Barley HVA1 Gene Confers Salt__ Tolerance in R3 Transgenic Oat
R e p r o d u c e d f r o m C r o p S c i e n c e . P u b l i s h e d b y C r o p S c i e n c e S o c i e t y o f A m e r i c a . A l l c o p y r i g h t s r e s e r v e d .
Barley HVA1Gene Confers Salt Tolerance in R3Transgenic Oat
Hesham F.Oraby,Callista B.Ransom,Alexandra N.Kravchenko,and Mariam B.Sticklen*
ABSTRACT
in the oat background,and the salt tolerance trait com-plexity (Cushman and Bohnert,2000).Crop improve-A major cause of oat crop yield loss worldwide is osmotic stress ment strategies that are based on the use of new technol-due to drought and/or salinity.This study investigated the third genera-tion of transgenic oat (Avena sativa L.)expressing barley HVA1stress ogies,such as biotechnology,can be used in conjunction tolerance,?-glucuronidase (uid A;gus )and bar herbicide resistance with traditional breeding efforts (Abebe et al.,2003;genes.Transgenic plants showed normal 9:7third generation inheri-Epstein et al.,1980;Ribaut and Hoisington,1998),offer-tance for glufosinate ammonium herbicide resistance.Molecular and ing a responsible way to enhance agricultural productiv-histochemical studies confirmed the presence and stable expression ity.Biotechnology could help eliminate many obstacles of all three https://www.sodocs.net/doc/4214725469.html,pared with the nontransgenic control plants,limiting crop production in developing countries.The transgenic R3plants exhibited greater growth and showed a significant development of crops with the internal capacity to with-(P ?0.05)increase in tolerance to salt stress conditions (200m M stand abiotic stresses would help to reduce the use of NaCl)for traits including number of days to heading,plant height,water (FAO,1999),thus promoting sustainable yields flag leaf area,root length,panicle length,number of spikelets/panicle,number of tillers/plant,number of kernels/panicle,1000-kernel weight,(Sharma et al.,2001).Also,modern plant genetic engi-and kernel yield/plant.
neering presents the possibility of rapid and precise introduction of a desirable trait from closely related plants without associated deleterious genes (Richards,E
nvironmental stresses such as drought and salinity 1996)because it avoids the transfer of unwanted chro-represent some of the most limiting factors for ag-mosomal regions (Cushman and Bohnert,2000;Sharma ricultural productivity worldwide (Boyer,1982;Roy and et al.,2002).Oat transformation using microprojectile Wu,2002).Not only do they greatly decrease the poten-bombardment was first reported by Somers et al.(1992).It tial yield of current crop species,but they also restrict is now possible to produce fertile transgenic lines of major the area where production can take place and hinder cereal crops including oat for different studies (Makare-the introduction of crop plants into new areas (Epstein vitch et al.,2003;McGrath et al.,1997;Pawlowski et al.,et al.,1980).Salinity in particular is a major problem,1998a,1998b;Svitashev et al.,2000,2002;Torbert et al.,affecting crop production on nearly one-third of the 1995,1998a,1998b,1998c;Zhang et al.,1999).
world’s irrigated agricultural land (Apse et al.,1999;Several promising abiotic stress tolerance candidate Schachtman and Lui,1999).There is a shortage of arable genes for crop transformation have been https://www.sodocs.net/doc/4214725469.html,nd area and this area is steadily decreasing,as farming Among those are the Late Embryogenesis Abundant practices cause cultivated land to become more saline (LEA)proteins,which are ubiquitous in plants.These (Qadir et al.,1998).Farmers are,therefore,being forced proteins accumulate during the late stage of seed forma-to cultivate in salinity-prone areas,but plants grown tion and in vegetative tissues under drought,heat,cold,in these areas experience severe salinity stress.This and salt stress conditions or with abscisic acid (ABA)situation will only worsen in the coming decades (Cherry application (Sivamani et al.,2000).LEA proteins are et al.,1999).
generally divided into five groups,and they appear to Oat is an important cereal crop used in human and protect cellular structures from dehydration stress;how-animal diets (Welch,1995).Osmotic stress due to drought ever,the exact functional role of these hydrophilic pro-and/or salinity is a major cause of global oat crop yield teins remains poorly understood.Proposed roles include loss (Frey,1998;Martin et al.,2001;Tamm,2003).Al-water binding or replacement,hydration buffers (Dure,though,compared with the other cereals,oat is consid-1993a;Ingram and Bartels,1996),ion sequestration (Dure,ered a moderately salt tolerant crop (Murty et al.,1984),1993b),osmotic adjustment or reverse chaperones (Close,soil salinity is responsible for decreasing oat seed germi-1996),and transport of nuclear-targeted proteins during nation and stunting subsequent development in a culti-stress (Goday et al.,1994).
var-dependent manner (Murty et al.,1984;Verma and HVA1,a barley (Hordeum vulgare L.)group III LEA Yadava,1986).
protein,is highly induced by ABA/stress.The HVA1Traditional breeding has limitations and cannot solve gene (Hong et al.,1992)has been used successfully to this problem alone,perhaps because of the inefficiency confer stable tolerance to osmotic stress in rice (Oryza of selection methods,the lack of the genetic variability
sativa L.)(Xu et al.,1996),oat (Maqbool et al.,2002),and wheat (Triticum aestivum L.)(Patnaik and Khur-Dep.of Crop and Soil Sciences,Michigan State Univ.,Plant and ana,2003).
Soil Science Bldg.,East Lansing,MI 48824.Received 14Oct.2004.This paper reports studies on the third generation of *Corresponding author (stickle1@https://www.sodocs.net/doc/4214725469.html,).transgenic oat expressing HVA1,?-glucuronidase (uid A;Published in Crop Sci.45:2218–2227(2005).
gus ),and the bar herbicide resistance genes under green-Genomics,Molecular Genetics &Biotechnology house conditions.The objectives were:(i)to determine doi:10.2135/cropsci2004-0605
the segregation of the linked bar and HVA1genes and ?Crop Science Society of America
677S.Segoe Rd.,Madison,WI 53711USA
verify their proper expression and stability of transmis-2218
Published online September 23, 2005
R e p r o d u c e d f r o m C r o p S c i e n c e . P u b l i s h e d b y C r o p S c i e n c e S o c i e t y o f A m e r i c a . A l l c o p y r i g h t s r e s e r v e d .
ORABY ET AL.:BARLEY HVA1GENE CONFERS SALT TOLERANCE IN R3TRANSGENIC OAT
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sion to progeny;(ii)to evaluate the effects of the trans-genes on plant growth;and (iii)to compare the perfor-mance of these transgenic lines against nontransgenic controls for common important agronomic traits under salt stress conditions.
MATERIALS AND METHODS
Plant Materials
R3seeds from five independent transgenic oat lines of the cultivar Ogle (BRA-8,BRA-17,BRA-19,BRA-41,and BRA-82)generated in a previous study (Maqbool et al.,2002)were used in this study.Transgenic lines were produced from a multimeri-stem transformation system with two plasmids,pBY520
and pAct1-D.The plasmid BY520contained the linked selectable Fig.1.R3transgenic seedlings after in vitro selection.
marker/herbicide resistance bar (phosphinothricin acetyl trans-ferase)gene (driven by cauliflower mosaic virus 35S promoter once per day ensuring adequate leaching,and preventing salin-and the nopaline synthase nos terminator)and the barley ity excess.Plants were given the salinity treatments for 14d,HVA1gene (driven by rice Act1promoter and terminated by followed by 1wk of irrigation with plain water to measure the potato protease inhibitor pin II).The plasmid Act1-D plant recovery after salt stress.Afterward,the salt treatments contained the nonlinked Escherichia coli -glucuronidase (gus )were resumed uninterrupted for 35d.
gene flanked by the Act1promoter and the nos terminator.The lines chosen for this work had one copy of the linked PCR Analysis
HVA1-bar genes.Bagged flowers of some R0,R1,and R2progenies were allowed to self-pollinate and produce seeds The detection of the bar and the HVA1genes in all the which were collected in bulk from each line.Bulked R3trans-studied R3transgenic plants by PCR amplification was per-genic and control (Ogle wild-type cultivar)seeds were germi-formed with leaf disk DNA as template and REDExtract-N-Amp nated;plants of R3generation were grown and evaluated in Plant PCR Kit (Sigma-Aldrich,St.Louis,MO,Cat #XNA-P)as two greenhouses under salt treatments.
per the manufacturer’s instruction using the following primers:bar F,5?-ATG AGC CCA GAA CGA CG-3?(forward primer);bar R,5?-TCA GAT CTC GGT GAC GG-3?(reverse Segregation of Herbicide Resistance of R3Progeny
primer)and HVA1F,5?-TGG CCT CCA ACC AGA ACC Seeds were surface-sterilized with 70%(v/v)ethanol for 2AG-3?(forward primer);and HVA1R,5?-ACG ACT AAA min,followed by 20%(v/v)commercial bleach (5.25%com-GGA ACG GAA AT-3?(reverse primer).DNA amplifi-mercial sodium hypochlorite)treatment for 10min,rinsed cations were performed in a thermo cycler (PerkinElmer/with sterile distilled water several times,and briefly blotted Applied Biosystem,Foster City,CA)using initial denatur-onto sterile filter paper.Transgenic and nontransgenic control ation at 94?C for 4min,followed by 35cycles of 1min at seeds were germinated on MS (Murashige and Skoog,1962)94?C,1min at 55?C,2min at 72?C,and a final 10min extension basal medium containing 15mg/L glufosinate ammonium for at 72?C.The reaction mixture was loaded directly onto a 0.8%selection.Control seeds were also germinated on the same (w/v)agarose gel,stained with ethidium bromide,and visual-medium lacking herbicide.Cultures were maintained under ized with UV light.The transgene product size was about 0.59continuous fluorescent light at 28?C for 1wk.The seedlings kb for the bar gene and 0.70kb for the HVA1gene.
that survived the selection were used for molecular and pheno-typic characterization of the lines.
Histochemical Analysis of GUS
Seeds,seed husks,and root segments from the transgenic Salinity Treatments
and nontransgenic plants were used to detect GUS activity Young seedlings were transferred into Baccto Professional by histochemical staining with 5-bromo-4-chloro-3-indoyl-Planting Soil Mix (700–800g kg ?1horticultural sphagnum ?-d -glucuronicacid salt (X-gluc).Samples were immersed in peat,200–300g kg ?1perlite,pH 5.5–6.5)in small pots (8?GUS substrate mixture and incubated at 37?C (Jefferson et 4?6cm),one plant per pot (Fig.1).The pots were kept in al.,1987).The tissues were thoroughly washed with 70%etha-water-filled flat-bottom trays for 1wk.Two-week-old seed-nol and examined under a Zeiss SV8stereomicroscope.
lings from each transgenic line and the control were trans-ferred to 7.3-L (2-gallon)pots for another week before starting DNA Isolation and Southern Blot
the salt stress treatments.
Hybridization Analysis
The environmental settings in the greenhouses were main-tained between 21and 25?C,with a relative humidity between Independence of the lines and confirmation of the HVA1transgene transmission into the oat R3transgenic plants were 80and 90%.Natural illumination was augmented for 16h per day with fluorescent light and light levels were between 150performed by Southern-blot hybridization using the HVA1-coding sequence as a probe.Genomic DNA from transgenic and 185?mol m ?2s ?1.
The soil surface around the seedlings was covered with and nontransgenic oat plants was isolated using the protocol of Saghai-Maroof et al.(1984).For Southern blots,10to 15?g redwood bark mulch to minimize the evaporation and prevent algae growth.A dilution of commercial 20:20:20fertilizer solu-of genomic DNA was digested with Hin dIII restriction enzyme,electrophoresed in 0.8%(w/v)agarose gel,transferred onto tion was added twice a week for nutritional needs.Sodium chloride (NaCl)was added in five concentrations of 0,50,100,Hybond-N ?(Amersham-Pharmacia Biotech)membranes,and fixed with a UV crosslinker (Stratalinker UV Crosslinker
150,and 200m M .Plants were watered with the saline solution
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Table 1.Segregation of herbicide resistance of R3transgenic Ogle 1800,Stratagene,CA)as recommended in the manufacturers’cultivar.
instructions.The HVA1gene-specific probe was generated by a Hin dIII-Bam HI digest of pBY520to isolate a 1.0-kb Germinated Not Line seeds?
germinated
?2?P value fragment.The restriction fragment was purified with the QIA-quick kit (QIAGEN).Probe labeling and detection were ob-BRA-8257430.02400.8769tained with the DIGHigh Prime DNA Labeling and Detection BRA-1760400.57830.4470BRA-86238 1.34340.2464Starter Kit II (Kit for chemiluminescent detection with CSPD,BRA-1960400.57830.4470Roche Co.)following the manufacturer’s protocol.
BRA-41
61
39
0.9168
0.3383
?Seeds bulked for each line through the R3generation were germinated RNA Isolation and Northern Blot
on MS basal medium containing 15mg/L glufosinate ammonium.
Hybridization Analysis
?No significant difference between the observed versus the 9:7expected segregation ratio in all the lines (P ?0.05)
Assay for transcriptional expression of the HVA1transgene was performed by Northern blots.Total RNA was isolated for each group.The greenhouses and blocks were considered from young leaves of oat plants (transgenic and nontransgenic)random factors.The statistical analysis for quantitative traits using the TRI Reagent (Sigma-Aldrich,St.Louis,MO)ac-was performed by PROC MIXED (SAS Institute Inc.,2003).cording to the manufacturer’s instructions.For the Northern The significance of the interaction between genotype and sa-blot,20?g of RNA were separated in a 1.2%(w/v)agarose-linity was tested and the line means were compared at each formaldehyde denaturing gels according to Sambrook et al.salinity level by t tests.
(1989)and blotted onto Hybond-N ?nylon membranes (Amer-sham-Pharmacia Biotech).Transcripts of HVA1were ana-RESULTS AND DISCUSSION
lyzed by a standard Northern-blotting method (Sambrook et al.,1989)using the HVA1-coding sequence as a probe labeled Here,the third generation of five independent trans-with ?-[32P]-dCTP with the Random Primer Labeling Kit genic lines obtained from a previous study (Maqbool et (Invitrogen,Carlsbad,CA)according to the manufacturer’s al.,2002)was tested for salinity tolerance under green-instructions.
house conditions.The independence of the lines was confirmed by Southern blot analysis.The molecular Measurements of Parameters
analyses,segregation for herbicide resistance,GUS his-Data were collected on kernel yield and its components tochemical assay,and salinity treatment were used to (number of tillers/plant,number of kernels/panicle,1000-ker-investigate transgene integration,transcription,and trans-nel weight).Additional measurements were taken on number lation in the R3progeny of the transgenic plants.
of days to heading,plant height,flag leaf area,root length,panicle length,and number of spikelets/panicle during the Segregation of Herbicide Resistance
experiment for each plant in the five replications under each of R3Progeny
salinity concentration.The whole experiment was also re-peated at the same time in two greenhouses.Number of days The transmission of the linked bar and HVA1genes to heading was recorded when panicles were extruded from was first observed by germinating a total of 100seeds the flag leaf sheath.Plant height was measured from the soil from each of the five lines of R3progeny and the control surface to the top of the main panicle at physiological maturity.in the presence of a high concentration of herbicide (15The area of each individual fully expanded flag leaf blade was mg/L glufosinate ammonium)in vitro.The seedlings of computed as length ?maximum width ?0.75.It was assumed that length and width did not change after full expansion the transgenic lines grew in the selection medium as (Elings,2000).Stems were counted on each plant at the 6to vigorously as in the absence of the herbicide.No seeds 7leaf stage.The number of spikelets and panicle length were of the nontransgenic control germinated on the same counted and measured at maturity before harvest.Plants were selection medium.The segregation ratio of the bar gene harvested at full maturity.Shoots were removed from roots (glufosinate ammonium resistance)was not significantly at the soil surface.The soil was carefully washed from the different from 9:7,the segregation ratio for one pair of roots and the root length was measured.The number of ker-hemizygous alleles in the third transgenic generation nels/panicle was determined by counting kernels on every (Table 1).The results of the PCR analysis (Fig.2)were panicle for each plant after harvest.Mean kernel weight was found to be consistent with that observed in the germi-calculated from the weight of three sets of 300kernels.Kernel nation test confirming the existence of the intact linked yield/plant was determined on the basis of the harvested plants.
bar and HVA1genes in the R3progeny.The herbicide-resistant progeny of the transgenic plants were used in the subsequent molecular and agronomic analyses.
Statistical Analyses
Chi square (?2)analysis,using the correction factor of Yates Histochemical Analysis of GUS
(Steel and Torrie,1980),was performed to determine if the observed segregation ratio of the third generation was consis-GUS expression pattern in R3transgenic seeds and tent with the expected ratio of a pair of hemizygous alleles.seed husks were visualized by histochemical staining To study the effects of salinity,the experimental design was with X-Gluc (Jefferson et al.,1987).GUS staining was a split plot with salinity as a whole-plot factor and lines as a observed also in younger root segments for all of the subplot factor.The experiment was conducted in two green-five lines with no expression in the control plants houses with five replications (blocks)in each greenhouse.In (Fig.3).The results revealed constitutive expression of each block,pots of each line (one plant/pot)were assigned at the gus gene in tissues of transgenic oat and indicated random to receive certain salinity level.Then,the pots were arranged in the whole plot groups with the same salinity level
successful inheritance of the gene to the R3progeny.
R e p r o d u c e d f r o m C r o p S c i e n c e . P u b l i s h e d b y C r o p S c i e n c e S o c i e t y o f A m e r i c a . A l l c o p y r i g h t s r e s e r v e d .
ORABY ET AL.:BARLEY HVA1GENE CONFERS SALT TOLERANCE IN R3TRANSGENIC OAT
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Fig.2.PCR amplification of the bar (0.59kb)and HVA1(0.7kb)genes shows the presence of the transgenes in R3for the five https://www.sodocs.net/doc/4214725469.html,ne 1:100-bp ladder marker,Lane 2:plasmid (positive control),Lane 3:nontransformed (negative control),and Lanes 4–8:transgenic lines.
Southern Blot Analysis of the HVA1Gene
that R3progeny of transgenic oat lines inherited the transcriptionally active HVA1gene (Lanes 1–5).As a in R3Progeny Plants
control in the RNA blotting experiment,RNA from Southern blot analysis was used to investigate the nontransformed oat leaves was included on the blot.stable incorporation,independence of the transgenic lines,The assay showed that the HVA1transcript was not and transmission of the HVA1gene in the R3progeny detectable in the control plants (Lane C).
(Fig.4a).The plasmid (Lane P),transgenic lines (Lanes 1–5),and nontransgenic control of Ogle cultivar (Lane Salinity Effects on Plant Growth,Yield,
C)DNA were included in the blot.
and Its Components
When hybridized with the HVA1gene probe,the nontransformed plants did not show any hybridization NaCl is a common salt that negatively influences plant band.The plasmid and the genomic DNAs of the five growth under natural conditions.NaCl solution was transgenic lines that hybridized with the same probe used in this study,although single salt solutions do not showed bands of different sizes larger than 3.67(Fig.4a)exist in nature (Bernstein,1962).
as expected.Line BRA-19showed a band size of about The analysis of variance of the salinity levels,geno-3.9kb,which is smaller than its size in R0(Maqbool et types (lines and control),and their interaction is dis-al.,2002).This could be explained by possible DNA played in Table 2.Greenhouses did not have a significant rearrangements resulting from deletion,translocation,effect on all the studied traits.Significant differences or crossing-over on the genomic DNA downstream of were observed among the salinity levels and genotypes the two transgenes cassettes.
for all the traits.Although the magnitude of the inter-These results provide evidence that the HVA1gene action between salinity and genotypes is very small has remained stably integrated in the plant genome with compared with the main effects of the treatments,the five independence events,and transmitted into the ge-ANOVA mean squares revealed significant genotype ?nomic DNA of the R3progeny.
environment interaction (P ?0.05)for all the studied characteristics.This significant interaction arises from Northern Blot Analysis of the HVA1Gene
the differential genotypic responses to different salinity in R3Progeny Plants
levels during the plant life cycle.
The magnitude of the main effect mean squares sug-Northern analysis was used to assay the transcription gested that the number of days to heading,plant height,of the HVA1regulated by the rice Act1constitutive panicle length,root length,number of kernels/panicle,promoter,whose expression were associated with differ-1000-kernel weight,and kernel yield were influenced ences in salinity tolerance between the transgenic lines by salinity more than genotype as it showed between and the control plants (Fig.4b).RNA isolated from 20and 85%of the total variation.Flag leaf area,number young leaves of the transgenic lines was probed with of spikelets/panicle,and number of tillers/plant appear the HVA1gene.A transcript of the expected size (ap-proximately 1kb)for this gene was detected,indicating
to be almost equally affected by salinity levels and
geno-
Fig.3.GUS expression in R3transgenic oat seed husks (a),seeds (b),and root segments (c)
R e p r o d u c e d f r o m C r o p S c i e n c e . P u b l i s h e d b y C r o p S c i e n c e S o c i e t y o f A m e r i c a . A l l c o p y r i g h t s r e s e r v e d .
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Fig.4.Top;pBY520partial map,bottom;Southern blot (a)and Northern blot (b)analyses showing bands for R3of transgenic oat plants digested with Hin dIII,M:ladder marker;P:plasmid BY520;Lanes 1–5:Ogle BRA-82,Ogle BRA-17,Ogle BRA-8,Ogle BRA-19,Ogle BRA-41respectively;C:nontransgenic control.
types which explained more than 40%of the total varia-entiation,which subsequently enables the plant to main-tain higher number of kernels/panicle (Blum et al.,1990).tion particularly for these traits.These results indicate that the chosen salinity levels were very efficient in The plant height,flag leaf area,and root growth pa-rameters may serve as yield attributes (Ulery et al.,imposing the stress.
Although transgenic plants showed better recovery 1998).The height was not significantly different among genotypes at 0and 50m M NaCl salinity.The transgenic and grew faster than the control plants after the salt-stress recovery period (data not shown)and also main-lines were able to retain their height until 100m M com-pared with control.The control plants tended to be tained more tolerance to salinity during and after the salt treatments,higher salinity levels (150and 200m M )shorter under salinity and the shortest height for most genotypes was obtained at 200m M NaCl (Fig.5b).Simi-significantly reduced plant growth of both transgenic and control plants (Table 3and Fig.5).The differences lar findings were reported by Xu et al.(1996)in rice.The minimum response of plant height to changes in in stress treatments revealed a progressive decrease in the average number of days to heading,plant height,salinity levels of transgenic lines may result in the ability of the plants to preserve current assimilates as a source flag leaf area,and root length (Fig.5a-d).
Early heading is one of the mechanisms that plants of carbon stored in the stem for grain filling (Blum,1998).In addition,this performance may improve the use to escape the damage effects caused by salinity stress (Bajji et al.,2004).In this study,mean days to heading response to nitrogen fertilization under salt stress con-ditions.
decreased as salinity level increased,although this de-crease did not vary among the genotypes (Fig.5a).Most The plant ability to maintain large leaves and delay leaf senescence under water limitation stresses is a stress of the transgenic lines headed significantly later than the control at early high salinity stress (?50m M NaCl).tolerance mechanism called “non-senescence”or “stay-green”(Rosenow,1977;Thomas and Smart,1993).One
This delay in flowering gives the opportunity of late differ-Table 2.Mean squares for kernel yield and its components and some other agronomic characters for the R3transgenic lines expressing the HVA1gene and the control.
Traits
Number of Number of Number Number days to Plant Flag leaf Panicle spikelets/Root of tillers/of kernels/1000-kernel Kernel Source
df heading height
area
length
panicle
length
plant
panicle
weight yield/plant Salinity 476.66*5785.25*2178.73*658.15*2779.52*1406.80*21.52*10451.00*850.83*615.19*Genotype
519.62*2850.95*2600.72*107.81*2702.18*408.11*10.21*4752.28*145.66*204.49*Salinity ?genotype 20 5.96*360.89*205.99*34.98*284.18*141.38* 2.15*436.94*16.29*41.01*Greenhouse
1 1.24NS 56.38NS 0.82NS 51.35NS 24.48NS 27.07NS 9.79NS 1.76NS 4.39NS 0.28NS Reps (greenhouse)8 1.57NS 17.38NS 18.83NS 14.33NS 7.97NS 10.33NS 2.50NS 6.72NS 1.87NS 0.81NS Reps ?salinity 36 1.14NS 52.60NS 21.37NS 7.95NS 23.51NS 5.22NS 1.33NS 3.67NS 3.41NS 0.60NS (greenhouse)Residual
214
1.24
39.81
23.40
6.48
21.27
3.65
1.26
3.80
3.32
0.62
*Indicates significance at P ?0.05
NS indicates not significant
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Table 3.Differences between the R3transgenic lines and the nontransgenic control under different salinity levels for the number of tillers/plant,number of kernels/panicle,1000-kernel weight,and kernel yield/plant.
Traits
Salinity level Line Number of tillers/plant
Number of kernels/panicle
1000-Kernel weight
Kernel yield/plant
m M g
BRA-82 5.70a?74.00a 33.48a,b 12.10a BRA-17 5.50a 92.00c 34.56a,b 15.44c BRA-8 5.90a 90.30b,c 32.88a 16.05c BRA-19 5.20a 75.00a 33.21a,b 11.75a BRA-41 5.20a 94.90d 34.64a,b 14.31b Control 5.90a 89.00b 35.19b 16.93d 50
BRA-82 5.30b 70.10b 31.33b 10.29b BRA-17 5.30b 92.00f 31.51b 14.13d BRA-8 5.90b 85.30d 31.90b 14.45d BRA-19 5.90b 73.00c 32.70b,c 11.74c BRA-41 5.20a,b 90.00e 34.58c 13.95d Control 4.30a 66.90a 26.84a 7.26a 100
BRA-82 5.20b 69.00b 31.06b 9.19b BRA-17 5.40b 90.00d 31.11b 13.66d BRA-8 5.60b 82.70c 31.87b,c 13.52d BRA-19 5.40b 70.00b 32.64b,c 10.59c BRA-41 5.30a,b 91.00d 33.47c 13.73d Control 3.80a 54.00a 24.20a 3.75a 150
BRA-82 4.80b 64.00b 27.48c 7.64b BRA-17 4.50b 72.00c 25.41b 10.04d BRA-8 4.10b 79.90d 25.77b,c 9.82c,d BRA-19 4.10b 65.00b 26.46b,c 8.72c,d BRA-41 4.70b 79.00d 27.46b,c 9.23c Control 2.84a 37.17a 19.85a 2.01a 200
BRA-82 4.50b 50.00b 22.88b 5.96c BRA-17 4.90b 54.00c 21.49b 5.90d BRA-8 4.80b 50.40b 22.50b 5.14b BRA-19 4.50b 51.00b 23.31b 6.85d BRA-41 4.70b 60.00d 25.52c 8.37e Control
1.83a 28.79a 16.78a 1.11a Standard error
0.49
0.79
0.76
0.32?In the same column under each salinity level,numbers followed by the same letter are not significantly different (P ?0.05).
of the most important points is whether or not retaining The effects of salinity levels on the panicle traits are shown in Fig.5e and f.In general,panicle length (Fig.5e)a green and large leaf area under salt stress increases kernel yield.Helsel and Frey (1978)reported a positive and number of spikelets/panicle (Fig.5f)were reduced by salinity levels 50m M and higher.While plants of trans-correlation between leaf area duration and kernel yield in the oat crop.Leaves of transgenic lines were normal genic lines maintained high panicle length and number of spikelets/panicle among the salinity levels,salinity and displayed no symptoms of wilting at 50m M salinity.The genotypic differences under salinity-induced changes treatments at 100and 50m M NaCl resulted in significant differences in panicle length and number of spikelets/in the flag leaf area were most prominent at 100m M and above concentrations.The flag leaf area was more panicle respectively,compared with the control.The panicle length and number of spikelets/panicle were severely reduced by salt stress in the control plants than the transgenic lines (Fig.5c).The data presented in reduced of approximately 23to 52%and 25to 77%at 200m M NaCl respectively (Fig.5e and f).The panicle Table 3confirmed that Line BRA-41,which retained the highest flag leaf area (Fig.5c),maintained the high-length of control plants was 50%shorter than the best transgenic line.The lack of panicle length extension est yield among the different salinity levels.This proba-bly resulted from increased photosynthesis when kernel under salinity might have contributed to plant height reduction (Milach et al.,2002).Moreover,Giunta et filling occurred under stress (Blum,1983;Van Oosterom and Acevedo,1992).
al.(1993)reported that,in wheat,severe water deficit around anthesis produces serious effects on yield,reduc-The length of oat roots was correlated with the toler-ance of juvenile plants to water stress in the field (Lars-ing the number of spikelets and therefore causing a decrease in plant fertility.
son and Gorny,1988).Root length of the control was not different from the lines in the salinity control treat-It was reported that variation in kernel yield poten-tials in cereals under water stress growing conditions ment (0m M )(Fig.5d).As salt concentration increased,root length of all transgenic lines increased relative to were predominantly associated with variations and se-quential development of yield components (Fischer and
the control.However,when salt concentrations in-creased beyond 100m M ,root growth declined.Since Maurer,1978;Garc?
′a del Moral et al.,1991,2003;Si-mane et al.,1993).Kernel yield was greater under salin-only the final root length of each line and the control was measured,no differences in root growth were detected ity stress for the transgenic plants than the control.The higher yield was due to increased number of tillers,between the lines but there is a significant difference between the lines and the control at 50m M and above number of kernels/panicle and heavier kernels (Table 3).Salinity stress at 200m M NaCl for a long period of time
(Fig.5d)(Sivamani et al.,2000).
R e p r o d u c e d f r o m C r o p S c i e n c e . P u b l i s h e d b y C r o p S c i e n c e S o c i e t y o f A m e r i c a . A l l c o p y r i g h t s r e s e r v e d .
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2005
Fig.5.The effect of increasing NaCl concentrations on number of days to heading (a),plant height (b),flag leaf area (c),root length (d),panicle length (e),and number of spikelets/panicle (f)for the transgenic lines and the nontransgenic control.
caused reduction in kernel yield estimated at ?40%for negative effect on number of kernels/panicle and kernel weight under salinity stress.The compensatory effect be-transgenic lines and 90%for the control plants.Under the conditions of this study (Table 3),number of kernels/tween tiller production and the other components may have resulted from the negative allometry between these panicle was the most sensitive yield component affected by salinity stress (reduced by ?30and 60%)followed traits during plant development (Hamid and Grafius,1978).Moreover,it is well documented that under stress by 1000-kernel weight (reduced by ?26and 50%for the transgenic lines and the control respectively).The number environments compensation could be mainly achieved by extensive tillering of surviving plants (Holena et al.,2001).of tillers/plant was less affected by salinity for the trans-genic lines (reduction of 10–20%),but the influence of Overall,transgenic plants of the R3generation devel-oped normal flowers,grew to maturity,and set seeds salinity was higher on the control plants (?60%reduc-tion).Among the transgenic lines,the line BRA-41ex-in a normal manner under greenhouse conditions,sug-gesting that expression of the linked bar-HVA1and non-pressed the highest yield under severe salinity stress (200m M )because of its lower reduction in number of linked gus genes had no deleterious effects on growth and fertility.
tillers/plant,1000-kernel weight,and number of kernels/panicle (Table 3).Evans and Wardlaw (1976)explained The correlation between accumulation of LEA group III proteins and stress tolerance is well studied in wheat yield component compensation as the allowance of sub-sequently occurring components of final kernel yield to and rice (Ried and Walker Simmons,1993;Rohila et al.,2002;Sivamani et al.,2000;Wise,2003).Moreover,compensate for restrictions and/or losses during earlier stages of development,or to maximize reproductive it is evident that genetic variability exists for stress re-sponses,and this could be due to the differential expres-growth in the plant life cycle.
On the basis of the transgenic line observations,the sion and regulation of stress responsive genes such as HVA1gene when the plants are exposed to stress (Jaya-
results indicate that number of tillers/plant may have a
R e p r o d u c e d f r o m C r o p S c i e n c e . P u b l i s h e d b y C r o p S c i e n c e S o c i e t y o f A m e r i c a . A l l c o p y r i g h t s r e s e r v e d .
ORABY ET AL.:BARLEY HVA1GENE CONFERS SALT TOLERANCE IN R3TRANSGENIC OAT
2225
Fig.7.R3transgenic (a)and nontransgenic (b)oat roots at 100m M NaCl
stress.
transcription,and translation of the intact linked bar -Fig.6.R3transgenic oat (a)and nontransgenic (b)at 150m M NaCl HVA1and the nonlinked gus genes in the R3progeny stress.
of transgenic oat plants.
Transgenic and nontransgenic plants performed simi-prakash et al.,1998;Uma et al.,1995).On the basis of larly under well-watered growing conditions (0m M NaCl).our experimental results,the positive significant rela-Appearance and development of the symptoms of dam-tionship between the present increase in growth of the age caused by salinity were delayed in transgenic plants.transgenic lines under stress over controls and the pres-Under salinity stress,differences in salinity tolerance ence of HVA1transcripts suggest more evidence that between transgenic lines and the control were associated constitutive expression of HVA1gene in transgenic with the flag leaf area growth,positive effects of plant plants can improve growth performance under salinity height,panicle length and number of spikelets/panicle stress conditions;however,the exact function of LEA (Fig.6)and development and maintenance of extensive proteins remains uncertain.
root system (Fig.7).
Kernel filling in cereals is considered to be one of the CONCLUSION
most sensitive growth stages to stresses (Nayyar and Walia,2004).Physiological stress expressed by salinity We previously characterized five transgenic oat lines during this period reduces the storage capacity of cereal that expressed the barley HVA1gene and showed in-kernels by decreasing the number of endosperm cells creased tolerance to salinity under in vitro conditions and/or the number of amyloplasts initiated (Jones et al.,at the seedling stage (Maqbool et al.,2002).In this study,1996).Salinity can reduce the kernel weight and number the molecular and agronomic characteristics of R3prog-of kernels by limiting the rate and duration of the filling eny of those lines were analyzed under salinity stress process and inhibiting photosynthesis (Gupta et al.,conditions in greenhouses.Molecular (PCR,Southern,2001;Vishwanathan and Khanna-Chopra,2001).As in-and Northern blots),segregation for herbicide,and GUS histochemical analyses confirmed the existence,
dicated by yield and yield components (number of til-
R e p r o d u c e d f r o m C r o p S c i e n c e . P u b l i s h e d b y C r o p S c i e n c e S o c i e t y o f A m e r i c a . A l l c o p y r i g h t s r e s e r v e d .
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Evans,L.,and I.F.Wardlaw.1976.Aspects of the comparative physiol-lers/plant,number of kernels/panicle and 1000-kernel ogy of kernel yield in cereals.Adv.Agron.22:301–359.
weight),the results demonstrate that control plants were FAO.1999.Biotechnology in food and agriculture [Online]http://more sensitive to salinity and experienced more severe https://www.sodocs.net/doc/4214725469.html,/unfao/bodies/COAG/COAG15/X0074E.htm;verified water stress than did the transgenic lines under high 8July 2005.
Fischer,R.A.,and R.Maurer.1978.Drought resistance in spring wheat salinity conditions.
cultivars:I.Kernel yield responses.Aust.J.Agric.Res.29:897–912.Although transgenic lines experienced a decrease in Frey,K.1998.Genetic responses of oat genotypes to environmental performance at the highest salinity stress level (200factors.Field Crops Res.56:183–185.
m M ),these lines showed better performance than non-Garc?
′a del Moral,L.F.,J.M.Ramos,M.B.Garc?′a Del Moral,and P.transgenic controls under continuous salt stress.Line Jimenez-Tejada.1991.Ontogenetic approach to kernel production BRA-41experienced the least and the control plants in spring barley based on path-coefficient analysis.Crop Sci.31:1179–1185.
the most decrease in performance through the salinity Garc?
′a del Moral,L.F.,Y.Rharrabtia,D.Villegas,and C.Royo.2003.levels for most of the studied traits.The results provide Evaluation of kernel yield and its components in durum wheat more evidence about the role of the HVA1protein in under Mediterranean conditions:An ontogenic approach.Agron.water deficit damage prevention (Xu et al.,1996)and J.95:266–274.
the protection of oats against salinity.This could benefit Giunta,F.,R.Motzo,and M.Deidda.1993.Effect of drought on yield and yield components of durum wheat and triticale in a farmers with healthier plants,potentially resulting in Mediterranean environment.Field Crops Res.33:399–409.
possible contribution to superior yield.
Goday,A.,A.Jensen,F.A.Culia ′n ?ez-Macia
`,M.M.Alba `,M.Figueras,J.Serratosa,M.Torrent,and M.Page
′s.1994.The maize abscisic ACKNOWLEDGMENTS
acid responsive protein rab17is located in the nucleus and cyto-plasm and interacts with nuclear localization signals.Plant Cell The authors wish to thank Dr.James Kelly,Dr.Russel 6:351–360.
Freed (Department of Crop and Soil Sciences,Michigan State Gupta,N.K.,S.Gupta,and A.Kumar.2001.Effect of water stress University,MI)and Dr.Farouk Oraby (Department of Plant on physiological attributes and their relationship with growth and Sciences,Higher Institute for Productivity Efficiency,Zagazig yield of wheat cultivars at different stages.J.Agron.Crop Sci.University,Egypt)for constructive criticism and helpful sug-186:55–62.
gestions for the manuscript.This research was supported by Hamid,Z.A.,and J.E.Grafius.1978.Developmental allometry and Consortium for Plant Biotechnology Research (CPBR)and its implication to kernel yield in barley.Crop Sci.18:83–86.
the Egyptian government that financially support Hesham Helsel,D.B.,and K.J.Frey.1978.Kernel yield variations in oats associated with differences in leaf area duration among oat lines.Oraby’s studies.
Crop Sci.18:765–769.
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