Cesar Augusto Zanello

Phalaenopsis is an orchid genus of great economic value in world floriculture. In vitro clonal propagation is the only large-scale feasible method for Phalaenopsis propagation, but it is difficult because of the low multiplication rate. The aim of this study was to evaluate the effect of types and concentrations of N⁶-benzyladenine (6-BA) and gibberellic acid (GA₃) on the in vitro multiplication of shoots from inflorescence nodal segments (INS) of Phalaenopsis hybrids. INS with one axillary bud were inoculated in New Dogashima Medium with different combinations of BA and GA₃. The results show that the treatment containing 1.0 mg L⁻¹ BA and 1.5 mg L⁻¹ GA₃ showed the higher percentage of live inflorescence segments (71.48%) and a number of shoots (1.68 shoots/INS). The highest 6-BA concentration (4 mg L⁻¹) tested in this study resulted in the best shoot multiplication rate (4.3). Contamination and browning of the INS tissues were the main difficulties identified for clonal propagation of Phalaenopsis. Successful in vitro rooting occurred on half-strength Murashige and Skoog medium (100%), and acclimatization (100%) was obtained independent of the substrates. However, the best gains in number of roots, leaves, chlorophyll content, and fresh weight of plantlets were achieved using vermiculite.

The process through induction, proliferation and regeneration of protocorm-like bodies (PLBs) is one of the most advantageous methods for mass propagation of orchids which applied to the world floricultural market. In addition, this method has been used as a tool to identify genes of interest associated with the production of PLBs, and also in breeding techniques that use biotechnology to produce new cultivars, such as to obtain transgenic plants. Most of the molecular studies developed have used model plants as species of Phalaenopsis, and interestingly, despite similarities to somatic embryogenesis, some molecular differences do not yet allow to characterize that PLB induction is in fact a type of somatic embryogenesis. Despite the importance of species for conservation and collection purposes, the flower market is supported by hybrid cultivars, usually polyploid, which makes more detailed molecular evaluations difficult. Studies on the effect of plant growth regulators on induction, proliferation, and regeneration of PLBs are the most numerous. However, studies of other factors and new technologies affecting PLB production such as the use of temporary immersion bioreactors and the use of lighting-emitting diodes have emerged as new tools for advancing the technique with increasing PLB production efficiency. In addition, recent studies on Phalaenopsis equestris genome sequencing have enabled more detailed molecular studies and the molecular characterization of plantlets obtained from this technique currently allow the technique to be evaluated in a more comprehensive way regarding its real applications and main limitations aiming at mass propagation, such as somaclonal variation.

Induction of Protocorm-like bodies (PLBs) from leaf segments is a clonally propagation technology that used somatic embryogenesis regeneration from in vitro buds of floral stalks, and is a potential alternative for replacement actual low-yield and high-cost clonal propagation of Phalaenopsis orchids. The objective of this work was to evaluate induction of PLBs in leaf segments of two commercial hybrids of Phalaenopsis, arranged in culture media with different combinations of plant growth regulators. The leaf segments (0.4–0.5 cm²) used for PLBs inductions were obtained from young in vitro-induced shoots from inflorescence stalks. They are cultivated in NDM culture media, supplemented with combinations of plant growth regulators Naphthaleneacetic Acid, Thidiazuron and Benzyladenine. The flasks were kept for 60-d in darkness and then for cold white light with photon flux density of 35–40 μmol m⁻² s⁻¹. The cultivar ‘Ph908’ showed a higher percentage of leaf segments with PLBs (45%) and number of PLBs (25 per leaf segment), in medium containing 0.25 mg L⁻¹ of TDZ, whereas ‘RP3’ showed only 10% containing PLBs and 2 PLBs per leaf segment in NDM with 1.0 mg L⁻¹ NAA, 20 mg L⁻¹ BA and 0.125 mg L⁻¹ TDZ.