Possibilities and constraints in the regeneration of trees from cotyledonary needles of Picea abies in vitro

Abstract

Although use of embryonic or seedling tissues for mass clonal micropropagation in vitro in conifer reforestation programmes is questionable, there is a potential application in the regeneration of plants from scarce and costly seed derived from controlled pollination. In addition, in vitro culture shortens considerably the lag phase in numbers during the initial stages of vegetative propagation via rooted cuttings. Successive steps of the present technique are described whereby cotyledonary needles (secondary explants) were subcultured on a hormone‐free medium after administration of cytokinin or auxin to 14‐day‐old seedling (primary) explants of Picea abies. For bud induction, N6‐benzyladenine (BA) was applied either as a short‐duration (3 h), high‐concentration (125 μM) pulse or by vacuum infiltration and incubation in a BA‐containmg (5 μM) infusion medium. Induced adventitious shoots were elongated with the aid of far‐red light and rooted in vivo after a long‐duration (12 h), high‐concentration (625 μM) application of indolebutyric acid. Pulse and infusion treatments resulted in the induction of greater numbers of adventitious buds (average of 12 per needle) over a three to four week shorter culture period than was the case with the conventional inclusion of growth regulators in the agarified medium. No exogenous auxin was required in the bud‐induction programme; its inclusion even at nanomolar levels promoted histo‐ rather than morphogenesis. In cotyledonary needles, to the primary explants of which BA was applied as a pulse or by infusion, the cell divisions which gave rise to the meristemoids from which adventitious buds were produced, appeared to commence mainly in undifferentiated hypodermal layer cells but also in the mesophyll immediately below. By contrast, where BA was incorporated in the agarified medium the first divisions occurred mainly in cells of the epidermal layer.A number of factors affected plantlet regeneration, for instance seed variability, age of seedlings, and mode of application of growth substances. It should also be accepted that the xeromorphic nature of the conifer leaf might impose physiological and morphological constraints on its culture in vitro that could militate against easy morphogenic manipulation. It is deemed essential that the current mean ratio of regenerated plants to cotyledonary needles of 1:1 be increased 10 to 20 fold in order to approach commercial feasibility.