This study evaluates resistance to witches' broom disease in flower cushions of Theobroma cacao under field conditions. The aim was to determine optimal inoculation methods to evaluate the disease incidence using flower cushions in the field. A segregating mapping population of 580 trees (cultivar TSH 1188 9 CCN 51) was analysed under two field conditions: high and low inoculum levels (in different years), corresponding respectively to trees with or without dried witches' brooms hanging on the trees and producing basidiocarps. The number of newly formed cushion brooms in each tree was counted by the conventional method, and also the healthy and infected flower cushions in three 30 cm-long regions along the trunk and the two main branches. The field inoculation methods discriminated between genotypes, with a 26% increase in disease incidence by Moniliophthora perniciosa at high inoculum. Two different segregation patterns were also observed: 27:27:9:1 under low, and 27:9:9:9:3:3:3:1 under high inoculum potential. It was also determined that at least 20 flower cushions were needed to accurately determine the percentage of infection. These methodologies allowed identification of the extreme phenotypes in this mapping population, and can therefore facilitate the detection of sources of resistance to witches' broom disease.
ABSTRACT. Theobroma cacao is a species of great economic importance with its beans used for chocolate production. The tree has been a target of various molecular studies. It contains many polyphenols, which complicate the extraction of nucleic acids with the extraction protocols requiring a large amount of plant material. These issues, therefore, necessitate the optimization of the protocols. The aim of the present study was to evaluate different methods for extraction of total RNA from shoot apical meristems of T. cacao 'CCN 51' and to assess the influence of storage conditions for the meristems on the extraction. The study also aimed to identify the most efficient protocol for RNA extraction using a small amount of plant material. Four different protocols were evaluated for RNA extraction using one shoot apical meristem per sample. Among these protocols, one that was more efficient was then tested to extract RNA using four different numbers of shoot apical meristems, subjected to three different storage conditions. The best protocol was tested for cDNA amplification using reverse transcription-polymerase chain reaction; the cDNA quality was determined to be satisfactory for molecular analyses. The study revealed that with the best RNA extraction protocol, one shoot apical meristem was sufficient for extraction of high-quality total RNA. The results obtained might enable advances in genetic analyses and molecular studies using reduced amount of plant material.
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