In angiosperms, the formation of the flower meristem is controlled by partially redundant flower meristem identity genes of which FLORICAULA (FLO)/LEAFY (LFY) plays a central role. It is not known whether formation of reproductive organs of pre-angiosperm species is similarly regulated. Recently, a FLO/LFY-like cDNA, NEEDLY (NLY), has been cloned in a conifer species Pinus radiata (D. Don). Here we report cloning of a different Pinus radiata FLO/LFY-like cDNA, PRFLL. PRFLL had two large regions of high similarity to angiosperm FLO/LFY orthologues: amino acids 61-126 and 247-406 (50% and 81% identity, and 75% and 88% similarity, respectively, to LFY) and shorter regions of local similarity. Overall identity was 53% to LFY and 61% to NLY. Phylogenetic analysis of deduced protein sequences including partial LFY-like sequences from Pseudotsuga menziesii indicated that conifer proteins constituted a separate clade that could be divided into two groups represented by NLY and PRFLL. In contrast to angiosperms, both conifers had two paralogous proteins resembling LFY. Northern hybridisation analysis revealed expression of PRFLL in vegetative buds of juvenile, adolescent and mature trees. The transcript was not detected in vascular cambium, roots or secondary needles. To follow PRFLL expression during the early stages of cone development we analysed a temporal series of buds containing cone primordia, and developing cones, using Northern hybridisation and confocal microscopy in parallel. PRFLL mRNA was detected in buds from dominant and subordinate branches, in which cone and shoot primordia develop, and in developing male cones but not in developing female cones. Expression was particularly high in buds containing axillary primordia prior to their differentiation as male cone primordia. This is consistent with PRFLL being involved in determination of the male cone primordium identity.

The shoot-forming capacity of Pinusradiata D. Don juvenile explants under tissue culture conditions was examined. Three types of explant were used: whole embryos; cotyledons excised from whole embryos which had been cultured for 1 week on the shoot initiation medium; and cotyledons excised from 1-week-old aseptically germinated seeds. The average number of rootable shoots produced per seed in 12–13 weeks was 9 for excised embryos, 18 for their separated cotyledons, and 180 for the cotyledons from germinated seeds. Induction of a smooth-surfaced, yellow–green, nodular meristematic tissue appears to be a requirement for the formation of large numbers of adventitious shoots. By extending the culture period from 12 to 24 weeks more than 1300 shoots were formed from the cotyledons of one germinated seed. To date, no other conifer tissue culture method has been reported to give such a large number of adventitious shoots of the same clone in a first generation.

Horgan (nee Reiliy), K. and Aitten, J. 1981. Reliable plantlet formation from embryos and seedling shoot tips of radiata pine. -Physiol. Plant. 53: 170-t75.A method has been devised for the reliable production of plantlets from embryos and seedling shoot tips otPinus radiata D.Don (radiata pine). Buds were induced on an agar or liquid Schenk and Hildebrandt (SH) medium containing 5.0 mg/1 benzylammopurme (BAF). Except tor some abnormal buds, the buds grew into elongated shoots on an agar SH medium without cytokinin. The transfer of shoots from a SH medium to a Gresshoff and Doy (GD) medium was found to be an important pretreatment which increased the survival of the shoots when they were placed in a peat and pumice mix for root formation. Elongated shoots were induced to form roots under non-sterile conditions in a humid environment with dccasionai misting. An intervening 5-day treatment of shoots in an agar medium containing 2.0 mg/1 indolebutyric acid (IBA) and 0.5 mg/1 napthaleneacetic acid (NAA) significantly increased the percentage of shoots forming roots and the number of roots formed per shoot over control shoots placed directly in the peat:pumice mis. An enhanced level of COT during root formation had no effect on the time of root formation or on the percentage of shoots forming roots. These results concerning the elongation, growth and rooting of adventitious shoots are now being applied to the development of very large numbers of plantlets starting from cotyledons from partially germinated seeds.

Maturation or phase change is a serious challenge in the deployment of superior trees of Pinus radiata D. Don because of the difficulties associated with propagation of cuttings from mature trees. We used an in vitro system to study 6-benzyladenine (BA)-induced reinvigoration of the fascicle meristems of mature buds during in vitro culture. Anatomical examinations revealed that BA inhibited the development of secondary needle primordia and 'rejuvenated' the fascicle meristems of the mature bud to produce primary needles, which are characteristic of the juvenile phase in P. radiata. Without BA supplement in the culture media, fascicle primordia continued developing secondary needles and quiescent fascicle meristems. BA metabolite analysis showed that the novel cytokinin pathway reported previously in P. radiata (H. Zhang, K.J. Horgan, P.H.S. Reynolds, G.E. Norris and P.E. Jameson. 2001. Novel cytokinins: The predominant forms in mature buds of Pinus radiata. Physiol. Plant. 112: 127-134) was mirrored in vitro, with BA converted into a variety of metabolites including 6-benzylamino-9-glucopyranosylribosyl-purine and its novel phosphorylated form, 6-benzylamino-9-glucopyranosylribosyl-purine. The culture of mature buds in the presence of BA caused a reduction in the level of endogenous cytokinins, suggesting a direct action of BA itself. Similar correlations are noted between levels of certain metabolites and the maturation status of buds from field-grown trees and buds in culture, indicating that this in vitro system may be a good model for studying the processes of maturation and reinvigoration.

No abstract