Protocol: Optimised methodology for isolation of nuclei from leaves of species in the Solanaceae and Rosaceae families.

In this study, a protocol is described for rapid preparation of an enriched, reasonably pure fraction of nuclear proteins from the leaves of tobacco (Nicotiana tabacum), potato (Solanum tuberosum) and apple (Malus domestica). The protocol gives reproducible results and can be carried out quickly in 2 hours. Tissue extracts clarified with filtration were treated with non-ionic detergent (Triton X-100) to lyse membranes of contaminating organelles. Nuclei were collected from a 60% Percoll layer of density gradient following low-speed centrifugation. Western blot analysis using antibodies to marker proteins of organelles indicated that the nuclear protein fractions were highly enriched and free or nearly free of proteins from the endoplasmic reticulum and chloroplasts.

Biolistic DNA delivery to leaf tissue of plants with the non-vacuum gene gun (HandyGun).

Non-vacuum gene guns such as HandyGun are flexible tools for bombardment of targets of varying size. Construction of HandyGun is simpler and cheaper than vacuum gene guns and will be described here. The conditions for maximal transient transformation efficiency of plant cells with plasmid DNA using HandyGun will be provided.

HandyGun: An improved custom-designed, non-vacuum gene gun suitable for virus inoculation.

Particle bombardment with a non-vacuum gene gun is an efficient method for transfection of plant cells with cloned viruses and initiation of virus infection. The HandyGun developed in this study is an improved version of a non-vacuum gene gun. Bombardment parameters were studied by inoculating an infectious, 35S promoter-driven cDNA of Potato virus A (PVA; Potyvirus) to the potato clone 'A6', Nicotiana benthamiana and N. tabacum as plasmid DNA coated on microprojectiles (gold particles). The large number of initial infection sites (necrotic local lesions) observed on inoculated 'A6' leaves and the high percentage of Nicotiana plants which were infected systemically with PVA following inoculation with HandyGun were not particularly sensitive to variation in the parameters tested (helium pressure and the amounts of plasmid DNA and gold particles). Data showed that HandyGun is a robust and reliable tool for obtaining high infection rates in plants reproducibly. It is easy and inexpensive to use and can be constructed from parts commonly available.