Ploidy Analysis Report

Oregon State University Seed Laboratory

18 Hoodia Samples Ploidy Results

Nov. 13, 2014

Objective: Determine differences in ploidy levels between several genotypes of Hoodia gordonii.

Materials and Methods: 1 cm² tissue segments were excised from 18 Hoodia gordonii plant samples and placed in 0.5 ml CyStain PI Absolute nuclei extraction buffer (Partec 05-5022) in a Petri dish. Using a fine razor, samples were thoroughly chopped and set aside to soak for 5 minutes. The sample/buffer solutions were then poured through 30 µm pore filters and 1.5 ml of CyStain UV Precise P staining buffer (Partec 05-5002) was added.  Samples were then analysed using a flow cytometer with a gain of 400 and speed of 0.5 µL/s.

Results: All but 3 samples had a Gap 1 (G1) peak mean within the range of 80-95 (relative size) (Table 1).  The other 3 samples (HGNC-10-10-08-02(11), HG-E5-HGNC-C-20, HG-E2C81) had a G1 peak mean in the range of 39-42 (relative size), approximately half that of the other 18 samples, suggesting they contain half the number of chromosomes.

The results indicated no close correlation between phenotype, e.g., typical of the species and unusually long shoots) and ploidy levels. For example, samples 1 and 2 labelled as “typical of the species”, but they have different ploidy levels (Table 1).

Table 1. Peak means of Hoodia gordonii samples measured by the flow cytometry.