Introduction

As the fifth most important staple crop in the world, the significance of bananas for food security cannot be overemphasized (Jain 2004). To sustain the world’s rising population, the application of plant tissue culture (PTC) techniques is a viable means of increasing food production (Caponetti et al. 2005). Indeed, micropropagation (PTC) has played a major role in the mass production of clonal material, conservation and breeding of bananas (Vuylsteke 1998; Jain 2004). Despite the success achieved on a global level with the use of PTC, some challenges are still encountered. These include low multiplication rates, rooting inhibition and somaclonal variations amongst others (Bairu et al. 2011a; Aremu et al. 2012). In addition, improved acclimatization and successful establishment in the field remain problematic, especially for large-scale application of PTC techniques (Pospíšilová et al. 1999). Often, acclimatization failures are due to poor development of photosynthetic capacity in vitro, coupled with the induced stress from the artificial environment (Valero-Aracama et al. 2006; Pospíšilová et al. 2007).

In contrast to primary metabolites which are required for basic life processes, plants synthesize and accumulate secondary metabolites in response to ecological and biochemical differentiation (Lewinsohn and Gijzen 2009). Secondary metabolites are useful for plant interaction and survival in their environment (Namdeo 2007). The important roles of secondary metabolites, especially the phenolic compounds in PTC as well as subsequent survival in the ex vitro environment, are well known (Curir et al. 1990; Wuyts et al. 2005; Wu et al. 2007; Buer et al. 2010). Studies have also indicated that CKs regulate the quantity of some secondary metabolites in micropropagated plants (Ramachandra Rao and Ravishankar 2002; Quiala et al. 2012).

In the past few decades, the use of topolins, especially the meta-form in several PTC protocols, has shown promising results in terms of growth as well as minimizing various physiological disorders (Aremu et al. 2012). However, reports on their effects on secondary metabolites are scanty. Findings from such investigations could provide clues and may elucidate additional physiological evidence to support the superiority of topolins over BA in micropropagation of some species. In this study, we investigated the effect of five topolins on the accumulation of the phenolic compounds and acclimatization competence in micropropagated ‘Williams’ bananas.

Materials and methods

Cytokinins

The aromatic cytokinins tested were benzyladenine (BA) and five topolins namely: meta-Topolin (mT), meta-Topolin riboside (mTR), meta-Methoxy topolin (MemT), meta-Methoxy topolin riboside (MemTR), and meta-Methoxy topolin 9-tetrahydropyran-2-yl (MemTTHP). All the topolins were obtained from the Laboratory of Growth Regulators, Palacký University and Institute of Experimental Botany, Academy of Sciences of the Czech Republic while BA was purchased from Sigma, USA.

Culture conditions and preparation of plant material

Aseptically maintained, in vitro banana plantlets (Musa spp. AAA cultivar ‘Williams’) regularly sub-cultured at 6-weekly intervals were used. Originally, the shoot-tip explants were initiated by surface sterilizing greenhouse-established banana plantlets purchased from Du Roi Laboratory, South Africa. Sterile plantlets at the fourth multiplication cycle were used for the experiment. Murashige and Skoog (MS) inorganic salts (Murashige and Skoog 1962) as modified for bananas by Vuylsteke (1998) were used. The medium was supplemented with varying concentrations of aromatic CKs (10, 20 and 30 μM) and 30 g l−1 sucrose. Thereafter, the medium was adjusted to pH 5.8 with either 0.1 M KOH or HCl prior to addition of 3 g l−1 gelrite (Labretoria, Pretoria, South Africa) and then autoclaved at 103 kPa and 121 °C for 20 min. Filter sterilized ascorbic acid (0.180 g l−1) was aseptically added to the medium before solidification (ca 50 °C). Shoot-tip explants (10 mm) were cut in half longitudinally and inoculated in culture tubes (100 × 25 mm, 40 ml volume) containing 12 ml of growth medium. Cultures were incubated in a growth room under 16 h light/8 h dark conditions and photosynthetic photon flux density (PPFD) of 45 μmol m−2 s−1 at 25 ± 2 °C.

Acclimatization competence experiment

In vitro plantlets derived from the 10 μM CK treatments, due to ease of rooting, were washed and potted in 12.5 cm pots containing sand, soil, vermiculite (1:1:1, v/v) treated with 1 % Benlate® (Du Pont de Nemours Int., South Africa). The plantlets were maintained for 3 months in the mist-house with day/night temperatures of 30/12 °C, relative humidity between 80 and 90 %, a misting interval of 15 min for 10-s duration. Ten well-established plantlets for the six CK treatments as well as the control were transferred to a greenhouse with 30–40 % relative humidity, day/night temperatures of 30/15 °C with an average PPFD of 450 μmol m−2 s−1. During the experiment, photoperiod was that of prevailing natural conditions during summer (12 h). After 2 months, plantlets were harvested and data on various quantitative parameters such as plant height, root length and number of leaves were collected.

Sample preparation for secondary metabolite quantification

After 6-week incubation, regenerated plantlets from the CK treatments were separated into aerial and underground parts. The plant materials were oven-dried at 50 ± 2 °C for 7 days and milled into powder form. Ground plant samples were extracted in 50 % methanol (MeOH) at 0.1 g/10 ml in an ultrasonic sonicator (Julabo GmbH, West Germany), that contained ice, for 20 min. The mixture was separated using a Benchtop centrifuge (Hettich Universal, Tuttlingen, Germany) to obtain the supernatant required for the phytochemical quantification.

Total phenolic, flavonoid and proanthocyanidin contents

Total phenolic content was evaluated using the Folin-Ciocalteu (Folin-C) assay as outlined by Makkar et al. (2007). Absorbance at 725 nm was measured using a UV–visible spectrophotometer (Varian Cary 50, Australia) against a blank consisting of 50 % MeOH. Total phenolic content was expressed in mg gallic acid (Sigma-Aldrich, USA) equivalents (GAE) per g dry weight (DW).

Total flavonoid content was determined by the aluminium chloride colorimetric assay (Zhishen et al. 1999) with slight modifications (Marinova et al. 2005). The absorbance at 510 nm was measured using a UV–visible spectrophotometer against a blank consisting of 50 % MeOH. Total flavonoid content was expressed in mg catechin (Sigma-Aldrich, Germany) equivalents (CE) per g DW.

The HCl/Butan-1-ol assay adapted from Porter et al. (1985) and described by Makkar et al. (2007) was used to quantify the proanthocyanidin content. The absorbance at 550 nm was measured using a UV–visible spectrophotometer against a blank consisting of 50 % MeOH. The proanthocyanidins content was expressed in μg cyanidin chloride (Carl Roth, Germany) equivalents (CCE) per g DW.

Data collection and analysis

After 2 months, the greenhouse-acclimatized plantlets were harvested, washed and air dried. The vegetative growth, such as number (leaf, shoot and root), length (shoot and root) and fresh weights (shoot and root), was measured. The shoot/root ratio was calculated as the ratio of the dry weights of the aerial part over that of the root. The leaf area was determined using an L1-3100 area meter (Li-Cor Inc., Lincoln, Nebraska, USA). Dry weights of the harvested plantlets were determined after the fresh materials were oven-dried at 50 ± 2 °C for 7 days.

Photosynthetic pigments of the acclimatized plantlets were measured spectrophotometrically. Fresh leaf samples (0.1 g) were homogenized in 5 ml acetone with addition of a few pinches of acid-washed sand (BDH Chemicals, England). The mixture was filtered through Whatman No. 1 filter paper and separated using a Benchtop centrifuge at 5,000 rpm for 5 min at room temperature. The absorbance of the resultant filtrate was measured at 470, 645, and 662 nm. The pigment content expressed as μg per g fresh weight (FW) was calculated based on the formulae of Lichtenthaler (1987).

$$ {\text{Chlorophyll }}a\,\left( {C_{a} } \right) = 11.24{\text{A}}_{662} - 2.04{\text{A}}_{645} $$
$$ {\text{Chlorophyll }}b\left( {C_{b} } \right) = 20.13{\text{A}}_{645} - 4.19{\text{A}}_{662} $$
$$ {\text{Total chlorophylls}} = 7.05{\text{A}}_{662} + 18.09{\text{A}}_{645} $$
$$ {\text{Total}}\;{\text{carotenoids}} = \left( {1,000{\text{A}}_{470} - 1.90C_{a} - 63.14C_{b} } \right)/214 $$

Data were subjected to one-way analysis of variance (ANOVA) using SPSS software package for Windows (SPSS®, version 10.0, Chicago, USA). Where there was statistical significance (P = 0.05), the mean values were further separated using the Duncan’s multiple range test (DMRT).

Results

Effect of the cytokinins on phenolic contents

Overall, total phenolics and flavonoids were higher in the aerial parts compared to underground parts (Table 1). However, total phenolic content was higher in the underground part in 30 μM MemTTHP treatment. In terms of the proanthocyanidin levels, the underground parts generally had an higher content with the exception of 20 and 30 μM mTR regenerants. Treatments with mT and MemTTHP indicated that increasing the concentration of these CKs decreased the quantity of total phenolic and flavonoid contents in aerial parts. On the other hand, an increase in quantity was observed with higher concentrations of MemTR for total phenolics and flavonoids (aerial and underground parts) as well as MemT for proanthocyanidin (underground part) content.

Table 1 Effect of aromatic cytokinins on the accumulation of secondary metabolites in micropropagated ‘Williams’ bananas
Full size table

Generally, mT and MemTTHP stimulated higher phytochemical production compared to BA-treated and control plants. Notably, mT and MemTTHP at 10 μM increased the proanthocyanidin content in the underground part by five- and threefold, respectively, compared to the controls. Furthermore, the same concentration of BA yielded approximately 2.7-fold lower content compared to mT. Similarly, MemTTHP plantlets had 1.4- and 3.7-fold more total phenolics than the corresponding BA and control ones, respectively, in the underground parts.

Effect of cytokinins on the growth of greenhouse-acclimatized banana plants

At the end of the experiment, the greenhouse-acclimatized plantlets from the six CK treatments are shown in Fig. 1a, b. Overall, the acclimatization was above 80 % in all the treatments (data not shown). The effects of the CKs were more pronounced in the underground compared to the aerial parts. Plantlets regenerated from MemTTHP were significantly better than the control and BA treatments, in terms of the length of the roots (Fig. 2a) as well as shoot/root ratio and plant height (Fig. 2f, g). In most of these parameters, however, MemTR treatment produced similar effects. The highest number of leaves was recorded in MemT-treated plants (Fig. 2d). On the other hand, similar responses were observed across all the treatments as well as the control plants for shoot length (Fig. 2c), number of new shoots (Fig. 2e) and total leaf area of individual plants (Fig. 2h).

Fig. 1
figure 1

Effect of six aromatic cytokinins on the morphology of greenhouse-acclimatized ‘Williams’ bananas. a aerial and b underground parts. Cont, control; mT, meta-Topolin; mTR, meta-Topolin riboside; MemT, meta-Methoxy topolin; MemTR, meta-Methoxy topolin riboside; BA benzyladenine; MemTTHP, meta-Methoxy topolin 9-tetrahydropyran-2-yl. Bar 10 cm

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Fig. 2
figure 2

Effects of six aromatic cytokinins on the growth of ‘Williams’ bananas. a root length; b number of root; c shoot length; d number of leaves; e number of new shoot produced; f shoot/root ratio; g plant height and h leaf area. In each graph, bars with different letter(s) are significantly different (P = 0.05) based on Duncan’s multiple range test. mT, meta-Topolin; mTR, meta-Topolin riboside; MemT, meta-Methoxy topolin; MemTR, meta-Methoxy topolin riboside; BA benzyladenine; MemTTHP, meta-Methoxy topolin 9-tetrahydropyran-2-yl

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Biomass accumulation for the various CK treatments is depicted in Fig. 3. Even though the shoot fresh weight had a wide range, from around 13 g in the control to 18 g in MemT regenerants, there was no statistical difference among the treatments (Fig. 3a). Root fresh weight was highest in MemT and lowest in mTR plants (Fig. 3b). Similarly, MemT-treated plants had higher dry weight than either the control or other CK treatments (Fig. 3c). In terms of root dry weighs, mT and MemTTHP were the most desired CKs as indicated in Fig. 3d.

Fig. 3
figure 3

Effects of six aromatic cytokinins on biomass of ‘Williams’ bananas. a Shoot fresh weight; b root fresh weight; c shoot dry weight and d root dry weight. In each graph, bars with different letter(s) are significantly different (P = 0.05) based on Duncan’s multiple range test. mT, meta-Topolin; mTR, meta-Topolin riboside; MemT, meta-Methoxy topolin; MemTR, meta-Methoxy topolin riboside; BA benzyladenine; MemTTHP, meta-Methoxy topolin 9-tetrahydropyran-2-yl

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Effect of cytokinins on photosynthetic pigment content of the greenhouse-acclimatized banana plants

The total carotenoids and chlorophyll (chlorophyll a and b) in the acclimatized banana plants are presented in Table 2. Generally, the lowest photosynthetic pigments were observed in mT treatments. The highest total chlorophyll and carotenoid contents were detected in control, mTR and MemT-treated plantlets, in a slightly decreasing pattern. Conversely, chlorophyll a/b ratio in both the control and mTR regenerated plants were significantly lower than MemT which had the optimal ratio. In fact, the control plants exhibited the lowest chlorophyll a/b ratio. The highest total chlorophyll/carotenoid ratio was observed with mTR while BA-regenerated plants had the lowest ratio.

Table 2 Effect of cytokinin on the photosynthetic pigment contents of greenhouse-acclimatized ‘Williams’ bananas
Full size table

Discussion

The regulatory role of CKs on different physiological and developmental processes is well-documented (Werner et al. 2001; Criado et al. 2007). Studies have shown that PGR’s signaling pathways are not isolated, they are interconnected with a complex regulatory network involving various defence signaling pathways and developmental processes (Bari and Jones 2009). In recent times, there has been numerous evidence of the increasing superiority of topolins over the commonly used CKs such as BA and kinetin in PTC (Aremu et al. 2012). For example, the advantages of the use of topolins over BA were clearly observed in the micropropagation of species such as Aloe polyphylla (Bairu et al. 2007), Spathiphyllum floribundum (Werbrouck et al. 1996) and Musa spp. (Escalona et al. 2003; Bairu et al. 2008). In addition, this study demonstrated the better stimulatory effects of topolins (mT and MemTTHP) on phenolic content in ‘Williams’ banana compared to BA. Generally, secondary metabolites such as phenolics and flavonoids play vital roles during the micropropagation of several species. The diverse functions of these compounds have been investigated in detail by several researchers in PTC (Wu et al. 2007; Buer et al. 2010; De Klerk et al. 2011). As postulated by Curir et al. (1990) and Buer et al. (2010), rooting is enhanced in the presence of certain types of flavonoids. The high accumulation of flavonoids could have accounted for the observed better ex vitro rooting in mT and MemTTHP regenerated plantlets. This result also shows the advantage of MemTTHP over BA, in that the formation of the metabolite 6-benzylamino-9-β-d-glucopyranosylpurine, known for inhibition of rooting and associated abnormalities (Bairu et al. 2011b), is blocked due to the conjugation of this molecule at the N9 position (Szücová et al. 2009). In the current study, BA negatively affected rooting as indicated in the number and length of the roots compared to the topolins. The detrimental effects of BA during the acclimatization of micropropagated plants are well-documented (Werbrouck et al. 1995; Baroja-Fernández et al. 2002; Valero-Aracama et al. 2010; Bairu et al. 2011b). Accumulation of the more stable and toxic metabolite 6-benzylamino-9-β-d-glucopyranosylpurine has been implicated in these acclimatization problems. Among the tested CKs, MemTTHP consistently remained the most promising by stimulating better acclimatization in the micropropagated ‘Williams’ bananas. Szücová et al. (2009) synthesised MemTTHP in an attempt to improve the biological activity of the parent structure (mT). Indeed, the compound maintained or improved CK activity when tested in the three classical CK bioassays. As postulated by the authors, the improved MemTTHP activity was due to higher resistance to enzymatic breakdown by CK oxidase. Hence, the stimulatory effect of the CK is probably due to their longer lifespan as well as availability in the plant. If confirmed with more similar studies on different species, MemTTHP would be an ideal CK for other plants with rooting and acclimatization problems.

The numerous advantages associated with the use of PTC for production of secondary metabolites remain of major interest to researchers (Ramachandra Rao and Ravishankar 2002). These authors highlighted the regulatory role of different CKs in the accumulation of secondary metabolites. The type and concentration of CKs affect the quantity of secondary metabolites produced. In Hypericum perforatum, e.g., BA-treated plantlets accumulated more hypericins compared to zeatin-treated ones (Liu et al. 2007). There was a reduction in the phenolic content of micropropagated Tectona grandis with an increase in BA concentrations (Quiala et al. 2012). The current findings demonstrate varying levels of the quantified phenolic compounds due to different CK types as well as concentration in micropropagated bananas. The topolins, particularly, mT and MemTTHP increased total phenolics, flavonoids and proanthocyanidins in both underground and aerial parts of regenerated banana plantlets. Consequently, the use of topolins could be an alternative method for the production of high-value secondary metabolites required for the pharmaceutical, agrochemical, flavor, fragrance, and food industries.

Plant survival depends on their interaction with the environment aided by their secondary metabolite content. During stress, the phenylpropanoid pathway is of critical importance as its products (phenolic compounds) protect the plant against abiotic and biotic factors (Dixon and Paiva 1995). Evidence has shown that phenylpropanoid-based polymers, such as lignin, and proanthocyanidin, contribute substantially to the stability and robustness of higher plants towards mechanical or environmental damage (Vogt 2010). Generally, phenolic compounds are essential in UV protection and modulating the levels of reactive oxygen species in plants (Peer and Murphy 2007; Buer et al. 2010). Particularly, flavonoids have been shown to be polar auxin transport modulators via different regulatory pathways (Peer and Murphy 2007). Recently, De Klerk et al. (2011) also reported that the presence of certain phenolic compounds protects auxins from decarboxylation which inevitably stimulated better rooting in Malus spp. Banana being a large herbaceous plant requires firm attachment thereby necessitating the development of a good root system. Plantlets regenerated from MemTTHP had the best shoot/root ratio, an indication of the plantlet survival potential during unfavorable conditions such as drought (Bernier et al. 1995). Furthermore, a positive correlation has been established between host resistance and products (mainly phenolic compounds) of the phenylpropanoid pathway (Binks et al. 1997; Collingborn et al. 2000). The biochemical basis for secondary metabolites and degree of resistance is not fully elucidated (Wuyts et al. 2005, 2007). In addition, different PGRs play positive or negative roles against various biotrophic and necrotrophic pathogens depending on the type of plant–pathogen interactions (Bari and Jones 2009). Unfortunately, most of the underlying molecular mechanisms involved in these interactions remain poorly understood.

Another important function of CK is the delay of senescence by accumulating and maintaining photosynthetic pigments in plants (Van Staden et al. 1988). The exogenous application of topolins such as mT and mTR delayed chlorophyll degradation in Pelargonium (Mutui et al. 2012), Triticum aestivum (Palavan-Ünsal et al. 2004) and Beta vulgaris (Čatský et al. 1996). In vitro studies have, however, demonstrated both stimulatory and inhibitory effects of different CKs on photosynthetic pigment content in micropropagated species, such as Phaseolus vulgaris (Adedipe et al. 1971; Yokoyama et al. 1980) and Dianthus caryophyllus (Genkov et al. 1997). In this study, the topolins generally had higher chlorophyll a/b ratios compared to the control or BA treatments. The higher ratio is an indication of the topolin-stimulated adaptive response required for effective photosynthesis by the acclimatized plants (Chow et al. 1990). High total chlorophyll/carotenoid ratio is related to the presence and maintenance of substantial total chlorophyll content in plants (Lichtenthaler 1987), and all the topolin treatments (except MemTR) had significantly higher total chlorophyll/carotenoid ratios and were greener than the BA-treated banana plants.

In conclusion, the importance of the right choice of CK has been further demonstrated in this study. Besides the obvious effect during the micropropagation phase, the CKs generally have other carry-over attributes which could either enhance or hinder the acclimatization of in vitro regenerated plantlets. In vitro treatment with mTR and MemT enhanced the photosynthetic pigments in the greenhouse-acclimatized banana plants. In addition to mT and MemTTHP increasing the phenolic compounds in micropropagated bananas, plantlets regenerated from most of the topolins were better acclimatized than the BA treatment. Clearly, MemTTHP, which is a new topolin derivative, has great promise as a CK which could be useful to circumvent rooting inhibition and acclimatization failure in PTC. In addition to the investigation of the effect of MemTTHP on other micropropagated plant species, we are also studying the effect of these topolins on the endogenous CK profiles of micropropagated ‘Williams’ bananas.

Author contribution

A.O. Aremu, M.W. Bairu, J.F. Finnie and J. Van Staden were responsible for the experimental design and conducted the experiment. L. Szüčová and K. Doležal were responsible for the synthesis of the topolins. A.O. Aremu drafted the manuscript and all authors read and approved the final manuscript.