Maternal Source Affects Seed Germination of a Rare Arabian Desert Species (Astragalus Sieberi)

Botany

Maternal source affects seed germination of a rare Arabian desert species (Astragalus sieberi)

Journal: Botany

Manuscript ID cjb-2020-0144.R2

Manuscript Type: Article

Date Submitted by the 28-Dec-2020 Author:

Complete List of Authors: Bhatt, Arvind; Lushan Botanical Garden Jiangxi Province and Chinese Academy of Sciences Carón, María ; Instituto Multidisciplinario de Biología Vegetal, IMBIV Souza-Filho, Paulo; Universidade Federal do Oeste da Bahia, Centro MultidisciplinarDraft de Barra Gallacher, David ; The University of Sydney, School of Life and Environmental Sciences

Keyword: Arid desert, Dormancy, Scarification, Seedling survival, Legumes

Is the invited manuscript for consideration in a Special Not applicable (regular submission) Issue? :

Maternal source affects seed germination of a rare Arabian desert species (Astragalus sieberi) Arvind Bhatta, María Mercedes Carónb, Paulo Roberto de Moura Souza-Filhoc, David J Gallacherd

a Lushan Botanical Garden, Chinese Academy of Science, Jiujiang, China b Instituto Multidisciplinario de Biología Vegetal (IMBIV), CONICET, Facultad de Ciencias Exactas, Físicas y Naturales, Universidad Nacional de Córdoba, CC 495, 5000 Córdoba, Argentina. c Centro Multidisciplinar de Barra, Universidade Federal do Oeste da Bahia, Centro Multidisciplinar da Barra, Brazil. d School of Life and Environmental Sciences, The University of Sydney, Australia.

Draft

Abstract Understanding variation of seed germination in wild populations can assist restoration projects through improved seed source selection. Recruitment of artificially distributed seed can be improved by selecting for suitable dormancy and germination characteristics. We investigated seed germination and emergence responses of three Astragalus sieberi populations (Abdali, Liya and Salmi) to in situ storage for 5 months at depths of 0 and 5 cm, pre-sowing hydration for 12 and 24 hours, and chemical scarification with concentrated sulfuric acid for 5, 10 and 15 minutes. Germination percentage was low for freshly collected seeds (< 3%) from Abdali and Salmi. In situ storage partially enhanced germination. Pre-sowing hydration did not affect seedling emergence, but acid scarification effectively broke seed dormancy. The longest scarification treatment of 15 min produced the greatest seedling emergence, though populations responded differently. Results indicate that selection of maternal seed sources can improve revegetation projects of desert areas with native seed sources. Knowledge of inter-population variation can improve the understanding of the mechanisms regulating seed germination,Draft thus optimizing restoration projects by selecting optimal seed sources. Keywords: Arid desert, Dormancy, Scarification, Seedling survival, Legumes

Introduction Restoration of native vegetation in arid regions is challenging. High temperatures, low and erratic rainfall, high soil salinity and low nutrient availability make it a difficult environment for vegetation establishment (Yasseen 2011; Podda et al. 2017). Native desert species that are adapted to these conditions are very effective for restoring ecosystems and reversing desertification (Abella et al. 2012; Phondani et al. 2016; Bhatt and Santo 2017). Generally, species revegetation via seed requires less handling and cost compared to vegetative propagation (Merritt and Dixon 2011). However, an effective conservation and restoration strategy using seed requires knowledge of inter-population variability (Eriksson 2014). Collecting seed from different populations preserves genetic variation (Westengen et al. 2013; Peres 2016), which improves resilience of the established population to future threats (Elzenga and Bekker 2017). Use of native, wild seed populations for restoring degraded areas is therefore preferable for long-term success (James et al 2011). Seed dormancy and germination varies significantly among populations of many species (Baskin and Baskin 2014). Larger seeds may exhibit greater seedling establishment and subsequent plant

fitness (Humara et al. 2000; Bischoff et al. 2006; Baskin and Baskin 2014). Consequently, restoration efforts must balance the need to maintain genetic diversity with the need to select seed from populations with a greater chance of reproductive success. Variation of germination characteristics among populations is poorly known for most Arabian species. Breaking seed dormancy is strongly affected by temperature, soil moisture and salinity (Baskin and Baskin, 2014). Understanding of seed dormancy and germination requirements improves vegetation restoration efforts (Bhatt and Pérez-García 2016; Bhatt et al. 2016a; Bhatt et al. 2019a), conservation and sustainable management (Baskin and Baskin 2014; Hoyle et al. 2015) by improving propagation success of targeted species (Merritt and Dixon 2011). Effective treatments for dormancy break can facilitate greater adoption of native species in Arabian desert restoration projects. More than 80% of desert species are reported to exhibit dormancy at the time of seed maturity (Baskin and Baskin 2014). Seeds can remain on the soil surface or be buried in the soil, maintaining dormancy until the appearance of favorable conditions that enhance the chances of seedling survival (Carta et al. 2014). PhysicalDraft dormancy linked to a hard water-impermeable seed coat (Büyükkartal et al. 2013) is common in species inhabiting arid and semiarid deserts (Baskin and Baskin 2014). Dormancy alleviation involves disruption of water-gap structures via an environmental signal (Baskin et al. 2000). Under natural conditions, factors such as dry heat, alternate wetting and drying, wet heat and low temperatures can break physical dormancy (Baskin and Baskin 1984; Norman et al. 2002; Van Assche et al. 2003; Van Klinken and Flack 2005; Van Klinken et al. 2006). The timing of increased seed permeability and thus the dormancy release is synchronized with favorable conditions for germination and seedling establishment (Baskin 2003; Jayasuriya et al. 2009). Germination requirements can vary among populations due to differences in the maternal environment or the population gene pool (Bischoff et al. 2006; Baskin and Baskin 2014). Dormancy and seed germination data that is population-specific enables the identification of naturally occurring seed sources for ecological restoration (Elzenga et al. 2019). Astragalus sieberi DC. (Fabaceae) is a suitable species for land rehabilitation for its capacity to stabilize sand and sequester atmospheric nitrogen (Bidak et al. 2015). It is a small perennial shrub, typically growing to 30 cm height, that favors sandy, gravelly and calcareous soils. Seeds are reniform-quadrangular in shape with a hard and impermeable coat. Foliage is highly palatable and frequently grazed by camels and sheep (Heneidy and Bidak 2004). Previous studies of the genus have indicated physical

seed dormancy via hard seed coats (Baskin et al. 2000; Long et al. 2012; Statwick 2016). Various pretreatments have been suggested for improving germination of Astragalus spp., including soaking in hot water, mechanical (nicking, sandpaper) or chemical scarification and dry storage (Acharya et al. 2006; Long et al. 2012; Albrecht and Penzagos 2012; Chou et al. 2012; Bhatt et al. 2020). Germination requirements of the genus are thus well known, though not of the species A. sieberi. The present study aims to identify the effect of dormancy breaking treatments in A. sieberi seed and thus increase this species inclusion in restoration projects. Specifically, we address (i) inter-population variation in seed dormancy, germination, and early seedling growth, and (ii) effectiveness of dormancy breaking methods; storage under natural conditions, pretreatment by soaking in water and acid stratification.

Material and methods Seed collection and storage Mature seed pods were collected June 2018Draft from three Kuwaiti inland locations that were similar in climate and altitude, but differed in soil texture and associated species (Table 1). The furthest distance among populations was 130 km between Abdali and Salmi. Pods were collected from at least 15 maternal plants per population along a 100-120 m transect, leaving at least 2 m between adjacent plants. Seeds from each population were extracted from pods and pooled, then divided into four batches. One batch (fresh) was tested for germination within one week of being collected, a second batch was set aside for dormancy breaking pre-treatment experimentation (see below), and two further batches were placed in nylon bags with a 0.2 mm size mesh for in situ storage. Seed mass was determined at time of collection from three 50-seed replicates per population, using a Sartorius electronic balance (Sartorius Co., Goettingen, Germany). Water imbibition was recorded on the same seeds by placing them with distilled water on two layers of Whatman No 1 filter paper for 24 hours. Water imbibition was calculated as the percentage of increase in mass from pre- to post-imbibition (Baskin and Baskin 2014). Seeds (stored surface and stored buried) from each population were kept at respective population where they originally been collected (in situ storage). Seeds contained in nylon mesh bags were placed in situ at the soil surface (stored surface) or buried to 5 cm (stored buried) until the first week of November, after which stored seeds were tested for germination. This date coincide with appropriate temperature and rainfall conditions for seed germination. Average minimum and

maximum temperatures during the storage period (June to October) were 29.7 and 44.1°C, respectively and average minimum and maximum relative humidity was 9.8 and 34.7%. There were no rainfall events during the in situ storage period. Five soil samples were collected randomly from each population at 10 cm soil depth then pooled together to form one composite sample. Soil pH and soil electrical conductivity (EC) were determined by the saturated paw method (AFNOR 1987). Kuwait is one of the world’s smallest countries in both land area (17,820 km2) and altitude (highest point being 306 m asl). Terrain is mostly flat and sandy. Climate is characterized by long, dry and hot summers in which temperatures can exceed 50°C, and with infrequent and unpredictable precipitation averaging 114 mm annually, concentrated during winter (Omar et al. 2007).

Population x Storage x Light Treatments consisted of three populations (Abdali, Liya and Salmi), three storage conditions (fresh, stored surface, stored buried) andDraft two photoperiod regimes (0 and 12 hours light per day, referred to as dark and light treatments, respectively). Fresh seeds were germinated within one week of collection, and stored seeds within a day of retrieval from in situ placement. Four 25-seed replicates were germinated per treatment. Seeds were germinated in a 9 cm Petri dish lined with one layer of Whatman No. 1 filter paper and kept moist with distilled water. Dishes were placed in incubators set at 12/12 hourly cycles of 20/25°C, in which the light treatment coincided with the higher temperature. Dishes in the dark treatment were wrapped in aluminum foil to prevent exposure to light. Germinated seeds were counted at the end of the 28-day trial, with germination defined as the emergence of a radicle by >1 mm.

Population x Pre-treatment and Seedling morphology Seeds from each population, which had been stored for one month at room temperature (20±2°C) were subjected to pre-sowing treatments to assess their effect on dormancy break. Pre-treatments included (i) soaking in distilled water for 12 and 24 hours, (ii) scarification with 100% sulfuric acid for 5, 10 and 15 minutes, and (iii) no pre-treatment. After exposure to sulfuric acid, seeds were rinsed five times in distilled water. Seed mass was recorded before and after pre-treatments to calculate water imbibition. Four 25-seed replicates for each pre-treatment and population were sown in plastic pots (8 cm in diameter and 11 cm in height) at 0.5 cm depth, using one pot per

replicate. The native soil (raw sand) was used to ﬁll the pots without adding any fertilizers and nutrients. The drainage outlet at the bottom of the pots was covered with nylon mesh in order to prevent the loss of sand while allowing drainage of excess water. Pots were placed in a greenhouse and the number of emerged seedlings was counted every second day for 28 days. Germination was considered to be complete at this point since the last seedling emerged on day 22. Pots were watered every second day using 50 ml of tap water. Air temperature and relative humidity were recorded using a Thermo-Hygrometer (Electronic Temperature Instrument Ltd, UK). The average temperature of the greenhouse was 27.8 °C (maximum) and 22.3 °C (minimum), whereas, the relative humidity varied from 64.6 % (maximum) to 44.8 % (minimum) respectively. After 90 days under same conditions as mentioned above, ten seedlings per population were randomly selected from pots of all pre-treatments, and pooled. Shoot and root lengths were measured with a ruler. Seedlings were separated into root and shoot components (stem and leaves), oven dried at 80°C to constant weight and the dried shoot and root biomass was recorded. Draft Data analysis Germination and emergence were evaluated using Generalized linear models (GLM) with binomial error structure and logit link function. Germination was evaluated as a function of storage condition, photoperiod, and maternal population. Emergence was analyzed as a function of the maternal population and the pre-sowing treatment with water and alternatively as a function of the maternal population and the pre-sowing treatment with acid. In all cases, the full GLM model was first fitted with all factors and interactions, and then simplified by dropping first the least significant interaction and then the least significant individual variable at each step. The comparison between models was based on the likelihood ratio test (LRT) until all the remaining terms were significant (Zuur et al. 2009). Seed mass, water imbibition and seedling morphology (seedling height, biomass, root:shoot biomass ratio) were evaluated as a function of the maternal population using one way-ANOVA and Tukey HSD test as post-hoc. Data was transformed to achieve normality and homogeneity of variance, using log (imbibition percentage, seedling height) and square root (biomass, root:shoot biomass ratio) transformations. All analysis were completed using R 3.5.2 (R Core Team 2018).

Results Seed mass and water imbibition Seeds from Abdali (0.75 ± 0.02 g) and Liya (0.72 ± 0.02 g) were significantly heavier than seeds from Salmi (0.68 ± 0.04 g; P = 0.001). Water imbibition of freshly collected seeds varied among populations (P = 0.0403), the higher imbibitions were recorded in seeds from Liya and Salmi 1.62 ± 0.44 % and 1.16 ± 0.29 % while the imbibition in seeds from Abdali was only of 0.71 ± 0.21 %. The acid scarification pretreatment improved the seed imbibition (P < 0.001), the 10 min (27.87 ± 17.50 % of seed weight) and 15 min (41.81 ± 29.40 %) leaded to higher imbibition than the 5 min pretreatment (22.15 ± 15.46 %).

Population x Storage x Light Germination under laboratory conditions was strongly influenced by storage conditions, the population of origin, and their interaction (Table 2, Figure 1). Germination percentage averaged 9.67, 12.67 and 19.67% in Abdali, SalmiDraft and Liya, respectively. The germination of freshly collected, stored at soil surface and stored buried was 5.7, 14.3 and 20.0%, respectively. The germination recorded under the combination of treatments ranged from 0% in fresh seeds from Abdali, to 26.5% in seeds stored buried from Liya (Figure 1). The interaction Storage x Population indicates that the treatments were more effective for some populations than others, e.g. both in situ storage treatments improved germination of Abdali and Salmi seeds, but were ineffective on Liya seeds. Germination of A. sieberi seeds was unaffected by light exposure.

Population x Pre-treatment and Seedling morphology Seedling emergence under greenhouse conditions was strongly influenced by population of origin, acid scarification, and their interaction (Table 2). Pretreatment by soaking in water had no effect on emergence. Emergence ranged from 21.5% in seeds of Salmi to 46.3% in seeds from Liya, and 11.0% in the pre-treatment control to 57.7% in the 15-minute sulfuric acid treatment, being the longest exposure to acid scarification. The interaction between population and scarification was also significant, with the highest emergence (72.0%) recorded in seeds from Liya exposed for 15 minutes to acid and the lowest emergence (0.0%) recorded in seeds from Abdali under the control treatment (Figure 2). Abdali seeds had lowest emergence with control or soaking, thus need the acid scarification to break the dormancy.

Seedling growth Among the seedling growth variables analyzed, only total biomass (F=6.59, P=0.005) and the root:shoot ratio (F=4.941, P=0.015) varied with the maternal population, but the seedling height was similar in all populations (F=0.952, P = 0.399). Biomass was highest in seedlings produced by seeds from Salmi while the root:shoot biomass ratio was highest in seedlings produced by seeds from Abdali, and both variables were lowest in seedlings produced by seeds from Liya (Figure 3).

Discussion Seed dormancy and germination can vary greatly among populations (Bhatt et al. 2019b, c), thus population selection can greatly improve germination. Germination responses varied significantly among the studied populations of A. sieberi despite being geographically close. Detected differences were likely caused by local environmental conditions but may also have been due to a differentiated gene pool. Draft In the present study, the freshly collected seeds of A. sieberi exhibited physical dormancy. This is commonly exhibited by members of the Fabaceae family (Baskin and Baskin 2014; Bhatt et al. 2016b, c) imposed by a water-impermeable layer of palisade cells in the seed coat (Smýkal et al. 2014; Janská et al. 2019). Seeds of A. sieberi mature in summer (June) when temperature is high and the possibility of rain is extremely low, then express dormancy until summer has passed. Physical dormancy is a common bet-hedging strategy in temporally variable environments (Venable 2007; Rubio de Casas et al. 2017) as found in Arabian deserts (Omar et al. 2007; Bhatt et al. 2016d).Intra-population variation of seed dormancy and germination has been described for plant species mainly in unpredictable, arid or water limited environments (Andersson and Milberg 1998; Gutterman 2000). (Baloch et al. 2001). In the present study, physical dormancy imposed by the presence of an impermeable seed coat varies between the populations. The seeds collected in Liya had more seeds with a permeable seed coat, therefore exhibiting higher germinability when freshly collected. Variable seed coat permeability among populations was observed for other species with physical dormancy such as Senna auriculata (Jaganathan et al. 2019). The differences in water imbibition and germination from seeds collected in different populations could be due to seed coat structure (such as hardseededness and/or lens) which can affect seed coat permeability. Variation in climatic factors, soil moisture and nutrient content among populations influences seed

size, dormancy and germination (Tremayne and Richards 2000; Santamaría et al. 2003; Baskin and Baskin 2014; Hudson et al. 2015; Li and Li 2015). In this study we collected seeds from populations exposed to similar climatic conditions. Variation in resource availability (water, nutrients) and in population gene pools may have caused the observed variation. In previous studies, competition and edaphic factors were more influential on seed dormancy and germination at smaller spatial scales and climatic factors were more influential at larger scales (Snaydon and Davies 1982; Macel et al. 2007; Becker et al. 2008; Carta et al. 2016). Further research is needed to correctly evaluate the role of genetic diversity and maternal environment. The microclimate imposed variable seed coat permeability and seed dormancy might be an adaptation strategy for spread germination in time, especially under harsh environmental conditions. The interpopulation variability in dormancy and germination exhibited by A. sieberi in this study could confer the species with an advantage to surviving in desert habitats by reducing the risk of synchronizing germination (Santo et al. 2015). Seed mass of A. sieberi varied among populationsDraft but was unrelated to germination percentage. The seed size is a trait that may reflect on the germination performance; however, such influence was not recorded in this work neither in other reports (Castro et al. 2006; Liu et al. 2007; Pivatto et al. 2014). It is possible that other seed characteristics such as endosperm quality or differences in the composition of the seed coat played a more important role than seed mass in affecting germination (Schmitt et al. 1992; Hereford and Moriuchi 2005). Usually, seed coat structure is determined by the maternal environment conditions (Hudson et al. 2015; Jaganathan 2016). Water soaking is well known pretreatment for breaking physical dormancy (El-Juhany et al. 2009) that proved to be ineffective in the study for A. sieberi seeds. These findings are in accordance with previous studies on legume species (Sy et al. 2001). However, pretreatment with acid, especially longer pre-treatment with sulphuric acid increase germination, though excessive exposure will damage the seed (Nourmohammadi et al. 2019). Acid pre-treatment softens the seed coat, providing permeability to water and oxygen and/or breaking the mechanical barrier for radicle protrusion (Baskin and Baskin 2014). Our findings in A. sieberi show the need for pretreatment for breaking dormancy to obtain high germination, as pointed out for other species of the same genus such as: A. arpilobus (Long et al. 2012), A. sinicus (Kim et al. 2008), A. cicer (Acharya et al. 2006; Statwick 2016), A. podolobus and A. adscendens (Tavili et al. 2014). The observed interpopulation variation in the response to acid scarification may be related to

differences in seed coat thicknesses (Richard et al. 2018) or lens structures (Hu et al. 2009) however, it is unclear which trait is the responsible in the case of A. sieberi. Under natural conditions, the physical dormancy of legumes may be broken by (i) constant low or high temperature, (ii) passage through the digestive tracts of animals and (iii) temperature fluctuations by the opening of the lens (Jayasuriya et al. 2009; Baskin and Baskin 2014; Smykal et al. 2014, Jaganathan et al. 2018). Temperature fluctuations are greatest at the soil surface; hence we might expect unburied seeds to germinate more readily (Baskin and Baskin 1989). However, we observed slightly greater germination in buried seeds, which may reflect adaptation to sandy habitats. Storage increased germination percentage, whether stored at 0 or 5 cm depth, but acid scarification was most effective at increasing both germination percentage and synchrony. Seedling vigor can influence growth, survival, and reproduction at later stages of the plant’s life. Maternal environment usually influences seedling traits via seed traits (Ortmans et al. 2016). Measures of seedling growth (total biomass and root:shoot ratio) varied among populations of the present study, such that seedlings producedDraft from Salmi seeds grew faster. These traits appeared unrelated to seed mass because the same population presented the smallest seeds. This seedling trait variation may be due to maternal environment and its effects on endosperm quality and nutrient concentration but further studies would be needed to clarify this point. However, these results should be carefully interpreted because age of the measured seedlings varied with germination timing. The evidence collected here indicates that the identification of seed sources of A. sieberi for restoration purposes cannot be based solely on seed size but should also consider variation in seedlings performance. A. sieberi is a species with potential for restoration projects in the Arabian desert that exhibits physical dormancy and persistence in a soil seed bank. Dormancy can be partially alleviated by in situ storage, especially when buried. Dormancy can be broken via sulfuric acid scarification for 15 minutes, increasing germination to 47%. Significant inter-population variation in A. sieberi seed traits and performance were observed in this study, with implications for effective germplasm selection for use in restoration projects. Thus, seeds from different populations should be tested prior to use in large scale restoration and rehabilitation projects.

Acknowledgements This work was support by Kuwait Institute for Scientific Research (KISR). MMC was supported by the FONCyT project PICT-2017-2245.

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Table 1: Astragalus sieberi population characteristics and habitat details.

Distance to Altitude EC Location Associated species Coordinates Habitat pH Kuwait Bay (m asl) (dS/m) (km)

Haloxylon salicornicum, 30° 2' 39.31'' N; Abdali Convolvulus cephalopodus, Sand 103 7.9 61 47° 40' 0.36'' E 2.72 Fagonia glutinosa

Fagonia bruguieri, 29° 36' 45.38 N; Liya Anastatica hierochuntica, Gravel 90 7.4 27 47° 34' 28.45'' E 1.97 Cornulaca monacantha

Stipagrostis plumosa, 29° 5' 25.55'' N; Salmi Plantago boissieri, Reseda Sand 112 86 46° 52' 37.80'' E 7.6 2.01 arabica Draft

Table 2: Influences of maternal population (Abdali, Liya, Salmi), storage (fresh, stored surface, stored buried) and light during germination (0, 12 hours per day) on germination, and pre-sowing treatments (acid scarification, soaking) on emergence of Astragalus sieberi. The Akaike information criterion (AIC), Likelihood Ratio Test (LRT) and chi-squared test probability (Pr (>Chi2)) informs the significance of each factor or interaction to the model performance. Degrees Pr Factor of Deviance AIC LRT (>Chi2) freedom Germination Storage 2 1436.4 1442.4 72.724 2.2 x10-16 Population 2 1397.3 1403.3 33.644 4.9 x10-8 Light 1 1328.0 1346.0 3.037 n.s. Storage x Light 2 1325.0 1349.0 0.006 n.s. Population x Light 2 1325.0 1345.0 0.022 n.s. Storage x Population 4 1363.7 1373.7 35.607 3.5 x10-7 Storage x Population x Light Draft 4 1325.0 1353.0 0.141 n.s.

Emergence Population 2 1346.7 1354.7 65.768 5.2 x10-15 Acid scarification 3 1473.1 1479.1 192.25 2.2 x10-16 Population x Acid scarification 6 1280.9 1292.9 29.668 4.5 x10-5

Population 2 610.58 616.58 114.777 2.2 x10-16 Soaking 2 496.37 502.37 0.564 n.s. Population x Soaking 4 495.81 505.81 4.230 n.s. n.s.: not significant (P > 0.05)

Figure 1: Germination percentage of seeds sourced from three populations (Abdali, Lia and Salmi) and germinated immediately (Fresh) after collection or after in situ storage for five months at soil surface (stored surface) or buried to 5 cm (stored buried).

Figure 2: Emergence percentage of seeds after pre-treatments of soaking (12 and 24 hours) and sulfuric acid scarification (5, 10 and 15 min).

Figure 3: Dry total biomass and biomass allocation of the A. sieberi seedlings from distinct seeds population origins (Addali, Liya and Salmi). The error bars indicate standard errors. Uppercase letters = Dry total biomass significant difference (P<0.05); Lowercase letters = Root:Shoot ratio significant difference (P<0.05). Draft

Draft

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Draft

Figure 2: Emergence percentage of seeds after pre-treatments of soaking (12 and 24 hours) and sulfuric acid scarification (5, 10 and 15 min).

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Figure 3: Dry total biomass and biomass allocationDraft of the A. sieberi seedlings from distinct seeds population origins (Addali, Liya and Salmi). The error bars indicate standard errors. Uppercase letters = Dry total biomass significant difference (P<0.05); Lowercase letters = Root:Shoot ratio significant difference (P<0.05).

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