Determination of ploidy and homozygosity of carrot plants obtained from anther cultures

Abstract

The purpose of the present work was to evaluate carrot plants obtained from anther cultures with respect to their ploidy and homozygosity. Ploidy was determined using flow cytometry. Homozygosity of analyzed plants was determined using isoenzymes, glucose-6-phosphate isomerase (PGI) and aspartate aminotransferase (AAT). The cytometric tests revealed that more than 90% of the carrot plants obtained from anther cultures were doubled haploid. In the initial assessment of polymorphism of the two enzymatic systems in selected androgenetic carrot plants of the cultivars: Berjo, Kazan F 1 , and Splendid F 1 , it was proven that 100% of those plants were homozygotes in respect to PGI and also with respect to AAT. In the second experiment, the obtained androgenetic progeny of the heterozygous donor plant of cultivar Narbonne F 1 was found to be 94% homozygotic with respect to PGI and 100% homozygotic in the case of AAT. For the androgenetic plants of the cultivar Kazan F 1 , 89% of them were homozygotic with respect to AAT but PGI enzyme system did not differentiate the homozygotic and heterozygotic androgenetic progeny. These results indicated that enzyme polymorphism depends on carrot genotype, therefore, analysis of some (more than one) isozymes was necessary to confirm homozygosity of plants. PGI and AAT can be a useful tool for determining homozygosity in androgenetic carrot plants.

Spontaneous and Chemically Induced Genome Doubling and Polyploidization in Vegetable Crops

“…In another study, regenerants obtained in a culture of isolated microspores had an SHGD rate ranging from 17.5% to 63.6% depending on carrot accession [37]. A total of 90% of carrot regenerants obtained from another culture were diploid [38]. The flow cytometric analysis of carrot plants obtained in an in vitro culture of unfertilized ovules in carrot showed that 97.7% of the regenerants were diploid [39].…”

Comparison of methods for obtaining doubled haploids of carrot

“…The ploidy of the acclimatized plants was determined by an indirect method involving measurements of the amount of nuclear DNA with a flow cytometer as described by Kiszczak et al [15] and by following a modified version of Galbraith's procedure [22]. The analysis was conducted with the use of a Partec CA-II flow cytometer (Partec GmbH, Germany).…”

“…The analysis of homozygosity of the donor plants and DH lines was performed using two isoenzymes, phosphoglucose isomerase (PGI, EC 5.3.1.9) and aspartate aminotransferase (AAT, EC 2.6.1.1), following the extraction protocol described by Kiszczak et al [15]. Electrophoresis was performed in 10% starch gel as described by Gottlieb [23].…”

“…The following year, Górecka et al [11] described the formation of secondary embryos and the direct conversion of embryos into plants derived from anther cultures, which accelerated and simplified the procedure for obtaining androgenetic plants. In 2011, Kiszczak et al [15] analyzed the ploidy and homozygosity in a population of carrot plants obtained through androgenesis in anther cultures. In these experiments, 90% of the carrot plants obtained from anther cultures were DH.…”

Characterization of ploidy levels in Chrysanthemum L. by flow cytometry

“…In addition, this method is non-destructive and only requires a small amount of tissue (Doležel 1997). Flow cytometry is a very successful and convenient method for analyzing ploidy level and/or estimating genome size in many plant species (Obidiegwu et al 2009(Obidiegwu et al , 2010Ono and Hosaka 2010;Schlaepfer et al 2010;Kiszczak et al 2011;Kumar et al 2011;Burson et al 2012;Scaldaferro et al 2012;Angeles et al 2013;Moon et al 2013).…”