Efficient disinfection of soursop (Annona muricata L.) explants for in vitro micropropagation

Abstract

Background: Soursop (Annona muricata L.) is a tropical fruit with important nutritional and medicinal properties. Clonal multiplication of soursop by in vitro tissue culture is the best alternative for genetic improvement and conservation purposes. Objective: To reduce the level of persistent microbial contamination in soursop explants to facilitate their introduction to in vitro tissue culture. Methodology: Leaf explants (LE) and internodal segments (IS) from ex vitro plants were disinfected with different concentrations of active chlorine and ethanol, and different combinations of antibiotics and fungicides: Treatment 1: Streptomycin:tetracycline with carbendazim (Strept100:Prozycar); Treatment 2: Streptomycin:tetracycline with benomyl (Strept100:Benomyl); Treatments 3, 4 and 5: Same as Treatment 2, with no ethanol wash and addition of 100, 200 or 300 ppm silver nanoparticles (AgNPs), respectively. Disinfected explants were cultivated on MS medium in the presence of different combinations of plant growth regulators (2,4-dichlorophenoxyacetic acid (2,4-D), benzyladenine (BA) or kinetin (Kin). Results: Strept100:Prozycar and Strept100:Benomyl combinations left ~96/98 % and ~60/54 % of LE/IS contaminated, respectively. The addition of AgNPs improved decontamination (~6% of LE and ~9% of IS, respectively). Simultaneous addition of 1.5 mg·mL-1 BA and 2.0 mg·mL-1 2,4-D, with a subsequent step with 0.5 mg·mL-1 Kin, led to the higher in vitro regeneration rates (~95.6% of LE and ~93.9% of IS). Implications: The reduction of internal contamination facilitates in vitro micropropagation of species of the Annonaceae family. Conclusion: The use of AgNPs was decisive to reduce internal contamination, improving conditions for the in vitro propagation of soursop tissues.