Key messageDepending on the capability for stress adaptation, the role played by glutathione in microspore embryogenesis consists of both antioxidative activity and stimulation of embryo-like structure development.AbstractThe efficiency of microspore embryogenesis (ME) is determined by the complex network of internal and environmental factors. Among them, the efficient defence against oxidative stress seems to be one of the most important. The present study confirms this hypothesis showing the positive effect of glutathione—the most abundant cellular antioxidant—on ME in isolated microspore cultures of triticale (× Triticosecale Wittm.). For the first time, low temperature (LT) pre-treatment of tillers was combined with the exogenous application of glutathione and associated with the total activity of low-molecular weight antioxidants, the endogenous content and redox status of glutathione, and the effectiveness of ME. The results indicate that efficient antioxidative defence is the first, although not the only, prerequisite for effective ME. In responsive genotypes, LT alone stimulated antioxidative defence and decreased cell redox status, which was associated with increased cell viability and high frequency (ca. 20%) of microspore reprogramming. Application of glutathione had no effect either on the microspore viability or on the initial number of embryogenic microspores. However, it increased the number of embryo-like structures, probably by stimulating the next phases of its development. In recalcitrant genotypes, the main role of glutathione seems to be its participation in cell protection from oxidative stress. However, even enhanced antioxidative activity, which sustained cell viability and increased the number of embryogenic microspores, was insufficient for efficient haploid/doubled haploid plant production. Evidently, there are still other defective elements in the complex network of factors that regulate the process of ME.Electronic supplementary materialThe online version of this article (10.1007/s00299-018-2362-x) contains supplementary material, which is available to authorized users.

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Androgenic responsiveness in anther (AC) and isolated microspore cultures (MC) was analysed using 15 lines of Polish winter rye (Secale cereale L.). The effect of low temperature (LT) alone or in combination with osmotic stress induced by mannitol treatment (MAN) and/or with reduced glutathione (GSH) on the effectiveness of the process was studied. Interestingly, each treatment had a different effect on microspore (mcs) vitality and capability to divide symmetrically. The first criterion for successful embryogenesis was to exceed the threshold number of at least 25% dividing microspores, which determined 'embryogenic suspension culture'. In some configurations a spectacular effect was achieved, especially in lines highly recalcitrant to androgenesis induction. Relatively high effectiveness of androgenesis induction (up to 4.58 AS per 10 5 mcs per spike in MC and 21.29 AS per spike in AC) showed that the developed protocol with GSH and/or MAN tiller pre-treatments overcomes the genotypic barrier for androgenesis initiation in rye. Moreover, relatively high, spontaneous genome diploidization (55%) of regenerated plants demonstrated that the described protocols could be effectively integrated into conventional rye breeding programmes. Key message Glutathione and mannitol break the barrier of androgenesis initiation by increasing the dividing and vital microspores ratio. This marker is useful for screening material in breeding programs utilizing DH technology. Communicated by Alison M.R. Ferrie.