Improved axillary and adventitious bud regeneration from Portuguese genotypes of Pinus pinaster Ait.

Axillary bud multiplication and direct adventitious bud regeneration were improved for clonal propagation of Portuguese genotypes of Pinus pinaster. Axillary buds induced in an ex vitro two year-old genotype by 6-benzylaminopurine (BAP) spraying and apex removal were used for the establishment of in vitro axillary bud cultures. Improved procedures were also developed for both axillary bud multiplication from four week-old seedlings and direct adventitious bud induction from cotyledons. The first in vitro multiplication cycle of buds derived from the ex vitro material resulted in an approximately five-fold multiplication rate. Regarding the axillary buds derived from four-week-old seedlings, the best induction medium for bud multiplication was based on a GD medium plus 1.11 mu M BAP and 0.27 mu M alpha-naphthaleneacetic acid (NAA). Induction of adventitious buds varied significantly among seed provenances. In the best performing provenance, the highest bud induction frequency (95%) after three multiplication cycles was achieved on a GMD basal medium supplemented with 4.45 mu M BAP and 0.05 mu M NAA for three weeks. The axillary bud multiplication protocol for seedlings is potentially useful for large-scale propagation of seeds coming from the Portuguese breeding program of P. pinaster.