Project description:Microspore embryogenesis (ME) is the most powerful tool for creating homozygous lines in plant breeding and molecular biology research. It is still based mainly on the reprogramming of microspores by temperature, osmotic and/or nutrient stress. New compounds are being sought that could increase the efficiency of microspore embryogenesis or even induce the formation of haploid embryos from recalcitrant genotypes. Among these, the mitogenic factor phytosulfokine alpha (PSK-α) is promising due to its broad spectrum of activity in vivo and in vitro. The aim of our study was to investigate the effect of PSK-α on haploid embryogenesis from microspores of oilseed rape (Brassica napus L., DH4079), one of the most important oil crops and a model plant for studying the molecular mechanisms controlling embryo formation. We tested different concentrations (0, 0.01, 0.1 and 1 µM) of the peptide and evaluated its effect on microspore viability and embryo regeneration after four weeks of culture. Our results showed a positive correlation between addition of PSK-α and cultured microspore viability and a positive effect also on the number of developed embryos. The analysis of transcriptomes across three time points (day 0, 2 and 4) with or without PSK-α supplementation (15 RNA libraries in total) unveiled differentially expressed genes pivotal in cell division, microspore embryogenesis, and subsequent regeneration. PCA grouped transcriptomes by RNA sampling time, with the first two principal components explaining 56.8% variability. On day 2 with PSK, 45 genes (15 up- and 30 down-regulated) were differentially expressed when PSK-α was added and their number increased to 304 by day 4 (30 up- and 274 down-regulated). PSK, PSKR, and PSI gene expression analysis revealed dynamic patterns, with PSK2 displaying the highest increase and overall expression during microspore culture at days 2 and 4. Despite some variations, only PSK1 showed significant differential expression upon PSK-α addition. Of 16 ME-related molecular markers, 3 and 15 exhibited significant differential expression in PSK-supplemented cultures at days 2 and 4, respectively. Embryo-specific markers predominantly expressed after 4 days of culture, with higher expression in medium without PSK, while on day 0, numerous sporophyte-specific markers were highly expressed.

Project description:SHOOTMERISTEMLESS (STM) is a well characterized key regulator of the shoot apical meristem (SAM) activity in diverse plant species. We used a transgenic approach to investigate how the altered expression of Brassica napus (Bn) STM affects the morphology and behavior of microspore-derived embryos (MDEs). Down-regulation of BnSTM repressed the formation of the SAM and reduced the number of MDEs able to regenerate viable plants at germination. This was in contrast to embryos over-expressing BnSTM which displayed enlarged SAMs with an improved regeneration frequency. To further investigate the regulatory mechanisms underpinning these different responses, global changes in transcript levels were analysed in laser micro dissected SAMs of the transformed MDEs. The induction of BnSTM up-regulated the expression of many transcription factors (TFs) some of which directly involved in the formation of the SAM, i.e. CUPSHAPED COTYLEDON1 and WUSCHEL, and regulatory components ofthe antioxidant response, hormone signalling, and cell wall synthesis and modification. Opposite expression patterns for some of these genes were observed in the SAMs of MDEs down-regulating BnSTM. These data were interpreted in relation to the ability of BnSTM to modulate the size and functionality of the SAM. Structural and expression studies were further conducted to confirm the role of BnSTM in mediating the regulatory network of cell wall and lignin synthesis. Besides lowering the transcript levels of early components of cell wall and lignin biosynthetic pathways in the SAM, BnSTM had a remarkable repressing effect on the expression of PHENYLALANINE AMMONIA LYASE2, CINNAMATE 4-4HYDROXYLASE, andCINNAMYL ALCOHOL DEHYDROGENASE. Since lignin formation is a feature of irreversible cell differentiation, these results suggest that one way in which BnSTM promotes indeterminate cell fate may be by preventing the expression of components of biochemical pathways involved in the accumulation of lignin in the meristematic cells. Overall these studies provide evidence for a novel function of BnSTM in enhancing the quality of in vitro produced meristems, and propose that this gene can be used as a potential target to improve regeneration of cultured embryos.

Project description:SHOOTMERISTEMLESS (STM) is a well characterized key regulator of the shoot apical meristem (SAM) activity in diverse plant species. We used a transgenic approach to investigate how the altered expression of Brassica napus (Bn) STM affects the morphology and behavior of microspore-derived embryos (MDEs). Down-regulation of BnSTM repressed the formation of the SAM and reduced the number of MDEs able to regenerate viable plants at germination. This was in contrast to embryos over-expressing BnSTM which displayed enlarged SAMs with an improved regeneration frequency. To further investigate the regulatory mechanisms underpinning these different responses, global changes in transcript levels were analysed in laser micro dissected SAMs of the transformed MDEs. The induction of BnSTM up-regulated the expression of many transcription factors (TFs) some of which directly involved in the formation of the SAM, i.e. CUPSHAPED COTYLEDON1 and WUSCHEL, and regulatory components ofthe antioxidant response, hormone signalling, and cell wall synthesis and modification. Opposite expression patterns for some of these genes were observed in the SAMs of MDEs down-regulating BnSTM. These data were interpreted in relation to the ability of BnSTM to modulate the size and functionality of the SAM. Structural and expression studies were further conducted to confirm the role of BnSTM in mediating the regulatory network of cell wall and lignin synthesis. Besides lowering the transcript levels of early components of cell wall and lignin biosynthetic pathways in the SAM, BnSTM had a remarkable repressing effect on the expression of PHENYLALANINE AMMONIA LYASE2, CINNAMATE 4-4HYDROXYLASE, andCINNAMYL ALCOHOL DEHYDROGENASE. Since lignin formation is a feature of irreversible cell differentiation, these results suggest that one way in which BnSTM promotes indeterminate cell fate may be by preventing the expression of components of biochemical pathways involved in the accumulation of lignin in the meristematic cells. Overall these studies provide evidence for a novel function of BnSTM in enhancing the quality of in vitro produced meristems, and propose that this gene can be used as a potential target to improve regeneration of cultured embryos. 2 mutants and 1 wild type

Project description:BackgroundMicrospore embryogenesis represents a unique system of single cell reprogramming in plants wherein a highly specialized cell, the microspore, by specific stress treatment, switches its fate towards an embryogenesis pathway. In Brassica napus, a model species for this phenomenon, incubation of isolated microspores at 32°C is considered to be a pre-requisite for embryogenesis induction.ResultsWe have developed a new in vitro system at lower temperature (18°C) to efficiently induce microspore embryogenesis throughout two different developmental pathways: one involving the formation of suspensor-like structures (52.4%) and another producing multicellular embryos without suspensor (13.1%); additionally, a small proportion of non-responsive microspores followed a gametophytic-like development (34.4%) leading to mature pollen. The suspensor-like pathway followed at 18°C involved the establishment of asymmetric identities from the first microspore division and an early polarity leading to different cell fates, suspensor and embryo development, which were formed by cells with different organizations and endogenous auxin distribution, similar to zygotic embryogenesis. In addition, a new strategy for germination of microspore derived embryos was developed for achieving more than 90% conversion of embryos to plantlets, with a predominance of spontaneous doubled haploids plants.ConclusionThe present work reveals a novel mechanism for efficient microspore embryogenesis induction in B. napus using continuous low temperature treatment. Results indicated that low temperature applied for longer periods favours an embryogenesis pathway whose first division originates asymmetric cell identities, early polarity establishment and the formation of suspensor-like structures, mimicking zygotic embryogenesis. This new in vitro system provides a convenient tool to analyze in situ the mechanisms underlying different developmental pathways during the microspore reprogramming, breaking or not the cellular symmetry, the establishment of polarity and the developmental embryo patterning, which further produce mature embryos and plants.

Dataset Information

Effects of altered expression of LEAFY COTYLEDON1 and FUSCA3 on microspore-derived embryogenesis of Brassica napus L.

Publications

Effects of altered expression of <i>LEAFY COTYLEDON1</i> and <i>FUSCA3</i> on microspore-derived embryogenesis of <i>Brassica napus</i> L.

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