Plant Cell, Tissue and Organ Culture 55: 1–8, 1999. © 1999 Kluwer Academic Publishers. Printed in the Netherlands. 1 In vitro recurrent selection of potato: production and characterization of salt tolerant cell lines and plants S.J. Ochatt 1 , P.L. Marconi 2,∗ , S. Radice 2 , P.A. Arnozis 2 & O.H. Caso 2 1 INRA, URGAP, Lab Physiologie & Culture in Vitro, BV 1540, 21034 Dijon Cedex, France. 2 Centro de Eco- fisiolog ´ ia Vegetal (CONICET), Serrano 669, 1414 Buenos Aires, Argentina. ( ∗ requests for offprints Tel./Fax: 54-1-856-7110) Received 13 September 1998; accepted in revised form 15 January 1999 Key words: Na + tolerance, plant regeneration, salt stress, Solanum tuberosum, somaclonal variation, RAPDs Abstract A stable salt-tolerant potato cell line, able to grow on media containing 60–450 mM NaCl (i.e. low to high salinity) was selected. Callus grown on 120 or 150 mM NaCl showed higher fresh weights than the rest of the treatments. Replacing NaCl by KCl or Na 2 SO 4 showed that reductions in fresh weight were mainly due to the presence of Na + ions. When PEG 6000 was added to the medium instead of salt, the salt tolerant cell lines were unable to overcome the PEG-induced water stress. Whole plants, regenerated from salt tolerant callus, exhibited salt stress tolerance as evidenced by their higher fresh and dry weights when watered with 90 mM NaCl, and they also produced more tubers per plant under salt stress. Salt-tolerant plants differed phenotypically from control plants both in terms of leaf shape, tuber flesh and skin colour, which was reddish. In addition, DNA fingerprinting by RAPDs, with 70 different primers, confirmed that the salt tolerant regenerants also differed genotypically from the control, salt sensitive Kennebec potato plants from which they had been selected. Abbreviations: BM – basal medium; DW – dry weight; FW – fresh weight; MS – Murashige and Skoog (1962); NAA – α-naphthaleneacetic acid; PCR – polymerase chain reaction; PEG 6000 – polyethylene glycol (average MW = 6000, Sigma); ϕw – water potential; RAPD – Randomly amplified polymorphic DNA Introduction The induction of salt-tolerance in hitherto sensitive plant genotypes has been studied for many species (Epstein and Rains, 1987; Maas, 1985; Tal, 1996). The mechanisms responsible for in vitro adaptation, and tolerance and/or resistance of plant cells to salt have also been examined (Bray, 1993; Hasegawa et al., 1990; Hurkman, 1992). Salinity is regarded as one of the most important stress factors in agricul- ture (Caldiz, 1994) but the number of in vitro studies that have actually resulted in the production of stably salt-tolerant plants is scanty (Tal, 1996). Solanum tuberosum L. has been classified as moderately salt-tolerant to moderately salt-sensitive (Maas, 1985). It is an autotetraploid (2n =4x = 48) with tetrasomic inheritance, and a species where com- mercial cultivars are often sterile. Potato improvement by traditional breeding methods is slow and unpre- dictible (Bajaj, 1987). Alternative strategies, espe- cially those based on biotechnology, offer more rapid means of improving existing cultivars. In potato, un- fortunately, although salt tolerance is known to be a polygenic trait, the mechanism responsible for salt tolerance has not been studied in detail (Haro et al., 1991). Tissue culture techniques that make use of somaclonal variation (Lee and Phillips, 1988) do not require previous knowledge of the genetic basis of res- istance, which facilitates in vitro selection of potato cells and tissues (Bajaj, 1987; Tal, 1996). The value of this approach for generating variation has already been demonstrated (Bajaj, 1987; Karp, 1991). How- ever, the acquisition of tolerance to salt has seldom been reported for potato (Sabbah and Tal, 1990; van