One Shade Too Dark

--- tale of condensed tannins in faba bean seed coats　

      The project was separated into two parts: a genotypic analysis for determining the genetic similarities and differences; and a seed coat chemical analysis for determining which line has condensed tannins in its seed coat and which condensed tannin precursor(s) exist in the seed coat.

      In the genotypic analysis, the supposed Earlibird and SSNS-1 seeds were mixed up with other seeds, they were renamed as Unknown 1 and Unknown 2 in this experiment (as show in Figure 2).

      The step-wise procedures for both experiments are:

Genotypic analysis:

1. Six plants of each line were grown for 2 weeks in pots containing soiless mix.

2. Leaf tissue was harvested and freeze-dried overnight.

3. Leaf tissue was ground into a fine powder using a paint shaker and glass beads.

4. DNA was extracted and purified using the cTAB method (reference 5).

5. DNA was quantified using a spectrophotometer.

6. DNA was amplified using PCR method with RAPD primers.

7. DNA bands were separated on agarose gels using electrophoresis and visualized using ethidium bromide and a UV transilluminator. Gel images were captured and saved for analysis.

8. The polymorphic bands were scored from the PCRs and the genetic similarity was analyzed using NTSYSpc software.

Seed coat chemical analysis:

1. Seed coats from selected faba bean lines were ground in liquid nitrogen.

2. The condensed tannins were extracted with 70% acetone, and the monomers of condensed tannins were then extracted with ethyl acetate while the polymers of condensed tannins remained in the aqueous fraction (Figure 4, reference 6).  

3. Ten microliters of extracts from both aqueous and ethyl acetate fractions were spotted onto a cellulose coated TLC plate, and the TLC plate was developed using sBAWC (s-butanol: H₂O: acetic acid: chloroform 70:20:10:10 v/v/v/v) (reference 7).

4. Dried TLC plate was sprayed with DMACA reagent (2% dimethylaminocinnamaldehyde, in 3M HCl/50% methanol) diluted 20-fold with methanol and analyzed for the presence of condensed tannins.