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Capsicum chinense is recalcitrant in in vitro morphogenesis. No efficient, reproducible somatic embryogenesis regeneration system exists for this species, impeding regeneration from transformed cells. An indirect somatic embryogenesis protocol is developed using mature C. chinense zygotic embryo segments (ZES). The ZES cultured in semi-solid Murashige-Skoog (MS) medium supplemented with 8.9 µM naphthaleneacetic acid, 11.4 µM indoleacetic acid and 8.9 µM 6-benzylaminopurine, developed an embryogenic callus and 8% of the calli developed somatic embryos. Torpedo-stage somatic embryos were detached from the callus and subcultured in semi-solid MS medium without growth regulators, producing a 75% conversion rate to plantlets with well-formed root tissue. Histological analysis showed the developed structures to have no vascular connection with the callus and to be bipolar, confirming that this protocol induced formation of viable somatic embryos from mature C. chinense ZES. All acclimated plantlets survived under greenhouse conditions. This protocol will facilitate regeneration of genetically transformed plants using either biolistics or Agrobacterium tumefaciens approach.

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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.

In our coconut laboratory micropropagation has been the subject of research for nearly three decades, as this plant species is highly recalcitrant for in vitro regeneration and so far only achieved through somatic embryogenesis as the sole path for coconut regeneration. Of all the explants tested, plumules have proved to be the most responsive and the process efficiency has been improved by indirect embryogenesis and thereafter secondary embryogenesis and callus multiplication, this strategy is currently applied in floral explants. Two different approaches have been used to find ways to have a more efficient protocol. The first one, a direct and practical method, included plant hormones and activated charcoal. On the other hand, the indirect approach consisted in basic studies on: morphohistological development, biochemical and physiological aspects such as uptake of exogenous auxin, levels of endogenous auxin; shoot apical meristem formation and maintenance (KNOX gene family); the occurrence and expression of genes related to the cell cycle control (Cyclin-Dependent Kinase), and somatic embryogenesis (Somatic Embryogenesis-Related Kinase); and the establishment of a transformation protocol. A better understanding of the somatic embryogenesis of coconut was achieved by these approaches. This way, in the short term there is no doubt that we will have mass propagation options based not only in plumule explants but also on rachillae, unfertilized ovary, and leaf explants.

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