Planta

, Volume 236, Issue 6, pp 1775–1790

Physiological responses and endogenous cytokinin profiles of tissue-cultured ‘Williams’ bananas in relation to roscovitine and an inhibitor of cytokinin oxidase/dehydrogenase (INCYDE) treatments

Authors

  • Adeyemi O. Aremu
    • Research Centre for Plant Growth and Development, School of Life SciencesUniversity of KwaZulu-Natal
  • Michael W. Bairu
    • Research Centre for Plant Growth and Development, School of Life SciencesUniversity of KwaZulu-Natal
  • Ondřej Novák
    • Laboratory of Growth Regulators, Institute of Experimental Botany AS CRPalacký University
  • Lenka Plačková
    • Laboratory of Growth Regulators, Institute of Experimental Botany AS CRPalacký University
    • Faculty of Science, Centre of the Region Haná for Biotechnological and Agricultural ResearchPalacký University
  • Marek Zatloukal
    • Laboratory of Growth Regulators, Institute of Experimental Botany AS CRPalacký University
    • Faculty of Science, Centre of the Region Haná for Biotechnological and Agricultural ResearchPalacký University
  • Karel Doležal
    • Laboratory of Growth Regulators, Institute of Experimental Botany AS CRPalacký University
    • Faculty of Science, Centre of the Region Haná for Biotechnological and Agricultural ResearchPalacký University
  • Jeffrey F. Finnie
    • Research Centre for Plant Growth and Development, School of Life SciencesUniversity of KwaZulu-Natal
  • Miroslav Strnad
    • Laboratory of Growth Regulators, Institute of Experimental Botany AS CRPalacký University
    • Faculty of Science, Centre of the Region Haná for Biotechnological and Agricultural ResearchPalacký University
    • Research Centre for Plant Growth and Development, School of Life SciencesUniversity of KwaZulu-Natal
Original Article

DOI: 10.1007/s00425-012-1721-z

Cite this article as:
Aremu, A.O., Bairu, M.W., Novák, O. et al. Planta (2012) 236: 1775. doi:10.1007/s00425-012-1721-z

Abstract

The effect of supplementing either meta-topolin (mT) or N6-benzyladenine (BA) requiring cultures with roscovitine (6-benzylamino-2-[1(R)-(hydroxymethyl)propyl]amino-9-isopropylpurine), a cyclin-dependent kinase (CDK) and N-glucosylation inhibitor, and INCYDE (2-chloro-6-(3-methoxyphenyl)aminopurine), an inhibitor of cytokinin (CK) degradation, on the endogenous CK profiles and physiology of banana in vitro was investigated. Growth parameters including multiplication rate and biomass were recorded after 42 days. Endogenous CK levels were quantified using UPLC–MS/MS while the photosynthetic pigment and phenolic contents were evaluated spectrophotometrically. The highest regeneration rate (93 %) was observed in BA + roscovitine while mT + INCYDE plantlets produced most shoots. Treatment with BA + roscovitine had the highest shoot length and biomass. Although not significant, there was a higher proanthocyanidin level in BA + roscovitine treatments compared to the control (BA). The levels of total phenolics and flavonoids were significantly higher in mT + roscovitine treatment than in the mT-treated regenerants. The presence of roscovitine and/or INCYDE had no significant effect on the photosynthetic pigments of the banana plantlets. Forty-seven aromatic and isoprenoid CKs categorized into nine CK-types were detected at varying concentrations. The presence of mT + roscovitine and/or INCYDE increased the levels of O-glucosides while 9-glucosides were higher in the presence of BA. Generally, the underground parts had higher CK levels than the aerial parts; however, the presence of INCYDE increased the level of CK quantified in the aerial parts. From a practical perspective, the use of roscovitine and INCYDE in micropropagation could be crucial in the alleviation of commonly observed in vitro-induced physiological abnormalities.

Keywords

Cyclin-dependent kinaseCytokinin metabolismMicropropagationMusa spp.PhotosynthesisPlant secondary metabolites

Abbreviations

ANOVA

Analysis of variance

BA

N6-Benzyladenine

BA9G

N6-Benzyladenine-9-glucoside

BAR

N6-Benzyladenosine

BAR5′MP

N6-Benzyladenosine-5′-monophosphate

CCE

Cyanidin chloride equivalents

CDK

Cyclin-dependent kinase

CE

Catechin equivalents

CK

Cytokinin

CKX

Cytokinin oxidase/dehydrogenase

cZ

cis-Zeatin

cZ9G

cis-Zeatin-9-glucoside

cZOG

cis-Zeatin-O-glucoside

cZR

cis-Zeatin riboside

cZR5′MP

cis-Zeatin riboside-5′-monophosphate

cZROG

cis-Zeatin-O-glucoside riboside

DHZ

Dihydrozeatin

DHZ9G

Dihydrozeatin-9-glucoside

DHZOG

Dihydrozeatin-O-glucoside

DHZR

Dihydrozeatin riboside

DHZR5′MP

Dihydrozeatin riboside-5′-monophosphate

DHZROG

Dihydrozeatin-O-glucoside riboside

DMRT

Duncan’s multiple range test

GAE

Gallic acid equivalents

IAC

Immunoaffinity chromatography

INCYDE

2-Chloro-6-(3-methoxyphenyl)aminopurine

iP

N6-Isopentenyladenine

iP9G

N6-Isopentenyladenine-9-glucoside

iPR

N6-Isopentenyladenosine

iPR5′MP

N6-Isopentenyladenosine-5′-monophosphate

IPT

Isopentenyltransferase

Kin

Kinetin

Kin9G

Kinetin-9-glucoside

KinR

Kinetin riboside

KinR5′MP

Kinetin riboside-5′-monophosphate

MRM

Multiple reaction monitoring

MS

Murashige and Skoog medium

mT

meta-Topolin

mT9G

meta-Topolin-9-glucoside

mTOG

meta-Topolin-O-glucoside

mTR

meta-Topolin riboside

mTR5′MP

meta-Topolin-5′-monophosphate

mTROG

meta-Topolin-O-glucoside riboside

oT

ortho-Topolin

oT9G

ortho-Topolin-9-glucoside

oTOG

ortho-Topolin-O-glucoside

oTR

ortho-Topolin riboside

oTR5′MP

ortho-Topolin-5′-monophosphate

oTROG

ortho-Topolin-O-glucoside riboside

PGR

Plant growth regulator

PPFD

Photosynthetic photon flux density

pT

para-Topolin

PTC

Plant tissue culture

pTOG

para-Topolin-O-glucoside

pTR

para-Topolin riboside

pTR5′MP

para-Topolin-5′-monophosphate

pTROG

para-Topolin-O-glucoside riboside

tZ

trans-Zeatin

tZ9G

trans-Zeatin-9-glucoside

tZOG

trans-Zeatin-O-glucoside

tZR

trans-Zeatin riboside

tZR5′MP

trans-Zeatin riboside-5′-monophosphate

tZROG

trans-Zeatin-O-glucoside riboside

UPLC

Ultra performance liquid chromatography

Introduction

Cytokinins (CKs) are N6-substituted purine derivatives. They constitute an essential class of plant growth regulators (PGRs) that together with auxins act at low concentration and regulate various physiological and developmental processes in plants (Letham and Palni 1983; Schmülling 2004). The absence or presence of hydroxyl groups as well as their stereoisomeric position is a common feature of CKs which account for their diverse activity (Strnad 1997; Mok and Mok 2001; Sakakibara 2006). Cytokinin homeostasis is an essential factor that determines the physiological activities, particularly the growth and development of plants (Kamínek et al. 1997). In plant cells, CKs act as normal adenylate compounds which exist in mixtures of free bases, N-glucosides, O-glucosides, nucleosides as well as the mono-, di-, and tri-nucleotides (Letham and Palni 1983; Bajguz and Piotrowska 2009). These compounds can be interconverted via reactions catalyzed by purine metabolizing enzymes such as glucosyltransferase, adenosine nucleosidase, and xylosyltransferase (Chen 1997). Based on their physiological metabolic pathways, CK inter-conversion, hydroxylation, conjugation and degradation remain important regulatory mechanisms in plant tissues (Strnad et al. 1997). The CK homeostasis is generally maintained by the regulation of both CK biosynthesis and catabolism, which are influenced by two main enzymes: isopentenyltransferase (IPT) and cytokinin oxidase/dehydrogenase (CKX), respectively (Frébort et al. 2011). In addition, CK homeostasis is regulated by several internal and external factors such as PGRs and inorganic nitrogen sources, which have been postulated to connect the nutrient signals and morphogenetic responses (Auer 1997; Sakakibara 2006).

In plant tissue culture (PTC), CKs are often used to stimulate the growth and development of plants in vitro (Zalabák et al. 2012). The exogenously applied CKs [N6-benzyladenine (BA)] interact with the endogenous level thereby affecting the balance in the cultured explants (Blakesley 1991; Blagoeva et al. 2004a). The uptake and metabolism of the applied CKs determine the resultant growth and development of the plant (Kamínek et al. 1997; Haberer and Kieber 2002). Cytokinins may be reversibly or irreversibly conjugated with sugars and amino acids (Bajguz and Piotrowska 2009). Often, the exogenous CK are converted into various forms of metabolites as a regulatory mechanism to maintain the CK homeostasis (Letham and Palni 1983). As suggested by the authors, these metabolites could be of less active (conjugates), translocation and storage forms which are readily available in the event of depletion of the free CKs pool or the detoxification products which are mostly unavailable to the plant tissue. In other words, since free CK bases remain the most active form, the various inter-conversion of the CK bases is essential for the regulation of CK levels and activities (Bajguz and Piotrowska 2009; Frébort et al. 2011).

The level of active CKs is down-regulated in plants mainly by side chain cleavage into simpler units by CKX. The enzyme is also useful for the maintenance of an optimal level of CKs required for growth and/or resetting a CK signalling system to a basal level (Werner et al. 2001; Frébort et al. 2011). In addition, the free CKs can undergo conjugation with glucose to produce either reversible forms such as O-glucosides or irreversible forms such as N-glucosides (Mok and Mok 2001). The ability of the glycoside to interact with the 3, 7 and 9 position of the purine ring probably make it the most common and abundant CK conjugates both in lower (Ördög et al. 2004) and higher plants (Van Staden and Crouch 1996; Auer 1997; Bairu et al. 2011). Although CK 7N- and 9N-glucosides are considered as non-active and detoxification products in plants, some authors have observed a contrary effect to this widely accepted assumption (Letham and Palni 1983; Upfold and Van Staden 1990).

In PTC, the presence of large amounts of 7N- and 9N-glucosylation products is a common event in several micropropagated species (Werbrouck et al. 1995; Blagoeva et al. 2004b; Dwivedi et al. 2010). Consequently, there is a decrease in the levels and availability of other CK forms especially the free bases which have more beneficial effects in plant tissues (Brzobohatý et al. 1994). Often, these ‘cause and effect’ phenomenon is responsible for the commonly observed physiological disorders in PTC. For example, there was a wide variation in the composition of CK metabolites of normal and necrotic shoots of Harpagophytum procumbens (Bairu et al. 2011), normal and hyperhydric shoots of Aloe polyphylla (Ivanova et al. 2006) as well as normal and variegated leaves of micropropagated bananas (Zaffari et al. 1998). The abundance of N-glucosides was also implicated in the inhibition of rooting and subsequent acclimatization failure observed in Spathiphyllum floribundum (Werbrouck et al. 1995, 1996). Among the commonly used CKs, BA has been associated with rapid formation of N-glucosides due to its structural limitation (absence of hydroxyl groups). The accumulation of BA N-glucosides particularly in the basal part of the shoots can have severe practical consequences in PTC (Bairu et al. 2007; Valero-Aracama et al. 2010).

The prospect of suppressing these detrimental metabolites could enhance CK activity as well as modulate the plant responses positively. Along this line, a number of potential N-glucosylation inhibitors such as papaverine, methylxanthines, theophylline and olomoucine have been tested (Letham et al. 1977; Tao et al. 1991; Blagoeva et al. 2004b). Recently, roscovitine (6-benzylamino-2-(R)-[1-(hydroxymethyl) propylamino]-9-isopropylpurine), was identified as the most potent inhibitor of N-glucosylation (Blagoeva et al. 2003, 2004b; Dwivedi et al. 2010). Roscovitine, a 2,6,9-trisubstituted purine is a well-known inhibitor of the cyclin-dependent kinases (CDKs), enzymes involved in the cell cycle progression that play a vital role in intracellular control of cell division (De Azevedo et al. 1997; Meijer et al. 1997). Roscovitine and similar compounds such as bohemine and olomoucine have also been found to be effective inhibitors of CDKs (Havlíček et al. 1997; Binarová et al. 1998; Spíchal et al. 2007).

Cytokinin oxidase/dehydrogenase is responsible for most of the CK catabolism and inactivation of the CKs in a single enzymatic step (Schmülling et al. 2003), the presence of inhibitor compound(s) could circumvent the activity of the enzyme. Recent research endeavors resulted in the discovery of a number of potent CKX inhibitors (Zatloukal et al. 2008). One such compound was INCYDE (2-chloro-6-(3-methoxyphenyl)aminopurine), which demonstrated CK activity in classical CK bioassays and was highly effective in the inhibition of Arabidopsis CKX.

Evidence from the limited studies indicates the great potential of roscovitine in PTC (Blagoeva et al. 2003; Stoynova-Bakalova and Petrov 2009; Dwivedi et al. 2010). The effectiveness of INCYDE, however, has not been studied in-depth as the compound was only discovered recently (Zatloukal et al. 2008). In the field of PTC and biotechnology, bananas are important species because of their huge economic contribution to food security. Although PTC has contributed significantly to improvement in the quality of planting materials and the increase in production globally, banana remains one of the most highly prioritized research crops. The presence of inherent physiological problems including the detrimental effect associated with the commonly used CK, BA necessitates further studies on the role of CK in the improvement of PTC protocols. Besides, stringent studies on the endogenous CKs and factors affecting their concentration would contribute to better understanding of the banana hormone physiology in vitro. In the current study, the role of roscovitine and INCYDE (with BA or mT) on the physiology (growth, photosynthetic pigment and phenolic contents) and CK metabolism of micropropagated ‘Williams’ bananas was investigated.

Materials and methods

Chemicals

Folin-Ciocalteu phenol reagent, gallic acid, catechin hydrate, ascorbic acid and BA were purchased from Sigma-Aldrich (Steinheim, Germany). The gelrite was procured from Labretoria, Pretoria, South Africa while cyanidin chloride was purchased from Carl Roth GmbH (Karlsruhe, Germany). The 23 deuterium-labeled CK internal standards were obtained from Olchemim Ltd (Olomouc, Czech Republic). Roscovitine, mT and INCYDE were prepared as described previously (Havlíček et al. 1997; Holub et al. 1998; Zatloukal et al. 2008). All chemicals used in the current study were of analytical grade.

Explant culture and growth conditions

Aseptically maintained, in vitro banana plantlets (Musa spp. AAA cultivar ‘Williams’) supplemented with either 30 μM mT or BA and regularly sub-cultured at 6-week intervals were used in this study. The sterile plantlets, from BA and mT CK lines, at the fifth multiplication cycle were used for the current experiments. Ideally, cultures at the fifth multiplication cycle are often the ones that go through acclimatization and field planting. Murashige and Skoog (MS) basal salt (Murashige and Skoog 1962) with modifications (Vuylsteke 1998) was used. The treatments included: mT, BA, BA + INCYDE, BA + roscovitine, BA + INCYDE + roscovitine, mT + INCYDE, mT + roscovitine and mT + INCYDE + roscovitine. Based on previous studies (Zatloukal et al. 2008; Dwivedi et al. 2010), roscovitine and INCYDE were tested at 5 and 100 μM, respectively, while mT and BA concentrations were at 30 μM. 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 and autoclaved at 103 kPa and 121 °C for 20 min. Filter-sterilized ascorbic acid (0.18 g l−1) was aseptically added to the medium before solidification (50 °C). Shoot-tip explants (10 mm) were cut in half longitudinally and inoculated in culture jars (110 × 60 mm, 300 ml volume) containing 36 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. After 42-day culture duration, plantlets were harvested. After parameters such as shoot number and plant height were measured, plantlets were separated into aerial (shoots and pseudostems) and underground (corms and roots) sections, then fresh weight was recorded immediately while dry weight was obtained after 7 days of oven drying at 65 °C.

Quantification of photosynthetic pigment and phytochemical content

After 42 days in culture, fresh leaf samples were extracted using acetone (0.2 g/ml) and the amount of chlorophyll a, chlorophyll b and the carotenoid content was quantified using a spectrophotometer (Varian Cary 50, Mulgrave, Victoria, Australia) as described by Lichtenthaler (1987) and outlined in Aremu et al. (2012a). The levels of photosynthetic pigment were expressed as μg/g FW for each treatment.

Oven-dried plant materials were extracted with 50 % methanol (MeOH) at a ratio of 0.01 mg/ml. The mixture was sonicated in an ice-bath for 20 min and filtered using Whatman filter paper. Based on the techniques outlined in our previous study (Aremu et al. 2012a), the total phenolics, flavonoids and proanthocyanidins were determined spectrophotometrically.

Extraction, purification and quantification of endogenous cytokinins

After 42 days, plantlets from each treatment were washed and separated into aerial and underground sections. Plant materials were immediately frozen in liquid nitrogen, freeze-dried and lyophilized. Prepared samples were extracted in 1 ml of Bieleski buffer (60 % MeOH, 25 % CHCl3, 10 % HCOOH and 5 % H2O) together with a cocktail of 23 deuterium-labeled CK internal standards at 1 pmol per sample, to check recovery during purification and to validate the determination (Novák et al. 2008). Purification of the samples was achieved using a combination of cation (SCX-cartridge), anion [DEAE-Sephadex-C18-cartridge] exchangers and immunoaffinity chromatography (IAC) based on wide-range specific monoclonal antibodies against CKs (Novák et al. 2003). The resultant eluates from the IAC columns were evaporated to dryness and dissolved in 20 μl of the mobile phase used for quantitative analysis.

Samples were analyzed using an ultra-performance liquid chromatography (UPLC) (Acquity UPLC™; Waters, Milford, MA, USA) coupled to a Quatro micro™ API (Waters, Milford, MA, USA) triple quadrupole mass spectrometer equipped with an electrospray interface [ESI(+)] and photodiode array detector (Waters PDA 2996). Thereafter, the purified samples were injected onto a C18 reversed-phase column (Waters Acquity UPLC BEH C18; 1.7 μm; 2.1 × 50 mm). Elution was performed using a methanolic gradient consisting of 100 % methanol (A) and 15 mM formic acid (B) adjusted to pH 4.0 with ammonium. With a flow rate of 0.25 ml/min and column temperature of 40 °C, the following protocol was applied: 0 min 10 % A + 90 % B—8 min linear gradient; 50 % A + 50 % B then column equilibration.

Endogenous CK quantification was obtained by multiple reaction monitoring (MRM) of [M + H]+ and the appropriate product ion. For selective MRM experiments, optimal conditions (dwell time, cone voltage, and collision energy in the collision cell) corresponding to exact diagnostic transition were optimized for each CK (Novák et al. 2008). Quantification was performed with Masslynx software using a standard isotope dilution method. The ratio of endogenous CK to appropriate labeled standard was determined and subsequently used to quantify the level of endogenous CKs in the original banana plantlet extract, based on the known quantity of added internal standard (Novák et al. 2003).

Data analysis

The growth, phytochemical and photosynthetic pigment contents data were measured and 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). Endogenous CK contents were quantified and expressed as mean ± standard deviation from three replicates. No statistical analysis was performed on the endogenous CK content because some of the CK levels were below the limit of detection.

Results

Effect of roscovitine and INCYDE on the growth of ‘Williams’ bananas

Regeneration rate was above 50 % in all the treatments. The 93 % response in BA + roscovitine-treated cultures was the highest rate (Fig. 1a). Plantlets treated with mT + INCYDE had the highest shoot number while BA + roscovitine + INCYDE treatment produced the lowest number of shoots (Fig. 1b). The combination of either mT or BA with roscovitine increases the shoot length compared to the CKs alone (Fig. 1c). However, the addition of INCYDE to BA-supplemented medium reduced the shoot length in the regenerants. Fresh and dry weights were highest in BA + roscovitine-treated and lowest in BA + INCYDE-treated plantlets (Fig. 1d and e). The negative effect of mT or BA + INCDYE on the fresh and dry weights was partially alleviated when roscovitine was added to the medium. Furthermore, BA + roscovitine regenerated plantlets had a significantly (P = 0.05) higher dry matter compared to other BA-containing cultures. Conversely, compared to their respective control (mT or BA) plantlets, the addition of roscovitine and/or INCYDE had no significant (P = 0.05) stimulatory effect on other growth parameters such as shoot number and regeneration rate. Apart from BA + roscovitine-treated plantlets with a few roots, all the other treatments inhibited rooting (data not shown).
https://static-content.springer.com/image/art%3A10.1007%2Fs00425-012-1721-z/MediaObjects/425_2012_1721_Fig1_HTML.gif
Fig. 1

Effect of roscovitine and INCYDE on growth of the micropropagated ‘Williams’ bananas. a Regeneration rate. b Shoot number. c Shoot length. d Fresh weight. e Dry weight. 1meta-topolin, 2 benzyladenine, 3 benzyladenine + INCYDE, 4 benzyladenine + roscovitine, 5 benzyladenine + INCYDE + roscovitine, 6meta-topolin + INCYDE, 7meta-topolin + roscovitine, 8meta-topolin + INCYDE + roscovitine. In each graph, bars with different letter(s) are significantly different (P = 0.05) based on Duncan’s multiple range test (DMRT)

Effect of roscovitine and INCYDE on endogenous cytokinin content

Generally, aromatic CKs were the major CK-type occurring in both aerial (Fig. 2a) and underground (Fig. 2b) parts in the regenerants. With the exception of the aerial part in which mT and mT + roscovitine treatments had (mainly cZ and its derivatives) 39 and 14 %, respectively, the isoprenoid CK constituent in all the treatment was generally low (<1 %). The relative abundance (%) as well as the distribution and/or transportation pattern of the isoprenoid and aromatic CKs within the plantlets in each treatment is shown in Fig. 3. The regenerated banana plantlets had more than 50 % of the isoprenoid CK contents in the aerial region with the exception of mT + roscovitine and mT + INCYDE treatments. Conversely, the controls (mT and BA) as well as mT + roscovitine and BA + roscovitine treatments had at least 90 % of their aromatic CK contents restricted to the underground region.
https://static-content.springer.com/image/art%3A10.1007%2Fs00425-012-1721-z/MediaObjects/425_2012_1721_Fig2_HTML.gif
Fig. 2

Total isoprenoid and aromatic cytokinin content (%) in aerial (a) and underground (b) parts of the micropropagated ‘Williams’ bananas. 1meta-topolin, 2 benzyladenine, 3 benzyladenine + INCYDE, 4 benzyladenine + roscovitine, 5 benzyladenine + INCYDE + roscovitine, 6meta-topolin + INCYDE, 7meta-topolin + roscovitine, 8meta-topolin + INCYDE + roscovitine

https://static-content.springer.com/image/art%3A10.1007%2Fs00425-012-1721-z/MediaObjects/425_2012_1721_Fig3_HTML.gif
Fig. 3

Relative abundance (%) and distribution pattern of the isoprenoid and aromatic cytokinins in aerial (a) and underground (b) parts of the micropropagated ‘Williams’ bananas. 1meta-topolin, 2 benzyladenine, 3 benzyladenine + INCYDE, 4 benzyladenine + roscovitine, 5 benzyladenine + INCYDE + roscovitine, 6meta-topolin + INCYDE, 7meta-topolin + roscovitine, 8meta-topolin + INCYDE + roscovitine

A total of 47 different CKs (aromatic and isoprenoid) were detected at varying concentrations across all the treatments in the aerial (Table 1) and underground (Table 2) parts. All the detected CKs were grouped into 9 CK-types namely: tZ, cZ, DHZ, iP, BA, mT, oT, pT and K. In mT containing treatments, mT-type was the most abundant CK and it was in the magnitude of mT + INCYDE > mT + INCYDE + roscovitine > mT + roscovitine > mT in the aerial part, while it was mT + roscovitine > mT + INCYDE > mT > mT + INCYDE + roscovitine in the underground parts. On the other hand with BA-treated plantlets, BA-type CKs were the most abundant and total CK was in the order of BA + INCYDE + roscovitine > BA + INCYDE > BA + roscovitine > BA (aerial part) and BA > BA + roscovitine > BA + INCYDE + roscovitine > BA + INCYDE (underground part). There was a high concentration of oT and its derivatives, especially in the underground region of BA-treated plantlets (Table 2). The total oT level in BA-treated plantlets was approximately 750 % higher than in mT-treated ones. Furthermore, oT9G constituted the bulk (≥50 %) of the total oT-type CK content. However, the addition of roscovitine and/or INCYDE to BA-supplemented medium reduced the total oT content by more than 50 %.
Table 1

Effect of roscovitine and INCYDE with aromatic cytokinins on endogenous cytokinins content (pmol/g FW) in ‘Williams’ banana aerial parts

Cytokinin type

Cytokinin treatment and resultant endogenous cytokinin content (pmol/g FW)

mT

BA

BA + INCYDE

BA + roscovitine

BA + INCYDE + roscovitine

mT + INCYDE

mT + roscovitine

mT + INCYDE + roscovitive

tZ

14 ± 0.7

10 ± 2.0

19 ± 7.1

2 ± 0.0

<LOD

<LOD

<LOD

<LOD

tZOG

8 ± 1.3

4 ± 0.6

10 ± 0.9

3 ± 1.4

7 ± 1.1

2 ± 1.1

<LOD

2 ± 1.0

tZR

2 ± 1.1

0.2 ± 0.2

3 ± 0.6

0.2 ± 0.0

3 ± 2.8

<LOD

<LOD

<LOD

tZROG

7 ± 0.5

3 ± 0.6

10 ± 0.8

7 ± 1.1

10 ± 2.0

4 ± 1.2

11 ± 2.3

9 ± 1.8

tZ9G

<LOD

4 ± 4.9

7 ± 0.0

<LOD

<LOD

72 ± 0.0

<LOD

<LOD

tZR5′MP

14±

<LOD

19 ± 4.0

<LOD

<LOD

<LOD

<LOD

<LOD

Total tZ

44 ± 3.6

21 ± 8.3

68 ± 13.4

12 ± 2.5

20 ± 5.9

79 ± 2.4

11 ± 2.3

11 ± 2.8

cZ

6 ± 0.5

1 ± 0.2

4 ± 1.9

1 ± 0.6

2 ± 0.0

<LOD

<LOD

<LOD

cZOG

11 ± 0.4

5 ± 0.4

16 ± 0.4

11 ± 2.1

13 ± 3.4

4 ± 0.3

10 ± 2.7

9 ± 0.3

cZR

97 ± 15.2

22 ± 3.7

128 ± 25.2

68 ± 4.2

59 ± 20.7

21 ± 5.7

62 ± 1.2

12 ± 3.3

cZROG

40 ± 1.3

13 ± 2.0

59 ± 5.0

46 ± 7.2

77 ± 6.1

11 ± 0.4

42 ± 7.7

9 ± 2.7

cZ9G

601 ± 386.1

1,048 ± 841.9

528 ± 273.8

1,116 ± 103.0

798 ± 485.5

474 ± 390.2

827 ± 253.6

1,759 ± 1,434.4

cZR5′MP

78 ± 28.0

47 ± 12.2

235 ± 49.9

209 ± 121.3

218 ± 12.0

61 ± 32.9

165 ± 42.0

23 ± 10.4

Total cZ

833 ± 431.5

1,137 ± 860.5

970 ± 356.1

1,450 ± 238.5

1,168 ± 527.7

571 ± 429.5

1,106 ± 307.2

1,812 ± 1,451.1

DHZ

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

DHZOG

2 ± 0.8

1 ± 0.3

3 ± 0.1

2 ± 1.3

3 ± 0.1

1 ± 0.1

5 ± 0.1

2 ± 0.8

DHZR

2 ± 0.2

1 ± 1.1

2 ± 1.1

0 ± 0.1

2 ± 1.3

<LOD

<LOD

1 ± 0.0

DHZROG

3 ± 1.1

1 ± 0.1

3 ± 1.8

3 ± 0.9

6 ± 1.3

1 ± 0.2

5 ± 0.2

2 ± 0.7

DHZ9G

3 ± 0.0

8 ± 5.1

41 ± 0.0

7 ± 0.0

27 ± 25.2

<LOD

3 ± 0.0

2 ± 0.0

DHZR5′MP

54 ± 0.0

20 ± 0.0

14 ± 14.8

2 ± 0.0

35 ± 0.0

11 ± 0.0

<LOD

30 ± 0.0

Total DHZ

64 ± 2.0

30 ± 6.6

64 ± 17.7

14 ± 2.3

74 ± 27.9

13 ± 0.3

12 ± 0.3

36 ± 1.4

iP

0.3 ± 0.0

0.5 ± 0.2

1 ± 0.3

<LOD

<LOD

<LOD

<LOD

<LOD

iPR

9 ± 0.2

7 ± 1.1

66 ± 8.2

24 ± 1.0

22 ± 3.5

3 ± 0.9

42 ± 2.0

<LOD

iP9G

26 ± 5.3

248 ± 16.7

72 ± 3.9

145 ± 23.8

194 ± 85.9

27 ± 8.2

110 ± 15.0

47 ± 0.6

iPR5′MP

12 ± 0.9

11 ± 2.1

96 ± 9.7

42 ± 9.1

39 ± 7.7

<LOD

66 ± 5.8

<LOD

Total iP

47 ± 6.4

266 ± 20.0

235 ± 22.0

212 ± 33.9

255 ± 97.0

30 ± 9.1

218 ± 22.9

47 ± 0.6

BA

18 ± 1.2

8,712 ± 900.0

11,846 ± 341.2

6,755 ± 4.2

10,637 ± 9,279.7

967 ± 57.5

92 ± 32.7

214 ± 17.1

BAR

1 ± 0.1

286 ± 35.5

425 ± 35.5

435 ± 41.6

2,353 ± 196.1

9 ± 1.0

0.5 ± 0.5

1 ± 0.3

BA9G

18 ± 3.4

8,730 ± 352.3

31,342 ± 810.2

30,693 ± 1,973.8

72,254 ± 16,570.7

3,096 ± 2.4

56 ± 8.2

125 ± 29.6

BAR5′MP

9 ± 0.5

926 ± 16.2

1,017 ± 64.8

979 ± 214.9

5,258 ± 756.2

16 ± 1.6

<LOD

<LOD

Total BA

46 ± 5.1

18,654 ± 1,304.0

44,630 ± 1,251.7

38,862 ± 2,234.5

90,501 ± 26,802.7

4,088 ± 62.5

148 ± 41.4

340 ± 47.0

mT

902 ± 60.3

33 ± 8.8

53 ± 0.7

41 ± 0.2

182 ± 221.0

36,579 ± 1,745.6

5,183 ± 89.6

22,894 ± 1,293.5

mTOG

294 ± 10.8

49 ± 11.3

120 ± 22.5

80 ± 24.7

699 ± 222.1

31,151 ± 2,236.7

2,249 ± 396.4

30,589 ± 115.8

mTR

39 ± 0.7

1 ± 0.1

2 ± 0.7

1 ± 0.4

5 ± 0.2

803 ± 52.3

28 ± 3.3

294 ± 39.0

mTROG

32 ± 8.1

4 ± 0.7

2 ± 0.9

5 ± 4.7

7 ± 3.4

7,804 ± 332.7

250 ± 26.9

5,850 ± 127.4

mT9G

63 ± 7.6

3 ± 1.5

17 ± 15.1

11 ± 0.6

19 ± 5.3

3,087 ± 3.6

224 ± 1.9

4,316 ± 547.0

mTR5′MP

108 ± 1.5

27 ± 25.3

3 ± 0.3

<LOD

<LOD

2,136 ± 628.3

64 ± 32.7

804 ± 7.7

Total mT

1,439 ± 88.9

117 ± 47.7

197 ± 40.2

138 ± 30.7

912 ± 452.2

81,560 ± 4,999.2

7,997 ± 550.9

64,748 ± 2,130.4

oT

<LOD

50 ± 7.8

80 ± 10.3

15 ± 0.6

191 ± 153.7

2 ± 0.3

0.2 ± 0.0

1 ± 0.2

oTOG

<LOD

26 ± 3.0

51 ± 0.1

23 ± 9.5

619 ± 117.8

24 ± 0.0

<LOD

<LOD

oTR

1 ± 0.0

1 ± 0.6

1 ± 0.6

1 ± 0.1

11 ± 0.6

<LOD

<LOD

1 ± 0.1

oTROG

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

oT9G

<LOD

63 ± 1.1

329 ± 0.7

142 ± 19.8

1,216 ± 213.3

9 ± 7.0

16 ± 0.0

14 ± 0.0

oTR5′MP

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

Total oT

1 ± 0.0

141 ± 12.5

461 ± 11.7

180 ± 30.0

2,036 ± 485.5

35 ± 7.4

16 ± 0.0

16 ± 0.3

pT

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

pTOG

2 ± 0.0

1 ± 0.9

6 ± 0.3

3 ± 3.0

10 ± 2.9

4 ± 2.1

5 ± 2.7

4 ± 3.0

pTR

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

pTROG

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

pTR5′MP

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

Total pT

2 ± 0.0

1 ± 0.9

6 ± 0.3

3 ± 3.0

10 ± 2.9

4 ± 2.1

5 ± 2.7

4 ± 3.0

K

43 ± 16.1

18 ± 11.1

39 ± 13.4

11 ± 1.8

11 ± 0.0

52 ± 10.1

27 ± 8.3

40 ± 14.7

KR

0.2 ± 0.0

0.1 ± 0.1

0.1 ± 0.0

0.1 ± 0.0

1 ± 0.3

<LOD

<LOD

<LOD

K9G

1 ± 0.7

2 ± 0.1

6 ± 0.1

7 ± 1.9

10 ± 2.8

<LOD

<LOD

<LOD

KR5′MP

<LOD

1 ± 0.0

2 ± 0.0

1 ± 0.0

<LOD

<LOD

<LOD

<LOD

Total K

44 ± 16.8

21 ± 11.2

47 ± 13.5

18 ± 3.7

22 ± 3.1

52 ± 10.1

27 ± 8.3

40 ± 14.7

Total CK

2,519 ± 554.4

20,388 ± 2,271.7

46,677 ± 1,727

40,887 ± 2,579.1

94,998 ± 28,404.9

86,432 ± 5,522.4

9,541 ± 936.0

67,053 ± 3,651.4

Values are given as mean ± standard deviation (n = 3); <LOD = below the limit of detection. Besides topolins, all the CKs abbreviations were based on Kamínek et al. (2000)

Table 2

Effect of roscovitine and INCYDE with aromatic cytokinins on endogenous cytokinins content (pmol/g FW) in ‘Williams’ bananas underground parts

Cytokinin type

Cytokinin treatment and resultant endogenous cytokinin content (pmol/g FW)

mT

BA

BA + INCYDE

BA + roscovitine

BA + INCYDE + roscovitine

mT + INCYDE

mT + roscovitine

mT + INCYDE + roscovitive

tZ

0.04 ± 0.03

2 ± 0.8

0 ± 0.3

2 ± 1.4

1 ± 0.3

1 ± 0.2

4 ± 0.2

1 ± 0.0

tZOG

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

tZR

<LOD

2 ± 0.1

1 ± 0.5

3 ± 1.1

2 ± 0.4

1 ± 0.2

3 ± 0.2

1 ± 0.0

tZROG

0.1 ± 0.01

1 ± 0.1

0 ± 0.2

1 ± 0.2

0.4 ± 0.01

0 ± 0.1

0 ± 0.1

2 ± 0.2

tZ9G

4 ± 0.4

13 ± 2.8

4 ± 1.4

7 ± 3.1

4 ± 0.2

3 ± 0.2

4 ± 2.9

2 ± 0.7

tZR5′MP

0.33 ± 0.2

6 ± 0.7

2 ± 1.0

31 ± 20.9

13 ± 8.1

4 ± 2.2

1 ± 1.1

20 ± 1.4

Total tZ

5 ± 0.6

24 ± 4.5

8 ± 3.3

44 ± 26.6

19 ± 9.1

9 ± 2.9

13 ± 4.4

26 ± 2.3

cZ

2 ± 0.9

7 ± 2.9

2 ± 0.2

4 ± 1.3

4 ± 0.2

21 ± 7.9

41 ± 6.6

2 ± 1.7

cZOG

1 ± 0.1

<LOD

0 ± 0.1

<LOD

<LOD

1 ± 0.3

2 ± 0.5

<LOD

cZR

14 ± 1.1

9 ± 2.4

4 ± 1.7

58 ± 17.3

56 ± 18.4

110 ± 3.3

288 ± 20.1

63 ± 7.0

cZROG

5 ± 0.3

2 ± 0.3

2 ± 1.0

59 ± 5.2

46 ± 12.7

41 ± 2.1

137 ± 17.4

20 ± 2.4

cZ9G

97 ± 33.6

199 ± 5.6

75 ± 21.8

175 ± 25.0

179 ± 42.7

217 ± 29.8

81 ± 6.8

21 ± 5.5

cZR5′MP

63 ± 1.8

45 ± 19.9

54 ± 17.2

490 ± 108.1

253 ± 40.5

394 ± 57.4

640 ± 126.2

194 ± 10.3

Total cZ

180 ± 37.8

262 ± 31.0

138 ± 42.0

785 ± 156.8

538 ± 114.6

784 ± 100.8

1,189 ± 177.6

300 ± 26.9

DHZ

<LOD

1 ± 0.3

0 ± 0.1

0 ± 0.2

0 ± 0.01

0 ± 0.1

1 ± 0.1

0.2 ± 0.03

DHZOG

0 ± 0.0

1 ± 0.2

0 ± 0.2

1 ± 0.5

1 ± 0.4

0 ± 0.1

0 ± 0.3

1 ± 0.3

DHZR

2 ± 0.2

2 ± 0.2

1 ± 0.2

2 ± 0.7

1 ± 0.5

17 ± 0.8

29 ± 0.8

7 ± 0.3

DHZROG

0 ± 0.1

1 ± 0.5

0 ± 0.1

2 ± 1.0

1 ± 0.4

5 ± 0.3

19 ± 3.5

3 ± 0.0

DHZ9G

1 ± 0.2

8 ± 1.3

2 ± 0.4

4 ± 0.8

4 ± 0.3

3 ± 0.2

11 ± 1.0

1 ± 0.2

DHZR5′MP

12 ± 0.8

7 ± 0.5

6 ± 1.1

110 ± 40.2

26 ± 16.6

114 ± 63.9

94 ± 1.8

57 ± 5.7

Total DHZ

16 ± 1.4

19 ± 3.0

9 ± 2.2

120 ± 43.3

33 ± 18.3

139 ± 65.5

155 ± 7.4

69 ± 6.6

iP

<LOD

12 ± 4.3

4 ± 1.3

8 ± 2.9

7 ± 0.8

5 ± 1.6

8 ± 3.7

4 ± 1.2

iPR

7 ± 2.1

19 ± 7.6

23 ± 9.5

54 ± 9.7

58 ± 6.3

10 ± 4.5

76 ± 4.7

6 ± 2.0

iP9G

49 ± 4.6

211 ± 10.0

109 ± 28.9

30 ± 7.6

42 ± 12.0

27 ± 5.0

218 ± 90.2

5 ± 2.7

iPR5′MP

36 ± 9.6

110 ± 11.1

56 ± 12.7

226 ± 57.5

132 ± 40.3

53 ± 18.2

131 ± 16.1

14 ± 4.0

Total iP

91 ± 16.3

352 ± 33.0

193 ± 52.3

319 ± 77.7

240 ± 59.4

96 ± 29.3

434 ± 114.7

30 ± 9.9

BA

218 ± 79.9

133,696 ± 34,010.0

58,646 ± 23,349.5

108,767 ± 40,570.4

90,962 ± 31,271.6

3,811 ± 313.5

610 ± 236.6

2,482 ± 1,088.7

BAR

1 ± 0.6

855 ± 54.8

941 ± 218.7

2,538 ± 77.4

3,656 ± 700.2

10 ± 2.5

9 ± 2.6

14 ± 2.1

BA9G

64 ± 46.4

231,971 ± 17,203.1

107,425 ± 37,160.8

192,241 ± 50,161.4

186,642 ± 17,584.5

2,958 ± 421.9

360 ± 29.8

837 ± 232.5

BAR5′MP

1 ± 0.4

6,904 ± 963.2

1,734 ± 861.0

3,288 ± 2,657.5

5,327 ± 2,536.5

40 ± 8.6

7 ± 3.1

30 ± 0.5

Total BA

284 ± 127.3

373,427 ± 52,231.1

168,746 ± 61,590.0

306,834 ± 93,466.6

286,587 ± 52,092.7

6,819 ± 746.5

986 ± 272.1

3,363 ± 1,323.8

mT

19,816 ± 1,715.2

666 ± 41.3

341 ± 179.4

2,062 ± 1,272.7

1,013 ± 24.4

58,057 ± 3,077.0

59,127 ± 539.7

38,593 ± 1,402.8

mTOG

3,928 ± 873.4

3,909 ± 670.3

1,501 ± 469.5

968 ± 538.5

748 ± 279.1

66,078 ± 9,963.9

145,144 ± 19,824.4

60,043 ± 5,169.3

mTR

167 ± 65.8

42 ± 9.2

38 ± 10.2

37 ± 8.7

46 ± 7.4

15,866 ± 2,822.0

10,996 ± 40.9

27,138 ± 465.7

mTROG

652 ± 71.7

116 ± 54.5

39 ± 22.3

79 ± 39.7

61 ± 3.7

17,305 ± 1,527.3

6,556 ± 824.8

38,652 ± 2,464.6

mT9G

5,020 ± 849.9

1,689 ± 137.8

952 ± 330.6

619 ± 374.5

513 ± 168.0

244,469 ± 22,819.4

425,229 ± 78,859.3

68,110 ± 11,401.7

mTR5′MP

332 ± 150.4

48 ± 31.2

77 ± 36.8

180 ± 105.0

86 ± 58.6

16,054 ± 481.1

1,597 ± 228.0

13,269 ± 4,864.2

Total mT

29,916 ± 3,726.4

6,469 ± 944.3

2,947 ± 1,048.9

3,945 ± 2,338.9

2,467 ± 541.2

417,830 ± 40,690.9

648,649 ± 100,317.0

245,805 ± 25,768.2

oT

6 ± 0.7

8,181 ± 1,549.0

2,900 ± 596.2

6,496 ± 723.5

7,168 ± 1,313.6

40 ± 19.6

7 ± 1.3

58 ± 3.0

oTOG

2 ± 0.9

5,034 ± 715.8

1,633 ± 958.0

209 ± 40.5

401 ± 229.9

15 ± 3.7

29 ± 14.5

23 ± 0.0

oTR

0 ± 0.2

89 ± 31.1

42 ± 15.6

83 ± 26.4

124 ± 22.9

<LOD

<LOD

<LOD

oTROG

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

oT9G

1 ± 0.3

19,766 ± 2,077.5

8,532 ± 2,629.2

7,166 ± 1,669.3

10,506 ± 3,139.8

115 ± 64.2

103 ± 14.8

38 ± 7.4

oTR5′MP

35 ± 37.8

49 ± 27.9

86 ± 70.5

116 ± 98.9

105 ± 92.3

<LOD

<LOD

<LOD

Total oT

44 ± 39.9

33,118 ± 4,401.2

13,193 ± 4,269.5

14,071 ± 2,558.6

18,305 ± 4,798.4

170 ± 87.4

139 ± 30.5

119 ± 10.4

pT

1 ± 0.1

4 ± 1.3

1 ± 0.3

5 ± 0.3

3 ± 0.6

0 ± 0.2

1 ± 0.5

1 ± 0.3

pTOG

<LOD

17 ± 5.8

7 ± 0.4

9 ± 0.7

6 ± 2.4

<LOD

<LOD

<LOD

pTR

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

pTROG

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

pTR5′MP

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

Total pT

1 ± 0.1

21 ± 7.1

8 ± 0.7

14 ± 1.0

10 ± 3.1

0 ± 0.2

1 ± 0.5

1 ± 0.3

K

58 ± 50.7

217 ± 129.1

85 ± 27.4

1,151 ± 250.9

450 ± 238.9

394 ± 121.1

93 ± 2.5

1,641 ± 2.2

KR

<LOD

3 ± 0.8

1 ± 0.2

4 ± 0.8

3 ± 1.6

2 ± 0.3

4 ± 0.7

3 ± 0.9

K9G

2 ± 0.1

831 ± 6.1

339 ± 51.5

486 ± 143.9

449 ± 95.5

35 ± 7.0

11 ± 1.5

47 ± 0.2

KR5′MP

<LOD

5 ± 0.7

2 ± 1.2

35 ± 32.0

12 ± 7.4

5 ± 0.8

<LOD

21 ± 1.3

Total K

60 ± 50.7

1,057 ± 136.8

427 ± 80.3

1,676 ± 427.6

914 ± 343.4

435 ± 129.2

108 ± 4.7

1,712 ± 4.5

Total CK

30,597 ± 4,000.6

414,749 ± 57,791.9

185,667 ± 67,089.1

327,808 ± 99,097.1

309,113 ± 57,980.2

426,282 ± 41,852.6

651,674 ± 100,929.1

251,425 ± 27,153.0

Values are given as mean ± standard deviation (n = 3); <LOD = below the limit of detection. Besides topolins, all the CKs abbreviations were based on Kamínek et al. (2000)

In the aerial part, the most abundant CK forms were the free bases for mT and mT + roscovitine, O-glucosides for mT + INCYDE and mT + INCYDE +roscovitine while it was 9-glucosides for the other treatments (Fig. 4a). Similarly, 9-glucosides were the most dominant CK form detected in the underground parts of majority of the treatments (Fig. 4b). However, mT and mT + INCYDE + roscovitine treatments had free bases and O-glucosides, respectively, as their major component. The levels of ribosides and ribotides were generally low, especially in BA-containing treatments. The pattern of the distribution and/or transportation of individual CK forms within the aerial and underground parts, respectively, is shown in Fig. 5a and b. The five CK forms were abundant in the underground region in all the treatments with the exception of O-glucosides, which were slightly higher (53 %) in aerial part of the BA + roscovitine + INCYDE-treated plantlets. In BA + roscovitine and/or INCYDE-treated plantlets, the quantity of O-glucosides was higher compared to BA-treated plantlets in aerial part. On the other hand, roscovitine and/or INCYDE in the presence of mT reduced the amount of 9-glucosides compared to the mT control in aerial part.
https://static-content.springer.com/image/art%3A10.1007%2Fs00425-012-1721-z/MediaObjects/425_2012_1721_Fig4_HTML.gif
Fig. 4

Total cytokinin pool (%) of the different cytokinin forms in aerial (a) and underground (b) parts of the micropropagated ‘Williams’ bananas. 1meta-topolin, 2 benzyladenine, 3 benzyladenine + INCYDE, 4 benzyladenine + roscovitine, 5 benzyladenine + INCYDE + roscovitine, 6meta-topolin + INCYDE, 7meta-topolin + roscovitine, 8meta-topolin + INCYDE + roscovitine

https://static-content.springer.com/image/art%3A10.1007%2Fs00425-012-1721-z/MediaObjects/425_2012_1721_Fig5_HTML.gif
Fig. 5

Relative abundance (%) and distribution pattern of the different cytokinin forms in aerial (a) and underground (b) parts of the micropropagated ‘Williams’ bananas. 1meta-topolin, 2 benzyladenine, 3 benzyladenine + INCYDE, 4 benzyladenine + roscovitine, 5 benzyladenine + INCYDE + roscovitine, 6meta-topolin + INCYDE, 7meta-topolin + roscovitine, 8meta-topolin + INCYDE + roscovitine

Effect of roscovitine and INCYDE on photosynthetic and phenolic contents

The interaction of mT or BA with roscovitine and/or INCYDE had varied effect on the phenolic content (Table 3). The combination of mT with roscovitine significantly increased the total phenolic and flavonoid levels in the plantlets, but there was a drastic reduction in the amount of proanthocyanidins compared to the use of mT alone. When incorporated with mT, INCYDE or its combination with roscovitine did not have a notable effect on the phenolic contents detected. When compared to BA treatment, plantlets treated with BA + INCYDE had a significantly lower amount of total phenolics and flavonoids as well as proanthocyanidins.
Table 3

Effect of roscovitine and INCYDE on the phenolic content of micropropagated ‘Williams’ bananas

#Treatment

Total phenolics (mg GAE/g DW)

Total flavonoids (mg CE/g DW)

Proanthocyanidins (μg CCE/g DW)

Control mT

58.4 ± 2.23 d

16.3 ± 0.13 f

534.8 ± 6.10 cd

Control BA

118.3 ± 5.83 a

34.3 ± 0.24 a

612.2 ± 13.00 ab

BA + INCYDE

91.7 ± 3.28 bc

23.5 ± 0.11 c

476.3 ± 8.37 e

BA + roscovitine

110.7 ± 7.80 a

30.2 ± 0.38 b

640.7 ± 7.59 a

BA + INCYDE + roscovitine

97.7 ± 6.50 b

21.8 ± 0.26 d

581.2 ± 15.64 bc

mT + INCYDE

67.9 ± 2.17 d

16.3 ± 0.16 f

463.9 ± 8.54 e

mT + roscovitine

83.8 ± 2.01 c

21.0 ± 0.23 e

405.4 ± 42.15 f

mT + INCYDE + roscovitine

59.4 ± 0.54 d

13.0 ± 0.04 g

500.6 ± 8.86 de

Mean values ± standard error (n = 5) in the same column with different letter(s) are significantly different (P = 0.05) based on Duncan’s multiple range test (DMRT)

#Treatment: mT, meta-topolin (30 μM); BA, N6-benzyladenine (30 μM); INCYDE 100 μM, roscovitine 5 μM

GAE gallic acid equivalents, CE catechin equivalents, CCE cyanidin chloride equivalents

Table 4 shows the effect of combining roscovitine and INCYDE (alone or together) with either mT or BA on the photosynthetic pigment content of micropropagated ‘Williams’ bananas. Generally, BA-treated plantlets had a higher pigment content compared to the mT treatments. Furthermore, the addition of roscovitine or INCYDE to BA medium resulted in the production of more pigment than the use of BA alone. The combination of BA, roscovitine and INCYDE was detrimental to the production of photosynthetic pigments. Conversely, treatment with mT alone had a higher pigment content compared to the presence of roscovitine or INCYDE. There was an increase in total chlorophyll content in mT + roscovitine + INCYDE-treated plantlets compared to the addition of mT + roscovitine or INCYDE treatments. The same combination (mT + roscovitine + INCYDE) had the highest chlorophyll/carotenoid ratio among all the treatments.
Table 4

Effect of roscovitine and INCYDE on the photosynthetic pigment contents of micropropagated ‘Williams’ bananas

#Treatment

Chlorophyll a (μg/g FW)

Chlorophyll b (μg/g FW)

Carotenoid (μg/g FW)

Total chlorophyll (μg/g FW)

Chlorophyll a/b

Total chlorophyll/carotenoid

Control mT

257.8 ± 27.78 abc

98.6 ± 11.39 abc

80.9 ± 8.84 ab

356.4 ± 39.13 abc

2.6 ± 0.03 abc

4.4 ± 0.08 b

Control BA

328.0 ± 42.40 abc

128.5 ± 17.59 abc

95.6 ± 10.83 ab

456.5 ± 59.94 abc

2.6 ± 0.04 ab

4.7 ± 0.14 ab

BA + INCYDE

363.8 ± 71.92 ab

137.3 ± 23.56 ab

98.7 ± 16.63 ab

501.0 ± 95.38 ab

2.6 ± 0.08 ab

5.0 ± 0.13 a

BA + roscovitine

389.5 ± 83.03 a

157.2 ± 32.14 a

117.2 ± 21.39 a

546.7 ± 115.16 a

2.5 ± 0.03 bc

4.6 ± 0.15 ab

BA + INCYDE + roscovitine

298.5 ± 45.86 abc

110.3 ± 18.93 abc

87.9 ± 12.01 ab

408.8 ± 64.72 abc

2.7 ± 0.06 a

4.6 ± 0.14 ab

mT + INCYDE

199.3 ± 37.16 c

73.3 ± 12.80 c

58.5 ± 8.42 b

272.5 ± 49.94 c

2.7 ± 0.04 a

4.6 ± 0.26 ab

mT + roscovitine

188.7 ± 14.00 c

78.6 ± 5.99 bc

61.2 ± 3.58 b

267.3 ± 19.89 c

2.4 ± 0.04 c

4.4 ± 0.21 b

mT + INCYDE + roscovitine

217.0 ± 35.33 bc

86.2 ± 13.18 bc

59.7 ± 9.60 b

303.3 ± 48.36 bc

2.5 ± 0.08 bc

5.1 ± 0.08 a

Mean values ± standard error (n = 5) in the same column with different letter(s) are significantly different (P = 0.05) based on Duncan’s multiple range test (DMRT)

#Treatment: mT, meta-topolin (30 μM); BA, N6-benzyladenine (30 μM); INCYDE 100 μM, roscovitine 5 μM

Discussion

Better understanding of the structure–activity relationship of CKs and their effect on plant physiology remains vital for improving the growth and development of plants in vitro (Chen 1997; Sakakibara 2006). From a commercial point of view, the multiplication rate of any plant species especially the economically important ones such as bananas should not be compromised. Thus, the possibility of any stimulatory or inhibitory effect of roscovitine and INCYDE on the growth parameters of the banana plantlets was evaluated. Roscovitine is a well-known potent and selective inhibitor of animal CDKs where it effectively blocks cells at G1/S and G2/M transitions (Meijer et al. 1997). Although, a similar observation was observed in Petunia hybrid culture (Tréhin et al. 1998), studies on the effect of roscovitine on plant cell cycle are limited (Planchais et al. 2000). In plant cells, roscovitine has been demonstrated as one of the potential synchronizing agents and used to characterize the requirements of different CDK activities for cell cycle progression (Planchais et al. 1997; Binarová et al. 1998; Tréhin et al. 1998; Blagoeva et al. 2003). The reversibility and specificity of roscovitine activities make it a suitable chemical inhibitor for synchronization in the plant cell cycle (Planchais et al. 2000). In the current study, the interaction of roscovitine with the CKs seems to play some vital role in the cell cycle as mT or BA with roscovitine treatments had a slightly higher shoot multiplication rate compared to the controls (mT or BA alone). Pasternak et al. (2000) postulated that CK play a regulatory role in the first cells of protoplast-derived cells of alfalfa at the G2/M border which may be related to the post-translational regulation of the CdcMsA/B kinase. Although INCYDE retained high CK activity in three classical CK bioassays (Zatloukal et al. 2008), the interaction of the compound with BA resulted in a slight reduction in the regeneration and multiplication rate of the explants. The opposite effect observed in the presence of mT is probably due to the presence of the hydroxyl group in mT. In addition, the effect of the increased total endogenous CK levels in mT + roscovitine and/or INCYDE-treated plantlets (Tables 1, 2) may have partially contributed to the better multiplication in these treatments.

The importance of exogenous application of CKs for in vitro shoot development cannot be overemphasized, particularly in the micropropagation of bananas (Bairu et al. 2008). The response of explants observed in terms of growth and development in vitro is regulated by the interaction and balance between the applied PGRs (type and concentration) and the endogenously produced ones (George 1993; Krikorian 1995). Factors such as the exogenous application of CKs are known to affect the biochemical pathways that regulate endogenous CK levels (Krikorian 1995; Blagoeva et al. 2004a). Since aromatic type CKs were used in the current study, as expected, the aromatic CKs were several fold more abundant compared to the isoprenoid CKs which are generally more common in plant tissue. When compared to the lower levels that ranged from approximately 0.2–39 % for the total isoprenoid CK pool in the regenerants, the aromatic CKs were more abundant with the highest level (99.8 %) observed in BA-treated plantlets (Fig. 2). Ivanova et al. (2006) reported a similar scenario whereby the application of exogenous BA to the media resulted in an alteration from isoprenoid to aromatic CKs in the newly formed Aloe polyphylla shoots compared to those grown on CK-free and Z-supplemented media. As only aromatic CKs were used in the current study, the detection of isoprenoid CKs indicates the occurrence of the de novo CK synthesis pathway in the regenerated banana in vitro. However, the high concentration of the exogenously applied aromatic CK probably suppressed the isoprenoid CK synthesis pathway in the cultured explants. In addition, CKX have been shown to exhibit preferences for the isoprenoid CKs while the aromatic CKs are more resistant (Zalabák et al. 2012). Interestingly, the current findings show that roscovitine and/or INCYDE when combined with mT or BA (with the exception mT + INCYDE) possibly prevented the breakdown of isoprenoid CK during the in vitro growth of ‘Williams’ bananas (Tables 1, 2).

Cytokinin uptake, metabolism and transport within the explants are vital parameters for the growth and development in PTC (Auer 1997). In the current study, the wide disparity between the aerial and underground parts is probably a function of the CK transport mechanism. In the eight treatments, the concentration of total CK detected in the underground parts was higher than in the aerial parts. Plantlets regenerated from mT + roscovitine had the highest total CK content (661,215 pmol/g FW) with the underground parts having approximately a 68-fold more than the aerial part. A general trend observed was that the addition of roscovitine and/or INCYDE with mT improved the total CK pool in both aerial and underground parts of the regenerants. A similar pattern was observed in the aerial part when BA was combined with roscovitine and INCYDE; however, both compounds reduced the total CK content in the underground sections as well as the sum total in the plantlets. It is noteworthy to highlight that the decrease in total CK pool was mainly due to the reduction in the quantity of 9-glucosides, which are generally detrimental to plant growth. This reduction in 9-glucosides levels could be attributed to the presence of either roscovitine or INCYDE when acting individually with BA.

Often, most of the physiological activity of Z as a free base has been attributed to tZ while the cis isomer is regarded as an inactive or weakly active form of CK (Kamínek et al. 1987; Haberer and Kieber 2002). Although a precise role for cZ-type CKs remains to be fully elucidated, recent studies have been identifying their potential functions in plants (Dwivedi et al. 2010; Gajdošová et al. 2011). In accordance with previous findings (Blagoeva et al. 2004b; Dwivedi et al. 2010), where olomoucine and its analogs such as roscovitine enhanced the cZ levels in vitro, roscovitine stimulated the production of more cZ in both aerial and underground regions of the regenerated plantlets compared to the controls (Tables 1, 2). Although, cZ was less abundant in the underground region, the concentrations were several folds higher than the tZ in the plantlets. In addition to the potential role of cZ and/or its derivatives in regulation of physiological processes, Gajdošová et al. (2011) postulated that these compounds may be relevant under growth-limiting conditions connected to developmental processes or external signals.

In BA-supplemented treatments, 9-glucosides account for approximately 60 % of total CK content (Fig. 4a, b). In terms of their distribution, an estimated 80 % were detected in the underground part of the plantlets (Fig. 5b). The quantity and distribution of CK derivatives are important information, which may explain some observed physiological disorders in PTC (Aremu et al. 2012b). It is generally known that most 9-glucosides particularly BA9G are formed and stored in the plant base and does not seem to be transported and have been implicated in problems such as acclimatization failure (Werbrouck et al. 1995), shoot-tip necrosis (Bairu et al. 2011) and high toxicity (Amoo et al. 2011). In the current study, when compared to BA alone, the presence of roscovitine and/or INCYDE with BA produced a substantial quantity of the 9-glucosides in the aerial parts (Fig. 5a, b). The enhanced transportation of 9-glucosides could be a regulatory mechanism to alleviate the detrimental effect in the underground region of the plantlets.

Even though the precise role of phenolic compounds in the processes of differentiation and morphogenesis is not fully understood (Schnablová et al. 2006), their importance in plant growth remains undisputed (Buer et al. 2010; De Klerk et al. 2011). It is well known that PGRs, especially CK influences the secondary metabolites in in vitro plantlets (Ramachandra Rao and Ravishankar 2002; Coste et al. 2011). In this study, the interaction of CKs with roscovitine and/or INCYDE exhibited diverse responses in terms of the amount of secondary metabolites quantified (Table 3). Overall, the combination of roscovitine and/or INCYDE with BA had higher total phenolic and flavonoid contents than mT-treatments. Remarkably, there was a direct relationship between the total amount of endogenous CK and total phenolic content depending on the type of aromatic CK used. When mT was combined with roscovitine and/or INCYDE, there was an increase in total endogenous CKs (Tables 1, 2) which correlated with an increase in total phenolics compared to the use of mT alone (Table 3). There was a reduction in the total endogenous CK content when BA was combined with roscovitine and/or INCYDE, with a decrease in the amount of phenolics compared to the BA-treated ones (Table 3). Evidence from the study by Schnablová et al. (2006) demonstrated that overproduction of endogenous CKs (CK) caused stress response in non-rooting Pssu-ipt transgenic tobacco grown in vitro. The authors observed the over-accumulation of phenolic compounds, synthesis of pathogenesis-related proteins, and increase in peroxidase activity. The use of these CK analogs could possible play vital role in the production of essential plant secondary metabolites as highlighted by Planchais et al. (2000) and Blagoeva et al. (2003).

Cytokinins are important for photosynthesis as they are known to influence the quantity of photosynthetic pigments among their numerous other functions. Furthermore, the protective action of CKs under stress conditions maintains the structure and function of the photosynthetic apparatus (Chernyad’ev 2009). In this study, the use of roscovitine and INCYDE in conjunction with the CKs did not cause any significant increase in the quantity of the photosynthetic pigments (Table 4). Nevertheless, there was a slight increase in the amount of pigments when roscovitine and/or INCYDE was used together with BA and a minimum reduction in quantity in the presence of mT compared to the controls (BA or mT alone). Particularly for BA, higher concentrations are known to have some detrimental effects as demonstrated in carrot and Arabidopsis cultures (Carimi et al. 2003). These authors observed that high concentrations of BA (27 μM) block cell proliferation and induce programmed cell death. Using a similar high concentration (30 μM) in this study, it was demonstrated that roscovitine or INCYDE could alleviate the possible detrimental effects of BA on the ability of the in vitro plantlets to produce more photosynthetic pigments. On the other hand, the use of mT with both roscovitine and INCYDE produced an opposite effect. The basis of the contrasting responsiveness of the CKs remains unclear and would require more stringent studies at a molecular level to fully elucidate the underlying mechanisms.

Conclusions

Often, the majority of available evidences of the molecular characterization and explanation of CK interactions are based on Arabidopsis mutants. However, the practical application of such findings on plants which are generally characterized by a more complex genome and physiology may exhibit different responses. From a practical perspective, current findings give valuable insights into the potential of roscovitine and INCYDE in micropropagation of ‘Williams’ bananas and possibly in other plant species. Depending on the types of CK, it has been demonstrated that the endogenous CK levels can be easily regulated with the addition of roscovitine and/or INCYDE. Particularly, the use of these compounds could be useful for controlling some tissue culture-induced physiological disorders related to endogenous CKs. In addition, roscovitine (in the presence of mT) enhanced the levels of the total phenolics and flavonoids in the micropropagated ‘Williams’ bananas. The use of both CK analogs for the manipulation of plant physiology to improve the various aspects of growth and synthesis of secondary metabolites remains a challenging research topic. Furthermore, the presence of a significant quantity of oT in BA-treated cultures raises important questions such as their source and origin? Why are they not available in mT-treated cultures? Currently, the role of oT and pT in plant growth and development is not fully understood by researchers, more in-depth studies with the use of radio-labeled compounds could possibly elucidate their metabolism and functions in due course.

Acknowledgments

The University of KwaZulu-Natal (Pietermaritzburg), South Africa and Centre of the Region Haná for Biotechnological and Agricultural Research, Palacký University (Olomouc), Czech Republic for providing financial support (Grant No. ED0007/01/01).

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© Springer-Verlag 2012