Plant Physiol. (1983) 71, 972-9740032-0889/83/7 1/972/03/$00.50/0

Short Commwication

Complete Release of Axillary Buds from Apical Donance inhtact, Light-Grown Seedlings of Pisum sativum L. following aSingle Application of Cytokinin1

Received for publication November 19, 1982

INDIREN PILLAY AND IAN D. RAILTON2Department ofPlant Sciences, Rhodes University, Grahamstown 6140, South Africa

ABSTRACT

Single applications of either 6-benzyladenine or zeatin to inhibitedaxillary buds of intact, light-grown seedigs of Pismn saivum L. cv Black-eyed Susan, resulted in the formation of rapidly elongating, leafy shoots.Similar treatment with kinetin or isopentenyladenine caused only limitedbut outgrowth which stopped 6 days after application.

The regulation of axillary bud outgrowth by cytokinins was firstreported by Wickson and Thimann (20) in excised, nodal segmentsof Pisum sativum. Subsequent studies using light-grown, intactplants demonstrated that the synthetic cytokinins K3 and BA,when applied directly to inhibited axillary buds, caused onlypartial release from apical dominance (1-3, 11, 16-18). Similarresults were obtained when BA was supplied via the transpirationstream, to derooted, but otherwise intact plants (8, 9). Furthertransitory growth of released lateral buds has been achieved bytreatment with IAA or GA3 following an initial application ofeither K (3, 17) or BA (2), although development of large leafyshoots was not demonstrated.

Surprisingly, most workers investigating the role of cytokininsin apical dominance have restricted themselves to the syntheticcompounds K and BA and only limited data are available con-cerning the effects of naturally occurring cytokinins on the growthof inhibited lateral buds. In one study, Kung Woo Lee et al. (4)employed IPA to stimulate lateral bud growth following treatmentwith the purine biosynthesis inhibitor hadacidin, but only decap-itated plants were used.We compared the ability of the naturally occurring cytokinins

IPA and Z and the synthetic compounds BA and K to stimulatelateral bud outgrowth in light-grown, intact plants of P. sativumL.

In this preliminary report, we demonstrate for the first time thedevelopment of a rapidly elongating leafy shoot following appli-cation of a cytokinin to an inhibited lateral bud ofan intact higherplant.

'Supported by a grant from Rhodes University.To whom requests for reprints should be sent.Abbreviations: K, kinetin; IPA, isopentenyladenine (6-y,y-dimethyl

allylaminopurine); Z, zeatin; Ad, adenine.

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FIG. 1. Effect of ethanol-carbowax solutions of BA (x-x), Z(x-x), K ( 0-), IPA (0-4), Ad (U-) and ethanol-Carbowaxalone (I-I) on the growth of axillary buds of intact, light-grown plantsof P. sativum L.

MATERIALS AND METHODS

Seeds of Pisum sativum L. cv Black-eyed Susan were soakedovernight in running tap water, sown in moist vermiculite, andmaintained under constant illumination (5,500 lux) at 25C.Seven-d-old seedlings were transplanted into trays containing amixture ofpotting soil and vermiculite and were maintained underidentical conditions to those described above throughout thecourse of the experiment.

BA, IPA, K, Z, and Ad were dissolved in a mixture of ethanol-Carbowax in an identical manner to that described by Sachs andThimann (17). An Eppendorf pipette was used to dispense 10-,ulaliquots (each containing 3.3 ,ug cytokinin) of these solutions to

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AXILLARY BUD RELEASE BY CYTOKININS

4-I

4

B C DI E FFIG. 2. Development of inhibited axillary buds at the second node of intact, light-grown plants of P. sativum L. after treatment with ethanol-

Carbowax (A) and solutions of ethanol-Carbowax containing Ad (B), IPA (C), K (D), BA (E), Z (F). Photograph taken 16 d after compounds wereapplied. All developing axillary buds facing right-hand side of photograph. Plants A to D were decapitated prior to photography.

the axillary bud at the second node of each seedling, and out-growth was measured at intervals with a plastic mm ruler.

RESULTS AND DISCUSSIONBA, K, IPA, Z, and Ad were each applied at a dosage of 3.3

gg to lateral buds as described (17), and bud growth was followedover a 20-d period.The growth kinetics (Fig. 1) show that the length of the lag

phase prior to axillary bud release was the same for all cytokinintreatments and that, during the first 3 d of growth, the growthrates of the released buds were similar. After this time, however,further growth of K- or IPA-treated buds was limited whereasthose buds treated with either BA or Z continued to elongaterapidly and produced large leafy shoots (Fig. 2).

Despite previous reports of slow growth by inhibited axillarybuds on intact, control plants (1, 11, 17, 18), no such growth wasobserved in the present study. Likewise, the lag period of 1 to 2 dbefore bud outgrowth following cytokinin treatment was signifi-cantly longer than that noted by others (e.g. 1, 17).The results obtained using K were similar to those reported

previously (17), and IPA-treated buds grew in a similar manner.Ad, which at high concentrations exhibits cytokinin activity inother systems (5), was inactive.Although BA has been applied directly to inhibited axillary

buds of intact, light-grown species other than Pisum (2, 18), onlylimited bud growth was achieved and the results were comparableto those observed originally using K (17). However, Usciati et al.

(19) showed in etiolated, intact seedlings of Cicer that BA appli-cation to lateral buds caused rapid growth which was maintainedfor almost 3 weeks. Likewise, Roussaux (14), using repeatedapplications of ng amounts of BA, reported a very small butconstant outgrowth ofaxillary buds ofintact, light-grown seedlingsof P. sativum cv Annonay. In other work where BA was suppliedto light-grown, derooted plants of Pisum via the transpirationstream (8, 9), only the earliest stages of lateral bud release wereinvestigated, and no detailed, long-term growth kinetic studieswere reported.The effect of Z on lateral bud growth was remarkable (Fig. 1)

and represents the first demonstration of axillary bud release bya naturally occurring cytokinin. The reasons for the inability ofKand IPA to maintain bud growth are unclear. Inasmuch as IPA isthe immediate precursor ofZ in some systems (6, 7) the possibilityexists that inhibited lateral buds were unable to efficiently hy-droxylate IPA to Z. Indeed, it has been convincingly demonstrated(4) that inhibited lateral buds lack the capacity for cytokininbiosynthesis, perhaps through inhibition of this terminal hydrox-ylation step. Recent studies have shown that the inability of Z,unlike BA, to retard leaf senescence resides in the differing me-tabolism of these two compounds (10), where BA was convertedinto a senescence delaying glucose-conjugate while Z was not. Asimilar mechanism might dictate the relative activities of cytoki-nins in releasing buds from apical dominance.The current idea of apical dominance maintains that, whereas

cytokinins are required for axillary bud release in intact plants,

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974 PILLAY AND RAILTON

continued bud growth requires the presence of auxin and gibber-elins (12, 13, 15). Axillary buds released from inhibition bydecapitation of the main shoot apparently achieve self sufficiencyin auxin and gibberellin production, whereas buds treated witheither K (3, 17) or BA (2) do not, and exogenous supplies of auxinand gibberellin are thus required for further growth.The present results clearly show that cytokinin application alone

can be sufficient to trigger previously inhibited lateral buds intorapidly elongating, fully differentiated shoots.

Acknowledgment-We wish to thank Nicole Railton for translating the Frenchpapers associated with this investigation.

LITERATURE CITED

1. ALI A, RA FLETCHER 1970 Hormonal regulation of apical dominance in soy-beans. Can J Bot 48: 1989-1994

2. Au A, RA FizrcEmR 1971 Hormonal interaction in controlling apical dominancein soybeans. Can J Bot 49: 1727-1731

3. CATALANO M, TA HIL 1969 Interaction between gibberellic acid and kinetin inovercoming apical dominance natural and induced by IAA, in tomato (Lyco-persicon esculentum Mill cv potentate). Nature (Lond) 222: 958-986

4. KUNG Woo LEE P, B KESSLER, KV THIMANN 1974 The effect of hadacidin onbud development and its implications for apical dominance. Physiol Plant 31:11-14

5. LETHAM DS 1978 Cytokinins. In DS Letham, PB Goodwin, TJV Higgins, eds,Phytohormones and Related Compounds: A Comprehensive Treatise, Vol 1.Elsevier/North Holland, Amsterdam, pp 205-251

6. MIuRA GA, RH HALL 1973 trans-Ribosyl zeatin. Its biosynthesis in Zea mays

Plant Physiol. Vol. 71, 1983endosperm and the mycorrhizal fungus Rhizopogon roseolus. Plant Physiol 51:563-569

7. MIURA GA, CO MILLER 1969 6-(y,y-dimethylallyl amino) Purine as a precursorof zeatin. Plant Physiol 44: 372-376

8. NAGAO MA, B RUBINSTEIN 1975 Relationship of cytokinin to lateral bud growthat early stages after decapitation. Bot Gaz 136: 366-371

9. NAGAO MA, B RUBINSTEIN 1976 Early events associated with lateral budoutgrowth of Pisum sativum. Bot Gaz 137: 39-44

10. PALNi LMS, GQ TAO, DS LETHAM 1982 Cytokinin metabolism in excised oatleaves. In Collected Abstracts I Ith Int Conf Plant Growth Substances, Aber-ystwyth, Wales, p 23

1 1. PANIGRAHI BM, LJ AUDUS 1966 Apical dominance in Viciafaba Ann Bot 30:457-473

12. PHILLIPS IDJ 1969 Apical dominance. In MB Wilkins, ed, Physiology of PlantGrowth and Development. McGraw-Hill, New York, pp 165-202

13. PHILLIPs IDJ 1975 Apical dominance. Annu Rev Plant Physiol 26: 341-36714. ROuSSAUX J 1970 Etude de quelques modalites de la croissance de bourgeons

axillaires de pois traites par la 6-benzylaminopurine. CR Acad Sci (Paris) 271:595-598

15. RuBINSTEIN B, MA NAGAO 1976 Lateral bud outgrowth and its control by theapex. Bot Rev 42: 83-113

16. SACHS T, KV THIMANN 1964 Release of lateral buds from apical dominance.Nature (Lond) 201: 939-940

17. SACHS T, KV THMANN 1967 The role of auxins and cytokinins in the release ofbuds from dominance. Am J Bot 54: 136-144

18. Schaeffer GW, FT Sharpe Jr 1969 Release of axillary bud inhibition withbenzyladenine in tobacco. Bot Gaz 130: 107-110

19. USCIATI M, M CODACCIONI, J GUERN 1972 Early cytological and biochemicalevents induced by a 6-benzylaminopurine application on inhibited axillarybuds of Cicer arietinum plants. J Exp Bot 23: 1009-1020

20. WICKSON ME, KV THIMANN 1958 The antagonism of auxin and kinetin in apicaldominance. Physiol Plant 11: 62-74

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