Introduction. Keywords: galactinol synthase, drought, transgenic rice, grain yield, confined field trial.

Transcription

1 Plnt Biotechnology Journl (217) 15, pp doi: /pi Overexpression of n Aridopsis thlin glctinol synthse gene improves drought tolernce in trnsgenic rice nd incresed grin yield in the field Michel Gomez Selvrj 1, Tkum Ishizki 2, Milton Vlenci 1, Stoshi Ogw 1,3, Bet Dedicov 1, Tkuy Ogt 4, Kyouko Yoshiwr 4, Kyonoshin Mruym 4, Miyko Kusno 5,6,7, Kzuki Sito 5,6,8, Fuminori Tkhshi 5,6, Kzuo Shinozki 5,6, Kzuo Nkshim 4 nd Mnu Ishitni 1, * 1 Interntionl Center for Tropicl Agriculture (CIAT), Cli, Colomi 2 Tropicl Agriculture Reserch Front (TARF), Jpn Interntionl Reserch Center for Agriculturl Sciences (JIRCAS), Ishigki, Okinw, Jpn 3 Jpn Society for the Promotion of Science, The University of Tokyo, Bunkyo-ku, Tokyo, Jpn 4 Biologicl Resources nd Post-hrvest Division, Jpn Interntionl Reserch Center for Agriculturl Sciences (JIRCAS), Tsuku, Irki, Jpn 5 RIKEN Center for Sustinle Resource Science, Yokohm, Kngw, Jpn 6 RIKEN Center for Sustinle Resource Science, Tsuku, Irki, Jpn 7 Grdute School of Life nd Environmentl Sciences, University of Tsuku, Tsuku, Irki, Jpn 8 Deprtment of Moleculr Biology nd Biotechnology, Grdute School of Phrmceuticl Sciences, Chi University, Chi, Jpn Received 12 August 216; revised 1 Ferury 217; ccepted 16 Mrch 217. *Correspondence (Tel ; fx ; emil m.ishitni@cgiar.org) Keywords: glctinol synthse, drought, trnsgenic rice, grin yield, confined field tril. Astrct Drought stress hs often cused significnt decreses in crop production which could e ssocited with glol wrming. Enhncing drought tolernce without grin yield penlty hs een gret chllenge in crop improvement. Here, we report the Aridopsis thlin glctinol synthse 2 gene (AtGolS2) ws le to confer drought tolernce nd increse grin yield in two different rice (Oryz stiv) genotypes under dry field conditions. The developed trnsgenic lines expressing AtGolS2 under the control of the constitutive mize uiquitin promoter () lso hd higher levels of glctinol thn the non-trnsgenic control. The incresed grin yield of the trnsgenic rice under drought conditions ws relted to higher numer of pnicles, grin fertility nd iomss. Extensive confined field trils using trnsgenic lines in Curing, tropicl jponic nd NERICA4, interspecific hyrid cross two different sesons nd environments reveled the verified lines hve the proven field drought tolernce of the Ui: AtGolS2 trnsgenic rice. The mended drought tolernce ws ssocited with higher reltive wter content of leves, higher photosynthesis ctivity, lesser reduction in plnt growth nd fster recovering ility. Collectively, our results provide strong evidence tht AtGolS2 is useful iotechnologicl tool to reduce grin yield losses in rice eyond genetic differences under field drought stress. Introduction Drought is mjor iotic stress condition criticlly limiting crop production nd yield (Edmedes, 28). Climte prediction models suggest tht iotic stresses will increse in the ner future ecuse of glol climte chnge (Ahuj et l., 21). The ever-rising world popultion nd recurrent glol climte chnge chllenge the griculturl system to produce sufficient food to feed the world (Godfry et l., 21). As the world s secondlrgest crop, rice plys criticl role in food security for more thn hlf of the world s popultion (FAO, 216: org/rowse/q/qc/e). Rice ccounts for out 27% of totl cerel production, with worldwide production of roughly million tons (FAO, 216). By 235, 26% increse in rice production will e required to feed the growing popultion (Cssmn et l., 23; Seck et l., 212). Glol wter shortge is mjor issue for cultivted rice, which needs lrge quntities of wter (Mnvln et l., 212). It ws reported tht the glol reduction in rice production due to drought verges 18 million tons nnully (O Toole, 24). Worldwide, drought ffects pproximtely 23 million h of rice production under rinfed conditions. Drought is prticulrly frequent in ununded uplnds, unded uplnds nd shllow rinfed lowlnd fields in mny prts of South nd South-Est Asi, su-shrn Afric nd Ltin Americ (Serrj et l., 211). To resolve those glol prolems, it is importnt to improve crop yields especilly within stple food crops like rice (Oryz stiv L.) through reeding-improved stress tolernce. Trnsgenic technologies re one of the numerous tools ville to plnt reeding progrmmes, which help to open new venues for crop improvement y developing crop cultivrs resistnt to vrious iotic nd iotic stresses (Younis et l., 214). Around million hectres of iotech crops were grown glolly nd trnsgenic crege grew 3% in 213, representing 35% of the glol seed mrket (Mrshll, 214). In rice, progress hs een mde in the genertion nd evlution of trnsgenic rice events ginst drought tolernce (Todk et l., 215). ª 217 The Authors. Plnt Biotechnology Journl pulished y Society for Experimentl Biology nd The Assocition of Applied Biologists nd John Wiley & Sons Ltd. This is n open ccess rticle under the terms of the Cretive Commons Attriution License, which permits use, distriution nd reproduction in ny medium, provided the originl work is properly cited. 1465

2 1466 Michel Gomez Selvrj et l. Plnts hve evolved severl mechnisms to ccustom to iotic stresses through chnges t the physiologicl levels nd moleculr levels (Todk et l., 212; Ymguchi-Shinozki nd Shinozki, 26). It is suggested tht overexpression of stress-relted genes could improve drought tolernce in rice (reviewed y Nkshim et l., 214 nd Todk et l., 215). Despite such efforts to develop drought-tolernt rice plnts, very few hve een shown to improve grin yields under the field environments (Gudin et l., 213). Encourging results include trnsgenic rice plnts expressing OsNAC5 (Jeong et l., 213), OsNAC9/SNAC1 (Redills et l., 212) or OsNAC1 (Jeong et l., 21), which ws shown to improve grin yield under field drought conditions. Mny genes tht my ply n importnt role under drought hve een mostly tested on single model rice genetic ckground (Nipponre) under lortory conditions, ut very few hve een tested vigorously in nturl trget environment using different commercil rice genetic ckgrounds. For improved rice to e ccepted y consumers, it is necessry to consider oth dpttion to the trget environments nd fulfilment of locl grin qulity nd tste preferences. This is predominntly importnt in trnsgenic studies in which the recipient genetic ckground is often chosen ccording to its ility to e trnsformed rther thn gronomic or culturl considertions (Gudin et l., 213). The ccumultions of metolite or osmoprotectnts re one of the key dptive mechnisms for plnts to hndle with dehydrtion stress nd cellulr injury (Hre et l., 1998). Solule sugrs, including those in the sucrose, trehlose nd rffinose fmilies lso known s oligoscchrides (RFOs), hve een found to ccumulte during drought stress in mny plnts (Collett et l., 24; Frrnt, 27; Peters et l., 27; Tji et l., 22). Glctinol synthse (GolS), key enzyme in the metolic pthwy leding to RFOs, synthesizes glctinol (from UDP-Gl nd myoinositol), which serves s glctosyl donor to form rffinose, stchyose nd verscose (Pnikulngr et l., 24). It hs een reported tht the production of enzymes involving the iosynthesis of RFOs, nd the resulting ccumultion of RFOs, plys criticl roles in cquired tolernce of Aridopsis thlin to drought nd het stresses (Tji et l., 22; Nishizw et l., 28; reviewed y Sengupt et l., 215). In Aridopsis, seven GolS genes nd three puttive GolS genes hve een identified, nd intricte induction ptterns were reported (Nishizw et l., 28). AtGolS1 ws inducile y drought, slinity (Tji et l., 22) nd temperture stresses (Pnikulngr et l., 24); AtGolS2 ws induced only y drought nd slinity stresses; nd AtGolS3 induction ws detected solely fter cold stress (Tji et l., 22). Overexpression of AtGolS2 cused the increse in glctinol nd rffinose contents in leves nd exhiited enhnced drought tolernce of trnsgenic Aridopsis (Tji et l., 22). Here, we descrie the production of trnsgenic rice events tht overexpress the AtGolS2 cdna driven y the mize uiquitin promoter () in the ckground of Curing ( Brzilin locl uplnd rice vriety) nd NERICA4 ( populr uplnd rice vriety in Africn countries) nd present the results of multiple confined field trils over two different environmentl conditions. Our extensive field test over different sesons nd environmentl conditions using multiple rice genetic ckgrounds clerly demonstrted tht AtGolS2 overexpression consistently incresed iomss nd grin yield under drought stress conditions. These findings implied tht the trnsgene plyed n importnt role in improving gronomic trits nd yield chrcteristics of rice nd tht overexpression would e n efficient wy to ccelerte the rice reeding progrmme for drought tolernce. Results Genertion nd moleculr nlyses of rice events expressing Our gol ws to produce nd select est-performing Curing nd NERICA4 trnsgenic rice lines for drought tolernce y expressing AtGolS2 from the constitutive mize uiquitin (Ui) promoter. We ccomplished this using Agrocterium-medited trnsformtion (Ishizki nd Kumshiro, 28; Zunig-Soto et l., 215). At lest 2 independent trnsgenic events were produced from ech vriety. T 3 or T 4 seeds tht possessed geneticlly fixed single copy of trnsgene were used for further nlyses. To link field performnce of the trnsgenic lines to the trnsgene expression levels nd metolite ccumultion levels. Therefore, quntittive PCR expression of trnsgenic Curing nd trnsgenic NERICA4 events ws nlysed y quntittive rel-time PCR (RT-PCR). All of the trnsgenic Curing lines expressed the trnsgene (Figure 1). Glctinol synthse (GolS) ctlyses the first committed step in the iosynthesis of rffinose fmily oligoscchrides (RFOs) including glctinol nd rffinose nd plys key regultory role in cron prtitioning etween sucrose nd RFOs (Srvitz et l., 1987; Tji et l., 22). We mesured glctinol content in the promising trnsgenic plnts grown in glsshouse under unstressed conditions. The NT Curing nd NERICA4 were used s the control. Under norml growth conditions, ech trnsgenic plnt showed significntly higher ccumultion of glctinol s compred with NT rice plnts (Figure 1). All of the trnsgenic Curing lines expressed the trnsgene nd the expression levels of the trnsgene in lines #325 nd #3214 were higher thn in the other lines (#258, #259, #2783 nd #32). In cse of NERICA4, the AtGolS2 gene ws overexpressed nd expression level of the gene nd ccumultion level of glctinol in line #1577 (NERICA4) were higher thn in other lines tested (Figure 1, ). We lso nlysed the expression of representtive drought mrker genes in the trnsgenic rice. Expression of genes for trnscription fctor OsNAC6, isocitrte lyse (ICL) nd lte emryogenesis-undnt protein LEA3 (Mruym et l., 214; Nkshim et l., 214) ws not induced in the events for without drought stress (Figure S1). These results indicte tht the ccumultion of glctinol is not relted to the expression of these droughtinducile genes. Drought tolernce of Curing lines t vegettive-stge stress Drought tolernce during the seedling growth period ws importnt for rice plnt estlishment in res where erlyseson drought overlpped with the vegettive stge. We conducted vegettive-stge drought experiments using homozygous trnsgenic rice lines overexpressing in Curing. The experiment ws conducted in rinout shelter fcility t CIAT, Colomi, nd iomss ws used s min criterion to select promising lines. To evlute the growth performnce of the - overexpressing Curing under drought conditions t the vegettive stge, three-week-old trnsgenic nd nontrnsgenic (NT) Curing control plnts were sujected to drought stress for up to 3 weeks in Novemer-Decemer 211 (Figure 2). Agronomic ª 217 The Authors. Plnt Biotechnology Journl pulished y Society for Experimentl Biology nd The Assocition of Applied Biologists nd John Wiley & Sons Ltd., 15,

3 AtGolS2 improves field drought tolernce in rice 1467 () 12 Reltive mrna level Expression of AtGolS2 1 Figure 1 Ectopic overexpression of AtGolS2 gene confers higher glctinol ccumultion on trnsgenic Curing nd NERICA4 rice. () Expression of AtGolS2 in trnsgenic rice ws nlysed y quntittive rel-time PCR. Expression of OsUi1 ws nlysed s n internl control to normlize the expression of AtGolS2. () Accumultion of glctinol in trnsgenic rice ws nlysed using GC-TOF-MS. The highest verge vlue of the smples ws set s 1, nd the reltive vlues were shown in () nd (). Five plnts were comined into one smple for ech line, nd the reltive vlues re men SD of three technicl replictes. Nontrnsgenic (NT) Curing nd NERICA4 were used s the control. () 12 Reltive ccumultion level NT NT Curing NERICA4 Glctinol NT NT Curing NERICA4 dt collected efore stress tretment showed tht there ws no significnt vrition mong the lines, which helps to explin the uniformity in the experiment. During pek stress, the trnsgenic lines significntly mintined more plnt height thn NT Curing (Figure 2e). The NT Curing strted to show visul symptoms of drought-induced lef rolling t n erlier stge thn the trnsgenic plnts (Figure 2). The trnsgenic lines showed low lef rolling score, 2-3 compred with the NT Curing plnts with (6.5) during pek drought stress (Figure 2). Only the event #259 ccumulted significntly higher dry iomss thn NT Curing t the end of drought stress (Figure 2c). However, dry iomsses of the trnsgenic plnts mesured fter rewtering were significntly higher thn those of NT Curing in ll ut one instnce (Figure 2d). Drought tolernce of the Curing lines under Mnged Drought Stress Environment (MDSE) In order to confirm the drought tolernce of Curing trnsgenic lines t the reproductive stge, three consecutive confined field trils were conducted under the removle rinout shelter fcility t CIAT, Plmir. As mentioned ove, we hd conducted two drought stress trils in the yer 212 over two cultivting sesons (212-riny seson-mdse-tril-1 nd 212-dry seson-mdse- Tril-2) nd one in 214 (214-riny seson-mdse-tril) (Figure S2). Up to eight independent T 4 single-copy homozygous trnsgenic nd NT Curing lines were used to conduct the drydown experiments. In ddition to the field drought experiments, one norml well-wtered pddy field tril (WW-field tril) ws lso conducted during the dry seson of 212 (Tle S1). In the WWfield tril, single plnt grin yield of ll the tested trnsgenic lines nd NT Curing ws not significntly different (Tle S1), which suggests no yield penlty in these trnsgenic lines. Bsed on these results, grin yield ws lso used s min prmeter to compre yield in dry fields etween these trnsgenic lines nd NT Curing. Drought intensity vried mong drought trils from mild to severe (Figure S2). Although similr levels of stress durtion nd environmentl conditions occurred in the riny seson drought experiments, it ws oserved tht verge single plnt yield of NT Curing ws shrply reduced compred to the other two riny seson experiments (Figure 3, nd c). This suggests dry seson effect. Additionlly, we found tht soil moisture rpidly decresed within the - to 4-cm soil lyer compred to other two riny seson experiments (Figure S2). The level of drought stress imposed under uplnd rinout shelter conditions ws equivlent to tht which cused n verge reduction of round 6% 7% in the single plnt grin yield otined in the NT Curing under well-wtered pddy field conditions (Tle S1 nd Figure 3, nd c). Sttisticl nlysis of yield nd yield-relted prmeters scored for three rinout shelter drought experiments reveled tht the overexpression consistently produced grin yield compred to NT cross the seson. Interestingly, in the Curing lines, the morphophysiologicl trit performnce ws significntly etter thn NT Curing (Tle S2 nd Figure 3). In ech tril, we found some trnsgenic lines tht hd significntly higher yield: three of eight trnsgenic ª 217 The Authors. Plnt Biotechnology Journl pulished y Society for Experimentl Biology nd The Assocition of Applied Biologists nd John Wiley & Sons Ltd., 15,

4 1468 Michel Gomez Selvrj et l. () 1 AquPro soil moisture (%) Dry iomss t the end of stress (g) Imposing drought stress Drought period Smpling t the end of stress Rewtering Smpling fter stress recovery Dys After Sowing (c) NT (e) 5 4 Plnt height (cm) cm 2-6 cm () Lef rolling score (d) 2.8 Dry iomss fter stress recovery (g) NT NT NT Figure 2 improves vegettivedrought tolernceintrnsgeniccuring.() Soilmoisture profileduringthevegettivedrought stressrinoutshelter experiment. Frgmented line nd dotted line indicte upper ( 2 cm) nd verge lower (2 6 cm) soil moisture, respectively. Arrowheds indicte drought stressschedulingndsmpling time. () Vritioninlefrolling scoremong thetrnsgeniclines duringpekstress (threeweeksfterstress). (c) Vritioninplnt dry iomss mong the trnsgenic lines t the end of the stress. (d) Vrition in plnt dry iomss mong the trnsgenic lines t the end of the hrvest fter stress recovery. (e) Vritionplntheightmongthetrnsgeniclinesttheendofthestress. Echdryiomssndplntheightvluerepresentsthemen SE(n = 3), in ech repliction dt point derived from three individul uniform plnts; lef rolling score sed on whole plot performnce nd represents the men SE (n = 3) from three replictions. Different letters in ech column denote significnt differences t P <.5 y Tukey Krmer method. lines in 212-riny-MDSE-Tril-1, four of six trnsgenic lines in 212-dry-MDSE-Tril-2 nd two of five trnsgenic lines in 214-riny-MDSE-Tril, respectively (Figure 3, nd c). We lso found two promising trnsgenic lines (#258 nd #259), which consistently outperformed NT Curing in terms of grin yield over the drought experiments (Figure 3). In these lines, the higher grin yield under severe drought stress (212- dry-mdse-tril-2) ws ssocited with significntly higher numer of pnicles, higher ccumultion of iomss, low lef rolling nd lef drying score, fster recovering ility, erly flowering (Tle S2), pnicle length nd grin fertility. Furthermore, under moderte stress (drought experiments in riny seson), no significnt difference in pnicle numer nd iomss etween trnsgenic nd nontrnsgenic lines ws oserved. Altogether, these results demonstrte tht the Ui: AtGolS2 expression incresed grin yield under drought stress conditions imposed t the reproductive stge through mechnism tht involves the mintennce of erly flowering, incresed vegettive iomss, higher numers of pnicles nd enhnced grin fertility (Tle S2). Anlysis of physiologicl prmeters in Curing lines Bsed on the results of the rinout shelter experiments in 212 (the 212-MDSE-Tril-1 nd 2), five promising T 4 Curing lines were selected for further nlysis of physiologicl prmeters nd field gene expression nlysis in the 214-riny- MDSE-Tril. During the dry-down experiment, the most uniform trnsgenic lines for physiologicl nlysis were tgged nd repeted mesurements were tken during drought stress. Reltive wter content (RWC) ws mesured efore nd fter sujecting the trnsgenic lines nd NT Curing to the drought stress tretment. Before stress, there were no ovious differences in the lef RWC etween NT Curing nd trnsgenic lines, nd the RWC ws within the rnge of 92% 95% (Figure 4). After the lines were sujected to wter stress for one week, the RWC of the NT Curing leves reduced shrply with respect to their first reding (efore stress) from 95% to 89 %, wheres the RWC of most of the trnsgenic lines declined very slowly (#258, 94%; #259, 94%; #2783, 94%; #32, 94%; nd #3214, 93%). After three weeks of drought stress, the RWC of the trnsgenic lines hd declined y just 18% 22% s compred to 3% in the NT Curing. Lines #258, #259, #2783, #32 nd #3214 mintined RWC very well even three weeks fter drought stress, with RWC percentges of 73, 77, 77, 72 nd 76, respectively. The rpid decline of the RWC (verge of 66%) ws oserved in the NT Curing fter three weeks of drought stress. To further verify the mechnism of drought tolernce, we mesured F v /F m vlues of the trnsgenic nd NT Curing during stress period using FluorPen-FP1, (Photon Systems Instruments, spol. s r.o., Czech Repulic) (Figure 4). The F v /F m vlues represent the mximum photochemicl efficiency of photo ª 217 The Authors. Plnt Biotechnology Journl pulished y Society for Experimentl Biology nd The Assocition of Applied Biologists nd John Wiley & Sons Ltd., 15,

5 AtGolS2 improves field drought tolernce in rice 1469 () Single plnt grin yield (SPY) Single plnt grin yield (SPY) (g/plnt) (g/plnt) (c) riny seson-mdse-tril-1 c c NT 214-riny seson -MDSE-Tril c c c () Single plnt grin yield (SPY) (g/plnt) (d) NT NT NT dry seson-mdse-tril-2 de e c d NT Figure 3 improves rice grin yield in Mnged Drought Stress Environment (MDSE) over the growing sesons rinout shelter experiments, CIAT, Plmir. Single plnt grin yield performnce of Curing trnsgenic lines in 212-riny seson-mdse-tril-1 (), 212-dry seson-mdse-tril-2 () nd 214-riny seson-mdse-tril (c). (d) Field performnce of NT Curing nd promising trnsgenic lines in 214-riny-MDSE-Tril. Photogrphs were tken next dy fter rewtering for recovery fter drought stress t the flowering stge. Ech single plnt yield vlue represents the men SE (n = 9 24). Different letters in ech column denote significnt differences t P <.5 y Tukey Krmer method. system (PS) II in drk-dpted stte, where F v stnds for vrile fluorescence nd F m stnds for mximum fluorescence. Initilly under unstressed conditions, the F v /F m vlues of oth NT nd trnsgenic plnts were similr, rnging from.7 to.74. After one week of drought stress, the F v /F m vlue of the NT Curing slightly decresed (.68), ut we did not find ny significnt differences etween NT Curing nd the trnsgenic lines. However, fter three weeks of drought stress, the F v /F m vlue for the trnsgenic lines #3214, #258, #259, #2783 nd #32 ws mintined t.59,.67,.65,.65 nd.61, respectively, while the NT Curing vlue significntly decresed to.56 (Figure 4). The promising trnsgenic lines #258 nd #259 showed significntly higher F v /F m vlues compred to NT Curing even fter three weeks of stress. Chlorophyll content ws mesured using SPAD-52 Chlorophyll Meter (Konic Minolt Inc., Tokyo, Jpn). Trnsgenic nd NT Curing plnts were mesured for their chlorophyll content efore nd during pek stress (Figure 4c). Before stress, the SPAD vlues of the trnsgenic nd NT Curing plnts were not significntly different rnging from 37 to 4. At pek stress (three weeks fter stress), the chlorophyll vlues of the NT Curing plnts were reduced (verge of 34) compred to the initil reding; in contrst, promising lines like #258 nd #259 mintined similr chlorophyll content fter three weeks of the stress (Figure 4c). A RT-PCR nlysis ws lso performed on the tested promising lines in the 214-riny-MDSE-Tril during different stges of the stress development to confirm the expression of the trnsgene under field conditions (Figure 4d). Drought tolernce of Curing in the different environments Trget Environment (TE) Tril Bsed on the initil vegettive nd reproductive drought stress experiments, up to six potentil trnsgenic Curing lines long with the NT Curing were chosen for uplnd rinfed field trils t CIAT Snt Ros sttion, Villvicencio, Colomi. To test the hypothesis of gene 9 environment interctions of, the three consecutive TE field trils were crried out from 212 to 215. The field tril conditions, design nd the plot size were well descried in the experimentl procedure section. The rinfll nd temperture pttern of this site during tril period is shown in Figure 5. Ten yers of rinfll dt from this site reveled tht nturl rinfll filure events usully occur in the months of Jnury Ferury, which coincides with the reproductive stge of the crop. For instnce, tril yers TE nd TE were very dry with continuous rin-free dys of 31 nd 39, respectively (Figure 5 nd ). However, tril yer TE hd rinfll on nd off during the reproductive stge (Figure 5c). In the first two rinfed trils (TE nd TE ), Curing trnsgenic lines reched 5% flowering significntly erlier thn NT Curing (4 nd 5 dys erlier), indicting tht Ui: AtGolS2 overexpression induced erliness in Curing lines (Tle S3). The continuous rin-free period during the reproductive stge cused mrked reduction in soil moisture in the - to ª 217 The Authors. Plnt Biotechnology Journl pulished y Society for Experimentl Biology nd The Assocition of Applied Biologists nd John Wiley & Sons Ltd., 15,

6 147 Michel Gomez Selvrj et l. () RWC % (c) SPAD Vlue BS 1 WAS 2 WAS 3 WAS NT NT () (d) Fv/Fm BS 1 WAS 2 WAS 3 WAS NT Figure 4 Vrition in physiologicl prmeters mong the Curing trnsgenic lines in 214-riny-MDSE-Tril. () Percentge of reltive wter content (RWC) vlues of trnsgenic nd NT lines t efore, first, second nd third weeks fter stress (WAS). () Chnges in chlorophyll fluorescence (F V /F M ) of trnsgenic nd NT lines t efore (BS), first, second nd third weeks fter stress (WAS). (c) SPAD chlorophyll vlues of trnsgenic nd NT lines t pek drought stress. (d) RT-PCR nlysis of trnsgenic nd NT lines evluted t different timing points: efore stress (BS), during pek stress (DS) nd fter stress (AS). RT-PCR nlyses were performed using RNAs from lef tissue t different points of drought development using genespecific primers. Ech physiologicl prmeter vlue represents the men SE (n = 9), three individul plnts from three replictions. Different letters in ech figure denote significnt differences t P <.5 y Tukey Krmer method. 4-cm soil lyer (Figure S3). Under these severe stress conditions, lines #258 nd #259 mintined higher pnicle numer nd showed significntly higher grin yield (GY) with reltive gins of 49%, 18%, 34% nd 17%, respectively, compred to NT Curing (Figure 6 nd Tle S3) in the first tril (TE ). We lso oserved yield gins continued in promising trnsgenic lines in the second (TE ) nd third (TE ) field trils (Figure 6 nd c). In the third rinfed tril (TE ), drought stress ws mild; there ws out 19 dys rin-free period tht coincided the grin-filling stge (Figure 5c). Field vegettive performnce of trnsgenic Curing (#258) ws much etter thn NT Curing s shown in Figure 6d. Thus, the verge grin yields of promising trnsgenic lines were significntly incresed compred to NT Curing in three field trils (Figure 6). Drought tolernce in the different genetic ckground expressing To understnd how the uiquitously works in rice genotype, we conducted drought tolernce experiments in homozygous trnsgenic lines overexpressing in the interspecific hyrid, NERICA4. We first tested seedling survivl rte in glsshouse pot experiment t Jpn Interntionl Reserch Center for Agriculture Sciences (JIRCAS) in Jpn. Seedling survivl of NERICA4 trnsgenic lines ws evluted through previously reported method (Ishizki et l., 213). Before drought stress tretment, no ovious phenotypic differences were oserved etween the NT NERICA4 plnts nd the trnsgenic NERICA4 lines. After nine dys of drought tretment nd susequent recovery for seven dys, the mjority of NT NERICA4 never recovered nd only 11.5% survived. By contrst, four of seven trnsgenic NERICA4 lines exhiited significntly higher survivl rtio, rnging from 26.2% to 34.5% (Tle S4). These results demonstrte tht cn significntly improve seedling survivl under drought in NERICA4. The -NERICA4 lines were lso evluted during the second (TE ) nd third (TE ) rinfed reproductive trils in Colomi long with Curing lines; NERICA4 lines were not included in the tril 1 (TE ) (Figure 7 nd Tle S5). AquPro soil moisture profiles indicted tht the conditions of NERICA4 plots were similr to those of Curing plots in TE Tril nd TE Tril (Figure S4 nd ). Under severe drought stress conditions in the second tril (TE ), the -NERICA4 lines #1577 nd #2344 showed significntly higher GY with reltive gins of 34% nd 49%, respectively, compred to NT NERICA4 (Figure 7). In the third tril (TE ), #1577, #2361 nd #2362 showed significntly higher grin yield thn NT NERICA4 (Figure 7). Interestingly, line 1577 consistently performed well in oth vegettive nd reproductive experiments (Tle S4 nd Figure 7). Correltion etween ccumultion level of glctinol nd grin yield To link field performnce of the trnsgenic lines to the expression level of trnsgene AtGolS2 nd the glctinol ccumultion levels, we clculted Person s coefficient of correltion etween ccumultion level of glctinol nd mrna level of AtGolS2, SPY nd GY in Curing nd NERICA4 evluted under field (Figure S5). The expression level of AtGolS2 correlted with the ccumultion level of glctinol: Person s coefficient of correltion etween those fctors ws.72 in Curing nd.94 in NERICA4, reveling ª 217 The Authors. Plnt Biotechnology Journl pulished y Society for Experimentl Biology nd The Assocition of Applied Biologists nd John Wiley & Sons Ltd., 15,

7 AtGolS2 improves field drought tolernce in rice 1471 () dys 2 4 rin-free period Dys After sowing () dys 2 rin-free 4 period Dys After sowing (c) dys 2 rin-free 4 period Dys After sowing Precipittion (mm) Precipittion (mm) Precipittion (mm) Irrigtion Terminted Irrigtion Terminted Irrigtion Terminted Figure 5 Rinfll nd temperture pttern during crop period in uplnd confined rinfed field tril, CIAT, Snt Ros uplnd sttion. () Climtic profile of TE Tril. () Climtic profile of TE Tril. (c) Climtic profile of TE Tril. Blck r shows mount of rinfll received during crop period, nd dotted line grph shows the mximum dily temperture during tril period. Temperture dt re dily verges nd rinfll is dily totl. Mximum temperture ( C) Mximum temperture ( C) Mximum temperture ( C) tht the expression of AtGolS2 certinly conferred the ccumultion of glctinol in rice plnt. However, SPY nd GY did not lwys correlte with the ccumultion level of glctinol: Person s coefficient of correltion etween those fctors rnged from -.5 to.65, suggesting the glctinol did not hve dose effects on yield under field. Discussion is verstile: improving drought tolernce cross stges of rice, genetic ckground, drought intensity nd environments While developing drought-tolernt crops, plnt productivity should e tken into considertion. Plnt productivity is widely ffected y nturl drought incidences under field conditions (Todk et l., 215). Droughts re rndom events nd dry spells cn occur t virtully ny time during the rice growing period in drought-prone res, leding to drought stress of vrying intensity. Although rice is highly sensitive to drought stress during the reproductive stge (Venuprsd et l., 27), drought t erly vegettive stge of rice growth cn considerly ffect plnt performnce. Commonly, drought survivl test during the vegettive stge is otined under lortory or glsshouse conditions nd is therefore not perfectly comprle to interprettions mde under rel-field conditions. Extensive field trils re thus criticl for the pproprite evlution of stresstolernt trnsgenic crops (Todk et l., 215). In this pper, we crried out field trils in CIAT, Colomi, nd demonstrted tht overexpression in rice ws effective t conferring drought tolernce during oth the vegettive nd the reproductive stges (Figures 2 nd 3). It is very rre phenomenon when the results otined from vegettive screening experiments concur with reproductive stge, indicting tht the overexpression cn e exploited for oth erly- nd mid-seson drought. This is very importnt in the perspective of trgeting rice vrieties to rinfed environments where rinfll uncertinty is expected. In rice, severl reports re ville tht exmine field drought tolernce cused y overexpression of trnsgenes (Xio et l., 29; You et l., 212; Yu et l., 213). However, most of these studies were conducted on plnts tht were grown under glsshouse conditions. There hve een instnces where trnsgene-medited trit expressed in the glsshouse ws unstle under field conditions (Brndle et l., 1995). As it ws reported tht the effect of trnsgene expression in whet vried from yer to yer sed on the climtic conditions of prticulr growing seson (Bhieldin et l., 25), it ws considered essentil to explore the yield stility of the ª 217 The Authors. Plnt Biotechnology Journl pulished y Society for Experimentl Biology nd The Assocition of Applied Biologists nd John Wiley & Sons Ltd., 15,

8 1472 Michel Gomez Selvrj et l. () Grin Yield (Kg/H) (c) Grin Yield (Kg/H) TE c c NT TE c c NT () Grin Yield (Kg/H) (d) TE c NT NT Curing c 258 Figure 6 improves Curing grin yield in Trget Environment (TE) Snt Ros rinfed Tril, CIAT uplnd rinfed sttion, Villvicencio. () Grin yield performnce of Curing trnsgenic lines in TE Tril. () Grin yield performnce of Curing trnsgenic lines in TE Tril. (c) Grin yield performnce of Curing trnsgenic lines in TE Tril. (d) Field performnce of NT Curing nd promising trnsgenic event 258 t TE Tril. Photogrphs were tken during stress t the grin-filling stge. Estimted grin yield (kg/h) ws derived from plot yield from three replictions, nd vlue represents the men SE (n = 3). Different letters in ech column denote significnt differences t P <.5 y Tukey Krmer method. () 6 Grin Yield (Kg/H) TE c c c c NERICA4 () Grin Yield (Kg/H) TE c c c NERICA NT NT Figure 7 improves NERICA4 grin yield in rinfed uplnd trils. () Grin yield performnce of NERICA4 lines in Trget Environment (TE) Tril t Snt Ros rinfed tril. () Grin yield performnce of NERICA4 lines in TE Tril t Snt Ros rinfed tril. Estimted grin yield (kg/h) derived from plot yield. Estimted grin yield (kg/h) ws derived from plot yield from three replictions, nd vlue represents the men SE (n = 3). Different letters in ech column denote significnt differences t P <.5 y Tukey Krmer method. trnsgenic rice in different sesons, for exmple riny nd dry nd under severl environmentl conditions such s rinfed nd wellwtered conditions. In this study, higher GY in the trnsgenic lines ws consistently oserved over the seson nd environments (Figures 3, 6 nd 7). The promising trnsgenic lines showed significntly enhnced drought tolernce in the field cross different genetic ckgrounds, Curing nd NERICA4, with grin yield of 17% 1% higher thn NT Curing under mild to severe drought stress, wheres the trnsgenic lines displyed no significnt differences under norml growth conditions (Figures 3, 6, 7 nd Tle S1). These improvements of grin ª 217 The Authors. Plnt Biotechnology Journl pulished y Society for Experimentl Biology nd The Assocition of Applied Biologists nd John Wiley & Sons Ltd., 15,

9 AtGolS2 improves field drought tolernce in rice 1473 yields under drought cn e considered greter thn wht hs een reported for other trnsgenic rice lines expressing genes conferring field drought tolernce, which hve often een chllenged with milder stresses s demonstrted y the grin yield reduction under drought of the control checks (Oh et l., 29). However, in this study, the drought intensity ws mild ecuse plnts were irrigted to evde lef rolling, which resulted in yield loss of round 32% in the WT. In nother study, rice plnts overexpressing OsNAC1 showed enhnced drought tolernce during the flowering stge nd incresed grin yield y 25% 42% compred to WT, ut gin milder drought stress conditions were pplied (Jeong et l., 21). A limited numer of studies pplied field drought conditions similr to our work (Todk et l., 215). Under severe stress, rice trnsgenic lines expressing OsCPI1 showed 2.5- to 3-fold greter GY over the control, for which yield dropped 9% (Hung et l., 27). Likewise, plnts overexpressing LOS5 nd ZAT1 exhiited gins etween 11% nd 36% compred to their controls which suffered 82% yield reduction (Xio et l., 29). Through this interntionl collortive project, we relized the importnce of conducting the initil screening efforts in frmerdpted vriety, ecuse these re populr over lrge growing res, loclly dpted nd ecuse reltively quick introgression of the trnsgene into other megvrieties is possile (Gudin et l., 213). In our study, we ttempted to improve two frmerdpted vrieties, one from Ltin Americ (Curing) nd nother one from Afric (NERICA4). The phenotype might e controlled y genes (G) including trnsgenes nd genetic ckground (genotypes) in trnsgenic plnts, nd plnt responses to drought re lso influenced y environment (E) including intensity, durtion nd frequency of the stress s well s y diverse plnt soil tmosphere interctions (Sint Pierre et l., 212). It is lwys suggested to test G 9 G nd G 9 E interctions/stility efore recommending potentil trnsgene into the reeding pipeline. In this study, we hve conducted drought experiments in two contrsting field sesons (riny nd dry). We found the response of the lines to e different thn the NT control sed on the seson. However, regrdless of the seson, the promising lines #258 nd #259 hd significntly greter plnt iomss nd pnicle numers. A similr result ws found within the trget environment. Our results provide strong evidence tht overexpression of AtGolS2 is useful iotechnologicl tool to reduce yield losses under field drought conditions under different environmentl conditions (E) nd in different rice genetic ckgrounds (G), which suggests tht AtGolS2 is n essentil gene to improve drought tolernce in rice regrdless of G 9 G nd G 9 E interctions. Mechnism of drought tolernce offered y the Ui: AtGolS2 trnsgene Even though mny stress resistnce genes hve een identified in noncrop species such s Aridopsis, evlution of the effect of these genes on improving field drought tolernce in given crop hs seldom een reported (Xio et l., 29). GolS plys key role in the ccumultion of glctinol under iotic stress conditions, conferring drought stress tolernce to plnts, ecuse glctinol my function s osmoprotectnts nd scvenger of hydroxyl rdicls (Nishizw et l., 28; Tji et l., 22). Bsed on our extensive evlution of trnsgenic lines, the trnsgene improved grin yield of rice under drought conditions. Although high expression of AtGolS2 nd ccumultion of glctinol were confirmed in events, no significnt chnge in the expression of drought mrker genes ws induced (Figure 1 nd Figure S1). These results suggest tht drought tolernce of trnsgenic lines with the ccumultion of glctinol ws not correlted with the expression of droughtresponsive genes. This could e contriuted through the following mechnisms. First, the trnsgenic lines re more tolernt to drought nd gin yield over the NT ecuse they re protected y elevted glctinol (RFOs) tht cn ct s osmoprotectnts nd scvenger of hydroxyl rdicls (Figure 1). The incresed trnscription of GolS genes during drought hs een reported in mny plnts nd crops. In Cucumis melo, it ws oserved tht GolS ctivtes ccumultion of RFO in plnts sumitted to drought stresses (Volk et l., 23). As second physiologicl perspective, the promising Curing trnsgenic lines hve etter mximum photochemicl efficiency (F v /F m ) nd lef chlorophyll content thn NT under drought stress (Figure 4). The decrese in F v /F m nd SPAD chlorophyll vlues under drought stress could e n indictor of oxidtive stress nd dmge in PSII (Frooq et l., 29). Under severe drought stress, we oserved high SPAD chlorophyll nd F v /F m vlues in the Ui: AtGolS2 Curing thn in NT Curing, nd the leves were greener thn those of the NT, which confirmed norml photosynthesis in trnsgenic rice. In ddition to photosynthetic-relted trits, stress-relted trits such s RWC, lef rolling nd drying of trnsgenic lines were significntly etter thn NT Curing (Tles S2, S3 nd S5). Mintennce of high plnt wter sttus, s expressed in high RWC of the rice, ws n good indictor of drought tolernce (s shown y Bu et l., 24), nd cpcity of trnsgenic lines mintined higher lef RWC compred with NT Curing under drought stress, which ws consistent with their ility to postpone dehydrtion (s indicted y Cstonguy nd Mrkhrt, 1992). In this study, we oserved tht the trnsgenic lines hd higher RWC vlues thn NT rice during pek drought stress nd hd lower lef rolling nd lef drying score (Figure 4; Tles S2, S3 nd S5). By contrst, we oserved tht NT rice quickly wilted nd dried s compred to those trnsgenic lines with higher RWC vlues under drought stress. Third, the AtGolS2 Curing trnsgenic lines showed erlier flowering thn NT Curing (Tle S3) under drought stress, ut displyed no difference in growth under norml growth conditions (Tle S1). In rice, erly mturtion is n escpe mechnism to ensure production under conditions of stress (Gur et l., 21). In this study, the Curing lines showed the erliest flowering nd exhiited higher grin fertility (82%) thn NT (7%), which my e due to the drought escpe mechnism of trnsgenic lines (dt not shown). However, erly flowering ws not oserved in the NERCA4 ckground (Tle S5). As NERICA4 is short-durtion vriety, erly flowering of the Ui: AtGolS2 lines my e profound in Curing due to the long durtion nture. Trnscription levels of the AtGolS2 gene of the trnsgenic lines correlted with the ccumultion level of glctinol; however, the ccumultion level of glctinol did not correspond with their field performnce (Figures 1 nd S5). These results suggest tht good field performnce might not lwys e ssocited with levels of gene expression nd of ccumultion of glctinol. The complexities of environments nd other fctors influencing performnce of rice plnts under drought my e resons for no dosge effects of glctinol on grin yield under field. ª 217 The Authors. Plnt Biotechnology Journl pulished y Society for Experimentl Biology nd The Assocition of Applied Biologists nd John Wiley & Sons Ltd., 15,

10 1474 Michel Gomez Selvrj et l. Conclusions nd prospects Our study reported extensive field evlution of trnsgenic rice plnts expressing the trnsgene under drought stress environments under field conditions in Colomi. We clerly oserved tht the expression nd the ccumultion of glctinol significntly enhnced grin yield under drought field conditions, ut did not ffect either grin yield or plnt growth under well-wtered pddy field conditions. Improved grin yield under stress ws ssocited with erly flowering, higher iomss ccumultion, higher numer of pnicles nd lower pnicle sterility. In this study, the sme gene construct, the, trnsgene ws tested on two different commercil genetic ckgrounds, Curing nd NERICA4, nd contrsting different sesons nd different environments. We presented the results of extensive confined field testing of trnsgenic rice overexpressing AtGolS2 nd the responses of these trnsgenic rice plnts to contrsting environments. Notly, we evluted the gronomic trits of these trnsgenic lines t ll stges of plnt growth in the field s function of the environment nd genetic ckground. As the trnsgene ws tested in the commercil rice genetic ckgrounds of Ltin Americ (Curing) nd Afric (NERICA4), we think it is esy to pyrmid the trnsgene into ongoing trnsgenic rice reeding progrmmes in Ltin Americ nd Afric. The promising NERICA4 trnsgenic lines selected from this study cn e integrted into ongoing NEWEST the NERICA4 (Nitrogen-use Efficient, Wter-use Efficient nd Slt Tolernt) rice project where extensive trnsgenic field trils re currently eing implemented in Ghn, Ugnd nd Nigeri through USAID feed the future progrmme. Development of this drought-tolernt rice through the trnsgene should hve significnt economic nd environmentl enefits in lowinput griculturl systems like Ltin Americ nd Afric. Experimentl procedures Genertion of Plnts To generte trnsgenic rice plnts overexpressing AtGolS2 encoding glctinol synthse 2 of Aridopsis thlin (Tji et l., 22), the pbig-ui vector ws used (Becker, 199; Ito et l., 26). AtGolS2 cdna ws mplified using BmHI linker primers. The resulting DNA frgment crrying BmHI sites t the 5 nd 3 termini ws inserted into pbig-ui t the BmHI site. The construct ws introduced into rice cv. Curing nd NERICA4 y Agrocterium-medited trnsformtion s descried previously (Ishizki nd Kumshiro, 28; Zunig-Soto et l., 215). The moleculr chrcteriztion of puttive trnsgenic events involved PCR nd Southern lot nlysis. The primers used for this study re reported in Tle S6. Expression nlysis of rice plnts expressing The trnsgenic nd nontrnsgenic (NT) rice plnts (Curing nd NERICA4) were grown in soil-filled, open-ottomed 5-mL plstic tues in the glsshouse. After the drought tretment, the leves from five plnts were collected, frozen in liquid nitrogen nd stored t 8 C. Totl RNA ws isolted from the lef smples using RNAiso Plus regent (Tkr Bio, Shig, Jpn). Extrcted RNA ws sujected to DNse tretment using RQ1 DNse (Promeg, WI), nd complementry DNA ws synthesized using PrimeScript RT Mster Mix (Tkr Bio). Rel-time quntittive RT- PCR ws performed with the QuntStudio 7 Flex rel-time PCR system (Thermo Fisher Scientific, MA) using SYBR Premix Ex Tq (Tkr Bio). Primers used for qrt-pcr re listed in Tle S6. Sugr metolite nlysis of rice plnts expressing Ui: AtGolS2 The trnsgenic nd control rice lines were grown in soil-filled, open-ottomed 5-ml plstic tues in the glsshouse. After the drought tretment, the leves from five plnts were collected, frozen in liquid nitrogen nd stored t 8 C. Sugr metolites were nlysed using GC-TOF-MS s descried previously y Mruym et l. (214). Seedling survivl test of NERICA4 trnsgenic events The ility of NERICA4 trnsgenic events to survive under rpid drying ws evluted y the reported method (Ishizki et l., 213). Vegettive drought stress experiment in confined field To evlute the drought tolernce of trnsgenic rice plnts t vegettive stge, single-copy independent homozygous lines of Curing trnsgenic lines, together with nontrnsgenic (NT) Curing controls, were direct-seeded in confined field conditions under rinout shelter t the Interntionl Center for Tropicl Agriculture (CIAT), Plmir, Colomi, in the dry seson, Novemer-Decemer 211. A rndomized lock design ws employed with three replictes with ech event sown in two rows plced 16 cm prt in rinout shelter where the depth of restructured soil ws 85 cm. Ech row ws 1 m long nd 4 plnts were ccommodted in ech row with equl spcing (5 cm) etween plnts. Drought stress ws imposed y withholding irrigtion t initil tillering stge (21 dys fter direct sowing) nd rewtered fter 21 dys (3 weeks) until severe wilting symptoms ppered in NT Curing (Figure 2). The intensity of drought ws monitored through AquPro soil moisture proes (AquPro sensors Inc, Cliforni, USA). Plnt height, the numer of tillers nd destructive plnt dry iomss dt were mesured from uniform tgged plnts t the efore, during pek stress nd t the end of the hrvest fter rewtering. Lef rolling ws determined t the time of pek drought stress. Mnged Drought Stress Environment (MDSE) Tril Rinout Shelter (RS) reproductive Stge tril t CIAT, Plmir, Colomi All rinout shelter reproductive stress experiments were crried out t our confined field fcility t CIAT, Plmir, Colomi. For Curing, three confined field drought trils (from 212 to 214) over two contrsting sesons were conducted under the movle semi-utomtic rinout shelter fcility. All three experiments were conducted using sme protocol with respect to designs nd field drought chrcteriztion. A rndomized lock design with three replictions ws followed to lyout the experiment under the rinout shelter fcility t CIAT. The seeds of T 4 homozygous lines were sown in the dry soil of the experimentl plots in rows (2 cm spcing etween rows). Ech event ws sown in two rows plced 2 cm prt where the depth of restructured soil ws 85 cm. Ech row ws 2 m long nd hd 2 plnts with equl spcing. The recommended fertilizer ppliction for uplnd rice ws used. Drought ws imposed y withholding irrigtion when pnicle initition ws round 1 mm long (6 66 dys fter sowing in the cse of Curing) for 3 4 weeks (or) until severe lef rolling nd drying ppered in the NT control. Then, the plnts were rewtered to 9% field cpcity until physiologicl mturity. The intensity of drought ws monitored through AquPro soil ª 217 The Authors. Plnt Biotechnology Journl pulished y Society for Experimentl Biology nd The Assocition of Applied Biologists nd John Wiley & Sons Ltd., 15,

11 AtGolS2 improves field drought tolernce in rice 1475 moisture proes tht were instlled to mesure moisture in the soil profile to depth of.85 m. Lef rolling (LR), lef drying (LD) nd drought recovery scores were recorded on 1-9 IRRI scle stndrdized for rice (IRRI, 22). The following gronomic trits were mesured sed on the criteri estlished in the Stndrd Evlution System for Rice (SES) (IRRI, 22): flowering dte, plnt height (cm), single plnt dry iomss (g), pnicle length (cm), the numer of tillers, the numer of fully emerged pnicles nd grin fertility (%). Single plnt yield (from five more uniform tgged plnts from ech lock with three replictions) nd plot yield were lso recorded. The degree of reltive chlorophyll content in the fully expnded flg lef ws determined while the plnt ws under stress, using SPAD-52 Chlorophyll Meter (Konic Minolt Co., Tokyo, Jpn). Chlorophyll fluorescence prmeters were lso mesured using FluorPen FP1 chlorophyll (Photon Systems Instruments, spol. s r.o., Czech Repulic). Reltive wter content ws clculted using the protocol sed on Schonfeld et l. (1988). Well-wtered experiment in confined field conditions To evlute the yield components of the trnsgenic Curing lines under norml well-wtered field conditions, selected independent T 4 homozygous lines of trnsgenic plnts together with NT controls were trnsplnted to rice pddy confined field t CIAT, Plmir (dry seson, Septemer Jnury 213). A rndomized design ws employed with three replictes of two 2-m-long rows per plot. For ech plot, 2 seedlings per line were rndomly trnsplnted with cm spcing 25 dys fter sowing. The recommended fertilizer ppliction for lowlnd rice ws used. Yield prmeters were scored for five tgged uniform plnts per plot. RT-PCR nlysis of field smples Rinout shelter Drought trils Totl RNA ws extrcted from the flg leves of tested promising trnsgenic lines t efore stress, pek stress nd fter rewtering from the 214-riny-MDSE-rinout shelter tril using the Trizol regent (Invitrogen). Reverse trnscription ws crried out using DNse (Promeg) nd SuperScript III (Invitrogen). Endpoint PCR ws conducted with primers listed in Tle S6, using stndrd protocols nd n nneling temperture of 55 C. PCR products were checked on 1% grose gel with SYBR-sfe stin. Trget Environment (TE) Tril Confined Field Evlution of Trnsgenic lines t Snt Ros, Colomi To evlute yield components of trnsgenic plnts under rinfed uplnd conditions with nturl drought condition, the most promising independent T 4 homozygous lines of Curing nd NERICA4 from the previous drought experiments long with their NT controls were evluted in replicted plot tril with rndomized lock design from 213 to 215 (three consecutive field trils) t CIAT Snt Ros rinfed uplnd sttion, Colomi. Promising NERICA4 lines were selected sed on the survivl test results from Jpn nd rinout shelter trils. An uplnd field tril ws lid out in rndom complete lock design with three replictes. The trnsgenic events long with NT rice were sown in m plots with cm spcing. Seeds were sown directly y hnd t the rte of 12 kg/h when soil moisture ws out 8% of field cpcity. The recommended fertilizer ppliction for uplnd rice ws used. The plnts were estlished in dry soil nd irrigtion ws provided until 5 dys fter sowing (DAS) vi sprinklers to estlish the crop. After plnt estlishment, irrigtion ws stopped nd plnts were totlly dependent on rinfll. The soil moisture ws monitored throughout the cropping period y Aqupro soil moisture device. Plnt growth nd development of ech of the trnsgenic events reltive to NT rice ws monitored regulrly nd plot yield (g) ws recorded. Grin yield (kg/h) ws estimted from plot yield sed on plnt density. Dt nlysis Dt were nlysed y one-fctor ANOVA t P <.5. When significnt differences were found, multiple comprisons y the Tukey Krmer method (P <.5) were mde. Acknowledgements We re grteful for technicl support provided y Emiko Kishi nd Miho Kishimoto of JIRCAS. We lso thnk Dr. Jgdish Rnne, AL.Chvez, Cesr Zulug, Sntigo Jrmillo nd Mri Recio of CIAT for the technicl ssistnce nd Dr. Joe Tohme, Director of Agroiodiversity Reserch Are of CIAT, for criticl discussion on field phenotyping. We lso thnk Richrd Bruton, Texs A&M, nd Angel Fernndo, CIAT, for their criticl English edition nd suggestions of the mnuscript. This work ws supported y the grnts from the Ministry of Agriculture, Forestry nd Fisheries (MAFF) of Jpn for project: Development of Drought-Tolernt Crops for Developing Countries. The uthors declre no conflict of interest. Author contriutions K.S., K.N. nd M.I designed the totl experiments. M. G. S., S.O. nd M.O.V. plnned, conducted nd nlysed drought field experiments. B.D. nd M.I. conducted trnsformtion experiments t CIAT. T.I. conducted trnsformtion experiments nd glsshouse experiments t JIRCAS. T.O. nd K.N. conducted gene expression experiments. K.Y., K. M., M. K. nd K. S. conducted sugr nlysis. F.T. mde the construct for trnsformtion. M.G.S. wrote the mnuscript, nd ll the uthors checked it. Ethicl stndrds The uthors declre tht the trnsgenic experiments comply with the current iosfety lws of the country in which they were performed. 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