Production of PMWaV-free pineapple plants using combined thermotherapy and etiolation during micropropagation

Mealybug wilt of pineapple (MWP) is an economically critical viral disease affecting global pineapple production. In Brazil, MWP is associated with pineapple mealybug wilt-associated viruses (PMWaV-1, PMWaV-2, and PMWaV-3), which are primarily disseminated via vegetative propagation material, as infected plantlets often remain asymptomatic. In vitro propagation techniques provide a reliable approach to generate pathogen-free planting material. This study aimed to combine thermotherapy and etiolation with shoot tip culture to establish an optimized protocol for producing virus-free ‘Vitória’ pineapple plants. In vitro shoots, multiplied from axillary buds of MWP-symptomatic ‘Vitória’ pineapple plants, were subjected to heat treatments at 35, 37, and 39 (± 1) °C for 35 days, or incubated in continuous darkness for 30, 60, and 90 days to induce etiolation. Following thermotherapy or etiolation treatments, shoot tips (≈ 2.0 mm) were excised and cultured in vitro for 90 days prior to leaf sampling for viral detection by reverse transcription-quantitative PCR (RT-qPCR). Etiolation induction for 60 and 90 days resulted in the eradication of PMWaV-3. Thermotherapy at 39 (± 1) °C achieved the elimination of PMWaV-1 and PMWaV-3 across all tested samples, and PMWaV-2 in 67% of samples, with a shoot tip regeneration of 37%. Virus-free shoots were subsequently multiplied in vitro and acclimatized in a greenhouse before field transplantation, where they remained healthy over two production cycles. The developed protocol provides a valuable method for large-scale production of virus-free planting material, meeting growers’ demands in a cost-effective manner.