Obtaining carrot (Daucus carota L.) plants in isolated microspore cultures

Abstract

Microspores were cultured on the modified B5 liquid medium containing 2.4D (0.1 mg L(-1)), NAA (0.1 mg L(-1)), L-glutamine (500 mg L(-1), L-serine (100 mg L(-1)), and sucrose (100 g L(-1)). The developmental stages of microspores and divisions were observed. Initially, the formation of binuclear and multicellular structures was noticed. Plants regenerated in the cultures in which the tetrad stage of microsporogenesis had predominated. Embryoids were still forming 24 weeks after the cultures were set up. Six weeks after the transfer of androgenetic embryos onto the B5 regeneration medium, they were converted into complete plants. Out of 90 androgenetic plants planted in a growth chamber, 42 plants adapted to the new conditions. All of those plants proved to be diploids in cytometric analysis.

Microspore embryogenesis and production of haploid and doubled haploid plants in carrot (Daucus carota L.)

“…The shortest time span was required by 900Q1 with only 38 days. This variation in rate of development was similar to findings by Górecka et al (2010), and much longer than those observed in Brassica species (Fang et al 2006;Zhang et al 2008;Bhowmik et al 2011;Takahira et al 2011). The ten different genotype accessions were selected for testing over different years (Table 2).…”

“…Agronomic characters measured included leaf color intensity, leaf size, leaf dissection, and petiole color of haploid, DH and triploid plantlets. Górecka et al (2010) noticed the first divisions of carrot immature microspores under a microscope about 2 weeks after culture initiation, while these structures could be observed with the naked eye after 3 weeks to 6 months. In our study, small embryos or calli were visible with the naked eye about 5 weeks after the IMC was established (Fig.…”

“…The ploidy level of young leaves was determined by a flow cytometer (FACSCalibur, BD, USA; Górecka et al 2010;. Approximately 2 cm 2 leaf tissue was chopped several times with a razor blade in the presence of 2 ml of lysis buffer (15 mM Tris, 2 mM Na 2 EDTA, 0.5 mM spermine, 80 mM KCl, 20 mM NaCl, 0.1 % v/v TRITON X-100), and incubated for 1 min.…”

“…Matsubara et al (1995) firstly obtained small calli from a few carrot-isolated microspores. Górecka et al (2010) observed 42 androgenetic carrot plants that were all diploids out of 90 plantlets. obtained 17 regenerated lines that produced seeds for field evaluation.…”

“…It was first envisioned as a technique to accelerate the breeding process and to use for practical and basic research in many crops (Dunwell 2010;Ferrie and Caswell 2010;Germanà 2011). While efficient protocols for microspore embryogenesis induction have been developed to obtain haploid and DH plants in several crops (Maluszynski et al 2003;Dunwell 2010;Ferrie and Caswell 2010), limited progress has been reported using anther cultures (Dohya et al 1997;Tyukavin et al 1999;Adamus and Michalik 2003;Górecka et al 2005;Smýka-lová et al 2009;), isolated microspore cultures (IMC; Dohya et al 1997;Górecka et al 2010;, or unfertilized ovule cultures (Kiełkowska and Adamus 2010) of Apiaceae species.…”