Báo cáo lâm nghiệp: "Internal levels of plant growth regulators during in vitro culture of wild cherry (Prunus avium L.)"

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Internal levels of plant growth regulators during in vitro culture of wild cherry (Prunus avium L.) P. Label 1 D. Cornu 2 B. Sotta E. Miginiac 1 INRA, Station dAm6lioration des Arbres Forestiers, Ardon, 45160 Olivet, and 2 des Plantes, 4, Université P.-et-M.-Curie, Laboratoire de Physiologie du pl. Dweloppement Jussieu, T53-E5, 75252 Paris Cedex 05, France Hormonal measurements were made during Introduction the multiplication and the rooting stages. For each measurement, 48 explants were divided In vitro micropropagation of wild cherry is into 3 parts: the apical part, including the apex sensu stricto and the youngest leaves inserted presently one of the main commercial in the short internodes of the stem tip; the ways to clonally propagate this species middle part of the explants, bearing the oldest (Cornu and Boulay, 1986). In order to leaves at the axis, whose axillary buds started extend this technique to a large number of to grow during multiplication treatment; and the clones, it seems necessary to improve our basal part including the portion of the stem inserted into the culture medium, where roots knowledge of the behavior of the explants were formed during the rooting stage. during the in vitro culture. Since plant For each series, explants were collected 0, 1, growth regulators (PGR) play an important 2, 4 and 8 d after their transfer into fresh role in this technique (Margara, 1961), our and medium. Frozen lyophilized samples were attention was drawn to the effect of exo- ground up with ball mill. Analytical measure- a ments were made following the procedure genous PGR on hormonal levels in the reported elsewhere (Label et al., 1989). Tech- explants. niques used were methanolic extraction, HPLC purification and fractionation, and immunolog- ical measurement (ELISA), (Leroux et aL, 1985; Maldiney et al., 1986; Sotta et al., 1987; Label Materials and Methods and Sotta, 1988). ELISA measurements were repeated 5 times. Mean values are given. Wild cherry explants were cultured according to the procedure described by Riffaud and Cornu (1981The micropropagation technique can be schematically divided into 3 stages: the multipli- Results cation stage, when axillary bud growth is promoted by an almost equal amount of indole- Morphological development 3-butyric acid (IBA, 4.9 pM) and benzyladenine (BA, 4.4 pM) in the culture medium; the elonga- tion phase which was not studied; and the Under standard multiplication conditions rooting phase, in which IBA (4.9 pM) alone promoted root formation. (Fig. 1 A, B and C), axillary buds located in
observable on d 5 and, after 3 wk of the middle part of the explants started to grow on d 4 and, after 4 wk of culture, the culture, 80% of the explants had at least multiplication rate was 3. When IBA was root. When IBA was omitted from this one omitted from this culture medium (Fig. 1 B culture medium (Fig. 1 D), 3% of the explants were rooted at the 3rd wk. and C), no multiplication was observable; moreover, 82% of the explants were necrotic at the 4th wk. When BA was Hormonal measurements omitted from the standard multiplication medium (Fig. 1 A), about 65% of the explants were rooted 4 wk after sub- hormonal levels are pre- Endogenous culture. the same unit scale in each sented on Under standard rooting conditions (Fig. figure (with and without exogenous PGR). 1 D), first primordia were histologically root This was done to point out some specta-
the ELISA technique. Thus, cular differences between hormonal levels quantified by clear depressive effect on in the explants according to treatment. For BA had a instance, in Fig. 1 C and D, without IBA in endogenous natural cytokinin metabolism. Although BA metabolism is well known the culture medium, IAA levels were very low (grey background), but the apico-basal (Letham and Palni, 1983), results on the effect of BA on endogenous cytokinin distribution of this hormone in the explants metabolism have never been published. In was, nevertheless, significant. Results are the future, the intensity of this depressive presented in nmol DW; to approxi- 1 g- ’ effect and the biological activity of this mately convert them into nmol!explant-!, measurements given in the apical, middle synthetic PGR should be explored. and basal part will be multiplied by 3, 2 and 1, respectively. Acknowledgments The technical assistance of P. Capelli and R. Discussion and Conclusion Camelin is gratefully acknowledged. This work was partly supported by a grant (no. 4473) from INRA. relationship between IBA and endo- A genous IAA be evoked. In each can experiment, when explants were cultured with IBA (multiplication, rooting) in the References culture medium, internal IAA was baso- apically distributed in the explants and IAA Cornu D. & Boulay M. (1986) La multiplication levels were 20-30 times higher than in vegetative. Techniques horticoles et culture in explants cultured without IBA, where IAA vitro. Rev. For. Fr. 38, 60-68 is apico-basally distributed. Epstein and Epstein E. & Lav6e S. (1986) Conversion of Lavee (1986) reported a transformation of indole-3-butyric acid to indole-3-acetic acid by cuttings of gravepine (Vitis vinifera) and olive IBA into IAA during in vitro culture of Vitis (Olea europea). Plant Cell Physiol. 253, 697- vinifera and Olea europea. The chemical 703 pathway could be a 0-oxidation of the bu- Label P. & Sotta B. (1988) An ELISA test for BA tyric side chain, but the biochemical mech- measurement: application to wild cherry culture anism of this process remains unknown. in vitro. Plant Cell Tissue Organ Culture 1, 155- 158 From the experiments run in the Label P., Maldiney R., Sossountzov L., Cornu presence and in the absence of BA in the D. & Miginiac E. (1989) Endogenous levels of culture medium (Fig. 1 B), we postulate acid, indole-3-acetic acid and abscisic that IBA might control the penetration of benzyladenine during in vitro bud growth induc- tion of wild cherry (Prunus avium L.). Plant BA into the explants. We still have to Growth Regul. 8, 325-333 investigate this point, and experiments Leroux B., Maldiney R., Miginiac E., Sos- using radiolabeled IBA and BA would be sountzov L. & Sotta B. (1985) Comparative of great interest in this perspective. quantitation of abscisic acid in plant extracts by gas-liquid chromatography and an enzyme- The last result was not illustrated linked immunosorbent assay using the avi- because of its strong clarity: when BA was din-biotin system. Planta 166, 524-529 present in the culture medium (with or Letham D.S. & Palni L.M.S. (1983) The bio- without IBA), no natural cytokinins could synthesis and metabolism of cytokinins. Annu. be detected in the explants, whereas each Rev. Plant Physiol. 34, 163-197 time BA was removed from the culture Maldiney R., Leroux B., Sabbagh L, Sotta B., medium, natural cytokinins could be Sossountzov L. & Miginiac E. (1986) A bio-
siers adultes (Prunus avium L.) s6lectionn6s tin-avidin-based enzyme immunoassay to en quantify three phytohormones: auxin, abscisic foret. Agronomie 1, 633-640 acid and zeatin riboside. J. Immunol. Methods Sotta B., Pilate G., Pelese F., Sabbagh I., Bon- 90, 151-158 net M. & Maldiney R. (1987) An avidin-biotin J. (1961) Les corr6lations d’inhibition. Margara solid phase ELIl3A for femtomole isopentenyl- Ann. Physiol. V6g. 8, 55-69 adenine and isopentenyladenosine measure- ments in HPLC-purified plant extracts. Plant Riffaud J.L. & Cornu D. (1981) Utilisation de la culture in vitro pour la multiplication de meri- Physiol. 84, 571-573