Location: Tropical Crops and Germplasm Research
Project Number: 6090-21000-061-002-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Jun 1, 2023
End Date: Oct 1, 2025
Objective:
The USDA-ARS cacao (Theobroma cacao) germplasm collection is maintained at the Tropical Agriculture Research Station (TARS), Mayaguez, PR. It includes includes strategically representative accessions belonging to the 10 genetic groups of cacao. During 2019-2020, symptoms reminiscent of Cacao Mild Mosaic Virus (CaMMV) were observed in the collection. Unexpected introductions of CaMMV and other viruses that are asymptomatic or cause mild symptoms in cacao may represent an emerging risk to cacao cultivation in the tropical Americas. Viruses are easily transmitted by vegetative propagation or by insect vectors such as mealybugs and aphids. Maintaining disease-free germplasm is one of the main goals of clonal germplasm repositories in the National Plant Germplasm System (NPGS). Eliminating viruses using thermotherapy, chemotherapy, cryotherapy, and meristem culture, are difficult requiring different sanitation efficiencies depending on the virus and plant genotype. Further, these methods can be ineffective for producing virus-free plants because viruses may not be detectable until after mature plants are established.
Somatic embryogenesis (SE) through cyclic somatic embryogenesis is one of the best methods for micropropagation of plants and for virus elimination from infected plant materials. Virus elimination using SE approaches occurs because of the absence of a connection between somatic embryos and the mother plant. Most viruses, including badnaviruses, are restricted to vascular tissues, while somatic embryos arise from non-vascular tissues, thereby increasing the likelihood that virus will be eliminated.
Upon the discovery of CaMMV trees in the USDA cacao germplasm collection, personnel at the USDA-TARS in Mayaguez, PR began deployment of SE to clean virus-infected accessions in the collection. Preliminary results have proven promising in that SE-derived plants produced from flowers (staminoids and petals) were virus-negative, despite evidence that the source tree tested positive for CaMMV infection using PCR amplification with the MPv2 primers.
The objectives of this research are 1) Screen SE-produced cacao plants for CaMMV presence and assess the effectiveness of SE to yield CaMMV-free, asymptomatic cacao plants; 2)Deploy the newly-developed Recombinase polymerase amplification assay for CaMMV detection and carry out PCR amplification with MPv2 primers.
Approach:
Immature flowers from virus-infected mother trees in the cacao germplasm collection in Mayaguez will be collected and transported on ice to the tissue culture laboratory. The flowers will be first sterilized then staminodes and petal bases will be dissected and cultured on tissue culture media for induction of primary somatic embryos. Primary somatic embryogenesis will be followed by secondary somatic embryogenesis using the methods of Maximova et al (2005), modified by Garcia et al (2018). Tissue from SE-derived plantlets will be shipped (APHIS permit) to the U-Arizona Brown lab for virus detection using newly designed RPA primers (39°C), and PCR amplification of representative positive and negative samples.
