The overall goal of this procedure is to determine in a high throughput manner the relative proportions of AMLO and amylopectin. In potato tuber starch, high AMLO starch is desirable due to its lower digestibility. Potatoes with high AMLO starch have a lower glycemic index and higher fiber content.
This technique is being demonstrated by Philip Smith, an undergraduate student at the University of Wisconsin Madison. In our lab, we're interested in breeding for more nutritional potato varieties. One way we can do that is to increase the AMLO content in potato starch.
High AMLO starch is more nutritional because it has a lower glycemic index, and in this assay, we have developed a procedure that allows us to look at a large number of samples fairly quickly, and it's a repeatable, high throughput, inexpensive assay. This assay is a modification of an iodine staining procedure. It is a colormetric assay, and it can be run using a 96 well plate fresh potato tubers are used for this method, dice the tubers into approximately one centimeter cubes and discard pieces containing skin.
A french fry cutter allows this step to be carried out efficiently. The cubes are placed into a labeled bag. We use nylon socks such as those used in shoe stores.
They're inexpensive and allow samples to dry quickly and evenly in the lyophilizer. The nylon bags containing the samples are placed into a paper bag and frozen overnight at negative 80 degrees C.The frozen samples are removed from the paper bag and placed into the freeze dryer. With our freeze dryer.
It takes about five days to completely dry the samples. After the cubes are dry, they're ready to be processed. The dried potato cubes are finely ground using a Wiley mill or with a motor and pestle.
The cubes or the powder can be stored until they're processed. However, be sure to seal them in plastic bags or containers to prevent them from taking up water. The powder is placed in a plastic bag for storage until the AMLO assays can be performed.
An analytical scale is used to weigh approximately 20 milligrams of potato powder, which is then placed into a 50 mil plastic tube. Samples weighing less than 20 milligrams or more than 30 milligrams do not give reliable results to make the starch soluble. 500 microliters of 45%per chloric acid is added to each tube.
Be careful when working with per chloric acid work in a fume hood and use personal protection equipment. After a four minute incubation period, the sample is diluted with 16 milliliters of Milli Q water while mixing on a vortex. Sir, After the non soluble material has settled to the bottom of the tube, transfer 40 microliters of the liquid to the microtiter plate.
Be careful to avoid transferring any non soluble material. 50 microliters of iodine solution are added to the sample and mixed by pipetting carefully. Do not allow any air bubbles to remain in the sample or on the walls of the well.
If air bubbles form, gently tap the plate on the bench top to dislodge them. Put the microtiter plate on the plate reader and read the absorbance of each sample at 550 nanometers and 620 nanometers. Once we have the output from the microplate reader, we can compare the data with the standard curve and determine the AMLO to amylopectin ratio in the starch sample table one.
This is a typical output of the standard curve as explained in the protocol. Figure one, the absorbance ratios are plotted and a regression line is calculated for the AMLO to determination in the potato samples. During standard curve construction, each point on the curve should be the absorbance value of the sample with the known AMLO concentration.
After subtracting the absorbance value of the blank. When calculating the value of an unknown sample, also subtract the absorbance value of the blank table.Two. This is a typical output of AMLO content in potato tubers.
Using this COLORMETRIC method, we have used this assay in our lab to determine the relative contributions of genotype and environment to AMLO Amylopectin ratio in potato starch. We hope ultimately to develop molecular markers associated with high AMLO starch and potato, and also to develop germplasm that breeders can use to develop high amylose potato varieties.
