The study of stomatal characteristics in Iranian wheat wild accessions and land races

 

Shahram Mohammady, Hamid Khazaei and Faiez Reisi

Faculty of Agriculture, University of Shahrekord, P.O. Box 115, Iran

Corresponding author: Shahram Mohammady

E-mail: shfaza@hotmail.com

Abstract

Wheat crops growing in arid and semi-arid regions generally experience some kinds of water-deficit. Water managing in the crop is partly depended on the stomatal characteristics. Therefore, reliable selection and breeding for suitable stomatal characteristics is of a great importance where water supplies are limited. This study was aimed to investigate different characteristics of stomata in Iranian wheat land races and wheat wild relatives not being studied so far. The results revealed that a huge variation exist between wheat land races and wheat wild relatives and even within the land races and wild relatives for different aspects of stomatal characteristics. Significant differences were found among the accessions of same ploidy level on the both adaxial and abaxial surfaces of flag leaf for stomatal frequency. Variation within diploid accessions was much higher than variation within either tetraploid or hexaploid wheats. The mean of stomatal frequency was the highest in diploids, intermediate in tetraploid and the least in hexaploids. Conversely, stomatal length and width was the lowest in diploids and highest in hexaploids indicating a negative relationship between stomatal frequency and stomatal size. Variations for stomatal area per unit leaf area were also found among and within ploidy levels. Nonetheless a particular trend was not found as the ploidy level changed. In general, this study revealed that wild species of wheat and landraces indicated variation for different aspects of stomatal characteristics and therefore they can be used in wheat breeding programs aiming to manipulate water-transpiration in wheat.

 

Introduction

Wheat is the first major world cereal and it has been cultivated within origin center of variation (southwest Asia) for over than 10,000 years. The wild species of wheat are still under cultivation in Iran, Iraq, east of Turkey, north of Palestine, Lebanon and Syria (Poehlman 1995). Braidwood et al. (1983), studying Zagros flanks of western Iran and northeastern Iraq reported that the cultivation of diploid and hexaploid wheats backed to 6500 years BC. Stomatal frequency and stomatal size have often been used as morphological markers for identifying ploidy level in many plant species, for example in Acacia mearnsii (Beck et al. 2003; 2005), Actinidia deliciosa (Przywara et al. 1989), Aegilops neglecta (Aryavand et al. 2003), Bromus intermis (Tan and Dunn 1973), Coffea L. (Mishra et al. 1991; 1997), Dactylis (Santen and Casler 1986), rye grass (Speckman et al. 1965), Triticale (Sapra et al. 1975), and Triticum spp. (Teare et al. 1971; Rajendra et al. 1978; Wang et al. 1989; Wang and Clarke 1993). Aryavand et al. (2003) reported that significant variation was found in stomatal frequency between the ploidy levels for basal leaves, the penultimate and flag leaves in Aegilops negelecta and stomatal frequency and size were highly negatively correlated. Selection and variation for stomatal characteristics has been reported in bread wheat (Bhgwat et al. 1993). In addition, stomatal characteristics have been also studied in wild species of other crops. It has been reported that stomatal size positively correlates with ploidy level and is negatively related to stomatal size in Bromus inermis, (Tan and Dunn 1975). Such a result has been also reported in Coffea L. (Mishra 1997).

Rajendra et al. (1978) reported that diploid, tetraploid and hexaploid wheats differ in stomatal frequency in which diploids having the highest and hexaploids the lowest number per unit area of leaf. Conversely, they reported that stomatal size increased with increasing ploidy level (2x<4x<6x). They also noticed that the adaxial flag leaf surface had a significantly higher stomatal frequency than the abaxial side.

Mohammady-D (2002) found significant differences for stomatal length in adaxial and abaxial surfaces of bread wheat leaves. However, he reported no differences between the adaxial and baxial surfaces of leaves for stomatal width. He also reported that stomatal length is more effective than stomatal width on water transpiration. It is widely accepted that stomatal frequency is negatively correlated with stomatal size. Tanzarella and Blanco (1979) found that stomatal frequency of the flag leaf is negatively correlated with stomatal length in durum wheat.

Stomatal size and frequency have been used as an indicator of water loss by many investigators (Singh and Sethi 1995; Venora and Calcagno 1991; Wang and Clarke 1993a) but no published work was found to use stomatal pore width as a trait which determines the capacity of stomata to reduce water loss. This is possibly due to difficulties which appear during the measurement of stomatal pores especially under unfavorable conditions. Venora and Calcagno (1991) measured and used stomatal width as an indicator of stomatal aperture. This application seems to explain differences between the varieties for water loss only under non-limiting conditions provided varieties with larger size of stomata have larger stomatal pores. Wang and Clarke (1993b) reported that SF was positively correlated with the rate of water loss. This indicates breeding for smaller and fewer stomata may lead to reduction in water loss.

Much of the genetic variation for improving stress tolerance has been lost during selection and modern breeding (Araus et al. 2002). Therefore, other genetic materials such as landraces and wild species rather than modern varieties should be used to obtain a large improvement in stress tolerance. Selected landraces and wild species can contribute to the enhancement of wheat production in dry regions by direct use for cultivation or by using in various methods of plant breeding in order to improve high yielding but drought susceptible varieties so that they can tolerate water-stress. Finding variation in stomatal characteristics and indirect selection for water-stress tolerance using these characters are among subjects that are of interest for scientists. The objective of this study was to determine stomatal frequency, stomatal size (length and width) and stomatal area per unit leaf area in adaxial and abaxial surfaces of leaves in some Iranian land races and wild species not being investigated so far.

Material and methods

Four diploid and 8 tetraploid accessions along with 5 hexaploid landraces and 3 cultivars, all except one collected from Iran, were used in this study. All the genotypes were provided by Cereal Research Department of Seed and Plant Improvement Institute, Iran. The vernalisation requirement for the genotypes and other descriptions were announced by the institute as cited in Table 1.

Seeds from accessions requiring vernalisation were germinated in petridishes and transferred into a growth chamber (2-4oC) for 5 weeks.  Four weeks later other genotypes not sensitive to vernalisation were also germinated. Three seedlings from each accession were planted in plastic pots filled with 1.1 kg of a soil containing 42% sand, 36% silt and 22% clay. Each pot was brought to water-holding capacity by adding 250 ml of water in each pot. Pots were weighted every 3 day and amounts of water equal to the loss in weight were added (Ehdaie and Waines 1993). Eight days after transplanting the seedlings, two seedlings were removed from each pot leaving the most vigorous one. Pots were arranged in a randomized complete-block design with three replications in an unheated glasshouse at the University of Shahrekord, Iran. When flag leaves were fully developed, stomatal frequency and stomatal size (length and width) were measured on the adaxial and abaxial surfaces by impression method (Wang and Clarke 1993). Impressions were taken from the middle of the both adaxial and abaxial surfaces of flag leaves. The number of stomata was counted from seven different microscopic fields of view at 160x magnification. To find the stomatal Frequency (SF), the number of stomata per field of view was converted to the number of stomata per one mm2 of leaf using a standard scale.

 

Stomatal length (SL) and stomatal width (SW) were measured on the both surfaces from the impressions using a scaled eyepiece of microscope and then stomatal size was converted to μm. Stomatal area per unit leaf area (SA) (μm of stomata/mm2 of leaf) was calculated using modified method of Wang and Clarke (1993) as a product of SF x SL x SW. The above measurements were made randomly on 20 stomata in each impression and the mean values of the 20 measurements were used for statistical analyses.

 

Data were analyzed using SAS, version 8.0 (copyright© 1999 by SAS Institute, Cary, NC, USA). Analyses of variance were preformed using the GLM procedure. Analyses of variance for diploid, tetraploid and hexaploid were also performed separately using non-orthogonal method. In addition, 3 contrasts, including 2x vs. 4x, 2x vs. 6x and 4x vs. 6x, were made in order to test variation between ploidy levels for the characters under study. Since none of the replications significantly affected the total variation, they were omitted from the tables of variance analyses. Correlation analysis was performed to determine the relationship between the traits using the CORR procedure and comparisons between means were made using LSD test.

 

Results

Stomatal frequency

Analysis of variance indicated that significant differences existed among the genotypes for stomatal frequency on the both adaxial and abaxial surfaces. However no significant difference was found among the accessions within ploidy levels for stomatal frequency on the abaxial surfaces (among the diploid, tetraploid and hexaploid accessions). The result of contrast between poloidy levels indicated that the variance between poloidy groups were highly significant (Table 2). All these significant variances imply that the genotypes used in the present study can be considered as a source of variation in selection for the stomatal frequency.

 

Mean stomatal frequency of the adaxial and abaxial surfaces of accessions is presented in Table 4. The range of difference between genotypes was considerable in all ploidy groups. Stomatal frequency of the adaxial surface ranged from 94.86 to 125.26 for diploid, from 56.18 to 81.51 for the tetraploid and from 45.59 to 64.47 for the hexaploid accessions. For the abaxial surface, it varied from 79.60 to 111.90 for diploid, from 42.83 to 63.55 for tetraploid and from 34.08 to 52.04 for hexaploid accessions (Table 5).

 

According to the data shown in Table 4, the mean of stomatal frequency was the highest in diploids (108.45 and 96.01, for adaxial and abaxial surfaces, respectively), intermediate in tetraploid accessions (66.31 and 52.09 for adaxial and abaxial surfaces, respectively) and the lowest in hexaploid accessions (55.78 and 43.46 for adaxial and abaxial surfaces, respectively). It is also clear from the Table that the diploid accessions showed non overlapping range with the other ploidy levels for stomatal frequency for both adaxial and abaxial surfaces, but tetraploid and hexaploid accessions showed overlapping range on the both adaxial and abaxial surfaces. The latter subject implies that stomatal frequency could not be considered as a morphological marker for poloidy identification as it was earlier reported by Beck et al. (2003; 2005) in Acacia mearnsii, Przywara et al. (1989) in Actinidia deliciosa, Aryavand et al. (2003) in Aegilops neglecta and Tan and Dunn (1973) in Bromus intermis. From the present study, in general, it can be deduced that stomatal frequency decrease with increasing in ploidy level. The similar results have been also reported by Teare et al. (1971), Rajendra et al. (1978), Wang and Clarke (1993) in wheat, Mishra (1997) in coffea and Aryavand et al. (2003) in Aegilops.

                                                              

For all accessions except 3829 the adaxial surface had higher stomatal frequency than the abaxial surface. Nonetheless, the differences between the 2 sides were not significant (t = 1.88ns, df = 38). Despite the present results, other studies have reported significant differences between adaxial and abaxial surfaces in wheat (Teare et al. 1971; Rajendra et al. 1978; Singht and Sethi 1995; Mohammady-D 2002).

 

The ratio of stomatal frequency on abaxial to adaxial surface was 0.81 over all studied genotypes. Mohammady-D (2002) obtained this ratio 0.69 for T. aestivum, and suggested that the ratio is possibly more stable that the absolute number of stomata in either surfaces of flag leaf. A highly positive correlation was observed between adaxial and abaxial surfaces for stomatal frequency (r = 0.915**). This indicates that selection based on analysis on one side of leaves is sufficient and there is no need to measure stomatal frequency on the both sides.

 

Stomatal length

Analysis of variance indicated significant differences for stomatal length existed among accessions and also among diploids and hexaploids on the adaxial surface and among diploid and tetraploid accessions in the abaxial surface (Table 3). The contrast analysis indicated significant difference for stomatal length existed between diploids and tetraploids, diploids and hexaploids and between tetraploids and hexaploids on the abaxial surface. A similar trend was observed for the character on the adaxial surface except that no significant difference observed between tetraploids and hexaploids (Table 3).

 

Mean stomatal length of the adaxial surface are presented in Table 4. Stomatal length of the adaxial surface ranged from 26.17 to 37.88 μm for diploid accessions, from 44.84 to 51.67 μm for tetraploid accessions and from 45.46 to 51.88 μm for hexaploid accessions (Table 4). For the abaxial surface (Table 5), it varied from 29.37 to 34.29 μm for diploid accessions, from 40.95 to 48.91 μm for tetraploid accessions and from 42.92 to 51.54 μm for hexaploid accessions.

 

The mean of stomatal length in diploid accession was the smallest (31.76 and 31.20 μm, for adaxial and abaxial surfaces, respectively) and it increased more or less with increasing ploidy level. These observations are similar to those reported by Rajendra et al. (1978), in Triticum, Borrino and Powel (1988) in Hurdeum, Przywara et al. (1988) in Actinida, Singh and Sethi (1995) in Triticale and Beck et al. (2003; 2005) in Acacia. The diploid accessions showed non-overlapping range with other ploidy level in both leaf sides, but tetraploid and hexaploid accessions had overlapping range. The adaxial surface had a higher stomatal length than abaxial surface in all accessions except 5172 (Table 4 and Table 5). However, the differences were not significant based on t-test (t = 1.38ns, df = 38).

 

Stomatal width

Significant differences were found for stomatal width among accessions, among diploid and tetraploid accessions for the adaxial and abaxial surfaces and among hexaploid accessions only in adaxial surface, but not among hexaploid accessions in abaxial surface (Table 3).

The contrast analysis indicated significant difference existed between diploid vs. hexaploid accessions and tetraploid vs. hexaploid accessions for the both surfaces. Similar to stomatal length, mean of stomatal width increased with increasing in ploidy level. Nonetheless, the similar data in diploid, tetraploid and hexaploid genotypes for SW indicates that this character is not a suitable marker for distinguishing between ploidy levels.

Stomatal area per unit leaf area (SA)

For Stomatal area per unit leaf area significant differences were found among accessions and within diploids and hexaploids for both surfaces and  within tetraploid group for the abaxial surface only (Table 3). Contrast analysis indicated significant difference between ploidy levels for the adaxial surface. Tetraploid accessions had highest stomatal area per unit leaf area (mean 84207.12), and hexaploids had intermediate amount (mean 78000.35) and finally diploid accessions had the lowest amount of SA (63170.69) (Table 4).

Discussion

It is theoretically expected that varieties with higher number of stomata per unit area and greater length and width of stomata lose more water during the growth period. This happens if stomata remain open during the water-stress period. Thus, any response of stomata to water-stress can be discussed in relation to stomatal resistance and Leaf Relative Water Content (LRWC). Reduction in water loss from leaf surfaces during periods of severe water-stress is an important drought tolerance indicator. Low rate of cuticle transpiration, therefore, may reduce leaf dehydration and promote leaf survival (Wang and Clarke 1993b). When water-stress develops, the response of stomata to water-stress seems to be of a great importance in reducing water loss comparing with stomatal characteristics. Mohammady-D (2002) studied the relationships between stomatal characteristics and water status in 2 wheat varieties including Falchetto (water-stress tolerance) and Oxley (water-stress susceptible). He reported that stomatal frequency of Falchetto was significantly higher than Oxley, but Falchetto had smaller stomata. On the other hand, his results revealed that Falchetto had a higher Leaf Relative Water Content (LRWC) and Stomatal Resistance (SR) than Oxley. These results indicated that SF is not always correlated with plant water status. This is because stomatal size, response of stomata to environmental stress and even cuticle resistance are also involved in determining plant water status particularly under water-stress conditions. In the case of cuticle resistance, Gupta et al. (2001) using diurnal observations, reported that a drought tolerant variety (C-306) had higher leaf diffusive resistance than a drought sensitive variety (Kalyansona). Since stomata are mostly closed at night, it can be concluded that differences in diffusive resistance are mostly due to differences in cuticle resistance.

 

The results of other workers concerning the relationship between stomatal characteristics and plant water status are inconsistent. McCaig and Romagosa (1989) found no consistent differences in SF between two durum genotypes with different water-retention capabilities. Wang and Clarke (1993b) reported that SF was not correlated with relative water loss and LRWC in field experiments. However, their results indicated that SF was positively correlated with the rate of water loss but not with LRWC under growth room experiments. The inconsistency of this relationship is possibly due to the influence of other characteristics of stomata rather than SF and due to negative relationships between stomatal size and frequency. In addition to SF and stomatal size, the stomatal responses to water-stress and cuticle resistance are other factors which influence water status of plants under water-stress conditions. Thus, the results presented in this section and those explained above indicate that stomatal characteristics are affecting water status of plants as a complex, and every component of this complex should be studied in relation to other components and with other factors which influence water status of plants.

 

The increase or decrease in transpiring area under stress conditions may not be achieved by selecting for high or low SF due to the negative correlation between SF and stomatal size (Venora and Calcagno 1991). For this reason, it seems that SA as a combination of SF, SL and SW is a better determination of water status in plants. The relationship between stomatal resistance and stomatal characteristics is also important in determining water status of crops under water-stress conditions. In a study carried out by Mohammady-D (2002), SR and SA were investigated in Varieties Falchetto (water-stress resistant) and Oxley (water-stress susceptible). He reported no significant differences between the two varieties for SA on the both surfaces of leaves but highly significant difference for stomatal resistance was observed between the two varieties. These results indicate that higher SR of Falchetto on the adaxial surface is not due to smaller SA but is possibly due to either differences in stomatal response to water-stress or differences in cuticle resistance.

 

The most important issue regarding water-stress tolerance is that the characteristics of stomata of the crop must match the pattern of water supply (Passioura 1996). When the water supply is insufficient from the onset of growth, less number of stomata and low stomatal area can lead to a conservative consumption of water and thus can be considered as a suitable adaptive trait. On the other side, when there is a small shortage of water supply happens at the end of growth cycle, low stomatal frequency or area, or even low stomatal transpiration, have no benefit to the crops due to low photosynthesis and thus enhancing yield reduction in crops.

 

In general, wild species of wheat and landraces used in the present study indicated variation for different aspects of stomatal characteristics and therefore they can be used in wheat breeding programs aiming to manipulate stomatal characteristics. There is also a need to evaluate these genotypes for other traits related to water statue in order to come to a conclusion about their promising for being involved in wheat breeding programs aiming to improve wheat cultivars for water statues in particular in dry regions.

 

References

Araus JL, Slafer GA, Reynolds MP, Royo C (2002) Plant breeding and drought in C-3 cereals: What should we breed for? Annals of Bot 89: 925-940.

 

Aryavand A, Ehdaie B, Tran B, Waines JD (2003) Stomatal frequency and size differentiate ploidy level in Aegilops neglecta. Genet Resources Crop Evol 50: 175-182.

 

Beck SL, Dunlop RW, Fossey A (2003) Stomatal length and frequency as a measure of ploidy level in black wattle, Acacia mearnsii (de Wild). Bot J Linnean Society 141: 177-181.

 

Beck SL, Visser G, Dunlop RW (2005) A comparison of direct (flow cytometry) and indirect (stomatal guard cell lengths and chloroplast numbers) techniques as a measure of ploidy level in black wattle, Acacia mearnsii (de Wild). South African J Bot 71: 354-358.

 

Bhagwat SG, Bhatia CR (1993) Selection for flag leaf stomatal frequency in bread wheat. Plant Breed 110: 129-136.

 

Borrino EM, Powell W (1988) Stomatal guard cell length as an indicator of ploidy in microspore plants of barley. Genome 30: 158-160.

 

Braidwood LS, Braidwood, JR, Howe B, Reed CA, Watson PJ (1983). Prehistoric archeology along the Zagros Flanks. Oriental Institute Publication, Chicago, USA.

 

Ehdaie B, Waines JG (1993) Variation in water use efficiency and its components in wheat: I. well water pot experiment. Crop Sci 33: 294-299.

 

Gupta NK, Gupta S, Kumar A (2001) Effect of water stress on physiological attributes and their relationship with growth and yield of wheat cultivars at different stages. J Agr Crop Sci 186: 55-62.

 

McCaig TN, Romagosa I (1989) Measurement and use of excised-leaf water status in wheat. Crop Sci 29: 1140-1145.

 

Mishra MK (1997) Stomatal Characteristics at different ploidy levels in Coffea L. Annals of Bot 80: 689-692.

 

Mishra MK, Prakash NS, Sreenivasan MS (1991) Relationship of stomatal length and frequency to ploidy level Coffea L. J Coffee Res 21: 32-41.

 

Mohammady-D S (2002) Inheritance of tolerance to water stress in wheat (Triticum aestivum). Ph D Thesis. University of Newcastle upon Tyne, UK.

 

Passioura JB (1996) Drought and drought tolerance. Plant Growth Regulation 20: 79-83.

 

Poehlman JM (1995) Breeding Field Crops. Iowa State University Press, Iowa, USA.

 

Przywara L, Pandeyf K.K, Sanders PS (1988). Length of stomata as an indicator of ploidy level in Actinidia deliciosa. New Zealand J Bot 26: 179-182.

 

Rajendra BR, Mujeeb KA, Bates LS (1978) Relationships between 2x Hordum sp., 2x Secale sp. And 2x, 4x, 6x Triticum sp. for stomatal frequency, size and distribution. Environ Exp Bot 18: 33-37.

 

Santen EV, Casler EV (1986) Evaluation of indirect ploidy indicators in Dactylis C. Sub species. Crop Sci 26: 848-852.

 

Sapra VT, Hughes JL, Sharma GC (1975) Frequency, size and distribution of stomata in Triticale leaves. Crop Sci 15: 356-358.

 

Singh S, Sethi GS (1995) Stomatal size, frequency and distribution in Triticum aestivum, Secale cereale and their amphiploids. Cereal Res Commun 23: 103-108.

 

Speckman G, Post J, Dijkstra, H (1965) The length of stomata as an indicator for polyploidy in rye grasses. Euphytica 14: 225-230.

 

Tan GY, Dunn G.M (1975) Stomatal length, frequency and distribution in Bromus inermis Leyss. Crop Sci 15: 283–286.

 

Tanzarella OA, Blanco A (1979) Stomatal frequency and size in durum wheat. Genet Agri 33: 355-362.

 

Teare ID, Peterson CJ, Law AG (1971) Size and frequency of leaf stomata in cultivars of Triticum aestivum and other Triticum species. Crop Sci 11: 496-498.

 

Venora G, Calcagno F (1991) Study of stomatal parameters for selection of drought resistant varieties in Triticum Durum DESF. Euphytica 57: 275-283.

 

Wang H, Clarke JM (1993a) Genotypic, intraplant, and environmental variation in stomatal frequency and size in wheat. Canadian J Plant Sci 73: 671-678.

 

Wang H, Clarke JM (1993b) Relationship of excised-leaf water-loss and stomatal frequency in wheat. Canadian J Plant Sci 73: 93-99.

 

Wang PY, Chen R, Fang R, Zhu Z (1989) Study of identification of ploidy in pollen plants and wheat using guard cells. Acta Agricultural Universities Pekinensis 15: 141-145.