Visualization of smaller chromosomes from unicellular microalgae

Abstract

Chromosome visualization is a technique used in many modern applications such as classification, in situ hybridization and genetic improvements, etc. Algal biotechnology grew steadily into an important global industry. Dunaliella salina (Teodoresco, 1905) is a model microalgae having tremendous potential for production of carotenoids for commercial use. The chromosomal size of microalgae is very small, and techniques for karyotype analysis are still unclear. In this work, it tried to simplify the techniques and establish a detailed step-by-step protocol using Dunaliella salina as a model and proved the technique in Tetraselmis chuii (Stein, 1878) which can be reproducible. The most important points were considered in the techniques as metaphase resting, fixation, dehydration and rehydration, hydrolysis and preparation of chromosomes for microscopic observations. During standardization, various concentration and duration of colchicine treatment were tested for metaphase arrest, replaced ethanol for methanol during fixation and dehydration, used a rehydration solution before hydrolysis and observed acid, base and enzymes impact on partial hydrolysis process of the cells. Colchicine treatment was found to be species dependent, and optimum concentration was 0.1% for 6 h (h) for Dunaliella salina, and methanol proved better to dehydrate for the preservation of algal cells. The low 500 mM concentration of HCl, 30 mM KOH and 5% enzyme proved to be suitable for the hydrolysis process for algal chromosome visualization. The technique permits clear visualization and counting of the smallest chromosome from unicellular algae. This technique described detail step by step without ambiguity and steps can be adapted on a species-specific manner.