Plant Cell, Tissue and Organ Culture 25: 69-74, 1991. © 1991 Kluwer Academic Publishers. Printed in the Netherlands.

Uptake and metabolism of 6-benzyladenine in shoot cultures of Musa and Rhododendron

David Blakesley Twyford Plant Laboratories Ltd., Baltonsborough, Glastonbury, BA6 8QG, UK (present address: School of Biological Sciences, University of Bath, Claverton Down, Bath BA2 7AY, UK)

Received 10 July 1990; accepted in revised form 10 December 1990

Key words: BA uptake, cytokinin metabolism, tissue culture

Abstract

Shoots of Musa and Rhododendron were cultured in vitro on a medium containing 0.5 mgl ~ BA. Shoots growing in the presence of [14C]BA were harvested at intervals during the culture period. Uptake of BA was linear throughout this culture period in Musa but slowed considerably in Rhododendron shoots after day 10. Rhododendron shoots absorbed 40% of the BA present in the medium and Musa shoots absorbed 52%. In each species the principal metabolite formed was [9G]BA. Benzyladenine was present in significant levels only in the pseudostem of Musa.

Abbreviations: BA - 6-benzyladenine, [3G]BA - 3-/3-glucopyranosyl-BA, [7G]BA - 7-/3-D-gluco- pyranosyl-BA, [9G]BA- 9-fl-D-glucopyranosyl-BA, [9R-G]BA- 9-(ribosylglucoside)-BA, [9R]BA- 9-/3-D-ribofuranosyl-BA, HPLC - high performance liquid chromatography, PAR - photosynthetically active radiation, TEAB - triethylammonium bicarbonate

Introduction

Cytokinins stimulate the initiation and growth of excised shoots in vitro. Consequently cytokinins are usually added to tissue culture media to stimulate auxiliary or adventitious shoot produc- tion. The synthetic cytokinin BA is the most frequently used compound in commercial mi- cropropagation (Thomas & Blakesley 1987). Few workers have addressed the long-term up- take and metabolism of BA in proliferating shoot cultures. Nordstr6m & Eliasson (1986) showed that approximately one-third of BA present in the medium was taken up at a linear rate by apple shoot cultures. In wild cherry shoot cultures Label et al. (1988) also reported a con- stant rate of BA uptake during an 8-day culture period. Biondi et al. (1984) investigated the metabolism of BA in elm shoot cultures, demon-

strating that BA was absorbed by the shoots and subsequently degraded by side chain cleavage. In Gerbera shoot cultures, Blakesley & Lenton (1987) demonstrated that all BA added to the medium was taken up within 24 days and rapidly metabolised. The 3- and 9-glucosides, 9-riboside and 9-ribosylglucoside were characterised as metabolites (Blakesley & Lenton 1987; Blakes- ley et al., in press). The major metabolite in basal callus was [9R-G]BA, whilst both [3G]BA and [9R-G]BA accumulated in apices, and [3G]BAq accumulated in laminae.

Musa and Rhododendron ponticum were chosen for investigation as examples of commer- cial crops for which production by micropropaga- tion is becoming increasingly important. Using shoot-tip explants, Wong (1986) successfully ob- tained proliferating shoot cultures of 22 Musa cultivars on media containing BA. Some

70

Rhododendron species have also been cultured on media containing BA although 2-isopen- tenyladenine is more often used (Norton & Nor- ton 1985).

The aim of this work was to make a prelimi- nary investigation into the uptake and metabo- lism of BA in shoot cultures of Musa and Rhododendron. Understanding the uptake and metabolic fate of cytokinins in shoot cultures should help to rationalise their use in tissue culture systems.

Materials and methods

Plant material

Shoot cultures of Musa cv Dwarf Cavendish were established from apical meristem explants isolated from lateral suckers. Shoot cultures of Rhododendron cv Anna Rose Whitney and YakushimanumxBashful were initiated from shoot tips. Shoots of both species were cultured on Linsmaier & Skoog (1965) medium, solidified with 0.8% agar (Tech. no. 3, Oxoid, UK) and supplemented with 0.5 mg 1-1 BA.

Shoots of both Musa and Rhododendron were subcultured every 25 days. Musa shoots were divided into single shoots from which laminae and basal callus were removed. Rhododendron 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. These trimmed shoots were then trans- ferred onto fresh medium and incubated at 25°C with a 16h photoperiod at 135 ~molm -2 s 1 PAR. For uptake studies, 14.7 kBq of [8-~4C]BA (specific activity 9.29 MBq mg 1, Amersham In- ternational, UK) was added with unlabelled BA (final concentration to 0.5mg1-1 in the medium). Six trimmed shoot clusters of each Rhododendron" cultivar and six individual trimmed Musa shoots were transferred into each container (65 mm x 65 mm x 75 mm deep magenta, Magenta Corp. Chicago, USA) con- taining 50ml of medium supplemented with radiolabelled BA. Three replicate containers were harvested at intervals during the subculture period.

Extraction and purification

When harvested, shoots were washed with dis- tilled water and divided as follows; Musa into the pseudostem and the laminae, Rhododendron into stem and laminae. The tissue was then frozen in liquid nitrogen and stored at -20°C. Tissue was homogenised in 80% aqueous methanol and filtered. The residue was re-extrac- ted in this solvent at 4°C for a further 15h, removed by filtration and the combined filtrates concentrated to dryness in vacuo.

The methanolic extract was dissolved in 5 ml of H20, adjusted to pH 3.5 with formic acid and applied to a SP Sephadex C-25 (H + form) col- umn (Redgwell 1980). This was washed with 50 ml H20, pH 3.5, yielding non-basic cytokinins (nucleotides and breakdown products such as ureides). Treatment of this fraction with alkaline phosphatase (intestinal mucosa, Sigma) failed to yield nucleosides in all extractions. Bases, in- cluding cytokinins were eluted with 50 ml 0.2M NH4OH and reduced to near dryness in vacuo. The residue was dissolved in 10 ml H20, pH 5.7 and applied to a C18 SepPak cartridge, which had been equilibrated with 10 ml methanol and 10 ml H20, pH 5.7. After washing with 3 x 5 ml H 2 0 , pH 5.7, cytokinins were eluted with 40% methanol and the eluate reduced to dryness in vacuo.

HPLC

Free bases were applied in 4% acetonitrile in dilute aqueous TEAB to a 5/~m Hypersil ODS column (250x4.5mm i.d.) and eluted at 2mlmin -1 in a linear gradient of 4% to 20% acetonitrile in dilute TEAB pH 7 over 35 min (Horgan & Kramers 1979). The absorbance of the eluate was measured at 268nm. Samples were cochromatographed with standard [3G]BA, [7G]BA, [9G]BA and [9R]BA, and i ml frac- tions collected. Radioactivity was measured by liquid scintillation counting after mixing sample aliquots from the HPLC eluate with Optiphase RIA scintillation fluid (LKB).

Results and discussion

Benzyladenine and its metabolites present in the

basic fraction from the ion exchange column were resolved by HPLC. Good separation was achieved between authentic standards of BA, [3G]BA, [7G]BA, [9R]BA and [9R-G]BA (Blakesley et al. in press). It was assumed that all radioactivity lost from the medium was taken up by the plants. However in both species the amount of radioactivity recovered from the shoots was less than the amount lost from the medium. This difference is due either to incorpo- ration of 14C into methanol-insoluble com- pounds, or its loss from the system a s 14CO2. No attempt was made to identify the metabolites in the methanol-insoluble fraction, or in the non- basic fraction from the ion exchange column.

Uptake and metabolism of BA in Musa

The fresh mass of the shoots increased 2-fold over the 25-day culture period and the shoot number increased 1.75-fold. Although greater rates of multiplication can be achieved on a medium containing higher levels of BA (D. Blakesley, unpubl.), the present investigation aimed to elucidate the uptake and metabolism of

71

BA rather than identify the optimum concen- tration of BA for shoot proliferation. After 25 days in culture, 52% of the radioactivity was taken up by the shoots and 34.6% of this was extracted from the shoots in compounds soluble in methanol. Benzyladenine and its metabolites were apparently relatively immobile since 86% of the methanol-soluble fraction was recovered from the pseudostem after 25 days (Fig. 1A). In the shoot explant as a whole, 89% of radioactivi- ty was associated with BA and its immediate metabolites contained in the basic ion exchange fraction (Fig. 1B). Only a small amount of radioactivity was associated with BA breakdown products present in the non-basic ion exchange fraction (Fig. 1B). Further analysis of the basic fraction by HPLC showed that BA was the major compound present over the first 5 days (Fig. 1B). Subsequently an increase in [9G]BA was found as the level of BA stabilised. Trace amounts of [9R]BA were also present. The dis- tribution of radioactivity between BA metabo- lites in the pseudostem was very similar to that in the laminae (Figs. 2A,B), although the amount of BA itself recovered from the laminae was very low in comparison to the pseudostem.

6 0 0

800

.= 0

400

1-1

O"

aoo

N

200

100

280 A

200

=o

150

tO0

| GO

o G lO 18 20 26

Time (days)

B

0 6 10 1G 20 28

Time (days) Fig. 1. Distribution of ]4C within each shoot explant of Musa over a 25-day culture period. (A) Recovery of ~4C from the methanol-soluble fraction of each shoot explant (&) and its partition between laminae (T) and the pseudostem (e ) . (B) Distribution of ~4C within the methanol-soluble fraction of each shoot explant between the non-basic ion exchange fraction ([]) and the metabolites within the basic ion exchange fraction; BA (©), [9G]BA (A) and [9R]BA (V).

72

40

i so 20

n -

A

I ~ I I I I I

0 8 10 18 20 28

Time (days)

A 1so

I

n o" m

100

_o

~ 5 0

B

0 6 10 18 20 28

Time (days)

Fig. 2. Distribution of 14C within the basic ion exchange fraction from laminae (A) and the pseudostem (B) of each shoot explant of Musa over a 25-day culture period; BA (O), [9G]BA (A) and [9R]BA (V).

Uptake and metabolism of BA in Rhododendron

The uptake and metabolism of BA in the two Rhododendron cultivars was very similar. Conse- quently only the data from the cv Anna Rose Whitney are presented. The fresh mass of the shoots increased 1.3-fold over the 25-day period and the shoot number increased 1.2-fold. Radioactivity was taken up from the medium over the first 10 days only. After 25 days in culture 40% of the radioactivity was taken up by the shoots and 70% of this was extracted from the shoots in compounds soluble in methanol. After 25 days 75% of the methanol-soluble frac- tion was recovered from the stem (Fig. 3A). In the shoot explant as a whole, 91% of radioactivi- ty was associated with BA and its immediate metabolites, contained in the basic ion exchange fraction (Fig. 3B). Only a small amount of radioactivity was associated with BA breakdown products present in the non-basic ion exchange fraction (Fig. 3B). The identity of the com- pounds present in the non-basic ion exchange fraction was not investigated further, although they are likely to be breakdown products of BA

such as ureides. Further analysis of the basic fraction by HPLC showed that [9G]BA was the major compound present after 8 days. Ben- zyladenine, [3G]BA and [9R]BA were also pres- ent but only in trace amounts. There was no difference in the pattern of BA metabolism be- tween the stem and the laminae, both being similar to the shoot as a whole (Fig. 3B), al- though the actual amount of [9G]BA present in the laminae was much lower than in stem tissue (Fig. 3A). These results contrast with the uptake and metabolism of BA in Gerbera shoot cultures (Blakesley et al. in press). Virtually all the BA supplied to Gerbera shoots via the culture media was absorbed, and after 24 days 70% of this had been degraded by side-chain cleavage to un- known compounds. The major conjugates formed were initially [9R]BA and subsequently [3G]BA and [9R-G]BA.

In the present study there was a significant difference in the amount and time of BA absorp- tion from the media by the two species (compare Figs. 1 and 3). Benzyladenine absorption by Rhododendron shoots ceased after just 10 days. This contrasts with the linear uptake of BA

73

1000

800

CO0

v

400

re 2OO

A

0 8 10 16 20 26

Time (days)

0

r ,

v

> , ( . , ( . )

re

6OO

B

600

400

300

200

100

0

/

0 6 10 18 20 28

Time (days)

Fig. 3. Distribution of ~4C within each shoot explant of Rhododendron over a 25-day culture period. (A) Recovery of 14C from the methanol-soluble fraction of each shoot explant (&) and its partition between laminae (T) and the stem (0) . (B) Distribution of 14C within the methanol-soluble fraction of each shoot explant between the non-basic ion exchange fraction (I~) and the metabolites within the basic ion exchange fraction; BA (O) and [9G]BA (A).

throughout the culture period by Musa shoots, and also in vitro shoots of Gerbera (Blakesley et al. in press) and apple (Nordstr6m & Eliasson 1986). Benzyladenine is toxic to in vitro shoots of some Rhododendron species (Anderson 1975, Norton & Norton 1985). The apparently self- limiting uptake of BA by Rhododendron shoots may protect the tissue from toxicity.

In both Rhododendron and Musa, B A was metabolised to [9G]BA, which has been iden- tified as a major metabolite in oat leaves (Tao et al. 1983) and a minor metabolite in Gerbera shoots in vitro (Blakesley et al. in press). The concentration of [9G]BA per individual shoot of Rhododendron and Musa was 3.2nmol and 1.5 nmol respectively after 25 days. Letham & Gollnow (1985) concluded that the 9-glucoside was a storage product, rather than a detoxifica- tion or inactivation product.

In conclusion, the uptake and metabolism of BA by in vitro shoots of Rhododendron and Musa differ markedly from Gerbera (Blakesley et al. in press). Further studies on the uptake and metabolism in a range of species should help

to elucidate the mechanism of action of BA in shoot cultures, and the role of BA metabolites.

Acknowledgements

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

References

Anderson WC (1975) Propagation of rhododendrons by tis- sue culture. Part 1. Development of a culture medium for multiplication of shoots. Comb. Proc. Int. Plant Prop. Soc. 25:129-135

Biondi S, Canciani L & Bagni N (1984) Uptake and metabo- lism of benzyladenine by elm shoots cultured in vitro. Can. J. Bot. 62:2385-2390

Blakesley D & Lenton JR (1987) Cytokinin uptake and metabolism in relation to shoot multiplication in vitro. British Plant Growth Regulator Group Monograph 16: 87-99

Blakesley D, Lenton JR & Horgan R (1991) Uptake and

74

metabolism of 6-benzylaminopurine in shoot cultures of Gerbera jamesonii. Physiol. Plant. (in press)

Horgan R & Kramers MR (1979) High-performance liquid chromatography of cytokinins. J. Chromatog. 173: 263- 270.

Label P, Maldiney R, Sossountzov L, Cornu D & Miginiac E (1988) Endogenous levels of abscisic acid, indole-3-acetic acid and benzyladenine during in vitro bud growth induc- tion of Wild Cherry (Prunus avium L.). J. Plant Growth Reg. 7: 171-180.

Letham DS & Gollnow BI (1985) Regulators of cell division in plant tissues XXX. Cytokinin metabolism in relation to radish cotyledon expansion and senescence. J. Plant Growth Reg. 4: 129-145.

Linsmaier EM & 8koog F (1965) Organic growth factor requirements of tobacco tissue culture. Physiol. Plant. 18: 100-127.

Nordstr6m A-C & Eliasson L (1986) Uptake and transloca- tion of (14C)-labelled benzylaminopurine in apple shoots

grown in vitro in relation to shoot development. Physiol. Plant. 68:431-435

Norton ME & Norton CR (1985) In vitro propagation of Ericaceae: a comparison of the activity of the cytokinins N6-benzyladenine and N6-isopentenyladenine in shoot pro- liferation. Scientia Hortic. 27:335-340

Redgwell RJ (1980) Fractionation of plant extracts using ion-exchange Sephadex. Anal. Biochem. 107:44-50

Tao G-Q, Letham DS, Palni LMS & Summons RE (1983) Cytokinin biochemistry in relation to leaf senescence I. The metabolism of 6-benzylaminopurine and zeatin in oat leaf segments. J. Plant Growth Reg. 2:89-102

Thomas TH & Blakesley D (1987) Practical and potential uses of cytokinins in agriculture and horticulture. British Plant Growth Regulator Group Monograph 14:69-83

Wong WC (1986) In vitro propagation of banana (Musa spp.): initiation, proliferation and development of shoot- tip cultures on defined media. Plant Cell Tiss. Org. Cult. 6: 159-166