Direct shoot regeneration from mature zygotic embryos of Pinus elliottii x P. caribaea hybrids and plantlets production assisted by temporary immersion bioreactors.

FORTES N.; DUARTE, E.; GONZÁLEZ A.; SANSBERRO P; LUNA C

Vitoria

Congreso; Third International Conference of the IUFRO unit 2.09.02: Somatic embryogenesis (SE) and other vegetative propagation (VP) technologies.; 2014

IUFRO

In order to develop biotechnological tools to produce in vitro plantlets of Pinus elliottii var. elliottii x P.caribaea var. hondurensis, vegetative buds were induced from mature zygotic embryos isolated from sterileseeds. The regenerative explants were transferred to a temporary immersion system for the elongationphase. Finally, adventitious rooting was induced and whole plants were obtained.Vegetative buds were induced through a direct pathway from cotyledons cultured on half strength Murashigeand Skoog (MS½) semisolid medium (plus sucrose 15 g∙L-1) containing different combinations of6-benzyladenine (BA 0.1 mg∙L-1) and thidiazuron (TDZ 0.1-5 mg∙L-1). The cultures were incubated in agrowth room at 27±2°C with a 14-h photoperiod (180 μmol∙m−2∙s−1, from fluorescent lamps) for 30 days.Consequently, regenerative explants from BA 0.1 mg∙L-1 + TDZ 0.1 mg∙L-1 were transferred to bioreactorscontaining 200 mL MS½ plus sucrose 15 g∙L-1 and indole-3-acetic acid (IAA, 0.1 mg∙L-1) for the elongationphase. For the temporary immersion program, the explants were in contact with the medium for 1 min every4 h. Finally, shoots were rooted by partial immersion in an aqueous solution of indole-3-butyric acid (IBA)500 mg∙L-1 for 1 h and transfer to either a semisolid medium (Phytagel® 3.5 g∙L-1) or vermiculite added withMS½ (sucrose 15 g∙L-1) and IBA 0.5 mg∙L-1.The regeneration frequency (93±3.3%) and the number of buds formed per responsive explants (6±0.8)was greater with explants cultured in MS½ with BA 0.1 mg∙L-1 and TDZ 0.1 mg∙L-1. Rooting of regeneratedshoots was observed in MS½ medium with vermiculite as the substrate and supplemented with IBA0.5mg∙L-1. All plants raised in vitro were phenotypically normal and successfully hardened to ex vitroconditions.These methodologies enable obtaining a great quantity of propagated material, which can be used in breedingprograms and forestation planes according to market.