Epsilon Open Archive

Abstract

Four different Agrobacterium rhizogenes wild-type strains were compared for the ability to induce transformed roots on micropropagated shoots of the apple rootstock Jork 9. The mannopine strain 8196 gave the best results in the production of chimeric plants compared to two agropine strains (A4 and 15834) and one cucumopine strain. From the transformed roots seven transgenic shoots were obtained, which rooted on a hormone-free medium. Furthermore the apple rootstock Jork 9 was transformed with Agrobacterium tumefaciens strain C58C1(pGV3850)(pB-B:GUS), containing the nptII, rolB and gus genes on the T-DNA. Transformation was confirmed by PCR and Southern blot analysis for all introduced genes. The rooting experiments showed that introduction of the rolB gene increased root percentage and root number, giving 13.8 roots per shoot compared to 2.3 for untransformed shoots. In addition, more than two copies of the rolB gene decreased the number of roots and percentage of rooted shoots. Auxin-induced expression of the ARRO-1 gene during the induction phase of adventitious root formation in both rolB transformed and untransformed Jork 9 stem discs and microcuttings has been studied. The higher expression of the ARRO-1 gene in the rolB transformed material suggests that its expression may be rooting-specific rather than auxin-specific. We have improved synchrony of root formation from Malus Jork 9 stem discs through temporarily blocking DNA synthesis by application of aphidicolin (AD). The effects of different treatments with and without AD were studied at the cellular level by visualising DNA replication through the thymidin analog 5-bromo-2’-deoxyuridine-5’-monophosphate (BrdU) labelling. The results show that DNA synthesis is effectively blocked by AD, and this blockage is overcome after AD withdrawal. It is concluded that AD treatment causes initial synchronization of the cell cycle and, thereby, more synchronized root initiation. BrdU labelling further revealed an increase in competence of the explants to respond to applied indole-3-butric acid (IBA) during the first day of cultivation.