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Differential patterns of germination and desiccation tolerance of mesic and xeric wild barley (Hordeum spontaneum) in Israel
ARTICLE IN PRESS Journal of Arid Environments Journal of Arid Environments 56 (2004) 95–105 www.elsevier.com/locate/jnlabr/yjare
Differential patterns of germination and desiccation tolerance of mesic and xeric wild barley (Hordeum spontaneum) in Israel Guoxiong Chena, Krugman Tamara, Tzion Fahimaa, Fengchun Zhangb, Abraham B. Korola, Eviatar Nevoa,* b
a Institute of Evolution, University of Haifa, Mount Carmel, Haifa 31905, Israel Wyler Department of Dryland Agriculture, Department of Life Science, Jacob Blaustein Institute for Desert Research, Ben-Gurion University of the Negev, Sede Boker 84990, Israel
Received 06 June 2002; received in revised form 29 November 2002; accepted 03 December 2002
Abstract Differential patterns of germination were identified in mesic (Maalot) versus xeric (Wadi Qilt) ecotypes of wild barley, Hordeum spontaneum C. Koch, in the following traits: afterripening (dormancy), seedling desiccation tolerance, and the effect of glumellae and ethanol on afterripening and seedling growth. The following results were indicated: (i) Dispersal unit afterripening average (75%) was identical, but its coefficient of variation was larger in the xeric than in the mesic ecotype (29.9% and 9.9%, respectively). (ii) Survival ratio of seedlings after 1-month dehydration was higher in the xeric ecotypes indicating that seedling desiccation tolerance was higher in the xeric ecotype. (iii) Inhibition of germination and seedling growth by glumellae were higher in the mesic ecotypes. Ethanol sterilization of naked caryopsis promoted root growth of mesic seedlings but not xeric seedlings. Seedlings originating from xeric ecotypes had longer roots than mesic seedlings. It appears that, in the germination stage, natural selection adapted wild barley to a xeric environment by increasing the diversity of afterripening, enhancing desiccation tolerance, and improving root length growth. r 2003 Elsevier Science Ltd. All rights reserved. Keywords: Afterripening; Drought resistance; Ethanol; Caryopsis; Point of no return; Survival ratio
*Corresponding author. Tel.: +972-4-824-0445; fax: +972-4-824-6554. E-mail address: [email protected] (E. Nevo). 0140-1963/03/$ - see front matter r 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0140-1963(02)00321-X
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1. Introduction Wild barley, Hordeum spontaneum C. Koch, like wild wheat, is one of the principal grain plants on which Neolithic food production in the Near East was founded. Wild barley is the indisputable progenitor of cultivated barley: this is indicated by its cross-compatibility, full fertility, and sporadic spontaneous hybridization with the cultivars (Nevo, 1992). The vast genetic resources of wild barley are the best, largely unexploited, resources for improving the narrowing genetic base of the cultivated barley (Nevo, 1992). Wild barley is an annual, brittle, two-row diploid (2n ¼ 14), and is predominantly self-pollinating (Brown et al., 1978). It is widespread in the Near East Fertile Crescent. In Israel, it is abundant, occupying an extraordinarily large diversity of habitats ranging from the mesic Mediterranean to the xeric southern steppes (Nevo, 1992). Populations of H. spontaneum involve abundant genetic variation against drought and salinity, and the highest resistant genotype is significantly correlated with the highest stress environment and with the highest genetic polymorphism (Nevo et al., 1984, 1997). An important survival strategy of wild barley under unpredictable small amounts and distribution of winter rain in the Israel xeric area is afterripening (Evenari et al., 1982; Gutterman, 1993, 1998). This primary dormancy effect prevents germination of mature caryopses after a late rain at the beginning of the long, dry summer (Evenari, 1965; Gutterman, 1993, 1996; Gutterman et al., 1996). Primary dormancy occurs when the intact seed is covered by the glumellae. The covering structures of the caryopses (seed-coat and pericarp) may limit the oxygen supply to the embryo (Lenoir et al., 1986; Gutterman et al., 1996) and thus inhibit germination. During the following rainy season, after summer dormancy, the caryopses are ready to germinate. If the rainfall is adequate to trigger germination, and is followed by a long period of drought, seedlings with desiccation tolerance may survive and develop into normal plants by developing new adventitious roots and leaves when rewetted (Friedman et al., 1981; Evenari et al., 1982; Gutterman and Gozlan, 1998, 1999). The survival ratio of seedlings of some ecotypes of H. spontaneum after 1 week of dehydration differed with respect to stages of root length at dehydration. It was higher in mesic Neve Yaar (annual rainfall 600 mm) than in xeric Sede Boker (90 mm) at the stage of root length less than 10 mm, and it was lower at the stage of a root length longer than 10 mm (Gutterman and Gozlan, 1998). Ethanol promoted seed germination of Zinnia elegans by removing the ethanolsoluble compounds in the pericarp that inhibited germination of the seed (Ogawa and Iwabuchi, 2001). Ethanol breaks dormancy of Hordeum distichum grain by causing a decrease in endogenous ABA (Wang et al., 1998) and may promote caryopsis germination of H. spontaneum. The aim of this work was to compare contrasting mesic (Maalot) and xeric (Wadi Qilt) ecotypes of wild barley, H. spontaneum, for (1) afterripening patterns, (2) seedling survival ratio after 1-month dehydration, and (3) the physical and chemical effects of glumellae and ethanol, respectively, on afterripening and seedling growth.
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2. Materials and methods 2.1. Plant material Two Israeli ecotypes of H. spontaneum were used in the present experiments: Maalot originating from mesic environments (western Upper Galilee) and Wadi Qilt from xeric environments (Judean Desert) (Fig. 1) (Table 1). Five genotypes from each ecotype were randomly chosen from the collection of the Gene Bank of the Institute of Evolution, University of Haifa. The Gene Bank of H. spontaneum includes 3500 genotypes, 122 populations from Israel, Jordan, Turkey, and Iran.
20 Km
Maalot Haifa
Me
dit
err
ane
an
sea
Sea of Galilee
Tel -Aviv
Wadi Qilt Jerusalem
Israel
Beer-sheva
Dead Sea
Negev Desert
Fig. 1. Geographic location of Maalot and Wadi Qilt.
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Table 1 Location of the two Hordeum spontaneum ecotypes and selected environmental data of sites of origin based on Nevo et al. (1984) and Van Rijn et al. (2000) Site of origin
Longitude (1E)
Latitude (1N)
Altitude (m)
Mean annual temperature (1C)
Mean annual rainfall (mm)
Mean humidity at 14:00 h (%)
Maalot Wadi Qilt
35.27 35.38
33.00 31.83
500 50
18 23
790 144
50 35
Caryopses of these genotypes were propagated in 1997 and have been stored since then at 101C with relative humidity ranging 20–25%. 2.2. Afterripening and desiccation tolerance Germination: caryopses were germinated on an ALBET filter paper wetted with 5 ml of distilled water in a 90 mm diameter Petri dish at 251C in the dark. Germination percentage was recorded after a 3-day incubation. Two methods of dehydration procedure were tested: (i) Sudden dehydration, post-germination seedlings were removed daily to Petri dishes with dry filter paper for a dehydration period of 1 month at room temperature in the dark (Gutterman and Gozlan, 1998). This method in which desiccation is being suddenly imposed on post-germination seedlings, is not like what’s found in nature or crop field. In order to make dehydration gradual and not contaminated by fungus in the Petri dishes, the second method was conducted as follows. (ii) Gradual dehydration, naked caryopses were first sterilized to prevent contamination of fungi with 70% ethanol for 5 min following five times rinsing in distilled water. Post-germinating seedlings were kept in their original place for gradual drying for 1 month in the dark at room temperature. Survival ratio: The dried seedlings were rehydrated with distilled water, and the survival ratio was calculated. Seedlings were considered viable when adventitious roots and coleoptile were developed and elongated (Gutterman and Gozlan, 1998). Four replicates of 30 caryopses of each genotype were used in this experiment. The mean values of all xeric and mesic genotypes were used to compare the effect of ecotypes and caryopses (intact caryopsis and naked caryopsis sterilized with ethanol) on afterripening (germination percentage) and desiccation tolerance. 2.3. The effects of glumellae and ethanol To find which factor, glumellae or alcohol, affects the germination percentage, two representative genotypes were chosen, a mesic genotype (Maalot 10-57) and a xeric genotype (Wadi Qilt 23-19). Four treatments were performed. (1) Caryopsis with glumellae—to reveal physical and chemical effects of glumellae. (2) Naked caryopsis together with the glumellae in the Petri dish—to reveal the chemical effect of glumellae. (3) Naked caryopsis sterilized with 70% ethanol—to reveal the effect of alcohol on germination and seedling growth. (4) Naked caryopsis—as the control.
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Four replicates of 15 caryopses in each treatment were placed in 90 mm diameter Petri dishes, on one ALBET filter paper, and wetted with 5 ml of distilled water. Distilled water was added to compensate the daily loss of water in Petri dishes. Germination percentage and shoot and root lengths were measured after a 3-day incubation at room temperature in the dark. Caryopses were considered to have germinated when the radicle protruded from the envelopes (Evenari, 1965; Gutterman and Gozlan, 1998). 2.4. Statistical analysis Statistical analysis of data was conducted using STATISTICA for windows 5.1D (StatSoft Inc., USA), Tukey honest significant difference (HSD) test was used to compare means (significance level, 5%).
3. Results 3.1. Germination patterns The xeric and mesic ecotypes showed the same germination percentage of caryopsis; however, the xeric ecotype had a larger coefficient of variation (ratio of standard deviation to the mean) than the mesic ecotype (Fig. 2), exhibiting higher diversity of afterripening in xeric than mesic ecotypes. The same germination pattern also happened in naked caryopsis. Germination percentage of caryopsis and naked caryopsis was similar in the xeric ecotype, whereas in the mesic ecotype germination percentage of naked caryopsis (sterilized with 70% ethanol) was higher than that of caryopsis (with glumellae). These results indicate that glumellae might inhibit caryopsis germination or alcohol might enhance naked caryopsis germination in the mesic ecotype or that glumellae and ethanol influenced germination of the xeric ecotype in different ways.
Fig. 2. Germination percentage of naked caryopses sterilized with 70% ethanol (NE) and caryopsis (G) from mesic and xeric ecotypes of Hordeum spontaneum in Israel. Four replicates of 30 caryopses of each of five genotypes for each ecotype were conducted in this experiment. Bars represent standard deviation. Letters above bars indicate significant differences (Po0:05; Tukey HSD test).
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3.2. Survival success The survival ratio of post-germination seedlings after the dehydration period of 1 month was higher in the xeric than mesic ecotypes in both caryopsis and naked caryopsis (Fig. 3), meaning that the xeric ecotype is more drought resistant compared to the mesic ecotype. The seedlings derived from naked caryopsis were much bigger than those derived from caryopsis with glumellae because the former seedlings were not removed from the original place and allowed to dry gradually, which would have given them more time to grow (Section 2.2). The seedlings derived from naked caryopsis had a lower survival ratio than that derived from caryopsis with glumellae in both xeric and mesic ecotypes (Fig. 3). One may infer that a bigger seedling has a lower ability to survive under drought stress. 3.3. The effects of glumellae and ethanol on germination In order to confirm the effect of ethanol and glumellae on germination (Fig. 3) and to separate the physical and chemical effects of glumellae on germination, two representative genotypes, genotype 10-57 from Maalot (mesic) and genotype 23-19 from Wadi Qilt (xeric), were chosen to conduct the next germination experiment. For genotype 10-57 (Fig. 4), germination percentage of naked caryopsis with glumellae was as high as naked caryopsis without glumellae indicating no glumellae chemical effect. Germination percentage of caryopsis was lower than naked caryopsis with glumellae revealing a glumellae physical effect on germination. No ethanol effect was tested because germination percentage of naked caryopsis sterilized with 70% ethanol was the same as that of naked caryopsis without sterilization. By contrast, for genotype 23-19, no significant glumellae and ethanol effects were observed in this germination experiment (Fig. 4). Therefore, the lower germination percentage of caryopsis compared with naked caryopsis sterilized with ethanol in the mesic ecotype showed in Fig. 2 was attributed to the physical
Fig. 3. Survival ratio after one-month dehydration of seedlings derived from naked caryopses sterilized with ethanol (NE) and caryopsis (G) from mesic and xeric ecotypes of Hordeum spontaneum in Israel. Bars represent standard deviation. Letters above bars indicate significant differences (Po0:05; Tukey HSD test).
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Fig. 4. Germination percentage of caryopses of genotype 10-57 (mesic) and 23-19 (xeric) of Hordeum spontaneum of four treatments. Caryopsis (G), naked caryopsis with glumellae (NG), naked caryopsis (N), and naked caryopsis sterilized with ethanol (NE). Four replicates of 15 caryopses of each of the four treatments were conducted in this experiment. Bars represent standard deviation. Letters above the bars indicate significant differences (Po0:05; Tukey HSD test).
Fig. 5. Coleoptile and seedling root length of genotype 10-57 (mesic) and 23-19 (xeric) of Hordeum spontaneum derived from caryopses of four treatments. Caryopsis (G), naked caryopsis with glumellae (NG), naked caryopsis (N), and naked caryopsis sterilized with ethanol (NE). Bars represent standard deviation. Letters above bars indicate significant differences (Po0:05; Tukey HSD test).
inhibition of glumellae. Without the glumellae and ethanol effect, the xeric ecotype exhibited the same level of germination percentage with caryopsis or naked caryopsis (Fig. 2). 3.4. The effects of glumellae and ethanol on seedling growth The growth of three-day-old post-germination seedling of genotype 10-57 (mesic) and 23-19 (xeric) was investigated. Coleoptile lengths of both genotypes were similar in all four treatments (Fig. 5). However, seedlings derived from caryopsis and naked caryopsis of the xeric genotype had longer roots than that of the mesic genotype. The root lengths of seedlings derived from both caryopsis and naked caryopsis with glumellae were the same, but were shorter than that of seedlings derived from naked caryopsis without glumellae indicating chemical not physical, inhibition of glumellae on root elongation. This chemical effect of glumellae was exhibited in both mesic and
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xeric genotypes. Ethanol improved root length growth in the mesic genotype, but showed no effect in the xeric genotype (Fig. 5).
4. Discussion 4.1. Ecogeographic variation in dormancy The dispersal units of wild barley used in the present study were harvested in 1997 and stored at 101C. The dormancy of xeric and mesic wild barley caryopses may be released at different speeds during storage. Within 4 years, they reached the same level on average. Germination percentage of caryopsis in the xeric ecotype was as high as in the mesic ecotype; however, the xeric ecotype had larger variation than the mesic ecotype in caryopsis germination percentage (Fig. 2). This indicates that higher diversity exists in the xeric ecotype than in the mesic ecotype, which is consistent with the levels of genetic diversity that correlates with ecological heterogeneity, niche breadth, and stress (Nevo et al., 1986; Li et al., 1999; Turpeinen et al., 2001). Caryopses of H. spontaneum have been shown to be dormant at harvest time (Gutterman and Nevo, 1994). However, this dormancy, the depth of which depends on conditions of caryopsis development, is progressively lost during the dry storage of 35–401C or under summer conditions; afterwards almost all caryopses germinate by the following winter rains (Gutterman et al., 1996). Differences in levels of dormancy are found to correlate with influences of the climatic environmental conditions of the habitats of the ecotypes. The highest levels of dormancy occur in the most severe conditions in the xeric Negev Desert Highlands, and the lowest levels of dormancy is found in caryopses collected from the north-facing slope of Nahal Oren, Mount Carmel, known as ‘‘Evolution Canyon’’, the most humid habitat tested (Gutterman and Nevo, 1994). Ethanol, like gibberellic acid and hydrogen peroxide, is able to stimulate the germination of intact grains, as well as isolated embryos of Hordeum distichum, by causing a decrease in endogenous ABA (Wang et al., 1998). Zinnia elegans seed germination is promoted by the removal of pericarp from seeds or by the removal of ethanol-soluble compounds from the seeds with pericarp. The ethanol-soluble compounds suppress the germination of seeds having no pericarp (Ogawa and Iwaguchi, 2001). However, the present work showed that ethanol had no effect on the germination percentage of naked caryopsis in both xeric (23-19) and mesic (1057) genotypes (Fig. 4). This may be attributed to the fact that the naked caryopses of both 23-19 and 10-57 genotypes had already reached a very high germination percentage, 100% and 98%, respectively. 4.2. Physical inhibition of glumellae on germination Freshly harvested wild barley dispersal units do not germinate, whereas naked caryopsis germinated indicating the inhibition of glumellae on germination (Gutterman and Nevo, 1994; Gutterman and Gozlan, 1998). However, there are
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no clues as to whether the inhibition is physical or chemical. The present work revealed that glumellae had a physical, not chemical, inhibition on germination only in the mesic ecotype (Fig. 4). This may be the main reason why the germination percentage of caryopsis was lower than that of naked caryopsis sterilized with 70% ethanol in mesic ecotype (Fig. 2). No glumellae and ethanol effect on xeric ecotype germination (Fig. 4) clarified the equal germination percentage between caryopsis and naked caryopsis sterilized with ethanol in the xeric ecotype (Fig. 2). 4.3. Survival ratio The survival ratio of post-germination seedlings subjected to severe drought is a good trait to measure drought resistance of wild barley in the germination stage. The xeric ecotype had a higher survival ratio than the mesic ecotype (Fig. 3). However, not all the genotypes of a xeric ecotype were more drought resistant than the genotypes of a mesic ecotype. Wild barley located in a creek in a xeric environment may have had better soil moisture than wild barley located on a slope in a mesic environment. In this case, wild barley that adapted to the microniche of a dry slope in a mesic area may have gained the ability to resist drought. Therefore, some genotypes of mesic wild barley may have similar or higher ability to resist drought than some genotypes of xeric wild barley. For example, seedlings of mesic genotype 10-30 and xeric genotype 23-38 had similar survival ratios in the present study (data not shown), and seedlings from mesic Neve Yaar have higher survival ratios than xeric Sede Boker after one week of dehydration at the stage of a root length less than 10 mm (Gutterman and Gozlan, 1998). Thus microniche adaptations may sometimes overcome large-scale habitat divergence of mesic/xeric environments. The importance of microscale divergence is dramatically emphasized in the ‘‘evolution canyon’’ model studies (Nevo, 1997, 2001). Naked caryopsis sterilized with 70% ethanol in this survival experiment derived bigger seedlings (10–60 mm long root) with lower survival ratios than caryopsis (4– 10 mm long root), suggesting that the seedling age (or seedling size) affected survival ratios. Likewise, possibly the protection of glumellae caused higher survival ratios of caryopsis. In the desert annual Anastatica hierochuntica, the true rose of Jericho, seedlings with roots 4–6 mm long renew their growth within 8 h of wetting after one week of dehydration (Friedman et al., 1981). Gutterman and Gozlan (1998) found that caryopses of H. spontaneum with the longest initial root of 1–4 mm continued to develop when rewetted, but in lower percentages than seedlings with initial roots of 5 mm or longer. One may infer from the above results that seedlings with initial 4– 10 mm roots had high survival ratios after one to four weeks of dehydration. 4.4. Chemical affecting germination and seedling growth There are water-soluble chemicals in glumellae inhibiting post-germination seedling growth. Water extracts from glumes of Sorghum bicolor contain watersoluble materials inhibitory to wheat seedling growth (Ben et al., 1995). Seedlings derived from naked caryopses with glumellae affected by water-soluble chemicals in
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glumellae had shorter roots than from naked caryopses without glumellae in both mesic and xeric ecotypes (Fig. 5). Ethanol improved seed germination, but inhibited seedling growth. Applying 1%, 5%, 10%, and 20% solutions of ethanol to the roots of Pseudotsuga menziesii seedlings three times a week is deleterious to their physiology and growth (Joseph and Kelsey, 2000). However, the present work revealed that seedlings derived from ethanol treated naked caryopses had longer roots than those derived from non-treated naked caryopses in the mesic genotype (Fig. 5). There was no effect of ethanol treatment on root growth in the xeric genotype. This may be because ethanol removed ethanol-soluble chemical inhibitors from the pericarp (Ogawa and Iwaguchi, 2001) or because ethanol decreased endogenous ABA content (Wang et al., 1998). One may infer that the xeric genotype had less ethanol-soluble inhibitors in the pericarp or less endogenous ABA.
5. Conclusion One of the strategies of the adaptation of H. spontaneum to xeric environments is increasing the diversity of caryopsis afterripening to prevent germination in inappropriate growing seasons, enhancing the desiccation tolerance of seedlings, and improving root length growth after germination. The alternative mesic and xeric ecotypes display adaptive complexes that may be uniquely selected to increase fitness in their respective contrasting environments.
Acknowledgements This work was supported by the following grants: the Israel Discount Bank Chair of Evolutionary Biology; the Ancell-Teicher Research Foundation for Molecular Genetics and Evolution; the German-Israel project Cooperation (DIP project funded by the BMBF and supported by BMBF’s International Bureau at the DLR), and the Graduate School of the University of Haifa, Israel. The authors thank Mrs. Ma Yan and Ms. Milade Naela for assistance in the experiment.
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Differential patterns of germination and desiccation tolerance of mesic and xeric wild barley (Hordeum spontaneum) in Israel Guoxiong Chena, Krugman Tamara, Tzion Fahimaa, Fengchun Zhangb, Abraham B. Korola, Eviatar Nevoa,* b
a Institute of Evolution, University of Haifa, Mount Carmel, Haifa 31905, Israel Wyler Department of Dryland Agriculture, Department of Life Science, Jacob Blaustein Institute for Desert Research, Ben-Gurion University of the Negev, Sede Boker 84990, Israel
Received 06 June 2002; received in revised form 29 November 2002; accepted 03 December 2002
Abstract Differential patterns of germination were identified in mesic (Maalot) versus xeric (Wadi Qilt) ecotypes of wild barley, Hordeum spontaneum C. Koch, in the following traits: afterripening (dormancy), seedling desiccation tolerance, and the effect of glumellae and ethanol on afterripening and seedling growth. The following results were indicated: (i) Dispersal unit afterripening average (75%) was identical, but its coefficient of variation was larger in the xeric than in the mesic ecotype (29.9% and 9.9%, respectively). (ii) Survival ratio of seedlings after 1-month dehydration was higher in the xeric ecotypes indicating that seedling desiccation tolerance was higher in the xeric ecotype. (iii) Inhibition of germination and seedling growth by glumellae were higher in the mesic ecotypes. Ethanol sterilization of naked caryopsis promoted root growth of mesic seedlings but not xeric seedlings. Seedlings originating from xeric ecotypes had longer roots than mesic seedlings. It appears that, in the germination stage, natural selection adapted wild barley to a xeric environment by increasing the diversity of afterripening, enhancing desiccation tolerance, and improving root length growth. r 2003 Elsevier Science Ltd. All rights reserved. Keywords: Afterripening; Drought resistance; Ethanol; Caryopsis; Point of no return; Survival ratio
*Corresponding author. Tel.: +972-4-824-0445; fax: +972-4-824-6554. E-mail address: [email protected] (E. Nevo). 0140-1963/03/$ - see front matter r 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0140-1963(02)00321-X
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1. Introduction Wild barley, Hordeum spontaneum C. Koch, like wild wheat, is one of the principal grain plants on which Neolithic food production in the Near East was founded. Wild barley is the indisputable progenitor of cultivated barley: this is indicated by its cross-compatibility, full fertility, and sporadic spontaneous hybridization with the cultivars (Nevo, 1992). The vast genetic resources of wild barley are the best, largely unexploited, resources for improving the narrowing genetic base of the cultivated barley (Nevo, 1992). Wild barley is an annual, brittle, two-row diploid (2n ¼ 14), and is predominantly self-pollinating (Brown et al., 1978). It is widespread in the Near East Fertile Crescent. In Israel, it is abundant, occupying an extraordinarily large diversity of habitats ranging from the mesic Mediterranean to the xeric southern steppes (Nevo, 1992). Populations of H. spontaneum involve abundant genetic variation against drought and salinity, and the highest resistant genotype is significantly correlated with the highest stress environment and with the highest genetic polymorphism (Nevo et al., 1984, 1997). An important survival strategy of wild barley under unpredictable small amounts and distribution of winter rain in the Israel xeric area is afterripening (Evenari et al., 1982; Gutterman, 1993, 1998). This primary dormancy effect prevents germination of mature caryopses after a late rain at the beginning of the long, dry summer (Evenari, 1965; Gutterman, 1993, 1996; Gutterman et al., 1996). Primary dormancy occurs when the intact seed is covered by the glumellae. The covering structures of the caryopses (seed-coat and pericarp) may limit the oxygen supply to the embryo (Lenoir et al., 1986; Gutterman et al., 1996) and thus inhibit germination. During the following rainy season, after summer dormancy, the caryopses are ready to germinate. If the rainfall is adequate to trigger germination, and is followed by a long period of drought, seedlings with desiccation tolerance may survive and develop into normal plants by developing new adventitious roots and leaves when rewetted (Friedman et al., 1981; Evenari et al., 1982; Gutterman and Gozlan, 1998, 1999). The survival ratio of seedlings of some ecotypes of H. spontaneum after 1 week of dehydration differed with respect to stages of root length at dehydration. It was higher in mesic Neve Yaar (annual rainfall 600 mm) than in xeric Sede Boker (90 mm) at the stage of root length less than 10 mm, and it was lower at the stage of a root length longer than 10 mm (Gutterman and Gozlan, 1998). Ethanol promoted seed germination of Zinnia elegans by removing the ethanolsoluble compounds in the pericarp that inhibited germination of the seed (Ogawa and Iwabuchi, 2001). Ethanol breaks dormancy of Hordeum distichum grain by causing a decrease in endogenous ABA (Wang et al., 1998) and may promote caryopsis germination of H. spontaneum. The aim of this work was to compare contrasting mesic (Maalot) and xeric (Wadi Qilt) ecotypes of wild barley, H. spontaneum, for (1) afterripening patterns, (2) seedling survival ratio after 1-month dehydration, and (3) the physical and chemical effects of glumellae and ethanol, respectively, on afterripening and seedling growth.
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2. Materials and methods 2.1. Plant material Two Israeli ecotypes of H. spontaneum were used in the present experiments: Maalot originating from mesic environments (western Upper Galilee) and Wadi Qilt from xeric environments (Judean Desert) (Fig. 1) (Table 1). Five genotypes from each ecotype were randomly chosen from the collection of the Gene Bank of the Institute of Evolution, University of Haifa. The Gene Bank of H. spontaneum includes 3500 genotypes, 122 populations from Israel, Jordan, Turkey, and Iran.
20 Km
Maalot Haifa
Me
dit
err
ane
an
sea
Sea of Galilee
Tel -Aviv
Wadi Qilt Jerusalem
Israel
Beer-sheva
Dead Sea
Negev Desert
Fig. 1. Geographic location of Maalot and Wadi Qilt.
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Table 1 Location of the two Hordeum spontaneum ecotypes and selected environmental data of sites of origin based on Nevo et al. (1984) and Van Rijn et al. (2000) Site of origin
Longitude (1E)
Latitude (1N)
Altitude (m)
Mean annual temperature (1C)
Mean annual rainfall (mm)
Mean humidity at 14:00 h (%)
Maalot Wadi Qilt
35.27 35.38
33.00 31.83
500 50
18 23
790 144
50 35
Caryopses of these genotypes were propagated in 1997 and have been stored since then at 101C with relative humidity ranging 20–25%. 2.2. Afterripening and desiccation tolerance Germination: caryopses were germinated on an ALBET filter paper wetted with 5 ml of distilled water in a 90 mm diameter Petri dish at 251C in the dark. Germination percentage was recorded after a 3-day incubation. Two methods of dehydration procedure were tested: (i) Sudden dehydration, post-germination seedlings were removed daily to Petri dishes with dry filter paper for a dehydration period of 1 month at room temperature in the dark (Gutterman and Gozlan, 1998). This method in which desiccation is being suddenly imposed on post-germination seedlings, is not like what’s found in nature or crop field. In order to make dehydration gradual and not contaminated by fungus in the Petri dishes, the second method was conducted as follows. (ii) Gradual dehydration, naked caryopses were first sterilized to prevent contamination of fungi with 70% ethanol for 5 min following five times rinsing in distilled water. Post-germinating seedlings were kept in their original place for gradual drying for 1 month in the dark at room temperature. Survival ratio: The dried seedlings were rehydrated with distilled water, and the survival ratio was calculated. Seedlings were considered viable when adventitious roots and coleoptile were developed and elongated (Gutterman and Gozlan, 1998). Four replicates of 30 caryopses of each genotype were used in this experiment. The mean values of all xeric and mesic genotypes were used to compare the effect of ecotypes and caryopses (intact caryopsis and naked caryopsis sterilized with ethanol) on afterripening (germination percentage) and desiccation tolerance. 2.3. The effects of glumellae and ethanol To find which factor, glumellae or alcohol, affects the germination percentage, two representative genotypes were chosen, a mesic genotype (Maalot 10-57) and a xeric genotype (Wadi Qilt 23-19). Four treatments were performed. (1) Caryopsis with glumellae—to reveal physical and chemical effects of glumellae. (2) Naked caryopsis together with the glumellae in the Petri dish—to reveal the chemical effect of glumellae. (3) Naked caryopsis sterilized with 70% ethanol—to reveal the effect of alcohol on germination and seedling growth. (4) Naked caryopsis—as the control.
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Four replicates of 15 caryopses in each treatment were placed in 90 mm diameter Petri dishes, on one ALBET filter paper, and wetted with 5 ml of distilled water. Distilled water was added to compensate the daily loss of water in Petri dishes. Germination percentage and shoot and root lengths were measured after a 3-day incubation at room temperature in the dark. Caryopses were considered to have germinated when the radicle protruded from the envelopes (Evenari, 1965; Gutterman and Gozlan, 1998). 2.4. Statistical analysis Statistical analysis of data was conducted using STATISTICA for windows 5.1D (StatSoft Inc., USA), Tukey honest significant difference (HSD) test was used to compare means (significance level, 5%).
3. Results 3.1. Germination patterns The xeric and mesic ecotypes showed the same germination percentage of caryopsis; however, the xeric ecotype had a larger coefficient of variation (ratio of standard deviation to the mean) than the mesic ecotype (Fig. 2), exhibiting higher diversity of afterripening in xeric than mesic ecotypes. The same germination pattern also happened in naked caryopsis. Germination percentage of caryopsis and naked caryopsis was similar in the xeric ecotype, whereas in the mesic ecotype germination percentage of naked caryopsis (sterilized with 70% ethanol) was higher than that of caryopsis (with glumellae). These results indicate that glumellae might inhibit caryopsis germination or alcohol might enhance naked caryopsis germination in the mesic ecotype or that glumellae and ethanol influenced germination of the xeric ecotype in different ways.
Fig. 2. Germination percentage of naked caryopses sterilized with 70% ethanol (NE) and caryopsis (G) from mesic and xeric ecotypes of Hordeum spontaneum in Israel. Four replicates of 30 caryopses of each of five genotypes for each ecotype were conducted in this experiment. Bars represent standard deviation. Letters above bars indicate significant differences (Po0:05; Tukey HSD test).
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3.2. Survival success The survival ratio of post-germination seedlings after the dehydration period of 1 month was higher in the xeric than mesic ecotypes in both caryopsis and naked caryopsis (Fig. 3), meaning that the xeric ecotype is more drought resistant compared to the mesic ecotype. The seedlings derived from naked caryopsis were much bigger than those derived from caryopsis with glumellae because the former seedlings were not removed from the original place and allowed to dry gradually, which would have given them more time to grow (Section 2.2). The seedlings derived from naked caryopsis had a lower survival ratio than that derived from caryopsis with glumellae in both xeric and mesic ecotypes (Fig. 3). One may infer that a bigger seedling has a lower ability to survive under drought stress. 3.3. The effects of glumellae and ethanol on germination In order to confirm the effect of ethanol and glumellae on germination (Fig. 3) and to separate the physical and chemical effects of glumellae on germination, two representative genotypes, genotype 10-57 from Maalot (mesic) and genotype 23-19 from Wadi Qilt (xeric), were chosen to conduct the next germination experiment. For genotype 10-57 (Fig. 4), germination percentage of naked caryopsis with glumellae was as high as naked caryopsis without glumellae indicating no glumellae chemical effect. Germination percentage of caryopsis was lower than naked caryopsis with glumellae revealing a glumellae physical effect on germination. No ethanol effect was tested because germination percentage of naked caryopsis sterilized with 70% ethanol was the same as that of naked caryopsis without sterilization. By contrast, for genotype 23-19, no significant glumellae and ethanol effects were observed in this germination experiment (Fig. 4). Therefore, the lower germination percentage of caryopsis compared with naked caryopsis sterilized with ethanol in the mesic ecotype showed in Fig. 2 was attributed to the physical
Fig. 3. Survival ratio after one-month dehydration of seedlings derived from naked caryopses sterilized with ethanol (NE) and caryopsis (G) from mesic and xeric ecotypes of Hordeum spontaneum in Israel. Bars represent standard deviation. Letters above bars indicate significant differences (Po0:05; Tukey HSD test).
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Fig. 4. Germination percentage of caryopses of genotype 10-57 (mesic) and 23-19 (xeric) of Hordeum spontaneum of four treatments. Caryopsis (G), naked caryopsis with glumellae (NG), naked caryopsis (N), and naked caryopsis sterilized with ethanol (NE). Four replicates of 15 caryopses of each of the four treatments were conducted in this experiment. Bars represent standard deviation. Letters above the bars indicate significant differences (Po0:05; Tukey HSD test).
Fig. 5. Coleoptile and seedling root length of genotype 10-57 (mesic) and 23-19 (xeric) of Hordeum spontaneum derived from caryopses of four treatments. Caryopsis (G), naked caryopsis with glumellae (NG), naked caryopsis (N), and naked caryopsis sterilized with ethanol (NE). Bars represent standard deviation. Letters above bars indicate significant differences (Po0:05; Tukey HSD test).
inhibition of glumellae. Without the glumellae and ethanol effect, the xeric ecotype exhibited the same level of germination percentage with caryopsis or naked caryopsis (Fig. 2). 3.4. The effects of glumellae and ethanol on seedling growth The growth of three-day-old post-germination seedling of genotype 10-57 (mesic) and 23-19 (xeric) was investigated. Coleoptile lengths of both genotypes were similar in all four treatments (Fig. 5). However, seedlings derived from caryopsis and naked caryopsis of the xeric genotype had longer roots than that of the mesic genotype. The root lengths of seedlings derived from both caryopsis and naked caryopsis with glumellae were the same, but were shorter than that of seedlings derived from naked caryopsis without glumellae indicating chemical not physical, inhibition of glumellae on root elongation. This chemical effect of glumellae was exhibited in both mesic and
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xeric genotypes. Ethanol improved root length growth in the mesic genotype, but showed no effect in the xeric genotype (Fig. 5).
4. Discussion 4.1. Ecogeographic variation in dormancy The dispersal units of wild barley used in the present study were harvested in 1997 and stored at 101C. The dormancy of xeric and mesic wild barley caryopses may be released at different speeds during storage. Within 4 years, they reached the same level on average. Germination percentage of caryopsis in the xeric ecotype was as high as in the mesic ecotype; however, the xeric ecotype had larger variation than the mesic ecotype in caryopsis germination percentage (Fig. 2). This indicates that higher diversity exists in the xeric ecotype than in the mesic ecotype, which is consistent with the levels of genetic diversity that correlates with ecological heterogeneity, niche breadth, and stress (Nevo et al., 1986; Li et al., 1999; Turpeinen et al., 2001). Caryopses of H. spontaneum have been shown to be dormant at harvest time (Gutterman and Nevo, 1994). However, this dormancy, the depth of which depends on conditions of caryopsis development, is progressively lost during the dry storage of 35–401C or under summer conditions; afterwards almost all caryopses germinate by the following winter rains (Gutterman et al., 1996). Differences in levels of dormancy are found to correlate with influences of the climatic environmental conditions of the habitats of the ecotypes. The highest levels of dormancy occur in the most severe conditions in the xeric Negev Desert Highlands, and the lowest levels of dormancy is found in caryopses collected from the north-facing slope of Nahal Oren, Mount Carmel, known as ‘‘Evolution Canyon’’, the most humid habitat tested (Gutterman and Nevo, 1994). Ethanol, like gibberellic acid and hydrogen peroxide, is able to stimulate the germination of intact grains, as well as isolated embryos of Hordeum distichum, by causing a decrease in endogenous ABA (Wang et al., 1998). Zinnia elegans seed germination is promoted by the removal of pericarp from seeds or by the removal of ethanol-soluble compounds from the seeds with pericarp. The ethanol-soluble compounds suppress the germination of seeds having no pericarp (Ogawa and Iwaguchi, 2001). However, the present work showed that ethanol had no effect on the germination percentage of naked caryopsis in both xeric (23-19) and mesic (1057) genotypes (Fig. 4). This may be attributed to the fact that the naked caryopses of both 23-19 and 10-57 genotypes had already reached a very high germination percentage, 100% and 98%, respectively. 4.2. Physical inhibition of glumellae on germination Freshly harvested wild barley dispersal units do not germinate, whereas naked caryopsis germinated indicating the inhibition of glumellae on germination (Gutterman and Nevo, 1994; Gutterman and Gozlan, 1998). However, there are
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no clues as to whether the inhibition is physical or chemical. The present work revealed that glumellae had a physical, not chemical, inhibition on germination only in the mesic ecotype (Fig. 4). This may be the main reason why the germination percentage of caryopsis was lower than that of naked caryopsis sterilized with 70% ethanol in mesic ecotype (Fig. 2). No glumellae and ethanol effect on xeric ecotype germination (Fig. 4) clarified the equal germination percentage between caryopsis and naked caryopsis sterilized with ethanol in the xeric ecotype (Fig. 2). 4.3. Survival ratio The survival ratio of post-germination seedlings subjected to severe drought is a good trait to measure drought resistance of wild barley in the germination stage. The xeric ecotype had a higher survival ratio than the mesic ecotype (Fig. 3). However, not all the genotypes of a xeric ecotype were more drought resistant than the genotypes of a mesic ecotype. Wild barley located in a creek in a xeric environment may have had better soil moisture than wild barley located on a slope in a mesic environment. In this case, wild barley that adapted to the microniche of a dry slope in a mesic area may have gained the ability to resist drought. Therefore, some genotypes of mesic wild barley may have similar or higher ability to resist drought than some genotypes of xeric wild barley. For example, seedlings of mesic genotype 10-30 and xeric genotype 23-38 had similar survival ratios in the present study (data not shown), and seedlings from mesic Neve Yaar have higher survival ratios than xeric Sede Boker after one week of dehydration at the stage of a root length less than 10 mm (Gutterman and Gozlan, 1998). Thus microniche adaptations may sometimes overcome large-scale habitat divergence of mesic/xeric environments. The importance of microscale divergence is dramatically emphasized in the ‘‘evolution canyon’’ model studies (Nevo, 1997, 2001). Naked caryopsis sterilized with 70% ethanol in this survival experiment derived bigger seedlings (10–60 mm long root) with lower survival ratios than caryopsis (4– 10 mm long root), suggesting that the seedling age (or seedling size) affected survival ratios. Likewise, possibly the protection of glumellae caused higher survival ratios of caryopsis. In the desert annual Anastatica hierochuntica, the true rose of Jericho, seedlings with roots 4–6 mm long renew their growth within 8 h of wetting after one week of dehydration (Friedman et al., 1981). Gutterman and Gozlan (1998) found that caryopses of H. spontaneum with the longest initial root of 1–4 mm continued to develop when rewetted, but in lower percentages than seedlings with initial roots of 5 mm or longer. One may infer from the above results that seedlings with initial 4– 10 mm roots had high survival ratios after one to four weeks of dehydration. 4.4. Chemical affecting germination and seedling growth There are water-soluble chemicals in glumellae inhibiting post-germination seedling growth. Water extracts from glumes of Sorghum bicolor contain watersoluble materials inhibitory to wheat seedling growth (Ben et al., 1995). Seedlings derived from naked caryopses with glumellae affected by water-soluble chemicals in
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glumellae had shorter roots than from naked caryopses without glumellae in both mesic and xeric ecotypes (Fig. 5). Ethanol improved seed germination, but inhibited seedling growth. Applying 1%, 5%, 10%, and 20% solutions of ethanol to the roots of Pseudotsuga menziesii seedlings three times a week is deleterious to their physiology and growth (Joseph and Kelsey, 2000). However, the present work revealed that seedlings derived from ethanol treated naked caryopses had longer roots than those derived from non-treated naked caryopses in the mesic genotype (Fig. 5). There was no effect of ethanol treatment on root growth in the xeric genotype. This may be because ethanol removed ethanol-soluble chemical inhibitors from the pericarp (Ogawa and Iwaguchi, 2001) or because ethanol decreased endogenous ABA content (Wang et al., 1998). One may infer that the xeric genotype had less ethanol-soluble inhibitors in the pericarp or less endogenous ABA.
5. Conclusion One of the strategies of the adaptation of H. spontaneum to xeric environments is increasing the diversity of caryopsis afterripening to prevent germination in inappropriate growing seasons, enhancing the desiccation tolerance of seedlings, and improving root length growth after germination. The alternative mesic and xeric ecotypes display adaptive complexes that may be uniquely selected to increase fitness in their respective contrasting environments.
Acknowledgements This work was supported by the following grants: the Israel Discount Bank Chair of Evolutionary Biology; the Ancell-Teicher Research Foundation for Molecular Genetics and Evolution; the German-Israel project Cooperation (DIP project funded by the BMBF and supported by BMBF’s International Bureau at the DLR), and the Graduate School of the University of Haifa, Israel. The authors thank Mrs. Ma Yan and Ms. Milade Naela for assistance in the experiment.
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