Comparison of shoot tip culture and cryotherapy for eradication of ampeloviruses associated withPineapple mealybug wiltin wild varieties

ThePineapple mealybug wilt-associated virus(PMWaV) provokes a disease that causes considerable losses to pineapple growers due to the reduced leaf tissue turgescence, resulting in leaf necrosis and death in severe cases. In this work, we describe the comparison of shoot tip culture and cryotherapy methods to eradicate the pineapple wilt disease-associated ampeloviruses. Plants from the accessionsAnanas comosusvar.comosus(AGB-009), var.bracteatus(AGB-119), and var.parguazensis(AGB-376) were indexed by RT-PCR, confirming mixed infections of PMWaV-1, PMWaV-2, and PMWaV-3. The accessions were culturedin vitroand their shoot tips treated by cryotherapy following a droplet vitrification protocol. The regeneration rate from shoot tip culture was 93% for AGB-376 and 100% for the other two accessions. After freezing, AGB-376 had 100% regeneration subsequent to exposure to PVS2 for 45 min, followed by 95% for AGB-009, while for AGB-119 the optimal exposure time was 60 min, with plant regeneration from nearly 80% of the shoot tips. For the accessions AGB-009 and AGB-376, all the recovered plants were virus free by the two methods, while 50% of the plants from accession AGB-119 remained infected. These results indicate that shoot tip culture alone or in association with cryotherapy is a promising routine method for virus removal from pineapple plant tissues and is useful to ensure that backup reserves of pineapple germplasm, conserved byin vitrobank and cryobank, are formed with virus-free plants.