Clonal stability, seasonal periodicity and transgenes: the lesson from long-term micropropagation of transgenic and non-transformed Populus tremula L. plants

Annual and diurnal rhythms of plant development are manifested as ordered changes in growth and regeneration, in physiological processes and metabolism, and in molecular events. This periodic behavior may reflect either external (environmental) stimuli, and/or endogenous (intrinsic) biological clocks of various types. Although exogenous stimuli can be controlled in vitro, the tissue also exhibits rhythmic behavior under environmentally controlled in vitro culture. A combination of in vitro culture and gene insertion via transformation could, therefore, be used to elucidate some of the potential interactions between culture growth rhythms and stability, the regeneration potential, and molecular control mechanisms such as silencing. These and related aspects were examined in our laboratory, using long-term cultures of transgenic and non-transformed aspen plants (Populus tremula L.). Stem segments have been routinely subcultured every 4 to 8 weeks under standard conditions, without any hormones, since 1990, resulting currently in plantlets representing about 80 to 120 passages in culture. In addition, two types of transgenic plants have been generated: (1) expressing the rol-T-DNA fragment from Agrobacterium rhizogenes LBA9402, carrying a Ri vir, and (2) expressing the water stress-regulated BspA cDNA. Seasonal periodicity in non-transformed aspen development was clearly evident with respect to adventitious root formation, shoot elongation and axillary bud break. Generally, these growth parameters were at a maximum when expiants were subcultured in the summer, and extremely low during the winter. This was also reflected in the considerably higher in vitro propagation coefficient in the summer vs. winter. This recorded pattern of development has persisted for over more than 10 years since the primary expiants were excised from the mother plant and cloned in vitro and no other phenotypic changes have been detected. The in vitro-cultured transformed lines, transgenic for rol-T-DNA and expressing the rolB and rolC genes, were characterized by accelerated regeneration and growth of roots and shoots, and by the loss of a seasonal growth rhythm. Seasonal fluctuation in the expression of the gene encoding an aspen water-stress-responsive boiling-stable protein (BspA) at the RNA and protein levels as also detected in the same plant material. The persistence of growth periodicity in long-term cultures suggests that the actual rate of phenotypic polymorphism in plants is often far less frequent than DNA and protein band polymorphism. It is proposed that specific existing and induced mechanisms contribute to clonal stability in tissue culture, and that these are at least as important as the suggested mechanisms of variation induction in vitro.