Báo cáo lâm nghiệp: "Polyamines and ethylene during of Prunus avium L. in vitro rooting"

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in vitro Polyamines and ethylene during rooting of Prunus avium L. 2 N. Bagni S. Biondi 1 Regionale Foreste dell’Emilia-Romagna, Bologna, and Azienda 2 Dipartimento di Biologia E.S., Universitj di Bologna, Bologna, Italy Prunus awum shoots. In addition, the Introduction effects of externally supplied polyamines, in particular SPM, of the ethylene pre- Polyamines widely occurring organic are 1-aminocyclopropane-1-carboxylic cursor polycations recognized as plant now acid (ACC) and of some inhibitors of poly- growth substances. They appear to be amine and ethylene biosyntheses, namely involved in cell division, to delay senes- a-difluoromethylornithine (DFMO), a-di- and usually accompany active cense inine 3 (DFMA), dicyclohexy- fluoromethylar< growth and metabolism (Bagni et al., (DCHA), lamine methylglyoxal-bis-guanyl- 1982). Although a requirement for poly- hydrazone (MGBG) and aminoetho- amines has been postulated in some in xyvinylglycine (AVG) and aminooxyacetic vitro morphogenetic processes, a full acid (AOA) were examined. understanding of their role in organo- genesis has yet to come. The interaction between ethylene and auxin is a well- known phenomenon. Our primary interest Materials and Methods in studying the action of ethylene in adventitious root formation stems from auxin usually being the key factor in root experimental material is part of a routine The induction. Secondly, since ethylene and micropropagation program for selected clones of P. avium L. The establishment of bud cul- polyamines share a common precursor tures and the shoot multiplication and elonga- (S-adenosylmethionine) in their biosynthe- tion phases have been described elsewhere tic pathways, there is some evidence for a (De Paoli and Flocchi, 1984). Rooting experi- possible interrelationship between them. ments were carried out by transferring elongat- ed shoots onto agar-solidified medium com- The aim of the present work was to begin of half-strength macro- and micro- posed examining the changes in the endogenous nutrients of Murashige and Skoog (1962) (MS), content of the polyamines, putrescine MS vitamins, 30 g/I sucrose, pH 5.9-6.0, prior (PUT), spermidine (SPD) and spermine to autoclaving. Iridole butyric acid (IBA) or indo- le acetic acid (IAA) was supplied at various (SPM) and in the ethylene production concentrations (2.5, 5, 10, 25, 50 uM) to induce accompanying the in vitro rooting of
no significant effect on rooting. levels, had rooting. Spermine, DFMO, DFMA, MGBG, AVG, AOA and ACC were filter-sterilized and added plus MGBG (0.5 mM each) DCHA to the autoclaved medium. Cultures were kept markedly reduced rooting but this inhi- in a growth chamber with a 16 h photoperiod at bition was only partially reversed by the 22 ± 2°C. Polyamine levels were individually simultaneous application of 0.5 mM SPD. monitored at intervals in the shoot apical and basal 3 mm portions, stem and leaves. Dansy- At higher concentration, the drugs provok- lated derivatives of the polyamines were sepa- ed visible toxicity symptoms. DFMO plus rated by thin-layer chromatography and fluoro- DFMA (1 mM each) drastically reduced metrically determined. The incorporation of rooting as well and 0.2 mM PUT again labeled PUT, the precursor of SPD and SPM, was evaluated by adding 185 kBq to 100 ml of partially reversed this effect. As expected, medium (15 shoots). Shoot portions were ex- treatment with these specific inhibitors of tracted on d 2, 7 and 15 and polyamines were PUT biosynthesis caused a severe decline analyzed as described above. Radioactivity was in endogenous PUT levels. DCHA plus measured in spots comigrating with SPD and MGBG, however, caused only a minor SPM. A system was developed to measure 3,4- [!4C]methionine (74 kBq/20 ml of medium) reduction in SPD content. ACC was sup- incorporation into ethylene. Ten shoots were plied only at 1 mM concentration, which placed in small flasks equipped with a side-arm, proved to be lethal and thus no rooting into which filter paper soaked in KOH was was observed. Finally, AVG (25 or 50 M) N I inserted, and a center well containing either 0.1 did not affect rooting percentages but M mercuric acetate (in methanol) or 0.25 M mercuric perchlorate in order to capture the enhanced the number of roots formed per labeled ethylene formed. The radioactivity in the rooted shoot. Results showed that mer- ethylene traps was measured after a 24 h incu- curic perchlorate is a more efficient and bation under normal culture conditions. The reliable ethylene trap than mercuric time course of methionine uptake was deter- mined by extracting the different shoot portions acetate. The latter, being dissolved in in 10% trichloroacetic acid. methanol, quickly evaporated and, due to the presence of water vapor in the flasks, formed a yellow precipitate which was dif- ficult to collect. The time course of labeled Results methionine uptake indicated that the com- pound was rapidly taken up (within 30 Of the two auxins and different concentra- min) and reached a plateau around 15 h in tions tested, 5 ,uM IBA gave the best root- all shoot portions. Preliminary data ing percentages after 12-15 d and was concerning ethylene biosynthesis indicate thus used in all subsequent experiments. that, upon transfer to a rooting medium Endogenous polyamine content during the containing 5 !M IBA, ethylene production rooting phase was characterized by a was of the order of 2.2 pmol/shoot/h. peak in PUT levels on d 9-11 in all shoot Finally, 0.5 mM AOA was shown to signifi- portions. Spermidine levels did not change cantly reduce ethylene biosynthesis and, significantly in leaves but showed maxi- to a lesser extent, so did a 48 h exposure mum accumulation on d 9 or 11 in other to DCHA plus MGBG. shoot portions. Spermine was always absent or present in traces. Although no labeled PUT incorporation into SPM was observed, it is worth noting the sharp peak Discussion in SPD synthesis observed in the basal portion of shoots on d 7. Exogenously The results outlined above suggest that supplied SPM (10, 50 or 100 pM), either in polyamines may be involved in the rooting the presence of optimal or suboptimal IBA
process, probably in the stages of active Further work the role of ACC, AVG and on cell division. In fact, increases in intracel- in adventitious root ethylene production lular PUT and SPD levels preceded root formation is in progress. protrusion and may have coincided with maximum primordium development. Also, a peak in SPD synthesis was observed on References d 7 in the basal portions, which are the site of root formation. Finally, although there does not seem to be a requirement Bagni N., Serafini-Fracassini D. & Torrigiani P. for SPM, either endogenous or exo- (1982) Polyamines and cellular growth pro- genous, specific inhibitors of PUT and cesses in higher plants. In: Plant Growth Sub- stances 1982. (Wareing P.F., ed.), Academic SPD biosyntheses had a clear inhibitory Press, New York, pp. 473-482 action on rooting. Our preliminary data De Paoli G. & Rocchi M. (1984) Propagazione seem to indicate that DCHA plus MGBG in vitro del ciliegio da legno. In: Propagazione do not enhance ethylene production; this in vitro: ricerche su alcune specie forestali. may be due to the fact that these drugs A.R.F.E.R., Boloona, pp. 100-102 were ineffective in blocking polyamine Murashige T & Skoog F. (1962) A revised synthesis or may suggest that the 2 bio- medium for rapid growth and bioassays with tobacco cultures. Physiol. Plant. 15, 473-497 competitive. not synthetic pathways are