OPTIMIZATION OF PRICKLY PEAR CACTI (Opuntia spp.) MICROPROPAGATION USING AN EXPERIMENTAL DESIGN METHOD

In Morocco, despite the adaptation of the prickly pear cactus (Opuntia spp) to edaphoclimatic conditions, differing from those that prevail in the countries from which it originates, this introduced species is regressing due to biotic and abiotic stresses that impact its survival. Hence, finding innovative technologies to preserve this species are essential. However, the success of in vitro micropropagation technologies is hinged upon the initial disinfection step. In this work we have tested several solutions and exposure time mixtures for disinfection across three different explants, as well as the adoption of hormonal combinations for the induction/regeneration step that allow for efficient micropropagation of cacti. We aimed to produce healthy regenerable vitroplants capable of effectively preserving themselves under aseptic conditions. We describe a specific protocol based on the type of areoles: (1) cladode without areolas (CWA); (2) areolas with shredded glochids (ASG); and (3) areolas with intact glochids (AIG). For each of these areoles, the disinfectant concentration and contact time were optimized and various hormonal combinations were tested for induction/regeneration. The optimal design meeting I-optimality criterion was used to predict the optimal combination by maximizing desirability. The disinfection combination that used 4.23% calcium hypochlorite (w:v), 0.4% Tween 20 (v:v), and 10 minutes of exposure time reduced the contamination frequency to 0.002% with a necrosis rate of less than 0.33% in explants with AIG. This type was the only one that gave responses in the induction/regeneration phase, which allowed us to predict the best rate of caulogenesis (71.74%) under a hormonal combination of 5.3 mg/L of BenzylAminopurine (BAP) and 0.59 mg/L of 1-Naphthaleneacetic acid (ANA). Furthermore, the highest callogenesis rate of 94.45% was obtained by a hormonal combination of 6 mg/L of BAP and 0.59 mg/L of ANA.  The explant’s rhizognesis was carried out with a concentration of 0.5 mg / l ANA, before acclimatization on a substrate containing soil and sand in a ratio of 3:1. In conclusion, these protocols provide easy and cost-effective means to multiply cacti in vitro to preserve them in areas where their culture has been destroyed by cochineal.