Protocol: transient expression system for functional genomics in the tropical tree Theobroma cacao L

Area of Science:

• Plant Biotechnology
• Molecular Biology
• Agricultural Science

Background:

• Theobroma cacao (cocoa) is economically significant.
• Gene function studies in cacao are crucial.
• A rapid Agrobacterium-mediated transient genetic transformation protocol was developed.

Purpose of the Study:

• To present a detailed methodology for cacao transformation.
• To develop an assay for pathogen inoculation of cacao leaves.
• To enable rapid gene function analysis in cacao.

Main Methods:

• Agrobacterium tumefaciens cultures vacuum-infiltrated into cacao leaves.
• Transformation success assessed via green fluorescent protein and qRT-PCR.
• Cacao leaf developmental stages characterized for optimal transformation.

Main Results:

• The protocol efficiently transforms developmental stage C leaves in four of eight genotypes.
• Overexpression of cacao chitinase decreased pathogen biomass and lesion size.
• Phytophthora tropicalis infection assays demonstrated functional gene analysis.

Conclusions:

• Transgene-expressing leaves facilitate downstream functional genetic assays.
• The protocol is rapid (1 day) and allows 5-7 days for analysis.
• This accelerates testing of candidate defense genes in cacao.