Abstract Blackwell Verlag GmbH and State of Victoria

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1 Plnt Breeding 14 Blckwell Verlg GmH nd Stte of Victori doi:.1111/pr A novel crop wter nlysis system: identifiction of wter stress tolernt genotypes of cnol (Brssic npus L.) using non-invsive mgnetic turgor pressure proes S URYA K ANT 1,DAVID B URCH 1,WILHELM E HRENBERGER 2,REBECCA B ITTER 2,SIMON R UG E R 2,JOHN M ASON 3,4,ULRICH Z IMMERMANN 2 nd G ERMAN S PANGENBERG 3,4, 1 Deprtment of Environment nd Primry Industries, Biosciences Reserch Division, Grins Innovtion Prk, 1 Ntimuk Rod, Horshm, Victori 3, Austrli; 2 ZIM Plnt Technology GmH, Neuendorfstr. 19, Hennigsdorf, D-16761, Germny; 3 Deprtment of Environment nd Primry Industries, Biosciences Reserch Division, AgriBio, Centre for AgriBioscience, Ring Rod, Bundoor, Victori 83, Austrli; 4 L Troe University, Bundoor, Victori 83, Austrli; Corresponding uthor, E-mil: germn.spngenerg@dpi.vic.gov.u With figures nd 2 tles Received Septemer 2, 13/Accepted Mrch, 14 Communicted y J. Leon Astrct Chnging glol climtic conditions nd irrigtion wter shortges impose wter stress conditions on crops. To develop genotypes tolernt to wter stress necessittes relile high-throughput methods to study plnt wter sttus nd wter stress tolernce mechnisms. We report the use of non-destructive, utomted, precise nd rpid system for ssessing rel-time wter sttus in cnol plnts. Lef ptch clmp pressure proes were clmped on the leves of four different genotypes of cnol grown under field conditions. The dt generted diurnl curves chrcterizing the pttern of turgor pressure mintennce within the leves. A novel methodology termed inverse hysteresis ws developed to mesure reltive wter stress levels in plnts using the proe-derived dt. The inverse hysteresis dt show tht genotypes CT12 nd CT hd higher ility to withstnd wter stress nd were more tolernt to wter stress thn DS23 nd DS3. The chlorophyll content nd seed yield were lso higher in CT12 nd CT. This novel nlyticl tool for monitoring wter sttus in cnol plnts will e of gret enefit in other crop species to efficiently screen genotypes for wter stress tolernce. Key words: oilseeds lef ptch clmp pressure proe ZIM-proe iotic stress reeding Glol climte chnge hs een forecsted to increse extreme wether events such s drought, errnt rins, rising temperture, hetwves nd incresed greenhouse gses (Rosenzweig et l. 1, IPCC 7, Schmidhuer nd Tuiello 7). Cropping is destined to occur in less suitle climtes thn t present, due to extreme wether conditions nd shrinking rle lnd (Rosenzweig et l. 1, Schmidhuer nd Tuiello 7). In comintion with predicted rise in world popultion to over 9 illion y, these fctors suggest tht glol food demnd is expected to doule during this period (Tilmn et l. 2, Godfry et l. ). One of the current mjor limiting fctors for crop production is wter vilility, which would e pronounced under the forecsted scenrios of climte chnge nd incresed humn popultion. Plnt scientists will inevitly need to develop genotypes tht re tolernt to wter stress conditions to comt growing food demnds. Cnol (Brssic npus L.) is n importnt oilseed crop in mny griculturl regions worldwide. It provides economic vlue to frmers s prt of crop rottion pln, due to n improved potentil for weed control, mngement of hericide resistnce nd improvement of cerel yield in the following yer. Wter stress studies on cnol show tht reduced wter vilility ffects seed yield, depending upon timing nd severity of wter stress (Chmpolivier nd Merrien 1996, Diepenrock, Ghodi et l. 6). In Austrli, cnol is predominntly grown in regions tht receive n nnul rinfll in the mm. If vrieties could e developed to dpt to n nnul rinfll < mm, productivity nd ccess to dditionl res of rle lnd would e incresed (Burton et l. 3). This reliztion hs encourged plnt reeders to select cnol genotypes tht re tolernt to wter stress conditions. A simple nd rpid method to identify such tolernt genotypes would increse the cpcity of plnt reeders to redily select for this vlule trit. For this to occur, detiled understnding of plnt wter reltions nd ility to perform, rpid ssessments of lef wter potentil nd turgor mintennce under different environmentl conditions re required. The lef wter potentil is often mesured using Scholnder om, which uses pressurized chmer to force sp from the xylem (Scholnder et l. 196). This technique is time-consuming, lorious, repetitive nd requires destructive smpling (Jones 4, Rueger et l. ). Other indirect methods of ssessing plnt wter sttus include mesurement of reltive wter content using wet nd dry plnt tissue weights, soil wter mesurements, stomtl conductnce nd visile ssessment. However, ll these methods hve limittions, mking them undesirle for ssessment of ccurte plnt wter sttus. The recent development of non-invsive lef ptch clmp pressure proe (LPCP proe; commercil nme ZIM-proe TM ) hs llowed plnt scientists nd horticulturists to mesure plnt wter sttus in rel time (Zimmermnn et l. 8). This technique is non-invsive, utomted nd precisely mesures reltime plnt wter sttus. The dt provide diurnl ptterns illustrting reltive lef turgor pressure chnges tht cn e used to study long-term (through the entire growing seson) crop responses to wter stress. This technology hs een tested successfully on severl orchrd tree species such s grpevines, olives nd nns (Zimmermnn et l. 8, 13,, Westhoff et l. 9, Fernndez et l. 11, Ehrenerger et l. 12) nd in whet grown under controlled conditions (Brmley et l. 13). The proe system hs lso een tested in comprison with sp flow, dendrometer, lef wter potentil nd soil moisture mesurements demonstrting tht this technology gives more precise informtion out the wter sttus of plnts thn the other methods (Ehrenerger et l. 12, Rodriguez-Dominguez et l. 12). The results in this pper, for the first time, descrie its ppliction to ny nnul crop plnt grown under

2 2 S. KANT,D.BURCH,W.EHRENBERGER et l. field conditions. The experiment ws conducted on cnol plnts using LPCP proes to identify genotype-specific responses to wter stress conditions. From the diurnl turgor ptterns, unique phenomenon termed inverse hysteresis hs een chrcterized, in which the time tken for lef to lose nd recover turgor pressure more thn 24-h period ecomes relile indictor of plnt wter stress conditions. The cnol genotypes tolernt to wter stress conditions tht were identified using LPCP proes exhiit positive correltion with higher chlorophyll content nd incresed yield. The interprettion of the results from this study my provide pplictions in other nnul crops, with the multiple ojectives of the following: (i) efficiently select desirle crop genotypes tolernt to wter stress conditions, (ii) identifying wter stress tolernce chrcteristics of crop genotypes, (iii) delineting criticl stges of plnt wter stress nd (iv) timely ssessment of severity of wter stress. Mterils nd Methods Experimentl detils: The experiment ws conducted under field conditions t the premises of the Deprtment of Environment nd Primry Industries t Horshm, Victori, Austrli. This re is one of Austrli s prime grin growing regions, with temperte climte providing moderte winter conditions with medium rinfll nd hot dry summers. Four genotypes of cnol (CT12, CT, DS23 nd DS3) were plnted in four replicted plots of 1 m 9 m dimension. CT12 nd CT re trnsgenic genotypes, while DS23 nd DS3 re sister null control non-trnsgenic genotypes. The trnsgenic plnts express chimeric isopentenyltrnsferse gene from Agrocterium tumefciens, under control of the AtMYB32 gene promoter. The rinfll pttern during the experiment is shown in Tle 1. Mesurements nd oservtions: The lef wter sttus ws monitored in rel time using the LPCP proes (commercil nme ZIM-proe TM ). The LPCP proes were clmped on leves of three representtive plnts from ech genotype, ensuring tht there ws no edge effect nd tht plnts were representtive of the developmentl stge nd vigour of the whole plot. The clmped lef ws the first min lef extending from the first secondry rnch derived from the primry stem, ensuring leves of similr physiologicl growth. The proes were clmped onto leves fter removl of dust nd moisture from the lef surfce to chieve homogenous contct. The lef ptch pressure redings were recorded in rel time over growth phses, strting from vegettive growth until physiologicl mturity. Mesurements of mient temperture (T) nd reltive humidity (RH) dt were lso tken simultneously from within the plot using T nd RH proes. Lef chlorophyll content ws mesured using Chlorophyll Meter SPAD-2 (Konic Minolt Optics, Inc, Tokyo, Jpn), which provides reltive chlorophyll content y mesurement of the mount of light trnsmitted (or sored) y lef t pek wvelengths of 6 nm (red) nd 9 nm (infrred). The resultnt dt vlues in the rnge of 1 provide reltive lef chlorophyll content (Chng nd Roison 3). Oservtions of chlorophyll level were tken during reproductive growth t 17 dys fter plnting (DAP). Crop iomss cover ws recorded using Crop Circle ACS-2 (Hollnd Scientific, Inc, Lincoln, NE, USA), which ws used to clculte the normlized difference vegettion Tle 1: Monthly rinfll (mm) t the experimentl site during the experiment nd long-term verge rinfll Rinfll My Jun Jul Aug Sept Oct Till hrvest (1 24 Nov) Totl Recorded Long-term verge index (NDVI). This unit mesures spectrl reflectnce, in the wvelengths of 9 nd 68 nm. NDVI ws clculted s (R 9 R 68 )/ (R 9 + R 68 ). This index cn e used s n indiction of the mount of photosynthetic iomss present within plot. Crop circle oservtions were tken t 17 DAP. Seed yield ws mesured y susmpling 1 m 2 re per plot t physiologicl mturity. Seeds were clened nd weighed seprtely for ech plot. Functionlity of LPCP proe: The principle of LPCP proes hs een descried in detil in previous pulictions (Zimmermnn et l. 8, Westhoff et l. 9). In rief, the lef ws clmped etween the two mgnetic pds of the proe, one of which contins pressure sensor chip. The clmp pressure, P clmp, tht is exerted y the two mgnets onto the lef ptch cn e djusted y vrition in the distnce etween the two mgnets, to mintin contct with the lef without cusing dmge through excess pressure. P clmp is kept constnt during turgor mesurements. The turgor pressure in the lef ptch is inversely proportionl to the mgnetic ptch pressure (P p ), mening tht P p is low t high turgor pressure nd increses s turgor pressure (P c ) drops. Theory hs shown (Zimmermnn et l. 8) tht the following eqution holds: P p ¼ 1 F P clmp ð1þ P c þ where nd re lef-specific elstic constnts equl or lrger thn unity; F is lef-specific ttenution fctor, which tkes into ccount tht prt of P clmp is explined y compression of ir-filled spces nd structurl elements such s cuticles nd cell wlls. F is prcticlly constnt even t very low turgor pressures (c. kp), even over long mesuring periods (Zimmermnn et l. 8, Westhoff et l. 9, Fernndez et l. 11). Eqution 1 represents power function indicting tht over lrge turgor pressure rnges from to 7 kp, P p exhiits liner correltion with turgor pressure. However, upon pproching the plsmolytic point (P c = ), P p increses drmticlly s smll turgor pressure decreses. Therefore, ssuming F remins constnt (even upon pproching the plsmolytic point) n increse in P p vlues cn e tken s evidence tht the turgor pressure is zero or very close to zero. If F is not constnt, reversl of the P p curves occurs (Fernndez et l. 11, Ehrenerger et l. 12). Proe signls trnsmit dt vi trnsmitter wirelessly to controller situted in the field, which is then trnsferred to n Internet server providing ccess to dt in rel time. All proes nd components for remote dt trnsfer were purchsed from the compny ZIM Plnt Technology, GmH, Hennigsdorf, Germny. Tests hve shown tht the proes operte independently to T, with chnges over rnge of C only resulting in vrition of c. 3 kp. Bsed on the P p signls recorded in the field, this noise level is non-significnt. The trnsmission time of the P p signls to the Internet server occurred every min throughout the experiment. As the dt provide mesurement for reltive lef turgor pressure chnges, therefore, the mgnitude of the initil mgnetic clmp pressure hs no effect on the diurnl profiles of the output signl of the LPCP proe in response to chnges in microclimte nd/or chnges in the irrigtion regime. Previously, experiments performed on leves of different plnt species hve demonstrted (Zimmermnn et l. 8, Westhoff et l. 9) tht proes clmped on nery leves responded lmost identiclly upon temporry chnges in trnspirtion nd/or wter supply even if the initil clmp pressure vlues were different due to differences in lef thickness nd deformility properties nd locl compressiility. Results Diurnl lef ptch pressure (P p ) pttern The P p on the leves clmped with LPCP proes nd temperture (T) nd RH in the microclimte within the plot were recorded in

3 Novel crop wter nlysis system 3 rel time. Figure 1 displys the diurnl P p cycles for selected time periods, to provide exmples of P p pek correltions with T, RH nd wter vilility. The P p peks during the dy nd night continue to increse with rising T during DAP (Fig. 1). At 2 DAP, there ws dytime T drop, ut the P p pek vlues remined high despite this T decrese, indicting tht incresing wter stress ws leding to higher turgor loss. Following rinfll on the night of 2 DAP, P p peked t lower level, indicting recovery of lef turgor (Fig. 1). The P p peks were consistent with T during 8 16 DAP (Fig. 1). Following rinfll on the night of 16 DAP, P p peks lowered riefly, followed y n increse, with the highest pek t 167 DAP (Fig. 1). During this period, P p mximum vlues during the dy nd minimum night-time P p records continued to rise due to wter stress, while () r Pp (kp) Pp (kp) T ( o C) () Dys fter plnting r 8 6 RH (%) T ( o C) Dys fter plnting 8 6 RH (%) Fig. 1: Typicl lef P p vlues in cnol, with corresponding chnges in mient temperture nd reltive humidity (RH). Rin (r) events re denoted with downwrd pointed rrows. Nocturnl hours re mrked y grey columns. () An exmple of P p vlues which were correlted predominntly y the increse in mient T (resulting in n increse in trnspirtionl wter loss). () P p peking t noon nd night P p vlues incresed continuously. The drmtic increse in P p peking t noon on 167 dys fter plnting indictes tht turgor pressure pproched to zero s modelled in Eqn (1). Lef senescence occurred nd ws mnifested y the drop of P p to zero in the following dy. Amient temperture (T; solid line) nd RH (dotted line).

4 4 S. KANT,D.BURCH,W.EHRENBERGER et l. H < % H 1 % H 2 % H 3 >% H 4 Lef senescence Fig. 2: Typicl dependencies of P p on T mesured on leves of cnol plnts. Closed squres represent the dependency of P p on T during the morning hours (from sunrise until P p peking t noon), nd open circles represent the reltionship etween P p nd T during the fternoon nd night hours (from noon to sunrise of the following dy). The different levels of inverse hysteresis were termed H to H 4, ech level indicting different degree of wter stress. The inverse hysteresis loops were determined y the clcultion of the re etween the two P p = f (T) curves. The re of the hysteresis loop ws normlized y referring to the coordintes of the lowest nd highest P p redings of the proes nd determining the rectngulr re which encompssed the ellipticl hysteresis loop (see inset in the H to H 3 grphs). ility of the plnt to return to full lef turgor ws inhiited. At 167 DAP, the P p pek incresed y out 43 kp when compred to the previous dy, indicting tht lef turgor pressure ws pproching zero (see Eqn 1). This is consistent with oservtions tht the P p dropped to very low vlues, within the ccepted noise level of the proe. At this stge, the photosynthetic cpcity of the lef ws irreversily degrded nd lef senescence ws initited. This ws confirmed y visile oservtions of the lef chnging from green to yellow s n indiction of lef senescence. The phenomenon of inverse hysteresis A plot of P p versus T gives insight into the turgor potentil recovery of the lef cells following trnspirtionl wter loss nd/or limited wter uptke through the roots. When P p is plotted ginst T (Fig. 2, with morning vlues in closed squres nd fternoon vlues in open circles), it otins chrcteristic ellipticl loop with fster P p decrese, s T declines during the evening hours, compred with morning increse in P p with rising T, indicting fster recovery of turgor pressure reltive to its loss. This phenomenon is referred to s inverse hysteresis. The intensity of inverse hysteresis could vry due to different fctors such s trnspirtionl wter loss, wter use for plnt growth nd cellulr development, wter stress levels nd the lef growth stge. The mjor contriuting fctors, however, re wter stress levels nd rpid turgor chnges during lef senescence. The intensity of the inverse hysteresis ws determined y clculting the re within the two curves from the morning nd evening hours s P p = f (T), which roughly forms n ellipticl

5 Novel crop wter nlysis system loop. The re of the hysteresis loop ws normlized y referring to the coordintes of the lowest nd highest P p redings of the proes, determining the rectngulr re which encompsses the ellipticl hysteresis loop (Fig. 2, inset H to H 3 ). The inverse hysteresis ws seprted into five ctegories, H,H 1,H 2,H 3 nd H 4, depending upon intensity level. The hysteresis H component contins loop re of <%, indicting the reduction in P p with T in fternoon correltes lmost exctly with the morning increse in P p with T. As lef cells return to full turgescence esily in evening hours, it is sfe to ssume tht there ws no or only mild wter stress imposed on the plnt t this point. H 1 nd H 2 hysteresis, with reltive loop res of etween % nd % nd %, respectively, disply typicl inverse hysteresis s the plnts recover P p more rpidly thn T decrese. Agin, this rpid return of full lef turgor indictes tht there is n dequte supply of wter from the plnt roots to replce ny wter loss through trnspirtion nd usge for growth during the wrmer dytime period. These H, H 1 nd H 2 hysteresis phses indicte tht there ws no wter stress or cellulr degrdtion preventing wter uptke, tht is, the leves were helthy, functionl nd photosynthesizing normlly with sufficient soil wter ville for plnt growth. H 3 hysteresis is the point t which the difference in wter stress tolernce etween the cnol genotypes ws oservle through the LPCP proes. The lrger loop re would indicte fster turgor recovery, ut disturnces in the lte evening P p suggest tht stress ehviour hs initited in the leves (Fig. 2). During period of wter stress, plnt leves cn experience xylem emolisms, in which ir spces fill the xylem where wter would normlly e present. Lck of wter in the extrcellulr mtrix of the lef tissues nd the xylem vessels is the first sign of wter stress within the plnt. Reduction in pressure of these emolisms is the cuse of rpid P p decreses, rther thn turgor recovery s seen in prior hysteresis stges. The indictions of xylem emolism were oserved in the evening when P p initilly decresed in liner fshion with T, ut then exhiited shrp increse in P p towrds the end of the dy, referred s undershoot hysteresis. This effect ws due to wter returning to the cells to replce the ir tht ws present, s the emolisms within the xylem relxed nd the gs volume reduced. Undershoot hysteresis ptterns re expected t this level of wter stress (Zimmermnn et l. 8, Fernndez et l. 11, Ehrenerger et l. 12). At this stge, wter stress is reversile with resumption of wter ppliction nd meliortion of het. The pronounced hysteresis of the H 4 ctegory is the inevitle result of wter stress leding to lef senescence. P p increses t much fster rte during the morning s compred to prior hysteresis phses, indicting pre-existing wter stress nd the rekdown of cellulr processes tht regulte wter loss. Following the P p pek t mid-dy depression, there ws rpid decrese in the evening, which eventully formed plteu t very low level (equivlent to the ckground Tle 2: Reltive frequency of hysteresis levels in cnol genotypes Hysteresis levels (%) CT12 CT DS23 DS3 Reltive frequency of hysteresis levels H H H H H noise level of the proes) with no indiction of further wter uptke s oserved in H 3 hysteresis. At this stge, the lef hs lost the cpcity to recover wter from its roots nd trnsport it to its photosynthetic structures, which re undergoing physiologicl degrdtion. Mesurements fter H 4 hysteresis did not follow ny discernile pttern, with only slight diurnl curve in scttered dt points due to smll T chnges in ir or trpped wter in the senesced lef tissue. Inverse hysteresis pttern-delineted wter stress tolernce in cnol genotypes Inverse hysteresis levels (H to H 4 ) were ssigned to ech genotype for ech dy during the nlysis time spn of 4 dys preceding physiologicl mturity. This ws sed on proe redings for ech 24-h period, providing three hysteresis vlues representing three LPCP proes per plot per dy. From this detiled mtrix of inverse hysteresis levels of four genotypes clmped with three proes ech for 4 dys (dt not shown), the reltive frequency, tht is, the percentge vlue of ech hysteresis level ws clculted s the numer of dys the prticulr hysteresis occurred for given genotype divided y 4 dys (Tle 2). H, H 1 nd H 2 hysteresis levels indicte miniml or no wter stress. There ws little difference in frequencies tht ll genotypes spent in comintion of H,H 1 nd H 2 hysteresis (88.2, 79., 86.1 nd 8.3% for CT12, CT, DS23 nd DS3, respectively). The criticl issue ws the occurrence of H 3 nd H 4 hysteresis, s H 3 is sustntil, ut reversile stge of wter stress nd H 4 is n irreversile rekdown of cellulr mechnisms leding to lef senescence. The vrious genotypes differed in frequency of H 3 nd H 4 hysteresis. Genotypes CT12 nd CT spent longer reltive time in H 3 hysteresis compred with DS23 nd DS3 (Tle 2). As H 3 hysteresis represents wter stress, the fct tht the former two genotypes were oserved in this phse for longer period indictes higher tolernce to wter stress. CT12 nd CT could retin sic cellulr functions nd mintin turgor recovery for longer period, in contrst to DS23 nd DS3 which filed to survive under such conditions for s long nd entered into H 4 hysteresis. This oservtion suggests, genotypes DS23 nd DS3 lck the cpcity to tolerte conditions of prolonged wter stress to the sme degree s CT12 nd CT, due to the short H 3 frequency efore initition of senescence. Genotypes CT12 nd CT lso hd lower frequencies of H4 hysteresis compred to DS23 nd DS3, indicting mintennce of turgor for longer period nd delyed onset of lef senescence. To illustrte the reltive time periods ech genotype spent in ech wter stress hysteresis ctegory, the H 3 :H 4 rtio ws clculted for ech genotype: 1.68, 1.9,.2 nd. for CT12, CT, DS23 nd DS3, respectively. This rtio indictes tht genotypes CT12 nd CT hd greter cpcity to survive wter stress conditions nd remin photosyntheticlly functionl for longer period in order to efficiently utilize stored photosynthtes for greter grin filling nd yield ttriutes. Seed yield nd chlorophyll level in genotypes tolernt to wter stress conditions Genotypes CT12 nd CT exhiited significntly higher (13 14%) reltive chlorophyll levels thn DS23 nd DS3 (Fig. 3). Similrly, for NDVI which is n estimte of green photosynthetic

6 6 S. KANT,D.BURCH,W.EHRENBERGER et l. iomss level, CT12 nd CT hd significntly greter NDVI vlues (13 14%) compred with DS23 nd DS3 (Fig. 4). Both methods used for mesuring cnopy chlorophyll nd greenness showed tht genotypes CT12 nd CT hd greter photosyntheticlly ctive green folige t reproductive stge (17 DAP) compred with DS23 nd DS3, n indiction for improved grin filling. Genotypes CT12 nd CT showed higher seed yield with significntly increse of 23 26% compred with DS23 nd DS3 (Fig. ). Higher chlorophyll levels (Fig. 3) nd NDVI vlues (Fig. 4) nd incresed wter stress tolernce (Tle 2) of genotypes CT12 nd CT would hve ccounted for improved seed yield (Fig. ) in these genotypes. The improved cpcity to photosynthesize under wter stress conditions is likely to led to NDVI Seed yield (g/m 2 ) CT12 CT DS23 DS3 Genotypes Fig. 4: Normlized difference vegettion index (NDVI) in cnol genotypes. Dt re mens SE (n = 4). Brs with different letters (, ) indicte significnt difference t P <.. SPAD chlorophyll level 4 3 CT12 CT DS23 DS3 Genotypes Fig. 3: SPAD chlorophyll levels in cnol genotypes. Dt re mens SE (n = 4). Brs with different letters (, ) indicte significnt difference t P <.. CT12 CT DS23 DS3 Genotypes Fig. : Seed yield in cnol genotypes. Dt re mens SD (n = 4). Brs with different letters (, ) indicte significnt difference t P <.. higher seed yield, trit of gret relevnce to frmers cropping under conditions of low wter vilility. Discussion Wter stress t ny stge of cnol life cycle cn negtively impct on seed yield, depending upon the severity of stress nd the growth stge of plnts. A shortge of wter during flowering nd grin filling cn e detrimentl for seed yield due to the following: (i) reduced florl development nd lower grin numer, (ii) premture loss of photosynthetic cpcity due to erly lef senescence nd (iii) n inility to remoilize stored metolites for grin filling due to reduced sink utiliztion cpcity nd smller seed size (Chmpolivier nd Merrien 1996, Diepenrock, Ghodi et l. 6). The totl rinfll received during the experimentl seson ws lower thn the long-term verge rinfll for the region (Tle 1). Specificlly, rinfll received during the month of Septemer ws remrkly low, nd lso elow verge during Octoer (Tle 1), which ws the criticl period for flowering, silique formtion nd grin filling. These conditions imposed wter stress on the plnts t the experimentl site, such tht tolernt genotypes displyed competitive dvntge over susceptile genotypes. Timely identifiction of tolernt genotypes during wter stress periods, rther thn dependence on seed yield dt nd n understnding of wter stress tolernce mechnisms, would e of genuine enefit to plnt scientists for effective screening of such genotypes. Relince on yield chrcteristics lone s n indiction of wter stress tolernce could e confounded y environmentl fctors, s well s plnt genotypic nd phenotypic trits. The technique pplied in this experiment demonstrtes direct correltion etween wter stress tolernce nd incresed yield. Additionlly, the timely wrning of the onset of criticl wter stress levels otined through use of the LPCP proes would ssist frmers to irrigte in mnner which provides high yield, while lso efficiently using vlule fresh wter resources. The development of novel technologies for the rpid selection of superior genotypes is vitl for plnt reeders. Climte chnge is expected to induce more intense drought events (Rosenzweig et l. 1, Schmidhuer nd Tuiello 7), nd so development of crop genotypes tht cn tolerte errtic wter vilility will provide significnt chllenge for plnt reeders. Efficient detection of wter stress in high-vlue nnul crops would e huge dvntge to plnt reserchers, providing greter cpcity to trget complex polygenic trit such s wter stress tolernce. The ility to cpture plnt wter reltion dt in rel time for n entire growing seson using LPCP proes will e n invlule tool for enhncement of understnding of how crops rect to wter stress, nd how to select the most tolernt genotypes nd develop efficient irrigtion regimes. In genotypic selection process for which decisions re sed upon sutle phenotypic differences, precise nd relile method of quntifiction of wter stress tolernce is vlule tool. As demonstrted in the present study, lef turgor mintennce cn e oserved using LPCP proes, which record reltime mesurements of chnges in lef wter sttus. Here, for the first time, it is reported tht plot of P p ginst T cn e used to determine plnt wter sttus nd diurnl turgor recovery. Wter stress ws mnifested in inverse hysteresis phenomen of vrying degrees when the reltionship etween P p nd T ws plotted for the morning nd fternoon hours. By delineting the frequencies of different hysteresis levels for the cnol genotypes tht were used in the field experiment, two genotypes (CT12

7 Novel crop wter nlysis system 7 nd CT) were identified tht displyed higher tolernce to wter stress under low rinfll vilility (Tle 2). Higher wter stress tolernce in these genotypes ws trnslted into mintennce of higher chlorophyll levels nd greener crop iomss cover during grin filling nd susequently greter seed yield (Figs 3 ). A plnt tht exhiited n incresed ility to mintin photosyntheticlly ctive source tissues during the crucil grin filling period llows for greter light energy cpture, generting greter seed yield. It would lso llow metolites contined in the source tissues to e systemticlly relesed for incresed grin filling. In conclusion, novel nlyticl tool for monitoring wter sttus in cnol plnts hs een developed using LPCP proes. These dt, comined with lef chlorophyll content nd seed yield, demonstrted tht the proes, which record rel-time plnt wter sttus in non-destructive mnner, cn precisely ssess whether nd when genotype is fcing wter shortge nd the intensity of wter stress conditions. By developing n index of inverse hysteresis, n esy nd rpid method for ssessment of n gronomicl importnt trit of wter stress tolernce in cnol hs een otined. This method, if pplied to other crops nd experiments, hs the potentil to ecome powerful tool for plnt scientists to efficiently select desirle genotypes tolernt to wter stressed environments. References Brmley, H., W. Ehrenerger, U. Zimmermnn, J. Plt, S. R uger, nd K. M. Siddique, 13: Non-invsive pressure proes mgneticlly clmped to leves to monitor the wter sttus of whet. Plnt Soil 369, Burton, W., P. Slisury, nd D. Potts, 3: The potentil of cnol qulity Brssic junce s n oilseed crop for Austrli. 13th Austrlin Reserch Assemly on Brssics, Chmpolivier, L., nd A. Merrien, 1996: Effects of wter stress pplied t different growth stges to Brssic npus L. vr. oleifer on yield, yield components nd seed qulity. Eur. J. Agron., Chng, S. X., nd D. J. Roison, 3: Nondestructive nd rpid estimtion of hrdwood folir nitrogen sttus using the SPAD-2 chlorophyll meter. For. Ecol. Mnge. 181, Diepenrock, W., : Yield nlysis of winter oilseed rpe (Brssic npus L.): review. Field Crops Res. 67, Ehrenerger, W., S. R uger, R. Fitzke, P. Vollenweider, M. G unthrdt- Goerg, T. Kuster, U. Zimmermnn, nd M. Arend, 12: Concomitnt dendrometer nd lef ptch pressure proe mesurements revel the effect of microclimte nd soil moisture on diurnl stem wter nd lef turgor vritions in young ok trees. Funct. Plnt Biol. 39, 297. Ehrenerger, W., S. Rueger, C. M. Rodriguez-Dominguez, A. Diz-Espejo, J. E. Fernndez, J. Moreno, D. Zimmermnn, V. L. Sukhorukov, nd U. Zimmermnn, 12: Lef ptch clmp pressure proe mesurements on olive leves in nerly turgorless stte. Plnt Biol. (Stuttg). 14, Fernndez, J. E., C. M. Rodriguez-Dominguez, A. Perez-Mrtin, U. Zimmermnn, S. Rueger, M. J. Mrtin-Plomo, J. M. Torres-Ruiz, M. V. Cuevs, C. Snn, W. Ehrenerger, nd A. Diz-Espejo, 11: Online-monitoring of tree wter stress in hedgerow olive orchrd using the lef ptch clmp pressure proe. Agric. Wter Mng., 2 3. Ghodi, M., M. Bkhshndeh, G. Fthi, M. H. Ghrineh, K. Almi- Sid, A. Nderi, nd M. E. Ghodi, 6: Short nd long periods of wter stress during different growth stges of Cnol (Brssic npus L.): effect on yield, yield components, seed oil nd protein contents. J. Agron., Godfry, H. C. J., J. R. Beddington, I. R. Crute, L. Hddd, D. Lwrence, J. F. Muir, J. Pretty, S. Roinson, S. M. Thoms, nd C. Toulmin, : Food security: the chllenge of feeding 9 illion people. Science 327, IPCC, 7: Climte chnge 7: the physicl science sis. In: S. Solomon, D. Qin, M. Mnning, Z. Chen, M. Mrquis, K.B. Averyt, M. Tignor nd H.L. Miller. 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