<i>Agrobacterium</i>-Mediated Transformation of Mexican Lime (<i>Citrus aurantifolia</i> Swingle) Using Optimized Systems for Epicotyls and Cotyledons

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.

Citrus Genetic Transformation: An Overview of the Current Strategies and Insights on the New Emerging Technologies

“…Another natural hybrid used as rootstock is rough lemon, which was genetically transformed by Savita et al (2011) and Ali et al (2012) . Other crops, such as Mexican lime ( Peña et al, 1997 ; Pérez-Molphe-Balch and Ochoa-Alejo, 1998 ; Domínguez et al, 2000 ; De Oliveira et al, 2015 ), Femminello siracusano lemon ( C. limon (L.) Burm. F) ( Gentile et al, 2007 ) and grapefruit ( Yang et al, 2000 ; Costa et al, 2002 ; Jia et al, 2017 ) have been efficiently transformed and regenerated.…”

“…A variety of explants, such as internodal stem segments, epicotyls, cotyledons, leaf segments, protoplasts, and embryogenic calli from different citrus species and relatives, have been assessed for Agrobacterium -mediated transformation ( Moore et al, 1992 ; Almeida et al, 2003a ; Kayim et al, 2004 ; Khawale et al, 2006 ; Omar et al, 2007 ; Chen et al, 2008 ; De Oliveira et al, 2009 , 2015 ; Ballester et al, 2010 ; Duan et al, 2010 ; Fu et al, 2011a , b ; Ali et al, 2012 ). All of them have their intrinsic advantages and drawbacks.…”

“…Lately, Costa et al (2002) and Febres et al (2003) , efficiently transformed C. paradisi cv. Duncan and De Oliveira et al (2015) also established a similar protocol using 30-day-old epicotyls for the transformation of Mexican lime.…”