Screening of cultivars for tissue culture response and establishment of genetic transformation in a high-yielding and disease-resistant cultivar of Theobroma cacao

Abstract

A highly efficient transformation protocol is a prerequisite to developing genetically modified and genome-edited crops. A tissue culture system spanning culture initiation from floral material to conversion of embryos to plants has been tested and improved in Theobroma cacao. Nine cultivars were screened for their tissue culture response and susceptibility to Agrobacterium transfer-DNA delivery as measured through transient expression. These key factors were used to determine the genetic transformability of various cultivars. The high-yielding, disease-resistant cultivar INIAPG-038 was selected for stable transformation and the method was further optimized. Multiple transgenic events were produced using two vectors containing both yellow fluorescent protein and neomycin phosphotransferase II genes. A two-fold strategy to improve both T-DNA delivery and secondary somatic embryogenesis rates was conducted to improve overall transformation frequency. The use of Agrobacterium strain AGL1 and cotyledon tissue derived from secondary somatic embryos ranging in size between 4 to 10 mm resulted in the highest T-DNA delivery efficiency. Furthermore, the use of higher concentrations of basal salts and cupric sulfate in the medium increased the frequency of explants producing greater than ten embryos by five-fold and four-fold during secondary somatic embryogenesis, respectively. Consequently, an optimal combination of all these components resulted in a successful transformation of INIAPG-038 with 3.7% frequency at the T0 plant-level. Grafting transgenic scions with undeveloped roots to non-transgenic seedlings with healthy roots helped make plantlets survive and facilitated quick transplantation to the soil. The presented strategy can be applied to improve tissue culture response and transformation frequency in other Theobroma cacao cultivars.

Geographic distribution, conservation, and genomic resources of cacao Theobroma cacao L

“…Elite cacao clones can directly be improved by CRISPR‐based gene editing, but it requires high quality reference genomes for optimizing specific gRNA constructs and to avoid off‐target products (Morrissey et al., 2019). In addition, cacao tissue culture needs to be optimized, although protocols for somatic embryogenesis using staminode explants were adapted in nine cacao cultivars (Jones et al., 2022). In addition, further gene editing studies can validate candidate gene functions (e.g., loss of the function mutations) in important biological processes of cacao, such as disease resistance, and SC and Cd uptake.…”

Improvement of Gene Delivery and Mutation Efficiency in the CRISPR-Cas9 Wheat (Triticum aestivum L.) Genomics System via Biolistics

“…The hpt, dsRED and Cas9 transgenes were confirmed by PCR using sequence specific primers ( Table S1 ). Amplifications were performed in a 25-μL reaction with DreamTaq PCR Master Mix (2X) (Thermo Fisher Scientific, Grand Island, NY, USA) as described [ 35 ]. For each PCR reaction, 23 μL were loaded onto a 0.8% agarose gel for electrophoresis.…”

Regeneration of cocoa (Theobroma cacao L.) via somatic embryogenesis: Key aspects in the in vitro conversion stage and in the ex vitro adaptation of plantlets.

“…For plant survival, the most important changes include the development of cuticles, epicuticular waxes, and effective stomatal regulation of transpiration leading to stabilization of water status 17 . From Aguilar et al (1992) and Figueira & Janick, (1995) 1 , to Jones et al (2022) 20 and Manlé et al (2021) 1 , the acclimation of cocoa vitro-plants is a difficult and crucial stage as it happens in most plantlets produced by in vitro tissue cultures 21 . Therefore, knowing the factors that affect the survival of vitroplantlets is essential when implementing the production of plant material on a large scale, whose main contribution is the development of an optimal propagation protocol.…”