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Section IIIC Vegetable Crops: Solanaceae Improving Crop Resistance to Abiotic Stress, First Edition. Edited by Narendra Tuteja, Sarvajeet Singh Gill, Antonio F. Tiburcio, and Renu Tuteja Ó 2012 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2012 by Wiley-VCH Verlag GmbH & Co. KGaA. j 1067

Improving Crop Resistance to Abiotic Stress (TUTEJA:PLANT STRESS OMICS O-BK) || Tomato: Grafting to Improve Salt Tolerance

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Section IIIC Vegetable Crops: Solanaceae

Improving Crop Resistance to Abiotic Stress, First Edition.Edited by Narendra Tuteja, Sarvajeet Singh Gill, Antonio F. Tiburcio, and Renu Tuteja� 2012 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2012 by Wiley-VCH Verlag GmbH & Co. KGaA.

j 1067

42Tomato: Grafting to Improve Salt TolerancePaloma Sanchez-Bel, Isabel Egea, Francisco B. Flores, and Maria C. Bolarin

Salinity is considered one of the main factors that limit crop productivity, anddevelopment of crop species tolerant to this abiotic stress is vital to meet the growingfood demand through sustainable agriculture. Therefore, the greatest challenge inthe coming years is to increase crop production in such abiotic stress-affected lands asoccur in areas affected by salinity. Tomato is considered one of themost economicallyimportant vegetable crops in the world, but unfortunately it is salt sensitive. Thecommercial success obtained through traditional breeding programs with regard tosalinity tolerance has been very scarce because of the complexity of the plant responseto the stress. One way of avoiding or reducing losses in production caused by salinityin high-yield genotypes would be to graft them onto rootstocks capable of reducingthe effect of external salt on the shoot. This strategy could also provide the plantbreeder with the possibility of combining good shoot characteristics with good rootcharacteristics and of studying the contribution of genes transcribed in the roots totheir performance on the shoot. This chapter gives an overview on the mainphysiological processes involved in the salt tolerance response of grafted tomatoplants and illustrates how grafting can enhance salt tolerance in tomato, determinedby fruit yield, a key agronomic parameter. Moreover, it is important to highlight thatthe salt tolerance conferred by the rootstock to the shoot genotype in terms of fruityield seems to be a heritable trait. However, it is still necessary to conduct a good dealof research work in order to simplify the process of rootstock selection, as differentresultsmay be obtained depending on the shoot and root genotypes, aswell as the saltlevels and exposure times of the grafted plants.

42.1Introduction

Tomato is considered one of the most economically important vegetable crops in theworld. Abiotic stresses, like those caused by soil salinity, have a huge impact ontomato production and mainly affect arid and semiarid regions. Development oftomato plants tolerant to stress is vital to meet the growing food demand through

j 1069

Improving Crop Resistance to Abiotic Stress, First Edition.Edited by Narendra Tuteja, Sarvajeet Singh Gill, Antonio F. Tiburcio, and Renu Tuteja� 2012 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2012 by Wiley-VCH Verlag GmbH & Co. KGaA.

sustainable agriculture. Therefore, the greatest challenge for the coming years will beto increase crop production in abiotic stress-affected lands [1].

In traditional breeding programs, commercial success has been very limited due tothe complexity of the trait: salt tolerance is complex genetically and physiologically [2].Even when halophytic species exist in the gene pool, as it is the case of tomato, thedevelopment of salt-resistant cultivars has been slow [3]. Two major problems areencountered in breeding for salt tolerance: the definition, or selection criteria, for salttolerance and the efficient use of the wild germplasm to increase the salt tolerance ofthe crop [4]. The selection of appropriate genes to obtain salt-tolerant varieties is adifficult task because salt resistance is a complex character controlled by a number ofgenes or groups of genes and involves a number of component traits that are likely tobe quantitative in nature. Thus, in the Solanum genus, the existence of accessions ofhalotolerant wild species (e.g., Solanum pennellii, S. cheesmaniae, and S. pimpinelli-folium), and their sexual compatibility with the cultivated species (S. lycopersicum),should have permitted the genetic dissection of the tolerance character by traditionalmethods. The studies carried out to date have provided valuable information(reviewed by Cuartero et al. [5]). Unfortunately, despite the wealth of genetic variationwithin the pool of tomato wild-related species, it is still not known which are the keygenes determining the high tolerance level in those plants, and it is not possible toconclude that true halotolerant cultivars have been obtained. Moreover, both thedistortion of the segregation, a common fact in interspecific crosses, and thedifficulties inherent to the evaluation of plants under saline conditions have madethe analysis difficult. As a consequence, we still do not know which are the maingenes determining salt tolerance in the wild species of tomato. The problem arisingwhile obtaining salt-tolerant varieties is the choice of the genetic material tointroduce, taking into account that new cultivars bred for salt tolerance not onlyhave to be salt tolerant but also have to achieve the same attributes of productivity andquality as observed in modern cultivars [6, 7]. Given the rapid increase in molecularbiology techniques, a key question is whether such techniques can aid the develop-ment of salt resistance in plants.

The introduction of genes conferring salt tolerance to elite cultivars or elite parentsof current hybrids, by transformation, is a very attractive idea because, hypothetically,susceptible but productive cultivars should be converted into tolerant cultivars whilemaintaining all the very valuable traits present cultivars possess. In recent years,transgenic approaches have been employed to produce plants with enhanced salinitytolerance by overexpression of genes controlling different tolerance-related physi-ological mechanisms [1]. However, given the nature of the genetically complexmechanisms of abiotic stress tolerance and the potential detrimental effects,approaching this strategy with reasonably successful possibilities is extremelydifficult [2, 8]. Therefore, more efforts are needed in developing transgenic tomatowith overexpression/silencing of specific genes in order to evaluate their putativepositive effect in enhancing drought and salt tolerance, with the final goal ofcircumventing the increasing problem of scarcity of water of good quality in tomatocultivation [9].

1070j 42 Tomato: Grafting to Improve Salt Tolerance

But solving a problem as complex as the profitable use of saline water in irrigatedagriculture requiresmore than one strategy. For generating tolerant cultivars, a set ofcultural techniques, each contributing to a certain degree to developing planttolerance to the deleterious effects of salt, need to be assessed to verify the positiveeffects of their application [3]. Some of those strategies, such as the application ofchemical fertilizers at levels somewhat above the optimum for freshwater irrigation,the application on the nutrition solution of chemical adjustment, or leaching salts todeeper soil layers have doubtful compatibility with preservation of the environment,seed priming, or seedling conditioning.

Finally, another possible cultural practice in order to avoid or reduce yield lossescaused by salinity would be to graft cultivars onto rootstocks able to reduce the effectof external salt on the shoot. This strategy of grafting could also provide growers theopportunity to combine the best shoot characters with the best root characters andresearchers the possibility of studying the contribution to the shoot performance ofgenes expressed in the roots and vice versa, and their interaction. In this respect,grafting provides an experimental means to juxtapose diverse genotypes, to test fortransport of hormones, signals, or metabolites [10], and to demonstrate the mobilityof RNAs and proteins through the phloem. It is important to point out that growerswould exploit immediately a technique like this if that allows them to use salineconditions in the culture, while retaining the yield and quality of crop varieties forwhich they already have established markets.

42.2Formation of the Rootstock–Scion Union

Grafting of two plants so that they grow as a single individual is an ancienthorticultural technique that can combine valuable traits of different genotypes.Grafting is an established method of vegetative propagation for many forest treesand iswidely used in horticulture to propagate ornamentals. Grafting is not limited towoody species, but it is also used for some vegetables crops [11, 12]. Among themostgrafted horticultural species are the cucurbits (watermelon, melon, and cucumber)and the Solanaceae (tomato, eggplant, and pepper).

The agricultural use of grafting is considerably restricted to closely related taxabecause of incompatibility. Grafting partnersmay belong to the same species, genus,or family. For successful grafting to take place, the vascular cambium tissues of therootstock and scion plants must be placed physically in contact with each other. Bothtissuesmust be kept alive until the graft has takenplace, usuallywithin a period of fewweeks [13, 14].

Several authors have defined the sequence of structural events during the healingof the graft in herbaceous plants. An overview of this sequencewould be as follows: (i)the scion tissuewithmeristematic activity is placed in intimate contact with rootstocktissue in such a manner that the cambial regions of both are able to interconnectthrough the callus bridge. Thus, new parenchymatous cells proliferate from both

42.2 Formation of the Rootstock–Scion Union j 1071

rootstock and scion producing the callus tissue and filling up the spaces between thetwo components connecting the scion and the rootstock. (ii) New cambial cellsdifferentiate from the newly formed callus, forming a continuous cambial connec-tion between rootstock and scion. Furthermore, prior to the binding of vascularcambium across the callus bridge, initial xylem and phloem may be differentiated.The wound-repair xylem is generally the first differentiated tissue to bridge the graftunion, followed by wound repair phloem. (iii) In the last step of the graft process, thenewly formed cambial layer in the callus bridge begins typical cambial activityforming new vascular tissues. Production of new xylem and phloem thus permits thevascular connection between the scion and the rootstock [13, 15]. Although the timein which each process of grafting occurs depends on various factors such as thegrafted species and variety, many authors observed that the differentiation of callusparenchyma to form new cambial cells begins between days 4 and 8 and is fullydeveloped after 15 days [14, 16].

Observation of the structure of the graft union in tomato showed formation ofxylem and phloem vessels through the scion–rootstock union 8 days after graft-ing [14]. In addition, root hydraulic conductance, L0, indicates that the graft bond isfully functional 8 days after grafting.

Many studies have suggested that peroxidases play a role in lignification [17, 18].Fernandez-Garcia et al. [14] showed that total peroxidase activity increased duringdevelopmentofcontrolandgraftedtomatoplants.However,graftedplantsshowedmoreactivity thancontrols,whichis inaccordancewiththe increased lignificationobservedinthe graft union by histochemical analysis. Moreover, grafted tomato plants showed asignificant increase inH2O2 at day 8 [14]. Lignification is a process that requires H2O2

andcellwallperoxidases tobringaboutpolymerizationof lignin. Inaddition,H2O2mayserveasanimmediatemechanismfordiseaseresistance inresponse topathogensanditcould play an important role in wound response and cell apoptosis [19–21].

Graft incompatibility includes failure to unite into a strong scion–rootstockconnection, failure of the grafted plant to grow in a healthy manner, or prematuredeath following grafting. Physiological incompatibility may be either due to lack ofcellular recognition, wounding responses, and growth regulators or due to incom-patible toxins, but the ultimate biological nature of this grafting incompatibility isnot known [22]. In tomato, our group observed in one graft combination betweentwo cultivars, P73 as scion and Pera as rootstock, that although grafting wassuccessful leaf morphological alterations began to appear after a certain period oftime (between 20–25 days after grafting), which seemed to be associated withhormonal imbalance (Figure 42.1). Thus, Aloni et al. [23] found that the disruptionof rootstock–scion connections in incompatible grafting occurred approximately 25days after grafting and proposed that the main cause of incompatibility is theoccurrence of hormonal imbalance, primarily of auxins and ethylene in the rootsystem following the establishment of grafting connections. Recently, Aloniet al. [13] showed a schematic presentation of a possible mechanism for graftingincompatibility in melon plants, indicating that incompatibility may result frombasipetal auxin transport to the rootstock where it induces ethylene production andoxidative stress. This oxidative stress may be activated also in compatible grafting.

1072j 42 Tomato: Grafting to Improve Salt Tolerance

These authors suggested that exogenous application of indole acetic acid (IAA)transport blockers, abscisic acid (ABA), ethylene antagonist agents, or antioxidantsmay reduce grafting incompatibility by reducing oxidative stress in the root andtherefore enabling its growth.

42.3The Use of Grafting in Tomato

The application of grafting began in the 1920s, initially to limit the effects of soilpathogens such as Fusarium oxysorum [24]. Application and use of grafting in diverseplant species of horticultural importance have risenwith the increaseduse of improvedsoil mix or substrate, farmer�s preferences for better seedlings, efficient managementof nursery systems, lower prices of grafted seedlings, and efficient nationwide delivery,and/or transportation system [25]. This technique has been widely used to enhancenutrient uptake [26, 27], to induce resistance against drought [28, 29], to boostresistance to low and high temperatures [29–31], to bring about resistance againstheavymetal contamination [27, 32], to improve alkalinity tolerance [33], and to increasesynthesis of endogenous hormones [13, 34]. Grafting has also been used as a tool tostudy various aspects of plant biology including apical dominance [35], nodulation [36],flowering [37], dwarfing [38], and characterization of mutants [39].

Inrelationtosalttolerance,itispossibletofindseveralstudiesthatusegraftingasatoolto improve salinity tolerance of tomato either through its direct application or indirectlythrough the detection of QTL in support of conventional breeding [14, 40–47]. These

Figure 42.1 Leaf and apex alterations observed in tomato-grafted plants (cv. P73 used as scion andcv. Pera used as rootstock) (b–e), compared to nongrafted plants of both cultivars that showed noabnormal characteristics on such vegetative organs (a and f).

42.3 The Use of Grafting in Tomato j 1073

studies suggest that grafting provides an alternative way to improve salt tolerance byreducing the ionic stress [41, 43, 44, 48, 49] and by improving the photosynthesisperformanceandtheantioxidantsystems[47].Cultivatedtomatospecies isaglycophyte,thus screening for rootstocks that confer resistance to salinity to the scion is mainlydirected to combinationsof interspecificgrafts. Tomato is compatiblewith awide rangeof genera and species. For example, it has been grafted on potato (S. tuberosum)producing tomato fruits and potato tubers [50]. Although intergeneric, interspecific,and intervarietal combinations are possible, the use of halophytes as rootstocks showmajor problems due to the different sizes and growth rates and the compatibilitybetween the genotypes used. Therefore, the use of interspecific hybrids as rootstocksmaybeabetter strategy to studyand integrate theagronomic,physiological, andgeneticcomponents of salt tolerance conferred by the rootstock to the scion [46]. Furthermore,graftingprovidesadirectwaytotransfersalttolerancetraitsfromthewildspeciesintothedomestic tomato by using recombinant inbred lines (RILs) derived from interspecificcrossesasrootstocks[43].Previousstudieswithtomatosuggestthatgraftingdidimproveplant adaptation to salt stress [40, 41].

Grafting could be a promising tool to raise fruit quality under both nonsaline andsaline conditions [32, 51, 52]. Althoughmore studies are necessary, the results obtainedby our group on this aspect are remarkable. Results obtained with tomato-graftedplants showed that grafting may be an effective agricultural approach to improve fruitquality under both control growth conditions and salinity, but careful screening foroptimal rootstocks is a key question, as the fruit quality of the shoot depends, at leastpartially, on the root system [53]. These results are very interesting, as it is known thatsimultaneous increase inboth fruit yield and soluble solids content, amain fruit qualityparameter in tomato, in commercial tomato cultivars is a difficult task [6]. This is due tothe inverse relationship generally found between both parameters, which seems tobecome stronger under saline conditions [54]. In this study, however, the beneficialeffect induced by the rootstock on the fruit quality of the shoot genotype was notassociated with any negative effect on the fruit yield under saline conditions. But therootstock was even able to induce significant increases in fruit soluble solids contentand titratable acidity not only under saline conditions but also when the grafted plantswere grown under unstressful conditions [53]. These results suggest that graftingmaybe a valid strategy to improve fruit quality.

42.4Physiological Processes Involved in Salt Tolerance of Grafted Tomato Plants

The salinity response of grafted plants may be different depending on the mainpredominant effect induced by salinity as this abiotic stress acts onplants in twoways:high concentrations of salts in soil make it harder for roots to absorb water, resultingin an osmotic stress the main symptom of which is dehydration; and high con-centrations of salts within the plant can be toxic, causing an ionic stress the mainsymptom of which is leaf chlorosis and swelling due to the excess of sodium ion.Moreover, if the plant is not able to reach homeostasis and adapt to these salinity

1074j 42 Tomato: Grafting to Improve Salt Tolerance

conditions, oxidative stress arises as a result of the excessive production of reactiveoxygen species in cells, leading to senescence and death. In a recent review of the roleof grafting in plant crops under saline conditions, it was shown that grafted plantsdeploy numerous and diverse physiological and biochemicalmechanisms in order tocope with salt stress [33].

The first phase of the plant growth response results from the effect of salt outsidethe plant. The immediate effect on the plant of the high concentrations of salinesolutes in soil is the loss of turgor, and the main plant morphological processdepending on turgor is cellular expansion. Therefore, leaf expansion and rootelongation are the morphological processes that are more sensitive to the osmoticstress caused by salinity in plants. The cellular and metabolic processes involved arecommon to drought-affected plants. To cope with this situation, one key mechanismdisplayed by stressed plants with the aim of restoring thewater uptake and cell turgoris osmotic adjustment. The plant needs to accumulate solutes tomaintain cell volumeand turgor, so the response to turgor reduction is osmotic adjustment, a majorcomponent of the response to salt stress in affected plants. The main solutescontributing to osmotic adjustment are inorganic solutes, which are taken up fromthe substrate and transported to the shoot, and organic solutes, which are synthesizedby the plant. Naþ and Cl� are energetically efficient osmolytes for osmotic adjust-ment, but they must be compartmentalized into the vacuole to minimize ioncytotoxicity. Within the cytoplasm, osmotic adjustment is achieved by accumulationof the so-called compatible osmolytes. Some compatible osmolytes are essentialelemental ions, such as Kþ , but the majority of these are organic solutes, especiallysugars (mainly fructose and glucose), organic acids, and other metabolites such astrehalose, proline, inositol, glycine, and betaine that have an osmoprotector role,too [55–57].

Since the long-term damage caused in cultivated tomato by salinity is ionic toxicitydue to the excessive accumulation of Naþ and Cl� in leaves [3], it is a reasonablesupposition that useful rootstocks should be able to reduce the uptake and transportrates of saline ions to the shoot (a trait often termed �salt exclusion�). The enhancedsalt tolerance of grafted plants has often been associated with lower Naþ and/or Cl�

content in the shoot [33], as has been observed in tomato [41, 43]. This indicates thattolerance induced by rootstock is related to the ionic stress rather than to the osmoticstress. However, in contrast to the cultivated species S. lycopersicum that generallyexcludes toxic ions [44, 58], most wild accessions seem to have an ionic inclusionmechanism because they accumulate higher concentrations of Naþ and Cl� in theirleaves [59]. It is necessary to keep in mind that the use of organic solutes for osmoticadjustment is energetically much more expensive than the use of saline ionsproceeding from the substrate [60]. The ATP requirement for the biosynthesisand/or transport for accumulation of solutes in leaves was assessed by Ravens [61]at 3.5 units for Naþ , 34 formannitol, 41 for proline, 50 for glycine-betaine, and about52 for sucrose. In this respect, salt tolerance may always be associated not only withlow Naþ concentration in the leaves but also with the capability of the tissue totolerateNaþ .While themost tolerant genotypes ofmany species are thosewith betterabilities to prevent excessive ion accumulation, the leaves of halophytes do contain

42.4 Physiological Processes Involved in Salt Tolerance of Grafted Tomato Plants j 1075

high salt concentrations [62], which are necessary to adjust the leaf water relation-shipswith low external potentials, and halophyte plants use the cheapest solutes froman energetic point of view [63]. Tissue tolerance toNaþ involves the storage ofNaþ invacuoles, to avoid its accumulation in cytosol, preventing alterations in the activitiesof cytosolic enzymes [64]. Electrochemical Hþ gradients, generated by Hþ -pumpslocated at the plasma membrane (Hþ -ATPase) and the tonoplast (Hþ -ATPase,Hþ -PPase), provide the energy used by the plasmamembrane- and tonoplast-boundNaþ /Hþ antiporters to couple the passivemovement ofHþ to the activemovementof Naþ out of the cell and into the vacuole, respectively [65]. Such a situation seemsalso to occur in grafted tomato plants when grown at low-mid levels of salt, as thehigher the fruit yield, the higher the contribution of inorganic solutes, including thesaline ions, to the osmotic potential [43, 44]. Then, breeding for Naþ accumulation,rather than exclusion, could be a more effective strategy for improving salt toleranceof conventional crop plants.

In addition to its known components of osmotic effect and ion toxicity, salt stress ismanifested by an oxidative stress, all of which contribute to its deleterious effects [66,67].When plants are not able to adapt or to tolerate salinity conditions, the availabilityof CO2 within the leaf is restricted and fixation of it is inhibited. This short supply ofCO2 is due to the high density of closed stomata of stressed plants. Plants tend to closestomas in response to drought and salt stress in order to avoid losing water byevaporation. The alteration inCO2fixation induces the impairment of ATP synthesis.Under these conditions, the concentration of the final electron acceptor NADPþ isgenerally very low, which leads to an excess of excitation energy in the photosystems.High-energy states may be dissipated by either nonphotochemical quenching (e.g.,xanthophyll cycle) or alternative processes, such as photorespiration [68]. If notdissipated, electrons accumulate in the electron transport chain and are transferred tooxygen (Mehler reaction), generating reactive oxygen species (ROS). Because of theirhigh reactive potential, ROS are harmful tomany cellular componentswhen a certainthreshold is exceeded (e.g., proteins, DNA, and lipids). This abnormal accumulationof ROS constitutes the starting point of oxidative stress. With regard to photosyn-thesis, under stressful conditions the electronic flow ceases and as a consequencephotoinhibition is favored, but ROS accumulation also induces the photooxidation ofphotosystems I and II [69]. Plants have defensive mechanisms and utilize severalstrategies to overcome salt-mediated oxidative stress. Plant enzymatic defensesagainst oxidative stress include antioxidant enzymes promoting ROS scavengingsuch as catalases, superoxide dismutases, and peroxidases participating in theglutathione and ascorbate cycles [66, 70]. The biochemical defense system alsoincludes nonenzymatic components such as carotenoids, ascorbate, glutathione, andtocopherols. A correlation between antioxidant capacity and salinity tolerance hasbeen reported in tomato through comparative studies between cultivated and wildspecies [67, 71, 72]. According to Colla et al. [33], antioxidants can be used asmarkersof salinity tolerance in grafted vegetables. In tomato, the alleviation of oxidativedamage in grafted tomato plants under NaCl stress originated from the increase inactivities of catalases and enzymes involved in the ascorbate–glutathione cycle suchas ascorbate peroxidase, dehydroascorbic reductase, and glutathione reductase [47].

1076j 42 Tomato: Grafting to Improve Salt Tolerance

Therefore, an efficient antioxidant system is an advantage for enhancing salttolerance of grafted plants. Nevertheless, in contrast to the negative significancegiven to the increased ROS production, implying a harmful process, recent studieshave shown that ROS play a key role in plants as signal transduction moleculesinvolved in mediating adaptive responses to abiotic stress, suggesting that ROSsignaling is an integral part of the response of plants to salinity [73, 74].

42.5The Rootstock Improves Salinity Tolerance at Agronomical Level

In most studies on the role of grafting in salinity tolerance of crop plants, the salinityresponses of the grafted plants have been studiedmainly on the basis of plant growthbut not on the basis of fruit yield [29]. Only in some cases, such as melon andcucumber, there are studies showing the salinity effect in grafted plants on fruityield [33].However, to our knowledge, the only results on the grafting effect in salinitytolerance of tomato on the basis of fruit yield have been obtained by our researchgroup [40, 43, 44]. The most important question to elucidate is whether fruit yieldmay be increased in grafted plants grown under salinity. For example, when acommercial tomato hybrid like Jaguar was grafted onto the roots of several tomatogenotypes, the positive effect of grafting on the fruit yield was found when electricalconductivity (EC) levels in the irrigationwater increased (because of its increasing saltcontents), with fruit yield significantly higher in all grafted combinations than that ofthe self-grafted cultivar (Figure 42.2). It is interesting to note that the important effectinduced by some rootstocks on the salt tolerance of the shoot genotype wasdetermined by means of fruit yield, as occurred in the plants grafted onto two ofthe four tomato cultivars assayed as rootstocks, Radja and Pera. Thus, while the self-grafted commercial hybrid plants reduced their fruit yield by 50% at 7.5 dS m�1, thegraft combinations onto Radja and Pera were able to maintain their yields around90%at this salt level, compared to the plants grown at 2.5 dSm�1 (control conditions).

As we said elsewhere, although intergeneric, interspecific, and intervarietal com-binations in grafting are possible, the use of halophytes as rootstocks show majorproblemsdue to the different sizes and growth rates and the compatibility between thegenotypes used. Therefore, the use of interspecific hybrids as rootstocks may be abetter strategy for investigating the agronomic, physiological, and genetic componentsof salt tolerance conferred by the rootstock to the scion. Different studies have beencarried out by using a commercial tomato hybrid S. lycopersicum cv. Boludo as scionand as rootstock twoRIL populations developed from a cross between a genotype of S.lycopersicum var. cerasiforme, as female parent, and as male parents two salt-tolerantlines belonging to the wild tomato species S. pimpinellifolium (123 lines) and S.cheesmaniae (100 lines). In these studies, our group corroborated the positive effect ofthe rootstockon fruit yield, as inboth populations therewere rootstock lines that raisedthe fruit yield of the commercial hybrid under saline conditions [46]. Taken together,the set of results obtainedbyour researchwork anddiscussed in this sectionhighlightsthe effectiveness of grafting to enhance fruit yield in tomato.

42.5 The Rootstock Improves Salinity Tolerance at Agronomical Level j 1077

42.6Genetic Basis of Salinity-Tolerant Rootstocks

The grafting strategy could also provide the plant breeder with the possibility ofcombining good shoot characters with good root characters and studying thecontribution of genes transcribed in the roots to their performance in the shoot.An interesting approach to clarify how genes govern the tolerance to salt is thecombination of segregation analysis of markers and phenotyping of lines to detectQTL. If it were possible to reveal molecular markers tightly linked to the genesgoverning salt tolerance, their favorable alleles could be selected in segregatingpopulations by those markers and eventually incorporated into salt-tolerantcultivars. These markers closely linked to QTL alleles may reveal masked allelesand facilitate the introduction of genetic material without the disadvantagesassociated with traditional methods. The prospects of modifying a phenotypethrough conventional breeding have more possibilities of succeeding if it isincorporated with one or few defined regions of crucial importance than ifgenerating a desired phenotype depends upon changes in a large number ofgenes, each with a small effect and scattered all over the genome. The identifi-cation of QTL has, therefore, practical importance to attempts to enhance stresstolerance.

CE (dS/m)R

elat

ive

frui

t yie

ld

JRVPC

(b)

(a)

30

40

50

60

70

80

90

100

2.5 5.0 7.5 10.0

Figure 42.2 Tomato plants grafted ontodifferent rootstocks when grafting wasestablished (a) and at the end of growth cycle(b). Relative fruit yield comparison between self-grafted plants of a commercial tomato hybrid(cv. Jaguar, J) and Jaguar grafted onto differentcultivars used as rootstocks (R, cv. Radja; V cv.Volgogradskij; P, cv. Pera; and C, commercial

rootstock) at increasing levels of electricalconductivity of the irrigation solution. At the firstlevel, the EC resulted from the sum of irrigationwater EC (1.0 dS m�1) plus the nutrientsolution; in the following levels (5.0, 7.5, and10.0 dS m�1), the EC was increased because ofthe addition of 25, 50, and 75mM NaCl,respectively, to the irrigation solution.

1078j 42 Tomato: Grafting to Improve Salt Tolerance

Efforts on salt tolerance dissection using tomato experimental populations havebeen made taking into account different kinds of traits; however, in the case of cropplants, it is ultimately the yield under specific field conditions that will determinewhether or not a gene or combination of genes (or QTL) is of agronomic importance.Experimental populations and the assays performed with them using grafting havealready been discussed in the previous section, when these were used to test graftedplants with tolerance to salinity [46]. As already mentioned, salt tolerance in terms offruit yield was studied by QTL analysis using the same RIL populations of F9 linesdeveloped from a salt-sensitive genotype ofS. lycopersicum var. cerasiforme, as femaleparent, and two salt-tolerant lines, as male parents, from S. pimpinellifolium and S.cheesmaniae [4]. Contrary to the expected, it was found that the wild allele (i.e., fromthe wild salt-tolerant genotype) was advantageous only at one total fruit yield QTL onchromosome 10 (tw10.1, near the salt-specific fn10.1). In fact, it was found that theadvantageous allele at all fruit yield QTL came from the cultivated, salt-sensitivespecies. Therefore, other approaches in raising tolerance to salt using wild germ-plasm need to be considered.

Next, the rootstock effect on the fruit yield of a grafted tomato variety wasgenetically analyzed under salinity using as rootstock the previous RIL popula-tions [46]. It is shown that the fruit yield increase induced by rootstock under salinityis a heritable trait governed by at least eight QTL. The most relevant component wasthe number of fruits. Thus, most of the detected QTL involved in salinity tolerancecorrespond to this component. In general, QTL genetic effects have a rather lowdegree, with contributions from 8.5 to 15.9% at most, and the advantageous allelecomes from the wild, salt-tolerant species. To our knowledge, this is the first QTLanalysis of the rootstock effect on the scion fruit yield. It is shown that the salttolerance alleles from wild species can be more easily used to improve salt toleranceof the cultivated species through their utilization in tomato rootstock breedingprograms [46].

42.7Conclusions and Future Perspectives

There are rootstocks able to induce salt tolerance in tomato-grafted plants, anobservationmade on the basis of the determination of fruit yield, a most importantagronomical trait. As discussed in this chapter, grafting practices offer the possi-bility of avoiding or reducing yield losses caused by salinity by means of graftingcultivars onto rootstocks able to reduce the effect of salt on the shoot. What is more,at the same time, is that it induces the development of salt tolerance to be conferredby a suitable rootstock and it allows to retain desired features of the shoot, such asfruit production and quality, which are essential parameters from the agrofoodindustry perspective. Themain challenge here is the selection of the right rootstock,that is, the one that counteracts the negative effects of salt on the scion withoutaffecting the levels of production and quality of the shoot. However, in order tosimplify the process of rootstock selection, it would be very interesting to identify

42.7 Conclusions and Future Perspectives j 1079

the main rootstock characteristics able to reduce the negative effects of salinity onthe shoot genotype in shorter time, in order to avoid crop losses in the grafted plantsat the initial period of stress. Moreover, screening for a trait associated with aspecific mechanism of the plant response to salinity is preferable to the screeningfor salt tolerance itself, as measuring the effect of salt on crop yield of a largenumber of lines is very difficult and complex. However, it is very important to takeinto account that the salinity response of tomato varies not only with genotype butalso with salt levels and exposure times. Probably, the different and even contra-dictory results found in the literature may be, at least partially, due to either thestress level or the exposure time applied was not sufficient to show net differencesamong distinct grafted plants. Finally, the importance of osmotic tolerance mech-anism to salt tolerance must be considered, which has not received as muchattention as the ion exclusionmechanism, as it could be equally crucial in providingsalt tolerance to tomato plants. From this perspective, more studies are required inorder to arrive at the selection of the rootstock traits able to induce salt tolerance inthe osmotic phase of this abiotic stress.

Acknowledgments

This work was supported by the Council of Science and Technology from the Regionof Murcia (Spain) (Fundación SENECA) [grant 04553/GERM/06].

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