Plantlet regeneration from subculturable nodular callus of Pinus radiata

Abstract

An efficient planlet regeneration system via nodular callus formation is described for Pinus radiata. Subculturable nodular callus was induced at its highest frequency (93%) on embryonic explants excised from seeds at an early stage of germination (radicle length 2–5 mm). The optimal medium for nodular callus tissue proliferation was $\frac{1}{2}$ LP basal medium that was modified by reducing the concentration of potassium nitrate to 500 mg l⁻¹ and supplemented with 22.2 μM 6-benzyladenine (BAP) and 2.85 μM indole-3-butyric acid (IBA). Bud differentiation from the nodules was achieved by reducing BAP and sucrose concentrations in the culture medium. The maximum frequency of adventitious bud formation occurred on $\frac{1}{2}$ LP basal medium containing 2% sucrose and 0.44 μM BAP on which about 61% of the transferred nodules formed buds. During the next 6 weeks of culture on the same cytokinin-free medium multiple shoots elongated from the buds. These shoots were excised and transferred to root initiation medium (RIM2.1), consisting of full-stregth SH macro- and micro-salts, 1000 mg l⁻¹ myo-inositol, 0.4 mg l⁻¹ thiamine-HCl, 2% sucrose and a combination of naphthaleneacetic acid (NAA), IBA and BAP at concentrations of 2.69, 4.93 and 0.11 μM, respectively. After 5–15 days, root meristems were initiated on the stem bases. The highest rooting frequency was achieved when shoots were treated for 10 days on RIM2.1 medium, before being transferred to half-strength Schenk and Hildebrandt medium with 1% sucrose and without growth regulators for root growth.