Instability of phenotype and gene expression in long-term culture of carrot hairy root clones

Hairy root clones were established from carrot root discs inoculated with a n agropine-type strain of Agrobacterium rhizogenes A4 harbouring the gus re porter gene on the TL-DNA. The clones were periodically examined for their phenotypic characteristics and for their ability to express the gus gene, t o produce opines and to grow in the presence of NAM. The presence of the gu s gene in the roots was confirmed by Southern blot hybridisation. The clone s displayed various morphologies which were generally not correlated with t he transformation events, and they were highly unstable throughout the succ essive subcultures, both for their phenotype and for their ability to expre ss the transgenes. Reversible inactivation of the gus gene expression was a ssociated with a high gus copy number. This could have some consequences fo r fundamental studies and practical uses of hairy roots.