Developing a method for the enhancement of the Beta Carotene content of Sweet Potato (cv BRONDAL) using Protoplast Technology

Abstract

Agronomically superior sweet potato (Ipomoea batatas) variety Brondal contains relatively low amounts of vitamin A as opposed to low yielding variety Nemagold, which is relatively rich in high amounts of the nutrient. The study focused on attempting to use protoplast technology, a tissue culture technique, to develop somatic hybrids with combined traits, such as high yield per unit area and high vitamin A content, from both varieties. For protoplast isolation, 10 enzymatic treatments were used, which were combinations of the enzyme cellulase with either macerozyme or hemicellulase. There were significant difference between treatments (P < 0.05) for protoplast yield and viability. The most effective enzyme concentration for sweet potato protoplast isolation was a combination of 2% hemicellulase and 2% cellulase to give the highest protoplast yield LSD0.05 (3.446) and viability LSD0.05 (4.242) at relatively low enzymatic concentrations. The isolated protoplasts were then tested for viability and the results of the analysis showed that an inverse relationship existed between protoplast yield and protoplast viability. There were significant differences between the ten-enzymatic treatments (P < 0.05) for protoplast viability. For protoplast fusion the CaCl2 concentrations ranged from 0.1 M to 1 M. Statistical analysis revealed that there was a significant difference between treatments (P < 0.05). Mean separation reveal that all treatment means for protoplast fusion were significantly different from each other (LSD0.05 = 2.591). CTAB 3% method was used to extract genomic DNA from both sweet potato varieties. In order to have a method for confirming successful fusions of the two lines, Random Amplified Polymorphic DNA method was used. Primers OPH5, OPH7, OPU5, OPU1 and OPH6 used in the experiment were able to detect polymorphisms in the two sweet potato genotypes. For every case, each given primer was able to produce bands in similar electrophoretic positions that matched in both sweet potato varieties (monomorphic bands). This revealed a level of similarity between the two genotypes. However, very few electrophoretic band positions produced by the same primer did not match (polymorphic) proving that the two sweet potato varieties had a degree of dissimilarity. The number of polymorphic bands produced per primer was between 2 and 3. Different fragment lengths were produced in the two varieties using the same primer, revealing that some of the banding patterns were similar in both sweet potato varieties. Visual assessment of the DNA was estimated from the intensity of UV-induced fluorescence emitted after ethidium bromide staining. However, quantitative analysis of electrophoretic banding patterns using sensitive such as the spectrophotometric method which is based on optical density (OD) was not done.