Inter-batch variability of hemagglutinin content transiently expressed in Nicotiana benthamiana plants grown under sole-source lighting and sunlight conditions before gene transfer

I investigated the effects of seasonal fluctuations in light availability for plants before gene transfer on the inter‑batch variation of target recombinant protein productivity in a viral vector‑based transient gene expression system. Nicotiana benthamiana plants were grown five times in different seasons, either in a growth chamber ( GC ) under sole‑source ( solely electric ) lighting or in a temperature‑controlled greenhouse ( GH ) under sunlight until agroinfiltration for gene transfer. The plants were then further grown in a shared growth chamber to allow accumulation of hemagglutinin ( HA ) until harvest. The coefficient of variation of leaf HA content per plant among GH batches was more than twice that among GC batches. The greater variation of leaf HA content per plant in GH was due to the higher coefficient of variation ( CV ) of leaf biomass and the slightly higher CV of leaf HA content per biomass, the former being primarily due to the variation in cumulative photosynthetic photon flux density before gene transfer. There was a significant linear regression between leaf HA content per biomass at harvest and leaf biomass at the timing of gene transfer across growth conditions ( GC or GH ) and seasons. This regression indicates that the variation of leaf biomass or any related variables immediately before gene transfer can account for a significant part of the observed variation of leaf HA accumulation per plant at harvest. Thus, strictly regulated plant growth conditions before gene transfer are crucial to reducing the inter‑batch variation of HA productivity. I conclude that indoor facilities with sole‑source lighting are more appropriate than greenhouses, not only for plant cultivation after gene transfer but also before gene transfer.