TITLE:
Agrobacterium-Mediated Transformation of Mexican Lime (Citrus aurantifolia Swingle) Using Optimized Systems for Epicotyls and Cotyledons
AUTHORS:
Maria Luiza P. de Oliveira, Gloria Moore, James G. Thomson, Ed Stover
KEYWORDS:
Agrobacterium tumefaciens, Embryo Cotyledons, Epicotyls Segments, Recombinase Mediated Exchange Cassette, Tissue Culture
JOURNAL NAME:
Advances in Bioscience and Biotechnology,
Vol.6 No.11,
November
16,
2015
ABSTRACT: Transgenic Mexican lime (Citrus aurantifolia Swingle) was produced through two explant sources,
each using systems previously optimized for each source. One used epicotyls segments, which was
the predominant explant for transgenic Citrus production following co-cultivation with Agrobacterium,
and has a well-established protocol. The other procedure used embryo cotyledons from mature
seeds, which was developed in our lab as an alternative for stable Citrus transformation. Cotyledon
transformation and regeneration protocols were optimized by comparing variables in
culture medium composition on shoot regeneration and four parameters in transient transformation.
The optimized protocols were compared, and frequency of regeneration, frequency of transgenic
plant-recovery and stable transformation efficiency indicated the superiority of the cotyledon
protocol for Agrobacterium-mediated genetic transformation in Mexican lime. The tissue
choice resulted in marked improvement in shoot regeneration (14.1% of explants producing
shoots in epicotyls; 55.8% in cotyledons), stable transformation frequency (11.4% of epicotyls explants;
40.2% in cotyledons), and frequency of transgenic plant-recovery (37.9% in epicotyl explants;
92.6% in cotyledons). Thus, easy availability of explants using embryo cotyledons from
mature seeds, technical simplicity, shortening of transformation time-course, and higher transformation
and regeneration frequencies makes this new system an attractive alternative over the
previously published Citrus transformation protocols. In the course of this project, we generated
Mexican lime with a Recombinase Mediated Exchange Cassette landing pad, which was designed
for stacking transgenes.