Impaginato 61 Adv. Hort. Sci., 2021 35(1): 61­72 DOI: 10.36253/ahsc­9212 Clonal propagation of Argania spinosa (L.) skeels: effects of leaf retention, substrate and cutting diameter A. Benbya 1, S. Cherkaoui 2, F. Gaboun 1, O. Chlyah 2, F. Delporte 3, M. Mdarhri Alaoui 1 1 Biotechnology Unit, Regional Center of Agricultural Research of Rabat, National Institute of Agronomy Research of Morocco (INRA), P.O. Box 6570, Rabat Instituts, Rabat, Morocco. 2 Physiology and Biotechnology Laboratory, Department of Biology, Faculty of Sciences (FSR), University Mohammed V of Rabat, P.O. Box 1014, Rabat, Morocco. 3 Department of Life Sciences, Bioengineering Unit, Walloon Agricultural Research Center (CRA‐W), Chaussée de Charleroi 234, P.O. Box 5030, Gembloux, Belgium. Key words: Argania tree, rooting, semi­hardwood, vegetative propagation. Abbreviations: IBA= Indole­3­Butyric Acid, FS= Fine sand substrate, PM= Peat moss substrate, FS/PM= mixture of fine sand and peat moss. Abstract: To evaluate the rooting ability and growth performance in semi­hard­ wood cuttings of Argania spinosa under non­mist greenhouse conditions, our experimentation was conducted with three cutting diameters (0.1­0.3, 0.3­0.6 and 0.6­0.9 cm), four leaf retention treatments (leafless, 2, 4 and 8 leaves) and three different rooting substrates (fine sand, peat moss, a 1:1 mixture of fine sand/peat moss). Significant effects of cuttings diameter, leaf retention and rooting substrate on sprouting, rooting and survival ability from A. spinosa semi­hardwood cuttings were observed. Among all diameters tested, a diame­ ter of (0.3­0.6 cm) showed maximum rooting and survival capacity, while cut­ tings with a diameter of 0.6­0.9 cm resulted in the greatest sprouting ability. Successful vegetative propagation was restricted to leafy stem cuttings. Moreover, it was observed that the highest rooting ability was reached in cut­ tings planted in fine­sand substrate. However, the highest sprouting ratio (85.0%) and survival rate (92.5%) were achieved in a mixture of sand and peat moss. Thus, argan trees vegetative propagation could be most effectively achieved using semi­hardwood cuttings with a 0.4 cm diameter and 4 leaves, planted in a fine sand substrate during the root initiation period and grown in a mixture of fine sand and peat moss for hardening. 1. Introduction The Argan tree [Argania spinosa (L.) Skeels] is the only species repre­ senting the tropical Sapotaceae family in Morocco (Emberger, 1925). It is (*) Corresponding author: meriem.malaoui@gmail.com Citation: BENBYA A., CHERKAOUI S., GABOUN F., CHLYAH O., DELPORTE F., MDARHRI ALAOUI M., 2021 ­ Clonal propagation of Argania spinosa (L.) skeels: effects of leaf retention, substrate and cutting diameter. ­ Adv. Hort. Sci., 35(1): 61­72 Copyright: © 2021 Benbya A., Cherkaoui S., Gaboun F., Chlyah O., Delporte F., Mdarhri Alaoui M. This is an open access, peer reviewed article published by Firenze University Press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. Received for publication 3 September 2020 Accepted for publication 27 January 2021 AHS Advances in Horticultural Science https://doi.org/10.36253/ahsc-9212 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ Adv. Hort. Sci., 2021 35(1): 61­72 62 a prodigious perennial thermophilic and xerophilic thorny species (Ehrig, 1974; Morton and Voss, 1987). The argan tree can be found in a wide array of envi­ ronments where soils are ranging from heavy clay to poor and rocky desert soils, it grows in altitudinal ranges extending from sea level (along the Atlantic coast) up to 1500 m (Msanda, 1993). Moreover, it has the ability to survive in very dry conditions at very hot temperatures up to 50°C and low rainfall levels between 150 and 400 mm/year, shedding foliage and remaining in a state of dormancy for sev­ eral years during prolonged drought (M’Hirit et al., 1998). Recently, A. spinosa has attracted worldwide attention as a source of highly valued oil (Charrouf and Guillaume, 2008). It’s becoming the most expen­ sive edible oil in the world, as the present supplies does not meet demand (Lybbert et al., 2011). Despite the ecological value and local economic importance of this species, its forest density has been divided by two during the twentieth century and the natural regeneration rate is very low and is worsened by cli­ mate changes and the negative impact of over e x p l o i t a ti o n ( K e n n y a n d D e Z b o r o w s k i , 2 0 0 7 ) . Besides, its cultivation is not sufficiently developed. In part, this is due to excessive utilization of kernels for oil production which hindered the conventional propagation through seed germination (Nouaim et al., 2002); on the other hand, it is due to the slow progress in propagation methods, mainly because of the generally low capacity of this species to form adventitious roots (Nouaim et al., 2002; Justamante et al., 2017). Therefore, research is required to devel­ op efficient vegetative propagation techniques for the production of more planting material and conser­ vation of this species. The rooting efficiency and growth performance of cuttings in many species have been found to be dependent of the cutting thickness (Foster et al., 2000). The rooting effectiveness in function of cuttings diameter could be explained by different factors (Foster et al., 2000). The variation observed among different cutting diameters could be influenced by physiological conditions (nutrients, car­ bohydrates, auxin, phenolics, lignification etc.), their position within the branch or their ontogenetic aging (Kaul, 2008; Wendling et al., 2014). Moreover, leaf retention was also reported to influence rooting behaviour of cuttings (Leakey and Coutts, 1989). The proportion of leaves retained by the cuttings during propagation is related to the percentage of cuttings t h a t f o r m a d v e n ti ti o u s r o o t s ( T c h o u n d j e u a n d Leakey, 1996; Mesén et al., 1997a). Another factor which creates a suitable environment for rooting is substrate (Tchinda et al., 2013). The importance of substrate type on the rooting capacity of cuttings is w i d e l y r e c o g n i z e d a s t h e s t a r ti n g p o i n t t o b e addressed for a successful vegetative propagation using stem cuttings (Hartmann et al., 2002; Leakey, 2004). Cuttings of many species root successfully in a variety of 50 propagation substrates, but the rooting performance may be greatly influenced by the kind of substrate which is linked to the hydromorphic or xeromorphic status of the species (Hartmann et al., 2002; Leakey, 2004). The present study aims to eval­ uate the effect of three independent factors (cuttings diameter, leaf retention and substrate) on sprouting, adventitious rooting and survival ability of Argania spinosa semi­hardwood cuttings to develop an effi­ cient technique for achieving large­scale production of superior clonal stock plants and conservation of genetic resources. It is a continuation of our previous research on Argan vegetative propagation, which concerned the nutrient solution influence (Benbya et al., 2018), and the effects of auxin type and genotype (Benbya et al., 2019) on the multiplication process. 2. Materials and Methods Experimental site and plant materials Vegetative propagation experiment was carried out during the vegetative period of the tree (May to September) at non­mist greenhouse of the biotech­ nology unit of the Regional Center of Agricultural Research of Rabat (INRA­Morocco), under natural photoperiod and a mean temperature of 32±2°C. The semi­hardwood cuttings of A. spinosa were collected from selected adult mature trees, naturally growing at the Arboretum of Oued Cherat in the province of Bouznika, Morocco (33° 81’ 96” N; 7° 11’ 03” W; 45 m Altitude), which is located within 2 km of the Moroccan Atlantic coast and with an average annual rainfall of 460 mm/yr. Preparation of cuttings and experimental establish‐ ment Plant material was placed in cold dark storage at 4°C for 48h before planting. Then, semi­hardwood cuttings were screened for uniform length (10±0.5 cm) and were divided into three groups by diameter: 0.1­0.3 cm with 10 to12 nodes; 0.3­0.6 cm with 8 to 10 nodes and 0.6­0.9 cm with 6 to 8 nodes using cali­ brated electronic digital Vernier caliper. Given cut­ tings were prepared as multiple nodes, without Benbya et al. ‐ Clonal propagation of Argania spinosa 63 leaves (leafless); or with two leaves, four leaves or eight leaves. The tip of shoot was removed and the lower leaves of each cutting were removed. Before planting, cuttings were treated with aqueous solution of 0.2% (w/v) fungicide (Dithane 750 g/kg Mancozeb) for 10 minutes and subsequently washed with dis­ tilled water to remove excess fungicide. Then, the basal end (5.0 cm) of the cuttings was soaked for 5 min in a freshly prepared aqueous solution of auxins (17.12 mM of IBA). Prepared cuttings were immedi­ ately inserted in different polyethylene (PE) pots (1000 cc) containing three different substrates: steril­ ized sieved fine sand (FS) (pH of 7.3, pH­KCl method; water retention of 160 ml/l; organic matter content of 0.2%, Walkley & Black method); peat moss (PM) pH of 6; water retention of 800 ml/l; organic matter content of 20%); mixture of fine sand and peat moss (1:1 v/v) (FS/PM). The rooting period for the experi­ ment was run for 12 weeks, and then cuttings were uprooted carefully without harming root with run­ ning water. Successfully rooted cuttings (at least one root with a length of at least 1 cm long) were trans­ planted into polyethylene pots (4500 cc) and were maintained for hardening at a spacing of 20 cm × 20 cm for 48 weeks. Given cuttings were regularly watered twice a week with tap water during the first twelve weeks; after a Hoagland & Arnon nutrient solution (Hoagland and Arnon, 1950) was used as irri­ gation source. Data collection The morphological characteristics assessments were realized after 12 weeks for the number of sprouts (NS), length of the longest sprout (SL, cm), sprouting percentage (SP, %), number of roots (NR), length of the longest root (RL, cm), and rooting per­ centage (RP, %), whereas the survival rate success (SR, %) was recorded at week 48 of the hardening phase. Experimental design and treatments The experiment was conducted in Randomized Complete Block Design (RCBD) with four blocks and three independent variables (cutting size, leaf reten­ tion and substrate) (Fig. 1A). For each treatment there were 32 cuttings (eight per block), randomly allocated in groups of two cuttings (forming four Fig. 1 ­ Clonal propagation of Argania spinosa semi hardwood cuttings. (A) Cuttings planted in peat moss substrate according to a ran­ domized complete block design (RCBD) under non mist greenhouse conditions (24 weeks). (B) Rooted plant cutting set in sand substrate (2 years). (C) Well rooted plant cutting set in a mixture of sand and peat moss (1:1 v/v) substrate (3 years). (D) Cutting with developed root primordia from fine sand substrate (12 weeks). (E) Vigorous adventitious roots of semi hardwood cuttings grown in a mixture of sand and peat moss substrate (2 years). (F) Argan plantlet in a mixture of fine sand and peat moss substra­ te under non­mist greenhouse conditions. (3 years). Scale bars: 10 mm. Adv. Hort. Sci., 2021 35(1): 61­72 64 3. Results Effect of the cutting diameter, leaf retention and sub‐ strate on number of sprouts and sprouts length of the Argania spinosa cuttings Cuttings with 0.6­0.9 cm diameter and four leaves set in FS/PM substrate showed the greater mean number of sprouts and the longest sprout of cuttings (1.81±0.10 cm and 17.25±0.96 cm respectively), while the lowest number of sprouts and the shortest sprout length occurred for cuttings with 0.3­0.6 cm diameter and eight leaves planted in FS substrate (1.06±0.09 cm and 8.19±1.11 cm, respectively). However, thinner cuttings (0.1­0.3 cm) and leafless cuttings for all diameters failed to produce any sprouts and are therefore not shown on the graphs (Fig. 2). The mean number of sprouts per rooted cutting showed a progressive increase with increasing cut­ ting diameter (r= 0.56***). A similar trend was also observed in sprouts length per rooted cutting (Table 1). Results showed that PM substrate as well as experimental units for thirty­six treatment combina­ tions). The experiment was established with three cutting diameters (0.1­0.3 cm; 0.3­0.6 cm and 0.6­0.9 cm), four leaf number treatments (leafless, 2, 4, and 8 leaves) and three different substrates (FS, PM and FS/PM). Statistical analysis The main effects and interactions of cutting diam­ eter, leaf retention and substrate were determined using a general linear model (GLM) procedure in SAS program version 9.1 (SAS Institute, Cary, NC) for all the evaluated parameters. The differences between the treatments were tested using Duncan’s Multiple Range Test (DMRT) with at least 95% level of statisti­ cal reliance, and as a result, homogenous groups were acquired and interpreted. Collected data were subjected to simple linear regression analysis. All data were reported as means ± standard error (SE). Sprouting, rooting and survival percentage data were arcsine transformed to ensure normal distribution and homogeneity of variances. Fig. 2 ­ Effects of cutting diameters, four leaf number treatments (leafless, 2, 4, and 8 leaves) and three different substrates (FS, PM and FS/PM) on mean values of number of sprouts (NS) and length of the longest sprout (SL) per rooted cutting of Argania spinosa. Values followed by a common letter within each column are not significantly different at P<0.05 using the Duncan’s Multiple Range Test (DMRT). A value represents mean ± standard error of means (n = 32). Table 1 ­ Simple linear regression for cutting diameter and number of sprouts or sprout length (cm) of Argania spinosa cuttings ***= Highly significant (P<0.001). n = 32. Dependent variable (Y) Independent variable (X) Correlation coefficient (r) Regression equation P­Value Number of sprouts Cutting diameter (cm) 0.56 Y= 0.59 X ­ 0.44 0.000 *** Sprout length (cm) Cutting diameter (cm) 0.58 Y= 5.42 X ­ 4.32 0.000 *** Benbya et al. ‐ Clonal propagation of Argania spinosa 65 FS/PM substrate exhibited higher means number of sprouts compared to FS substrate. However, the FS/PM substrate showed the longest sprouts, fol­ lowed with PM substrate, while FS exhibited the shortest sprout per cutting (Table 1). Over all the combined data for leaf retention treatments, it showed that with cutting diameter increase, there was an increase in the number of sprouts and sprout length (r=0.58***) (Table 1). Indeed, cuttings with 0.6­0.9 cm diameter showed the greater mean num­ ber of sprouts and sprout length of cuttings, followed by cuttings with 0.3­0.6 cm. Among leaf retention tested, cuttings with 4 leaves produced the longest sprouts compared with 2 and 8 leaves (Table 2). Based on the analysis of variance (ANOVA), cut­ ting diameter and substrate had a significant effect (P<0.05) on the mean number of sprouts, while leaf retention was not found significant on the number of sprouts per cuttings (Table 3). Moreover, cutting diameter, leaf retention and substrate had a highly significant effect (P<0.001) on the sprout length, whereas leaf retention had only significant effect on sprout length (Table 3). Neither mean number of the sprouts per cuttings nor the sprout length was signifi­ cantly (P > 0.05) affected by the two­way and three­ way interaction between the three factors tested (Table 3). Effect of the cutting diameter, leaf retention and sub‐ strate on number of roots and length of the longest root of the A. spinosa cuttings The highest number of roots and root length were observed in cuttings with 4 leaves, followed by 2 leaves though not significantly, while lowest number Table 2 ­ Main effects of cutting diameter, leaf retention and substrate on the number of sprouts and length of the longest sprout (cm) of Argania spinosa cuttings Values followed by the same letter(s) are not significantly diffe­ rent at P<0.05 using the Duncan’s Multiple Range Test (DMRT). A value represents mean ± standard error of means (n = 32). Treatments Sprouts number Sprout length Cutting diameter (cm) 0.1­0.3 ­ ­ 0.3­0.6 1.40 ± 0.05 b 11.65 ± 0.46 b 0.6­0.9 1.58 ± 0.05 a 14.46 ± 0.44 a Leaf retention Leafless ­ ­ 2 1.48 ± 0.06 a 13.34 ± 0.58 ab 4 1.56 ± 0.05 a 13.98 ± 0.55 a 8 1.43 ± 0.06 a 11.85 ± 0.56 b Substrate Fine sand (FS) 1.27 ± 0.07 b 11.30 ± 0.63 c Peat moss (PM) 1.58 ± 0.06 a 13.13 ± 0.52 b Mixture of FS and PM 1.62 ± 0.05 a 14.74 ± 0.51 a Table 3 ­ Analysis of variance (ANOVA) for the effects of cutting diameter, leaf retention and substrate and their interactions on number of sprouts and length of the longest sprout cm per Argania spinosa cuttings NS= Not significant (P>0.05); *= Significant (P<0.05); ***= Highly significant (P<0.001). df= degrees of freedom. ×= Interaction between treatments. Source of variance Dependent variable No. of sprouts Sprout length df F­value P­value df F­value P­value Cutting diameter 2 7.36 0.0071 * 2 20.36 <0.0001 *** Leaf retention 3 1.30 0.2736 NS 3 4.08 0.0179 * Substrate 2 10.08 <0.0001 *** 2 10.15 <0.0001 *** Cutting diameter × Leaf retention 6 0.03 0.9702 NS 6 0.9559 0.9559 NS Cutting diameter × Substrate 4 0.39 0.6750 NS 4 0.01 0.9896 NS Leaf retention × Substrate 6 0.35 0.8451 NS 6 0.23 0.9232 NS Cutting diameter × Leaf retention × Substrate 12 0.20 0.9399 NS 12 0.03 0.9983 NS Block 1 0.10 0.7534 NS 1 2.43 0.1200 NS of roots and root length were mostly recorded in cut­ tings with 8 leaves. Best response in term of number of roots was obtained when cuttings were set in FS substrate with four leaves and diameters of 0.3­0.6 cm for which the mean number of adventitious roots was 45.06±1.14 (Fig. 1E), followed by cuttings with four leaves set in FS substrate with diameters of 0.6­ 0.9 cm (44.130±1.21). The greatest value of root length was recorded when cuttings were grown in FS substrate with 0.3­0.6 cm thickness and with four leaves (33.63±2.12), while cuttings set in a PM sub­ cutting, while leaf retention had only a significant effect (P<0.05). Root length was significantly (P<0.05) affected by the interaction between cutting diameter and substrate, while the other interactions between the three factors studied were not significant on the number of roots and root length of cuttings (Table 5). 66 Adv. Hort. Sci., 2021 35(1): 61­72 strate didn’t exceed 23 cm (Fig. 3; Fig. 1E). However, the incidence of necrosis was the greatest among thinner cuttings (0.1­0.3 cm) and leafless cuttings, indeed these cuttings failed to produce any adventi­ tious roots and are therefore not on the graphs (Fig. 3). The mean number of roots was dependent on type of substrate; cuttings grown in FS and FS/PM substrates rooted significantly better, whereas the lowest mean number of adventitious roots per cut­ tings was recorded in cuttings propagated in PM sub­ strate (Table 4). Over the entire experimental period, the three substrates showed pronounced differences in the root length: cuttings planted in FS substrate were found to produce significantly longest adventi­ tious roots, followed by FS/PM substrate then cut­ tings set in PM substrate (Table 4). Among the cutting diameters studied, (0.3­0.6) cm showed the highest number of roots and the longest root, followed by the cutting diameter (0.6­0.9) cm, while cuttings with (0.1­0.3) cm were the least performing and failed to produce roots (Table 4). Regarding the ANOVA test values, the effects of cutting diameter, leaf retention and substrate were found highly significant (P<0.001) on the number of adventitious roots formed per cutting (Table 5). Moreover, cutting diameter and substrate had also a highly significant effect (P<0.001) on root length of Table 4 ­ Main effects of cutting diameter, leaf retention and substrate on the number of roots and the length of the longest root (cm) of Argania spinosa cuttings Values followed by the same letter(s) are not significantly diffe­ rent at P<0.05 using the Duncan’s Multiple Range Test (DMRT). A value represents mean ± standard error of means (n = 32). Treatments Roots number Root length Cutting diameter (cm) 0.1­0.3 ­ ­ 0.3­0.6 38.38 ± 0.74 a 27.24 ± 0.73 a 0.6­0.9 35.61 ± 0.90 b 24.42 ±0.69 b Leaf retention Leafless ­ ­ 2 36.95 ± 0.94 b 25.65 ± 0.93 ab 4 38.97 ± 1.04 a 27.47 ± 0.83 a 8 35.07 ± 1.03 b 24.36 ± 0.85 b Substrate Fine sand (FS) 42.07 ± 0.66 a 30.76 ± 0.76 a Peat moss (PM) 27.44 ± 0.82 b 18.25 ± 0.63 c Mixture of FS and PM 41.48 ± 0.75 a 28.47 ± 0.65 b Fig. 3 ­ Effects of cutting diameters, four leaf number (leafless, 2, 4, and 8 leaves) and three different substrates (FS, PM and FS/ PM) on mean values of number of adventitious roots (NR) and length of the longest root cm (RL) per rooted cutting of Argania spinosa. Values followed by a common letter within each column are not significantly different at P < 0.05 using the Duncan’s Multiple Range Test (DMRT). A value represents mean ± standard error of mean (n = 32). Benbya et al. ‐ Clonal propagation of Argania spinosa 67 Effect of the cutting diameter, leaf retention and sub‐ strate on sprouting percentage, rooting percentage and survival rate of the Argania spinosa cuttings Although the thinner cuttings gave no sprouts, among the other different treatments, the highest sprouting percentage was 85.00 ± 3.76% when cut­ tings with 4 leaves and 0.6­0.9 cm diameter were planted in FS/PM substrate, while cuttings with 8 Table 5 ­ Analysis of variance (ANOVA) for the effects of cutting diameter, leaf retention and substrate and their interactions on the number of adventitious roots and the length of the longest root (cm) of Argania spinosa cuttings NS= Not significant (P>0.05); *= Significant (P<0.05); ***= Highly significant (P<0.001). df= degrees of freedom. ×= Interaction between treatments. Source of variance Dependent variable No. of roots Root length df F­value P­value df F­value P­value Cutting diameter 2 11.27 0.0009 *** 2 13.29 0.0003 *** Leaf retention 3 7.43 0.0007 *** 3 5.42 0.0049 * Substrate 2 134.30 <0.0001 *** 2 98.88 <0.0001 *** Cutting diameter × Leaf retention 6 0.18 0.8318 NS 6 0.46 0.6345 NS Cutting diameter × Substrate 4 5.81 0.0034 * 4 0.11 0.8990 NS Leaf retention × Substrate 6 0.72 0.5809 NS 6 0.30 0.8766 NS Cutting diameter × Leaf retention × Substrate 12 0.14 0.9657 NS 12 0.58 0.6775 NS Block 1 2.46 0.1178 NS 1 0.26 0.6133 NS Fig. 4 ­ Effects of cutting diameters, four leaf number treatments (leafless, 2, 4, and 8 leaves) and three different substrates (FS, PM and FS/PM) on mean values of sprouting percentage (SP), rooting percentage (RP), and survival rate (SR) of Argania spinosa cuttings. Values followed by a common letter within each column are not significantly different at P<0.05 using the Duncan’s Multiple Range Test (DMRT). A value represents mean ± standard error of mean (n = 32). leaves and 0.3­0.6 cm diameter propagated in FS sub­ strate gave the poorest sprouting percentage (61.50± 5.68%). However, leafless cuttings performed least and failed to produce any sprouts (Fig. 4). The pro­ portion of cuttings forming new shoots during growth varied between the three substrates. Cuttings plant­ ed in PM and FS/PM substrates were the most suc­ cessful in sprouting, while cuttings propagated in FS The data revealed that the substrate caused sig­ nificant (P < 0.05) variations in sprouting, rooting and survival percentage of the rooted cuttings, while cut­ ting diameter and leaf retention were not significant on the survival rate (Table 7). However, there was no significant effect (P>0.05) of treatment interactions between the three independent factors on the three parameters studied. 4. Discussion and Conclusions The present investigation revealed that cutting diameter (0.1­0.3, 0.3­0.6 or 0.6­0.9 cm), leaf reten­ tion (0, 2, 4 or 8 leaves) and substrate (FS, PM or FS/PM) were found to be important factors for suc­ cessful adventitious rooting, sprouting and survival ability of Argania spinosa semi­hardwood cuttings. This experiment on A. spinosa cuttings concerning cutting diameter indicated a significant effect on the sprouting, rooting and survival performances. Among the different cutting diameters tested, cuttings with 0.3­0.6 cm diameter tended to root better and devel­ op more rooting capacity compared with thinner diameter (0.1­0.3 cm), which failed to produce any sprouts or roots. Moreover, larger cuttings (0.6­0.9 cm) showed the best sprouting ability but did not result in any significant improvement in root growth and development. The greatest rooting potential of cuttings with 0.3­0.6 cm diameter could be due to their good storage capacity of carbohydrates and Adv. Hort. Sci., 2021 35(1): 61­72 68 substrate showed the lowest sprouting ability (Table 6). Results showed that cuttings with 0.3­0.6 cm diameter with two and four leaves set in a FS sub­ strate showed maximum rooting: (65.00 ± 5.15% and 65.00 ± 5.87% respectively). Compared with the cut­ tings of 0.1­0.3 cm thickness and leafless cuttings which failed to root, all cutting diameters and leaf retention treatments enhanced the rooting percent­ age. Moreover, percentage of cuttings developing adventitious roots decreased when the leaf number exceeds 4 leaves (Fig. 4). The maximum rooting rates were obtained for cuttings grown in FS substrate, fol­ lowed by FS/PM substrate, while the cuttings grown on PM substrate showed the lowest rooting capacity (Table 6). The survival rate ranged from 57.00 ± 4.53% for plantlets with 0.6­0.9 cm diameter and 8 leaves grown in FS substrate, to 92.50 ± 1.94% for plantlets derived from cuttings with 0.3­0.6 cm diam­ eter and 4 leaves potted on substrate containing a mixture of fine sand and peat moss. However, cutting mortality was the greatest in thinner cuttings (0.1­ 0.3) cm and leafless cuttings which showed 100% mortality rate within forty­eight weeks (Fig. 4). The rooted plantlets were successfully hardened and acclimatized under non­mist greenhouse conditions (Table 6). These plants showed the best survival rate and performed better growth and development when they were grown in a FS/PM substrate com­ pared with cuttings raised in PM and FS substrates (Fig. 1F). Table 6 ­ Main effects of cutting diameter, leaf retention and substrate on sprouting percentage, rooting percentage, and survival rate of Argania spinosa cuttings Values followed by the same letter(s) are not significantly different at P<0.05 using the Duncan’s Multiple Range Test (DMRT). A value represents mean ± standard error of means (n = 32). Treatments Sprouting (%) Rooting (%) Survival (%) Cutting diameter (cm) 0.1­0.3 ­ ­ ­ 0.3­0.6 70.69 ± 1.58 b 55.56 ± 2.22 a 78.14 ± 1.72 a 0.6­0.9 75.42 ± 2.52 a 49.55 ± 1.75 b 75.25 ± 2.20 a Leaf retention Leafless ­ ­ ­ 2 72.96 ± 1.87 ab 52.54 ± 2.03 ab 75.75 ± 1.04 ab 4 77.92 ± 1.93 a 56.33 ± 1.82 a 80.04 ± 2.16 a 8 68.29 ± 1.52 b 48.79 ± 1.90 b 74.29 ± 2.03 b Substrate Fine sand (FS) 67.54 ± 1.94 b 59.21 ± 2.27 a 65.08 ± 2.37 c Peat moss (PM) 74.08 ± 2.01 a 44.42 ± 2.19 b 76.96 ± 2.44 b Mixture of FS and PM 77.54 ± 2.17 a 54.04 ± 2.41 a 88.04 ± 2.83 a Benbya et al. ‐ Clonal propagation of Argania spinosa 69 other reserves for adventitious root formation (Leakey and Storeton­West, 1992; Tchoundjeu and Leakey, 1996). The good sprouting response may be due to the presence of adequate sugar reserves such as fructose, glucose and sucrose (Gehlot et al., 2015). Moreover, the level of endogenous auxins and other rooting cofactors might be lower in cuttings with a small diameter, which leads to reduced rooting per­ centage or even the absence of rooting (Wilson and Van Staden, 1999). The rooting efficiency of these cuttings could be also due to the lower content of mineral elements, especially nitrogen which is posi­ tively correlated with rooting (Budiarto et al., 2006). In addition, the effect of cutting diameter on rooting capacity could also be attributed to the origin of a cutting within shoots, and to its position of the stock­ plant (Leakey, 2004). Indeed, thin cuttings are pro­ duced from shoots which arise in sub­dominant posi­ tions of the mother plants which contain a low amount of auxin, grow slowly and stop growth early (Howard and Ridout, 1991). The lower rooting capaci­ ty of cuttings with larger diameters (0.6­0.9 cm) may probably be due to changes in the extent of lignifica­ tion and the degree of secondary thickening along their stems (Girouard, 1969; Hartmann et al., 1990). These results are consistent with other studies that found a positive effect of cutting diameter on sprout­ ing, rooting and survival success of rooted cuttings. Palanisamy and Kumar (1997) showed that the high­ er rooting efficiency of Picea abies was obtained by cuttings of 0.3­0.4 cm diameter. OuYang et al. (2015) confirmed the highest rooting efficiency of Picea abies obtained by cuttings of 0.3­0.4 cm diameter. Moreover, Foster et al. (2000) observed that the cut­ tings of Pinus taeda with an average diameter of 0.2­ Table 7 ­ Analysis of variance (ANOVA) for the effects of cutting diameter, leaf retention and substrate and their interactions on sprout­ ing percentage, rooting percentage and survival rate of Argania spinosa cuttings NS= Not significant (P>0.05); *= Significant (P<0.05); ***= Highly significant (P<0.001). df= degrees of freedom. ×= Interaction between treatments. Source of variance Dependent variable Sprouting (%) Rooting (%) Survival (%) df F­value P­value df F­value P­value df F­value P­value Cutting diameter 2 4.28 0.0434 * 2 6.70 0.0124 * 2 1.96 0.1668 NS Leaf retention 3 5.93 0.0047 * 3 3.53 0.0363 * 3 2.80 0.0694 NS Substrate 2 6.60 0.0027 * 2 13.98 <0.0001 2 41.37 <0.0001 Cutting diameter × Leaf retention 6 0.02 0.9849 NS 6 0.06 0.9444 NS 6 0.23 0.7918 NS Cutting diameter × Substrate 4 0.22 0.8072 NS 4 0.17 0.8411 NS 4 0.13 0.8775 NS Leaf retention × Substrate 6 0.27 0.8937 NS 6 0.12 0.9745 NS 6 0.29 0.8855 NS Cutting diameter × Leaf retention × Substrate 12 0.15 0.9633 NS 12 0.09 0.9860 NS 12 0.14 0.9655 NS 0.3 cm tended to root better and develop more roots. Concerning leaf retention, this study showed that successful vegetative propagation of Argania spinosa was restricted to leafy stem cuttings. Indeed, defoli­ ated cuttings failed to produce any sprouts / roots and showed a mortality rate of 100% within forty­ eight weeks after planting. This result could be explained by the direct influence of the presence of leaves on the primary shoots, because initial growth of shoots depends on assimilates supplied by leaves and also through their influence on the cutting’s water status (Newton et al., 1992; Van Labeke et al., 2001). Moreover, the leaf retention could also exert a strong influence on root initiation and development because it allows post­severance carbon assimilation (Leakey and Coutts 1989; Hartmann et al., 1990; Thomas and Schiefelbein, 2004). Leafy cuttings pro­ vide a continuous supply of photosynthates besides their reserves after implementing them into the sub­ strate (Tchoundjeu et al., 2002; Leakey, 2004). On the other hand, the inability of leafless cuttings to root has been associated with the rapid depletion of stored carbohydrates in stem tissues after excision from the stock plants (Hoad and Leakey, 1996; Druege et al., 2000). In addition, the leaf retention may also influence the endogenous auxins of the cut­ ting. Since rooting is stimulated by the level of auxins available, it is expected that adventitious rooting response of a cutting will be proportional to the num­ ber of leaves retained (Tchoundjeu and Leakey, 1996). In fact, our results showed that the ability of cuttings to develop adventitious roots decreased when the leaf number exceeds 4 leaves. This could be explained by the balance between the positive Adv. Hort. Sci., 2021 35(1): 61­72 70 effects of assimilate production through allowing suf­ ficient photosynthesis and the negative effects of water loss via transpiration (Leakey and Coutts, 1989; Mesén et al., 1997a). However, cuttings with eight leaves exhibited early root growth and delayed sprouting and subsequently a decreased rooting effi­ ciency. Indeed, cuttings with a high number of leaves appear to suffer from water deficit and a consequent reduction in photosynthetic activity as reflected by higher transpiration rates and leaf shedding com­ pared to cuttings with an optimal number of leaves (Leakey and Coutts, 1989; Newton et al., 1992; Aminah et al., 1997). Thus, the balance between pho­ tosynthesis, transpiration and nutrient transport is an important factor influencing rooting (Leakey and Coutts, 1989). The positive effect of leaf retention on cutting success has been reported across a wide range of species including Triplochiton scleroxylon (Leakey and Coutts, 1989), Cordia alliodora (Mesén et al., 1997a), Vitis vinifera (Thomas and Schiefelbein, 2004), Eucalyptus hybrids (Trueman and Adkins, 2013), Santalum austrocaledonicum (Tate and Page, 2018). Finally, the study of the substrate effect showed that a significant difference in sprouting and rooting as well as in survival ability was found between the three substrates tested. It was observed that the number of roots, root length and rooting percentage reached significantly higher values in cuttings set in the FS substrate compared to the PM substrate and to the mixture of fine sand and peat moss. This effec­ tiveness of adventitious rooting by rooted cuttings set in FS substrate could be related to its optimal vol­ ume of gas­filled pore­space and oxygen diffusion rate which create an adequate aerated environment for increased transpiration and respiration and enhanced adventitious root formation (Andersen, 1986). Moreover, sand is a porous substrate that lim­ its the development of microbial pathogens and where roots can be settled well without damage (Tchinda et al., 2013). Though, the poor aeration and high water holding capacity of peat moss substrate is a fundamental problem for adventitious root forma­ tion, leading to enhance the rate of fungal infection and decay of cuttings before root initiation (Schmitz et al., 2013). Furthermore, the higher water content in the peat moss substrate induces closing of the stomata and inhibits oxygen diffusion. This anoxia caused by oxygen deficiency or by accumulation of toxic substances, including bicarbonate and carbon dioxide, is suspected of being linked to the rooting problems due to tissue death, wilting, severe defolia­ tion, and reduced water absorption and leaf water potential (Mesén et al., 1997b; Drew, 1983; Veen, 1988). The results also showed that cuttings set in a mixture of sand and peat moss performed better than those set in sand or peat moss substrate alone, in terms of sprouting and survival rate. It has been suggested that the addition of peat moss to fine sand improved the water holding capacity and promoted mineral nutrients absorption (Leakey et al., 1990). The uptake of water by cuttings is positively related to the water content of the substrate and this may enhance survival rate by reducing water deficits, leaf abscission and cutting necrosis (Newton et al., 1992). A number of comparative studies between different substrates have indicated that cuttings set in a sand substrate performed better in term of rooting and survival capacity. Atangana et al. (2006) reported that the higher rooting percentages were observed in sand for Allanblackia floribunda. Gehlot et al. (2014) also recommended that higher rooting percentages of Azadirachta indica were recorded for cuttings planted in sand. Moreover, Adugna et al. (2015) stat­ ed that in Vanilla planifolia, the cuttings set in sand performed the highest rooting percentage. In conclusion, the results of the present study clearly indicate that cuttings with thinner diameters (0.1­0.3 cm) and defoliated cuttings have failed to produce any sprouts or roots and showed a 100% mortality rate within forty­eight weeks after planting. The greatest mean number of sprouts, sprout length and sprouting capacity as well as survival success was performed by cuttings with diameter of 0.3­0.6 cm and 4 leaves set in a mixture of sand and peat moss. The best mean number of roots, root length and rooting ability was achieved by cuttings with diame­ ter of 0.3­0.6 cm and 4 leaves in a fine sandy sub­ strate. This study also reveals that Argania spinosa is amenable to clonal propagation using sand­based rooting substrate which could provide a promising p r o s p e c t f o r t h e c o n s e r v a ti o n o f t h i s e n d e m i c species. In addition, it is suggested that this low­cost propagation techniques could greatly facilitate the domestication and the development of superior tree crops as a commercial agroforestry species with genetically homogenous plant material. The cuttings used in the experiments were taken during the spring season. It is therefore recommended that further works should be done to determine the optimal peri­ od for cutting collections for successful clonal propa­ gation of A. spinosa trees. Benbya et al. ‐ Clonal propagation of Argania spinosa 71 Acknowledgements This work is supported by public funds through the National Institute for Agronomic Research (INRA), Ministry of Agriculture ­ Morocco. References ADUGNA M., BELEW D., TILAHUN D., 2015 ­ Influence of rooting media and number of nodes per stem cutting on nursery performance of vanilla (Vanilla planifolia Andr. syn. Vanilla fragrans). ­ J. Hort. For., 7(3): 48­56. 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