Ontogenesis and Ploidy Level of Plantlets Regenerated from Populus trichocarpa x deltoides cv. Hunnegem Root, Leaf and Stem Explants

Poplar is a useful model system for biotechnologies on woody species; several poplar genotypes have already been shown to be amenable to both bud regeneration and transformation. The processes of direct or indirect bud regeneration from various organs of micropropagated in vitro plants of the hybrid Populus trichocarpa x deltoides cv. Hunnegem have been compared. Efficient and rapid regeneration was obtained after in vitro culture on Murashige and Skoog (MS) medium containing 0.5 µM NAA and 1 µM BAP, the best explants being excised internodes. In contrast to previous reports, the time required for bud regeneration was short. The anatomy of direct shoot organogenesis was studied under light microscopy to investigate the differential competence of tissues. After 4 to 6 days, various regions of meristematic activity were apparent in both internodes and excised petioles. In the wounded regions, most of the tissues underwent unorganized cell divisions giving rise to a very small callus from which shoot meristems were regenerated. In the non-wounded regions of the internodes, rapid periclinal divisions of vacuolated outer cortical cells formed superficial meristematic bumps prior to bud regeneration. DNA flow cytometric analyses indicated that cell differentiation occurred without endopolyploidisation and that all the nuclei of mature organs were in a 2C presynthetic condition. All the regenerants arising from direct regeneration were typically diploid and never displayed phenotypic variation after in vitro rooting and acclimatization. It was also possible to obtain indirect shoot regeneration following callus induction on MS medium containing 10 µM 2,4-D or NAA with subsequent subculture in the presence of 10 µM NAA and 5 wM BAP, but some of the regenerants were tetraploid. The implications of developmental patterns in direct and indirect bud regeneration are relevant in developing suitable regeneration-transformation systems.