Enabling biocontained plant virus transmission studies through establishment of axenic whitefly (Bemisia tabaci) colony on plant tissue culture

Abstract

A colony of axenic ‘sweet potato’ whiteflies (i.e., free from culturable microorganisms) was established on tissue-cultured plants and was used to demonstrate in vitro begomovirus transmission. A co-culture system based on non-viral host cabbage (Brassica oleracea) provides for efficient maintenance of a whitefly (Bemisia tabaci) colony. The recovery of emerging whiteflies through coupled tissue culture vessels to new axenic plants allows robust subculturing and facilitates the production of hundreds of whitefly adults per month in a 1.1 L tissue culture vessel. This novel ‘communal axenic’ plant-insect-virus (aP-I-V) protocol provides high-level biocontainment to allow for work with genetically modified viruses. Versatility is illustrated by achieving whitefly proliferation on more than a dozen plant species as well as methods for in vitro testing of whitefly preference for different plants – although there were large differences in whitefly accumulation and ease of host use. Although initial colony establishment was achieved with surface sterilization of detached whitefly eggs on vegetatively propagated sweet potato (Ipomoea batatas), a much more reliable approach is described based on non-axenic whitefly egg-lay onto axenic phototrophically tissue-cultured plants, followed by surface sterilization of the pedicel-attached eggs. Axenic whitefly adults were able to acquire and transmit tomato mottle virus (ToMoV) (Genus: begomovirus) from Nicotiana benthamiana tissue cultured material. This indicated the absence of a role for culturable gut microorganisms in the transmission of this begomovirus. Acquisition occurred from tissue-cultured plants initially inoculated with ToMoV via Agrobacterium infective clones as well as from a gamma irradiated sachet of parafilm. An autoclavable membrane device was developed to facilitate a simpler approach to undertake virus transmission in biocontained, axenic plant tissue culture. The methodology described here could be used to address a wide range of questions regarding whitefly feeding on plants as well as their transmission of viruses without the expense, facilities, and contamination risks associated with standard approaches.