Zygotic embryos (ZE): Cacao pods were obtained by hand pollination of a genotype Scavina6 (Sca6) plants with pollen from a genotype West African Amelonado plant in the greenhouse at The Pennsylvania State University. Developing fruits were harvested during maturation from 14 weeks after pollination (WAP), which corresponded to the Torpedo stage (T-ZE), 16 WAP (Early-Full, EF-ZE), 18 WAP (Late-Full, LF-ZE) to 20 WAP (Mature, M-ZE), when pods are fully ripe. Zygotic embryos were extracted from each pod, cotyledon tissue was excised, frozen in liquid nitrogen and stored at -80°C for RNA extraction. Secondary SEs were generated in temporary immersion system bioreactors from flower parts of genotype Sca6 as previously described (Niemenak N et al. Plant Cell Rep 27:667–76:2008) with minor modifications as follows: glass beads (2 mm diameter) were used to support the embryos in the bioreactors. SEs at early torpedo developmental stage were selected and transferred to embryo development (ED) medium (20 embryos per bioreactor). Tissue at 2 different developmental stages was harvested after 2 weeks on ED medium: 1. Whole SEs at Late Torpedo (LT-SE) stage cultured on 30 g/L sucrose and 2. Cotyledon tissue from mature SEs (M-SE) cultured on 60 g/L sucrose. One gram of tissue per biological replicate was frozen in liquid nitrogen and stored at -80°C for RNA extraction.( Alemanno, L. et al. Planta 227, 853–66 :2008)
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from frozen samples that were ground in liquid nitrogen and the powder was suspended in extraction buffer containing 4 M guanidinium isothiocyanate, 0.24 M sodium acetate, 0.03M N-lauroyl sarcosine sodium salt, 22.5 mM PVP-40 (MW 40,000)and 14 mM β-mercaptoethanol. After centrifugation at 14,000 g for 30 min at 4°C, RNAs were chloroform extracted, precipitated with isopropanol, washed with 70% ethanol and then re-suspended in RNase-free sterile water. The RNA was then treated with DNase l (Invitrogen) and integrity was assessed using a RNA 6000 Nano assay bioanalyzer (Aglient), RNA with a RNA integrity number of 7 or above was used.
The R programming environment and Bioconductor software were used to perform the analysis. The LIMMA package was used to perform the background adjustment, normalization and summarization. The RMA procedure, which performs a convolution background correction, quantile normalization and summarization based on a multi-array model fit robustly using the median polish algorithm, was used to obtain the average Log2 expression values for each gene (average of all probes) ranging from 2 to 16. Background noise was calculated as the average intensity levels of the control probes and a value of 6 (Log2) was used as a cutoff value to eliminate any signals due to noise. Statistical significance was using a moderated t-statistic implemented in the ‘limma’ package in Bioconductor. It is based on an empirical Bayes approach. The moderated t-statistics and their corresponding p-values were computed for statistical analysis. In this study, this analysis was performed with a > 2-fold +/- change and p-value <0.01 as our regular cutoff in statistical analysis
Genome-Wide Analysis Reveals Divergent Patterns of Gene Expression During Zygotic and Somatic Embryo Maturation of Theobroma cacao L., The Chocolate Tree
