Effects of zeatin concentration and cultivar on growth indices at proliferation stage
Benelli and De Carlo (2018) reported that zeatin (10 µM) not only affected axillary shoot induction of olive but also it stimulated shoot length. Another study reported that 18.4 µM of zeatin caused a negative effect on olive growth indices (Haddad et al., 2018). Ali et al. (2009) showed that 4 mg/L zeatin caused an acceptable microshoot formation of olive in comparison with 1, 2 and 3 mg/L zeatin. Zeatin is the best cytokinin enhancing shoot formation and even length development in olive which affects cell expansion. High zeatin concentration reduced shoot proliferation and elongation which are genotype-dependent (Haddad et al., 2018). Since supra optimal concentration of cytokinins had a low interaction with minerals in the media, shoot length depression occurred in sub optimal and supra optimal concentrations (Ali et al., 2009).
It is showed that combined cytokinins in olive increased the proliferation rate (Moradnezhad et al., 2017), zeatin in combination with other cytokinins like BAP, a fixed compound of media in this study was used to have synergistic effect on proliferation improvement as well. In ˈMoraioloˈoraioloation improvementsed to havetokinins like BAPPgth depression occurˈ cultivs betweemicroshoot formation (Ali et al., 2009). In other subspecies of olive (Laperrine olive), regeneration was to a high extent related to media and PGRs and only in case of not applying PGRs, microshoot formation did not develop (Haddad et al., 2018). Zeatin plus carbon source might have an effect on complex plant signaling network (Leva et al., 2013).
Interactive effect of culturing system and genotype at proliferation stage
Apical dominance and oxidized tannins from phenolic compounds in semi-solid media inhibited the normal regeneration in olive (Rugini, 1984; Sadder, 2002). Using bioreactor is advantageous because of lower production cost due to reducing the labor, zeatin concentration and increased efficiency of proliferation rate plus removing gelling agent (Benelli and De Carlo, 2018). A novel temporary immersion system was reported to be a suitable method in olive micropropagation in comparison with other liquid systems such as Erlenmeyer flasks, filter paper bridges in test tubes and LifeReactor (Grigoriadou et al., 2005). The advantage of liquid system is due to lowering the toxic compound aggregation, well aerated plantlets (Benelli and De Carlo, 2018; Sadder, 2002) and horizontal position of plantlets. This method allows reduction in carbon dioxide, ethylene gases and relative humidity in the vessel’s headspace and thus reduces morphogenesis abnormality (Benelli and De Carlo, 2018). They showed using plantform™ bioreactor, desired zeatin concentration in olive was reduced to 25–50%. In the present study, PMB functioned the way that TIS did. Sadder, 2002 reported that in olive cultivar ˈNabaliˈ suffocation was also reported in liquid OM (Sadder, 2002).
Analysis of 4–5 cm of olive shoot tip by Rugini (1984) resulted in introducing the best medium for olive micropropagation called OM which is high in Ca, Mg, S, Cu and Zn. Mg which is triple higher in OM compared to MS and VS (Duchefa, NL) increased shoot elongation by affecting enzymes positively, especially those involved in phosphate transfer as modulator of shoot initiation and growth promoter (Shaul, 2002). Nevertheles, it has as twice Boron as MS and VS have which evidently plays important roles in nucleic acid synthesis, cell differentiation and elongation (Läuchli, 2002).
VS medium contains macro elements as described by Murashige and Skoog (1962) and contains chelated Fe form EDDHA instead of EDTA as described by Van der Salm et al. (1994) which provides a better iron supply at higher pH and phosphate content since it is much stronger than Fe-EDTA (Duchefa, NL). It is showed that OM in olive cv. ˈArbequinaˈ was more effective rather than WPM and MS in producing total number of shoots and healthy plantlets (Moradnezhad et al., 2017). Different fundamental formulations are required for olive’s genotypes (Moradnezhad et al., 2017; Rugini, 1984), as minerals may sensitize cells to PGRs and nutrient mobilization occurs in PGRs existence by creating new source sink relationship. High calcium (CaCl2 and Ca (NO3)2) in OM plays a role in cytokinin signal transduction in cells which leads to better cell division (Ali et al., 2009). Folic acid co-enzymes are involved in carbon transfer, which are vital for methionine, serine, deoxythimidylic acid and purines synthesize that are necessary for cell differentiation (Ali et al., 2009).
Carbon and light sources
Leva et al. (2013) showed more undifferentiated parenchyma-like callus growth in medium containing sucrose rather than medium enriched with mannitol. Haddad et al. (2018) reported mannitol resulted in greener foliage and morphologically healthy plantlets similar to their mother plants. It is proved that shoot development pattern, plantlet survival, growth quality and secondary shoot formation in olive are affected by carbon sources (Leva et al., 2013). Starch, mannitol and sucrose are the most carbohydrates in olive and soluble sugar mannitol is predominant in leaves and branches twice as abundance as sucrose (Bustan et al., 2011) which contains 30% of olive total carbohydrates (Flora and Madore, 1993) and varies according to the year of bearing (on or off year), season as mannitol peaks is related to high temperature in summer, olive’s organs and even fruit harvesting time (Bustan et al., 2011). In olive, subsequent sub-culturing (seven times) in media containing mannitol increased the survival rate approximately twice compared with sucrose (Leva et al., 2013). Mannitol promoted bud sprouting and secondary shoot uniformity rather than sucrose regardless of its concentration (Leva et al., 2013). Mannitol may directly and indirectly influence endogenous hormonal status protecting against detrimental compounds in media (Leva et al., 2013) in contraction with sucrose in higher concentrations. As mannitol is involved in activating osmotic effect, plants with high amount of mannitol or well-acclimatized in mannitol enriched media have negligible osmotic stresses (Moradnezhad et al., 2017). Moreover, mannitol transfer in intracellular metabolism aids olive against abiotic stresses by acting as a supportive agent against olive cell suspension dehydration (Leva et al., 2013). In catabolism of mannitol in sink cells, hexose phosphate generates 2 ATP molecules, while initial generation of hexose phosphate in catabolism of sucrose spends ATP, so that energetic advantages of mannitol might be used for exponential growth (Leva et al., 2013; Moradnezhad et al., 2017).
In stevia, shoot induction occurred more frequently in 6000 cd·sr·m−² intensity rather than 2000 and 4000 cd·sr·m−² under the controlled situation. Light intensity affected stevia organogenesis as well and 6000 cd·sr·m−² was suitable for axillary shoot formation (Roshandel et al., 2013). In Ternstroemia gymnanthera three light intensities (500, 1000, 2000 cd·sr·m−²) were compared and the results showed 1000–2000 cd·sr·m−² were more acceptable in growth quality and in case of LED light and fluorescent lamp the most proper luminous intensity was 2000 cd·sr·m−² (Ahn and Choi, 2016) similar to this study findings.
Callus is normally regenerated due to zeatin application in the proximal end of plantlets and at the end of petioles which is a common reaction to in vitro stresses. It was reported that OM induced callus in olive without additional PGRs (Sadder, 2002) however, callus organogenesis only occurred in special olive cultivars as it happened often in ˈAminˈ, ˈMeshkatˈ and ˈArbequinaˈ cultivars in comparison with two Iranian cultivars ˈDireˈ and ˈTokhmekabkiˈ (Mirzaei et al., 2020 under submission data) and two commercial cultivars ˈConserveoleaˈ and ˈKoroneikiˈ .
Callus formation at proliferation stage
Krishna et al. (2016) reported that making wound in cutting samples, auxin and auxin analogs, sterilization and PGRs can cause mutagenesis and also, salts, temperature and light changes can result in oxidative stresses. Consequently, superoxide, hydrogen peroxide, hydroxyl, peroxyl and alkoxyl compounds can cause hyper/hypo methylation of DNA, DNA base deletion and substitution, chromosomal rearrangement and number change, which in turn resulting in mutation and somaclonal variation in in vitro culture (Bradaï et al., 2016; Krishna et al., 2016).
Molecular assessment of CIS and MIS in three cultivars
In order to further assess the olive genome coverage, several primers should be tested to increase the number of loci. It was previously stated that phenotypical, cytological and biochemical methods can be used in this purpose, though molecular assessment repetition in another growth phase is desirable (Bradaï et al., 2019) because somatical genetic stability of olive scarcely was studied so far. SSR was not informative in olive genetic variation assessment since replication slippage was more frequent that might ignore single nucleotide mutation (Bradaï et al., 2019), so that AFLP was chosen because its discriminating capacity was proved in analyzing olive cultivars (Belaj et al., 2003).
Nearly 97% resemblance between ˈAminˈ MIS and ˈArbequinaˈ MIS was seen proving that the resemblance between these genotypes for stem cutting in vitro culture was considered stable (Brito et al., 2010), consequently they were genetically close to each other (Bradaï et al., 2019 and 2016).
Embryogenic culture-induced olives from the same cotyledon maintained in in vitro conditions for 3 years showed genetic instability due to the long term maintenance (Bradaï et al., 2019). Although variation in olive was reported slightly higher in aged plants in vitro subcultured continually, it was highly genotype-dependent and biometric analysis of quantitative traits also showed intraclonal variation. Haddad et al. (2018) reported all amplicons in ISSR marker between regenerated diploid and triploid endangered olive in comparison with donor plants were monomorph, so that all the regenerants in high PGRs and their mother plants were grouped in the same category (Haddad et al., 2018). Nevertheless, as the stability of in vitro-raised olives in OM and zeatin enriched media was proved (Haddad et al., 2018), in this study MIS was considered as donor plants and CIS was compared to MIS. On the other hand, Guillaume et al., (2009) reported that meristem mutation position was the first important factor in chimerism maintenance and mutation in apical dome normally resulted in permanent chimera (Besnard and Baali-Cherif, 2009). In marker investigations, DNA is usually extracted from leaves and so, the mutation level in diploids might be underestimated (Besnard and Baali-Cherif, 2009). Nevertheless sampling after 6 subcultures could resolve such a problem as the results of this study showed.
No correlation between genetically somaclonal varied plants with phenotypic changes was found as phenotypical characteristics are controlled by several genes (Bradaï et al., 2019), moreover special part of this genuine variation might be due to the differences in non-coding portion of genome, which might not be expressed and revealed in olive appearance and performance. To reduce such controversial issues, complementary phenotypic analysis is recommended (Bradaï et al., 2016). Provoked changes in cells might also result in developmental reprogramming in plants (Belaj et al., 2003; Bradaï et al., 2019). The problem of undesired callus induction at the basal end and petiole end part in olive propagation was reported frequently. Callus phase is a prerequisite of variations. Studies on genetic stability in somaclonal varied olives are scarce (Bradaï et al., 2019), therefore this is the first study that compared plantlets originated from MIS and CIS as genetic stability and ploidy level fidelity in olive. Belaj et al. (2003) stated that the combination of two different genetic change assessments in olive is another approach and so, evaluation of ploidy level changes by FCM was used.
FCM assessment CIS and MIS
In this study DNA amounts were similar to olives measured previously (2.90–3.07 pg) (Loureiro et al., 2007). There was no significant nuclear DNA content difference among cultivars. Nonetheless, in the previous studies DNA amount fluctuation might be attributed to the cytosolic compounds like phenols causing tannic acid effect as a result of interference with DNA-PI staining since there is an abundant amount of such materials in woody plants (Loureiro et al., 2007). These interfering phenolic compounds are genotype-dependent and normally micropropagated plants accumulate higher amounts of cytosolic compounds which do not allow proper PI staining resulting in lower DNA amount (Loureiro et al., 2007). Even though olive is extremely high in such mentioned interferer compounds and its discrepancy in DNA 2C value should be considered cautiously, fortunately in the present study interferer materials were not problematic. PVP was added to nuclei isolation buffer to solve such problematic issues in secondary metabolites (Loureiro et al., 2007). Not severely chopped leaves by razor blade can also equilibrate interferer material (Loureiro et al., 2007).
Loureiro et al. (2007) reported that 2C DNA content of O. europaea ssp. europaea var. europaea ranged between 2.90 ± 0.020 pg/ 2C and 3.07 ± 0.018 pg/ 2C and for wild olive, it was 3.19 ± 0.047 pg/ 2C. In this study, 84% of samples had lower DNA amounts. Ploidy level changes are prominent processes in plant evolution. Due to reduction in inbreeding depression effect, triploids are much more vigorous than diploids especially in extreme situations and as ˈAminˈ was highly resistance to salinity stress and other abiotic stresses (data are not shown), it was suspected to be a polyploid which was not proven in this study as previously reported for Iranian olive cultivars and checking other genetic variation disorder in this genotype is recommended.