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This protocol describes a high through put colorimetric method that relies on the formation of a complex between iodine and chains of glucose molecules in starch. Iodine forms complexes with both amylose and long chains within amylopectin. After the addition of iodine to a starch sample, the maximum absorption of amylose and amylopectin occurs at 620 and 550 nm, respectively. The amylose/amylopectin ratio can be estimated from the ratio of the 620 and 550 nm absorbance values and comparing them to a standard curve in which specific known concentrations are plotted against absorption values. This high throughput, inexpensive method is reliable and reproducible, allowing the evaluation of large populations of potato clones.
This protocol describes a high through put colorimetric method that relies on the formation of a complex between iodine and chains of glucose molecules in starch. Iodine forms complexes with both amylose and long chains within amylopectin. After the addition of iodine to a starch sample, the maximum absorption of amylose and amylopectin occurs at 620 and 550 nm, respectively. The amylose/amylopectin ratio can be estimated from the ratio of the 620 and 550 nm absorbance values and comparing them to a standard curve in which specific known concentrations are plotted against absorption values. This high throughput, inexpensive method is reliable and reproducible, allowing the evaluation of large populations of potato clones.
Approximately 80% of the fresh weight of a potato tuber is water; nearly all of the remaining dry matter is starch1. Most of the starch (70%) is composed of amylopectin, while the remainder is amylose. The ratio between amylose and amylopectin is the most important property influencing the physical properties of starch. Amylose is a linear alpha 1-4 glucose chain, while amylopectin is a linear alpha 1-4 chain with alpha 1-6 branches2. Methods such as iodine binding, differential scanning calorimetry (DSC), high performance size exclusion chromatography (HPSEC) and concanavalin A interactions have been developed for starch determination in differe....
1. Amylose Determination from Potato Tuber Starch
The standard curve constructed from blanked data of different amylose/amylopectin concentration solutions shows approximate absorbance ratios ranging from 0.7 to 1.6 (Table 1). A linear regression trend line from these data is later used for inferring amylose content in freeze dried potato samples (Figure 1).
The plant production environment has some effect on the amylose content in the potato tuber7. Replicated amylose content determinations in two.......
Amylose content in potato tubers typically ranges from 20-35%7,8. If most values are outside this range, consider these possible errors: 1) raw data were used instead of blanked data for the construction of the amylose/amylopectin standard curve; 2) raw data were used instead of blanked data for the determination of the amylose/amylopectin ratios from the potato samples; or 3) the water used for the analysis was not pure enough. We noticed that by using distilled water and not ultra-pure water in the analysis,.......
We have nothing to disclose.
Funding for this research was provided in part by the USDA-ARS Research Associate Program and the USDA Crop Germplasm Committee.
....Name | Company | Catalog Number | Comments |
Reagents | |||
Amylose | Sigma | 859656 | |
Amylopectin | Sigma | A8515 | |
Perchloric Acid 60% | Sigma | 311413 | Very hazardous to skin and eyes |
Iodine | Sigma | 23214TD | |
Potassium iodide | Sigma | 08625JE | |
Equipment | |||
Plate Reader | Bio Tek | ELx800 |
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