In vitro androgenesis process under salt pressure is recognized as being a valuable method for quick production of salt stress tolerant doubled haploid lines, enabling yield improvement of plants cultivated in dry climates. This study aimed to investigate the effect of salt stress on in vitro androgenetic regeneration, and the production of salt tolerant barley doubled haploid lines. Moroccan barley varieties, Arig, Asni and Tamelalt were used as RS0, and after one round of selfing (RS1) or two (RS2). These successive generations made possible to verify genetic stability or possible accidental heterozygosity, and to obtain plants presumably more tolerant to salinity. After culture of 11,340 anthers on induction medium, 2,270 calluses or embryos were generated. According to the induction rate of these formations, the three generations of each variety were not significantly different, pointing out the genetic stability after two selfings. Tamelalt and Arig had similar in vitro response, while for Asni, the three generations behaviour differed from the previous ones and constitute a statistically separate group. After transfer of the formations on regeneration medium either salt free, as control, or supplemented by increasing concentrations of NaCl, ranging from 2.5 to 13 g.L-1, 170 regenerants were produced. Asni (RS1) showed to be the most salt tolerant, bearing the maximal NaCl concentration of 12 g.L-1, but the single regenerated plant obtained was albino, as most of the regenerated plants from any genotype and generation. However, RS1 and RS2 generations of Asni regenerated one green plant each, with NaCl concentration of 5 g.L-1, resulting in two new Asni doubled haploid fertile lines.
The anthers in vitro culture is a useful technique for plant improvement as to create new varieties. However, the success of this technique is limited by several factors. This study was carried out to identify factors as the influence of spikelet position in the spike, position of donor tiller on plate tillering and anther orientation onto the medium as well as cold pretreatment and the genotype that affect the callusing induction in anthers in vitro culture of three cultivars of Moroccan barley (Arig 8, Asni and Tamelalt). Among genotypes, the callusing response was significantly higher in Asni (21.42%). The callusing response was significantly decreased when spikes stemmed from tillers far from the main tiller on the same tillering plate. However, anthers coming from the spikelets of the middle part of the spike showed the highest callusing response (29.85%) compared with the basal (14.83%) and the upper (6.72%) parts. It was also shown that anther cold pretreatment at 4°C for 14 days had a beneficial effect only on the callusing response but it decreased the regeneration of green plants, due to the interaction between cold pretreatment and genotypes to which spring barley is recalcitrant and more predisposed to albinism, especially six-row type (Arig 8). Moreover, anther orientation onto the surface medium had a marked effect on callusing response, anthers placed in upper orientation developed more calli (60.22%) than those in flat position (18.99%). Our findings suggest that the studied factors control the callusing response. Especially, the Anthers orientation that must be taken into account during androgenesis through anthers in vitro culture.
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