Molecular variation in plant cell populations evolving in vitro in different physiological contexts

Previous work has shown the fixation of context-specific random amplified p olymorphic DNA (RAPD) patterns in tomato cell cultures grown for 2 years in different hormonal contexts. In this work, RAPD sequences were characteris ed and RAPD-derived molecular markers used for a further study of variation between and within auto- and auxo-trophic tomato cultures grown in differe nt hormonal equilibria. Results were then compared with those obtained usin g microsatellite markers located in noncoding regions of differentiation- a nd hormone-related genes and with those obtained with the external transcri bed spacer (ETS) from tomato rDNA. Hybridisation of RAPDs on a tomato genom ic DNA bank, or on total DNA after enzymatic digestion, suggested that the markers were repetitive in nature. Sequence analysis, however, showed that the homology between different fragments was due mainly to the presence of homo-AT nucleotide stretches. Moreover, a series of computational methods, such as an information-theory algorithm coupled with DeltaG estimates, sugg ested that the RAPD fragments isolated in our experiments are noncoding. Th e amplification of SSR-containing RAPD-derived markers, and of other SSRs l ocated in noncoding regions of tomato functional genes, consistently showed polymorphism between auxo- and auto-trophic somaclones (the latter being e ither habituated or transgenic for Agrobacterium tumefaciens oncogenes) but not within these same clones. Differences were also found between auxotrop hic clones and the differentiated tissue. These findings were confirmed by restriction fragment length polymorphism (RFLP) analysis with the REII repe titive element of the ETS from tomato rDNA, which was isolated during this study. The results obtained suggest a possible role for physiological conte xt in the selection of RAPD patterns during the evolution of tomato cells w ith different endogenous hormonal equilibria. The results are discussed in terms of a possible role for variation in noncoding regions of hormone-rela ted genes in the adaptation to different physiological contexts.