Efficient Biolistic Transformation of Immature Citrus Rootstocks Using Phosphomannose-isomerase Selection

Abstract

This research utilized the E. coli manA gene encoding phosphomannose isomerase (PMI) selection on sucrose/mannose medium to increase transformation efficiencies after biolistic transformation of two immature citrus rootstock cultivars. Plasmid DNA, containing the manA gene and the enhanced green fluorescent protein (egfp) reporter gene, was bombarded into epicotyl explants of immature Carrizo citrange and Swingle citrumelo. GFP positive shoots were micro-grafted onto in vitro grown immature Carrizo rootstocks. Nineteen transgenic Carrizo shoots were obtained from ten paired shots, and eight Swingle shoots from five paired shots. The mean transformation efficiency of Carrizo was 1.9 transgenics/paired shot while the transformation efficiency of Swingle was comparable at 1.6 transgenics/paired shot. The transformants were analyzed by PCR for the presence of transgenes. Southern blot analysis of eight representative Carrizo transgenic events and four Swingle transgenic events showed that all transgenics had one to three copies of the manA gene. The PMI enzyme activity in the transgenic lines was confirmed using the chlorophenol red assay.

Development of genetically modified citrus plants for the control of citrus canker and huanglongbing

“…Particle bombardment is a relatively new direct DNA integration technique for citrus (Wu et al 2019(Wu et al , 2016. In this technique, gold or tungsten particles are coated with naked DNA molecules and delivered into the cell by a high-pressure burst of helium gas (Klein et al 1992).…”

Recent Advances of In Vitro Culture for the Application of New Breeding Techniques in Citrus

“…In addition, biolistic methods, recently applied for the transformation of epicotyl explants of Carrizo citrange with low TE (0.3-1.9 transgenic shoots per paired shot), can be optimized and become a valid alternative to Agrobacterium-mediated transformation [64,65].…”

“…It was first used by Boscariol et al [23] in the transformation of sweet oranges, with TEs of 3-23% depending on the cultivar (Valencia 23.8%, Natal 12%, Pera 7.6%, and Hamlin 3%), and Ballestrer et al [79] concluded that it was an excellent candidate for citrus transformation, yielding TEs of 30% for citrange epicotyls and 13% for sweet orange mature internodes. Recently, PMI selection has been applied for the biolistic transformation of Carrizo citrange to increase the TE obtained with kanamycin selection, 0.7%, to 1.9% transgenic shoots per shot, avoiding the introduction of antibiotic resistance in plants [64,65].…”

Citrus Taste Modification Potentials by Genetic Engineering

“…In addition, agrobacterium-mediated genetic transformation is a well-developed approach for direct introduction of gene of interest into citrus genome, and some advances have been made in using genetic element of plant origin as driving promoter or selection marker to avoid foreign gene introduction [72]. Other transformation methods such as poly ethylene glycol mediated (PEG-mediated) plasmid DNA uptake by protoplasts and biolistic DNA delivery also made success in citrus genetic modification [73,74]. Generally, antibiotic or herbicide resistance is used as selection marker for transgenic plantlets production.…”