A research study was performed with four individual experiments to investigate and evaluate responses of desert teak ovary explants to various PGRs and also sucrose concentrations in in vitro culture medium and to uncover their probable potentiality in induction of SE.
3.1 In vitro ovary morphogenesis under influence of 2,4-D and NAA
As it is evident from Table 1, NAA showed obvious and significant superiority over 2,4-D in callus induction almost in all the concentrations tested. Cent percent callogenesis was achieved with 2.7 µM NAA though generally there were no significant differences among most of the NAA levels. However, the best callus induction were in the range of 2.7 to 21.5 µM. The most suitable 2,4-D concentration inductive of maximum callusing was 0.45 µM, the lowest level applied. Increasing 2,4-D beyond 0.45 µM decreased callus induction drastically so that the callus production was ceased at ≥ 13.5 µM totally. The quantity of NAA-induced callus manifested a rising trend with increase in the concentration used. The maximum amount was produced by the ovaries cultured on the medium supplemented with 16.1 µM NAA though the concentrations of 10.8, 21.5 and 27 µM NAA yielded also high callus amount. Regarding 2,4-D, the callus quantity was assessed as poor with 0.45 µM which was dropped to the scale of very poor when the concentration was raised up to 9 µM. Enhancing 2,4-D level beyond 9 µM suppressed callogenesis entirely.
Considering callus texture and color, all NAA concentrations as well as 2,4-D at 0.45 µM caused light green friable callus. Such calli were recognized as competent ones for SE (Fig. 1). Those inductive level of 2,4-D levels (above 0.45 µM) resulted in formation of friable callus but brownish in color which are usually unsuitable material to be used for further development particularly for the purpose of SE.
Apart from the dominant response of callogenesis, observations revealed some evidence of SE occurrence, eight weeks after the culture initiation, but only with NAA supplemented media. NAA at 5.4 and 10.8 µM could induced PEMs at rate of 40 and 20 percent, respectively, exhibiting few somatic embryos at globular stage, while 21.5 µM NAA induced PEMs revealing a larger number of globular stage somatic embryos (Fig. 1 a,b). Surprisingly, nil embryogenesis was noticed with 16.1 µM NAA. On the other side, the highest NAA concentration resulted in and unexpected adventitious root induction and development while no visible SE could be traced (Fig. 2). 2,4-D contained media, the inducers of poor callogenesis, made no progress towards any type of morphogenesis.
Overall, the collected data and observations indicated that the best outstanding outcomes can be obtained through application of NAA in the culture medium ranging 5.4 to 21.5 µM so that the induced ECs not only are satisfactory in callogenesis quantity and quality, but also exhibit their potential for SE through developing PEMs and globular stage somatic embryos.
Various PGRs mainly auxins and CKs were used for callus induction in tree species (Giri et al., 2004). Auxins are the key plant PGRs that control in vitro morphogeneis including SE (Cook et al., 1993; Hazubska-Przybył et al., 2020). In general, the most commonly route for inducing SE is through initial exposure of cultures to a medium containing higher doses of auxins, which in most cases leads to formation of EC, followed by subsequent transfer to a medium either devoid of auxin, with drastic reduction of auxin concentration or supplemented with optimal levels of CKs (Mithila et al., 2003; Fehér et al., 2001; Isah, 2016; Feher et al., 2002). However, some species tend to express SE even on auxin contained medium, the same medium used as their embryogenesis induction medium (Adil et al., 2018; Isah, 2016). According to Kawahara and Komamine (1995), exogenous auxins play role in gene expression at the early stages of SE.
Here, in the first experiment, 2,4-D and NAA as auxin sources in the culture medium were tested for induction of EC and SE. The results revealed superiority of NAA over 2,4-D in the the concentration range applied. Meanwhile, there are numerous reports on woody as well as herbaceous species that support superiority of one or efficiency of both of these auxins in induction of direct or indirect (callus-mediated) SE.
A report by Xu et al. (2019) showed successful induction of direct SE on different explants of Ranunculus sceleratus by using high concentration of NAA (10 mg l− 1 = 54 µM). In another study, in order to regenerate Fraxinus mandshurica through indirect SE, Liu et al. (2020) found increasing percentage of EC induction on ½-strength MS medium containing only low NAA ranging 0 to 0.15 mg l− 1 (~ 0.75 µM). Recently, it has been shown that auxin type (2,4-D, NAA and picloram) had no significant effect on embryonic tissue initiation in two spruce species, but the same report has illustrated significantly better EC quantity with NAA than 2,4-D in Picea abies but not for P. omorika (Hazubska-Przybył et al., 2020). My results are fully in agreement with these findings, emphasizing the prominent role of NAA in promotion of process of SE. In the study of Bonneau et al. (1994) on induction of SE in European spindle tree, expression of SE was only observed in the presence of NAA or IAA in the induction medium. They affirmed no somatic embryos were observed in the absence of a CK or in the presence of 2,4-D or IBA. In accordance with their results, here also ovary explants did not respond positively to 2,4-D.
Nevertheless, if 2,4-D would be used at still lower concentrations, or alternatively, the duration of explant incubation to the 2,4-D contained medium would be shortened (preferably less than 4 weeks), this auxinic herbicide might appear inductive of SE in Tecomella Undulata. There are also numerous reports supporting highly positive influential effect of 2,4-D in inducing SE (Lambardi, 2000; Fehér et al., 2001, 2002; Singh and Chand, 2003; Sathyanarayana et al., 2004). Studies have shown that exogenously applied 2,4-D can regulate and increase the level of endogenous auxin, which results in an increase in cell division and the establishment of a hormonal gradient that is optimal for the induction of embryogenesis from the somatic cells of treated explants (Cook et al., 1993, Hazubska-Przybył et al., 2020). But the critical point to succeed in the process is to discover the best inductive concentration(s) of 2,4-D for each specific genotype. In Terminalia chebula Retz. the frequency of callus induction from mature zygote embryos was less and non-embryogenic when auxins (2,4-D, NAA, IBA) were used singly in the medium (Anjaneyulu and Giri, 2018). Compared to the present results, this finding is true with respect to 2,4-D but not for NAA.
Successful protocols for induction of SE using a combination of 2,4-D and a cytokinin (kinetin or BA) have also been previously reported with nodal segments of Santalum album (Peeris and Senarath, 2015), immature zygotic embryos of Alnus glutinosa (Corredoira et al., 2013), cotyledon explants (Singh and Chand, 2003) of Dalbergia sissoo, and anthers and ovaries of several Vitis species (Martinelli et al. 2003; Gambino et al. 2007; Gambino et al. 2021). Similarly, there are also reports showing that a combination of NAA and a cytokinin (kinetin or BA) was inductive of EC in woody species such as Azadirachta indica (Sue et al., 1997), Hardwickia binata (Das et al., 1995) and Quercus suber (Fernandez-Guijarro et al., 1995). Discrepancy between these results and my findings refer to the differences in species genetic makeup, explant type, season of explant collection, mother tree age and its growing conditions, and other factors that could affect explant's endogenous phytohormones levels.
Increase in endogenous auxin content of cells during primary stages of SE has been well-documented (Fehér et al., 2001; Tran et al., 2016). Hence, employing exogenously supplied auxin through culture medium has been practiced routinely to gain somatic embryos from various explants types in a wide variety of plant species. However, many of these attempts have failed since both the endogenous auxin level, and its exogenous application can be considered as critical factors during the induction and expression of SE. Two mechanisms have been considered as the logic behind such failures; firstly, a critical level of endogenous auxin in cells may be necessary for embryogenesis. Any auxin exogenously supplied would then increase the auxin level in the cells above this critical level can lead to inhibition of embryogenesis. Secondly, a polarized distribution of endogenous auxin in the cells may be essential for embryogenesis. Disturbance of the polarized distribution of endogenous auxin in the cell clusters or callus tissue by an inappropriate exogenous auxin would then inhibit the action of endogenous auxin and cause inhibition of embryo formation (Fujimura and Komamine, 1979; Cook et al., 1993; Fehér et al., 2001).
In a research article by Patel and Patel (2013) on callus induction in internodal explants of Tecomella undulata (desert teak), both NAA and 2,4-D individually at 2.5-3 mg l− 1 induced good callogenesis mostly with friable texture. Their results are partially in agreement with the present results. The most likely reason for unresponsiveness/poor response of the ovary explants to 2,4-D in induction of EC, could be supra-optimal concentrations of 2,4-D added to the induction media. The present results suggest that 2,4-D at less than 0.45 µM might be highly inductive of callogenesis in ovary explants. Another alternative is the use of a cytokinin like kinetin at relatively low concentrations in combination with 2,4-D. There was no report of SE by Patel and Patel (2013). Apart from genotypic difference, it seems the explant type used in the two studies had a crucial role in achieving SE. There has been a dominant assumption for a long time that auxin is produced exclusively in young growing parts, such as leaves, flowers and root tips, and is delivered to action sites by a combination of long distance transport and short cell-to-cell polar auxin transport. However, Robert et al. (2015) highlighted the importance of local auxin biosynthesis for reproductive organ and embryo development processes. According to them, local auxin biosynthesis occurs in very few cells in specific developmental windows in stamens, gynoecia, ovules and embryos. These recently detected sources of auxin influence the flow of auxin within the reproductive tissues and embryos, and thus the formation of auxin gradients which play key roles in organ morphogenesis. Therefore, local auxin biosynthesis might be an upstream component and trigger of all auxin-mediated developmental programs. Keeping these facts in the mind, ovary explants are logically richer in auxin content compared to other vegetative tissue-originated explants especially those which are not known to be auxin producing plant parts such as mature internodal segment explants used by Patel and Patel (2013). Therefore, we can strongly assume that ovary explant possesses more cells capable of SE.
Previously Tarre et al. (2004) illustrated that SE and adventitious root initiation had a common origin histologically. The findings obtained in the present experiment is highly compatible with theirs, confirming positive effect of relatively high NAA in inducing SE as well as initiating adventitious rooting. Occurrence of adventitious rooting during SE induction using an auxin-fortified medium have also been reported earlier in several species such as Dalbergia sissoo (Singh and Chand, 2003), Olea europaea (Pires et al., 2020) and Koelreuteria paniculata (Yang et al., 2018). This phenomenon uncovers that fact that non optimal concentration(s) and type of auxin in culture medium could divert partially or totally the SE path within the explant.
3.2 Ovary callogenesis affected by BA, TDZ and high NAA
In order to know how ovary explant respond to CKs, BA and TDZ were used as medium supplements at different concentrations. NAA was also tested at higher concentrations to find out perception of the explant response at elevated levels. BA induced callogenesis at rate of 47 to 94 percent and the best concentration was found 44.4 µM (Table 2). Similarly, TDZ was able to induce callusing in the whole range used. Increase in TDZ level resulted in significant decline in the callogenesis rate. The cent percent callogenesis was achieved with 0.45 µM TDZ though not significantly different from that obtained with 44.4 µM BA. NAA applied at concentrations as high as 27 to 54 µM, yielded the poorest callus induction compared to both CKs used.
Raising BA level from 22.2 to 66.6 µM reduced callus quantity drastically. The same trend was also observed with TDZ. While in the first experiment the highest callus quantity was achieved in the range of 10.8 to 21.5 µM NAA, here the callus quantity reduced to the scale of moderate even poor by the rise in NAA level to 27 µM and more.
Regenerated calli by all the three PGRs had compact texture. Callus color varied from light to dark brown for BA supplemented media, brownish green to dark brown for TDZ and light brown to brownish green for NAA. The common feature observed with all three PGS was that, the higher concentration of the PGS in the medium, the darker the callus color became. Taking into account all callogenesis traits, the medium fortified with 0.45 µM TDZ was concluded as the best inductive treatment. Altogether, though the best callogenesis was obtained with TDZ at low concentrations but none of the treatments, neither the CKs nor NAA, could bear EC as there was no sign of formation of PEMs or pre-embryos. Another tip that led us to this conclusion was the fact that somatic embryos mostly originate from soft friable callus (Matsuoka and Hinata, 1979) or granular, white or yellow calli (Gambino et al., 2021) which was not observed in this experiment. Therefore, the whole induced calli were considered as non-embryogenic.
CKs are a group of phytohormones derived from adenine, which consist of a variety of molecular structures. The most abundant CKs in plants are adenine-type species (Li et al., 2021). As mentioned above, apart from auxins, various CKs both natural and synthetic including BA, Kinetin, TDZ, 2iP and Zeatin have been used for callus induction in numerous tree species (Giri et al., 2004). In Tecomella undulata, previously, profuse callusing with fewer shoot buds per explant was announced by Tiyagi and Tomar (2013), Rastgoo (2014) and Chhajer and Kalia (2017) who tried to propagate it through in vitro organogenesis pathway using BA.
TDZ, a substituted phenylurea compound with strong cytokinin-like activity, originally used as a cotton defoliant (Dinani et al., 2018), has received remarkable attention as a potent regulator of explant morphogenesis in in vitro regeneration systems and been used in a wide variety of plants (Huetteman and Preece, 1993; Lu, 1993, Dinani et al., 2018). Reviewing literature manifests that TDZ has proved to be a great inducer of a diverse array of cultural responses ranging from induction of callus to formation of somatic embryos in a wide range of plant species including both herbaceous and woody species (Murthy et al., 1998, Huetteman and Preece, 1993, Lu, 1993, Dinani et al., 2018). Murthy et al. (1998) listed a number of species stimulated for in vitro SE through inclusion of TDZ in the culture media. TDZ is a substitute for the auxin/cytokinin requirement that is needed during SE, thereby increasing the number of formed somatic embryos (Murthy et al., 1998, Dinani et al., 2018). There are numerous reports showing employment of TDZ for SE in many woody species such as white ash, eastern black walnut, Rubus, and Vitis vinifera (Huetteman and Preece, 1993), Azadirachta indica (Murthy and Saxena, 1998), Coffea spp. (Giridhar et al., 2004), and blueberry (Vaccinium corymbosum × V. angustifolium) cultivars (Ghosh et al., 2018). Several reports indicated TDZ at very low levels could induce direct SE (Qureshi and Saxena, 1992; Iantcheva et al., 1999; Lin et al., 2004). Chhajer and Kalia (2017) found addition of TDZ in the culture medium unsuitable for shoot multiplication of T. undulata as it led to proliferation of excessive callus. Despite difference in explant type (nodal segment used by them), profuse callus-inducing properties of TDZ is a common feature between Chhajer and Kalia's results and the results of the present work. TDZ has also been capable of shoot organogenesis in various species (Lu, 1993; Dinani et al., 2018).
Higher levels of CKs are known to induce programmed cell death in cell cultures (Carimi et al., 2003). CKs are also known to enhance biosynthesis of ethylene (Abeles et al., 1992) thereby applying them at relatively high levels can adversely affect the growth of cultures and lead towards aging. Testing lower concentrations of this PGRs category might bear positive results.
3.3 Auxins influenced callus proliferation and triggered SE variably
Upon successful induction, ECs continue to grow to produce new masses when regularly subcultured on fresh maintenance medium amended with auxins or CKs at lower or same concentration to the induction medium till cultures are established (Isah, 2016). Hence, in order to gain sufficient EC for the next step viz. induction of somatic embryos, as well as evaluating response of NAA-induced ECs to a wider range of auxins, the ECs produced using low NAA, were transferred onto the media containing NAA, IBA and IAA at variable concentrations and combinations. A PGR-free medium was also included as the control. The results have been displayed in Table 3. The highest explant survival rates were recorded in the medium devoid of PGR (80%) and the media containing 40.5 µM NAA with and without 12.3 µM IBA (77.7% and 66.7%, respectively). The percentage of proliferated explants recorded the highest values in the media with 40.5 and 27 µM NAA, each combined with 12.3 µM IBA (77.7% and 56.3%, respectively). The medium containing 54 µM NAA alone showed the lowest figures for both explant survival rate and proliferation rate.
Having looked at the quantity of callus proliferation, the best results belonged to the media with NAA alone (40.5 µM) as well as with NAA + IBA (27 + 12.3) µM. Callus texture was friable in the control as well as the medium with 27 µM NAA alone whereas the remaining media proliferated compact callus. Callus color was light green to light yellow or lemon-colored in almost all the media excepting the medium fortified with NAA 54 µM that changed the color slightly brownish.
Apart from callus proliferation, some embryogenic cases were observed in some of the media. The control treatment protruded several individual yellow-colored globular and heart-shaped somatic embryos in a few explants (Fig. 3a). All those media contained NAA alone, exhibited development of PEMs and revealed numerous greenish/yellowish globular and heart-shaped somatic embryos in most of their survived explants (Fig. 3b). The strongest demonstration of SE was observed with 54 µM NAA that could induce and develop a high number of globular and heart-shaped embryos, up to 34 somatic embryos per explant in some cultures.
Combination of IBA and IAA with NAA, in the concentrations used here, had inhibitory effect on induction and development of somatic embryos in the proliferated EC so that it could be inferred, cautiously, both IBA and IAA might have negative impact on somatic embryo formation in this species under the conditions prevailing in this research. Previously, similar comment about negative impact of IBA in culture media on somatic embryo induction in European spindle tree has been reported by Bonneau et al. (1994).
Overall, a conclusion drawn from this experiment is that, for the purpose of proliferation of friable EC, the PGR-free medium is the best choice. This outcome is in accordance with some earlier reports (Adil et al., 2018; Hernandez et al., 2003; Mauri and Manzanera, 2005; Estabrooks et al., 2007; Fernandez-Guijarro et al., 1995). In the meantime, looking for induction of SE led to the medium containing 54 µM NAA alone. However, a scrutinized look on the results disclosed that the medium received 40.5 µM NAA could also yield an intermediate output as it showed reasonable rates of explant survival and callus proliferation as well as satisfactory percentage of embryo development. Announcing NAA as a suitable growth regulator for proliferation of embryogenic tissue has been previously mentioned in some other species such as Picea abies (Hazubska-Przybył et al., 2020) and Fraxinus mandshurica (Liu et al., 2020).
While in this experiment the satisfactory proliferation of EC as well as somatic embryo development was obtained in the medium containing 40.5 µM NAA, Corredoira et al. (2013) introduced MS medium supplemented with a cytokinin (0.44 µM BA) as the best proliferation medium for induction of secondary somatic embryos in alder tree (Alnus glutinosa). They also found that adding NAA (0.54 µM) to the medium, dropped secondary embryogenesis coefficient significantly. Similarly, Valladares et al. (2006) and Mallo´n et al. (2012) maintained embryogenic lines of mature oak trees (Quercus robur) on MS medium supplemented with 0.44 µM BA and 0.27 µM NAA. Such distinct and even contradictory results can only be interpreted by considering all probable internal and external factors that influence the endogenous hormonal balance of the explant employed for each species. It is, therefore, important to find a reliable formula for culture medium composition, particularly with respect to PGRs, that can support proliferation of the already produced EC which in turn needed to ensure sufficient availability of explant material for induction of somatic embryos at the next phase, concurrently insuring maintenance of its embryogenic potential which is even of higher importance.
3.4 Low BA positively affected further development of the ECs
In order to advance SE and development of PEMs and early stages somatic embryos into mature somatic embryos, the proliferated ECs obtained during the experiment 3 carrying embryonic structures, were transferred onto MS modification No.2 medium devoid of growth regulator (Control), and also onto the media supplemented with GA3 and BA at different concentrations individually. Sucrose was also tested at two levels; standard and high dose (30 and 70 g l− 1) when GA3 was used as the sole source of PGR.
According to Table 4, the media containing 30 g l− 1 sucrose whether with no PGR or with GA3 (1.45 and 2.9 µM) could not advance the path of SE and did not yield any promising outcomes. There are reports with similar results of inhibitory effects of GA3 in somatic embryo formation and subsequent development in some other systems (Chen et al., 2010; Hutchinson et al., 1997). However, the stimulatory effect of GA3 in somatic embryo induction, formation or germination has also been reported in other species like Medicago sativa (Rudus et al., 2000), Cocos nucifera (Montero-Co´rtes et al., 2010). While a supporting report of Rudus et al. (2000) theorized that the level of endogenous GAs is presumably sufficient for callus induction and growth but not optimal for the induction and particularly for the differentiation of embryos, an opposing report by Hutchinson et al. (1997) concluded that the presence of GAs during both the induction and expression phases of SE was significantly detrimental to somatic embryo formation. It seems species- as well as explant-dependent endogenous hormonal balance especially intrinsic content of gibberellins would ultimately determines the need or no need for using exogenous gibberellins in the developmental phases of SE process.
Regardless of GA3 concentration, elevating sucrose level to 70 g l− 1 not only did not improve further development of EC but also paralyzed explant response entirely, ceased the minute callus proliferation which occurred with 30 g l− 1 sucrose, absolute failure was observed and the explants degenerated gradually. This result is contradictory to Dennis Thomas's report (2006) that showed beneficial influence of 200 mM (~ 70 g l− 1) sucrose incorporated into the GA3-supplemented medium in embryogenesis response of Tylophora indica.
CKs have been shown to promote secondary SE in several woody species (Jime´nez 2005). Fortifying the media with BA at low concentrations (0.9 and 4.44 µM) led to recurrent callus proliferation and more importantly induction of secondary SE in a number of the explants (Fig. 4a). Quantity of newly proliferated callus with the low levels of BA was assessed as good to excellent, exhibiting light green to brownish color and compact texture. Raising BA level to 22.2 µM though maintained callus proliferation status (Fig. 4b) but inhibited occurrence of secondary SE which was observed at low concentrations.
The ultimate encouraging conclusion drawn from this experiment was that for the goal of maintenance of SE capability of the beforehand produced ECs, the optimum medium is the one that its sucrose level is kept at 30 g l− 1 and supplemented with BA in the range of 0.9–4.44 µM. This conclusion is in agreement with that of Corredoira et al. (2013) with embryogenic lines of Alnus glutinosa and Liu et al. (2020) with cotyledon-derived EC of Fraxinus mandshurica. Substitution of sucrose by other carbohydrate sources or osmotic agents has been proposed as an effective alternative tool for pushing forward SE process for some species not responding satisfactorily with sucrose (Santos et al., 2002).