 0 Views