Studies on adventitious root formation in Pinus radiata : biochemical and molecular aspects.

Abstract

A reliable in vitro rooting system was established for further studies on biochemical and molecular aspects during adventitious root formation in Pinus radiata hypocotyls. High root counts and rooting efficiency were observed in a medium comprising ½ strength of the Murashige & Skoog's basal medium (1962), 20 g/l sucrose, 9 mg/l IBA and solidified with 8 g/l agar. In this system, the first cell division, formation of root primordium initial (a cluster of meristematic cells), well-organized root primordium (a dome shaped structure) and root emergence were typically observed at around day 4, 7, 10 and 13, respectively. The increase of buffer soluble total proteins resulted in a peak at day 7 in the treatment with IBA (rooting treatment), but this peak was not observed in the other non-rooting treatments (including IBA+kinetin, kinetin alone and hormone-free control). Rooting related proteins were not observed using one-dimensional SDS polyacrylamide gel electrophoresis (PAGE). However, 19 proteins associated with the IBA treatment were observed by two-dimensional PAGE although their magnitudes were very small so that it was difficult to quantify them and establish them as novel proteins. Changes of peroxidase (PO), IAA-oxidase (IAA-O), amylase (Amy) and succinic dehydrogenase (SDH) activities assayed using tissue extracts were not significantly different between the rooting and the non-rooting treatments during the experiment. By contrast, the increase of polyphenol oxidase (PPO) activity was more significant at day 7 in the rooting treatment than in the other treatments. Nevertheless, histochemical localization of PO, PPO and SDH indicated that these enzymes were somehow associated with adventitious root formation. In the IBA treatment, the increase of starch content in the rooting region reached its maximum at day 4 and 7. This increase coincided with the histological observation ofaccumulation of starch grains at the sites where root primordia initiated. To identify the molecular signals that initiate adventitious root formation, subtracted cDNA library was made from IBA-treated hypocotyls following polymerase chain reaction (PCR) amplification of subtracted single strand-cDNAs. Inserts of 5 bacterial clones (Rl-72, R2-7, R2-22, R2-35 and R3-24) were digoxigenin-labelled and used to detect their corresponding transcripts using RNA dot blotting. Using RNA dot blotting analysis, Rl-72 detected an equivalent level of transcript in the hypocotyls at day 0 and day 7 in the treatments with and without IBA; whereas the other 4 clones, especially R2-7 and R2-35, detected different levels of transcripts in the hypocotyls during adventitious root initiation. Southern blotting analysis indicated that the R2-7 transcript is likely to be represented by a single gene or a member of a smaller gene family. Adventitious root formation in P. radiata was significantly improved using Agrobacterium rhizogenes. Compared with the strain A4T, strain LBA9402 was very effective in increasing rooting percentage and root number. High rooting percentages and root numbers were obtained in the trials with hypocotyl segments, intact seedlings and adventitious shoots. The findings in this research can assist further studies to shed some light on why some clonal cuttings of radiata pine root more easily than others do, and why mature cuttings are difficult for adventitious root induction