A new liquid selection system for mature citrus transformation

Abstract

Gravity Wells are attachments for We-V liquid culture vessels designed to gradually dispense liquid medium into the vessels during in vitro plant propagation. The objective of this study was to compare Agrobacterium-mediated mature citrus rootstock selection in We-V vessels with Gravity Wells to the standard transformation protocol using semi-solid medium and to determine which kanamycin concentrations (0, 50, 100, 150, 200 mg/l) were best for liquid selection. Two mature citrus rootstocks were each transformed with a binary vector: Kuharske rootstock was transformed with pGNA, and US-942 rootstock was transformed with pGNS. The in vitro plant growth period was for a total of seven weeks, in which the explants were incubated in the dark on semi-solid medium for three weeks, followed by four weeks in the light in liquid or semi-solid medium. In total, 108 Kuharske transgenics and 40 US-942 transgenics were obtained over all treatments. The two cultivars were analyzed separately using ANOVAs. The response variables analyzed were the mean number of shoots longer than 2 mm (MSL>2), the mean transformation efficiency based on the number of explants (TEE), and the mean transformation efficiency based on the number of screened shoots (TES). For Kuharske and US-942, there were significant differences among kanamycin concentrations and between liquid vs semi-solid medium with liquid medium being superior to semi-solid medium because of an increased TES while also lowering MSL>2. The TEE variable was also significant for Kuharske, but it was not for US-942. US-942 formed fewer organogenic shoots in all other treatments. Selection at higher kanamycin concentrations (150 and 200 mg/l) yielded more transgenics and fewer shoots had to be screened using We-V vessels with gravity wells. PCR confirmed the presence of the transgenes, and Southern blots showed integration of the nptII transgene into the genome.