In vitro assay of native Iranian almond species (Prunus L. spp.) for drought tolerance
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- Sorkheh, K., Shiran, B., Khodambshi, M. et al. Plant Cell Tiss Organ Cult (2011) 105: 395. doi:10.1007/s11240-010-9879-1
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Eight native Iranian almond species from three sections, ‘Euamygdalus’ (Prunus communis; Prunus eleagnifolia and Prunus orientalis); ‘Lycioides’ (Prunus lycioides and Prunus reuteri) and ‘Spartioides’ (Prunus arabica, Prunus glauca and Prunus scoparia) were in vitro screened for drought tolerance using sorbitol and polyethylene glycol (PEG) as an osmoticum. Different levels of water stress were induced using five concentrations of either sorbitol or polyethylene glycol in Woody Plant Medium (WPM). Water potential of various media ranged from −0.80 to −2.05 MPa and water stress in culture medium adversely affected plantlet growth. Wild species from ‘Spartioides’ were less affected than ‘Lycioides’ and ‘Euamygdalus’. At the same level of water potential, sorbitol had lower adverse effects than PEG; the latter being severe. Prunus × sorbitol and Prunus × PEG interactions were significant. At 0.2 M sorbitol and 0.003 M PEG, ‘Spartioides’ produced significantly more roots with higher total root length and root volume, as well as root-dry weight than those of ‘Lycioides’ and ‘Euamygdalus.’ It is concluded that in vitro screening of native Iranian almond species under specific and limited water-stress conditions may provide a system for effectively differentiating the wild species of almond for their expected root mass production under field conditions.
KeywordsDrought toleranceIn vitro rootingPrunus L. spp.Water stress
Woody Plant Medium (Lloyd and McCowen 1981)
The identification of plant germplasm capable of tolerating a water deficit environment is an important research objective for numerous annual and perennial crops (Ledbetter et al. 1998). Non-domesticated germplasm and exotic landraces represent sources of genetic diversity from which important physiological characters, such as drought tolerance, can be identified and then utilized in a breeding program. Exotic germplasm has been screened successfully in wheat (Ehdaie et al. 1991), pegon pea (Sharma et al. 1991), cowpea (Atokple et al. 1993), tomato (Saranga et al. 1993), blueberry (Ehlenfeldt 1994), millet (Ouendeba et al. 1995), maize (Matheka et al. 2008), date palm (El-Sharabasy et al. 2008), Arabidopsis (de Klerk and Pumisutapon 2008; Zhao et al. 2009), tomato (Aazami et al. 2010), and Pelargonium (Hassanein 2010) for a variety of biotic and abiotic stresses. Various osmotic agents have been employed in appropriate nutrient media to screen germplasm in vitro for drought tolerance. Although specific in vitro methods vary with plant types being screened, researchers have been able to control the drought environment more precisely using in vitro or artificial selection techniques (Krizek 1985; Maruyama et al. 2008; He et al. 2009; Srinivasan et al. 2010).
Water is essential for plant growth and a necessary component of most physiological processes. Improvement in root traits is considered important for developing drought-tolerant genotypes (Rossouw and Waghmarae 1995; Iwama and Yamaguchi 2006; Maruyama et al. 2008; Barreto et al. 2010). However, measurement of the root traits in field-grown plants is laborious and time-consuming (Ingram et al. 1994; Erusha et al. 2002; Iwama and Yamaguchi 2006; Gopal and Iwama 2007). As a result, little progress has been made in the development of drought-tolerant varieties/cultivars. The normal method of root measurements in field-grown plants requires deep (>1 m) digging, collecting soil cores from various depths, and washing soil from the roots in order to record root characteristics.
An in vitro approach may be an alternative to overcome the problems associated with field evaluation of almond species for root characteristics. The effectiveness of these techniques has also been studied in potato to facilitate selection for tuber characters including yield (Alasdon et al. 1988; Gopal et al. 1998; Donnelly et al. 2003; Gopal and Iwama 2007; He et al. 2009), maturity (Lentini and Earle 1991), and disease resistance (Platt 1992a, b).
Snow (1905) was among the first researchers to observe the inhibitory effect of mannitol as used in plant growth assays. General plant growth inhibition in response to mannitol has led numerous researchers to develop screening techniques using mannitol as an osmotic agent (Wright and Jordan 1970; Erb et al. 1988; Karunaratne et al. 1991; Hsissou and Bouharmont 1994; Hassanein 2010). However, a short-term study using excised wheat embryos, rape seedlings and potato stem segments demonstrated the uptake of mannitol from nutrient medium (Lipavská and Vreugdenhil 1996). These authors indicated that mannitol uptake in these plant species might be mainly apoplastic and that mannitol-supported growth could neither be proven nor estimated. In micropropagated Prunus germplasm, significant growth differences were observed between control and mannitol-treated ‘Marianna 2624’ explants only 14 days after the onset of the in vitro culture period (Rajashekar et al. 1995). Using this screening technique, significant differences in explant growth were observed among five clonally propagated plum rootstock candidates and ‘Marianna 2624’ when challenged with the inclusion of mannitol in the proliferation medium (Ledbetter et al. 1996).
Heat and drought tolerance have not been evaluated in vitro in Prunus L. spp. Progressive reduction in plant growth occurs as NaCl levels increase, but there is some inconsistency in the literature concerning the precise level suitable for effective in vitro screening. Thus, to arrive at credible results, growth evaluation from a range of salinity levels has been suggested (Ashraf 1994; Zhang and Donnelly 1997; Khrais et al. 1998; Zhao et al. 2009). Osmotic solutions of NaCl, mannitol/sorbitol, and polyethylene glycol (PEG) have been used as in vitro stress factors for selecting salt- and drought-tolerant genotypes in screening procedures for seed germination of wheat (Almansouri et al. 2001), sunflower (Punia and Jain 2002) and potato (Gopal and Iwama 2007), embryo germination in peach and almond (Ledbetter et al. 1998), sprouting percentage in mulberry (Tewary et al. 2000), nodule development in alfalfa (Djilianov et al. 2003), and growth of nodal cuttings in cassava (Ng and Ekanayake 1997).
There is little information on the effect of various cultural factors on in vitro root growth. Medium supplemented with NaCl is reported to adversely affect root growth in potato (Zhang and Donnelly 1997). In wheat genotypes, drought-tolerant genotypes were found to have better-developed root systems under in vitro water stress mediated through PEG (Ye et al. 2002). Many decades of field experience have demonstrated clearly that almond and peach–almond hybrid rootstocks are more tolerant of drought than seed-propagated peach rootstocks (Day 1953). Root systems of almond tend to penetrate directly downward and be of a larger caliber as compared with the more fiberous and horizontal root mat of peach root systems. Rootstock breeding efforts at Shahrekord University are now including hybrids with wild species of almond that can be utilized for seed-propagated rootstocks. Hence, we are currently examining the use of sorbitol and polyethylene glycol as osmotic agents in cultures of seed-propagated Prunus germplasm.
Our objectives in this current study were to examine shoot and root growth of the in vitro plantlets derived by growing seeds on Woody Plant Medium (WPM) medium (Lloyd and McCowen 1981) with and without sorbitol or polyethylene glycol. Eight Prunus species of almond that are native to Iran were examined with the objective of developing in vitro screening methods for drought tolerance according to Gopal and Iwama (2007) and Ledbetter et al. (1998).
Materials and methods
Wild almond species and collecting regions
The wild almond species utilized, belonging to the genus Prunus, subgenus Amygdalus, were Prunus communis (L.) Archang, Prunus elaeagnifolia (Spach) Fritsch, Prunus orientalis Mill. (syn. Prunus argentea Lam.) in section ‘Euamygdalus’ Spach; Prunus lycioides Spach, Prunus reuteri Boiss. et Bushe in section ‘Lycioides’ Spach; and Prunus arabica (Olivier) Neikle, Prunus glauca (Browicz) A.E. Murray, Prunus scoparia Spach in section ‘Spartioides’ Spach. The number of accessions sampled per site ranged from one to five, depending on habitat diversity and availability at collection time.
Field expeditions were carried out in 2007 and 2008 on the basis of recent literature (Moradi 2005; Gorttapeh et al. 2005; Sorkheh et al. 2007, 2009), indigenous information, or conspicuous presence. Collections came from both wild and cultivated habitats, which were concentrated in two different regions in Iran. The first region (Azerbaijan and Kordestan, 36°00′–38.28′N, 44°51′–45°46′E, mean elevation 1,473 m a.s.l.) is characterized by relatively lush environment, mean annual rainfall of 507 mm, high biological diversity, and relatively low agricultural development. The second region (Shahrekord and Shiraz, 27°32′–32.26′N, 49°50′–56°50′E, mean elevation 2,030 m), with a mean annual rainfall of 436 mm, is in a more xerophytic area with widespread agriculture.
Employed medium and cultural requirements
Medium used and their water potential according to Gopal and Iwama (2007)
Water potential (MPa)
With PEG 6000
The pH of the media was adjusted to 5.7 ± 0.1, and the media solidified with 7 g l−1 agar (Ridel-deHan). Test tubes (150 mm × 25 mm; Sigma–Aldrich), each containing 20 ml of the medium and closed with cotton plugs, were used for culturing.
Explant and culture conditions
WPM Medium was utilized as above with an inclusion of sorbitol and PEG (Table 1). Treatment media were adjusted to a pH 5.7 prior to autoclaving and dispensing into 150 mm × 25 mm culture tubes. Permeable membrane caps (Biomedical Polymers, Leominster, MA, USA) were used to cover culture tubes. Media sterilization was accomplished with an autoclave treatment of 121°C at 108 kPa for 15 min. Fruit from the eight Prunus L. spp. were harvested on 2 July 2008. Fruit were split and the immature seeds were removed from the stones. Immature seeds were surface disinfested with a 4.0% solution of Amphyl (National Laboratories, L & F Products, Montvale, NJ, USA) prior to three successive rinses with sterile distilled water. The surface disinfested immature seeds were then left overnight at 4°C prior to culturing. Flasks containing the immature seeds were then brought to the laminar flow hood for culturing. Cultured seeds were placed in a growth environment with a 16-h photoperiod (Vita-Lite 40 w, 47.1 μmol m−2 s−1) and an average temperature of 24 ± 1°C prior to evaluation.
Water potential, shoot and root character measurements
Water potential of all media was measured using an isopiestic thermocouple psychrometer (Boyer and Knipling 1965). Measurements were repeated to arrive at a reliable consistent value according to Gopal and Iwama (2007). When cultures were 90 days old (without subculturing) and fully grown, having stout stems and broad leaves in the control treatment (i.e., WPM medium without sorbitol or PEG), data were recorded for various shoot and root characters. At the time of observation, the plantlets, in general, had just started showing signs of senescence (browning of leaf edges and/or shoot tips). Akin to procedures of Gopal and Iwama (2007), plantlets were removed from tubes and numbers of roots were counted. Shoots were cut from the roots, and plantlet height was measured as the length of the main stem from base to the meristematic tip. Foliage (stems and leaves) was cut into pieces and weighed. Roots were washed to remove any attached medium and preserved in 70% alcohol, and later measured for root length, root diameter, root volume, and root dry weight. The former three characters were recorded using an image analysis system (WinRhizo; Regent Instruments, Canada) and root-dry weight was determined after oven-drying the samples to a constant weight at 80°C.
Both experiments were conducted in a completely randomized two-factor (8 wild almond species × 5 levels of osmoticum) factorial design with eight replications. As explant growth was not normal in some replications, the effective number of replications used for data recording was reduced to six according to Gopal and Iwama (2007) and Ledbetter et al. (1998). Data were analyzed and treated by analysis of variance (ANOVA) using SAS software (SAS Institute 1989) in order to detect significant differences among the different treatments (PROC GLM) at P ≤ 0.05.
Water potentials of the various media used are presented in Table 1. The control semisolid WPM medium with 30 g l−1 sucrose had a water potential of −0.80 MPa. As expected, the water potential of the media decreased with the addition of sorbitol as well as PEG. Medium with the highest concentration (0.4 M) of sorbitol had a water potential of −2.05 MPa, whereas it was −1.30 MPa for the medium with the highest concentration (0.012 M) of PEG.
Effect of sorbitol levels on various wild species of almond (Prunus L. spp.)
Effect of PEG levels on various wild species of almond (Prunus L. spp.)
Prunus × PEG interaction was not significant for number of roots per plantlet. Mean number of roots over media were more in Prunus arabica (6.3/plantlet), Prunus Scoparia (5.1/plantlet) and Prunus glauca (4.6/plantlet) than Prunus lycioides (4.5/plantlet), Prunus reuteri (4.1/plantlet), Prunus communis (3.6/plantlet), Prunus eleagnifolia (3.7/plantlet) and Prunus oreintalis (3.5/plantlet), and the latter wild species in sections ‘Lycioides’ and ‘Euamygdalus’ were on a par with each other.
The addition of sorbitol or PEG to the WPM medium decreased the water potential of the media, inducing water stress that adversely affected both shoot and root growth of the plantlets. Water stress under field conditions is also known to adversely affect plant growth including stem height, foliage weight, root number, and root dry weight along with a corresponding decline in tuber yield (Munns and Pearson 1974; Lynch and Tai 1989; Deblonde and Ledent 2001; Tourneux et al. 2003; Lahlou and Ledent 2005; Gopal and Iwama 2007). So, the general effect of water stress on in vitro plant growth as observed in the present study was similar to that observed under the field conditions (results not shown). These similarities in the effects of water stress under in vitro and in vivo conditions suggest that the in vitro system can be used as an alternative to field evaluations for studying the general effect of water-stress on plant growth and development.
Prunus lycioides and Prunus reuteri are late maturing, while Prunus arabica, Prunus glauca and Prunus scoparia mature even later (Sorkheh et al. 2009). Prunus L. spp. with longer fruit developmental periods, in general, have high root dry weight under field conditions, but in species with early fruit maturity, root growth stops earlier. The wild species of almond used in the present study represented the different possible combinations of root dry weight and foliage maturity. A Prunus L. spp. with high or medium root dry weight and early maturity is perhaps very difficult to find. The results of the present study show that root dry weight of the Prunus species in three sections under field conditions were very well reflected by their root growth in vitro on WPM medium supplemented with 0.2 M sorbitol or 0.003 M PEG. In these media, Prunus arabica, Prunus glauca and Prunus scoparia had larger numbers of roots with higher total root length and root volume, as well as root dry weight than those of wild species in sections ‘Lycioides’ and ‘Euamygdalus’, and the latter two sections did not differ significantly for any of these characters (Figs. 1 and 2). Similar patterns of differences were also observed for plantlet height and foliage fresh weight.
Prunus L. spp. in section ‘Spartioides’ also had higher root dry weight than ‘Lycioides’ and ‘Euamygdalus’ in media supplemented with other concentrations of sorbitol or PEG. However, in the medium without sorbitol and PEG, or having more than 0.2 M sorbitol or more than 0.003 M PEG, species differences for one or more of the root or shoot characters were not significant. Under extreme water stress conditions as induced by 0.4 M sorbtiol and 0.012 M PEG, all species had little shoot or root growth (Figs. 1 and 2) and species differences were not significant for any character. Thus, the proposed in vitro system for screening wild species from different sections against water stress has the limitation that water stress can be induced to only a limited level for the purpose of differentiating Prunus L. spp.
These results are similar to studies with single-node potato cuttings screened for salinity tolerance where the use of a relatively high level of NaCl failed to quantify differences among cultivars in vitro (Morpurgo 1991). Water stress beyond the limit of tolerance by a plant will naturally prove fatal, as is also observed under field conditions. Differential growth responses observed at different water stress levels in vitro is consistent with similar observations found in vivo (Iwama et al. 1999), and suggests the involvement of different genes at different levels of stress (Tal 1994). This and significant species × water stress (sorbitol/PEG) interaction for a majority of the characters as observed in the present study suggest that ranking of a Prunus L. spp. at a particular level of water stress would not be predictive of its water stress tolerance at other levels applied in culture or in the field. Relative vigor is an important component of tolerance to abiotic stress (Munns 1993). Higher foliage weights of Prunus L. spp. in ‘Spartioides’ section in medium without sorbitol or PEG suggests that relative plantlet growth may be sufficient to select for both vigor and root geometry such as root length, both of which are useful under water stress conditions. However, keeping in view the consistent pattern of Prunus differences for all characters, WPM medium with 0.2 M sorbitol or 0.003 M PEG can be considered as the best for reliably differentiating the wild species of almond for their expected root dry weight under field conditions. This can be based on the in vitro root dry weight or related characters, i.e., root number, total root length, or root volume. Among these, root dry weight, which reflects the overall root growth, is perhaps also the easiest to measure reliably. Since species difference for root diameter did not follow the pattern observed for other root characters in various media tested, this parameter is not useful for differentiating Prunus L. spp. for their overall root growth. The results also demonstrated that the observed adverse effect of water stress on shoot and root growth was, in general, lower on ‘Spartioides’ than on ‘Lycioides’ or ‘Euamygdalus’ sections over a range of stress levels, thus indicating higher drought-tolerance ability of Prunus species in ‘Spartioides’ section. Under water stress field conditions, too, some Prunus in ‘Spartioides’ had higher root dry weight than Prunus species in ‘Lycioides’. However, ‘Euamygdalus’ was not included in the study. Root fresh weight in vitro (Morpurgo 1991; Gopal and Iwama 2007) as well as in vivo (Iwama et al. 1982) is reported to be positively correlated with tuber yield in vivo. Under field conditions, larger root dry weight delays leaf senescence and thus prolongs tuber bulking (Iwama et al. 1982). So, the observed relationship of growth under mild water stress (induced by 0.2 M sorbitol or 0.003 M PEG) in culture in three sections with their relative growth under both non-stress and water stress field conditions is due to the inherent higher drought-tolerance ability of ‘Spartioides’ compared with ‘Lycioides’ and ‘Euamygdalus’. The nature of the water stress-inducing agent is also important for root growth. It is desirable to use a compound that does not interact with plants in any way other than lowering the water potential of the medium. Thus, slowly penetrating osmotica such as mannitol or sorbitol (Hohl and Schopfer 1991) or inorganic salts (Termaat and Munns 1986) are not ideal, especially for experiments extending beyond a few hours. Polymers of PEG have been used for many years, principally because PEG molecules with molecular weight ≥6,000 cannot penetrate the cell wall pores (Carpita et al. 1979). Because PEG does not enter the apoplast, water is withdrawn not only from the cell but also from the cell wall. Therefore, PEG solutions mimic dry soil more closely than solutions of low molecular weights osmotica, which infiltrate the cell wall with solute (Verslues et al. 1998). However, PEG has its own drawbacks. Some studies have indicated that PEG could contain toxic contaminants that inhibit plant growth (Plaut and Federman 1985; Maruyama et al. 2008). Another potential disadvantage is that the high viscosity of PEG solutions limits the movement of O2, thereby increasing the likelihood of root O2 deficiency. In the present study, the water potential of the medium with 0.2 M sorbitol was −1.35 MPa, and, though it was similar to that caused by 0.012 M PEG (Table 1), the latter medium had higher adverse effect on plant growth, and genotypes did not differ for various measured characters in this medium. PEG being very viscous, the media supplemented with this were found to very sticky and hard, and also difficult to wash from the roots. Thus, it is not only the water potential imposed by the different stress inducing agent but also the consistency of the medium that affects the plant growth, especially the root development. Keeping in view the ease with which the medium can be washed off the roots, and lower adverse effect on plant growth at the same level of water potential, sorbitol would be a better choice for in vitro induction of water stress.
Ledbetter et al. (1998) reported that the embryo culture osmotic screen can be used as a preliminary tool to select osmotically tolerant parents in a seed-propagated Prunus rootstock breeding program. In vitro selection of osmotically tolerant clonally propagated Prunus explants has been demonstrated previously (Ledbetter et al. 1996). Establishing the relative osmotic tolerance of seed-propagated Prunus germplasm is particularly important in the United States since the Prunus nursery industry functions primarily with seed-propagated rootstocks. Prunus breeders might select those particular accessions having relatively high vigor in WPM and where plantlet growth reduction is minimized when embryos are cultured in osmotically modified WPM.
An in vitro screening system using WPM medium with 0.2 M sorbitol and immature seeds as explants may have the potential to differentiate Prunus species for their root dry weight production under field conditions. The effectiveness of this system, however, should be further tested on a greater number of Prunus species with known performance for root characteristics related to drought tolerance under field conditions. One issue in comparing effectiveness of screening methods in relation to field rankings for drought tolerance is to determine the reliability of the rankings. In such comparisons, data from field evaluations must be based on well-conducted trials repeated over years. Unfortunately, such evaluations for root characters are available for only a few species. Nevertheless, the results of the present study clearly showed that screening of Prunus L. spp. under cultural conditions that simulate the in vivo conditions might provide a high efficiency as an in vitro screening method for abiotic stresses like drought tolerance.
The authors offer grateful thanks to Shahrekord University for financial assistance, as well as to the Agriculture and Natural Resources Research Center of Shahrekord for access to almond trees. Thanks to Professor Craig A. Ledbetter for helpful commentary on the manuscript. We are grateful to Mr. S. Mosavi for helping arrange the facilities and to Ms. F. Tavakoli, Mr. R. Amiri Fahliani and Mr. A. Arminian for their kind help in undertaking this study.