Selection of embryogenic sugarcane callus by image analysis

In the cultivation of plant calli on solid media, two kinds of calli such as compact and friable calli, which are a bright yellow and a whitish clump, respectively, are often obtained. Distinction of these calli is of much importance in the regeneration step. The image analysis system associated with a Charge Coupled Device (CCD) camera and microscopy were used to distinguish sugarcane calli. The original images from compact and friable calli were input to a computer via an image analysis board. At first, the brightnesses of trichromatic colors, red (R), green (G) and blue (B), of each pixels were extracted and the average brightness value for each color was calculated. From these values of the trichromatic colors, compact and friable calli could not be clearly distinguished. Next, the brightness of yellow, Br(Y), and white, Br(W), were defined using Br(R), Br(G) and Br(B), and the difference between Br(Y) and Br(W), Br(Y-W), which can be used to express the yellowish grade, was calculated. When Br(Y-W) was determined from all pixels of the original images of both calli, the compact calli were found to be clearly distinguished from the friable calli by the frequency distributions of Br(Y-W). Average brightness center value, Av(C(Y-W)), was calculated from the frequency distributions. It was found that the calli with less than 10 units of Av(C(y-w)) was never regenerated and a proportional relationship between Av(C(Y-W)) and the regeneration frequency of the callus line was obtained.