A high throughput <Emphasis Type="Italic">Brassica napus</Emphasis> microspore culture system: influence of percoll gradient separation and bud selection on embryogenesis

Microspore culture for the purpose of developing doubled haploid plants is routine for numerous plant species; however, the embryo yield is still very low compared with the total available microspore population. The ability to select and isolate highly embryogenic microspores would be desirable for high embryo yield in microspore culture. To maximize the efficiency of canola microspore culture, a combination of bud size selection and microspore fractionation using a Percoll gradient was followed. This approach has consistently given high embryo yields and uniform embryo development. Microspores isolated from buds 1.5 to 4.4 mm in length of Brassica napus genotypes Topas 4079, DH12075, Westar and 0025 formed embryos at different frequencies. The most embryogenic bud size range varied with each cultivar: Topas 4079 3.5–3.9 mm, DH12075 2.0–2.4 mm, and Westar and 0025 2.5–2.9 mm. When the microspores from 2.0 to 2.4 mm buds of DH12075 were carefully layered on top of a discontinuous Percoll gradient of 10, 20 and 40%, and subsequently spun through the Percoll layers by centrifugation, bands were formed containing populations of microspores of uniform developmental stage. The middle layer of the gradient contained the late uninucleate and early binucleate microspores that were the most embryogenic. In addition, the relationship between the bud size, developmental stage of isolated microspores, Percoll gradient concentration and the embryogenic frequency of each cultivar were studied. Optimization of these factors is required for each genotype evaluated.     

Identification of potentially embryogenic microspores in Brassica napus

Studies were undertaken with Brassica napus L. cv. Topas to identify buds containing microspores predisposed to embryogenesis in vitro and to investigate bud and microspore development in relation to this process. No significant correlation was found between the final embryo number and bud components. There appears to be a developmental window of less than 8 h duration during which microspores are very likely to form embryos: over 70% of the microspores can undergo division and up to 70% of these can form embryos. Embryos were mainly obtained from late uninuucleate to early binucleate microspores: the former contained mainly a G2 or M phase nucleus located at the microspore periphery and the latter a generative nucleus (associated with the intine) and a vegetative nucleus. Observations indicated that only the vegetative nucleus contributed to embryo formation. The first embryogenic division occurred between 8 and 16 h for uninucleate- and between 8 and 48 h for binucleate-derived embryos.     

NO, ROS, and cell death associated with caspase-like activity increase in stress-induced microspore embryogenesis of barley

Under specific stress treatments (cold, starvation), in vitro microspores can be induced to deviate from their gametophytic development and switch to embryogenesis, forming haploid embryos and homozygous breeding lines in a short period of time. The inductive stress produces reactive oxygen species (ROS) and nitric oxide (NO), signalling molecules mediating cellular responses, and cell death, modifying the embryogenic microspore response and therefore, the efficiency of the process. This work analysed cell death, caspase 3-like activity, and ROS and NO production (using fluorescence probes and confocal analysis) after inductive stress in barley microspore cultures and embryogenic suspension cultures, as an in vitro system which permitted easy handling for comparison. There was an increase in caspase 3-like activity and cell death after stress treatment in microspore and suspension cultures, while ROS increased in non-induced microspores and suspension cultures. Treatments of the cultures with a caspase 3 inhibitor, DEVD-CHO, significantly reduced the cell death percentages. Stress-treated embryogenic suspension cultures exhibited high NO signals and cell death, while treatment with S-nitrosoglutathione (NO donor) in control suspension cultures resulted in even higher cell death. In contrast, in microspore cultures, NO production was detected after stress, and, in the case of 4-day microspore cultures, in embryogenic microspores accompanying the initiation of cell divisions. Subsequent treatments of stress-treated microspore cultures with ROS and NO scavengers resulted in a decreasing cell death during the early stages, but later they produced a delay in embryo development as well as a decrease in the percentage of embryogenesis in microspores. Results showed that the ROS increase was involved in the stress-induced programmed cell death occurring at early stages in both non-induced microspores and embryogenic suspension cultures; whereas NO played a dual role after stress in the two in vitro systems, one involved in programmed cell death in embryogenic suspension cultures and the other in the initiation of cell division leading to embryogenesis in reprogrammed microspores.     

Embryogenesis and plant regeneration from isolated microspores of Brassica rapa L. ssp. Oleifera

Summary Conditions favourable to embryogenesis from isolated microspores of Brassica rapa L. ssp. oleifera (canola quality) were identified. A population with enhanced responsiveness for microspore embryogenesis (C200) was synthesized by crossing individual plants showing microspore embryogenic potential. For optimal microspore embryogenesis, buds (2–3mm in length, containing mid-late uninucieate microspores) were collected from older plants (2 months old) and microspores isolated and washed in iron-free B5 medium. NLN medium with its iron content reduced to half was beneficial for initial microspore culture. An elevated temperature(33–35°C) during the first day of culture, followed by maintenance at 25°C resulted in dozens of embryos from each isolation (about 100 buds). Seeds were obtained from plants regenerated from microsporederived embryos after colchicine treatment.     

Identification and characterization of genes expressed in early embryogenesis from microspores of <Emphasis Type="Italic">Brassica napus</Emphasis>

To understand the mechanism in induction of embryogenesis from microspores of Brassica napus, we isolated exhaustively the genes expressed differentially during the early stage of microspore culture. A subtracted cDNA library composed of up-regulated genes during androgenic initiation was produced by suppression subtractive hybridization followed by differential screening by dot blot hybridization, and a total of 136 non-redundant expressed sequence tags were identified. Analysis of the potential functions of the genes showed that 64% of these genes were homologous to known genes, and the remaining ones have not been previously reported to participate in embryogenesis. Many embryo-specific genes were contained in the isolated genes, for example, genes cording lipid transfer protein, napin, cruciferin, oleosin, and phytosulfokine. Real-time RT-PCR analysis for 15 selected genes, which are understood to not be related with embryogenesis, demonstrated that all genes were expressed highly in the early stage of microspore embryogenesis. A few genes also showed higher expression in microspores cultured in non-embryogenic condition or in later stages of embryos. A principal component analysis based on expression profiles of the 15 genes demonstrated that these genes were classified into 2 groups, one characterized by their high expression in initiation of embryogenesis, and the other characterized by their expression in the early to middle stage of embryogenesis. The expressions of these genes were confirmed in zygotic embryos. The identification and characterization of the genes isolated in the present study provide novel information on microspore embryogenesis in Brassica.     

Selection of Brassica napus L. embryogenic microspores by flow sorting

Flow cytometry can be used to select and sort microspore subpopulations of Brassica napus cv. Topas. Data obtained from embryogenic microspore populations were used to identify potentially embryogenic microspores from developmentally heterogeneous microspore populations based on differences in forward light scatter and green autofluorescence. Culture enrichment for embryogenic microspores is possible. Frequencies of 8 and 14% microspore embryogenesis were obtained when selected 16 h and 72 h after culture initiation. This represents 5- and 13-fold increase in microspore embryogenesis compared to non-sorted controls.     

甘蓝型油菜小孢子胚状体发生的细胞学观察

应用甘蓝型油菜ＤＨ系保６０４为材料研究小孢子胚发生过程,结果表明,在小孢子离体培养１～５ｄ内,随培养天数增加,小孢子的存活率迅速下降,部分小孢子培养后出现细胞膨大和分裂,并沿２－细胞。“ｆ”形３细胞,多细胞原体,胚柄球形胚,心形胚最终发育成鱼雷形胚,一般在心形胚阶段,胚柄脱离胚主体部分游离到培养基中,大多数膨大的细胞不能分裂或分裂后停止发育或发育异常。     

The 35S-CaMV promoter is silent during early embryogenesis but activated during nonembryogenic sporophytic development in microspore culture

Summary The cauliflower mosaic virus 35S (35S-CaMV) promoter, which is generally used as a constitutive promoter in plants, is known to be silent during microspore and pollen development. Here we analyzed whether the 35S-CaMV promoter fused to thegus (-glucuronidase) gene can be used as a marker for early sporophytic development in embryogenic microspore cultures of tobacco andBrassica napus. In microspore culture ofB. napus, the 35S-CaMV promoter remained off from the start of embryogenic culture up to the mid-cotyledonary embryo stage. 35S-CaMV promoter activity was only present in those microspores that initiated sporophytic development, but failed to enter embryogenic development. Similar results were also obtained with shed-microspore cultures of tobacco, in which rapid, direct embryogenesis takes place. In isolated-microspore cultures, in which embryogenesis is delayed, an intermitting period of sporophytic development was observed, characterized by extensive 35S-CaMV promoter activity. Therefore, the 35S-CaMV promoter discriminates between two classes of sporophytic development: it is activated in microspores which change fate from gametophytic into (temporarily) nonembryogenic sporophytic development, whereas the promoter is silent in sporophytic microspores that enter embryogenic development directly. This mirrors our observation that the 35S-CaMV promoter is also silent in young zygotic embryos.     

Mapping of quantitative trait loci for microspore embryogenesis-related traits in the oilseed rape doubled haploid population DH4069 × Express 617

Oilseed rape (Brassica napus L.) is among dicotyledonous plants, a model species for microspore embryogenesis. Tremendous differences exist among oilseed rape genotypes in their embryogenic response and direct embryo to plant conversion. Despite some attempts to identify relevant genes, the genetic basis of these traits remains largely unknown. The objective of this work was to develop and to provide to the scientific community a doubled haploid (DH) population derived from a cross of the reported highly embryogenic genotype DH4079 and the low embryogenic inbred line Express 617. A population of 198 DH-lines was generated and genotyped with the Brassica 60 K Illumina Infinium? SNP array. The parental and the F1 genotypes as well as between 81 and 107 DH-lines were characterized for their number of microspores, number of microspore-derived embryos, embryo survival rate, direct embryo to shoot conversion, and related traits. The results obtained for the F1 genotype were mostly in between the two parents. SNP markers in the DH population showed to 49% distorted segregation and of those 63% were in favor of DH4079. Significant genotypic differences were found for all traits and heritabilities ranged from 66 to 88%. Together, 13 quantitative trait loci (QTL) for the different traits were identified on linkage groups A01, A02, A05, A10, C04, and C06, and candidate genes were identified within their QTL confidence intervals.     

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).     

Stress-induced microspore embryogenesis in tobacco: an optimized system for molecular studies

Summary Specific stress treatments applied to isolated tobacco (Nicotiana tabacum L.) microspores efficiently induced haploid embryo formation in vitro. A heat shock at 33 or 37°C in the presence of sugar, as well as sucrose-starvation at 25°C, resulted in the formation of embryogenic microspores. A combination of both treatments had an additive effect. Under optimal induction conditions all viable microspores in the culture were embryogenic and developed subsequently into pollen embryos by culture at 25°C in a sugar-containing medium, with induction frequencies of more than 70% with respect to the initial microspore population. A high fraction of the early pollen embryos continued their development in vitro, giving rise to haploid plants. In contrast to other available systems for microspore/pollen embryogenesis, the new protocol allows the production of homogeneous populations of embryogenic microspores and early globular embryos in large-scale cultures, without any purification step, and is therefore well suited for biochemical and molecular work.Abbreviations EDTA ethylenediaminetetraacetate - DAPI 4,6-diamidino-2-phenylindole     

Efficient embryogenesis and regeneration in freshly isolated and cultured wheat (Triticum aestivum L.) microspores without stress pretreatment

The major advantage of doubled haploids in plant breeding is the immediate achievement of complete homozygosity. Desired genotypes are thus fixed in one generation, reducing time and cost for cultivar or inbred development. Among the different technologies to produce doubled haploids, microspore embryogenesis is by far the most common. It usually requires reprogramming of microspores by stress such as cold, heat, and starvation, followed by embryo development under stress-free conditions. We report here the development of a simple and efficient isolated microspore culture system for producing doubled haploid wheat plants in a wide spectrum of genotypes, in which embryogenic microspores and embryos are formed without any apparent stress treatment. Microspores were isolated from fresh spikes in a nutrient-free medium by stirring and cultured in medium A2 in the dark at 25°C. Once embryogenic microspores were formed, ovaries and phytohormones were added directly to the cultures without changing the medium. The cultures were incubated in the dark at 25–27°C until the formation of embryos and then the embryos were transferred to regeneration medium. The regeneration frequency and percentage of green plants increased significantly using this protocol compared to the shed microspore culture method.Communicated by W. Harwood     

Improvement in efficiency of microspore culture to produce doubled haploid lines of Ethiopian mustard

Factors affecting microspore embryogenesis of Ethiopian mustard (Brassica carinata A. Braun) were evaluated, including flower bud length, pollen developmental stage, and microspore density. An embryogenic frequency of 300 embryos per Petri plate was observed with NLN (Nitsch-Lichter-Nitsch) medium supplemented with 13% sucrose, 3.0–3.4-mm-long buds, and a plating density of 65,000 microspores/ml. About 65% of the microspores from buds 3.0–3.4-mm long were at the late uninucleate stage. Microspore-derived embryos were successfully transferred to solid medium for germination. After 4 wk, the resulting plantlets were transplanted to a soilless potting mixture and grew well under greenhouse conditions.     

Modification of cell development in vitro: The effect of colchicine on anther and isolated microspore culture in Brassica napus

In an attempt to discover the biological basis of microspore derived embryogenesis, the effect of the antimicrotubule agent colchicine on anther and free microspore embryogenesis was investigated. The microtubule inhibitor colchicine promoted embryogenesis from cultured anthers, both with regard to the number of anthers responding and the number of embryos being produced per anther. A similar promotional response was also observed with cultured microspores. Although the parameters for cultured anthers and free microspores differed, administration of the drug for a short period immediately prior to pollen mitosis I seems to exert the maximum promotional effect. Of the five cultivars of Brassica napus studied, all responded to colchicine treatment. However, the drug did release more embryogenic potential in poor-responding varieties (i.e. Lirawell and Optima) than in the highest responding variety (Topas). Colchicine also resulted in increased embryogenic response in microspores cultured at lower temperatures.These results are considered in terms of models proposed to explain the switch in microspore development from a gametophytic to a sporophytic pathway. The use ofcolchicine as agent to promote embryogenesis in previously recalcitrant species other than Brassica is also discussed.     

Effects of aluminum on DNA synthesis, cellular polyamines, polyamine biosynthetic enzymes and inorganic ions in cell suspension cultures of a woody plant, Catharanthus roseus

Individual buds of Brassica napus cv. Topas, near the first pollen mitosis, were used for microspore culture. Bud and petal lengths were recorded. Microspores isolated from the individual buds were plated and small samples were fixed for cytology. Following embryo induction and three weeks of culturing, numbers of embryos were scored. Bud and petal lengths did not accurately indicate which buds would supply microspores that would form embryos at high frequencies. Fluorescence microscopy was used to examine nuclei stained with Hoechst 33258 and vacuolar morphology of microspores was revealed by the weaker fluorescence due to glutaraldehyde fixation. Following isolation, nuclear and vacuolar characteristics were used to stage the microspores as miduninucleate, late uninucleate vacuolate, late uninucleate, mitotic, or binucleate. The relationship of developmental stage to the frequency of microspore-derived embryos was evaluated. A classification scheme was developed which uses the relative proportions of microspores at each of the stages to identify microspore isolations that would form embryos at high frequencies. It was found that when 1 to 87% of the isolated microspores were binucleate, 21.4 ± 3.0% of the viable microspores developed into embryos. This was a significant ( P < 0.001) increase over the other 3 classes. The ability to select highly embryogenic microspore isolations is of great advantage for developmental cell biology studies.     

Early markers of in vitro microspore reprogramming to embryogenesis in olive (Olea europaea L.)

Microspore embryogenesis to form haploid and double-haploid embryos and regenerated plants is an efficient method of producing homozygous lines for crop breeding. In trees, the process is of special interest since classical methods are impractical in many cases, as in Olea europaea L. Recently, a convenient method has been developed for microspore embryogenesis induction by stress in olive isolated microspores in vitro cultures. In the present work, the switch of the microspore developmental pathway and the formation of microspore-derived multicellular proembryos have been achieved and a cytochemical and immunocytochemical analysis was performed in the early stages. The young microspore proembryos displayed defined features different to both, the in vivo gametophytic, and the in vitro non-responsive microspores. Reprogrammed microspores showed an absence of starch, the occurrence of a first symmetrical division and cytokinesis, the presence of an abundant ribosomal population, and changes in cellulosic and pectic cell wall components which constituted early markers of the embryogenic microspore process. They provided new insights on the molecular and cellular events associated with the microspore reprogramming of woody plants, and specifically in olive, providing interesting knowledge which could guide future selection and regeneration strategies in this fruit tree of high economic interest.