Haploid embryogenesis and molecular detection of somatic embryogenesis receptor-like kinase (TcSERK) genes in sliced ovary cultures of cocoa (Theobroma cacao L.)

Somatic embryos were induced from sliced unpollinated ovaries of cocoa. The influence of genotypes and cold pre-treatment were studied on the induction of callus and haploid embryogenesis. Among the five cocoa genotypes studied, CCRP 5 and CCRP 1 had recorded a maximum callus induction frequency of 66.00% and 62.00%, respectively, from sliced ovaries on WPM medium supplemented with 2-iP (1.0 mg L−1), Zeatin (1.0 mg L−1), and AgNO3 (5.0 mg L−1). Sliced ovaries isolated from cold pre-treated (4 °C for 1 day), 4–6 mm long (containing mature ovule) flower buds recorded the maximum callus induction frequency (67%). The highest percentage of embryogenic calli was noticed from ovaries of pre-treated (4 °C for 1 day) flower buds of CCRP 5 (31.00%). During proliferation and sub-culturing, callus morphogenesis such as white compact, light creamy nodular, proliferative beige shaded embryogenic, and light brown watery spongy non-proliferative calli were observed. Induction of globular and heart stage somatic embryos was noticed in WPM medium supplemented with ascorbic acid (35.2 mg L−1), Zeatin (1.0 mg L−1), Kinetin (3.0 mg L−1), and sucrose (30.0 g L−1). Further, the cotyledonary stage embryos and shoot conversion were observed in WPM medium supplemented with MgSO4 (4.0 g L−1), K2SO4 (12.0 g L−1), glucose (1.0 g L−1), and sucrose (30.0 g L−1). Histological and scanning electron microscopic studies revealed an asynchronous pattern of somatic embryos development (globular, heart, and torpedo stage) from embryogenic calli. The molecular confirmation of embryogenic competence with different types of ovary callus at different stages was confirmed with the detection of the TcSERK gene through semi-quantitative RT-PCR. The TcSERK gene expression was higher in embryogenic friable calli and lower in callus with early embryo induction. Flow cytometry analysis revealed that cells from ovary calli were haploids (1n = 10). This study would be a starting step for the induction of haploid embryogenesis from sliced ovaries of the well-adapted regional genotypes of cocoa, for obtaining rapid homozygosity; as induction of haploids through androgenesis in an earlier study could not yield fruitful results.