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.
Highlights
The genetic transformation of Citrus has been widely studied as a tool to generate transgenic plants with enhanced tolerance of biotic [1]-[4] and/or abiotic stresses [5]
The cotyledons were extracted from mature seeds and conditions for regeneration and Agrobacterium transformation were optimized
The cotyledons from seeds of Mexican lime were cultured on different media formulations containing different concentrations of BAP, Kin, naphthalene acetic acid (NAA), and Indole acetic acid (IAA) (Figure 2)
Summary
The genetic transformation of Citrus has been widely studied as a tool to generate transgenic plants with enhanced tolerance of biotic [1]-[4] and/or abiotic stresses [5]. These protocols have relied on somatic embryogenesis from nucellar calli [6] or from protoplast-derived cultures [7], or more commonly, shoot organogenesis from epicotyl or internodal stem segments [8]-[11]. The resources required to maintain a continuous supply of explants at the correct developmental stage for transformation to be substantial
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