Obtaining calli and regenerated plants in anther cultures of pea

Abstract

Pea (Pisum sativum L.) is a species for which there is no efficient method for the recovery of haploid plants yet. This research investigated the influence of various genotypes, nutrient media, and stress treatments on callus formation, embryogenesis and plant regeneration in anther cultures of pea. A wide range of pea genotypes and nutrient media was studied. Morphogenic calli were initiated on media supplemented with α-naphthaleneacetic acid (NAA), 6-benzyladenine (BA), and 2,4-dichlorophenoxyacetic acid (2,4-D) without application of stress treatments. Embryogenic calli and embryos were regenerated on media with low sucrose content in the presence of 2,4-D or indole-3-butyric acid (IBA) after cold stress (4°C) of isolated buds, alone or in combination with in vitro treatment of isolated anthers at higher temperatures (35–38°C). The efficiency of regeneration via shoot morphogenesis on different nutrient media and the peculiarities of regeneration from embryogenic calli were investigated. Green embryogenic calli initiated on 2,4-D were able to develop through shoot morphogenesis on a medium supplemented with BA and NAA. This process led to regeneration of hypertrophic embryos at various developmental stages. The origin of regenerated plants (i.e. from microspores or somatic anther cells) was estimated using marker alleles determining morphological traits. Almost all R₀ regenerants derived from morphogenic calli originated from anther somatic cells.