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Salt tolerance varies between species and genotypes of plants, but evaluation of these differences is cumbersome, because whole plants that are highly complex systems show a variety of responses depending on the applied methodology. However, focusing on plant roots, which are in direct contact with the soil, could offer a simpler and more efficient model for analyzing salt stress tolerance in different species. This study explores whether root growth under salt stress is associated with genotypic differences in Prunus species with different degrees of salt tolerance. Excised root cultures were grown in vitro under increasing salt concentrations (0, 20, 60, and 180 mM NaCl). Root tips taken from in vitro-rooted shoots of Prunus species with different salt tolerance were measured after 3 weeks of culture in a shaker, and changes in their anatomy were examined. Both growth and starch content of in vitro root cultures were affected by salt concentration. Root length increments were related to salt stress tolerance at 60 mM NaCl, in which significant differences were also found between species. A significant inverse correlation was found between salt tolerance and starch accumulation in the maturation zone of root tips. Genotypic differences were observed in agreement with species’ salt stress tolerance in vivo. These results suggest the use of excised root cultures for rapid, early detection of salt stress tolerance in plants. Chemical names: sodium chloride (NaCl). Salinity is a problem increasing facing agriculture, especially in irrigated lands located in semiarid zones. These agricultural zones account for 100 to 110 million hectares, of which 20 to 30 million hectares are seriously damaged by salt accumulation and an estimated 0.25 to 0.5 million hectares are lost from production every year as a result of salt accumulation (FAO, 2002). Only 1% of the world’s flora is considered halophyte, and the most important agricultural crops are not among the most tolerant species, making salinity a serious human as well as ecological concern (Byrt and Munns, 2008). Hence, efforts to increase salt tolerance of crop plants could potentially improve crop yield and support agriculture on marginal lands (Türkan and Demiral, 2009). The response of plants to saline environments has generally been determined by measuring emergence, survival, growth, phenology, maturity, aboveground biomass, and commodity yields. However, the responses of crops to salinity often vary with different plant growth stage, across varieties, and for each measure of plant growth, development, and product yield (Steppuhn and Wall, 1999). Discrepancies arise from the fact that whole plants, as highly complex systems, show a variety of responses depending on the methodologies used in evaluation: different growth stage, evaluation criteria, or salt stress application. Focusing on plant roots, which are in direct contact with the soil, could offer a simpler, more direct, and more efficient model for analyzing salt stress tolerance in different species. Fruit tree rootstock selection would benefit from research strategies that shorten the long times required for growing woody plants. Selection studies with fruit trees have been accelerated in recent years with the use of potting and hydroponic techniques, thus reducing the duration needed for field studies. However, generating a sufficient number of clonal plants and measuring the effects of applied stresses still require a great deal of time and effort. In vitro shoot cultures of cherry (Erturk et al., 2007), peach, and the almond · peach hybrid GF677 (Biricolti and Pucci, 1995) have proven to be applicable for the study of salt stress on plant tissues. In vitro studies represent a practicable strategy for selection studies, because promising results have been achieved in the screening of different genotypes of grapevine (Troncoso et al., 1999) and mulberry rooted shoots (Vijayan et al., 2003), in which the salinity tolerance of selected genotypes in vitro were correlated to those ex vitro. Recently, an in vitro approach was used for the selection of mung bean and tomato plantlets regenerated from cotyledons under salt stress (Hassan et al., 2008). It is clear that different genetically determined mechanisms for salt tolerance exist, but these remain largely unknown. Comparisons between species that are genetically closely related but vary in terms of salt tolerance would speed the progress of understanding the mechanisms involved in resistance of salinity, as suggested by Byrt and Munns (2008). This is an objective of the present study, which examines different degrees of salt stress tolerance in the related species of the genus Prunus. Abiotic stress tolerance is an important trait for fruit tree rootstock selection. Although resistant to important abiotic stresses such as drought or alkalinity, many Prunus species are included in the group of saltsensitive species (Day, 1953; Kotuby-Amacher et al., 2000). Nevertheless, Prunus species display different degrees of tolerance to salt stress. Past studies of different Prunus species have allowed us to estimate their relative salt tolerance with plum type species being among the more tolerant and cherry type species among the least tolerant. Specifically, local selections of P. insititia (‘‘Pollizo’’) in the southeast of Spain grew better than apricot seedlings or Myrobalan (P. cerasifera) in heavy and saline soils (Egea, 1970). Mexican plum (P. mexicana) and the Titan almond · Nemaguard hybrid showed more relative tolerance than Nemaguard or Nemared, and these were more tolerant than Myrobalan or bitter almond when cultured in sand cultures watered with saline solutions (Ottman and Byrne, 1988). The almond · peach hybrid GF677 showed an intermediate salt tolerance compared with the relatively more tolerant GF655/ 2 (P. insititia) and the less tolerant Myrobalan hybrid MrS. 2/5 or to peach seedlings (Massai and Gucci, 1998). These studies roughly agree with that of Kotuby-Amacher et al. (2000), which ranked different species according to the salinity (expressed as conductivity) needed to reduce yield by 50%. This work positioned cherry species (sweet and tart) at the more sensitive end with only 3.1 dS m, whereas plum needed 4.3 dS m. Rootstocks can play an important role in salt tolerance; thus, the use of the adequate rootstock allowed Japanese plums to withstand low salt levels when grafted onto Marianna 2624 (P. cerasifera · munsoniana) (Ziska et al., 1991) and loquat (Eriobotrya japonica) was unaffected under salt stress when grafted on Angers rootstock (Cydonia oblonga) (López-Gómez et al., 2007). Here, we propose a novel approach using excised roots as a simplified experimental model (Marı́n and Marı́n, 1998), because roots are the plant organs directly in contact with soil. Instead of studying the effect of salt stress in whole plants or in plant parts distant from the origin of salt source, we examined the responses of isolated roots to salt stress in vitro in controlled culture conditions without the influence of substrates and microorganisms. Excised roots have been used in the past in short-duration (few hours) experiments for the study of ion uptake mechanisms of Received for publication 9 Sept. 2010. Accepted for publication 28 Oct. 2010. This work was partially supported by INIAFEDER projects RF2008-00029-C02-01 and RF200800028-C02-02 and by the Grupo de Excelencia A-43 (Gobierno de Aragón). We thank Marı́a Herrero for her critical review of the manuscript. To whom reprint requests should be addressed; e-mail andreu@eead.csic.es. 80 HORTSCIENCE VOL. 46(1) JANUARY 2011 woody plants from saline solutions (Altman and Mendel 1973). Besides, the use of longduration (3 weeks) aseptic cultures allowed us to avoid the possible effects of the rhizosphere (Yang et al., 2009). A simple model, instead of studying whole plants, can yield results more quickly, which would shorten the selection processes. In vitro root cultures either attached to or detached from plants have previously shown a certain correlation to whole organism salt stress tolerance (Prakash and Widholm, 1993; Vijayan et al., 2003), in addition, root cultures showed an accumulation of proline as a response to salt stress similar to that of shoot cultures in vitro (Marı́n et al., 2009; Sotiropoulos, 2007). However, more in-depth studies of the root response to salt stress are important, mainly in fruit tree species, where the plant characteristics make these studies difficult. Besides root growth, we studied the starch content of the root tissues, because starch formation and accumulation in vitro were related in tobacco to the metabolic activity of the callus tissue (Thorpe et al., 1986), and the metabolic activity could be affected by salt, as recently reported in tomato, in which salinity stress enhanced carbohydrate accumulation as starch during the early development stages of tomato fruits (Yin et al., 2010). Both root growth and starch content are parameters relatively easy and quick to determine, allowing a quick test of genotypes. In this work, the effect of increasing salt concentrations on excised root cultures of different Prunus rootstocks is studied to determine the validity of the hypothesis that root responses to salinity are related to those of whole plants and then to confirm this technique as an early selection method. A patent application has been filed to protect the procedure (Spanish application P200803727). Materials and Methods Plant material. Following already published data on salt stress tolerance in Prunus (see previously for references), we have chosen different Prunus genotypes from groups that cover a relatively broad range: ‘Adesoto 101’ (P. insititia L.) and ‘Marianna 2624’ (P. cerasifera Ehrh. ·munsoniana Wight & Hedr.) from the plum-type group that was more tolerant; ‘Masto de Montañana’ and CAB 6P (P. cerasus L.) cherry rootstocks that were less tolerant;

Early Detection of Salt Stress Tolerance of Prunus Rootstocks by Excised Root Culture