A new procedure for in vitro propagation of vanilla (Vanilla planifolia) using a double-phase culture system

• A new procedure for propagation of vanilla was investigated.
• We analyzed the effect of a thin layer of liquid medium for propagation of vanilla.
• Improvements in the proliferation rate require a thin liquid layer over a semi-solid medium.
• This new procedure can be a simple, low-cost and efficient method for vanilla propagation.

We have developed an efficient and simplified method for shoot proliferation of vanilla (Vanilla planifolia), using a Double-Phase Culture System (DPS) (semi-solid medium with a layer of liquid medium on the top). In vitro proliferating shoot cultures were the source of nodal explants (approximately 1.0 cm in height and at least one axillary bud) used in all experiments. Three experiments were conducted: the first determined the influence of cultivation time (30, 60 and 90 days) and concentrations of 6-benzylaminopurine (BA) (0, 1, 2 and 3 mg l−1) on in vitro multiplication of vanilla; the second evaluated the influence of conventional (semi-solid medium, or SS) and DPS systems on in vitro multiplication of vanilla. Finally, we evaluated the effect of indole-3-butyric acid (IBA) (0, 1, 2 and 3 mg l−1) concentrations on the rooting of shoots multiplied in vitro and on survival rates after acclimatization. BA concentration in the culture medium and DPS for shoot multiplication had significant effects on success. The multiplication rate, which reflects axillary shoot multiplication, was greatest when DPS with 1 mg l−1 BA was used, representing more than a 2.5-fold increase over the rates obtained with SS medium, after 90 days of cultivation. Shoots taken from DPS were rooted at a frequency of 100%, in full-strength Murashige and Skoog medium without IBA. The plantlets were then transferred to soil, and survived acclimatization with 100% success. The results indicate the establishment of a new procedure for vanilla micropropagation through DPS, with a high production of plantlets at the end of the process and great potential for agriculture application. In addition, the presented system is more economic than the single-phase during the subcultures.