Adventitious rooting in microcuttings of Malus rootstocks MM106 was studied as regards their histological and biochemical aspects. Microcuttings from shoots raised in Murashige and Skoog's (1962) medium were transferred into a rooting medium containing IBA in the dark, then fixed 0, 3, 5, 7 and 10 days after. Some cambial zone and adjacent phloem cells became dense cytoplasm, nuclei with prominent nucleoli and the first cell divisions were observed at day 3. Meristemoids became individualized, consisting of densely staining cells (with enlarged nucleoli) formed outside the xylem by day 5. Identifiable root primordia with a conical shape and several cell layers were present at day 7. Roots with organized tissue system emerged from the stem 10 days after the root induction treatment. From these histological observations, it can be established that the rooting induction stage ended before day 3. The initiation stage, with the first histological modifications to the formation of meristemoids, would correspond to the transient increase of our biochemical marker (peroxidase activity) until day 5. The best rooting percentage obtained with cultures in the presence of auxin during 5 days confirms this hypothesis. The expression of rooting can then take place.

Apple rootstock MM106 shoots, raised in vitro, rooted at 96.7% after culture on a medium supplemented with an auxin for 5 d in darkness followed by culture on a second medium without growth regulators for 25 d in light. In control conditions (in absence of auxin in the first medium), these shoots did not root. Putrescine (PUT), spermidine (SPD), cyclohexylamine (CHA), and aminoguanidine (AG) enhanced rooting when applied during the first d of culture in the absence of IBA; on the contrary, α-difluoromethylornithine (DFMO) added to the first medium with IBA inhibited rooting. The endogenous levels of indole 3-acetic acid (IAA) and indole 3-acetylaspartic acid (IAAsp) increased up to a maximum concentration at days 2 and 3, respectively, in initial rooting conditions. PUT, when added with IBA, did not affect the typical IAA and IAAsp increase; when applied alone, it provoked an increase of their levels. Similar results were recorded with CHA. SPD, AG, and DFMO did not induce an increase of IAA and IAAsp in nonrooting conditions. The levels of endogenous PUT increased to a maximum at day 2 in rooting conditions; it was slightly affected by exogenous PUT and CHA application but reduced by SPD, AG, and DFMO. In rooting conditions, if the first medium was supplemented with SPD or AG, a small increase in peroxidase activity was observed, similar to that obtained with PUT treatment. The present work indicates an involvement of polyamines in the control of rooting and an interaction with auxins during the physiological phase of rooting. The consequence of this relationship was a different rooting expression, according especially to the content of these regulators in the culture medium.

Somatic embryogenesis is a useful tool of plant breeding. In this context, a procedure for inducing somatic embryogenesis in Prunus incisa leaf explants had been previously developed. The original in vitro protocol relies on picloram treatments and exposure to darkness as inductive conditions, the best frequency of embryogenesis being obtained on the second leaf (F(2)) exposed to 4 μM picloram during 30 days. The morphological and biochemical changes observed during somatic embryogenesis occur in response to alterations in gene expression regulation patterns. A molecular study was conducted in order to provide deeper insight into the fundamental biological factors involved in the induction of this process using a gene candidate strategy and semi-quantitative reverse transcription polymerase chain reaction analysis. So far, no sequence data related to somatic embryogenesis has been available in cherry. In the present study, we cloned and sequenced cDNA fragments of putative genes encoding auxin-binding protein, cell cycle regulator and somatic embryogenesis receptor kinase. Time-course differential transcript accumulations were observed for all investigated genes in leaves or derived callus tissues during the observation period (first month of culture). Their possible involvement in the sequential steps of the embryogenic pathway (dedifferentiation, cell proliferation, differentiation through somatic embryogenesis) is presented and discussed.

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