Impaginato 185 Adv. Hort. Sci., 2022 36(3): 185­191 DOI: 10.36253/ahsc­12488 Impact of cultural conditions on germi­ nation of olive (Olea europaea L.) somatic embryos and plantlets develop­ ment from the Algerian cultivar Chemlal K. Titouh 1, 2 (*), K. Hadj Moussa 2, N. Boufis 1, L. Khelifi 2 (*) 1 National Institute of Agronomic Research of Algeria (INRAA), Research Center of Baraki, Algiers, Algeria. 2 National Higher School of Agronomy (ENSA, ES1603), Laboratory of Genetic Resources and Biotechnologies (LRG‐B), Algiers, Algeria. Key words: Germination, micropropagation, olive, plant regeneration, somatic embryo. Abstract: The in vitro propagation techniques are currently a commercial alter­ native for the production of plants with good quality in several plant species, including the olive tree (Olea europaea L.). Somatic embryogenesis is the process practically used for the application of several biotechnological tools of improvement and in vitro plant regeneration via the germination of somatic embryos. Our work aims to evaluate the effect of the chemical and hormonal composition of the culture medium on the germination of olive somatic embryos (cv. Chemlal) as well as the micropropagation of the obtained plantlets before their acclimatization to natural conditions. The results indicat­ ed that the production of olive plants by somatic embryogenesis depends strongly on the genotype of the somatic embryos (cell line) and more on the culture conditions, particularly the presence of growth regulators. Indeed, a solid OM medium supplemented with hormones (BA and IBA) permitted an advanced root emergence and germination allowing the production of well­ developed plants with several leaves. In addition, an OM medium supplement­ ed with Zeatin and IBA allowed better reactivity of micro­cuttings producing well­developed shoots with several emitted roots which facilitates their further acclimatization to natural conditions. 1. Introduction The olive tree (Olea europaea L.) a diploid dicotyledonous species of the Oleaceae family, includes several cultivars selected and multiplied ini­ tially by farmers mainly for the size of their fruits and the oil content (Besnard et al., 2018). Conventional methods of breeding and multiplica­ tion represent an important solution to the crop problems, especially the increasing demand for plants needed for new orchards. However, these (*) Corresponding authors: titouhkheireddine@gmail.com khelifi.lakhdar@gmail.com Citation: TITOUH K.,HADJ MOUSSA K., BOUFIS N., KHELIFI L., 2022 ­ Impact of cultural conditions on germi‐ nation of olive (Olea europaea L.) somatic embryos and plantlets development from the Algerian cultivar Chemlal. ­ Adv. Hort. Sci., 36(3): 185­191. Copyright: © 2022 Titouh K., Hadj Moussa K., Boufis N., Khelifi L. 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 28 December 2021 Accepted for publication 13 June 2022 AHS Advances in Horticultural Science https://doi.org/10.36253/ahsc-12488 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., 2022 36(3): 185­191 186 techniques have become unable to achieve signifi­ cant results because of the long juvenile period of the species (Rugini et al., 2020) as well as the recalci­ trance of certain main cultivars, such as ‘Chemlal’ in Algeria, to semi­hardwood cuttings (Fabbri et al., 2004). In this context, in vitro propagation techniques are currently a commercial reality for the multiplica­ tion of several olive cultivars (Rugini et al., 2020) due to the sanitary quality and the confirmed genetic sta­ bility of the regenerated plants (Lopes et al., 2009) despite some phenotypic changes and somaclonal variations observed after several subcultures (Leva, 2009; Bradaï et al., 2016, 2019). Somatic embryogenesis is a morphogenetic process through which somatic cells produce a bipo­ lar structure morphologically similar to the zygotic embryo called a ‘somatic embryo’ able to develop into a whole plant (Neumann et al., 2009). Actually, this process has become a common technique for in vitro regeneration allowing the application of several biotechnological tools of improvement and conserva­ tion such as genetic transformation, in vitro selection and cryo­preservation of various species and inter­ esting genotypes (Sánchez­Romero, 2021). Most of the established works on somatic embryogenesis in olive consider three main steps starting with the establishment of embryogenic cultures combining the induction and proliferation of calli, followed by a phase of expression and development of structured embryos ready to be converted into whole plants. However, few works have been done about the ger­ mination conditions of olive somatic embryos (Mazri et al., 2020) although regeneration of plantlets has frequently been achieved by introducing the embryos under photoperiod on a standard culture medium based on the chemical compositions ‘MS’ (Murashige and Skoog, 1962) or ‘OM’ (Rugini, 1984). In addition, the low conversion rates remain the great obstacle of the process in many species, includ­ ing in olive tree, are caused mainly by deficiencies in the development and maturation of the used embryos (Merkle et al., 1995; Sánchez­Romero, 2019) but also by unfavorable conditions to their ger­ mination (Bradaï et al., 2016). The objective of our study is to evaluate the effect of the chemical and hormonal composition of the cul­ ture medium on the germination of somatic embryos of the main Algerian olive cultivar ‘Chemlal’. In addi­ tion, the micropropagation of the obtained plantlets as well as their acclimatization to natural conditions were tested. 2. Materials and Methods Establishment of the embryogenic cultures The embryogenic cultures used were induced from radicles of zygotic embryos of the cultivar ‘Chemlal’ according to a modified method of Cerezo et al. (2011). Radicles isolated after disinfection of the seeds extracted from the stones of mature olives were cultured on solid OMc medium (Cañas and Benbadis, 1988) supplemented with 0.5 mg l­1 of Zeatin and 5 mg l­1 of indole­3­butyric acid (IBA) for three weeks. Subsequently, the explants were trans­ ferred to the same OMc medium without Zeatin and containing 0.5 mg l­1 of IBA for four weeks. Finally, the obtained calli were maintained by monthly sub­ cultures on a solid ECO basal medium (Cerezo et al., 2011) supplemented with 0.1 mg l­1 of Zeatin, 0.1 mg l­1 of Benzylaminopurine (BA) and 0.05 mg l­1 IBA in addition to 0.55 g l­1 of glutamine and 1 g l­1 of casein hydrolyzate. All the media were supplemented with 20 g l­1 of sucrose, 50 mg l­1 of Myo­Inositol and solid­ ified with 6 g l­1 of agar after adjusting the pH to 5.74 with NaOH or HCl (1 N). The cultures were incubated in total darkness at a temperature of 25±2°C. Pro­embryos exceeding 2 mm in size were isolat­ ed from calli of two embryogenic lines (C2 and C3) after one month of culture in suspension on a liquid ECO medium supplemented with hormones in dark­ ness with stirring at 100 rpm. These immature embryos were transferred for maturation on solid ECO free­hormones medium and supplemented with 1 g l­1 of activated charcoal for two months in total darkness at 25±2°C. Germination of somatic embryos Mature embryos with a perfectly bipolar or cotyledonary structure, were germinated individually in test tubes over two different media: OM (Rugini, 1984) and MS (Murashige and Skoog, 1962) solidified with 6 g l­1 of agar. In addition, the effect of a hor­ monal combination consisting of 1 mg l­1 BA and 0.1 mg l­1 IBA was tested. The two media were supple­ mented with 20 g l­1 of sucrose, 100 mg l­1 of Myo­ Inositol. At least 16 mature somatic embryos were incubated for more than eight weeks under a 16 h light photoperiod (35 µmol m­2 s­1) at a temperature of 25±2°C. Germination, root emergence, shoot Titouh et al. ‐ Somatic embryogenesis in Algerian olive cultivars 187 length as well as the number of leaves per obtained plantlet after eight weeks of culture were observed. Micropropagation of shoots and acclimatization of regenerated plantlets In order to maintain the maximum number of plants in culture; micropropagation of the shoots resulting from the germination of somatic embryos was applied. Therefore, the shoots were divided into uni­nodal micro­cuttings and cultured on two differ­ ent culture media: OM and DKW (Driver and Kuniyuki, 1984) as modified by Revilla et al. (1996). The two culture media were supplemented with 20 g l­1 of sucrose, 100 mg l­1 of Myo­Inositol in addition to hormonal balances composed of 2 mg l­1 of BA or Zeatin combined with 0.1 mg l­1 of IBA. The different media were solidified with 6 g l­1 of agar. At least 16 micro­cuttings having two leaves were cultured indi­ vidually in test tubes containing 15 ml of the micro­ propagation medium for eight weeks under a 16 h light photoperiod at a temperature of 25±2°C. The reactivity of the micro­cuttings and the number of reactive buds, shoot length, number of leaves as well as the number of roots emitted were observed. The obtained plants showing an acceptable length with several leaves and well­developed roots were acclimatized in the laboratory under a photoperiod for about two months on a humidified mixture of sand/potting soil/perlite at a rate of 2/2/1 (v/v/v). Subsequently, the reactive plantlets were transferred to natural conditions under greenhouse on a sub­ strate rich in organic matter and frequently irrigated before being permanently planted in the field. Data analysis Statistical analyses of the data (Analysis of vari­ ance and tests) were carried out using the “XLSTAT” program version 2016.02.27444. In case of a signifi­ cant difference, the separation of means was per­ formed by Fisher’s LSD (Least Significant Difference) test. The percentages were analyzed by the chi­ square test. The results were presented as a mean ± standard deviation or as a percentage relative to the total of introduced explants. A significance level of 5% was considered in all analyses. The letters in the tables indicate homogeneous groups. 3. Results Germination of somatic embryos From the first days under photoperiod, the white­ opaque somatic embryos of olive showed greening of their stem part and yellowing of the root part (Fig. 1 A) followed by its elongation preceding an increase in size of the two cotyledonary leaves and their separa­ tion each one from the other (Fig. 1 B) before the emergence of a small shoot (Fig. 1 C). Indeed, the germination capacity of embryos and the plants development varied from one cell line to another and were significantly influenced by the culture condi­ tions, particularly the presence of growth regulators (Table 1). Thus, more embryos of the C2 line germi­ nated on OM medium while the germination of the two lines was similar on the MS medium. However, the presence of hormones allowed a significant improvement in the embryos germination of both lines. In fact, the best germination rates were obtained on OM medium supplemented with BA and IBA (OM1) with 56.3 and 37.5% respectively for C2 and C3 while the low germination rate of 25% was recorded on MS without hormones (MS0) (Table 1). In addition, embryos of C2 germinated with root emergence from the first week of culture (Fig. 1 A) while no reactivity was observed before two weeks for C3 embryos. Likewise, the germination and the root emergence were faster on the OM medium than Fig. 1 ­ Germination of mature somatic embryos from two lines of embryogenic olive callus, cv. Chemlal, and micropropa­ gation of the obtained shoots after 8 weeks of culture on different culture media. (A) Somatic embryo germinated on solid medium. (B) Swelling of cotyledonary leaves of embryos. (C) Different stages of embryos germination. (D and E) Plantlets obtained after germination. (F and G) Plantlets obtained after the micropropagation of shoots obtained from germination of somatic embryos. (H) Acclimated plantlets. (→: the arrows indicate the emer­ gence of the root, shoot and the two cotyledonary lea­ ves. Bar corresponds to 1 cm. Cot: Cotyledons). Adv. Hort. Sci., 2022 36(3): 185­191 188 on MS, especially in the presence of growth regula­ tors which accelerated significantly germination of embryos. Therefore, the embryos emitted their roots after 10.5 and 15.4 days and germinated after 14 and 18.7 days on the OM1 medium respectively for C2 and C3 (Table 1). Consequently, early germination and rooting of C2 embryos resulted in well­devel­ oped plants with an average length of 1.6 and 2.3 cm with 6 and 6.3 leaves per plantlet respectively on OM0 and OM1 (Table 1, Fig. 1 D) while the MS1 medi­ um was more beneficial for the C3 plants reaching 1.9 cm in length with 4.5 leaves (Table 1, Fig. 1 E). Micropropagation of shoots and acclimatization of regenerated plantlets Reactivity of micro‐cuttings and shoot develop‐ ment. The micropropagation of shoots resulting from the germination of somatic embryos was significantly influenced by the callus line as well as the chemical and hormonal composition of the culture medium (Table 2). In fact, micro­cuttings of the C2 line were more reactive than those of C3 regardless of the cul­ ture conditions, although the presence of hormones was essential for the development of the shoots given the low reactivity recorded on the control media. In addition, the best result of reactivity (100%) of the explants of both lines was obtained with the balance Zeatin/IBA and also the C2 cuttings on the combination BA/IBA in presence of which only 62.5 and 87.5% of the C3 cuttings reacted respective­ ly on OM and DKW (Table 2). Elsewhere, the DKW medium in particular supplemented with hormones (Zeatin/IBA) accelerated the bud reaction and improved the number of reactive buds per explant while the cuttings introduced particularly on the free­ hormones OM medium reacted late with less sprout­ ed buds (Data not shown). The development of the obtained shoots depend­ ed directly on the culture conditions and was signifi­ cantly influenced by the reactivity degree of the cut­ Table 1 ­ Effect of the chemical composition (OM and MS) of the culture medium and the presence of hormones (without hormones ‘0’ or with hormones ‘1’) on germination and root emergence of somatic embryos of two lines of embryogenic olive calli, cv. Chemlal, after eight weeks of culture The different small letters of the same format in columns indicate the homogeneous groups of a significant difference at level of 5%. Table 2 ­ Effect of the micropropagation medium (OM and DKW) and the presence of hormones (BA/IBA or Zeatin/IBA) on the reactivi­ ty, development and rooting of shoots from uni­nodal micro­cuttings obtained after germination of somatic embryos of two lines of embryogenic olive calli, cv. Chemlal, after eight weeks of culture The different small letters of the same format in columns indicate the homogeneous groups of a significant difference at level of 5%. Germination medium Germination rate (%) Average time of germination (Days) Average time of root emergence (Days) Average length of plantlet (cm) Average number of leaves/plantlet C2 C3 C2 C3 C2 C3 C2 C3 C2 C3 OM0 50.0 b 31.3 d 16.8±3.8 b’ 21.0±0.0 de’ 12.3±3.5 a’’ 17.5±4.9 cd’’ 1.6±0.1 b 0.6±0.1 d 6.0±1.6 a’ 2.0±0.0 c’ OM1 56.3 a 37.5 c 14.0±4.4 a’ 18.7±4.0 bc’ 10.5±4.0 a’’ 15.4±3.1 b’’ 2.3±0.8 a 0.9±0.2 c 6.3±1.6 a’ 2.3±0.6 c’ MS0 25.0 e 25.0 e 24.5±4.9 f’ 21.7±1.2 e’ 21.0±0.0 e’’ 18.7±4.0 d’’ 0.8±0.1 cd 0.8±0.1 cd 2.5±0.7 c’ 2.0±0.0 c’ MS1 31.3 d 31.3 d 21.0±0.0 de’ 19.3±4.7 cd’ 17.5±0.7 cd’’ 16.3±4.0 bc’’ 0.9±0.1 c 1.9±0.1 b 2.5±0.7 c’ 4.5±0.7 b’ Composition of the micro­ propagation medium Hormonal combina­ tions Reactivity and shoot development Rooting Reactivity rate (%) Average length of shoot (cm) Average number of leaves/shoot Rooting rate (%) Average number of roots C2 C3 C2 C3 C2 C3 C2 C3 C2 C3 OM Control 25.0 d 12.5 d 0.7±0.3 g 0.9±0.0 fg 1.5±0.7 d’ 2.0±0.0 cd’ 0.0 e’ 0.0 e’ 0.0±0.0 f’’ 0.0±0.0 f’’ BA/IBA 100.0 a 62.5 c 2.0±0.7 de 1.9±0.2 de 2.9±1.0 cd’ 3.1±0.9 cd’ 31.3 d’ 31.3 d’ 1.0±0.0 e’’ 1.5±0.7 bc’’ Zeatin/IBA 100.0 a 100.0 a 3.7±1.0 b 5.2±2.1 a 6.9±3.2 b’ 9.9±3.1 a’ 62.5 a’ 43.8 c’ 1.5±0.0 bc’’ 1.7±0.6 b’’ DKW Control 18.8 d 12.5 d 0.5±0.0 g 0.7±0.0 g 2.0±0.0 cd’ 2.0±0.0 cd’ 0.0 e’ 0.0 e’ 0.0±0.0 f’’ 0.0±0.0 f’’ BA/IBA 100.0 a 87.5 b 1.7±0.3 ef 3.1±1.0 bc 3.4±1.1 c’ 2.7±0.4 cd’ 43.8 c’ 31.3 d’ 1.3±0.6 cd’’ 1.5±0.7 bc’’ Zeatin/IBA 100.0 a 100.0 a 2.7±0.2 cd 3.3±1.4 bc 6.6±1.1 b’ 6.8±2.4 b’ 56.3 a’ 50.0 b’ 1.3±0.5 d’’ 2.0±0.8 a’’ Titouh et al. ‐ Somatic embryogenesis in Algerian olive cultivars 189 ting particularly the number of reactive buds. In cut­ tings with two active buds; one shoot regularly grown more than the other (Fig. 1 F and G). Moreover, the cuttings cultured on OM medium supplemented with hormones especially Zeatin/IBA and which sprouted early, produced well­developed shoots of 3.7 and 5.2 cm in length with 6.9 and 9.9 leaves respectively for C2 and C3 while the shoots obtained on the control media were the least developed, with less than 1 cm of length and a maximum of 2 leaves per plantlet (Table 2, Fig. 1 F and G). Rooting of developed shoots. The presence of hor­ mones in the culture medium was essential for the formation of a basal callus on the micro­cuttings before the emission of roots, while the chemical composition contributed more in the development of the induced calli due that the majority of calli gener­ ated on OM were generally larger compared to those obtained on DKW (Data not shown, Fig. 1 F and G). Root emission was significantly influenced by the genotype (cell line) as well as chemical and hormonal composition of the propagation medium (Table 2). Indeed, more rooting was observed with micro­cut­ tings of the line C2 particularly on the OM medium supplemented with Zeatin/IBA allowing 62.5 and 43.8% rooting with 1.5 and 1.7 roots emitted by cut­ ting respectively for the two lines while the chemical composition of DKW was more beneficial to C3 explants with 50% of rooting and an average of 2 roots per plantlet (Table 2). However, the appear­ ance of roots occurring from the 3rd to the 7th week of culture was often faster on DKW compared to OM medium especially in the presence of BA with IBA. Furthermore, the substitution of BA by Zeatin in the added hormonal combination reduced the rooting time by more than a week in the cuttings of both lines, which improved the length of the emitted roots (Data not shown). Therefore, the plantlets showing well­developed shoots and roots were easily acclima­ tized in the laboratory and exhibit normal growth and phenotype even after transfer to natural field conditions (Fig. 1 H). 4. Discussion and Conclusions Germination of somatic embryos Germination of olive somatic embryos was fre­ quently achieved on media based on the chemical formulations OM and MS with a reduced concentra­ tion of mineral salts, similar to those used for the cul­ ture of zygotic embryos (Sánchez­Romero, 2019). Rugini (1988) indicated that germination and devel­ opment of olive plantlets from somatic embryos is faster on OM medium than on MS. Indeed, Rugini and Caricato (1995) observed the germination of embryos of cultivars ‘Canino’ and ‘Moraiolo’ after 1 to 2 weeks on OM free­hormones medium, whereas Shibli et al. (2001) didn’t note reactivity in embryos of the cultivar ‘Nabali’ before two weeks of incuba­ tion on MS medium, which agree with our results indicating a faster germination on OM medium com­ pared to MS one. Several studies reported low rates of embryo ger­ mination varying with genotype but depending more on the quality of the used embryos. Therefore, Jafarzadeh­Bajestani et al. (2011) obtained less than 6% of germination on MS medium with embryos of the cultivar ‘Zard’ while a desiccation step for three days improved conversion to 50% and formation of rooted plantlets with 6 to 8 leaves. Furthermore, cul­ turing embryos of the cultivar ‘Picual’ on a cellulose acetate semi­permeable membrane during the first month of maturation (Cerezo et al., 2011) allowed an adequate dehydration, good structuring of the embryos and a significant improvement in germina­ tion and quality of the obtained plantlets. Therefore, the low germination capacity observed in this study was probably due to the use of embryos directly after their maturation without passing a desiccation phase allowing the synchronization of their germination (Merkle et al., 1995). Germination of olive somatic embryos was often achieved in the absence of hormones, although Rugini (1995) recommended the addition of Zeatin to solid MS medium to boost emergence. Thus, embryos of the cultivars ‘Chetoui’, ‘Chemlali’ and ‘Arbequina’ (Trabelsi et al., 2003) and ‘Picholine Marocaine’ (Brhadda et al., 2008) germinated easily on OM and MS media supplemented with 0.5 mg l­1 of Zeatin and resulted to well­developed plantlets, while a free­ hormones medium rich in sucrose induced cell prolif­ eration of the embryos. Conversely, Toufik et al. (2017) observed that the presence of growth regula­ tors, in particular Zeatin alone in the OM­based medi­ um, was not essential for the germination of embryos of the cultivar ‘Dahbia’ and can even inhibit the root emergence and cause a strong explant necrosis although the addition of NAA with GA3 allowed up to 45% conversion from mature embryos according to Mazri et al. (2020). Therefore, the inclusion of growth regulators especially cytokinins to the germination 190 Adv. Hort. Sci., 2022 36(3): 185­191 medium is directly determined by the genotype and degree of maturity of the embryos (Merkle et al., 1995). In this sense, Bradaï et al. (2016) indicated that well­matured and structured or cotyledonary embryos of ‘Picual’ germinated easily in the absence of hormones, unlike globular embryos whose germi­ nation varies between 30 and 70%. The regenerated plants in this study showed a nor­ mal phenotype (phyllotaxis, leaf shape, etc.) during their in vitro maintenance as well as an easy acclima­ tization to natural conditions. Leva (2009) indicated that plantlets regenerated by somatic embryogenesis show a stable phenotype similar to that of the moth­ er plants. Moreover, despite their fragility, these plants acclimatize easily to natural conditions and show normal growth under greenhouse and good development after transfer to the field. Micropropagation of shoots and acclimatization of regenerated plantlets The in vitro multiplication of plants regenerated by somatic embryogenesis was rarely practiced because their acclimatization was usually carried out directly after germination (Bradaï et al., 2016). In fact, the DKW medium (Driver and Kuniyuki, 1984) as modified by Revilla et al. (1996) and supplemented with a hormonal balance rich in cytokinins was often used although the chemical formulation of OM medi­ um containing Zeatin was commonly recommended for the rapid stimulation of axillary buds of several olive cultivars (Lambardi et al., 2013) due to its nutri­ tional content, particularly in microelements (Rugini et al., 2020). Cerezo et al. (2011) indicated that the micro­cuttings taken after germination of embryos of the cultivar ‘Picual’ respond easily on DKW medium supplemented with BA and IBA by developing shoots of about 1.4 cm in height with formation of a basal callus often accompanied by the emission of one or more roots. Nevertheless, Bradaï et al. (2016) observed that shoots multiplication from somatic embryos was influenced more by the age of the cell culture than by its genotype (callus line). These authors obtained more developed rooted shoots from young cultures while less sprouted buds with older lines giving small shoots often without roots. According to Bhojwani and Razdan (1996) in vitro shoots need to reach a minimal size to root easily as small plants may not survive during the acclimatiza­ tion period. Therefore, our results confirm the impor­ tance of the genotype, the significant effect of the DKW chemical formulation and the presence of Zeatin in addition to an auxin for the multiplication and rooting of shoots resulting from the germination of somatic embryos. In conclusion, our study is a contribution to the optimization of the in vitro regeneration of olive tree by somatic embryogenesis and describes for the first time an efficient regeneration of whole plants with­ out morphological abnormalities in the main olive cultivar in Algeria ‘Chemlal’ via embryogenic cultures induced from juvenile material, radicles of zygotic embryos. 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