Abscisic Acid and the Rooting of Runner Bean Cuttings

Originalarbeiten . Original Papers

Botanisches Institut der Universidit Wiirzburg, Lehrstuhl Botanik I, Federal Republic ofGermany

Abscisic Acid and the Rooting of Runner Bean Cuttings

WOLFRAM HARTUNG, BRUNHILDE OHL and VOLKER KUMMER

With 4 figures

Received 1 October 1979 . Accepted 27 October 1979

Summary

Abscisic acid promotes the formation of adventitious roots in cuttings of Phaseoluscoccineus L. The optimum concentration is 10-5 M. ABA antagonises the GAa inducedinhibition of rooting. The ABA effect is accompanied by a stimulation of the growth of the2nd internode. No ABA action is observed on cuttings kept continuously in the dark or ondefoliated cuttings. Treatment of cuttings with ABA causes an accumulation of assimilates,especially sucrose, which are basipetally transported from the primary leaves to the basalparts of the cuttings.

Key words: Abscisic acid, assimilates, sugars, auxin, gibberellic acid, hormone-directedtransport, root formation, Phaseolus coccineus.

Introduction

The role of ABA for the rooting of cuttings is not understood. Some authorsreported that ABA promotes the rooting of cuttings (CHIN et aI., 1969, Phaseolusaureus and Hedera helix; BLAZICH and HEUSER, 1977, Phaseolus aureus ; BASU et aI.,1970, Lycopersicon esculentum; JUILLARD, 1970, Vitis vinifera; SCHMID, 1972, Populus alba). Other cuttings did not respond to ABA (READ and HOYSLER, 1971,Euphorbia pulcherrima; KRELLE and LIBBERT, 1969, Phaseolus vulgaris and Calysetgia sepium; COLEMAN and GREYSON, 1976, Lycopersicon esculentum) and KRISHNAMOORTY (1972, Cucumis melo) and DELEGHER-LANGOHR (1974, Zebrina pendula)even demonstrated inhibition of rooting by ABA.

Abbreviations: ABA, abscisic acid, GA: gibberellic acid, HDT: hormone directedtransport, IAA: indole acetic acid.

Z. Pflanzenphysiol. Bd. 98. S. 95-103. 1980.

96 WOLFRAM HARTUNG, BRUNHILDE OHL and VOLKER KUMMER

The inhibitor fJ fraction which is believed to contain mainly ABA stimulates

rooting of mung beans (HESS, 1962 j BASU et aI., 1968) but not of Vitis vinifera (TIZIO

et aI., 1968).These contradictory observations caused us to reinvestigate the problem.

The work of BREEN and MURAOKA (1973) and ALTMAN and WAREING (1975) has

shown that auxin directed assimilate transport from the leaves to the cutting base is

involved in the process of adventitious root formation. We were interested to know

whether ABA is also involved in the regulation of assimilate transport in the cutting.

Materials and Methods

Seeds of Phaseo/us coccineus cv. WeiBer Riese were soaked in tap water for 3h andgrown in the glasshouse in compost soil under long day conditions at temperatures between295-297 K. After 9-11 days when the plants were about 13 cm long, cuttings with a 10 cmlong 1st and a 1 cm long 2nd internode were made. Rooting experiments were done asdescribed by CHIN et al. (1969). Cuttings in 15 ml aqueous hormone solution or H20 (control)in test tubes wrapped with aluminium foil were kept in darkness at 295 K for 24 h. Theywere subsequently washed, transferred into distilled water and kept in a controlledenvironment chamber in a 14 h long day at 295 K. Light intensity was 10,000 Ix. Water waschanged every day. The number of visible roots was recorded after 8 days.

6 h after hormone treatment soluble sugars were determined with anthrone according toROE (1954) after extracting the homogenised basal end (30 mm) of the cutting in 80 0/0

methanol.14C02 was applied as described by SCHOPFER (1976). 925 KBq e4C)bicarbonate (specific

activity 1.85 GBq mmol-I ) + 0.3 ml 1 % Na2COS were added to a test tube (40 mm lengthand 10 mm diameter) which was then attached with vaseline to the lower side of a primaryleaf in an controlled environment chamber at conditions as described above. 14C02 wasreleased by injecting 1 ml 2 M sulfuric acid from an aperture in the side of the tube whichwas sealed with parafilm. Cuttings were allowed to fix 14C02 for 15 min. After 6 h in thelight they were exposed to an Agfa Curix RP1 X-ray film in an deep freeze refrigerator at243 K for 7 days or divided in several sections, homogenised in liquid N2 and extractedwith 80 % methanol. Radioactivity was determined in the extracts by fl scintillation counting. In other experiments 2.59 KBq uniformly labelled (14C)sucrose (specific activity 13 GBqmmol-I ) were applied in a 5 ,ul drop to a 3 mm long incision to the midrib of one of the primaryleaves at the end of the dark phase. Radioactive compounds were supplied by the Radiochemical Centre Amersham. Scintillation counting was carried out in a Berthold 8000 Betascintscintillation counter using a water soluble scintillation fluid on dioxane base. The patternof (14C)labelled assimilates was determined by thinlayer autoradiochromatography accordingto FEIGE et al. (1969). Each experiment was performed 3-5 times with similar results, foreach treatment 5 replicates were used.

Results and Discussion

ABA effects on rooting of cuttings

After treatment of 10 days old runner bean cuttings with ABA of different

concentrations in the dark (24 h) root formation was stimulated. The optimum concentration was 10-5 M. Higher concentrations did not affect or were even inhibitory

Z. Pjlanzenphysiol. Bd. 98. S. 95-103. 1980.

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ABA and rooting 97

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Fig. 1 a: Effect of ABA on the rooting and the growth of the 2nd internode of a runnerbean cutting 8 days hormone treatment. -e- number of roots, -0- increase oflength of the 2nd internode.Fig. 1 b: The root formation of runner bean cuttings treated with H 20 (control) and 10-5 MABA up to the 8th day after hormone treatment. Age of the plants at begin of the experiment: 9 days.

to rooting. The promotion of rooting was accompanied by promotion of the growthof the 2nd internode or of lateral buds, if the cuttings were decapitated. Such apositive correlation could not be found after application of IAA and GAa• Herestimulation (IAA) or inhibition (GAa) of rooting was parallelled by an inhibition orpromotion respectively of internode or lateral bud growth (VAN OVERBEEK, 1969).

Earlier experiments, not described here in detail, using radioactive hormonesindicated that C4C)IAA was not translocated acropetally from the incubation

z. Pflanzenphysiol. Ed. 98. S. 95-103. 1980.


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Fig. 2: Decapitated runner bean cuttings treated with H 20 (control) and 10-5 M ABA 10days after the experiment. Age of the plants at the start of the experiment: 10 days.

ABA and rooting 99

solution to the top of the cutting. 21 Ofo of total applied radioactivity from (3H)GAl,however, was recovered in the apical bud and 4.7 Ofo of total applied radioactivitymoved up to the apex after (14C)ABA treatment. This indicates that in contrast toIAA small amounts of ABA are transported to the apex. They may be responsible forthe growth stimulation there.

At low concentrations ABA can act as a growth promoter (HARTUNG and STEIGERWALD, 1977). The stimulation of rooting by ABA may be explained by theobservation that ABA is able to promote cell division (ALTMAN and GOREN, 1971;MINOCHA, 1979).

Fig. 1 b shows that it is obviously not the time of rooting but the number of rootsthat is affected by the ABA treatment. In both, the ABA plants and the controls,roots appear between the 4th and 5th days after ABA application.

The rooting of some other plants such as Phaseolus species (P. aureus, P. lunatus, P. semiereetus, P. vulgaris and the wild bean P. aborigineus), Pisum sativum,Lens culinaris and Lycopersicon esculentum, Lupinus luteus and Helianthus annuuswas also promoted after application of 10-5 M ABA. However, in accord withobservations of KRISHNAMOORTY (1972) rooting of Cucumis sativus was inhibited.Inhibition was also observed with cuttings from Vicia faba.

If 10-4 M IAA, which was previously found to be optimal for rooting of runnerbean cuttings, is combined with 10-5 M ABA, a very small additional increase in thenumber of roots occurs, less than one would expect if both hormones act additively(Tab. 1). There is a similar situation in the case of Phaseolus vulgaris (BAsu et al.,1970) whereas ABA and auxin act additively in tomato- and mung bean cuttings(BAsu et al., 1970). Synergistic action of ABA and indolebutyric acid has beenobserved in Dahlia variabilis cuttings (BIRAN and HALEVY, 1973) and in tomatocuttings (BAsu et al., 1970). With gibberellin rooting was strongly inhibited, inagreement with BRIAN et al. (1960). Inhibition was completely overcome bysimultaneously added 10-5 M ABA. CHIN et al. (1969) reported similar results for

Table 1: The rooting of runner bean cuttings after treatment with different phytohormonesunder different conditions.

Hormone treatment

control10-5 M ABA10-4 M IAA10-8 M GAs10-5 M ABA + 10-4 M IAA10-5 M ABA + 10-8 M GAs10-5 M ABA10-5 M ABA

conditions other thandescribed in materialsand methods

cuttings defoliatedcontinuously dark

number of roots

11±22S±32S±33±2

37±417±34±23±1



Phaseolus aureus and so did COLEMAN and GREYSON (1976) in the case of tomatoleaf cuttings and SCHMID (1972) concerning Populus alba cuttings. Rooting ofPhaseolus vulgaris, however, was synergistically inhibited by ABA in the presence ofGAs (KRELLE and LIBBERT, 1969).

ABA, sugar content and assimilate transport in runner bean cuttings

Phytohormone-induced rooting is accompanied by alterations of sugar content inthe regeneration parts of the cuttings. The presence of sugars, especially sucrose,increases the auxin-induced root formation in cuttings (PIEREK and SEGERS, 1973;NANDA et al., 1968). STUART (1939) demonstrated that the content of solublecarbohydrates is increased in the base of auxin-treated french bean cuttings. Similarly,in our experiments content of soluble sugars in the basal 30 mm of runner beancuttings is enhanced by 10-4M IAA pretreatment (+ 75 0/0). ABA increased solublesugars by 45 Ofo and GAs had no effect. The question arises whether that increasedsugar content is due to a basipetal hormone-directed assimilate transport. ALTMANand WAREING'S (1975, Phaseolus vulgaris) as well as BREEN and MURAOKA'S data(1973, plum cuttings) suggest that auxin causes an accumulation of necessaryassimilates in the basal sections of the cuttings. Nothing is known, however, whetheralso ABA and GAs playa role in HDT in cuttings. The fact that rooting of ABAtreated cuttings is prevented by darkness or defoliation may indicate that assimilatetransport from leaves to the base could also be involved in ABA-induced rooting(Tab. 1).

We have therefore performed transport experiments. Leaves of cuttings weresupplied 15 min. with uC02 and the appearence of labelled compounds in the baseof the stem was measured after 6 h. Most of the label was found in sucrose (65.9 Ofoof total radioactivity) less in glucose and fructose (14.2 Ofo and 13.2 Ofo respectively).Experiments were also carried out with (uC)sucrose. The distribution of radioactivitywas measured after extracting stem segments by p-scintillation counting and byautoradiography (Fig. 3, 4). Our experiments show that after (uC)sucrose and 14C02application to nhe primary leaves the strongest accumulation of label in thebasal parts takes place after auxin treatment (+ 840 % of (uC)-assimilates).In the case of ABA an enhanced accumulation of uC-assimilates can also beseen in the cutting base (+ 150 Ofo), GAs on the other hand prevents basipetalassimilate and sucrose translocation. These data suggest that ABA can playa role inHDT similar to IAA. While data agree with observations of ALTMAN and WAREING(1975) and BREEN and MURAOKA (1973) who found increased assimilate translocationunder the influence of IAA, they disagree with reports of MULLINS (1970, frenchbean internodes), WAGNER (1974, barley grains) and SCHNEIDER (1977, apple andpeach fruit) according to whom ABA inhibits assimilate accumulation. However, theexperiments of the latter authors are related to apical dominance, grain ripening andfruit abscission which are difficult to compare with adventitious root formation.KARMOKER and VAN STEVENINCK (1979), on the other hand observed enhanced sugar

Z. Pjlanzenphysiol. Bd. 98. S. 95-103. 1980.

ABA and rooting 101

14C-SUCROSE APPUCATION

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Fig. 3: Distribution of radioactivity in stems of runner bean cuttings pretreated with 10-5 MABA, 10-4 M IAA, 10-6 M GAa and H 20 (control) after application of 2.57 KBq (14C)sucroseto the midrib of a primary leaf or of 925 KBq 14C02 for 15 min to the lower side of aprimary leaf. At the end of a transport period of 6 h in the light cuttings were divided insections as shown in the insert and the radioactivity per segment was measured. For conditionsof experiment, see materials and methods. Age of plants at begin of experiment: 10 days.

and assimilate transport from the leaves of Phaseolus vulgaris to the root systemwhen the roots were preincubated with abscisic acid.

The possibility that basipetal transport of hormonal rooting factors could beinfluenced by hormone pretreatment of the cutting base could be excluded for



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Fig. 4: Autoradiograms of cuttings pretreated with 10-5 M ABA, 10-4 M IAA and 10-6 MGA3 compared to a water control after application of 925 KBq 14C02 for 15 min to aprimary leaf. Transport period: 6 h in the light. Experimental conditions, see materials andmethods. Age of plants at the begin of the experiment: 10 days.

runner beans. We were not able to observe changed transport and distribution pattern

6h after application of radioactive IAA, ABA and GAl to the apex, neither after

IAA- nor after ABA- or GAs-pretreatment.

Acknowledgements

We are grateful to Miss CORNELIA FUNFER and Mr. THOMAS ROSE for help and technicalassistance. This project was supported by the Deutsche Forschungsgemeinschaft.

References

ALTMAN, A. and R. GOREN: Plant Physio!. 47, 844 (1971).ALTMAN, A. and P. F. WAREING: Physio!. Plant. 33, 32 (1975).BASU, R. N., B. GHOSH, and P. K. SEN: Indian Agriculturist 12,194 (1968).BASU, R. N., B. N. Roy, and T. K. BOSE: Plant & Cell Physio!. 11, 681 (1970).BIRAN, 1. and A. H. HALEVY: Physio!. Plant. 28, 436 (1973).BLAzIeH, F. A. and C. W. HEUSER: Hort Science 12, 392 (1977).BREEN, P. J. and T. MURAOKA: J. Amer. Soc. Hort. Sci. 98, 436 (1973).BRIAN, P. W., H. G. HEMMING, and D. LOWE: Ann. Bot. 24, 407 (1960).

Z. Pjlanzenphysiol. Ed. 98. S. 95-103. 1980.

ABA and rooting 103

CHIN, T. Y., M. MEYER, and L. BEEVERS: Planta 88, 192 (1969).COLEMAN, W. and R. I. GREYSON: J. expo Bot. 27,1339 (1976).DELEGHER-LANGOHR, V.: Bull. Soc. Roy. Bot. Belg. 107, 73 (1974).FEIGE, B., H. GIMMLER, W. D. JESCHKE, and W. SIMONIS: J. Chromatogr. 41, 80 (1969).HARTUNG, W. and F. STEIGERWALD: Planta 134,295 (1977).HESS, C. E.: XVIth Int. Hort. Congr. 4, 375 (1962).JUILLARD, B.: C. R. Acad. Sc. Paris, Ser. D. 270, 2795 (1970).KARMOKER, J. L. and R. F. M. VAN STEVENINCK: Planta 146, 25 (1979).KRELLE, E. and E. LIBBERT: Flora (A), 160,299 (1969).KRISHNAMtOORTY, H. N.: Biochem. Physiol. Pflanzen 163, 513 (1972).MINOCHA, S. c.: Z. Pflanzenphysiol. 92, 431 (1979).MULLINS, M. G.: Ann. Bot. 34, 897 (1970).NANDA, K. K., A. N. PUROHIT, and K. MEHROTRA: Plant & Cell Physiol. 9, 735 (1968).PIERIK, R. L. M. and TH. A. SEGERS: Z. Pflanzenphysiol. 69, 204 (1973).READ, E. P. and V. HOYSLER: Hort. Science 6,350 (1971).ROE, Z. H.: J. BioI. Chern; 212,335 (1954).SCHNEIDER, G. W.: J. Amer. Soc. Hort. Sci.: 102, 179 (1977).SCHOPFER, P.: Experimente zur Pflanzenphysiologie, Berlin, Heidelberg, New York, 1976.SCHMID, A.: Ber. Schweiz. Bot. Ges. 82, 14 (1972).STUART, W.: Bot. Gaz. 100,298 (1939).TIZIO, R., I. S. MOYANO, and H. MORALES: Phyton 25,123 (1968).VAN OVERBEEK, J.: Sci. Amer. 219, 75 (1968).WAGNER, H.: Angew. Bot. 48, 331 (1974).

WOLFRAM HARTUNG, Botanisches Institut der Universitat, Lehrstuhl Botanik I, MittlererDallenbergweg 64, D-8700 Wiirzburg.

Note added in proof

Since this paper was submitted, RASMUSSEN and ANDERSEN (1980) have shown that ABAstimulated rooting of pea cuttings, especially when they were kept under high irradiance.Their data support the results and suggestions of the present paper.

Reference: RASMUSSEN, S. and A. S. ANDERSEN: Physiol. Plant. 48, 150 (1980).


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Abscisic Acid and the Rooting of Runner Bean Cuttings