In vitro morphogenesis
In vitro morphogenesis offer potentials for study of plant transdifferentiation developmental events, based on biosynthetic capacity to produce tissues and organs with application in the production of high-quality and disease-free plants and phytochemicals [9–11]. The system involves formation of meristemoids from single or globular mass of cells to produce organized structures such as somatic embryos, shoot or root primordium, and has been achieved with many plant species . In the case of in vitro morphogenesis that involves a woody species, explant juvenility plays key role in the morphogenic response, and only possible with many species when a juvenile is used [9, 8, 23], the reason callus cultures derived from zygotic embryo explants were cultivated on MS medium supplemented with cytokinins or their combinations with NAA at lower levels in the present experiment. Callus initiation was achieved with 2,4-D +BAP (3.0 +0.5 mg L-1) using explants of 3–4 weeks old axenic seedlings (Fig. 1d–j). With most of the explants, the induction was observed within second week of culture at differential frequency and intensity. Formation of the callus was more and earlier in seed embryos followed by cotyledons, leaves, hypocotyls and root explants, and within 4–6 weeks of cultivation, most of the explants transformed to white friable callus of faster biomass accumulation (Fig. 1k). In the course of maintaining induced callus cultures, formation and leaching of latex into cultivation medium that resulted in callus browning and necrosis occasionally occurred (Fig. 1x). Such brownish callus cultures (Fig. 1x) remained brown and non-morphogenic over several subcultures. However, addition of ascorbic acid (0.5 mg L-1) overcame the condition in most of these cultures, without organogenesis achieved from the callus in subsequent cultivation duration.
For morphogenic effects of BAP, TDZ or Kin on callus cultures of C. fragrans, the callus was cultivated on MS medium supplemented with their various concentrations or when in combination with NAA (0.5 mg L-1). Callus cultures were cultivated on the same PGRs-amended medium for at least three passages, and in vitro morphogenesis in the form of shoot bud/primordial or somatic embryogenesis (SE) not stimulated. However, variable growth and degree of callus morphologies were observed in most of the cultures (Fig. 1l–w). Due to its high cytokinin activity, TDZ is regarded most effective PGR for promoting in vitro morphogenesis in woody plants, and even at low concentration can facilitate efficient in vitro response of recalcitrant plants by initiating shoot bud primordial formation, SE or callus induction [24, 25]. The intricate morphogenesis signal routes induced by PGRs are yet unclear but, treatment with their concentrations had stimulated formation of nodular-green morphogenic callus in many plants [e.g, 26, 27]. In the present experiment, cultivation of C. fragrans callus cultures on solid medium added with 0.5 mg L-1 of the cytokinins could not induce differential callus morphology about PGRs-free media cultivation. However, with the TDZ slight change in morphology of the callus cultures into dark brown was observed. The dark browning showed increased intensity accompanied by increased compactness of callus cultures with the increase in concentration of TDZ amended in the cultivation medium (Fig. 1t–w). In many reported studies in the literature lower levels of the TDZ was found sufficient for stimulating shoot organogenesis from cotyledonary explant while higher concentrations promoted SE . Cultivation of rhizogenic callus cultures on medium supplemented with lower levels of the TDZ stimulated shoot formation and SE at low frequency  while it promoted formation of embryogenic callus from root tip, stem and leaf segments explants . Thidiazuron at low or high concentration was found efficient in stimulating high frequency shoot morphogenesis from shoot tip-derived callus cultures . It was also found efficient for the induction of shoot from seed embryo-derived callus cultures [32, 33]. However, in an instance, cultivation on TDZ-supplemented medium induced semi-compact callus without organogenesis .
For other cytokinins, cultivation of C. fragrans callus cultures on medium supplemented with BAP or Kin concentrations resulted in differential callus morphologies from friable, compact to nodular-green; most of the concentrations stimulated pigmentation at variable intensity without organogenesis, and BAP was the most efficient over Kin (Fig. 1l–o: p–s). The green pigmentation showed increased intensity at higher concentration of the cytokinins, and after three subcultures of the callus on solid medium supplemented with the same concentration of the cytokinins, in vitro morphogenesis was not observed. Due to unhindered diffusion of media components permitted in liquid medium cultivation, morphogenic response of in vitro cultures can be initiated by an enhanced uptake of nutrients and supplements, as found efficient in stimulating in vitro morphogenesis with many species [e.g, 35, 36]. In the present study, when green non-organogenic callus cultures were cultivated in liquid medium (Fig. 1y–z, 1) with a weekly replenishment of medium nutrients, in vitro morphogenesis was not stimulated after four weeks and subsequent subcultivations, suggesting that C. fragrans is recalcitrant to indirect organogenesis, at least from seed embryo-derived callus cultures under the experimental conditions of the present study. The fact was supported by additional experiment to assess synergistic action of cytokinins with auxin and its capacity to stimulate formation of organized structures. Callus cultures were cultivated on solid medium amended with low levels of NAA in addition to the cytokinins concentrations. Such cultivation could not result in substantial difference in morphogenic response observed with the other cytokinins concentrations. Even at low levels, the addition of BAP or Kinetin (Kin) has been found efficient for shoot initiation with many medicinal and woody plants [16, 36–39] and with some of the plants, it promoted cell growth along with morphogenic callus formation without organogenesis. For instance, it occurred at higher rate in liquid over solid medium cultivation with synergistic response obtained when auxin was supplemented along with the cytokinins [37, 40–42]. In this study, transfer of the morphogenic callus cultures into liquid medium were ineffective in stimulating formation of organized structures from callus cultures. Browning effect due to latex and phenolics release from the callus cultures into cultivation medium that resulted in callus necrosis was observed but, amending ascorbic acid (1.0 mg L-1) into the cultivation medium overcame the effect without organogenesis capacity shown by the cultures (Fig. 1x, 1–2). In other reported studies in the literature, indirect shoot morphogenesis from callus induced from various explants involved formation of hard, green and compact calli on cytokinins-amended medium, and occurred at very low frequency to absent even after second subcultivation of callus due to hardening and habituation [38, 43]. In Garcinia mangostana, BAP stimulated formation of nodular callus before shoot organogenesis from leaf-derived callus cultures  while shoot morphogenesis and SE were stimulated by its different concentrations when cotyledonary leaf segment-derived callus cultures of Digitalis lamarckii were cultivated on medium added with the PGR . Cultivars-based differential organogenesis was observed when mature seed embryo-derived callus cultures were cultivated on medium amended with different concentrations of BAP [46, 47] while genotypes-dependent differential regeneration efficiency occurred with callus cultivated on medium amended with combinations of BAP and Kin . Although Kin has been used to stimulate indirect organogenesis with many species, it is less efficient than BAP in most of the reported literature due to higher efficiency of plant tissues to metabolize BAP over Kin and enhanced stimulating effect it has on biosynthesis of endogenous hormones in plant tissues [44, 49]. Superior degree of callus pigmentation stimulated by BAP over Kin concentrations observed in the present experiment supports the explanation. However, low and higher concentrations of the two cytokinins was found insufficient in stimulating formation of organized structures from the callus cultures, possibly due to genetic background of C. fragrans that lack capacity for indirect regeneration from seed embryo-derived callus cultures as competence for indirect organogenesis is species and explant-specific in some plants. Ability of in vitro cultivated plant cells, tissues or organ to respond to exogenous applied PGRs in a cultivation medium with resultant morphogenic response is dependent on interaction between endogenous and exogenous factors of culture conditions [9, 48]. Therefore, inherent capacity to synthesize and respond to exogenous applied cytokinins is essential for successful induction of in vitro morphogenesis in plants; higher capacity to synthesize cytokinins imply more responsiveness to shoot organogenesis. Increasing concentration of the PGRs in cultivation medium is irrelevant when cultivated tissue is unresponsive to the in vitro morphogenesis .
Somatic embryogenesis, an in vitro morphogenic program by which somatic plant cells acquire competence for the formation of embryogenic tissue that further differentiates into somatic embryos through its various developmental stages [51–53]. Strategies that involve manipulating culture medium composition and conditions can be employed to switch somatic program to the embryogenic in a plant in vitro cultures [54–57]. In this study, callus cultures were cultivated on solid medium amended with 2,4-D (3.0 mg L-1) for at least three passages before used in the SE study. Cultivation of the callus cultures on PGRs-free medium (control), lower concentrations of 2,4-D or NAA (0.25, 0.5, 1.0 mg L-1) and their combinations with BAP, TDZ or Kin (0.25, 0.5, 1.0 mg L-1) were found insufficient for stimulating embryogenic tissue formation or SE, suggesting that at least for mature seed embryo-derived callus cultures and under the experimental condition of the study, SE is not attainable in C. fragrans. Although indirect in vitro plant regeneration is the most appropriate developmental pathway for genetic improvement of high-value medicinal plants, the explant and genotype, PGRs type and concentration amended in the cultivation medium, and conditions of physical environment determine the morphogenic response. With some of the plants, the response is not attainable, as observed in an experiment with mature seed embryo-derived callus cultures of C. fragrans in the present study.
Production of camptothecin
Production of camptothecin in explants and resultant callus cultures
Capacity to produce a bioactive molecule produced by ex vitro grown plant when cultivated in the in vitro condition at differential yield was recognized decades ago [11, 58]. However, exploiting endangered plant sources of CPT is relied upon for its supply to pharmaceutical industry for anticancer drugs production [6, 11]. Production of the alkaloid from plant sources is yet to reach commercial application using PCTOC techniques, which has resulted in an endangered status to most plant sources and increasing interest to apply biotechnological strategies of PCTOC in producing CPT [6, 11, 59, 60]. Biosynthesis of the plant secondary metabolites that include CPT shows close correlated expression with morphological and cytological differentiation. However, the degree of the production and its dependence on development is unknown, whether it is genetic or physiological phenomenon in plant cells and tissues [61, 62]. In this study, evaluation of CPT content in the various explants of 4–weeks–old aseptic seedling, mature seed embryo axes and resultant callus cultures revealed that maximum production of the alkaloid was in roots explant (0.019 %). This was followed by mature seed embryos (0.0053 %), cotyledons (0.0039 %), hypocotyls (0.0024 %) and leaves (0.0017 %). Yield of the alkaloid showed significant difference between most of the explants, except in seed embryos and cotyledons (Fig. 2A). However, production of the alkaloid was reduced in callus induced from the explants, and no variation in CPT content of callus cultures derived from various explants was observed. In the reported literature, many studies have shown variations in biosynthetic capacity for alkaloids in differentiated and undifferentiated in vitro-raised cells and tissues [59, 60, 62, 63] and includes anticancer alkaloids such as CPT [64–67]. For instance, biosynthesis of cardenolides and benzylisoquinoline alkaloids in the callus cultures was reported to be dependent on organ redifferentiation [63, 64, 68], as in morphine alkaloid production as well . Green callus cultures that differentiated epidermis and vascular bundles produced alkaloids while cells with limited degree of differentiation contained codeine as the principal alkaloid but, level of morphine production increased as the tissue differentiate , similar to observation made in the present study.
Influence of PGRs on production of camptothecin in seed embryo-derived callus biomass
Plant growth regulators are instrumental composition of culture medium that have profound effect on cell growth, cyto-differentiation and alkaloid production in the in vitro plant cell cultures  and their type, concentration and modification is an effective tool for optimizing cultures condition and improved in vitro biosynthesis of alkaloid [41, 62]. In this study, production of CPT from callus cultures (after dried to constant mass) and in the culture medium were evaluated to assess its influence on biosynthetic capacity about solid medium cultivation. Variation in biomass production and CPT content yield observed with the solid medium cultivation was similarly obtained in the liquid. However, yield of CPT in the callus cultures were reduced to about half the productivity obtained on solid medium cultivation system but, overall productivity relatively higher (Fig. 2B–F), despite the possibility of degradation of the leached CPT in liquid medium cultivation system [20, 71]. For evaluation of the influence of PGRs on biomass production and biosynthesis of CPT in C. fragrans callus cultures, absolute biomass production in callus obtained from in vitro morphogenesis study and CPT production were evaluated by assessment of fresh and dried weight, alkaloid extracted and HPTLC analysis performed . The PGRs amended in the cultivation medium had influential effect on biomass and CPT biosynthetic capacity of callus cultures over the control, and the substantial effect was based on type and concentration of PGRs amended in the cultivation medium (Fig. 2D–E). Control callus cultures cultivated on PGRs-free medium showed lowest biomass and CPT production when compared with the cytokinins-amended medium cultures, suggesting substantial effect of PGRs on the production of biomass and CPT biosynthesis in C. fragrans (Fig. 2B–F). In all the tested concentrations, BAP had more enhancing effect on biomass and CPT biosynthesis over Kin and TDZ. However, the production was dependent on the culture medium type, i.e., solid or liquid medium cultivation system. Corresponding higher production of biomass was achieved in liquid medium cultivation of the callus over the solid added with BAP (4.0 mg L-1) and its combination with NAA (0.5 mg L-1). In the case of solid medium cultivation of the callus cultures, maximum of 1.72g and 2.11g absolute callus biomass was produced on BAP-amended medium after 21 days culture over the lowest with the TDZ where 1.31g and 1.51g were produced (respectively) on medium added with their concentrations. Liquid medium cultivation enhanced production of the biomass with maximum of 2.39g and 2.49g absolute biomass generated by BAP-amended cultures. Correspondingly, TDZ produced 1.81g and 1.95g absolute biomass at similar amended concentration to the BAP and combinations, and culture duration (Fig. 2B–C). Yield of the alkaloid showed similar productivity trend to that of the callus cultures cultivated on solid and in the liquid medium systems. However, the productivity of callus cultures grown in liquid medium (intracellular CPT) was relatively lower than that of solid medium cultivation, which can be explained by the leaching effect of the alkaloid (extracellular) in the case of liquid medium cultivation systems. However, the overall CPT produced by liquid medium cultures were relatively higher (Fig. 2D–F); maximum production of CPT was achieved with BAP and its combination with the NAA where combined intracellular (0.00063+0.00072 mg/g calli DW) and extracellular (0.00071+0.00079 mg/2 mL medium) were biosynthesized, which is relatively higher to that of the solid medium cultivation. Solid medium cultures produced maximum of 0.00093 mg/g calli DW and 0.00098 mg/g calli DW respectively. Correspondingly, TDZ and its combination with NAA produced lowest with combined intracellular (0.00035 mg/g calli DW, 0.00047 mg/g calli DW) and extracellular CPT (0.00044 mg/2 mL medium, 0.00053 mg/2 mL medium) yield, been higher than that produced by solid medium cultivation of the callus cultures (0.00064 mg/g calli DW and 0.00077 mg/g calli DW), respectively (Fig. 2D–F). Cytokinins have enhancing effect on secondary metabolites production [41, 42] and enhance the biosynthesis in the plant in vitro cell cultures by their type and concentration amended in the cultivation medium. Purine-type of the cytokinins have beneficial effect on alkaloids production in the in vitro cultures without adverse effect on callus growth while phenylurea derivatives either inhibit growth of cultures or highly improve biomass production at the expense of alkaloids biosynthesis . Therefore, modification of their combination and ratio is an effective strategy for studying plant secondary metabolites biosynthetic capacity of cultures during in vitro morphogenesis [41, 71]. For instance, in the tender-stem induced callus cultures of Securinega suffruticosa, production of biomass and virosecurinine alkaloids was influenced by sucrose concentration and PGRs amended in the cultivation medium, and combination of Kin with NAA was the best for the productivity . Biosynthesis of indolizidine alkaloids in the cultivated callus cultures of the same species was also enhanced when cytokinins were amended in the cultivation medium, and when in combination with auxins . Kinetin enhanced production of the alkaloids but, no correlation between concentration amended in the culture medium and alkaloids production was established. In the study, about two-fold enhanced biosynthesis of securinine was stimulated in the callus cultures by TDZ treatment. However, allosecurinine level was negatively affected while PGR-free medium cultivation resulted in low production of the indolizidine alkaloids, suggesting the substantial effect of PGRs on biosynthesis of alkaloids [42, 73]. In previous study with N. nimmoniana callus cultures, biomass and CPT production were influenced by the culture medium type and cultivation duration . In the study, significant proportion of the synthesized CPT was leached into the culture medium, resulting into lower yield in its callus cultures. However, cultivating callus cultures on cytokinins supplemented-medium such as Kin or BA influenced biosynthetic capacity of alkaloids, and higher concentrations inhibited the yield . Biosynthesis of indolizidine alkaloids in the callus cultures of S. suffruticosa was enhanced by the type and concentration of cytokinins amended in the cultivation medium, as well as its synergistic action with auxins . In Phyllanthus glaucus shoot cultures, cytokinins types, their combination with auxins or auxins-alone and concentrations supplemented in the cultivation medium influenced the levels of securinega-type alkaloids and biomass production in differential manner . Only BAP enhanced production of the alkaloids while other cytokinins showed inhibitory effect to the securinega-type alkaloids biosynthesis. In the present study, the observed low production of CPT in C. fragrans callus cultures cultivated in liquid medium could be explained by the leaching effect of the alkaloid into cultivation medium and its possible degradation. In callus cultures of Cinchona pubescence, PGRs and culture conditions affected biosynthesis of alkaloids  while culture medium of cultivation and PGRs influenced CPT yield in Camptotheca acuminata . Application of BA enhanced yield of the alkaloid and morphogenic parameters in C. acuminata callus cultures . Rhizome explant-derived morphogenic callus cultures of Alpinia galanga obtained on BAP-added medium produced significant phytoconstituents at higher level over the non-morphogenic .