An improved protocol for plant regeneration from leaf- and hypocotyl-derived protoplasts of carrot

Abstract

An easy and effective regeneration system from leaf-and hypocotyl-derived protoplasts was established for carrot. The protoplast isolation efficiency after preplasmolytic treatment and digestion of source material in enzyme mixture consisted of 1% cellulase Onozuka R-10 and 0.1% pectolyase Y-23 reached on average 3 9 10 6 and 10 6 protoplasts per g of leaf and hypocotyl tissue, respectively. A modified thin alginate layer technique was applied for the protoplast culture. Direct somatic embryogenesis on a simplified Kao and Michayluk medium in the presence of 2,4-D and zeatin occurred during cultivation of both leaf-and hypocotyl-derived protoplasts for all accessions used. Morphologically normal plants were produced at very high efficiency within two months after initiation of the protoplast culture. Ninety three percent of in vitro derived plants were diploids. Pollen viability and seed set after self-pollination were similar to those of plants obtained from seeds. Keywords Carrot Á Daucus carota Á Plating efficiency Á Protoplast viability Á Somatic embryogenesis Abbreviations CPP Carrot petiole protoplast medium 2,4-D 2,4-Dichlorophenoxyacetic acid ETAF Extra thin alginate film FDA Fluorescein diacetate MES 2-(N-Morpholino)ethanesulfonic acid MS Murashige and Skoog medium (1962) NAA a-Naphthaleneacetic acid PEM Proembryonic mass TAL Thin alginate layer

Plant regeneration from leaf-derived protoplasts within the Daucus genus: effect of different conditions in alginate embedding and phytosulfokine application

“…1a), respectively. All steps of protoplast isolation were performed according to a previously described protocol (Grzebelus et al 2012a). Briefly, about 1 g of source tissue was cut in 9 9 1.5 cm glass Petri dish into small pieces in the presence of a 0.5 M mannitol solution (pH = 5.6-5.8), and incubated for 1 h at 26 ± 2°C in the dark to plasmolyze the cells.…”

“…Therefore, before applying this procedure to plant breeding programs, successful protocols for plant regeneration from protoplasts of both partners should be elaborated. For carrot, well known as a model species for plant tissue culture systems, efficient procedures for protoplast isolation and plant regeneration from different types of source tissue, such as suspension cultures, petioles, leaves, and hypocotyls have been presented (Grambow et al 1972;Dirks et al 1996;Grzebelus et al 2012a). However, to our knowledge, similar studies with regard to wild Daucus species do not exist.…”

“…Protoplasts embedded in alginate matrix can be dispensed as beads (Damm and Willmitzer 1988), blocks (Pan et al 2003), or layers with different thickness such as thin alginate layers (TAL, Damm et al 1989) and extra thin alginate films (ETAF, Pati et al 2005), and then cultured in liquid media. Alginate has been successfully used as a gelling agent in protoplast cultures of many species; among others, in Lotus covniculatus and Nicotiana tabacum (Pati et al 2005), Citrus sinensis (Niedz 2006), Helianthus annuus (Rákosy-Tican et al 2007), D. carota (Grzebelus et al 2012a), Beta vulgaris (Grzebelus et al 2012b), and Brassica oleracea (Kiełkowska and Adamus 2012).…”

“…Protoplasts were isolated from in vitro grown shoots produced from seeds. In order to obtain aseptic material, donor seeds were disinfected using a three-step procedure, as described by Grzebelus et al (2012a). To make the germination of wild accessions possible, decontaminated seeds were left to swell in sterile water.…”

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

“…These species are considered to be recalcitrant to DH technologies, although carrot is a model plant for tissue culture and regeneration protocols are available for several species Grzebelus et al 2012). Practical utilization of the DH technique is hindered by the low efficiency of embryogenesis in carrot anther cultures.…”