Introduction Directory UMM :Data Elmu:jurnal:P:PlantScience:PlantScience_Elsevier:Vol160.Issue1.2000:

Plant Science 160 2000 165 – 176 Resistance to bacterial wilt in somatic hybrids between Solanum tuberosum and Solanum phureja I. Fock a , C. Collonnier a , A. Purwito b , J. Luisetti c , V. Souvannavong d , F. Vedel e , A. Servaes a , A. Ambroise a , H. Kodja f , G. Ducreux a , D. Sihachakr a, a Morphogene`se Ve´ge´tale Expe´rimentale, Baˆt. 360 Uni6ersite´ Paris Sud, 91405 Orsay Cedex, France b Plant Biomolecular and Cellular Laboratory, IUC Biotechnology, Bogor Agricultural Uni6ersity IPB , JL Puspa Kampus IPB Darmaga, Bogor 16610 , Indonesia c Laboratoire de Phytopathologie, 7 chemin de l ’ IRAT, Ligne Paradis, 97410 Saint-Pierre, La Re´union, France d Groupe Endotoxines, UMR 8619 , CNRS-UPS, Baˆt. 430 Uni6ersite´ Paris Sud, 91405 Orsay Cedex, France e Laboratoire d ’ Ecologie, Syste´matique et E6olution, UPRESA-CNRS 8079 , Baˆt. 360 Uni6ersite´ Paris Sud, 91405 Orsay Cedex, France f Laboratoire de Biologie et Physiologie Ve´ge´tales, Ge´ne´tique Mole´culaire et E6oluti6e, 15 A6enue Rene´ Cassin, BP 7151 , 97715 Saint-Denis, La Re´union, France Received 2 March 2000; received in revised form 25 August 2000; accepted 4 September 2000 Abstract Somatic hybrid plants were produced after protoplast electrofusion between a dihaploid potato, cv. BF15, and a wild tuber-bearing relative, Solanum phureja, with a view to transferring bacterial wilt resistance into potato lines. A total of ten putative hybrids were selected. DNA analysis using flow cytometry revealed that six were tetraploids, two mixoploids, one amphiploid and one octoploid. In the greenhouse, the putative hybrids exhibited strong vigor and were morphologically intermediate, including leaf form, flowers and tuber characteristics. The hybrid nature of the ten selected plants was confirmed by examining isoenzyme patterns for esterases and peroxidases, and analysis of RAPD and SSR markers. Analysis of chloroplast genome revealed that eight hybrids possessed chloroplast ct DNA of the wild species, S. phureja, and only two contained Solanum tuberosum ct type. Six hybrid clones, including five tetraploids and one amphiploid, were evaluated for resistance to bacterial wilt by using race 1 and race 3 strains of Ralstonia solanacearum, originating from Reunion Island. Inoculations were performed by an in vitro root dipping method. The cultivated potato was susceptible to both bacterial strains tested. All somatic hybrids except two were tolerant to race 1 strain, and susceptible to race 3 strain. Interestingly, the amphiploid hybrid clone showed a good tolerance to both strains. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords : Solanum tuberosum; Solanum phureja; Electrofusion; Somatic hybrids; Bacterial wilt; Ralstonia solanacearum www.elsevier.comlocateplantsci

1. Introduction

Bacterial wilt, a severe and devastating plant disease caused by Ralstonia solanacearum, occurs in most tropical, subtropical and warm temperate areas and even in some cool temperate regions [1]. It affects more than 200 plant species distributed in more than 50 families, particularly potatoes and tomatoes [2]. The bacterium invades plant vascular tissues from wounded roots or natural openings sec- ondary roots. The colonisation of the stem results in browning of the xylem and frequently leads to partial or complete wilting [3]. Potatoes and toma- toes are affected by two of the three major races of R. solanacearum. Under cool temperate condi- tions, particularly in north-western Europe or at high altitude in some tropical countries such as Peru or Reunion Island, only race 3 strains induce wilting of potatoes [4]. Race 1 strains also damage potato crops under tropical and subtropical condi- tions [5]. Corresponding author. Tel.: + 33-1-6915-4690; fax: + 33-1-6985- 5490. E-mail address : dara.sihachakrmve.u-psud.fr D. Sihachakr. 0168-945200 - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 0 0 0 0 3 7 5 - 7 Resistant or tolerant cultivars are mainly uti- lized for disease management since chemicals are not effective and sanitation measures difficult to apply. Therefore, the worldwide control strategy has consisted of plant breeding for resistance to bacterial wilt [6]. Using resistant cultivars to con- trol bacterial wilt has been successful for tobacco and peanut. Since immunity was not identified in potato, only tolerant cultivars were selected [7], such as cultivar Prisca mainly cropped in Mada- gascar, cultivar Ndinamagara in Burundi, Rwanda and Zaire and cultivar Achat in Brazil [4,8]. To better control bacterial wilt, continuous develop- ment of resistant or tolerant varieties is needed. Some wild or related cultivated species are known to be resistant or highly tolerant to bacte- rial wilt and thus are potential sources for resis- tance. Unfortunately, hybrids of potato with resistant genotypes of Solanum chacoense, Solanum sparsipillum and Solanum multidissectum revealed some traits of wildness such as a high glycoalkaloid content besides a moderate level of resistance to bacterial wilt [4]. Unlike these wild species which have been classified as wild Tuberosa [9], Solanum phureja cultivated Tuberosa is phyl- logenetically close to Solanum tuberosum and dis- plays resistant traits, dominant and readily transmitted to progeny. Some clones of S. phureja with high degree of resistance to bacterial wilt have been used for crossing with commercial culti- vars of S. tuberosum [4,10]. Resistance to bacterial wilt derived from S. phureja was first described as dominant and controlled by three unlinked genes [6,11,12]. More recently, at least four major genes have been reported to be involved in potato resis- tance to bacterial wilt [13]. The introgression of multiple resistance genes from wild Solanum species into S. tuberosum by classical breeding methods is time-consuming, la- borious and may encounter difficulties because of sexual incompatibilities, particularly due to differ- ences in ploidy level or in endosperm balance numbers. Somatic fusion is expected to provide a new possibility for increasing genetic variability, and also a means of transferring desirable agro- nomic traits into potato. The potential use of somatic hybridisation has been demonstrated by the successful introduction of traits such as resis- tance to viruses [14,15] and frost [16] from Solanum bre6idens, resistance to Phytophtora infes- tans and Globodera pallida from Solanum circaei- folium [17], insect resistance from Solanum berthaultii [18], and resistance to bacterial wilt from Solanum commersonii [19], into the cultivated potatoes. In other studies of fusion between potato and S. phureja, partial elimination of chro- mosomes was reported in the resulting somatic hybrids. The nuclear chromosomes of S. phureja were preferentially eliminated [20,21]. So far, no information has been available about evaluation of the somatic hybrid clones for the introgression of resistance against bacterial wilt from S. phureja into potato. In this study, somatic hybridisation between the dihaploid S. tuberosum cv. BF15 and the diploid S. phureja was performed. The somatic hybrids were identified and characterised as well as evalu- ated for resistance to race 1 and race 3 strains of R. solanacearum.

2. Materials and methods