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

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.

Screening of Cultivars for Tissue Culture Response and Establishment of Genetic Transformation in a High-yielding and Disease-resistant Cultivar of Theobroma cacao

A highly efficient transformation protocol is a prerequisite to developing genetically modified and genome-edited crops. A tissue culture system spanning the initiation of floral material to the regeneration of plantlets into soil has been tested and improved in cacao. Fourteen cultivars were screened for their tissue culture response and transfer DNA (T-DNA) delivery efficiency via Agrobacterium. 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 immature somatic embryos ranging in size between 4-10 mm resulted in the highest T-DNA delivery efficiency. Furthermore, the use of higher concentrations of basal salts and cupric sulfate in secondary callus growth medium increased the percentage of explants producing greater than ten embryos by 504% and 443%, 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 wild-type seedlings with strong, healthy roots helped make plantlets survive and facilitated quick transplantation to the soil. The present methods can be applied to improve tissue culture response and transformation frequency in other cacao cultivars.Key messageTissue culture and genetic transformation methods for a high-yielding, disease-resistant cultivar of Theobroma cacao were established while factors affecting T-DNA delivery and somatic embryogenesis were identified.