Development of an Efficient Protocol to Obtain Transgenic Coffee, Coffea arabica L., Expressing the Cry10Aa Toxin of Bacillus thuringiensis

Abstract

This report presents an efficient protocol of the stable genetic transformation of coffee plants expressing the Cry10Aa protein of Bacillus thuringiensis. Embryogenic cell lines with a high potential of propagation, somatic embryo maturation, and germination were used. Gene expression analysis of cytokinin signaling, homedomains, auxin responsive factor, and the master regulators of somatic embryogenesis genes involved in somatic embryo maturation were evaluated. Plasmid pMDC85 containing the cry10Aa gene was introduced into a Typica cultivar of C. arabica L. by biobalistic transformation. Transformation efficiency of 16.7% was achieved, according to the number of embryogenic aggregates and transgenic lines developed. Stable transformation was proven by hygromycin-resistant embryogenic lines, green fluorescent protein (GFP) expression, quantitative analyses of Cry10Aa by mass spectrometry, Western blot, ELISA, and Southern blot analyses. Cry10Aa showed variable expression levels in somatic embryos and the leaf tissue of transgenic plants, ranging from 76% to 90% of coverage of the protein by mass spectrometry and from 3.25 to 13.88 μg/g fresh tissue, with ELISA. qPCR-based 2−ΔΔCt trials revealed high transcription levels of cry10Aa in somatic embryos and leaf tissue. This is the first report about the stable transformation and expression of the Cry10Aa protein in coffee plants with the potential for controlling the coffee berry borer.

Osmotic stress-induced somatic embryo maturation of coffee Coffea arabica L., shoot and root apical meristems development and robustness

“…Typica with a high potential of propagation, plantlet conversion and genetic transformation. Three months were required to induce the development of SEs to be ready for stable genetic transformation 8 . Briefly, this protocol was followed: leaf explants of coffee cultured in callus induction medium (CIM medium, Van Boxtel et al 14 ) provided proembryogenic masses (PEM) obtained after one month with 93% efficiency of cultivated explants.…”

“…SE induced from leaf explants has been the most widely used target tissue in coffee micropropagation and stable genetic transformation 1 . Published protocols have shown that SE induction and proliferation is time consuming, ranging from 9 to 20 months until they can be used for stable genetic transformation 8 , 20 , 21 . In most of these reports, SE regeneration to plants in C. arabica has been shown to be more difficult and time consuming than in C. canephora P .…”

“…SEs at early torpedo stage, symmetric and having a suspensor-like structure were separated, and individually subcultured to the corresponding fresh medium (SE-M3 or SE-M9), 25 SEs in each plate, for one month until cotyledonary stage developed according to Valencia-Lozano et al . 8 .…”

“…Regenerated plantlets were subcultured in flasks with SE-G medium until plants reached 10 leaves and 27 cm root length. Regenerated plants were potted in vermiculite and soil (1:1 v/v), incubated in a plant growth chamber at 50 µmol/m 2 -s irradiance provided by T8 Phillips P32T8/TL850 fluorescent lamps with a photoperiod of 16/8 h and temperature of 19 to 23ºC, as described by Valencia-Lozano et al 8 . Efficiency of SEs maturation was calculated according to the number of SEs converted to plantlets derived from SEs at cotyledonary stage produced at the end of the second stage of maturation.…”

Plant Cell and Organism Development

“…The article by Valencia-Lozano [11] is another paper on coffee (Coffea arabica). It demonstrates an efficient protocol that enables the particle bombardment-mediated stable genetic transformation of coffee plants that express the Cry10Aa protein of Bacillus thuringiensis (Bt) for the first time.…”