Abstract

In this work, we describe an improved protocol for induced parthenogenesis and ovule culture of carrot (Daucus carota L.). The effects of pollination with parsley pollen and/or 2,4-dichlorophenoxyacetic acid (2,4-D) treatment on the stimulation of parthenogenesis were studied using heterozygous donor plants of 30 varieties and breeding populations of carrots. Isolated ovules, cultured in vitro, enlarged and developed embryos or calli. The application of 2,4-D on pollinated flowers stimulated callus development but did not increase the frequency of embryo development from ovules and, thus, was not useful for increasing the frequency of haploid plant recovery. The efficiency of embryo development was accession-dependent and varied from 0 to 24.29%. In optimized conditions, most accessions responded by embryo development exclusively. The highest frequency of embryo development was observed from ovules excised from ovaries 20–22 d after pollination with parsley pollen. Among several media used for ovule culture, 1/2-strength Murashige and Skoog medium with 0.06 μM indole-3-acetic acid (IAA) was the best. It allowed the production of embryos at a similar frequency as on the media supplemented with kinetin, gibberellic acid, putrescine, or thidiazuron, but restricted callus development. Most plants obtained were haploids and diploids derived from parthenogenesis, as evidenced by homozygosity at three independent loci based on isozyme and PCR analyses. In total, considering haploids and embryo-derived homozygous diploids together, 72.6% of regenerated plants were of gametic origin.

Highlights

  • Carrot is one of the top ten most economically important vegetables in the world (FAO 2010)

  • The discovery and application of genetic-cytoplasmic male sterility (CMS) systems opened new perspectives in carrot breeding (Simon et al 2008), and the development of F1 hybrid varieties has relied on the use of CMS lines

  • We examined the effects of modifications of the conditions used for in vitro culture of carrot ovules in increasing the frequency of embryo development and validated the results using a wide selection of carrot accessions

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Summary

Introduction

Carrot is one of the top ten most economically important vegetables in the world (FAO 2010) It is an outcrossing, insect-pollinated diploid (2n=2x=18). Haploids develop as a result of stimulation by application of irradiated pollen or pollen of other species or genera (wide pollination; ForoughiWehr and Wenzel 1993). Haploid and diploid plants were obtained from unfertilized ovules cultured in vitro. The efficiency of this laborious and time-consuming process was low: only 1.85% of cultured ovules responded and more than half of them showed callus development rather than embryo development. We examined the effects of modifications of the conditions used for in vitro culture of carrot ovules in increasing the frequency of embryo development and validated the results using a wide selection of carrot accessions

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