Micropropagation of Pinus oocarpa Schiede and Cupressus lusitanica Miller

The in vitro induction of organogenesis on two tropical gymnosperms, Pinus oocarpa Schiede and Cupressus lusitanica Miller, was the main objective in this study. Bud induction, shoot elongation, and rooting of the elongated shoots were achieved on both species. Adventitious bud induction on P. oocarpa was achieved when entire embryos, dissected embryos, or cotyledons derived from 7 to 14 day-old seedlings were cultured on MS modified medium or GD modified medium containing BAP (10.0, 25.0, 44.4, and 50.0 μM) alone or in combination with NAA (10.0, 25.0, and 53.7 μM). Initiation of bud formation on entire and dissected embryos was observed after three weeks in the bud induction media. Embryos showed well organized buds and a few elongated shoots by the seventh week. Cotyledons derived from 14 day-old seedlings formed buds mainly at their distal end. Cotyledons derived from 10 day-old seedlings formed buds along their entire surface, but the concentration of buds was higher at the distal end. Cotyledons derived from 7 day-old seedlings formed buds along their entire surface, but the buds had less organization and elongated slower than the buds formed on older cotyledons. Needle-like structures were formed on cotyledons derived from both, 7 and 10 day-old seedlings. Bud development and shoot elongation on P. oocarpa embryos and cotyledons was stimulated by transferring the explants to MS modified medium supplemented with 2% sucrose or to half strength GD modified medium supplemented with 2% sucrose. Shoots of P. oocarpa longer than 5 mm were rooted on solidified agar nutrient medium containing 0.1 or 1.0 μM NAA and 0.5 or 1.0% sucrose. Shoots were also rooted on solidified agar nutrient medium containing a combination of auxins (NAA combined with lAA or IBA), a cytokinin (BAP), and 4% sucrose. Adventitious buds were produced on the cotyledons, hypocotyls, and primary leaves of stem tips derived from 4 week-old seedlings of C. lusitanica. The stem tips were cultured on a defined nutrient medium supplemented with BAP (1.0 and 5.0 μM), 2iP (5.0 μM), and kinetin (5.0 μM) alone or in combination with IBA (5.0, 50.0, and 500.0 nM). BAP alone or in combination with IBA was most effective in inducing adventitious bud formation. Subsequent bud development and shoot elongation was accomplished by transferring the explants to half strength nutrient medium supplemented with 2% sucrose. Shoots longer than 5 mm were rooted on a mixture of peat, vermiculite, and sand (4-2-1). The shoots were treated two times a week for the first two weeks with a water solution containing 15 μM NAA.