Regeneration of Plants from Isolated Cotyledons of SalgareñO Pine (Pinus nigra Arn. Ssp. Salzmannii (Dunal) Franco)

Plantlet regeneration of salgareño pine (Pinus nigra Arn. ssp. salzmannii (Dunal) Franco) was achieved from cotyledons. The data showed that the best differentiation response occurred when cotyledons, from 1-d-old embryos germinated in darkness, were cultured on Murashige and Skoog medium (half-strength macroelements) with 22 μM N⁶-benzyladenine (BA) and 0.05 μM α-naphthaleneacetic acid (NAA) for 2-3 wk. Shoot development was obtained by subculturing treated explants on the same medium without growth regulators. Shoots were successfully micropropagated by sequential subculturing them on medium containing growth regulators (5 μM BA and 0.005 μM NAA) and on hormone-free medium for 2 and 12 wk, respectively. To induce adventitious roots, shoots were treated with 3 μM NAA and 8 μM indole-3-butyric acid for 2 wk, followed by transfer to Murashige and Skoog medium (¼-strength macroelements,$20\text{g}\cdot \text{L}^{-1}$sucrose) without growth regulators. After 3-4 wk, almost all the rooted shoots (65%) could be successfully transplanted and acclimatizated in the greenhouse, where the plants exhibited normal growth habit.