Indirect regeneration from in vitro grown leaves of three pear cultivars and determination of ploidy level in regenerated shoots by flow cytometry

Abstract

The aim of the present investigation was to optimize a protocol for indirect regeneration in three commercially important pear cultivars: “Bartlett”, “Dargazi” and “Harrow delight”. Effects of different concentrations of thidiazuron (TDZ) and naphthaleneacetic acid (NAA) in modified Murashige and Skoog culture medium (1/2 strength micro elements, 1/4 strength macro elements and full strength vitamins and mio-inositol for induction phase & 1/2 strength macro and micro elements and full strength vitamins and mio-inositol for regeneration phase), explant types (upper and lower leaf sections) and cultivars were examined based on the quality and quantity of callus formation, indirect regeneration rate and mean number of shoots per explant. Three kinds of callus were detected during the induction phase. Only white and puffy callus (type I) proceeded to shoot regeneration while callus type II (similar to callus type I, becoming green and watery in some parts) and callus type III (green or creamy in color and watery) did not show regeneration potential. The highest percentage of explants producing callus type I (100%) and regeneration (64%) and the highest mean number of shoots per explant (10.6) was obtained from cv Dargazi on medium containing 16μM TDZ. The lower section of leaves was significantly more effective than the upper section regarding the parameters studied. Flow cytometry analysis indicated that regenerated shoots and donor shoots were identical with respect to the ploidy level. Shoot regeneration from organogenic callus was successfully achieved and regeneration capacity varied depending on genotype, explant type and plant growth regulator combinations.