Optimization of methods for using polyethylene glycol as a non-permeating osmoticum for the induction of microspore embryogenesis in the Brassicaceae

Abstract

The objective of this work was to enhance the quality and quantity of microspore-derived embryos of cruciferous species by using polyethylene glycol (PEG) to replace sucrose in the culture medium. The main advantage in using PEG is that it produces embryos that are morphologically more similar to zygotic embryos and have enhanced germination capabilities. When microspores were cultured in full strength NLN medium supplemented with 25% (w/v) PEG, the addition of 3 ml of full strength NLN with 13% (w/v) sucrose at 14 d was beneficial for embryo quality and quantity. Experiments showed that this PEG system could be used for a number of Brassica napus cultivars, as well as a number of other cruciferous species. PEG enhanced microspore embryogenesis in B. nigra, Crambe abyssinica, and Raphanus oleifera. Microsporederived embryos were obtained from all cruciferous species evaluated (B. alboglabra, B. carinata, B. juncea, B. rapa, B. nigra, R. oleifera, Crambe abyssinica, Sinapis alba) using either sucrose or PEG as the osmoticum. Microspore embryogenesis was induced in B. napus in PEG-based cultures without a 32°C heat shock (i.e., 4, 15, 18, and 24°C). These temperature conditions were non-inductive when sucrose was used as the osmoticum. Spontaneous chromosome doubling occurred in 64-92% of the regenerated plants when PEG was used in the NLN culture medium, whereas in culture medium containing sucrose, the spontaneous doubling rate was 2-18%.

Isolated microspore culture techniques and recent progress for haploid and doubled haploid plant production

“…Prem et al (2008) reported a four-fold increase in B. juncea microspore embryogenesis when silver nitrate and activated charcoal were included in the culture medium. Other recent improvements include the addition of the anti-auxin, p-chlorophenoxyisobutyric acid (PCIB), to enhance microspore embryogenesis in B. juncea (Agarwal et al 2006), and substitution of polyethylene glycol for sucrose in B. nigra (L.) Koch, Crambe abyssinica Hochst., Raphanus oleifera L., and several genotypes of B. napus (Ferrie and Keller 2007). The B. napus microspore protocol has been successfully extended to another cruciferous species with the report by Leskovšek et al (2008) of DH plantlet production from rocket or arugula (Eruca sativa Mill.…”

Bamboo Regeneration via Embryogenesis and Organogenesis

“…Osmotic pressure is correlated with plant development and differentiation, appropriate treatment by the common osmoticums such as sucrose, mannitol and polyethylene glycol (PEG) enhances the embryogenesis and organogenesis in Solanum melongena (Mukherjee et al,1991), Brassica napus (Ferrie & Keller, 2007) and white spruce (Picea glauca) (Misra et al, 1993).…”

Haploids and doubled haploids in Brassica spp. for genetic and genomic research

“…Mapping genes related to microspore embryogenesis Tremendous differences among B. napus genotypes in their embryogenic response of cultured microspores have been reported (Ferrie 2003;Ferrie and Keller 2007). However, the genetic basis of the genotypic differences in microspore culture response remains largely unknown.…”