Transient genetic transformation of embryogenic callus of Cocos nucifera

Abstract

Coconut palm (Cocos nucifera) is a plant species recalcitrant to in vitro morphogenesis and no protocols for the genetic transformation of coconut tissues have been published. The present study aimed to develop a protocol for genetic transformation of this palm species; evaluating reporter genes, transformation methods, and conditions for the use of antibiotics to select transformed plant cells. The gene gusA was first used for Agrobacterium tumefaciens mediated transformation of coconut embryogenic calli. However, endogenous GUS-like activity was found in calli not co-cultured with bacteria. Then essays for Agrobacterium-mediated transformation were developed using green and red fluorescent genes. Both genes are suitable as reporter genes for coconut transformation. In order to establish a protocol for coconut genetic transformation, an approach was used that combined biobalistics to generate micro-wounds in explants, vacuum infiltration and co-culture with Agrobacterium tumefaciens (C58C1 + pER10W-35SRed containing the embryogenesis related gene WUSCHEL). Calli treated with the combined protocol showed red fluorescence with greater intensity and greater area than calli treated with either biobalistics or infiltration, followed by bacteria co-culture. PCR amplification of DNA extracts from transformed embryogenic callus produced a band with the expected size using WUSCHEL primers (862 bp). No band was obtained using the VirE2 primers. This is the first report of transient genetic transformation of C. nucifera and it is the first step toward a protocol that will be useful for the study of the role of genes of interest and for practical applications, such as the improvement of coconut micropropagation via somatic embryogenesis.

Tissue culture and associated biotechnological interventions for the improvement of coconut (Cocos nucifera L.): a review

“…The constitutively expressed promoters Act1 and Ubi were found to produce the strongest transient expression, suggesting that these promoters could be used in future work. More recently, Andrade-Torres et al (2011) have reported the Agrobacterium-mediated transformation of a number of coconut explant tissues such as immature anthers, excised zygotic embryos, plumule-derived embryogenic calli, and somatic embryogenesis-derived roots and leaves. They tested a number of reporter genes and evaluated the techniques used in antibiotic selections of transformants.…”

“…Furthermore, it is now possible to cryopreserve, and then recover coconut embryos for in long-term conservation programs, without inducing morphological, cytological or molecular changes in the regenerated plants (Sisunandar et al 2010a). Although genetic transformation in coconut has been attempted Andrade-Torres et al 2011), achievements have been quite limited to date.…”

Somatic Embryogenesis in Cocos nucifera L.

“…PCR amplification of the extracted DNA from the transformed embryogenic callus, using WUSCHEL primers, showed a band with the expected size (862 bp). Oppositely, no bands were observed when using VirE2 primers (Andrade-Torres et al 2011). …”

“…The essays for Agrobacterium-mediated transformation were developed using green and red fluorescent protein genes, which are both suitable as reporter genes in coconut transformation. The established protocol for coconut genetic transformation combined biobalistics that generates micro-wounds on the explants followed by vacuum infiltration and coculture with Agrobacterium tumefaciens (C58C1 + pER10W-35SRed containing the embryogenesis-related gene WUSCHEL) (Andrade-Torres et al 2011). Calli treated with the combined protocol showed red fluorescence with greater intensity and greater area than calli treated with either biobalistics or infiltration, followed by bacteria coculture.…”

Potential of Omics to Control Diseases and Pests in the Coconut Tree

“…Some bottlenecks have been solved, such as the establishment of tissue culture procedures [60], but it is necessary to improve the transformation of the coconut crop. Currently, there is a single report on the transformation of embryogenic calli by Agrobacterium tumefaciens in combination with biobalistics, but transformation was transient [60]. It is necessary to achieve stable transformation to be capable of targeting genes in this crop.…”