Plant Cell, Tissue and Organ Culture 28: 183-186, 1992. © 1992 Kluwer Academic Publishers. Printed in the Netherlands.

Uptake and metabolism of 6-benzyladenine in shoot cultures of a range of species

David Blakesley ~ & David Constantine 2 ~School of Biological Sciences, University of Bath, Claverton Down, Bath BA2 7AY, UK; 2Twyford Plant Laboratories Ltd, Baltonsborough, Somerset, BA6 8QG, UK

Received 11 February 1991; accepted in revised form 1 October 1991

Key words: cytokinin metabolism, tissue culture, BA uptake

Abstract

Shoots of a range of species were cultured in vitro on a medium containing 2.22 IzM benzyladenine (BA). After 25 days in culture the amount of BA taken up differed between species. The conjugates of BA present in the plant tissues were identified by HPLC. The pattern of BA metabolism varied considerably between species as did the actual levels of each conjugate. Benzyladenine accumulated in Hippeastrum, Alstroemeria and Ficus with little conjugation. In Musa, Rhododendron and Fuchsia BA also accumulated along with its ribosyl and glucoside conjugates. In contrast, very low levels of BA were detected in Gerbera and Dendranthema, but various glucosides did accumulate.

Abbreviations: BA - 6-benzyladenine, [3G]BA - 3-/3-D-glucopyranosyl-BA, [7G]BA - 7-/3-D- glucopyranosyl-BA, [9G]BA - 9-/3-D-glucopyranosyl-BA, [9R-G]BA - 9-/3-D-(ribosylglucoside)-BA, [9R]BA - 9-/3-ribofuranosyl-BA, HPLC - high performance liquid chromatography, PAR - photo- synthetically active radiation, T E A B - triethylammonium bicarbonate

Introduction

The synthetic cytokinin BA is routinely added to tissue culture media to stimulate shoot prolifer- ation and is the most commonly used cytokinin commercially (Thomas & Blakesley 1987). Sev- eral workers have identified metabolites of BA in plant tissue (Letham & Palni 1983), but few have addressed the long-term uptake and metab- olism of BA in proliferating shoots in vitro. Nordstr6m & Eliasson (1986) demonstrated that BA was taken up from the medium by apple shoots, and Biondi et al. (1984) showed that in vitro elm shoots absorbed BA and subsequently degraded the cytokinin by side chain cleavage. More detailed studies on the uptake and metabo- lism of BA in shoot cultures of Gerbera (Blakes- ley et al. 1991) and Rhododendron and Musa (Blakesley 1991) demonstrated that both the rate

and amount of BA absorbed from the medium, and the pattern of BA metabolism can vary considerably between species. It is not clear from these studies whether BA or its riboside is active per se in shoot proliferation. The aim of this short communication is to indicate the range of metabolic responses to applied BA by different species under identical in vitro conditions.

Materials and methods

Plant material

Proliferating shoot cultures of each species were established for at least two transfers on Lin- smaier & Skoog (1965) medium, solidified with 0.8% agar (Tech. No. 3, Oxoid, UK) and sup- plemented with 2.22 IxM BA. Shoots were sub-

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cultured every 25 days. Musa cv Dwarf Caven- dish and Hippeastrum x hybrida shoots were di- vided into single shoots from which laminae and basal callus were removed. Rhododendron cv Anna Rose Whitney, Gerbera jamesonii, Fuch- sia x hybrida, Dendranthema grandiflora and Ficus lyrata shoots proliferate from axillary buds at the base of the shoots. Consequently they were divided into small clusters of 2-3 shoots from which the apical part of the stem and basal callus were removed. Alstroemeria produce new shoots from a rhizome. These trimmed shoots were transferred onto fresh medium and incu- bated at 25°C with a 16-h photoperiod at 135 txmolm -z s -~ PAR. For uptake studies, 14.7 KBq of [8-~4C]BA (specific activity 9.29 MBq mg -1, Amersham International, UK), was added with unlabelled BA (final concen- tration to 2.22 IxM in the medium). Depending on the species, 6-10 trimmed shoots were trans- ferred into each of 3 containers (65mm× 65 mm × 75 mm deep Magenta, Magenta Corp. USA) containing 50 ml of medium supplemented with radiolabelled BA. The 3 replicate contain- ers were harvested after 25 days.

Extraction and purification

When harvested, shoots were washed with dis- tilled water and divided to give a preliminary indication of the movement of BA within the shoot explants:

Dendranthema: callus and shoot Hippeastrum: laminae base (including basal plate) and laminae Ficus: stem and laminae Fuchsia: stem and laminae Alstroemeria: rhizome and shoot Musa: pseudostem and laminae Rhododendron: stem and laminae Gerbera: callus, petioles plus apices and laminae.

The tissue was then frozen in liquid nitrogen and stored at -20°C. The extraction and purification procedures employed have been described in detail previously (Blakesley et al. 1991). Essen- tially plant tissue was extracted in 80% methanol and concentrated in vacuo. The residue was applied to a SP Sephadex C-25 (H + form) col- umn, which separated non-basic cytokinins (nu-

cleotides and breakdown products) from the bases, including cytokinins. Bases were further purified on a C18 Sep-Pak cartridge prior to HPLC separation. Free bases were applied to a 5 p,m Hypersil ODS column (250 × 4.5 mm i.d.) and eluted at 2 ml min -1 in a linear gradient of 4-20% acetonitrile in dilute TEAB, pH 7 over 35 min (Horgan & Kramers 1979). Samples were cochromatographed with standard [3G]BA, [7G]BA, [9G]BA and [9R]BA, and fractions collected for determination of radioactivity using a liquid scintillation counter. Good separation was achieved between authentic BA conjugates (Blakesley et al. 1991). Absolute levels of BA conjugates were determined from this data. Total uptake of BA was determined by the loss of radioactivity from the medium. Not all of this was recovered in the methanol extraction, in- dicating that s o m e 14C was contained in methanol-insoluble compounds or had been lost a s 14CO2.

Results and discussion

The aim of this work was to consider how differ- ent species take up and metabolise BA, a poten- tially damaging synthetic compound, under iden- tical conditions in vitro. No attempt was made to identify the metabolites in the methanol-insolu- ble fraction or in the non-basic fraction from the ion exchange column.

Uptake and metabolism in the whole shoot

After 25 days in culture 41% of the radioactivity was taken up by the shoots of Rhododendron and Fuchsia in contrast to 90% by Gerbera shoots (Table 1). There was also considerable variation in the amount of radioactivity associ- ated with BA and its conjugates (Table 1). More than 60% of the radioactivity recovered from Dendranthema and Rhododendron shoots was associated with this fraction in contrast to less than 30% in Gerbera and Hippeastrum, Further analysis of the basic fraction by HPLC indicated several different patterns of metabolism (Table 2); -accumulation of BA with lower levels of

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Table 1. Percentage uptake of [14C]BA into shoot explants (as measured by loss of 14C from the medium) and the distribution of that between the non-basic and basic ion exchange fractions and the fraction that was not recovered (*radioactivity assumed to have been incorporated into methanol-insoluble compounds or released as CO2). Values given are means of three replicates, harvested after 25 days.

Species Uptake (%) Distribution of 14C ( • )

Non accounted* Methanol-soluble

Non-basic Basic

Hippeastrum 52 51 20 29 Ficus 77 51 15 34 Alstroemeria 32 48 2 l 3 ! Fuchsia 41 11 41 48 Musa 52 64 4 32 Gerbera 90 32 41 27 Dendranthema 68 21 7 72 Rhododendron 41 31 6 63

Table 2. Benzyladenine and its conjugates in shoot explants after 25 days (nmol per g fresh mass). Values given are means of three replicates.

Species Conjugate

BA [9R]BA [3G]BA [TG]BA [9GIBA [9R-G]BA

Hippeastrum 2.37 0.35 ND ND ND ND Ficus 2.46 0.59 0.08 ND ND ND Alstroemeria 3.37 0.31 ND ND ND ND Fuchsia 2.32 2.76 2.2 ND ND ND Musa 1.14 0.44 ND ND 3.28 ND Gerbera 0.04 0.09 0.66 ND 0.20 1.0 Dendranthema 0.52 0.18 ND 9.07 9.21 ND Rhododendrom 1.54 0.64 0.64 ND 16.9 ND

ND = not detected

[9R]BA and virtually no glucosides (Hippeas- trum, Alstroemeria and Ficus),

- accumulation of BA, [9R]BA and glucoside con- jugates (Musa, Rhododendron and Fuchsia),

- very low levels of BA and [9R]BA; accumula- tion of glucosides (Gerbera and Dendran- thema).

The actual amount of free BA in each shoot explant varied considerably from 0.04 nmol per g fresh mass in Gerbera shoots to 2.46 nmol per g fresh mass in Ficus shoots (Table 2). In those species where BA was metabolised to a glucoside conjugate, the concentrations were much higher; for example 9.0nmol per g fresh mass of [7G]BA and [9G]BA in Dendranthema shoots and 16.9 nmol per g fresh mass of [9G]BA in Rhododendron shoots (Table 2).

When shoots were harvested, they were sub- divided as indicated earlier. There were very few major differences between the pattern of BA

conjugation within different organs of the same shoot explant. Only in Gerbera were differences found between callus, petioles plus apices and laminae. In callus [9R-G]BA accumulated, in laminae [3G]BA and approximately equal amounts of these two conjugates in apices plus petioles.

The results clearly show that under identical conditions in vitro, there is a marked variation in the uptake and metabolism of BA supplied in the medium. In spite of this, BA is used for each species to stimulate shoot proliferation. Studies with tobacco cell cultures (Laloue & Pethe 1982) suggested that only the free base is active, whilst Letham et al. (1982) suggested that the 9- riboside and 9-ribotide might also be active in radish cotyledons. The data presented here do not resolve that question. More detailed studies on Gerbera (Blakesley et al. 1991) showed that throughout the culture period BA was only pres-

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ent in trace amounts within the shoot. There was, however, an early accumulation of [9R]BA, which had declined to a very low level by day 24, the time that Gerbera shoots in the present experiment were harvested. In contrast, the levels of BA in other species accumulated dramatically. Further studies using time course, differing concentrations of BA and competitive inhibitors of BA should help to resolve these questions.

Acknowledgements

The authors thank Twyford Plant Laboratories Ltd. for their support in this work, and Dr. R Horgan for a gift of BA standards.

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