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.

高效的转化方案是开发转基因和基因组编辑作物的先决条件。已在可可可可中测试并改进了一种组织培养系统，该系统从花材料的培养开始到胚胎转化为植物。筛选了九个栽培品种的组织培养反应和通过瞬时表达测量的对农杆菌转移 DNA 递送的敏感性。这些关键因素用于确定各种栽培品种的遗传转化能力。选择高产抗病品种INIAPG-038进行稳定转化，进一步优化方法。使用含有黄色荧光蛋白和黄色荧光蛋白的两种载体产生了多个转基因事件新霉素磷酸转移酶II 基因。进行了提高 T-DNA 递送和次级体细胞胚胎发生率的双重策略，以提高整体转化频率。使用土壤杆菌菌株 AGL1 和来自次生体细胞胚胎的子叶组织，大小在 4 到 10 毫米之间，导致最高的 T-DNA 传递效率。此外，在培养基中使用更高浓度的基础盐和硫酸铜使次生体细胞胚胎发生过程中产生超过 10 个胚胎的外植体的频率分别增加了 5 倍和 4 倍。因此，所有这些成分的最佳组合导致 INIAPG-038 在 T ₀处以 3.7% 的频率成功转化厂级。将具有未发育根的转基因接穗嫁接到具有健康根的非转基因幼苗有助于使小植株存活并促进快速移植到土壤中。所提出的策略可用于提高其他可可可可栽培品种的组织培养反应和转化频率。