Callus induction and plantlet regeneration in Bixa orellana L., an annatto-yielding tree

A protocol has been developed for plantlet regeneration from seed callus of Bixa orellana L. Seeds demonstrated a high percentage of callus induction (63.8 +/- 7.3%) and a high yield (356.3 +/- 14.7 mg per seed) of white friable callus on Murashige and Skoog (MS) medium containing 5.0 muM 1-naphthaleneacetic acid (NAA) and 2.5 muM N-6-benzyladenine. (BA) within 6 wk of culture in the dark. Callus induction frequency was greater under 24 h dark as compared to 16 h light/8 h dark photoperiod or 24 h light photoperiod. Increased myo-inositol (MI; 200 mg l(-1)) and addition of ascorbic acid (AA; 200 mg l(-1)) to the culture medium positively improved callus induction frequency and growth. Shoot differentiation from white friable seed callus was best using 10.0 muM BA and 5.0 muM NAA, where the highest percentage of calluses forming shoots (74.9 +/- 4.8%), the highest number of shoots per callus (six or seven) and the highest shoot-forming index (5.0) were obtained within 6 wk. Shoots elongated to 4 cm within 4 wk of transfer onto MS medium devoid of growth regulators. Shoots were rooted using half-strength MS medium containing 5.0 muM indole-3-butyric acid (IBA). About 85% of these plants were established in pots containing pure garden soil and organic manure after 3 wk of hardening. Regenerated plants were morphologically uniform with normal leaf, shape and growth patterns. These plants are currently being screened for the presence of agronomically useful genetic variants.