Keywords

6-Benzylaminopurine (BAP, also known as N6-benzyladenine: BA) was long the most widely used cytokinin (CK) in micropropagation systems, due to its efficacy and affordability (Holub et al. 1998). However, its adverse effects on the growth, rooting, and acclimatization of some recalcitrant species and induction of other physiological disorders (Aremu et al. 2012a) prompted the search for and subsequent discovery of viable alternatives: the choice of CK remains critical to the success or failure of any newly developed micropropagation protocol (Bairu et al. 2007; Werbrouck 2010; Aremu et al. 2012a; Valero-Aracama et al. 2010). The increasing importance of hydroxylated derivatives commonly referred to as topolins (Strnad et al. 1997) in micropropagation has been critically reviewed more recently (Aremu et al. 2012a, 2017).

Following description of the basic aspects of CK activity of topolin ribosides (Kaminek et al. 1987) and unambiguous identification of 6-(3-hydroxybenzylamino)purine (meta-topolin, mT) and its derivatives as naturally occurring CKs in planta (Strnad 1996, 1997; Strnad et al. 1997), pioneering work was published on the metabolism and in vitro effects of mT in micropropagated Spathiphyllum floribundum (Werbrouck et al. 1996). mT was compared to BAP, 6-benzylamino-9-(2-tetrahydropyranyl)purine (BAP9THP), and 6-benzylaminopurine-9-β-D-riboside (BAPR) using an HPLC separation system, coupled to a tandem quadrupole mass spectrometer (MS/MS) equipped with an electrospray interface (ESI). In vitro, BAP and its 9-substituted derivatives BAP9THP and BAPR were mainly converted into the stable and inactive metabolite, 6-benzylamino-9-β-d-glucopyranosylpurine (BAP9G) (Fig. 6.1), located mostly in the basal part of the micropropagated plant (Werbrouck et al. 1995). In contrast, 6-(3-O-β-d-glucopyranosyl)benzylaminopurine-9-β-D-riboside (mTROG) (Fig. 6.1) was identified as the main metabolite of mT. This new cytokinin-O-glucoside, which was present in all plant parts, was formed much faster than BAP9G during acclimatization process. The effect of BAP and mT on in vitro shoot and root production and ex vitro rooting was then compared (Werbrouck et al. 1996). Only mT combined sufficient shoot production with acceptable in vitro root formation. The plants developed on medium with 10 μM or more mT rooted better also during acclimatization in comparison with those grown on medium with comparable BAP concentrations.

Fig. 6.1
figure 1

Chemical structure of 6-benzylamino-9-β-D-glucopyranosylpurine (BAP9G) and 6-(3-O-β-D-glucopyranosyl)benzylaminopurine-9-β-D-riboside (mTROG)

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Very similar results were later obtained by Bairu et al. (2011) in micropropagated Harpagophytum procumbens tissues, where changes in endogenous CK profiles and the physiological implications of this in relation to shoot-tip necrosis (STN) and CK treatments were also studied. Generally, necrotic shoots contained more total CKs compared to normal shoots, and CK accumulation was higher in the basal section. More importantly, further analysis of structural and functional CK forms revealed the inability of BAP to form O-glucosides as well as excessive accumulation of 9-glucosides (irreversible deactivation product) in necrotic and basal callus like tissues of BAP-treated shoots (Bairu et al. 2011). The addition of IAA enhanced the formation of 9-glucosides in BAP-treated cultures. The symptom of STN could therefore be attributed to conversion of active CK to other forms such as 9-glucoside (Bairu et al. 2011). On the other hand, the presence of a hydroxyl group in their molecule gives topolins a structural advantage over BAP. This was reflected in the presence of a generous amount of O-glucosides in topolin-treated samples and hence little or no CK shortage (Bairu et al. 2011). Moreover, the level of irreversible inactivation (9-glucoside formation) of mT was found to be even lower when exogenously applied in the form of 9-riboside, compared with free base application.

As the most efficient plant growth regulator (PGR), 10 μM 6-(3-hydroxybenzylamino)purine-9-β-d-ribofuranoside (mTR, Fig. 6.2) treatment also produced the highest number of shoots (approximately five shoots per explant) during clonal propagation of Lachenalia montana, a species endemic to Southern Africa and extensively traded as ornamental plants in the international floriculture industry (Aremu et al. 2017b). Based on the concentrations of endogenous CKs subsequently determined, 10 μM mTR regenerants also had the highest CK levels which were mainly of the aromatic type (98%). In terms of the functional role of the CKs, O-glucosides were again the dominant CK metabolites in the regenerants of the 10 μM mTR treatment. On the other hand, the insufficient rooting, predominantly in regenerants of the BAP treatments, was closely related to the high accumulation of N9-glucosides compared to regenerants from other treatments. These findings provided further evidence of the interrelationship between exogenous topolin application, positive phenotypic responses, and endogenous CK levels in the in vitro regenerants (Aremu et al. 2017b).

Fig. 6.2
figure 2

Chemical structure of 6-(3-hydroxybenzylamino)purine-9-β-d-ribofuranoside (mTR) and 6-(3-hydroxybenzylamino)-9-tetrahydropyran-2-ylpurine (mT9THP)

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Based on these studies, a series of attempts was made to prepare other topolin derivatives substituted at the 9-position using various protective groups to improve the specific biological functions of the CKs already routinely used in the plant micropropagation industry. For example, inspired by discovery of the protective role of ribose in the 9-position (Bairu et al. 2009). Szüčová et al. (2009) prepared several substituted 6-benzylamino-9-tetrahydropyran-2-ylpurine (THPP) and 9-tetrahydrofuran-2-ylpurine (THFP) derivatives, with hydroxy and methoxy functional groups at various positions on the benzyl ring. The new compounds were synthesized by condensation of 6-chloropurine with 3,4-dihydro-2H-pyran or 2,3-dihydrofuran and then by the reaction of these intermediates with the corresponding benzylamines (in n-propanol or n-butanol, in the presence of triethylamine). Identity and purity of the prepared compounds were confirmed by CHN analysis, TLC, HPLC, melting point determinations, CI+ MS, and 1H NMR spectroscopy. The CK activity of the prepared derivatives was determined in three classical cytokinin bioassays (tobacco callus, wheat leaf senescence, and Amaranthus bioassay).

In another study (Podlešáková et al. 2012), in contrast to canonical CKs, the 9-tetrahydropyranyl derivative of mT (Fig. 6.2) and its methoxy counterpart showed negative effects on root development at only three orders of magnitude higher concentration. The methoxy derivative also demonstrated a positive effect on lateral root branching and leaf emergence in nanomolar concentration range in comparison with untreated plants. Tetrahydropyranyl substitution at the N9-position of CK purine ring was also found to significantly enhance acropetal transport of a given CK. Together with the methoxy substitution, inhibition of the formation of non-active CK glucosides in roots allows gradual release of the active base and has a significant effect on the distribution and amount of endogenous isoprenoid CKs in different plant parts (Podlešáková et al. 2012). These results provided a basis for anticipating that the use of novel aromatic CK derivatives could improve the expected hormonal effects in plant propagation methodology in the future.

This led to 9-THP topolin derivative (Fig. 6.2) being successfully used in various micropropagation systems, for example, two widely used medicinal plants, Aloe arborescens and Harpagophytum procumbens (Amoo et al. 2014). In terms of A. arborescens shoot multiplication, mTTHP and mT (at equimolar level) showed similar effects, and both were comparably better than the control and BAPR. In H. procumbens, mT-treated cultures were the most responsive to treatment at 2.5 μM compared to the control. At 5.0 μM concentration, mT9THP and mTR demonstrated a similar activity on shoot proliferation. Particularly at low concentrations, mT9THP had a better rooting stimulatory activity than the other CKs in both plant species. It is conceivable that mT9THP is another viable alternative topolin with the added advantage of inducing rooting at low concentrations (Amoo et al. 2014). Later (Amoo et al. 2015), this compound was also successfully used to improve micropropagated Merwilla plumbea shoot production without rooting inhibition as well as its positive carry-over effect on ex vitro growth. Unlike mTTHP treatments, an increase in concentration of mTR or TDZ, other tested CKs, beyond 0.5 μM resulted in a significant decrease in the concentrations of all the photosynthetic pigments quantified. Even after 6 months of ex vitro growth, regenerated plants of the 0.5 μM mTTHP treatment had the significantly higher total leaf area, total leaf fresh weight, and bulb size compared to all mTR- and TDZ-treated plants (Amoo et al. 2015).

In a similar study on Merwilla plumbea (Lindl.) Speta, a popular and highly sought-after South African medicinal plant with diverse therapeutic uses (Aremu et al. 2014), the effect of another meta-topolin derivative, 6-(3-methoxybenzylamino)-9-tetrahydropyran-2-ylpurine (MemTTHP), was evaluated on the growth and level of endogenous CKs during micropropagation and acclimatization stages. A total of 37 (22 isoprenoid and 15 aromatic) CKs were determined in both in vitro and ex vitro acclimatized plants. Based on their metabolic function, these were separated into five different groups, including free bases, ribosides, ribotides, and O- and 9-glucosides. In addition to enhancing our understanding of the hormone physiology in M. plumbea, the current findings were discussed in line with the effect of exogenously applied CK on the observed differences in growth before and after the important stage of acclimatization. The observed dynamics in endogenous CK can provide an avenue to optimize the in vitro growth and development of investigated species.

Another successful attempt to enhance the anti-senescence properties of topolins was described by Doležal et al. (2017) and Matušková et al. (2020), by preparing their 9-β-d-arabinofuranosyl or 9-β-d-2′-deoxyribofuranosyl derivatives (Fig. 6.3) via a one-step reaction. The starting material, optically pure unprotected 9-(2′-deoxy-β-d-ribofuranosyl)hypoxanthine (2′-deoxy-9-β-d-inosine) or 9-(β-d-arabinofuranosyl)hypoxantine, and BOP [(benzotriazol-1yloxy)tris(dimethylamino)phosphonium hexafluorophosphate] were dissolved in dry DMF (3 mL) under nitrogen or argon atmosphere at 55–60 °C, and DIPEA was added, followed by appropriate substituted benzylamine (1.2 eq.) as the last component. The resulting white solid was isolated by filtration and re-crystallized from EtOH. The synthesized compounds were characterized by CHN and melting point analysis, analytical thin layer chromatography, high-performance liquid chromatography, ES+ MS spectrometry, and 1H NMR. The positive effect of selected derivatives on shoot proliferation in Harpagophytum procumbens and Amelanchier alnifolia as well as control of shoot-tip necrosis in in vitro cultures of the medicinal plant Gymnosporia buxifolia was then demonstrated (Doležal et al. 2017).

Fig. 6.3
figure 3

Chemical structure of 6-(3-hydroxybenzylamino)purine-9-β-d-arabinofuranoside (mT-ara) and 6-(3-hydroxybenzylamino)purine-9-(2′-deoxy-β-d-ribofuranoside) (mTdR)

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Modulating the CK status with inhibitors of CK perception and/or degradation may affect the general physiology of the plant (Dwivedi et al. 2010; Motte et al. 2013; Zatloukal et al. 2008). For this reason, regulation of phytohormone metabolism may be another potential way to improve plant growth and development during micropropagation.

The effect of supplementing either mT or BAP requiring cultures with INCYDE (2-chloro-6-(3-methoxyphenyl)aminopurine) (Fig. 6.4), an inhibitor of CK degradation (Spichal et al. 2012), on the endogenous CK profiles and physiology of banana in vitro was hence investigated (Aremu et al. 2012b).

Fig. 6.4
figure 4

Chemical structure of 2-chloro-6-(3-methoxyphenyl)aminopurine (INCYDE) and 2-(1-ethyl-2-hydroxyethylamino)-6-benzylamino-9-isopropylpurine (roscovitine)

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Another interesting alternative approach for decreasing levels of irreversible topolin deactivation in banana tissue cultures was developed by Aremu et al. (2012b). An inhibitor of 9-glucosylation, roscovitine 2-(1-ethyl-2-hydroxyethylamino)-6-benzylamino-9-isopropylpurine (Fig. 6.4), which was previously discovered among a number of 2,6,9-trisubstituted purines, tested as potential N-glucosylation inhibitors (Blagoeva et al. 2004; Letham et al. 1977; Dwivedi et al. 2010). Aremu et al. (2012b) demonstrated that its application simultaneously with exogenous CK(s) in the cultivation media has the potential to change endogenous CK pools, thereby influencing the rooting and ex vitro acclimatization of in vitro-derived Musa spp. It was observed that plantlets regenerated from mT + roscovitine media produced the most shoots. They also had the highest total CK content (661 pmol/g FW) with the roots having approximately 68-fold more than the shoots (Aremu et al. 2012b).

A general trend observed was that the addition of roscovitine and/or INCYDE with mT improved the total CK pool in both roots and shoots of the tissue-cultured ‘Williams’ banana regenerants (Aremu et al. 2012b). A similar pattern was determined in the shoots when BAP was supplemented with roscovitine and INCYDE; however, both compounds reduced the total CK pool in the roots as well as the sum total in the plantlets. It is noteworthy to highlight that the reduction in total CK pool was mainly due to the decrease in the level of 9-glucosides, which are generally detrimental to plant growth (Aremu et al. 2012b).

6.1 Future Perspectives: Application of Fluorinated Compounds

Fluorination has a long tradition in nucleoside chemistry. It was demonstrated that replacement of the 2′ or 3′ hydroxyl groups of a nucleoside with a fluorine atom causes only a minor change in the total structure, but significantly affects the stereoelectronic properties of the sugar moiety (Thibaudeau et al. 1998). Fluorine substitution has been extensively investigated in drug research and biochemistry as a means of increasing biological activity and enhancing chemical or metabolic stability. However, to date only a few fluorinated CK derivatives have been prepared and their biological activity tested (Clemenceau et al. 1996; Doležal et al. 2006, 2007). Later, Murvanidze et al. (2019) evaluated the impact of 6-(3-fluorobenzylamino)purine (mF-BAP) and its 9-β-d-riboside (mF-BAPR) (Fig. 6.5) on in vitro cloning of Phalaenopsis hybrids, which are usually characterized by slow growth and low multiplication rates. The plantlets formed significantly more but smaller new shoots when treated with mF-BAPR (25.3) compared to mF-BAP (14.6) and BAP (7.0). The results suggest the following strategy: massive micropropagation of small shoots for a number of cycles on mF-BAPR in closed containers, followed by a final step with BAP in filter vessels to produce large shoots with roots. For this reason, the use of fluorinated topolins might present a breakthrough in the in vitro micropropagation of Phalaenopsis (Murvanidze et al. 2019). Another class of N6-substituted-2′-deoxy-2′-fluoro-9-β-d-arabinofuranosylpurine derivatives (Fig. 6.5) was very recently prepared, and their biological activity is currently being evaluated (Bryksová et al. 2020).

Fig. 6.5
figure 5

Chemical structure of 6-(3-fluorobenzylamino)purine-9-β-d-ribofuranoside (mF-BAPR) and 6-(3-hydroxybenzylamino)purine-9-(2′-fluoro-2′-deoxy-β-d-arabinofuranoside) (mT-Fara)

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