Mitogen-activated protein kinases are developmentally regulated during stress-induced microspore embryogenesis in Brassica napus L

Plant mitogen-activated protein kinase (MAPK) cascades are involved in extracellular stress signalling pathways, leading to different cellular responses. Stress-induced microspore embryogenesis involves the internalization of an extracellular stress signal, generating a number of cellular responses where MAPK cascades might be involved. These responses include a change of the developmental programme, the entry into an early proliferative stage and, subsequently, into differentiation stages during haploid embryogenesis. In this work we studied the expression during microspore embryogenesis of several kinases, to assess their putative role in these events. The known Brassica napus MAP kinase kinase kinases (MAP3Ks BnMAP3K1, BnMAP3K1 and BnMAP3K, the BnBSK kinase and B. napus extracellular signal-regulated kinase (ERK) homologues were analysed by electron microscope (EM) in situ hybridization, immuno-gold labelling, immunofluorescence and western blotting. The differential in situ expression of these kinases suggests a role for them during embryogenesis. Two different expression patterns were observed, indicating a different regulation. BnMAP3K1, BnMAP3K, and the ERKs showed a pattern consistent with a role mainly in proliferative events. Conversely, BnMAP3K1 and BnBSK, presented a pattern that suggested an involvement in differentiation stages. In addition, ERK homologues migrate to the nucleus immediately after induction, being found in a phosphorylated state in a larger amount.     

Arabinogalactan protein profiles and distribution patterns during microspore embryogenesis and pollen development in Brassica napus

Arabinogalactan proteins (AGPs), present in cell walls, plasma membranes and extracellular secretions, are massively glycosylated hydroxyproline-rich proteins that play a key role in several plant developmental processes. After stress treatment, microspores cultured in vitro can reprogramme and change their gametophytic developmental pathways towards embryogenesis, thereby producing embryos which can further give rise to haploid and double haploid plants, important biotechnological tools in plant breeding. Microspore embryogenesis constitutes a convenient system for studying the mechanisms underlying cell reprogramming and embryo formation. In this work, the dynamics of both AGP presence and distribution were studied during pollen development and microspore embryogenesis in Brassica napus, by employing a multidisciplinary approach using monoclonal antibodies for AGPs (LM2, LM6, JIM13, JIM14, MAC207) and analysing the expression pattern of the BnAGP Sta 39–4 gene. Results showed the developmental regulation and defined localization of the studied AGP epitopes during the two microspore developmental pathways, revealing different distribution patterns for AGPs with different antigenic reactivity. AGPs recognized by JIM13, JIM14 and MAC207 antibodies were related to pollen maturation, whereas AGPs labelled by LM2 and LM6 were associated with embryo development. Interestingly, the AGPs labelled by JIM13 and JIM14 were induced with the change of microspore fate. Increases in the expression of the Sta 39–4 gene, JIM13 and JIM14 epitopes found specifically in 2–4 cell stage embryo cell walls, suggested that AGPs are early molecular markers of microspore embryogenesis. Later, LM2 and LM6 antigens increased progressively with embryo development and localized on cell walls and cytoplasmic spots, suggesting an active production and secretion of AGPs during in vitro embryo formation. These results give new insights into the involvement of AGPs as potential regulating/signalling molecules in microspore reprogramming and embryogenesis.     

Cold pretreatment enhances microspore embryogenesis in oilseed rape (Brassica napus L.)

Stress is an essential component during embryogenesis induction in microspore culture. Cold pretreatment has been used in cereal microspore culture but very seldom attempted in Brassica microspore culture. The effect of cold pretreatment of flower buds subjected to a liquid medium on microspore embryogenesis was investigated in spring and winter Brassica napus, as well as in B. rapa and B. oleracea. Cold pretreatment significantly enhanced microspore embryogenesis (by 1–7 fold) compared to commonly used microspore culture protocol in B. napus, while it was less effective in B. rapa or even negative in B. oleracea. The appropriate duration of cold pretreatment was found to be 2–4 days, which stimulated the best microspore embryogenesis. Cold pretreatment was also able to promote embryo development including the improvement of embryo quality and acceleration of embryogenesis. When incorporating with medium refreshing, cold pretreatment could initiate the most microspore embryogenesis than any other treatment used. With further improvement cold pretreatment method may have a positive potential in Brassica breeding programmes.     

Cytoskeletal changes and induction of embryogenesis in microspore and pollen cultures of Brassica napus L.

Microspores and pollen of Brassica napus were cultured under conditions leading to embryo formation. Concomitant changes in cytoskeletal configurations were analysed. The microfilamental cytoskeleton exhibited a loss of polarity in embryogenic cells but cytochalasin treatment revealed that microfilaments do not influence embryogenesis. Two embryogenic pathways started from microspores and were either characterized by turned division planes or by division when the nucleus was in the cell centre. In both cases microtubules clearly exhibited new arrangements and likely played a major role in newly induced symmetrical division. In pollen, embryogenic development started in the vegetative cell provided the generative cell was arrested near the pollen wall. The concomitant disappearance of defined microtubular arrays is likely to be responsible for the positioning of the cell.     

Effects of ascorbic acid,alpha-tocopherol,and glutathione on microspore embryogenesis in Brassica napus L.

The impact of culture conditions and addition of antioxidants to media on microspore embryogenesis in rapeseed (Brassica napus cv. ‘PF704’) was investigated. Different concentrations of ascorbic acid (0, 5, 10, 20, 50, 100, and 200 mg l^?1) and alpha (α)-tocopherol (0, 5, 10, 20, 50, 100, and 200 mg l^?1) were evaluated along with two temperature pretreatments (18 d at 30°C; 2 d at 32.5°C followed by 16 d at 30°C). In addition, combinations of reduced glutathione (0, 10, 50, and 100 mg l^?1) and ascorbic acid (5 and 10 mg l^?1) were tested. Microspore embryogenesis was significantly enhanced using 10 mg l^?1 ascorbic acid (334 embryos per Petri dish) compared with untreated cultures (184 embryos per Petri dish) at 30°C. α-Tocopherol (5 and 10 mg l^?1) enhanced (312 and 314 embryos per Petri dish, respectively) microspore embryogenesis relative to untreated cultures (213 embryos per Petri dish) at 30°C, although there were no significant differences among cultures treated with 5–50 mg l^?1 α-tocopherol. When 50 mg l^?1 α-tocopherol was combined with 5 or 10 mg l^?1 ascorbic acid, embryogenesis was significantly enhanced (308 and 328 embryos per Petri dish, respectively) relative to other ascorbic acid levels. Moreover, 10 mg l^?1 of reduced glutathione and 5 mg l^?l ascorbic acid enhanced microspore embryogenesis (335 embryos per Petri dish) compared to cultures without reduced glutathione (275 embryos per Petri dish). Microspore embryogenesis could be improved by adding ascorbic acid, α-tocopherol, and reduced glutathione when the appropriate combination and temperature pretreatment were selected.     

Characterization of a pollen-specific gene family from Brassica napus which is activated during early microspore development

In this paper we describe the isolation and characterization of a genomic clone (Bp4) from Brassica napus which contains three members of a pollen-specific multigene family. This family is composed of 10 to 15 closely related genes which are expressed in early stages of microspore development. The complete nucleotide sequence of the clone Bp4 and of three homologous cDNA clones is reported. One of the genes (Bp4B) contained in the genomic clone is believed to be non-functional because of sequence rearrangements in its 5 region and intron splicing sites. The remaining genes (Bp4A and Bp4C), as well as the cDNA clones, appear to code for small proteins of unique structure. Three different types of proteins can be predicted as a result of the deletion of carboxy or amino terminal portions of a conserved core protein. These proteins all share a common alternation of hydrophobic and hydrophilic domains. A fragment of the genomic clone containing the gene Bp4A, as well as the non-functional gene Bp4B, was introduced into tobacco plants via Agrobacterium-mediated transformation. The functional gene Bp4A is expressed in transgenic tobacco plants and shows spatial and temporal regulation consistent with the expression patterns seen in Brassica napus.     

Induction of microspore embryogenesis in Brassica napus L. is accompanied by specific changes in protein synthesis

Culture temperature determines the developmental fate of isolated microspores from Brassica napus L. At 18°C, tricellular pollen develops, whereas culture at 32°C for 8 h leads to the quantitative and synchronous induction of embryogenesis, and ultimately to the formation of embryos. We investigated the changes in protein synthesis that are associated with this 8-h inductive period by using in-situ [³⁵S]methionine labeling, followed by two-dimensional (2-D) gel electrophoretic analysis of the radiolabeled proteins. Qualitative and quantitative computer analyses of 2-D [³⁵S]methionine protein patterns showed six polypeptides specifically labeled under embryogenic culture conditions. Eighteen polypeptides incorporated [³⁵S]methionine at a statistically significant higher rate under embryogenic culture conditions (32°C) than in the controls (18°C), whereas one protein was preferentially labeled under non-embryogenic culture conditions (18°C). These results indicate that only a limited number of proteins detectable in the 2-D gels of microspore extracts are associated with the early induction of embryogenesis. The reproducible identification of the differentially radiolabeled proteins in the 2-D gels allow the sequencing of representative peptides and the isolation of the corresponding cDNAs. This may lead to the identification and characterization of proteins associated with the very first stages of plant embryogenesis.Abbreviations 2-D two-dimensional We would like to thank Dr. H. Van Steeg (Rijks Instituut voor Milieubeheer (RIVM), Bilthoven, The Netherlands) for use of the PhosphorImager apparatus. This research was carried out as part of the EC-Bridge project Regulation of the inductive phase of microspore embryogenesis and EC-Science project The role of mitotic and cytoskeletal genes in the induction of plant cell division.     

Improved microspore embryogenesis induction and plantlet regeneration using putrescine,cefotaxime and vancomycin in Brassica napus L.

The effects of three periods of exposure (12, 24 and 48 h) to different levels of putrescine (0, 0.2, 0.5, 1.0, 2.0 and 5.0 mg l^?1), as well as three incubation periods (24, 48 and 72 h) to different levels of cefotaxime and vancomycin (0, 50, 100, 200 and 500 mg l^?1) on microspore embryogenesis of rapeseed cv. ‘Hyola 401’ were assessed. Microspore embryogenesis was enhanced about threefold compared with untreated culture following 48 h treatment with 0.2 mg l^?1 putrescine. Putrescine treatment at 0.5 mg l^?1 for 48 h effectively induced root formation and increased normal plantlet regeneration by 92 % when microspore-derived embryos (MDEs) were transferred to regeneration medium. The highest embryo yield (184.2 embryos Petri dish^?1) was possible when induction medium was supplemented with 50 mg l^?1 cefotaxime for 24 h and the highest normal regeneration was observed in cultures exposed to 50 and 100 mg l^?1 at all durations tested. More abnormal MDEs (76 and 82 %) were observed when microspores treated with 200 and 500 mg l^?1 cefotaxime many of which failed to regenerate normally and resulted in callusing. Vancomycin at 100 mg l^?1 during the 48 h exposure increased the number of MDEs (181.6 embryos Petri dish^?1) in contrast to untreated cultures (93.6 embryos Petri dish^?1) but, normal plantlet regeneration decreased as vancomycin level increased and high callusing (84 and 90 %) was observed with 200 and 500 mg l^?1 for 72 h. Microspore embryogenesis and plant regeneration could be improved by putrescine, cefotaxime and vancomycin when appropriate levels and durations of incubation were selected.     

Efficient induction of microspore embryogenesis using abscisic acid,jasmonic acid and salicylic acid in Brassica napus L

The stress hormones abscisic acid (ABA), jasmonic acid (JA) and salicylic acid (SA) play an important role in the regulation of physiological processes and are often used in tissue culture to promote somatic embryogenesis and to enhance the quality of somatic embryos. Despite many studies on Brassica napus microspore culture, the effects of stress hormones (ABA, JA and SA) on microspore embryogenesis are not well explored. In this study, the effects of three incubation periods (6, 12 and 24 h) at different levels of ABA, JA and SA (0, 0.2, 0.5, 1.0, 2.0 and 5.0 mg l^?1) on microspore embryogenesis of rapeseed (B. napus L.) cv. ‘Regent’ were investigated. ABA (0.5 mg l^?1 for 12 h) enhanced microspore embryogenesis by about threefold compared with untreated cultures and increased normal plantlet regeneration by 68 %. ABA treatment also effectively reduced secondary embryo formation at all concentrations tested but enhanced callusing at high levels, for example 67 % at 1.0 mg l^?1 for 24 h. Highest embryo yield (286.0 embryos Petri dish^?1) was achieved using 1.0 mg l^?1 JA for 24 h and highest normal plantlet regeneration (54 %) was observed in cultures exposed to 0.5 mg l^?1 JA for 12 h. JA (5.0 mg l^?1 for 24 h) also reduced the germination of microspore-derived embryos on regeneration medium by 21 %. SA at 0.2 and 0.5 mg l^?1 for 6 h increased microspore embryogenesis (184.0 and 193.4 embryos Petri dish^?1) relative to the control (136.2 embryos Petri dish^?1). However, SA did not improve normal regeneration, secondary embryo formation or callusing. Microspore embryogenesis and plant regeneration could be improved by ABA, JA as well as SA when the appropriate level and duration of incubation were selected.     

Hsp70 and Hsp90 change their expression and subcellular localization after microspore embryogenesis induction in Brassica napus L

A stress treatment of 32 degrees C for at least 8h was able to change the gametophytic program of the microspore, switching it to embryogenesis in Brassica napus, an interesting model for studying this process in vitro. After induction, some microspores started symmetric divisions and became haploid embryos after a few days, whereas other microspores, not sensitive to induction, followed their original gametophytic development. In this work the distribution and ultrastructural localization of two heat-shock proteins (Hsp70 and Hsp90) throughout key stages before and after embryogenesis induction were studied. Both Hsp proteins are rapidly induced, localizing in the nucleus and the cytoplasm. Immunogold labeling showed changes in the distribution patterns of these proteins, these changes being assessed by a quantitative analysis. Inside the nucleus, Hsp70 was found in association with RNP structures in the interchromatin region and in the nucleolus, whereas nuclear Hsp90 was mostly found in the interchromatin region. For Hsp70, the accumulation after the inductive treatment was accompanied by a reversible translocation from the cytoplasm to the nucleus, in both induced (embryogenic) and noninduced (gametophytic) microspores. However, the translocation was higher in embryogenic microspores, suggesting a possible additional role for Hsp70 in the switch to embryogenesis. In contrast, Hsp90 increase was similar in all microspores, occurring faster than for Hsp70 and suggesting a more specific role for Hsp90 in the stress response. Hsp70 and Hsp90 colocalized in clusters in the cytoplasm and the nucleus, but not in the nucleolus. Results indicated that stress proteins are involved in the process of microspore embryogenesis induction. The differential appearance and distribution of the two proteins and their association at specific stages have been determined between the two systems coexisting in the same culture: embryogenic development (induced cells) and development of gametes (noninduced cells).     

Significance of preprophase bands of microtubules in the induction of microspore embryogenesis of Brassica napus

Microspores of Brassica napus L. cv. Topas, undergo embryogenesis when cultured at 32.5 °C for the first 18–24 h and then at 25 °C. The first division in heat-treated microspores is a symmetric division in contrast to the asymmetric division found after the first pollen mitosis in-planta or in microspores cultured continuously at 25 °C. This asymmetric division is unique in higher plants as it results in daughter cells separated by a non-consolidated wall. The cytoskeleton has an important role in such morphological changes. We examined microtubule (MT) organization during the first 24 h of heat induction in the embryogenic B. napus cv. Topas and the non-embryogenic B. napus breeding line 0025. Preprophase bands (PPBs) of MTs appeared in cv. Topas microspores in late uninucleate microspores and in prophase figures after 4–8 h of heat treatment. However, more than 60% of the PPBs were not continuous bands. In contrast, PPBs were never observed in pollen mitosis; MT strands radiated from the surface of the nuclear envelope throughout microspore maturation to the end of prophase of pollen mitosis I, during in-planta development and in microspores cultured at 25 °C. Following 24 h of heat treatment, over 95% of the microspores appeared to have divided symmetrically as indicated by the similar size of the daughter nuclei, but only 7–16% of the microspores eventually formed embryos. Discontinuous walls were observed in more than 50% of the divisions and it is probable that the discontinuous PPBs gave rise to such wall abnormalities which may then obstruct embryo development. Preprophase bands were not formed in heat-treated microspores of the non-embryogenic line 0025 and the ensuing divisions showed discontinuous walls. It is concluded that the appearance of PPBs in heat-induced microspores marks sporophytic development and that continuous PPBs are required for cell wall consolidation and embryogenesis. It follows that induced structures with two equally condensed nuclei, do not necessarily denote symmetric divisions. Received: 22 October 1998 / Accepted: 28 November 1998     

14-3-3 isoforms and pattern formation during barley microspore embryogenesis

The members of the 14-3-3 isoform family have been shown to be developmentally regulated during animal embryogenesis, where they take part in cell differentiation processes. 14-3-3 isoform-specific expression patterns were studied in plant embryogenic processes, using barley (Hordeum vulgare L.) microspore embryogenesis as a model system. After embryogenesis induction by stress, microspores with enlarged morphology showed higher viability than non-enlarged ones. Following microspore culture, cell division was only observed among the enlarged microspores. Western blot and immunolocalization of three barley 14-3-3 isoforms, 14-3-3A, 14-3-3B and 14-3-3C were carried out using isoform-specific antibodies. The level of 14-3-3C protein was higher in enlarged microspores than in non-enlarged ones. A processed form of 14-3-3A was associated with the death pathway of the non-enlarged microspores. In the early embryogenesis stage, 14-3-3 subcellular localization differed among dividing and non-dividing microspores and the microspore-derived multicellular structures showed a polarized expression pattern of 14-3-3C and a higher 14-3-3A signal in epidermis primordia. In the late embryogenesis stage, 14-3-3C was specifically expressed underneath the L(1) layer of the shoot apical meristem and in the scutellum of embryo-like structures (ELSs). 14-3-3C was also expressed in the scutellum and underneath the L(1) layer of the shoot apical meristem of 21 d after pollination (DAP) zygotic embryos. These results reveal that 14-3-3A processing and 14-3-3C isoform tissue-specific expression are closely related to cell fate and initiation of specific cell type differentiation, providing a new insight into the study of 14-3-3 proteins in plant embryogenesis.     

Initiation and development of microspore embryogenesis and plant regeneration of Brassica nigra

Brassica nigra is generally regarded as a recalcitrant species for microspore culture among Brassica crops. Conditions for reliable induction of microspore embryogenesis of B. nigra were studied in this context. Flower bud length and microspore developmental stage were correlated with further embryogenesis. The optimal bud size range was 2.0–2.5 mm for the highest proportion of totipotent, late uninucleate microspore and the highest frequency of microspore embryogenesis. Treatment of a short heat shock by incubating the microspore culture at 32°C for 24 h was suitable for the microspore survival, sustained cell divisions, and further induced embryogenesis. Subsequently, the use of NLN medium with the addition of 13% sucrose and 0.1% activated charcoal (AC) provided the optimal conditions for the development of microspore-derived embryos (MDEs). The early cotyledonary (EC) stage embryos cultured on MS medium fortified with 4.6 μM zeatin (ZT) and 0.12 μM indole-3-acetic acid (IAA) resulted in the most efficient rates of plantlet regeneration. The ploidy levels of regenerated plants of B. nigra were determined by flow cytometry, revealing that 50.6% were diploid. The results enable the advancement of breeding programs and genetic studies in B. nigra.     

Uptake and distribution of sinigrin in microspore derived embryos of Brassica napus L

In Brassica napus, glucosinolates are transported from all parts of the plant into the embryo during seed development. In this study we describe the uptake of the alkenyl glucosinolate sinigrin by microspore derived embryos from high and low glucosinolate genotypes. Microspore derived embryos develop completely isolated from maternal tissues unlike zygotic embryos, which contains glucosinolates transported into the embryo synthesised in the vegetative tissues. The sinigrin in the culture medium was almost completely absorbed by the embryos after three days of culture. The embryos of high and low glucosinolate genotypes were equally capable of absorbing sinigrin from the medium. A significant increase in different alkenyl glucosinolates following feeding of sinigrin suggests induction of biosynthetic enzymes in the embryos. Following excess feeding of sinigrin, we found a strong uptake against a concentration gradient and stable accumulation by the embryos. The glucosinolate was detected in single dissected cotyledons by a photometric test and by HPLC. This test could potentially be useful for screening mutants defective in glucosinolate uptake into the embryo.     

Effects of culture density,conditioned medium and feeder cultures on microspore embryogenesis in Brassica napus L. cv. Topas

Summary In microspore cultures of Brassica napus L. cv. Topas, embryo yield increases with culture density up to about 40,000 microspores per ml. A much higher density (100,000 per ml) appears inhibitory to embryogenesis. A relatively high culture density (30,000 or 40,000 per ml) for the first 2–4 days of culture is crucial for embryogenesis, after which cultures may be diluted to allow better embryo growth.Medium conditioned by culturing microspores at 30,000 or 40,000 per ml for 1 day improved microspore-embryo yield in low density cultures (3,000 or 4,000 per ml) more than 3-fold. In contrast, media conditioned with microspores from 1–4 days or 0–4 days of culture were inhibitory.Use of feeder cultures resulted in up to 10-fold increase of embryo yield in low density microspore cultures, depending on the method used. Filter papers and other membranes placed on top of feeders greatly inhibited embryogenesis in the feeder layer as well as microspores cultured on the feeder, possibly due to poorer gaseous exchange.