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 A4 RSII induced transformed roots on the hypocotyls of Pinus conforta fol lowing inoculation, LBA 9402 being more effective. The developmental s equence of root formation and morphology following infection were stud ied. Furthermore, the pattern of gene expression was studied during ro oting and in roots using the uidA reporter gene driven by the 35S prom oter. Morphologically most of the roots were normal, whether or not th ey expressed the reporter gene, but extensive proliferation of lateral roots was observed in some roots with beta-glucuronidase (GUS) activi ty. All roots originated from tissues inside the endodermis, often sim ilar to auxin-induced rooting in hypocotyl cuttings as described by Gr onroos and von Arnold (1987). Where the origin of GUS-positive roots c ould be traced, they developed from callus forming inside the endoderm is. GUS activity was often observed along the root inside the endoderm is, 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 veri fied using Southern blot analysis. To investigate whether transgene in activation occurs in conifer plants, root segments and calluses initia ted from them were treated with 5-azacytidine. Treatment with 5-azacyt idine increased the frequency of GUS-positive roots from about 20% to 50%. The effect of 5-azacytidine on calluses, however, varied among ca llus lines. To investigate whether methylation was the cause of transg ene inactivation, DNA from 5-azacytidine-treated and untreated calluse s 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 e xamined.