One Shade Too Dark

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

Genotypic analysis:

     The genomic DNA was successfully extracted from 16 lines of faba bean plants. PCRs with 19 primers showed different DNA banding patterns between different lines (Figure 5). Analysis of the gel images identified 72 polymorphic bands across the PCRs with 19 primers. The genetic similarity between the 16 faba bean lines was then analyzed using NTSYSpc software to generate the dendrogram (Figure 6). Unknown 1 was identified as Snowbird (more than 96% similarity) and Unknown 2 was most closely related to UKKO (72% similarity). The two lines with small black seeds, IPK 251/78 and IPK 296/78, were the most similar and grouped together, while the two lines with medium brown seeds, Puebla and Icarus, were also grouped together. Overall, this experiment shows that DNA fingerprinting is a powerful technique in identifying the genetic similarities and differences between different plant lines and individual plants.

Seed coat chemical analysis:

     Staining the seeds with DMACA reagent (used to signify the presence of condensed tannin and its precursors) showed that, out of the 16 lines tested, only FB6F3-3-1 and Snowbird had no condensed tannins in their seed coat (Figure 7). The composition of condensed tannins in the seed coat were further analyzed by TLC (thin layer chromatography) method (Figure 8) (reference 6 and 7). Analysis of the aqueous fraction (where condensed tannin polymers usually stay with the extraction method) and ethyl acetate fraction (where condensed tannin monomers or dimers  usually are with the extraction method) of seed coat extracts from selected lines indicated that catechin and epicatechin, monomers from both branches of the pathway existed in faba beans, but epicatechin was the more abundant monomer compared to catechin.

     The results I obtained support two of my hypothesis: 1. Genetic differences exist in the faba bean lines with different seed size and seed coat color, and DNA fingerprinting can be used to determine the genetic similarities and differences between these line. 2. The black seeded lines (IPK 296/78 and IPK 251/78) are most genetically similar. But the chemical analysis of the condensed tannin composition didn't support the third hypothesis--the black seeded lines have more condensed tannins than the other lines. The amount of catechin or epicatechin extracted from the two black seed lines looks less, not more, than the other lines that have condensed tannins in their seed coat (Icarus and UKKO in Figure 8). This could be because some of the condensed tannins are non-extractable from the mature seeds with the method used in this project since the remains of the seed coat of IPK 251/78 and IPK 296/78 after the 70% acetone extraction were still black, which was their original seed coat color (Figure 9). This phenomenon was observed in Brassica napus seeds before (reference 8).