rajinder kumar jain navinder saini and randhir...

Indian Journal of BiotechnologyVol 2, January 2003, pp 121-137

Rajinder Kumar Jain 1, Navinder Saini t, Sunita Jain2 and Randhir Singh2*IDepartment of Biotechnology and Molecular Biology, 2Department of Biochemistry, CCS Haryana Agricultural University.

Hisar 125 004, India

Salinity is one of the most important abiotic stresses for agricultural crops. High concentrations of salts causehyperosmotic and ionic stresses, which, in turn, may generate secondary stresses such as oxidative stress, etc. Thecomplexity and polygenic nature of salt tolerance trait has seriously limited the efforts to develop salt-tolerant cropvarieties. This paper reviews new molecular strategies that have been or can be used for the molecular dissection ofplant responses to salt stress, discovery of novel structural and regulatory genes involved in stress adaptation, andtransgenic and molecular marker strategies used for engineering salt tolerance in plants. Application of noveltechniques such as genome sequencing, high-throughput analysis of genomic-scale expressed sequence tags (ESTs),DNA chips/cDNA microarray analyses, targeted or random mutagenesis, knockouts, molecular mapping and gain-of-function or mutant complementation, is expected to accelerate the discovery of the new genes involved in stressadaptation as well as improve understanding of stress biology. A number of stress-related genes have beencharacterized including the ones that encode for important enzymes or a biochemical pathway, participate insignaling pathways or act as transcriptional regulators for coordinated regulation of stress related genes. Some ofthese genes have been successfully transferred in model plant species including Arabidopsis, rice and tobacco, and amarginal to significant improvement in salt-tolerance has been reported. In addition, molecular markers can be usedfor linkage mapping of genes/QTLs for salinity tolerance trait, marker-assisted transfer an~ pyramiding of suchQTLs into agronomically desirable genotypes and/or for map-based cloning of genes. Application of transgenic andmolecular marker research coupled with rapid gene discovery via functional genomic research in plants shallprovide effective means for designing salt-tolerant crops.

Keywords: Salt-tolerance, stress genomics, genetic engineering, transformation, molecular markers, marker-assisted selection

IntroductionSoil salinity is a major abiotic factor that limits

plant growth and productivity (Boyer, 1982; Maas,1986). About 20% of the world's cultivated land and50% of all irrigated lands are affected by salinity(Rhoades & Loveday, 1990). Despite literaturegrowth on plant salt tolerance in last five decades,practical progress in breeding for salt tolerance hasnot been significant. Salt tolerance is one of the least

*Author for correspondence:Tel/Fax: 91-1662-233884E-mail: [email protected]:ABA: Abscisic acid; AFLP: Amplified fragment lengthpolymorphism; HAC: Bacterial artificial chromosome; DHLs: Di-haploid lines; ESTs: Expressed sequence tags; MAS: Marker-assisted selection; OA: Osmotic adjustment; ORF: Open readingframe; QTLs: Quantitative trait loci; RAPD: Random amplifiedpolymorphic DNA; RFLP: Restriction fragment lengthpolymorphism; RILs: Recombinant inbred lines; ROS: Reactiveoxygen species; SOS: Salt overly sensitive; SSR: Simple sequencerepeat; YAC: Yeast artificial chromosome.

understood genetic traits and is considered intractable.Traditional breeding efforts made to introgress suchcomplex traits from related salt-tolerant germplasmhave met with limited success and only a few stress-tolerant plant varieties have been developed andreleased for commercial cultivation (Ashraf, 1994;Flowers & Yeo, 1995). Some 30 cultivars in 12different plant species have been released for salttolerance (Flowers et ai, 2000, Flowers & Yeo, 1995).The breeding for salt tolerance is limited by thecomplex polygenic nature of the salt tolerance trait,insufficient knowledge about the genetics of tolerancecomponents, little or no correlation among toleranceat different developmental stages, lack of efficient andeconomic selection techniques, low genetic varianceof yield and yield components, and largeenvironmental effects (Ashraf, 1994; Bohnert ef ai,1995; Chowdhury et aI, 1993; Frova et ai, 1999;Munns, 2002; Yeo, 1998). The complexity of the taskcombined with a lack of real urgency lay behind thelow success rate. Plant genetic improvement for salt

mailto:[email protected]

122 INDIAN J BIOTECHNOL, JANUARY 2003

tolerance requires the rigorous application ofmolecular biology and genomics to carry out themolecular dissection of cellular responses to saltstress, and various salt-tolerance mechanismsoperating In the available germplasm beforeattempting any crop improvement programs(Cushman & Bohnert, 2000; Flowers et al, 1997,2000; Hasegawa et al, 2000; Yeo, 1998; Zhu et al,1997). Besides improving the salt tolerance of variouscrops, research is also needed for improvingunderstanding of stress biology and other subjectsranging from gene regulation, signal transduction toion transport, and mineral nutrition. Although, themolecular techniques are being applied widely tocharacteristics attributable to single genes, they havenot been used much to tackle the problem of salinitytolerance, a quantitative trait. In this mini-review, theauthors have discussed the utility of transgenic,genomic and molecular marker research in improvingunderstanding of existing mechanisms of salt-tolerance and for designing the salt-tolerant crops.

Some Basic Aspects of Salt ToleranceHigh concentration of salts in soil results in ion

imbalance/toxicity and hyperosmotic stress in plants,which, in turn, leads to oxidative damage, growtharrest and programmed cell death. Salt tolerancedeterminants in plants could be broadly grouped intwo categories:

(a) The effectors that directly modulate stressetiology or attenuate stress effects, e.g. enzymesinvolved in rate-limiting steps of ion homeostasis,osmotic homeostasis, ROS scavenging, etc.(Bartels, 2001; Hasegawa et al, 2000; Munns,2002; Nuccio et al, 1999; Zhu et ai, 1997; Zhu,2001). For salt tolerance, both ionic and osmotichomoeostasis must be restored. Since Na+ inhibitsmany enzymes, there should be some mechanismsto prevent their accumulation in cytoplasm ororganelles at higher levels by theircompartmentation into vacuoles, by restrictedNa+ influx into plants cells and/or by Na+exclusion (Niu et ai, 1995; Serrano et al, 1999). ASOS pathway has been suggested for ionhomeostasis (Gong et al, 2001; Liu et al, 2000;Qiu et al, 2002; Shi et al, 2000; Wu et al, 1996;Zhu et al, 1998b). The high-affinity K+transporter, HKTl, and low-affinity cationtransporter, ECl l, from wheat and non-selectivecation channels may also mediate Na+ influxinto plant cells (Amtmann & Sanders 1999; Rubio

et al, 1995; Rus et al, 2001; Schactman et ai,1997). Another class of cation transporters,HALl, HAL2, HAL3, and calcineurin (a Ca"+-activated protein phosphatase) has been identifiedin yeast, which modulates K+ and Na+ transport(Serrano & Gaxiola, 1994; Cervera et al, 2000).Certain genes such as tonoplast Na+/H+ anti portergene, AtNHXl (Gaxiola et al, 1999) and vacuolartype H+-ATPase, V-ATPase (Golldack & Dietz,2001), which have a function incompartmentalizing Na+ into the vacuole havebeen identified in Arabidopsis thaliana andMesembryanthemum crystallinum (common iceplant) respectively. The ions stored within thevacuole, in turn, act as an osmoticum to maintainwater flow into the cell (Glenn et al, 1999). Na+efflux could be another mechanism to keep Na+ ata lower level in the cytoplasm. Unlike animal,fungal or algal cells, plant cells do not appear tocontain Na+K+· or Na+ ATPases (Niu et al, 1995).In plants, proton motive force created by H+-ATPases would drive Na" efflux from plant cellsthrough plasma membrane Na+/H+ anti porters(Schachtman & Liu, 1999). Several newtransporters and channels for regulation of iontransport in plants have recently been reported(Kim et al, 1998; Liu & Zhu, 1997; Liu et ai,2000; Rus et al, 2001; Shi et al, 2000; Zhang etal, 1999b; Zhu et al, 1998a; Zhu, 2000).

Plants accumulate various compatibleosmolytes in the cytosol, thus lowering theosmotic potential to sustain water absorption fromsaline soil solutions. A mitogen activated-proteinkinase (MAPK) cascade has been proposed tomediate osmotic homoeostasis (Kovtun et al,2000; Zhu et al, 1997; Zhu, 2001). The ionic andosmotic stresses result in generation of ROS,which cause the oxidative damage. A number ofstress proteins and ROS scavenging enzymeshave been proposed to function in detoxificationor alleviation of damages (Bartels, 2001; Allen,1995). Some of the organic osmolytes have alsobeen shown to help in protecting cellularstructures by detoxifying ROS. Water channelproteins might be involved in controlling thewater flux across cellular membranes under saltstress (Chrispeels et al, 1999).

(b) There could be a large number of regulatorymolecules that are involved in stress perception.signal transduction, regulation or modulation ofthe function of effectors, e.g. transcription factors

JAIN et a/: DEVELOPING SALT TOLERANT CROPS

and signal pathway intermediates (Hasegawa etal, 2000, Winicov, 1998; Yamaguchi-Shinozaki& Shinozaki, 1999). Basic Leu zipper motifs,MYB and MYC, and zinc finger transcriptionfactors, including rd22BPI (MYC), AtMYB2(MYB), DREBIA and DREBIA2A (AP2domain), Alfinl (zinc finger), etc. have beenreported to interact with promoters of osmotic-regulated genes (Abe et al, 1997; Liu et al, 1998;Hasegawa et al, 2000). The function of some ofthese regulatory genes in stress tolerance has beenconfirmed by their ectopic expression intransgenic plants (see section transgenicstrategies). Several regulatory intermediates likeSOS3 (Ca2+-binding protein), SOS2 (Sue non-fermenting-like), Ca2+-dependent protein kinases,and mitogen activated protein kinases (MAPK)have been shown to modulate plant salt responses(Hasegawa et al, 2000; Zhang & Klessig, 2001).The biochemical function of target stress-responsive genes (e.g. leas, dehydrins, etc) is stillnot clear. Both ABA-dependent and ABA-independent signaling pathways have beensuggested for the activation/transcription of theseeffectors and regulatory/signaling molecules.

It is now widely recognized that many salt-responsive genes do not contribute to tolerance,rather, their induction reflects salt stress damage. Itmust also be noted that many genes, that areimportant for salt tolerance, may not be induced bysalt stress and may escape detection even by powerfulmolecular analytical techniques. It is also important toknow what type of molecular changes/signalingcontrols the cell division and plant growth undersalinity stress. Molecular basis of morphologicalfeatures such as rates of photosynthesis andtranspiration, stomatal closure, general inhibition ofshoot growth with continued root growth, etc undersalinity stress, is not known. The link between saltstress and control of cell expansion, division andgrowth has not been examined carefully. It will not besurprising if stress inhibits cell growth via hormonehomeostasis i.e. by changing the concentrations ofgrowth hormones such as abscisic acid, auxins,cytokinins, gibberellins, brassinolides, etc. (Zhu,2001).

Gene DiscoveryIn the pre-genomic era, genetic engineering for

stress tolerance was greatly limited by the non-availability of structural genes and regulatory

123

elements, which positively contribute to stresstolerance improvement (Bartels & Nelson, 1994;Bohnert & Jensen, 1996; Zhu et al, 1997). Differentstrategies have been used to discover/isolate the genesimportant for stress tolerance (Bartels & Nelson,1994; Bray, 1993). With rapid developments ingenomics, it has been possible to study many genessimultaneously on a genomic wide-scale with respectto their structure and function with several-foldincrease in the efficiency of gene discovery (Bohnertet al, 2001a,b; Cushman & Bohnert, 2000; Sasaki,2001;Tyagi & Mohanty, 2000). Several new toolssuch as high-throughput analysis of genomic-scaleESTs, genome sequencing, DNA chips/cDNAmicroarray analyses, targeted or random mutagenesis,knockouts, sense- and anti-sense suppression andgain-of-function or mutant complementation, are nowavailable for the discovery of the novel structural andregulatory genes involved in stress adaptation ortolerance in glycophytes (Arabidopsis, rice, etc.),halophytes (M. crystallinum, Thellungiella halophiliaiand yeast (Saccharomyces cerevisiaei (Bevan et al,1999; Bohnert et al, 2001a,b; Cushman & Bohnert,2000; Goff, 1999; Walbot, 1999; Zhu, 2001).

A random sequencing of ESTs from rice cells led tothe identification of several genes of the enzymes ofglycolysis and TCA cycles, which were up-regulatedin response to the salt stress (Umeda et al, 1994). InArabidopsis, sequencing of 220 randomly chosenESTs from a subtracted Arabidopsis cDNA library,led to the identification of 15 osmotic-stress inducedgenes with early, late or continuous patterns ofexpression (Pih et al, 1997). Halophytes such as M.crystallinum (ice plant) and T. halophilia (a closerelative of Arabidopsis that shares a similarmorphology and plant cycle), which have evolved avariety of structural and regulatory mechanism(s) totolerate severe osmotic or ionic stresses, haveemerged recently as excellent model systems to studythe stress-related DNA sequences (Cushman et al,1999; Gokhman et al, 1998; Very et al, 1998).Extensive work is in progress on the identificationand sequencing of the stress-relevant ESTs inglycophytes and halophytes (for an updateinformation visit http://www.biochem.arizona.edu/Bohnertlfunctgenomics/front2.ht!l11; http://www .zmdb.iastate.edulzmdb/EST project.html; http://www.ncbi.nlm.nih.gov/dbEST/dbESTsummary. html;http://www.nsf.gov/bio/pubs/awards/genome99.htm;http://stress-genomics.orgl).


To understand the function of stress-relevant DNAsequences, cDNA microarray offers a high-throughput approach to examine comprehensive geneexpression profiles (Lemieux et al, 1998; Kehoe,1999; Baldwin et al, 1999). Yeast cells exposed tosalt-stress led to >two-fold increase in abundance of-300 transcripts (-5% of all ORFs), whereas -200genes were down regulated (Cushman & Bohnert,2000). Several genes involved in energy metabolism,ion homeostasis, cell defence, chaperone functionsand transport facilitation were most strongly upregulated. cDNA libraries have been developed fromroot, leaf and shoot tissues of the salt- and drought-stressed plants (rice vars. Pokkali, IR29, IR64; maizevar. B73, barley var. Tokak and M. crystallinum)(Kawasaki et al, 2001; Bohnert et al, 2001b). Fromthe initial non-subtracted 2,000 ESTs, expressionprofiles were generated that represents the set ofexpressed genes in different functional categoriesincluding a large number of unknown transcripts(-40%) and highly transcribed transcripts. Among themost highly transcribed genes in salt-stressed maizeroots are isoforms for a metallothionein-like protein, aglutathione S-transferase, glutamine synthetase,several putative water channel proteins, and severalORFs, for which no other sequence has been foundbefore ("no hit" category). Such studies are expectedto provide information about the role of severalunknown genes in cellular stress adaptation processes.Similar large-scale cDNA microarray analyses of theexpression profiles of salinity-stress induced genesare in progress for M. crystallinum, Arabidopsis, andrice (http://www.biochem.arizona.edu/Bohnertlfunctgenomics/front2.html). Further, the comparisonbetween such single-celled, halophyte and glycophytemodel organisms shall help in the identification ofcellular tolerance mechanisms that are evolutionaryconserved.

Further application of novel techniques such as T-DNA, transposon and/or retrotransposon insertionalmutagenesis, may be required to generate taggedmutants with defective stress tolerance responses(Cushman & Bohnert, 2000; Enoki et al, 1999; Tissieret aI, 1999; Winkler et al, 1998; Zhu, 2001). Thesemutant populations can be screened using bothforward and reverse genetic screens to isolate "knock-out" mutants that are either stress-tolerant orhypersensitive to stress. Using this approach, severalArabidopsis mutants with altered stress signaling orsensitivity have been isolated (Ichimura et al, 1998;Lee et al, 1999; Lippuner et al, 1996). The selected

stress-related genes/DNA sequences can be tested fortheir contribution in building stress tolerance by over-expressing them in transgenic plants, or bysuppression of salt-sensitive mutants as has been donein case of Arabidopsis. Mining of thesedatasets/information and genetic analysis combinedwith large-scale sequencing, proteomics andmetabolomics activities shall maximize the efficiencyof gene discovery research for stress adaptation.

Transgenic StrategiesThe transgenic approach offers powerful means of

incorporating a broad spectrum of genes withprofound ability to up- or down-regulating specificmetabolic steps associated with stress response.Improving salinity tolerance, which is a complexpolygenic trait, by genetic transformation has beenconsidered difficult by many scientists. Nonetheless,results obtained from transfer of single genes thatencode either biochemical pathways or endpoints ofsignaling pathways, have been quite encouraging. Adetailed and in-depth account of plant transformationfor abiotic stresses is available in numerous reviews(Bajaj et al, 1999; Bartels, 2001; Bartels & Nelson,1994; Bohnert & Sheveleva, 1998; Cheng et al, 2001;Hasegawa et al, 2000; Holmberg & BUlow, 1998; Itoet al, 1999; Jain, 2002; Jain & Jain, 2000; Jain &Selvaraj, 1997; Tyagi et aI, 1999; Winicov, 1998;Yeo, 1998; Zhang et al, 2000; Zhu, 2001). A fewexamples of the genes transferred in plantsspecifically for improving salinity tolerance (Table 1)are briefly described below:

Osmoprotectants and Molecular ChaperonsPlants engineered to synthesize and accumulate

osmolytes such as mannitol (Tarczynski et al, 1993;Karakas et al, 1997; Shen et al, 1997; Thomas et al,1995; Wang et al, 2000), sorbitol (Wang et al, 2000),glucose and fructose (Fukushima et al, 2001), fructans(Pilon-Smits et al, 1995), ononitol (Sheveleva et al,1997), trehalose (Holmstrom et al, 1996; Romero etal, 1997), proline (Kishor et al, 1995; Hong et al,2000; Zhu et al, 1998b) and glycine betaine(Holmstrom et al, 2000; Mohanty et al, 2002; Prasadet al, 2000; Sakamoto et al, 1998), have shownmarginally improved tolerance against dehydration,salinity and/or cold stresses (Table 1). Theaccumulation of these osmolytes is believed tofacilitate 'osmotic adjustment', by which the internalosmotic potential is lowered and may then contributeto abiotic stress tolerance (Hasegawa et al, 2000:

Category

Table I-Major targets, which have been engineered for improving salt tolerance in plants with specific examples of transgenic plants.

Possible mode(s) of action Examples of transgenic plants showing marginal to significant* increase in salinity tolerance

Gene, gene product Transgenic Response Referenceplant sp.

Osmoprotectants Osmotic adjustment; betA, choline dehydrogenase Tobacco Glycine betaine synthesis Lilius et at, 1996protein/membrane protection; codA, choline oxidase Arabidopsis Alia et at, 1998reactive oxygen scavenging Hayashi et al, 1997

B.juncea Prasad et at, 2000Rice Sakamoto et al, 1998

Mohanty et at, 2002 '-<;J>

IMTl, myo-inositol O-methyl- Tobacco D-ononitol synthesis Sheveleva et al, 1997 -Ztransferase ~

~MtLD, rnannitol-l-phosphate Arabidopsis Mannitol synthesis Tarczynski et al, 1993 & t:lDehydrogenase Thomas et al, 1995 tTi

<Tobacco Karakas et al, 1997 m

l'Maize Shen et al, 1997 0

"d-ZCl

MtlD and gutD genes Rice Mannitol and sorbitol Wang et al, 2000 o:;J>

synthesis t;Apoplastic yeast-invertase Tobacco Accumulation of glucose and Fukushima et at, 2001 >-l

fructose 0P5CS, ,i'-pyrroline -5- Tobacco Proline synthesis Kavi-kishor et al, 1995 r-

tTicarboxylate synthetase Hong et al, 2000 :::0

;J>Rice Zhu et at, 1998 Z>-l

Reactive oxygen Detoxification of reacti ve oxygen NtI07, glutathione-S- Tobacco Increased in the activity of Roxas et al, 1997,2000n:::0

scavengers species transferase enzymes encoded by 0"d

transgene and improves [/J

scavengingFeSod, Fe superoxide Tobacco** Van Camp et al, 1996dismutaseMn-SOD, Mn superoxide dismutase Rice chloroplasts Tanaka et at, 1999

Stress proteins Unknown, protein stabilization, Group 3 Lea, late embryo-genesis- Rice Lea 3 protein synthesis Xu et al, 1996 &water binding/ abundant protein Rohila et at, 2002slow desiccation rates; chaperones;protein/membrane stabilization; ionsequestration

Contd-

-NV1

Category Possible mode(s) of action

>-N0\

Ion/protontransporters

Transcri ptionfactors, ,gnalingcomponents

Table I-Major targets, which have been engineered for improving salt tolerance in plants with specific examples of transgenic plants-Contd

K+/Na+ uptake and transport;establishment of proton protongradients; removal andsequestration of (toxic) ions fromthe cytoplasm and organelles

Ca2+_

sensors/upregulation/activation oftranscription

Gene, gene product

Examples of transgenic plants showing marginal to significant* increase in salinity tolerance

Reference

HKTl, high-affinity K+ transporter

AtNHX1, a vacuolar Na+/H+ antiport

A VPI H+-pump, a vacuolarif pyrophosphatase

HALl,

HALl

HAL2SOSl

SOS2

OsCDPK7, ca2+-dependentprotein kinase

DREBIA, transcription factor

DREB2A & DREB2B, DRE/CRTbinding proteins, transcriptionalactivatorsAlfin l , a transcription factor

** Does not con!er tolerance to salt stress at the whole plant level.

Transgenicplant sp.

Response

Yeast Confers low-affinity Na+uptake

Rubio et ai, 1995

Arabidopsis* Enhanced vacuolar Apse et al. 1999 ZTomato* concentration of Na+ ions Zhang & Blumwald, tJ.....B. napus* 2001 >Z

Zhang et al, 200 1 '--'Arabidopsis* Enhanced vacuolar Gaxiola et al, 2001 t:t:I

0concentration of cations .....,

Altered Na+ and K+tn

Melon Bordas et ai, 1997 o~

tomato homoeostasis Gisbert et al, 2000 Z0

tomato Enhances K+/Na+ selectivity Rus et al, 2001 r'"under salt stress '--'>

Citrus ? Cervera et al, 2000 ZC

Arabidopsis Transport of Na+ across Qiu et al, 2002 >;;0plasma membrane >-<:

Rice Improved salt tolerance and Verma et al, 2002N00

less chlorophyll loss w

Rice Induction of stress Saijo et al, 2000responsive genes

Arabidopsis Activates the expression of Kasuga et al, 1999many stress tolerance genes

Arabidopsis Activated by both Nakashima et al, 2000dehydration and salt stresses

Alfalfa Possibly regulates the Winicov & Bastola,expression of NaCI inducible 1999gene MsPRP2

JAIN et al: DEVELOPING SALT TOLERANT CROPS

Huang et al, 2000; McNeil et al, 1999; Nuccio et al,1999: Sakamoto & Murata, 2000). These osmolytes athigher concentrations can reduce the inhibitory effectsof ions on enzyme activity (Solomon et al, 1994),increase the thermal stability of enzymes (Galinski,1993), and prevent dissociation of enzyme complexes(Papageorgiou & Murata, 1995). Proline deficiency intransgenic anti-sense P5CS Arabidopsis plantsspecifically adversely affects the biosynthesis of cellwall structural proteins (Nanjo et al, 1999). Many ofthese osmolytes may also have a function indetoxification by scavenging ROS or prevent themfrom damaging cellular structures (Shen et al, 1997).In addition, polyamines have also been implicated in avariety of stress responses including salt and droughtstresses in plants (Lee et al, 1999; Galston et al, 1997;Capell et al, 1998).

Reactive Oxygen Scavenging/DetoxificationEnzymes

Protection of sensitive metabolic reactions throughmaintaining the structures of protein complexes ormembranes by an increased capacity for scavengingreactive oxygen intermediates (singlet oxygen,superoxide, hydrogen peroxide, hydroxy radicals,etc)) has been an important strategy to engineer stresstolerance in plants (Allen, 1995; Bartels, 2001;Bohnert & Shevelva, 1998; Foyer et al, 1994; Nocter& Foyer, 1998). Transgenic plants over-synthesizingenzymes including cytosolic ascorbate peroxidase(Torsethaugen et al, 1997), glyoxalase I (Veena et al,1999), superoxide dismutase (Gupta et al, 1993;Mckersie et al, 1996, 1999), glutathione-S-transferase/glutathione peroxidase (Nocter & Foyer,1998; Roxas et al, 1997, 2000), aldose-aldehydereductase (Bartels, 2001) and iron-binding proteinferritin (Deak et al, 1999; Goto et al, 1999), haveshown greater tolerance to oxidative damage, saltstress, drought, etc. Support for the importance ofoxidative protection in salt tolerance also comes fromthe characterization of the Arabidopsis pst 1 mutants.The pst 1 mutant plants were more resistant to high-salt concentrations as well as had greater capacity totolerate oxidative stress (Tsugane et al, 1999). Itshould be noted that ROS enzymes, for example,ascorbate peroxidase, and superoxide dismutase, existin several iso-forms and are found in cytosol,mitochondria, plastids, etc (lshitani et al, 1997;Noctor & Foyer, 1998). Hence, transgenicmodifications of single ROS enzyme are likely tohave marginal effects because of the multitude ofcompartments that require protection.

127

Late-embryogenesis Abundant Protein (LEA) GenesLEA proteins are specifically produced in aleurone

tissues and embryos during seed maturation stage andtheir synthesis is induced by ABA, drought, salinitystress, or cold temperatures (Close, 1997; Hong et al.1992). The second-generation transgenic rice plantscontaining barley LEA3 (HVAl) gene, driven byconstitutive and ABA-inducible promoters, showedincreased tolerance to water deficit and salt-stress (Xuet al, 1996; Cheng et al, 2001; Rohila et aI, 2002).Imai et al (1996) reported enhanced tolerance againstsalt stress and freezing in yeast cells transformed witha tomato LEA-class gene. The precise mode of actionof LEA proteins under osmotic stresses is still notclear. LEA proteins may have a function in waterbinding or retention, protein/membrane stabilization,ion sequestration, and as molecular chaperones (Bajajet at, 1999). It may also affect cellular metabolismand thus influence the production of ATP viaglycolysis or photosynthesis.

Ion/Proton TransportersSoil salinity can lead to sodium toxicity in plants

mainly because of their adverse effects on K+nutrition, cytosolic enzyme activities, photosynthesisand metabolism. Plants have evolved variousmechanisms for the compartrnentation of Na+ invacuoles or its exclusion. Na+ compartmentationprovides economical means not only for preventingNa+ toxicity in the cytosol but also helps in osmotichomoeostasis and water uptake. Several groups haveidentified and characterized the Na+-H+ antiportersequences (Apse et al, 1999; Gaxiola et al, 1999) ofwhich, AtNHX1, which has already been transferred inplants, appeared to improve the salt tolerancesubstantially. The transgenic salt-tolerant Arabidopsis(Apse et al, 1999), tomato (Zhang & Blumwald,2001) and Brassica napus (Zhang et al, 2001) plantsover-expressed AtNHX1, a tonoplast Na+/H+antiporter gene from A. thaliana. These transgenicplants grew and/or produced fruits even at 200 mMNaCl, which is equivalent to 40% salt concentrationof seawater. The high Na+ content in the leaf tissuesof transgenic plants grown in saline environment wasdue to enhanced vacuolar accumulation of Na+ ions,indicating an alteration in the ion-homeostasis.Transgenic canola plants grown at 200 mM NaClgave seed yield and oil quality and quantity similar tothat of wild type plants grown at low salinity (Zhanget al, 2001). The transgenic plants accumulated Na+ atvery high levels suggesting their use for thereclamation of saline soils. Gaxiola et at (2001)


reported improved resistance against NaCI stress andwater deprivation in transgenic Arabidopsis plantsover-expressing the vacuolar H+-pyrophosphatase(A VP 1 H+ -pump) gene. The transgenics accumulatedmore Na+ and K+ in their leaf tissues and cations weresequestered in the vacuoles. Expression of a yeastcation transporter gene, HALl, in transgenic melonand tomato plants led to the alteration in Na+ and K+homoeostasis and significantly improved the salttolerance of several transgenic lines (Bordas et al,1997; Pardo et al, 1998; Gisbert et al, 2000).

Genetic analysis has linked components of the SOSpathway (SOS 1-3) to salt tolerance in A. thaliana(Zhu, 2001). The predicted SOS 1 protein sequenceand comparison of Na+ accumulation in wild type andtransgenic SOS 1 plants suggest that SOS 1 is directlyinvolved in the transport of Na+ across the plasmamembrane; while SOS2 and SOS3 regulate SOS 1transport activity (Qiu et al, 2002). Mutations in 5052cause Na+ and K+ imbalance and render plants moresensitive towards growth inhibition by high Na+ andlow K+ environments (Liu et al, 2000). TransgenicSOS2 rice plants were more tolerant to salinity stressas evaluated by seedling growth parameters andchlorophyll bleaching (Verma et al, 2002).

Water Channels/AquaporinsCertain proteins (also termed as aquaporins), that

act as water channels, might be involved in transportof water and mineral nutrients across the membranesof plant cells under salt stress (Weig et al, 1997;Chrispeels et al, 1999). The Arabidopsis genomecontains at least 23 genes, that encode proteins of thewater channel family, several of which have beenfunctionally characterized and are located either in theplasma membrane or in the tonoplast membrane(Weig et al, 1997). These aquaporins have beensuggested to be involved in metabolite, ion or watertransport. Stress-dependent altered expression andtranscript amounts, both up and down, of several ofthese aquaporins have been reported in Arabidopsisand other plant species. The stress-dependentregulation of aquaporins indicates their involvementas water channels during the stress responses andsome may also have a function in metabolite or iontransport. Regulation of activity of these aquaporinsmay be due to changes in the oligomerization, byphosphorylation or possibly by cycling through theendomembrane system as has been reported in animalsystems (Yang & Verkman, 1997). There are noreports on the manipulation of water channel proteinsup till now, but this area of research is being pursued

actively. Molecular dissection of the role ofaquaporins under stress is likely to provide moreinsight into plant water relations.

Transcription Factors and Signaling ComponentsThe process of adaptation to stresses such as

salinity and drought, involves certain protein factors(transcription factors, protein kinases, etc), whichregulate the expression of a group of functionallyrelated genes or are involved in signal transduction.The molecular mechanisms for coordinated transcriptaccumulation and signaling pathways have beenreviewed (Hasegawa et al, 2000; Knight & Knight,2001; Winicov, 1998; Yamaguchi-Shinozaki &Shinozaki, 1999). The transformation of plants usingsuch regulatory genes, which can activate theexpression of many stress-tolerant genessimultaneously, could be more rewarding. Kasuga etal (1999) reported over-expression of a regulator geneencoding OREBIA (a homologue of CBFItranscriptional activator gene, Jaglo-Ottosen et aI,1998) in transgenic Arabidopsis plants, whichactivated the expression of many stress-tolerant genes,rd17 and rd29A (LEA like proteins), Kin 1, Cor 6.6,Cor 15a, and P5CS. These transgenic plants showedgreater tolerance to drought, salt and freezing stresses.The CBF/OREB transcription factors can bind to theORE and CRT elements that are found in thepromoters of some stress-responsive genes (Liu et aI,1998). Transfer of a single rice gene encoding Ca2+_dependent protein kinase (COPK), OsCDPK7, intransgenic rice plants has been reported to improvetolerance against cold and salt/drought stresses (Saijoet al, 2000). Winicov & Bastola (1999) improvedsalinity tolerance in transgenic alfalfa plants by over-expressing the Alfin 1 gene, a putative transcriptionregulator, which regulates the expression of NaCl-inducible gene, MsPRP2. Transgenic plants with A(fin1 in antisense orientation were more sensitive to NaCIinhibition. A number of transcriptional activators ofkinases and phosphatases respond to salinity anddrought stresses, but their mechanism(s) of actionremain unresolved.

Genetic engineering of salt tolerance in plants viadifferent transgenic strategies has been quitepromising though it may vary greatly with the targettraits and the choice of gene(s) used. No systematicefforts have been made to prioritize or determine therelative importance of different strategies. However. itis amply clear from the above discussion that it ispossible to manipulate a variety of target traits leadingto a marginal to significant increase in salt-tolerance.

JAIN et at: DEVELOPING SALT TOLERANT CROPS

With the improvement in transformation technology,especially the (i) improvements in the planttransformation methods (Roy et al, 2000; Tyagi et al,1999), (ii) development of binary bacterial artificialchromosome (BIBAC) vectors for transferring largesized (-150 kb) DNA fragments (Hamilton, 1997),(iii) use of stress-inducible promoters (Cheng et al,2001; Kasuga et al, 1999; Su et al, 1998), (iv) use ofspecific DNA sequences (e.g. matrix attachmentregions) known to reduce position effect and genesilencing (Holmes-Davis & Comai, 1998), and (v)introduction of genes via chloroplast transformationor targeting of trans gene products into chloroplasts(Daniell, 1999; Jang et al, 1999), it should be possibleto effectively utilize the transgenic technology forgene transfer, gene pyramiding and simultaneousmanipulation of two or more mechanisms of salt-tolerance.

Molecular Breeding for Salinity ToleranceMolecular marker technologies have revolutionized

the genetic analysis of crop plants (Caetano-AnoIl6s& Gresshoff, 1998; Ribaut & Betran, 2000; Staub etal, 1996; Young, 1999). It is now possible to dissectcomplex physiological traits such as salt toleranceusing improved methods of identifying and measuringthe physiological components (Yeo et al, 1990) andmolecular markers (Flowers et al, 2000; Forster et al,2000; Jansen & Stam, 1994; Kearsey & Farquhar,1998; Zhang et aZ, 1999a). In several crops,comprehensive molecular marker/linkage maps with avariety of DNA markers have been developed (fordetailed information visit http:/probe.nalusda.gov).Most of the maps have been developed using mappingpopulations, which include the DHLs, RILs, andbackcross/Fj/F, families. It is not surprising thatmolecular mapping of genes controlling salt tolerance,osmotic adjustment and drought stress lags behindthat for disease resistance. Salt tolerance, OA anddrought are quantitative in nature and governed byseveral minor genes or QTLs, which do show theMendelian inheritance but are greatly influenced bythe environment. Using molecular linkage maps andQTL mapping technology, it is possible to estimatethe number of loci controlling genetic variation in asegregating population and to characterize these lociwith regard to their map positions on the genome,gene action, phenotypic effects, pleiotropic effect andepistatic interaction between the QTLs (Paterson et al,1988). These maps allow the selection of a largenumber of markers for any region of the genome withmany potential applications. ranging from fine

129

mapping, MAS and comparative mapping to mapbased cloning for the genetic improvement of plants,etc. In MAS, individuals carrying target genes areselected in segregating population based on linkedmarkers rather than on their phenotype. Thus, thepopulation can be screened at any stage of growth andin various environments. MAS increases theefficiency of a breeding program by selecting formarkers linked to target traits or QTLs. MAS alsoprovides new opportunities to transfer and pyramidQTLs into agronomically desirable genotypes. Thehigh-density genetic map coupled with thedevelopment of genomic DNA libraries (e.g. BAC,YAC), can be used for the isolation of specific genes(e.g. Xa21 gene for bacterial blast resistance in rice).

Genes/QTLs have been mapped for severalagronomically important traits such as disease andinsect resistance, yield, quality and abiotic stresstolerance traits (drought, sub-mergence tolerance,salinity tolerance, etc.) (Flowers et al, 2000; Forster etal, 2000; Jansen & Starn, 1994; Kearsey & Farquhar,1998; Khush & Brar, 2001; Zhang et ai, 1999a).Among the abiotic stresses, maximum progress hasbeen made towards the mapping of drought-relatedtraits and there have been only a few studies to mapQTLs for salinity tolerance. Most research onmapping for salt tolerance has been carried out in rice.Low Na/K ratio is governed by both additive anddominance gene effects in rice (Gregorio &Senadhira, 1993). Zhang et al (1995) used 130 RFLPprobes distributed throughout the rice genome andidentified a salt-tolerant gene using the probe 'RG4'that was located on chromosome 7. Seven QTLs,which control various rice seedling traits conferringsalt tolerance, were mapped by RFLP analysis using adi-haploid population derived from a cross betweenIR64 and Azucena (Prasad et al, 2000). Four of theseQTLs were located on chromosome number 6. A QTLfor root length on chromosome 6 flanked by RFLPmarker, RGI62-RG 653, has been identified. Ali et al(2000) mapped QTLs for root traits using RFLP andAFLP markers using a RIL population derived fromtwo lndica rice ecotypes. Tripathy et al (2000)identified QTLs for cell membrane stability underdrought stress in rice using RFLP, AFLP andmicrosatellite (SSR) markers. Xie et al (2000)compared the genetic diversity of salt-tolerant ricevarieties (Pokkali, Nona-Bokra, Bicol) and a saltsensitive rice variety (lR29) using RAPD markers.Four primers-amplified specific fragments appearedin all the salt tolerant varieties but not in the salt


susceptible variety. Flowers et al (2000) haveidentified four putative markers for sodium andpotassium ion transport and selectivity by AFLPmarker analysis using a custom-made mappingpopulation (designated as IR55178; the two parentsIR4630- and IRIS324- show extreme phenotypes forsodium transport and for tissue tolerance) of rice andin two near-isogenic lines of IR36 that differed insodium transport. However, none of these markersshowed any association with similar traits in a closelyrelated population of RILs or in selections of acultivar. This result cautions against any expectationof a general applicability of markers for physiologicaltraits and highlight the necessity of identification ofgenes involved. They also highlight the need of anefficient screening procedure for salinity tolerance,which is designed to cope with expected degree ofvariation in segregating population and reduce theenvironmental effects. This is particularly important,as the mapping is only as good as the quality ofquantitative phenotypic data. The same mappingpopulation has also been used for the identification ofQTLs, which govern Na+ and K+ uptake, and Na+:K+selectivity using AFLP, SSR and RFLP markers(Koyama et ai, 2001). QTLs for Na+ and K+ uptakewere on different chromosomes. Lang et al (2001)identified several QTLs for seedling survival in saltsolution, shoot and root dry weight, Na+ and K+absorption and for Na/K ratio by RFLP and SSRmarker analysis of 108 Fg Tesanai 2xCB RILs.

Six genetic markers linked to QTLs involved insalinity tolerance in terms of yield in tomato havebeen identified using a F2 segregating population of206 plants (Bret6 et ai, 1994). In barley, physiologicaltraits associated with salt tolerance were mapped tochromosomes 1 (7H), 4 (4H), S(lH) and 6 (6H) (Elliset al, 1997). The long arm of chromosome 4H hasalso been implicated for water use efficiency(Handley et al, 1994), and drought tolerance(Chalmers et at, 1992). The QTLs associated with salttolerance were identified on all the seven barleychromosomes but the QTLs with greatest effectsclustered around the dwarfing genes (Thomas et at,1998). Foolad & Chen (1998) identified 13 RAPDmarkers at eight genomic regions that were associatedwith QTLs affecting salt tolerance during germinationusing a UCTS (L. esculentum, salt-sensitive) x LA716(L. pennell ii, salt-tolerant) F2 population of 2000individuals. QTLs for OA, which is an importantcomponent of both drought and salinity tolerance, hasalso been mapped in rice (Ahn et ai, 1993; Lilley et

at, 1996; Zhang et at, 1998), wheat (Morgan & Tan1996), barley (Teulat et at, 1998) and sunflower(Jamaux et ai, 1997) (for review see Zhang et at,1999a).

Abundant germplasm sources for salinity toleranceexist in nature and they are yet to be characterized andutilized effectively. In some plant species, progresshas been made to enhance the salt tolerance with afew new cultivars released. Perhaps the mostspectacular progress has been made in rice, wheresalt-tolerant rice varieties (CSRlO, CSR30, etc.) havebeen developed at CSSRI, Karnal, India. Applicationof molecular marker technology can greatly enhancethe efficiency and accuracy of the breeding process. Amolecular marker strategy for linkage mapping, andbreeding for salinity tolerance in rice is being used atCCS Haryana Agricultural University, Hisar, India(Fig. 1). Current progress in the QTL mapping for salttolerance is although promising, but a little slow. Careshould be taken while selecting donor genotypes, andthat introduction of salt tolerance traits should notalter the established traits. Marker-assisted selectionwould be particularly effective for pyramiding ofdifferent tolerant genes/QTLs in order to provideeffective and potentially stable resistance in cropplants, where simultaneous or even sequentialscreening of plants is difficult or impractical. Majorchallenges in this approach remains the fine mappingof all important salt-stress related QTLs/genes, toknow their structure and function, and to find outwhat genes underlie biochemical pathways and thephysiology causing quantitative variation.Developments in genomics, especially in Arabidopsisand rice, shall complement and provide newopportunities to solve these problems.

Conclusions and Future ProspectsIt remains a challenge to improve the agricultural

crops for complex, polygenic traits like salinitytolerance. Developments in the area of genomics,transformation and molecular mapping have providednew tools for the molecular dissection of complextraits like salinity tolerance, improving ourunderstanding of stress perception/responses, rapiddiscovery of genes/QTLs specifically involved in salttolerance and molecular designing of salt tolerantcrops. A spectacular progress has already been madein the last ten years. A variety of salt-stress relatedgene(s) from different metabolic and regulatorypathways have been identified and successfullytransferred in some model plant species resulting in amarginal to significant increase in salt tolerance. The

JAIN et al: DEVELOPING SALT TOLERANT CROPS 131

Stress tolerantindica!japonica

Marker-assistedselection (MAS)

Assessment of molecularpolymorphism amongthe parental varieties

TraditionalBasmatiX rice variety e.g. Taraori

Basmati

l

,.Phenotypic evaluations for F3,F4

IBasmati rice traits and stresstolerance/ stress relatedparameters

~tLinkage mapping

F6-F8Genetic linkage map Recombinant inbredQTL identification lines (RlLs)

Use ofpolymorpbic markers forthe identification of bybridsIDHLs

Large segregating pop--ulation at early stagesof recombination

Development of elitehigh-yielding, stresstolerant lines with intactBasmati rice traits

Fig. I-Potential use of molecular markers for linkage mapping and Basmati rice breeding for salt-tolerance

transgenic strategies, specifically employing the genesinvolved in ion transportlcompartmentation, signaltransduction and co-ordinated regulation of manystress-responsive genes, look quite promising. It nowseems plausible to engineer salt-tolerant crops withfar fewer genes/target traits than had been anticipated.Discovery of stress-relevant structural genes andregulatory elements, which has been major limitingfactor, is likely to gain momentum by the applicationof genomics including the development of large scaleESTs, genome sequencing, cDNA microarrayanalysis, insertional mutagenesis, knockouts, etc.Molecular markers are being used for linkagemapping of stress-tolerant genes/QTLs and thistechnology could be highly useful for pyramiding ofstress-tolerant genes/QTLs in commercially importantcultivars. All these studies shall eventually provide uswith insights into the cellular processes that arecrucial for salt tolerance and to manipulate theessential protective mechanism(s) in crop plants. Inthe coming years, it will be interesting to see howmolecular approaches rationally improves ourunderstanding of salt tolerance mechanisms in plantsand improves the efficacy and accuracy of breedingprograms for this complex, intractable trait.

AcknowledgementResearch grant from the Rockefeller Foundation,

New York, USA, for rice biotechnology research at

CCS Haryana Agricultural University, Hisar (India),is gratefully acknowledged.

ReferencesAbe H, Yarnaguchi-Shinozaki K, Urao T, Iwasaki T, Hosokawa D

& Shinozaki K, 1997. Role of Arabidopsis MYC and MYBhomo logs in drought- and abscisic acid-regulated geneexpression. Plant Cell, 9,1859-1868.

Ahn S, Anderson J A, Sorrells M E & Tanksley S D. 1993.Homoeologus relationships of rice, wheat and maizechromosomes. Mol Gen Genet, 241, 483-490.

Ali M L, Path an M S, Zhang J, Bai G, Sarkarung S & NguyenHT, 2000. Mapping QTLs for root traits in a recombinantinbred population from two indica ecotypes in rice. TlieorAppl Genet, 101, 756-766.

Alia, Hayashi H, Chen T H H & Murata N, 1998. Transformationwith a gene for choline oxidase enhances the cold toleranceof Arabidopsis during germination and early growth. PlantCell Environ, 21, 232-239.

Allen R D, 1995. Dissection of oxidative stress tolerance usingtransgenic plants. Plant Physiol, 107, 1049-1054.

Amtmann A & Sanders D, 1999. Mechanisms of Na+ uptake byplant cells. Adv Bot Res, 29, 75-112.

Apse M P, Aharon G S, Snedden W A & Blumwald E, 1999. Salttolerance conferred by overexpression of a vacuolar Na+/H+antiport in Arabidopsis. Science, 285,1256-1258.

AshrafM, 1994. Breeding for salinity tolerance in plants. Crit RevPlant Sci, 13, 17-42.

Bajaj S, Targolli J, Liu L-F, Ho T-H D, Wu R, 1999. Transgenicapproaches to increase dehydration-stress tolerance inplants. Mol Breed, 5, 493-503.

Baldwin D, Crane V & Rice D, 1999. A comparison of gel-based.nylon filter and microarray techniques to detect differentialRNA expression in plants. Curr Opin Plant Bioi, 2, 96-103.


Bartels D, 2001. Targeting detoxification pathways: an efficientapproach to obtain plants with multiple stress tolerance?Trends Plant Sci, 6, 284-286.

Bartels D & Nelson D, 1994. Approaches to improve stresstolerance using molecular genetics. Plant Cell Environ, 17,659-667.

Bevan M, Bancroft I, Mewes H W, Martienssen R & McCombieR, 1999. Clearing a path through the jungle: Progress inArabidopsis genornics, Bioessays, 21, 110-120.

Bohnert H J, Ayoubi P, Borchert C, Bressan R A, Burnap R L,Cushman J C, Cushman M A, Deyholos M, Fischer R,Galbraith D W, Hasegawa P M, Jenks M, Kawasaki S,Koiwa H. Kore-eda S, Lee B-H, Michalowski C B, MisawaE, Nomura M, Ozturk N, Postier B, Prade R, Song C-P,Tanaka Y, Wang H & Zhu J-K, 2001a. A genomicsapproach towards salt stress tolerance. Plant PhysiolBiochem, 39, 295-311

Bohnert H J & Jensen R G, 1996. Metabolic engineering forincreased salt tolerance - The next step. Aust J PlantPhysiol, 23, 661-667.

Bohnert H J, Kawasaki S, Michalowski C B, Wang H, Ozturk N,Deyholos M & Galbraith D, 2001b. Isolation of candidategenes for tolerance of abiotic stresses. in Rice Genetics IV.Proc Fourth International Rice Genetics Symposium, IRRI,Los Banos, Philippines, edited by G S Khush, D S Brar &B Hardy. IRRl, Science Publishers, Inc., New Delhi, LosBanos. Pp 345-363.

Bohnert H J, Nelson D E & Jensen R G, 1995. Adaptations toenvironmental stresses. Plant Cell, 7,1099- 111l.

Bohnert H J & Sheveleva E, 1998. Plant stress adaptations -Making metabolism move. Curr Opin Plant Bioi, I, 267-274.

Bordas M, Montesinos C, Dabauza M, Salvador A., Roig L A,Serrano R & Moreno V, 1997, Transfer of yeast salttolerance gene HALl to Cucumis melo L. cultivars and invitro evaluation of salt tolerance. Transgenic Res, 6, 41-50.

Boyer J S, 1982. Plant productivity and environment. Science,218, 443-448.

Bray E A, 1993. Molecular responses to water deficit. PlantPhysiol, 103, 1035-1040.

Bret6 M P, Asins M J & Caronell E A, 1994. Salt tolerance inLycopersicon species. III. Detection of quantitative traitloci by means of molecular markers. Theor Appl Genet, 88,395-401.

Caetano-Anollos G & Gresshoff P M, 1998. DNA markers:Protocols, Applications and Overviews. Wiley- VCH, NewYork, Weinheim, Singapore, Toronto.

Capell T, Escobar C, Liu H, Burtin D, Lepri 0 & Christou P,1998. Overexpression of the oat arginine decaroxylasecDNA in transgenic rice (Oryza sativa L.) affects normaldevelopmental patterns in vitro and results in putrescineaccumulation in transgenic plants. Theor Appl Genet, 97,246-254.

Cervera M, Ortega C, Navarro A, Navarro L & Pena L, 2000.Generation of transgenic citrus plants with the tolerance-to-salinity gene HAL2 from yeast. J Hortic Sci Biotechnol, 75,26-35.

Chalmers K J, Waugh R, Watters J, Forster B P, Nevo E, AbbottR J & Powell W, 1992. Grain isozyme and ribosomal DNA

variability in Hordeum spontaneum populations fromIsrael. Theor Appl Genet, 84, 313-322.

Cheng Z Q, Targolli J, Su J, He CK, Li F & Wu R, 2001.Transgenic approaches for generating rice tolerant ofdehydration stress. in Rice Genetics IV. Proc FourthInternational Rice Genetics Symposium, IRRI, Los Banos.Philippines, edited by G S Khush, D S Brar & B Hardy.IRRI, Science Publishers, Inc., New Delhi, Los Banos.Pp 423-438.

Chowdhury J B, Jain S & Jain R K, 1993. Biotechnologicalapproaches for developing salt tolerant field crops. J PIWlt

Biochem Biotechnol, 2, 1-7.Chrispeels M J, Crawford N M & Schroeder J I. 1999. Proteins

for transport of water and mineral nutrients across themembranes of plant cells. Plant Cell, 11, 661-675.

Close T, 1997. Dehydrins: A commonality in the response ofplants to dehydration and low temperature. Physiol Plant.100, 291-296.

Cushman .T C & Bonhert H .T, 2000. Genomic approaches to plantstress tolerance. Curr Opin Plant Bioi, 3, 117- I24.

Cushman M A, Bufford D, Fredrickson M, Ray A, Akselrod I.Landrith D, Stout L, Maroco J & Cushman J. 1999. Anexpressed sequence tag (EST) database for the common iceplant Mesembryanthemum crystallinum. Plant Phvsiol .120S, 145.

Daniell H, 1999. New tools for chloroplast genetic engineering.Nature Biotechnol, 17, 855-856.

Deak M, Horvath GV, Davletova S, Torok K, Sass L, Vass I,Barna B, Kiraly Z & Dudits D, 1999. Plants ectopicallyexpressing the iron-binding protein ferritin, are tolerant tooxidative damages and pathogens. Nature Biotechnol, 17,192-196.

Ellis R P, Forster B P, Waugh R, Bonar N, Handley LL. RobinsonD, Gordon DC & Powell W, 1997. Mapping physiologicaltraits in barley. New Phytologist, 137, 149-157.

Enoki H, Izawa T, Kawahara M, Komatsu M, Koh S, Kyozuka J& Shimamoto K, 1999. Ac as a tool for the functionalgenomics of rice. Plant J, 19, 605-613.

Flowers T J, Garcia M, Koyama M & Yeo A R, 1997. Breedingfor salt tolerance in crop plants - The role of molecularbiology. Acta Physiol Plant, 19,427-433.

Flowers T J, Koyama M L, Flowers S A, Sudhakar C. Singh K P& Yeo A R, 2000. QTL: Their place in engineeringtolerance of rice to salinity. J Exp Bot, 51, 99-106.

Flowers T J & Yeo A R, 1995. Breeding for salinity resistance incrop plants: Where next? Aust J Plant Physiol, 22,875-884.

Foolad M R & Chen F Q, 1998. RAPD markers associated withsalt tolerance in an interspecific cross of tomatotLycopersicon esculentum x L. Pennelliii. Plant Cell Rep.17,306-312.

Forster B P, Ellis R P, Thomas W T B, Newton A C, Tuberosa R.This D, EI-Enein RA, Bahri MH & Ben- Salem M. 2000.The development and application of molecular markers forabiotic stress tolerance in barley. J Exp Bot, 51, 19-27.

Foyer C H, Descourvieres P & Kunert K J, 1994. Protectionagainst oxygen radicals: An important defence mechanismstudied in transgenic plants. Plant Cell Environ. 17.507-523.


Frova C, Caffulli A & Pallavera E, 1999. Mapping quantitativetrait loci for tolerance to abiotic stresses in maize. J ExpZool, 282, 164-170.

Fukushima E, Arata Y, Endo T, Sonnewald U, Sato F, 2001.Improved salt tolerance of transgenic tobacco expressingapoplastic yeast-derived invertase. Plant Cell Physiol, 42,245-249.

Galinski E A, 1993. Compatile solutes of halophilic eubacteria:Molecular principles, water-solute interaction, stressprotection. Experientia, 49, 487-496.

Galston A W, Kaur-Sawhney R, Altabella T & Tiburcio A F,1997. Plant polyamines in reproductive activity andresponse to abiotic stress. Bot Acta, 110, 197-207.

Gaxiola R A, Li J, Undurraga S, Dang L M, Allen GJ & Alper SL, 2001. Drought- and salt-tolerant plants result from over-expression of A VP1F-pump. Proc Natl Acad Sci USA, 98,11444-11449.

Gaxiola R A, Rao R, Sherman A, Grisafi P, Alper S L & Fink GR, 1999. The Arabidopsis thaliana proton transporters,AtNhxl and Avpl, can function in cation detoxification inyeast. Proc Natl Acad Sci USA, 96, 1480-1485.

Gisbert C, Rus A M, Bolarin M C, Lopez-Coronado J M,Arrillaga 1, Montesinos C, Caro M, Serrano R & MorenoV, 2000. The yeast HALl gene improves salt tolerance oftransgenic tomato. Plant Physiol, 123, 393-402.

Glenn E, Brown J J & Blumwald E, 1999. Salt-tolerantmechanisms and crop potential of halophytes. Crit RevPlant Sci, 18, 227-255.

Goff S A, 1999. Rice as a model for cereal genomics. Curr OpinPlant Bioi, 2, 86-89.

Gokhman I, Fisher M, Pick U & Zamir A, 1998. New insights intothe extreme salt tolerance of the unicellular green algaDunaliella. in Micro Biogeochem Hypersaline Environ.CRC Press. Boca Raton, F L. Pp 203-213.

Golldack D & Dietz K-J, 2001. Salt-induced expression of thevacuolar H+-ATPase in the common ice plant isdevelopmentally controlled and tissue specific. PlantPhysiol, 125, 1643-1654.

Gong Z, Koiwa H, Cushman M A, Ray A, Bufford D, Kore-eda S,Matsumoto T K, Zhu J, Cushman J C, Bressan R A &Hasegawa P M, 2001. Genes that are uniquely stressregulated in salt overly sensitive (sos) mutants. PlantPhysiol, 126, 363-375.

Goto F, Yoshihara T, Shigemoto N, Toki S & Takaiwa F, 1999.Iron fortification of rice seeds by the soybean ferritin gene.Nature Biotechnol, 17,282-286.

Gregorio G B & Senadhira D, 1993. Genetic analysis of salinitytolerance in rice (Oryza sativa L.). Theor Appl Genet, 86,333-338.

Gupta A S, Heinen J L, Holaday AS, Burke J J & Allen R D,1993. Increased resistance to oxidative stress in transgenicplants that overexpress chloroplastic Cu/Zn superoxidedismutase. Proc Natl Acad Sci USA, 90, 1629-1633.

Hamilton C M, 1997. A binary-BAC system for planttransformation with high-molecular-weight DNA. Gene200,107-116.

Handley L L, Nevo E, Raven J A, Martinez-Carrasco R,Scrimgeour CM, Pakniyat H & Forster BP, 1994.Chromosome 4 controls potential water use efficiency inbarley. J Exp Bot, 45, 1661-1663:

133

Hasegawa P M, Bressan R A, Zhu J-K & Bohnert H J, 2000. Plantcellular and molecular responses to high salinity. Ann RevPlant Physiol Plant Mol Bioi, 51, 463-499.

Holmberg N & Bi.iIow L, 1998. Improving stress tolerance inplants by gene transfer. Trends Plant Sci, 3, 61-66.

Holmes-Davis R & Comai L, 1998. Nuclear matrix attachmentregions and plant gene expression. Trends Plant Sci 3,91-97.

Holmstrom K-O, Mantyla E, Welin B, MandaI A, Paiva E T.Tunnela 0 E & Londesborough J, 1996. Drought tolerancein tobacco. Nature, 379, 683-684.

Holmstrom K-O, Somersalo S, Mandai A, Paiva T E & Welin B,2000. Improved tolerance to salinity and low temperaturein transgenic tobacco producing glycine betaine. J Exp B01,51,177-185.

Hong B, Barg R & Ho T-H D, 1992. Development and organ-specific expression of an ABA- and stress-induced proteinin barley. Plant Mol Bioi, 18,663-674.

Hong Z, Lakkineni K, Zhang Z & Verma D P S, 2000. Removalof feedback inhibition of @l -pyrroline-5-carboxylatesynthetase results in increased proline accumulation andprotection of plant from osmotic stress. Plant Physiol, 122.1129-1136.

Huang J, Hirji R, Adam L, Rozwadowski K L, Hammerlindl J K,Keller W A & Selvaraj G, 2000. Genetic engineering ofglycine-betaine production toward enhancing stresstolerance in plants: Metabolic limitation. Plant Physiol,122,747-756.

Hayashi H, Alia, Mustardy L, Deshnium P, Ida M & Murata N,1997. Transformation of Arabidopsis thaliana with codAgene for choline oxidase: Accumulation of glycine betaineand enhanced tolerance to salt and cold stress. Plant J. 121133-142.

Ichimura K, Mizoguchi T, Irie K, Morris P, Giraudat J.Matsumoto K & Shinozaki K, 1998. Isolation ofATMEKKI (a MAP kinase, kinase, kinase) - interactingproteins and analysis of a MAP kinase cascade inArabidopsis. Biochem Biophys Res Commun, 253, 532-543.

Imai R, Chang L, Ohta A, Bray E & Takagi M, 1996. A lea-classgene of tomato confers salt and freezing tolerance whenexpressed in Saccharomyces cerevisiae. Gene, 170, 243-248.

Ishitani M, Xiong L, Stevenson B & Zhu J-K, 1997. Geneticanalysis of osmotic and cold stress signal transduction inArabidopsis: Interactions and convergence of abscisic acid-dependent and abscisic acid-independent pathways. PlautCell, 9, 1935-1949.

Ito 0, O'Toole J & Hardy B,1999. Genetic Improvement of Ricefor Water-Limited Environments, IRRI, Los Banos,Laguna, Philippines.

Jaglo-Ottosen K R, Gilmour S J, Zarka D G, Schaben berger 0,Thomashow M F, 1998. Arabidopsis CBFI overexpressioninduces COR genes and enhances freezing tolerance.Science, 280,104-106.

Jain RK, 2002. Transgenic rice. in Plant Genetic Engineering Vol2, Improvement of Food Crops, edited by P K Jaiwal & R PSingh, Sci-Tech Pub, Houston Texas, USA (in Press).


Jain R K & Jain S, 2000. Transgenic strategies for the geneticimprovement of Basmati rice. Indian J Exp Bioi, 38, 6-17.

Jain R K & Selvaraj G, 1997. Molecular genetic improvement ofsalt tolerance in plants. Biotech Annu. Rev, 3, 245-267.

Jamaux I, Steinmetz A & Belhassen E, 1997. Looking formolecular and physiological markers of osmotic adjustmentin sunflower. New Phytol, 137,117-127.

Jang I-C, Nahm B H & Kim J-K, 1999. Subcellular targeting ofgreen fluorescent protein to plastids in transgenic riceplants provides a high-level expression system. Mol Breed,5,453-461.

Jansen R C & Starn P, 1994. High resolution of quantitative traitsinto multiple loci via interval mapping. Genetics, 136,1447-1455.

Karakas B, Ozias-Akins P, Stushnoff C, Suefferheld M & RiegerM, 1997. Salinity and drought tolerance of mannitol-accumulating transgenic tobacco. Plant Cell Environ, 20,609-616.

Kasuga M, Liu Q, Miura S, Yamaguchi-Shinozaki K & ShinozakiK, 1999. Improving plant drought, salt and freezingtolerance by gene transfer of a single stress-inducibletranscription factor. Nature Biotechnol, 17,287-291.

Kawasaki S, Oeyholos M, Borchert C, Brazille S, Kawai K,Galbraith 0 W & Bohnert H J, 2002. Temporal successionof salt stress responses in rice by microarray analysis. PlantCell (in press).

Kearsey M J & Farquhar A G L, 1998. QTL analysis in plants;Where are we now? Heredity, 80,137-142.

Kehoe 0 M. Villand P & Somerville S, 1999. DNA microarraysfor studies of higher plants and other photosyntheticorganisms. Trends Plant Sci, 4, 38-41.

Khush G S & Brar 0 S, 2001. Rice genetics from Mendel tofunctional genomics. In Rice Genetics IV. Proc FourthInternational Rice Genetics Symposium, IRRI, Los Banos,Philippines, edited by G S Khush, 0 S Brar & B Hardy.IRRI, Science Publishers, Inc., New Delhi, Los Banos.Pp 3-25.

Kim E J, Kwak J M, Uozumi N & Schroeder J I, 1998. AKUP1:An Arabidopsis gene encoding high-affinity potassiumtransport activity. Plant Cell, 10, 51-62.

Kishor P B K, Hong Z, Miao G-H, Hu C A & Verma 0 P S, 1995.Overexpression of pyrroline-5-carboxylate synthetaseincr ses proline production and confers osmotolerance intran genic plants. Plant Physiol, 108, 1387-1394.

Knight H & Knight M R, 2001. Abiotic stress signaling pathways:specificity and cross talk. Trends Plant Sci, 6: 262-267.

Kovtun Y, Chiv W L, Tena G & Shee J, 2000. Functional analysisof oxidative stress-activated mitogen-activated proteinkinase cascade in plants. Proc Natl Acad Sci USA, 97,2940-2945.

Koyama M L, Levesley A, Koebner R M 0, Flowers T J & Yeo AR, 2001. Quantitative trait loci for component physiologicaltraits determining salt tolerance in rice. Plant Physiol, 125,406-422.

Lang N T, Yanagihara S & Buu B C, 2001. QTL analysis of salttolerance in rice (Oryza sativa L.). SABRAO J BreedGenet, 33. 11-20.

Lee J H, Van Montagu M & Verbruggen N, 1999. A highlyconserved kinase is an essential component of stresstolerance in yeast and plant cells. Proc Natl Acad Sci USA,96, 5873-5877.

Lemieux B, Aharoni A & Schena M, 1998. Overview of DNAchip technology. Mol Breed, 4, 277-289.

Lilley J M, Ludlow M M, McCouch S R & O'Toole J C, 1996.Locating QTL for osmotic adjustment and dehydrationtolerance in rice. J Exp Bot, 47, 1427-1436.

Lilius G, Holmberg N & BUlow L, 1996. Enhanced NaCI stresstolerance in transgenic tobacco expressing bacterial cholinedehydrogenase. Bio/Technology, 14, 177-180.

Lippuner V, Cyert M A & Gasser C S, 1996. Two classes of plantcONAs differentially complement calcineurin mutants andincreases salt tolerance of wild-type yeast. J Bioi Cheni.271, 12859-12866.

Liu J, Ishitani M, Halfter U, Kim C-S & Zhu J-K, 2000. TheArabidopsis thaliana SOS2 gene encodes a protein kinasethat is required for salt tolerance. Proc Natl Acad Sci USA.97,3730-3734.

Liu Q, Kasuga M, Sakuma Y, Abe H, Miura S, Yamaguchi-Sinozaki K & Shinozaki K. 1998. Two transcriptionfactors, OREBl and OREB2, with an EREP/AP2 DNAbinding domain separate two cellular signal transductionpathways in drought- and low-temperature-responsive geneexpression, respectively, in Arabidopsis. Plant Cell. 10.1391-1406.

Liu J & Zhu J-K, 1997. An Arabidopsis mutant that requiresincreased calcium for potassium nutrition and salttolerance. Proc Natl Acad Sci USA, 94, 14960-14964.

Mass E V, 1986. Salt tolerance of plants. Appl Agric Res. 1.12- 26.

McKersie B 0, Bowley S R, Harjanto E & Leprince O. 1996.Water-deficit tolerance and field performance of transgenicalfalfa overexpressing superoxide dismutase. Plant Physiol,111,1l77-1181.

McKersie B 0, Bowley S R & Jones K S, 1999. Winter survivalof transgenic alfalfa overexpressing superoxide dismutase.Plant Physiol, 119, 839-847.

McNeil S 0, Nuccio M L & Hanson A 0, 1999. Betaines andrelated osmoprotectants. Targets for metabolic engineeringof stress resistance. Plant Physiol, 120,945-949.

Mohanty A, Kathuria H, Ferjani A, Sakamoto A. Mohanty P.Murata N & Tyagi AK, 2002. Transgenics of an elite indicarice variety Pusa Basmati 1 harbouring the codA gene arehighly tolerant to salt stress. Theor Appl Genet (in press).

Morgan J M & Tan M K, 1996. Water use, grain yield, andosmoregulation in wheat. Aust J Plant Physiol, 13. 523-532.

Munns R, 2002. Comparative physiology of salt and water stress.Plant Cell Environ, 25, 239-250.

Nakashima K, Shinwari Z K, Sakuma Y, Seki M, Miura S.Shinozaki K & Yamaguchi-Shinozaki K, 2000.Organization and expression of two Arabidopsis OREB2genes encoding ORE-binding proteins involved indehydration- and high-salinity-responsive gene expression.Plant Mol Biol, 42, 657-665

Nanjo T, Kobayashi M, Yoshiba Y, Sanada Y. Wada K. TsukayaH, Kakubari y, Yamaguchi-Shinozaki K & Shinozaki K.1999. Biological functions of proline in morphogenesis andosmotolerance revealed in antisense transgenic Arabidopsisthaliana. Plant J, 18, 185-193.


Niu X, Bressan R A, Hasegawa P M & Pardo J M, 1995. Ionhomeostasis in NaCI environments. Plant Physiol, 109,735-742.

Noctor G & Foyer C H, 1998. Ascorbate and glutathione: keepingactive oxygen under control. Annu Rev Plant Physiol PlantMol Bioi, 49, 249-279.

Nuccio M L, Rhodes D, McNeil S D & Hanson A D, 1999.Metabolic engineering of plants for osmotic stressresistance. Curr Opin Plant Bioi, 2, 128-134.

Papageorgiou G & Murata N, 1995. The unusually strongstabilizing effects of glycine betaine on the structure andfunction of oxygen-evolving photo system II complex.Photosynth Res, 44, 243-252.

Pardo J M, Reddy M P, Yand S, Maggio A, Huh A-H, MatsumotoT, Coca MA, Paino-D'Urzo M, Koiwa H, Yun DJ, WatadAA, Bressan RA & Hasegawa PM, 1998. Stress signalingthrough Ca 2+/calmodulin-dependent protein phosphatasecalcineurin mediates salt adaptation in plants. Proc NatlAcad Sci USA, 95, 9681-9686.

Paterson A H, Lander E S, Hewitt J D, Paterson S, Lincoln S E &Tanksley S D, 1988. Resolution of quantitative traits intoMendelian factors by using a complete linkage map ofrestriction fragment length polymorphism. Nature (Lond),335,721-726.

Pih K Y, lang H J, Kang S G, Piao H L & Hwang I, 1997.Isolation of molecular markers for salt stress responses inArabidopsis thaliana. Mol Cells, 7, 567-571.

Pilon-Smits E A H, Ebskamp M J M, Paul M J, Jeuken M J W,Weisbeek P J & Smeekens S C M, 1995. Improvedperformance of transgenic fructan-accumulating tobaccounder drought stress. Plant Physiol, 107, 125-130.

Prasad K V S K, Sharmila P, Kumar P A & Saradhi PP, 2000.Transforamtion of Brassica juncea (L.) Czern withbacterial codA gene enhances its tolerance to salt stress.Mol Breed, 6, 489-499.

Qiu Q-S, Guo Y, Dietrich M A, Schumaker K S & Zhu 1-K, 2002.Regulation of SOSI, a plasma Na+!H+ exchanger inArabidopsis thaliana, by SOS2 and SOS3. Proc Natl SciAcad USA, 99, 8436-8441.

Rhoades J D & Loveday J, 1990. Salinity in irrigated agriculture.in American Society of Civil Engineers, Irrigation ofAgricultural Crops, Monograph 30, edited by B A Steward& D R Nielsen. American Society of Agronomists, USA.Pp 1089-1142.

Ribaut J M & Betran J, 2000. Single large-scale marker-assistedselection (SLS-MAS). Mol Breed,S, 531-541.

Rohila J S, Jain R K & Wu R, 2002. Genetic improvement ofBasmati rice for salt and drought tolerance by regulatedexpression of a barley HVAI cDNA. Plant Sci, 163, 525-532.

Romero C, Bellees J M, Vaya J L, Serrano R, Culiafiez-Macia FA, 1997. Expression of the yeast trehalose-6-phosphatesynthase gene in transgenic tobacco plants: Pleiotropicphenotypes include drought tolerance. Planta, 201,293-297.

Roxas V P, Smith R K Jr, Allen E R & Allen R D, 1997.Overexpression of glutathione-S-transferase/glutathioneperoxidase enhances the growth of transgenic tobaccoseedlings during stress. Nature Biotechnol, 15, 988-991.

l35

Roxas V P, Lodhi S A, Garrett D K, Mahan J R & Allen R D,2000. Stress tolerance in transgenic tobacco seedlings thatover-ex press glutath ione- A-transferase/ glutath ioneperoxidase. Plant Cell Physiol, 41, 1229-1234.

Roy M, Jain R K, Rohila J S & Wu R, 2000. Production ofagronomically superior transgenic rice plants usingAgrobacterium transformation methods: present status andfuture perspectives. Curr Sci, 79.• 954-960.

Rubio F, Gassmann W & Schroeder 11, 1995. Sodium-drivenpotassium uptake by plant potassium transporter HKTl andmutations conferring salt tolerance. Science, 270, 1660-1663.

Rus A M, Estaf M T, Gisbert C, Garcia-Sogo B, Serrano R, CaroM, Moreno V & Bolarin MC, 2001. Expressing the yeastHALl gene in tomato increases fruit yield and enhancesK+/Na+ selectivity under salt stress. Plant Cell Environ, 24.875-880.

Rus A, Yokoi S, Sharkhuu A, Reddy M, Lee B-H, Matsumoto TK, Koiwa H, Zhu J-K, Bressan R A & Hasegawa P M,2001. AtHKTI is a salt tolerance determinant that controlNa+ entry into plant roots. Proc Natl Acad Sci USA, 98,14150-14155.

Saijo Y, Hata S, Kyozuka J, Shimamoto K & Izui K, 2000. Over-expression of a single Ca2+-dependent protein kinaseconfers both cold and salt/drought tolerance on rice plants.Plant J, 23, 319-327.

Sakamoto A, Alia & Murata N, 1998. Metabolic engineering ofrice leading to biosynthesis of glycine betaine and toleranceto salt and cold. Plant Mol Bioi, 38,1011-1019.

Sakamoto A.& Murata N, 2000. Genetic engineering of glycinebetaine synthesis in plants: Current status and implicationsfor enhancement of stress tolerance. J Exp Bot, 51, 81-88.

Sasaki T, 2001. The progress in rice genomics. Euphytica, 118,103-111.

Schactman D P, Kumar R, Schoeder J I & Marsh E L, 1997. Thestructure and function of a novel cation transporter (LCT 1)in higher plants. Proc Natl Acad Sci USA, 94, 11079-11084 ..

Schactman D P & Liu W, 1999. Molecular pieces to the puzzle ofthe interaction between potassium and sodium uptake inplants. Trends Plant Sci, 4, 281-287.

Serrano R, Gaxiola R, 1994. Microbial models and salt stresstolerance in plants. Crit Rev Plant Sci, 13, 121-138.

Serrano R, Mulet 1 M, Rios G, Marquez J A, de Larrinoa I F.Leube M P, Mendizabal I, Pascual-Ahuir A, Proft M, RosR & Montesinos C, 1999. A glimpse of the mechanisms ofion homeostasis during salt stress. J Exp Bot, 50, 1023-1036.

Shen B, Jensen R G & Bohnert H J, 1997. Mannitol protectsagainst oxidation by hydroxy radicals. Plant Physiol. 115.527-532.

Sheveleva E, Chmara W, Bohnert H J & Jensen R G, 1997.Increased salt and drought tolerance by D-ononitolproduction in transgenic Nicotiana tabacuin L. PlantPhysiol,115,1211-1219.

Shi H, Ishitani M, Kim C & Zhu JK, 2000. The Arabidopsisthaliana salt tolerance gene SOS] encodes a putativeNa+/W antiporter. Proc Natl Acad Sci USA, 97, 6896-6901.

Solomon A, Beer S, Waisel v, Jones G P & Paleg L G. 1994.Effects of NaCI on carboxylating activity of Rubisco from


Tamarix jordanis in the presence and absence of proline-related compatible solutes. Plant Physiol, 90, 198-204.

Staub J E, Serquen F C & Gupta M, 1996. Genetic markers, mapconstruction and their application in plant breeding. HorticSci, 31, 729-740.

Su J, Shen Q, Ho T-H D & Wu R, 1998. Dehydration- stress-regulated transgenic expression in stable transformed riceplants. Plant Physiol, 117,913-922

Tanaka Y, Hibino T, Hayashi Y, Tanaka A, Kishitani S, TakabeT, Yokota S & Takabe T, 1999. Salt tolerance of transgenicrice overexpressing yeast mitochondrial Mn-SOD inchloroplasts. Plant Sci, 157,131-138.

Tarczynski M C, Jensen R G & Bohnert H J, 1993. Stressprotection of transgenic tobacco by production of theosmolytes mannitol. Science, 259: 508- 510.

Teulat B, This D, Khairallah M, Borries C, Ragot C, Sourdille P,Leroy P, Monneveux P & Charrier A, 1998. Several QTLsinvolved in osmotic-adjustment trait variation in barley(Hordeum vulgare L.). Theor Appl Genet, 96, 688-698.

Thomas W T B, Baird E, Fuller J D, Lawrence P, Young G R,Russell J, Ramsay L, Waugh R & Powell W, 1998.Identification of a QTL decreasing yield in barley linked toMlo powdery mildew disease. Mol Breed, 4 381-393.

Thomas J C, Sepahi M, Arendall B & Bohnert H J, 1995.Enhancement of seed germination in high salinity byengineering mannitol expression in Arabidopsis thaliana.Plant Cell Environ, 18, 801-806.

Tissier A F, Marillonnet S, Klimyuk V, Patel K, Torres M A,Murphy G & Jones J D G, 1999. Multiple independentdefective suppressor-mutator transposon insertions inArabidopsis: A tool for functional genomics. Plant Cell,11, 1841-1852.

Torsethaugen G, Pitcher L H, Zilinskas B A & Pell E J, 1997.Overproduction of ascorbate peroxidase in the tobaccochloroplast does not provide protection against ozone.Plant Physiol, 114, 529-537.

Tripathy J N, Zhang J, Robin S & Nguyen T N, 2000. QTLs forcell-membrane stability mapped in rice (Oryza sativa L.)under drought stress. Theor Appl Genet, 100, 1197-1202.

Tsugane K, Kobayashi K, Niwa Y, Ohba Y, Wada K &Kobayashi H, 1999. A recessive Arabidopsis mutant thatgrows photoautotrophically under salt stress showsenhanced active oxygen detoxification. Plant Cell, 11,1195-1206.

Tyagi A K & Mohanty A, 2000. Rice transformation for cropimprovement and functional genomics. Plant Sci, 158,1-18.

Tyagi A K, Mohanty A, Bajaj S, Chaudhury A & Maheshwari SC, 1999. Transgenic rice: A valuable Monocot System forCrop Improvement and Gene Research. Critical RevBiotechnol, 19,41-79.

Umeda M, Hara C, Matsubayashi Y, Li H-H, Liu Q, Tadokoro F,Aotsuka S & Uchimiya H, 1994. Expressed sequence tagsfrom cultured cells of rice (Oryza sativa L.) under stressedconditions: analysis of transcripts of genes engaged in ATPgenerating pathways. Plant Mol Bioi, 25, 469-478.

Van Camp W, Capiau K, Van Montagu M, Inze D, Siooten L,1996. Enhancement of oxidative stress tolerance intransgenic tobacco plants overproducing Fe-superoxidedismutase in chloroplasts. Plant Physiol, 112, 1703-1714.

Veena, Reddy V S & Sopory S K, 1999. Glyoxalase 1 fromBrassica juncea: Molecular cloning, regulation and itsover-expression confer tolerance in transgenic tobaccounder stress. Plant J, 17, 385-395.

Verma D, Madan M, Jain RK & Wu R, 2002. Agrobacteriummediated transformation of rice with SOS2 gene encodingprotein kinase that promotes salt tolerance. ill 10'hIAPTC&B Congress, Plant Biotechnology 2002 andBeyond- A Celebration and a Showcase, June 23-28,Orlando, Florida, USA. Pp 5.

Very A A, Robinson M F, Mansfield T A & Sanders D, 1998.Guard cell cation channels are involved in Naf-inducedstomatal closure in a halophyte. Plant J, 14,509-521.

Walbot V, 1999. Genes, genornes, genornics. What can plantbiologists expect from the 1998 national science foundationplant genome research program? Plant Physiol, 119, 1151-1155.

Wang H Z, Huang D N, Lu R F, Liu J J, Qian Q, Peng X X, 2000.Salt tolerance of transgenic rice (Oryza sativa L.) withmtlD gene and gutD gene. Chinese Sci Bull, 45,1685-1690.

Weig A, Deswarte C & Chrispeels M J, 1997. The major intrinsicprotein family of Arabidopsis has 23 members from threedistinct groups with functional aquaporins in each group.Plant Physiol, 114,1347-1357.

Winicov I, 1998. New molecular approaches to improving salttolerance in crop plants. Ann Bot, 82, 703-710.

Winicov I & Bastola D R, 1999. Transgenic overexpression of thetranscription factor Alfin 1 enhances expression of theendogenous MsPRP2 gene in alfalfa and improves salinitytolerance of the plants. Plant Physiol, 120,473-480.

Winkler R G, Frank M R, Galbraith D W, Feyereisen R &Feldman K A, 1998. Systematic reverse genetics oftransfer-DNA-tagged lines of Arabidopsis. Plant Phvsiol,118,743-750.

Wu S-J, Ding L & Zhu J-K, 1996. SOSJ, a genetic locus essentialfor salt tolerance and potassium acquisition. Plant Cell. 8.617-627.

Xie J H, Zapata-Arias F J, Shen M & Afza R, 2000. Salinitytolerant performance and genetic diversity of four ricevarieties. Euphytica, 116, 105-110.

Xu D, Duan X, Wang B, Hong B, Ho T-H D & Wu R, 1996.Expression of a late embryogenesis abundant protein gene.HVAJ, from barley confers tolerance to water deficit andsalt stress in transgenic rice. Plant Physiol, 110,249-257.

Yarnaguchi-Shinozaki K & Shinozaki K, 1999. Improvingdrought, salt, and freezing stress tolerance using a singlegene for a stress-inducible transcription factor in transgenicplants. in Genetic improvement of rice for water-limitedenvironments, edited by 0 Ito, J O'Toole & B Hardy. IRRI,Los Banos, Laguna, Philippines. Pp 173-179.

Yang B & Verkrnan AS, 1997. Water and glycerol perrneabilitiesof aquaporins 1-5 and MIP determined quantitatively byexpression of epitope-tagged constructs in Xenopusoocytes. J Bioi Chem, 272,16140-16146.

Yeo A, 1998. Molecular biology of salt tolerance in the context ofwhole-plant physiology. J Exp Bot, 49, 915-929.

Yeo A R, Yeo M E, Flowers S A & Flowers T J, 1990. Screeningof rice (Oryza sativa L.) genotypes for physiologicalcharacters contributing to salinity resistance, and their


relationship to overall performance. Theor Appl Genet, 79,377-384.

Young N D, 1999. A cautiously optimistic vision for marker-assisted breeding. Mol Breed,S, 505-510.

Zhang H-X & Blumwald E, 200l. Transgenic salt-tolerant tomatoplants accumulate salt in the foliage but not in the fruits.Nature Biotechnol, 19, 765-768.

Zhang G Y, Guo Y, Chen S L & Chen S Y, 1995. RFLP taggingof a salt tolerance gene in rice. Plant Sci, 110,227-234

Zhang H-X, Hodson J, Williams J P & Blumwald E, 200l.Engineering salt-tolerant Brassica plants: Characterizationof yield and seed oil quality in transgenic plants withincreased vacuolar sodium accumulation. Proc Natl AcadSci USA, 98, 12832-12836.

Zhang S & Klessig D F, 200l. MAPK cascades in plant defensesignaling. Trends Plant Sci, 6, 520-527.

Zhang J, Klueva N Y, Wang Z, Wu R, Ho T-H D & Nguyen H T,2000. Genetic engineering for abiotic stress resistance incrop plants. In Vitro Cell Dev Bioi Plant, 36, 108-114.

Zhang J, Nguyen H T & Blum A, 1999a. Genetic analysis ofosmotic adjustment in crop plants. J Exp Bot, 50, 291-302.

137

Zhang J S, Xie C, u Z Y & Chen S Y, 1999b. Expression of theplasma membrane H+-ATPase gene in response to saltstress in a rice salt-tolerant mutant and its original variety.Theor Appl Genet, 99, 1006-10 1l.

Zhang J, Zheng H G, Tripathy J N, Aarti, Sarial A K, Sarkarung S.Blum A & Nguyen H T, 1998. Mapping QTLs for osmoticadjustment in rice tOryza sativa L.). Abstr Plaut AnimalGenome VI, January 18-22, 1998, California. Pp 117.

Zhu J-K, 2000. Genetic analysis of plant salt tolerance usingArabidopsis. Plant Physiol, 124,941-948.

Zhu J-K, 200l. Plant salt tolerance. Trends Plant Sci, 6, 66-71.

Zhu J K, Hasegawa P M & Bressan R A, 1997. Molecular aspectsof osmotic stress in plants. Crit Rev Plan! Sci, 16, 253-277.

Zhu J-K, Liu J, Xiong L, 1998a. Genetic analysis of salt tolerancein Arabidopsis: Evidence for a critical role of potassiumnutrition. Plant Cell, 10,1181-1191.

Zhu B, Su J, Chang M C, Verma D P S, Fan Y L & Wu R, 1998b.Overexpression of a ",,1-pyrroline-5-caiboxylate synthetasegene and analysis of tolerance to water- and salt-stress intransgenic rice. Plant Sci, 139,41-48.

rajinder kumar jain navinder saini and randhir...

Documents