Molecular and biochemical studies of cassava (Manihot esculenta Crantz): seed coat phenolics, seed storage proteins and DNA finger printing for storage root color hydrocyanic acid (HCN) content and bacterial blight resistance

Proteins of fresh cassava (Manihot esculenta Crantz) seeds were found to be localized uniformly in the endosperm. Epifluorescence microscopy of the cassava seed coat revealed a compact structure of columnar cells which coherent with the seed coat's probable impermeability to water and gases. Hence, seed coat-imposed dormancy may be dominant type of dormancy in the cassava seed. Proximate analysis of the cassava testa revealed that almost 80% of its dry matter is still unaccounted for. The phenolics in cassava testa were found to be 2.28 ug/100 mg seed testa, wherein 0.49 ug of which are tannins. Moreover, the phenolics in the cassava testa accounts for 0.23% of its dry matter. The total hydrolysable tannins is 0.80 ug/100 mg seed testa, as determined by potassium iodate assay. The amount of protein-precipitable tannins was measured but none was detected. Seed storage proteins were fractionated and partially purified from cassava seeds. The mature seeds of cassava were found to contain 8.21% (w/w) total soluble proteins extracted by PBS. Storage proteins were fractionated into globulin, albumin, prolamin and glutelin using the modified Osborne procedure and low molecular weight peptides were present in all fractions. It was found that the globulin and glutelin fractions are the major storage proteins of the cassava seed. The globulin fraction was purified by gel filtration chromatography to separate the globulins from the low molecular weight peptides. The globulin isolate was found to be composed of 11 polypeptides with molecular weights 77, 64, 59, 54, 50, 38, 32, 25, 20, 19 and 17.5 kD. The peptide isolate had the smallest size, 11 kD. The glutelin fraction, on the other hand, had four polypeptides with molecular weights of 38, 33, 21 and 17.5 kD. PAS showed that all the glutelin polypeptides and seven of the eleven (77, 64, 50, 38, 32, 25, and 20 kD) globulin were glycosylated. However, the glutelin fraction, globulin isolate and low molecular weight peptides were all negative for anti-bacterial activity. Lunasin gene and prolamin gene primers were used for DNA finger printing of Philippine M. esculenta varieties. PCR amplicons ranging from 180 bp to 1.65 kb which exhibited band polymorphism among samples of different accessions. Phylogenetic analyses showed that the PROL 01/02 primer pair was able to group the accessions according to storage root color while the PROL 03/04 primers were able to differentiate the M. esculenta varieties according to resistance to cassava bacterial blight. On the other hand, the LUNA 01/02 primer was able to differentiate accessions according to root HCN content. Four DNA sequences were chosen for analyses and designated as Prol 800, Prol 700, Prol 425 and Luna 320. Blast search results of these DNA sequences showed the similarity of Prol 800 with Manihot esculenta Ty3/Gypsy-like retrotransposons and the Luna 320 with Manihot esculenta clone E19M42B1 AFLP marker genomic sequence. Genome browsing using Phytozone v6.0 showed that Prol 700 and Prol 425 were present in the annotated genome of M. esculenta.