Agrobacterium rhizogenes mediated induction of adventitious rooting from Pinus contorta hypocotyls and the effect of 5-azacytidine on transgene activity

Bipartite constructs of Agrobacterium rhizogenes strain LBA 9402 or A4RSII induced transformed roots on the hypocotyls of Pinus conforta following inoculation, LBA 9402 being more effective. The developmental sequence of root formation and morphology following infection were studied. Furthermore, the pattern of gene expression was studied during rooting and in roots using the uidA reporter gene driven by the 35S promoter. Morphologically most of the roots were normal, whether or not they expressed the reporter gene, but extensive proliferation of lateral roots was observed in some roots with beta-glucuronidase (GUS) activity. All roots originated from tissues inside the endodermis, often similar to auxin-induced rooting in hypocotyl cuttings as described by Gronroos and von Arnold (1987). Where the origin of GUS-positive roots could be traced, they developed from callus forming inside the endodermis. GUS activity was often observed along the root inside the endodermis, at the base of the lateral roots and at the root apex, but not in a region behind the apex. Stable integration of the transgene was verified using Southern blot analysis. To investigate whether transgene inactivation occurs in conifer plants, root segments and calluses initiated from them were treated with 5-azacytidine. Treatment with 5-azacytidine increased the frequency of GUS-positive roots from about 20% to 50%. The effect of 5-azacytidine on calluses, however, varied among callus lines. To investigate whether methylation was the cause of transgene inactivation, DNA from 5-azacytidine-treated and untreated calluses was digested using the two isoschizomeric restriction enzymes, HpaII and MspI, which differ in their sensitivity to methylation. There was no evidence for methylation and demethylation at the cleavage sites examined.