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Published online 23 September 2005
Published in Crop Sci 45:2218-2227 (2005)
© 2005 Crop Science Society of America
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GENOMICS, MOLECULAR GENETICS & BIOTECHNOLOGY

Barley HVA1 Gene Confers Salt Tolerance in R3 Transgenic Oat

Hesham F. Oraby, Callista B. Ransom, Alexandra N. Kravchenko and Mariam B. Sticklen*

Dep. of Crop and Soil Sciences, Michigan State Univ., Plant and Soil Science Bldg., East Lansing, MI 48824

* Corresponding author (stickle1{at}msu.edu)


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSION
 REFERENCES
 
A major cause of oat crop yield loss worldwide is osmotic stress due to drought and/or salinity. This study investigated the third generation of transgenic oat (Avena sativa L.) expressing barley HVA1 stress tolerance, ß-glucuronidase (uidA; gus) and bar herbicide resistance genes. Transgenic plants showed normal 9:7 third generation inheritance for glufosinate ammonium herbicide resistance. Molecular and histochemical studies confirmed the presence and stable expression of all three genes. Compared with the nontransgenic control plants, transgenic R3 plants exhibited greater growth and showed a significant (P < 0.05) increase in tolerance to salt stress conditions (200 mM NaCl) for traits including number of days to heading, plant height, flag leaf area, root length, panicle length, number of spikelets/panicle, number of tillers/plant, number of kernels/panicle, 1000-kernel weight, and kernel yield/plant.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSION
 REFERENCES
 
ENVIRONMENTAL STRESSES such as drought and salinity represent some of the most limiting factors for agricultural productivity worldwide (Boyer, 1982; Roy and Wu, 2002). Not only do they greatly decrease the potential yield of current crop species, but they also restrict the area where production can take place and hinder the introduction of crop plants into new areas (Epstein et al., 1980). Salinity in particular is a major problem, affecting crop production on nearly one-third of the world's irrigated agricultural land (Apse et al., 1999; Schachtman and Lui, 1999). There is a shortage of arable land area and this area is steadily decreasing, as farming practices cause cultivated land to become more saline (Qadir et al., 1998). Farmers are, therefore, being forced to cultivate in salinity-prone areas, but plants grown in these areas experience severe salinity stress. This situation will only worsen in the coming decades (Cherry et al., 1999).

Oat is an important cereal crop used in human and animal diets (Welch, 1995). Osmotic stress due to drought and/or salinity is a major cause of global oat crop yield loss (Frey, 1998; Martin et al., 2001; Tamm, 2003). Although, compared with the other cereals, oat is considered a moderately salt tolerant crop (Murty et al., 1984), soil salinity is responsible for decreasing oat seed germination and stunting subsequent development in a cultivar-dependent manner (Murty et al., 1984; Verma and Yadava, 1986).

Traditional breeding has limitations and cannot solve this problem alone, perhaps because of the inefficiency of selection methods, the lack of the genetic variability in the oat background, and the salt tolerance trait complexity (Cushman and Bohnert, 2000). Crop improvement strategies that are based on the use of new technologies, such as biotechnology, can be used in conjunction with traditional breeding efforts (Abebe et al., 2003; Epstein et al., 1980; Ribaut and Hoisington, 1998), offering a responsible way to enhance agricultural productivity. Biotechnology could help eliminate many obstacles limiting crop production in developing countries. The development of crops with the internal capacity to withstand abiotic stresses would help to reduce the use of water (FAO, 1999), thus promoting sustainable yields (Sharma et al., 2001). Also, modern plant genetic engineering presents the possibility of rapid and precise introduction of a desirable trait from closely related plants without associated deleterious genes (Richards, 1996) because it avoids the transfer of unwanted chromosomal regions (Cushman and Bohnert, 2000; Sharma et al., 2002). Oat transformation using microprojectile bombardment was first reported by Somers et al. (1992). It is now possible to produce fertile transgenic lines of major cereal crops including oat for different studies (Makarevitch et al., 2003; McGrath et al., 1997; Pawlowski et al., 1998; Pawlowski and Somers 1998; Svitashev et al., 2000, 2002; Torbert et al., 1995, 1998a, 1998b, 1998c; Zhang et al., 1999).

Several promising abiotic stress tolerance candidate genes for crop transformation have been identified. Among those are the Late Embryogenesis Abundant (LEA) proteins, which are ubiquitous in plants. These proteins accumulate during the late stage of seed formation and in vegetative tissues under drought, heat, cold, and salt stress conditions or with abscisic acid (ABA) application (Sivamani et al., 2000). LEA proteins are generally divided into five groups, and they appear to protect cellular structures from dehydration stress; however, the exact functional role of these hydrophilic proteins remains poorly understood. Proposed roles include water binding or replacement, hydration buffers (Dure, 1993a; Ingram and Bartels, 1996), ion sequestration (Dure, 1993b), osmotic adjustment or reverse chaperones (Close, 1996), and transport of nuclear-targeted proteins during stress (Goday et al., 1994).

HVA1, a barley (Hordeum vulgare L.) group III LEA protein, is highly induced by ABA/stress. The HVA1 gene (Hong et al., 1992) has been used successfully to confer stable tolerance to osmotic stress in rice (Oryza sativa L.) (Xu et al., 1996), oat (Maqbool et al., 2002), and wheat (Triticum aestivum L.) (Patnaik and Khurana, 2003).

This paper reports studies on the third generation of transgenic oat expressing HVA1, ß-glucuronidase (uidA; gus), and the bar herbicide resistance genes under greenhouse conditions. The objectives were: (i) to determine the segregation of the linked bar and HVA1 genes and verify their proper expression and stability of transmission to progeny; (ii) to evaluate the effects of the transgenes on plant growth; and (iii) to compare the performance of these transgenic lines against nontransgenic controls for common important agronomic traits under salt stress conditions.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 CONCLUSION
 REFERENCES
 
Plant Materials
R3 seeds 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 multimeristem transformation system with two plasmids, pBY520 and pAct1-D. The plasmid BY520 contained the linked selectable marker/herbicide resistance bar (phosphinothricin acetyl transferase) gene (driven by cauliflower mosaic virus 35S promoter and the nopaline synthase nos terminator) and the barley HVA1 gene (driven by rice Act1 promoter and terminated by the potato protease inhibitor pin II). The plasmid Act1-D contained the nonlinked Escherichia coli-glucuronidase (gus) gene flanked by the Act1 promoter and the nos terminator.

The lines chosen for this work had one copy of the linked HVA1-bar genes. Bagged flowers of some R0, R1, and R2 progenies were allowed to self-pollinate and produce seeds which were collected in bulk from each line. Bulked R3 transgenic and control (Ogle wild-type cultivar) seeds were germinated; plants of R3 generation were grown and evaluated in two greenhouses under salt treatments.

Segregation of Herbicide Resistance of R3 Progeny
Seeds were surface-sterilized with 70% (v/v) ethanol for 2 min, followed by 20% (v/v) commercial bleach (5.25% commercial sodium hypochlorite) treatment for 10 min, rinsed with sterile distilled water several times, and briefly blotted onto sterile filter paper. Transgenic and nontransgenic control seeds were germinated on MS (Murashige and Skoog, 1962) basal medium containing 15 mg/L glufosinate ammonium for selection. Control seeds were also germinated on the same medium lacking herbicide. Cultures were maintained under continuous fluorescent light at 28°C for 1 wk. The seedlings that survived the selection were used for molecular and phenotypic characterization of the lines.

Salinity Treatments
Young seedlings were transferred into Baccto Professional Planting Soil Mix (700–800 g kg–1 horticultural sphagnum peat, 200–300 g kg–1 perlite, pH 5.5–6.5) in small pots (8 x 4 x 6 cm), one plant per pot (Fig. 1) . The pots were kept in water-filled flat-bottom trays for 1 wk. Two-week-old seedlings from each transgenic line and the control were transferred to 7.3-L (2-gallon) pots for another week before starting the salt stress treatments.



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  		Fig. 1. R3 transgenic seedlings after in vitro selection.

 
The environmental settings in the greenhouses were maintained between 21 and 25°C, with a relative humidity between 80 and 90%. Natural illumination was augmented for 16 h per day with fluorescent light and light levels were between 150 and 185 µmol m–2 s–1.

The soil surface around the seedlings was covered with redwood bark mulch to minimize the evaporation and prevent algae growth. A dilution of commercial 20:20:20 fertilizer solution was added twice a week for nutritional needs. Sodium chloride (NaCl) was added in five concentrations of 0, 50, 100, 150, and 200 mM. Plants were watered with the saline solution once per day ensuring adequate leaching, and preventing salinity excess. Plants were given the salinity treatments for 14 d, followed by 1 wk of irrigation with plain water to measure plant recovery after salt stress. Afterward, the salt treatments were resumed uninterrupted for 35 d.

PCR Analysis
The detection of the bar and the HVA1 genes in all the studied R3 transgenic plants by PCR amplification was performed with leaf disk DNA as template and REDExtract-N-Amp Plant PCR Kit (Sigma-Aldrich, St. Louis, MO, Cat # XNA-P) as 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 primer) and HVA1 F, 5'-TGG CCT CCA ACC AGA ACC AG-3' (forward primer); and HVA1 R, 5'-ACG ACT AAA GGA ACG GAA AT-3' (reverse primer). DNA amplifications were performed in a thermo cycler (PerkinElmer/Applied Biosystem, Foster City, CA) using initial denaturation at 94°C for 4 min, followed by 35 cycles of 1 min at 94°C, 1 min at 55°C, 2 min at 72°C, and a final 10 min extension at 72°C. The reaction mixture was loaded directly onto a 0.8% (w/v) agarose gel, stained with ethidium bromide, and visualized with UV light. The transgene product size was about 0.59 kb for the bar gene and 0.70 kb for the HVA1 gene.

Histochemical Analysis of GUS
Seeds, seed husks, and root segments from the transgenic and nontransgenic plants were used to detect GUS activity by histochemical staining with 5-bromo-4-chloro-3-indoyl-ß-D-glucuronicacid salt (X-gluc). Samples were immersed in GUS substrate mixture and incubated at 37°C (Jefferson et al., 1987). The tissues were thoroughly washed with 70% ethanol and examined under a Zeiss SV8 stereomicroscope.

DNA Isolation and Southern Blot Hybridization Analysis
Independence of the lines and confirmation of the HVA1 transgene transmission into the oat R3 transgenic plants were performed by Southern-blot hybridization using the HVA1-coding sequence as a probe. Genomic DNA from transgenic and nontransgenic oat plants was isolated using the protocol of Saghai-Maroof et al. (1984). For Southern blots, 10 to 15 µg of genomic DNA was digested with HindIII restriction enzyme, electrophoresed in 0.8% (w/v) agarose gel, transferred onto Hybond-N+ (Amersham-Pharmacia Biotech) membranes, and fixed with a UV crosslinker (Stratalinker UV Crosslinker 1800, Stratagene, CA) as recommended in the manufacturers' instructions. The HVA1 gene-specific probe was generated by a HindIII-BamHI digest of pBY520 to isolate a 1.0-kb fragment. The restriction fragment was purified with the QIAquick kit (QIAGEN). Probe labeling and detection were obtained with the DIGHigh Prime DNA Labeling and Detection Starter Kit II (Kit for chemiluminescent detection with CSPD, Roche Co.) following the manufacturer's protocol.

RNA Isolation and Northern Blot Hybridization Analysis
Assay for transcriptional expression of the HVA1 transgene was performed by Northern blots. Total RNA was isolated from young leaves of oat plants (transgenic and nontransgenic) using the TRI Reagent (Sigma-Aldrich, St. Louis, MO) according to the manufacturer's instructions. For the Northern blot, 20 µg of RNA were separated in a 1.2% (w/v) agarose-formaldehyde denaturing gels according to Sambrook et al. (1989) and blotted onto Hybond-N+ nylon membranes (Amersham-Pharmacia Biotech). Transcripts of HVA1 were analyzed by a standard Northern-blotting method (Sambrook et al., 1989) using the HVA1-coding sequence as a probe labeled with {alpha}- [32P]-dCTP with the Random Primer Labeling Kit (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions.

Measurements of Parameters
Data were collected on kernel yield and its components (number of tillers/plant, number of kernels/panicle, 1000-kernel weight). Additional measurements were taken on number of days to heading, plant height, flag leaf area, root length, panicle length, and number of spikelets/panicle during the experiment for each plant in the five replications under each salinity concentration. The whole experiment was also repeated at the same time in two greenhouses. Number of days to heading was recorded when panicles were extruded from the flag leaf sheath. Plant height was measured from the soil surface to the top of the main panicle at physiological maturity. The area of each individual fully expanded flag leaf blade was computed as length x maximum width x 0.75. It was assumed that length and width did not change after full expansion (Elings, 2000). Stems were counted on each plant at the 6 to 7 leaf stage. The number of spikelets and panicle length were counted and measured at maturity before harvest. Plants were harvested at full maturity. Shoots were removed from roots at the soil surface. The soil was carefully washed from the roots and the root length was measured. The number of kernels/panicle was determined by counting kernels on every panicle for each plant after harvest. Mean kernel weight was calculated from the weight of three sets of 300 kernels. Kernel yield/plant was determined on the basis of the harvested plants.

Statistical Analyses
Chi square ({chi}2) analysis, using the correction factor of Yates (Steel and Torrie, 1980), was performed to determine if the observed segregation ratio of the third generation was consistent with the expected ratio of a pair of hemizygous alleles. To study the effects of salinity, the experimental design was a split plot with salinity as a whole-plot factor and lines as a subplot factor. The experiment was conducted in two greenhouses with five replications (blocks) in each greenhouse. In each block, pots of each line (one plant/pot) were assigned at random to receive certain salinity level. Then, the pots were arranged in the whole plot groups with the same salinity level for each group. The greenhouses and blocks were considered random factors. The statistical analysis for quantitative traits was performed by PROC MIXED (SAS Institute Inc., 2003). The significance of the interaction between genotype and salinity was tested and the line means were compared at each salinity level by t tests.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
CONCLUSION
 REFERENCES
 
Here, the third generation of five independent transgenic lines obtained from a previous study (Maqbool et al., 2002) was tested for salinity tolerance under greenhouse conditions. The independence of the lines was confirmed by Southern blot analysis. The molecular analyses, segregation for herbicide resistance, GUS histochemical assay, and salinity treatment were used to investigate transgene integration, transcription, and translation in the R3 progeny of the transgenic plants.

Segregation of Herbicide Resistance of R3 Progeny
The transmission of the linked bar and HVA1 genes was first observed by germinating a total of 100 seeds from each of the five lines of R3 progeny and the control in the presence of a high concentration of herbicide (15 mg/L glufosinate ammonium) in vitro. The seedlings of the transgenic lines grew in the selection medium as vigorously as in the absence of the herbicide. No seeds of the nontransgenic control germinated on the same selection medium. The segregation ratio of the bar gene (glufosinate ammonium resistance) was not significantly different from 9:7, the segregation ratio for one pair of hemizygous alleles in the third transgenic generation (Table 1). The results of the PCR analysis (Fig. 2) were found to be consistent with that observed in the germination test confirming the existence of the intact linked bar and HVA1 genes in the R3 progeny. The herbicide-resistant progeny of the transgenic plants were used in the subsequent molecular and agronomic analyses.


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  		Table 1. Segregation of herbicide resistance of R3 transgenic Ogle cultivar.

 


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  		Fig. 2. PCR amplification of the bar (0.59 kb) and HVA1 (0.7 kb) genes shows the presence of the transgenes in R3 for the five lines. Lane 1: 100-bp ladder marker, Lane 2: plasmid (positive control), Lane 3: nontransformed (negative control), and Lanes 4–8: transgenic lines.

 
Histochemical Analysis of GUS
GUS expression pattern in R3 transgenic seeds and seed husks were visualized by histochemical staining with X-Gluc (Jefferson et al., 1987). GUS staining was observed also in younger root segments for all of the five lines with no expression in the control plants (Fig. 3) . The results revealed constitutive expression of the gus gene in tissues of transgenic oat and indicated successful inheritance of the gene to the R3 progeny.



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  		Fig. 3. GUS expression in R3 transgenic oat seed husks (a), seeds (b), and root segments (c)

 
Southern Blot Analysis of the HVA1 Gene in R3 Progeny Plants
Southern blot analysis was used to investigate the stable incorporation, independence of the transgenic lines, and transmission of the HVA1 gene in the R3 progeny (Fig. 4a) . The plasmid (Lane P), transgenic lines (Lanes 1–5), and nontransgenic control of Ogle cultivar (Lane C) DNA were included in the blot.



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  		Fig. 4. Top; pBY520 partial map, bottom; Southern blot (a) and Northern blot (b) analyses showing bands for R3 of transgenic oat plants digested with HindIII, M: ladder marker; P: plasmid BY520; Lanes 1–5: Ogle BRA-82, Ogle BRA-17, Ogle BRA-8, Ogle BRA-19, Ogle BRA-41 respectively; C: nontransgenic control.

 
When hybridized with the HVA1 gene probe, the nontransformed plants did not show any hybridization band. The plasmid and the genomic DNAs of the five transgenic lines that hybridized with the same probe showed bands of different sizes larger than 3.67 (Fig. 4a) as expected. Line BRA-19 showed a band size of about 3.9 kb, which is smaller than its size in R0 (Maqbool et al., 2002). This could be explained by possible DNA rearrangements resulting from deletion, translocation, or crossing-over on the genomic DNA downstream of the two transgenes cassettes.

These results provide evidence that the HVA1 gene has remained stably integrated in the plant genome with five independence events, and transmitted into the genomic DNA of the R3 progeny.

Northern Blot Analysis of the HVA1 Gene in R3 Progeny Plants
Northern analysis was used to assay the transcription of the HVA1 regulated by the rice Act1 constitutive promoter, whose expression were associated with differences in salinity tolerance between the transgenic lines and the control plants (Fig. 4b). RNA isolated from young leaves of the transgenic lines was probed with the HVA1 gene. A transcript of the expected size (approximately 1 kb) for this gene was detected, indicating that R3 progeny of transgenic oat lines inherited the transcriptionally active HVA1 gene (Lanes 1–5). As a control in the RNA blotting experiment, RNA from nontransformed oat leaves was included on the blot. The assay showed that the HVA1 transcript was not detectable in the control plants (Lane C).

Salinity Effects on Plant Growth, Yield, and Its Components
NaCl is a common salt that negatively influences plant growth under natural conditions. NaCl solution was used in this study, although single salt solutions do not exist in nature (Bernstein, 1962).

The analysis of variance of the salinity levels, genotypes (lines and control), and their interaction is displayed in Table 2. Greenhouses did not have a significant effect on all the studied traits. Significant differences were observed among the salinity levels and genotypes for all the traits. Although the magnitude of the interaction between salinity and genotypes is very small compared with the main effects of the treatments, the ANOVA mean squares revealed significant genotype x environment interaction (P < 0.05) for all the studied characteristics. This significant interaction arises from the differential genotypic responses to different salinity levels during the plant life cycle.


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  		Table 2. Mean squares for kernel yield and its components and some other agronomic characters for the R3 transgenic lines expressing the HVA1 gene and the control.

 
The magnitude of the main effect mean squares suggested that the number of days to heading, plant height, panicle length, root length, number of kernels/panicle, 1000-kernel weight, and kernel yield were influenced by salinity more than genotype as it showed between 20 and 85% of the total variation. Flag leaf area, number of spikelets/panicle, and number of tillers/plant appear to be almost equally affected by salinity levels and genotypes which explained more than 40% of the total variation particularly for these traits. These results indicate that the chosen salinity levels were very efficient in imposing the stress.

Although transgenic plants showed better recovery and grew faster than the control plants after the salt-stress recovery period (data not shown) and also maintained more tolerance to salinity during and after the salt treatments, higher salinity levels (150 and 200 mM) significantly reduced plant growth of both transgenic and control plants (Table 3 and Fig. 5) . The differences in stress treatments revealed a progressive decrease in the average number of days to heading, plant height, flag leaf area, and root length (Fig. 5a-d).


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  		Table 3. Differences between the R3 transgenic 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.

 


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  		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.

 
Early heading is one of the mechanisms that plants use to escape the damage effects caused by salinity stress (Bajji et al., 2004). In this study, mean days to heading decreased as salinity level increased, although this decrease did not vary among the genotypes (Fig. 5a). Most of the transgenic lines headed significantly later than the control at early high salinity stress (>50 mM NaCl). This delay in flowering gives the opportunity of late differentiation, which subsequently enables the plant to maintain higher number of kernels/panicle (Blum et al., 1990).

The plant height, flag leaf area, and root growth parameters may serve as yield attributes (Ulery et al., 1998). The height was not significantly different among genotypes at 0 and 50 mM NaCl salinity. The transgenic lines were able to retain their height until 100 mM compared with control. The control plants tended to be shorter under salinity and the shortest height for most genotypes was obtained at 200 mM NaCl (Fig. 5b). Similar findings were reported by Xu et al. (1996) in rice. The minimum response of plant height to changes in salinity levels of transgenic lines may result in the ability of the plants to preserve current assimilates as a source of carbon stored in the stem for grain filling (Blum, 1998). In addition, this performance may improve the response to nitrogen fertilization under salt stress conditions.

The plant ability to maintain large leaves and delay leaf senescence under water limitation stresses is a stress tolerance mechanism called "non-senescence" or "stay-green" (Rosenow, 1977; Thomas and Smart, 1993). One of the most important points is whether or not retaining a green and large leaf area under salt stress increases kernel yield. Helsel and Frey (1978) reported a positive correlation between leaf area duration and kernel yield in the oat crop. Leaves of transgenic lines were normal and displayed no symptoms of wilting at 50 mM salinity. The genotypic differences under salinity-induced changes in the flag leaf area were most prominent at 100 mM and above concentrations. The flag leaf area was more severely reduced by salt stress in the control plants than the transgenic lines (Fig. 5c). The data presented in Table 3 confirmed that Line BRA-41, which retained the highest flag leaf area (Fig. 5c), maintained the highest yield among the different salinity levels. This probably resulted from increased photosynthesis when kernel filling occurred under stress (Blum, 1983; Van Oosterom and Acevedo, 1992).

The length of oat roots was correlated with the tolerance of juvenile plants to water stress in the field (Larsson and Gorny, 1988). Root length of the control was not different from the lines in the salinity control treatment (0 mM) (Fig. 5d). As salt concentration increased, root length of all transgenic lines increased relative to the control. However, when salt concentrations increased beyond 100 mM, root growth declined. Since only the final root length of each line and the control was measured, no differences in root growth were detected between the lines but there is a significant difference between the lines and the control at 50 mM and above (Fig. 5d) (Sivamani et al., 2000).

The effects of salinity levels on the panicle traits are shown in Fig. 5e and f. In general, panicle length (Fig. 5e) and number of spikelets/panicle (Fig. 5f) were reduced by salinity levels 50 mM and higher. While plants of transgenic lines maintained high panicle length and number of spikelets/panicle among the salinity levels, salinity treatments at 100 and 50 mM NaCl resulted in significant differences in panicle length and number of spikelets/panicle respectively, compared with the control. The panicle length and number of spikelets/panicle were reduced of approximately 23 to 52% and 25 to 77% at 200 mM NaCl respectively (Fig. 5e and f). The panicle length of control plants was 50% shorter than the best transgenic line. The lack of panicle length extension under salinity might have contributed to plant height reduction (Milach et al., 2002). Moreover, Giunta et al. (1993) reported that, in wheat, severe water deficit around anthesis produces serious effects on yield, reducing the number of spikelets and therefore causing a decrease in plant fertility.

It was reported that variation in kernel yield potentials in cereals under water stress growing conditions were predominantly associated with variations and sequential development of yield components (Fischer and Maurer, 1978; García del Moral et al., 1991, 2003; Simane et al., 1993). Kernel yield was greater under salinity stress for the transgenic plants than the control. The higher yield was due to increased number of tillers, number of kernels/panicle and heavier kernels (Table 3). Salinity stress at 200 mM NaCl for a long period of time caused reduction in kernel yield estimated at >40% for transgenic lines and 90% for the control plants. Under the conditions of this study (Table 3), number of kernels/panicle was the most sensitive yield component affected by salinity stress (reduced by >30 and 60%) followed by 1000-kernel weight (reduced by >26 and 50% for the transgenic lines and the control respectively). The number of tillers/plant was less affected by salinity for the transgenic lines (reduction of 10–20%), but the influence of salinity was higher on the control plants (>60% reduction). Among the transgenic lines, the line BRA-41 expressed the highest yield under severe salinity stress (200 mM) because of its lower reduction in number of tillers/plant, 1000-kernel weight, and number of kernels/panicle (Table 3). Evans and Wardlaw (1976) explained yield component compensation as the allowance of subsequently occurring components of final kernel yield to compensate for restrictions and/or losses during earlier stages of development, or to maximize reproductive growth in the plant life cycle.

On the basis of the transgenic line observations, the results indicate that number of tillers/plant may have a negative effect on number of kernels/panicle and kernel weight under salinity stress. The compensatory effect between tiller production and the other components may have resulted from the negative allometry between these traits during plant development (Hamid and Grafius, 1978). Moreover, it is well documented that under stress environments compensation could be mainly achieved by extensive tillering of surviving plants (Holena et al., 2001).

Overall, transgenic plants of the R3 generation developed normal flowers, grew to maturity, and set seeds in a normal manner under greenhouse conditions, suggesting that expression of the linked bar-HVA1 and nonlinked gus genes had no deleterious effects on growth and fertility.

The correlation between accumulation of LEA group III proteins and stress tolerance is well studied in wheat and rice (Ried and Walker Simmons, 1993; Rohila et al., 2002; Sivamani et al., 2000; Wise, 2003). Moreover, it is evident that genetic variability exists for stress responses, and this could be due to the differential expression and regulation of stress responsive genes such as HVA1 gene when the plants are exposed to stress (Jayaprakash et al., 1998; Uma et al., 1995). On the basis of our experimental results, the positive significant relationship between the present increase in growth of the transgenic lines under stress over controls and the presence of HVA1 transcripts suggest more evidence that constitutive expression of HVA1 gene in transgenic plants can improve growth performance under salinity stress conditions; however, the exact function of LEA proteins remains uncertain.


   CONCLUSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSION
REFERENCES
 
We previously characterized five transgenic oat lines that expressed the barley HVA1 gene and showed increased tolerance to salinity under in vitro conditions at the seedling stage (Maqbool et al., 2002). In this study, the molecular and agronomic characteristics of R3 progeny of those lines were analyzed under salinity stress conditions in greenhouses. Molecular (PCR, Southern, and Northern blots), segregation for herbicide, and GUS histochemical analyses confirmed the existence, transcription, and translation of the intact linked bar-HVA1 and the nonlinked gus genes in the R3 progeny of transgenic oat plants.

Transgenic and nontransgenic plants performed similarly under well-watered growing conditions (0 mM NaCl). Appearance and development of the symptoms of damage caused by salinity were delayed in transgenic plants. Under salinity stress, differences in salinity tolerance between transgenic lines and the control were associated with the flag leaf area growth, positive effects of plant height, panicle length and number of spikelets/panicle (Fig. 6) and development and maintenance of extensive root system (Fig. 7) .



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  		Fig. 6. R3 transgenic oat (a) and nontransgenic (b) at 150 mM NaCl stress.

 


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  		Fig. 7. R3 transgenic (a) and nontransgenic (b) oat roots at 100 mM NaCl stress.

 
Kernel filling in cereals is considered to be one of the most sensitive growth stages to stresses (Nayyar and Walia, 2004). Physiological stress expressed by salinity during this period reduces the storage capacity of cereal kernels by decreasing the number of endosperm cells and/or the number of amyloplasts initiated (Jones et al., 1996). Salinity can reduce the kernel weight and number of kernels by limiting the rate and duration of the filling process and inhibiting photosynthesis (Gupta et al., 2001; Vishwanathan and Khanna-Chopra, 2001). As indicated by yield and yield components (number of tillers/plant, number of kernels/panicle and 1000-kernel weight), the results demonstrate that control plants were more sensitive to salinity and experienced more severe water stress than did the transgenic lines under high salinity conditions.

Although transgenic lines experienced a decrease in performance at the highest salinity stress level (200 mM), these lines showed better performance than nontransgenic controls under continuous salt stress. Line BRA-41 experienced the least and the control plants the most decrease in performance through the salinity levels for most of the studied traits. The results provide more evidence about the role of the HVA1 protein in water deficit damage prevention (Xu et al., 1996) and the protection of oats against salinity. This could benefit farmers with healthier plants, potentially resulting in possible contribution to superior yield.


   ACKNOWLEDGMENTS
 
The authors wish to thank Dr. James Kelly, Dr. Russel Freed (Department of Crop and Soil Sciences, Michigan State University, MI) and Dr. Farouk Oraby (Department of Plant Sciences, Higher Institute for Productivity Efficiency, Zagazig University, Egypt) for constructive criticism and helpful suggestions for the manuscript. This research was supported by Consortium for Plant Biotechnology Research (CPBR) and the Egyptian government that financially support Hesham Oraby's studies.

Received for publication October 14, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSION
 REFERENCES
 


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