Microspore Embryogenesis In Wheat Research Articles

Impact of Ionic Liquids on Induction of Wheat Microspore Embryogenesis and Plant Regeneration

Ionic liquids are novel compounds with unique chemical and physical properties. They can be received based on synthetic auxins like 2,4-dichlorophenoxyacetic acid or dicamba, which are commonly used hormones in microspore embryogenesis. Nevertheless, ionic liquids have not been adapted in plant in vitro culture thus far. Therefore, we studied the impact of ionic liquids on the ability to undergo microspore embryogenesis in anther cultures of wheat. Two embryogenic and two recalcitrant genotypes were used for this study. Ten combinations of ionic liquids and 2,4-dichlorophenoxyacetic acid were added to the induction medium. In most cases, they stimulated induction of microspore embryogenesis and green plant regeneration more than a control medium supplemented with only 2,4-dichlorophenoxyacetic acid. Two treatments were the most favorable, resulting in over two times greater efficiency of microspore embryogenesis induction in comparison to the control. The effect of breaking down the genotype recalcitrance (manifested by green plant formation) was observed under the influence of 5 ionic liquids treatments. Summing up, ionic liquids had a positive impact on microspore embryogenesis induction and green plant regeneration, increasing the efficiency of these phenomena in both embryogenic and recalcitrant genotypes. Herbicidal ionic liquids can be successfully used in in vitro cultures.

Analysis of wheat microspore embryogenesis induction by transcriptome and small RNA sequencing using the highly responsive cultivar "Svilena".

BackgroundMicrospore embryogenesis describes a stress-induced reprogramming of immature male plant gametophytes to develop into embryo-like structures, which can be regenerated into doubled haploid plants after whole genome reduplication. This mechanism is of high interest for both research as well as plant breeding. The objective of this study was to characterize transcriptional changes and regulatory relationships in early stages of cold stress-induced wheat microspore embryogenesis by transcriptome and small RNA sequencing using a highly responsive cultivar.ResultsTranscriptome and small RNA sequencing was performed in a staged time-course to analyze wheat microspore embryogenesis induction. The analyzed stages were freshly harvested, untreated uninucleate microspores and the two following stages from in vitro anther culture: directly after induction by cold-stress treatment and microspores undergoing the first nuclear divisions. A de novo transcriptome assembly resulted in 29,388 contigs distributing to 20,224 putative transcripts of which 9,305 are not covered by public wheat cDNAs. Differentially expressed transcripts and small RNAs were identified for the stage transitions highlighting various processes as well as specific genes to be involved in microspore embryogenesis induction.ConclusionThis study establishes a comprehensive functional genomics resource for wheat microspore embryogenesis induction and initial understanding of molecular mechanisms involved. A large set of putative transcripts presumably specific for microspore embryogenesis induction as well as contributing processes and specific genes were identified. The results allow for a first insight in regulatory roles of small RNAs in the reprogramming of microspores towards an embryogenic cell fate.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0782-8) contains supplementary material, which is available to authorized users.

Effect of ovary induction on bread wheat anther culture: ovary genotype and developmental stage, and candidate gene association.

Ovary pre-conditioned medium and ovary co-culture increased the efficiency of green doubled haploid plant production in bread wheat anther culture. The positive effect of this medium led to a 6- and 11-fold increase in the numbers of embryos and green plants, respectively, having a greater effect on a medium-low responding cultivar. Ovary genotype and developmental stage significantly affected microspore embryogenesis. By the use of Caramba ovaries it was possible to reach a 2-fold increase in the number of embryos and green plants, and to decrease the rate of albinism. Mature ovaries from flowers containing microspores at a late binucleate stage raised the number of embryos and green plants by 25–46% as compared to immature ovaries (excised from flowers with microspores at a mid-late uninucleate stage). The highest numbers of embryos and green plants were produced when using mature Caramba ovaries. Ovaries from Galeón, Tigre, and Kilopondio cultivars successfully induced microspore embryogenesis at the same rate as Caramba ovaries. Moreover, Tigre ovaries raised the percentage of spontaneous chromosome doubling up to 71%. Attempts were made to identify molecular mechanisms associated to the inductive effect of the ovaries on microspore embryogenesis. The genes TAA1b, FLA26, and WALI6 associated to wheat microspore embryogenesis, the CGL1 gene involved in glycan biosynthesis or degradation, and the FER gene involved in the ovary signaling process were expressed and/or induced at different rates during ovary culture. The expression pattern of FLA26 and FER could be related to the differences between genotypes and developmental stages in the inductive effect of the ovary. Our results open opportunities for new approaches to increase bread wheat doubled haploid production by anther culture, and to identify the functional components of the ovary inductive effect on microspore embryogenesis.

Induction medium osmolality improves microspore embryogenesis in wheat and triticale

The objective of this study was to determine the effect of induction medium osmolality on embryogenesis and green plant production in wheat and triticale. Isolated microspores of wheat and triticale were subjected to a range of osmolality (300–500 mOsm kg−1) using mannitol. In both species, the maximum number of embryo-like structures (ELS) and green plants were obtained at 350 mOsm kg−1 when the induction medium was supplemented with 9.1 g L−1 of mannitol. A sharp decline in microspore response was observed at higher osmolality. These results demonstrate the effect of osmolality on induction of ELS and production of green plants indicating that the process of microspore embryogenesis can be improved in wheat and triticale by increasing osmolality of the induction medium to 350 mOsm kg−1.

Cefotaxime prevents microbial contamination and improves microspore embryogenesis in wheat and triticale

Cefotaxime (100mg/l) mitigate occasional gram negative bacterial contamination in wheat and triticale microspore culture and most importantly it increases cell growth and green plant production. Isolated microspore culture is a promising option to rapidly fix the product of meiotic recombination of F1 hybrids, in the process of varietal development. Clean culture and high embryogenesis rate are essential to commercial triticale and wheat microspore cultures. So, this study investigated (1) contaminants from isolated microspores cultures, (2) two antibiotics to control bacterial growth, and (3) the contribution of antibiotics to increased microspore-derived embryo-like structures (ELS), green and albino plants. Five species of bacteria were identified in contaminated cultures (Erwinia aphidicola, Pantoea agglomerans, Pseudomonas sp., Staphylococcus epidermis and Staphylococcus warneri) using fatty acid analysis and 16S ribosomal RNA sequences analysis, and yeast. Antibacterial susceptibility test using Cefotaxime and Vancomycin resulted in strong inhibition of 24 bacterial isolates, using Cefotaxime at 100mg/l, but not Pseudomonas sp. Other antibiotic treatments inhibited bacterial growth at least partially. Microspore induction medium supplemented with the same antibiotics treatments resulted in successful microspore embryogenesis and green plant production. Antibiotic treatments were first tested in triticale and then validated in wheat cultivars AC Carberry and AC Andrew. Induction medium supplemented with Cefotaxime at 50 and 100mg/l substantially increased the formation of ELS and green plants in triticale and wheat, respectively. Incidentally, it also affected the occurrence of albinism in all genotypes. Our results demonstrated dual purpose of Cefotaxime for isolated microspore culture, most importantly it increases cell growth and success of microspore cultures in triticale and wheat genotypes, but would also prevent accidental loss of cultures with most common bacterial contaminants.

Microspore embryogenesis in wheat: new marker genes for early, middle and late stages of embryo development

Microspore embryogenesis involves reprogramming of the pollen immature cell towards embryogenesis. We have identified and characterized a collection of 14 genes induced along different morphological phases of microspore-derived embryo development in wheat (Triticum aestivum L.) anther culture. SERKs and FLAs genes previously associated with somatic embryogenesis and reproductive tissues, respectively, were also included in this analysis. Genes involved in signalling mechanisms such as TaTPD1-like and TAA1b, and two glutathione S-transferase (GSTF2 and GSTA2) were induced when microspores had acquired a 'star-like' morphology or had undergone the first divisions. Genes associated with control of plant development and stress response (TaNF-YA, TaAGL14, TaFLA26, CHI3, XIP-R; Tad1 and WALI6) were activated before exine rupture. When the multicellular structures have been released from the exine, TaEXPB4, TaAGP31-like and an unknown embryo-specific gene TaME1 were induced. Comparison of gene expression, between two wheat cultivars with different response to anther culture, showed that the profile of genes activated before exine rupture was shifted to earlier stages in the low responding cultivar. This collection of genes constitutes a value resource for study mechanism of intra-embryo communication, early pattern formation, cell wall modification and embryo differentiation.

Assessment of various stresses and carbohydrates for their effect on the induction of embryogenesis in isolated wheat microspores

Isolated microspores of flowering plants can undergo embryogenesis when cultured in vitro under appropriate conditions. This process is triggered by various stresses including cold, heat and starvation. We have studied the effects of these stresses on the induction of embryogenesis in wheat microspores isolated from freshly cut or cold pretreated tillers. We show that the stress can be applied directly to microspore cultures, and efficient induction of embryogenesis can be obtained from microspores using a variety of stresses, namely cold (4°C), heat (33°C) and carbohydrate/nitrogen starvation. Cold pretreatment of tillers was able to replace stresses applied to microspores. For the first time we report on embryo formation in true ab initio wheat microspore cultures, not requiring any pretreatment of anthers or tillers prior to culture, in a maltose-containing medium. The effect of maltose on the induction of wheat microspore embryogenesis was specific and can possibly be attributed to its slow hydrolysis by plant cells, exerting starvation conditions early in culture and stable osmolarity of the medium later on. A comparison of three protocols for the induction of wheat microspore embryogenesis and plant regeneration has revealed no significant difference in plant yield, but the ploidy of the plants obtained was affected.

Impact of Explant Type, Duration and Initiation Time on the Co-Culture Effect in Isolated Microspore Culture of Wheat Triticum aestivum L)

Summary The effect of co-culture explant type, duration and initiation time on the culture of blender-isolated microspores of the spring wheat ( Triticum aestivum L.) genotypes and Hja 24201 was investigated. Microspore-derived embryo-like structures (ELS) were obtained only with co-culture. Wheat ovary, glume, floret and anther co-cultures produced ELS in both genotypes. Among the explant types, ELS formation and plant regeneration were highest with ovary co-culture. Five days of ab initio ovary co-culture were sufficient to induce embryogenesis, although prolonged co-culture durations resulted in improved ELS and plant production. Addition of ovaries to the culture medium 5 days after microspore isolation still produced ELS but addition after 10 days gave ELS only in . We conclude that the positive co-culture effect on wheat microspore embryogenesis was obtained with non-embryogenic glume explants, indicating that the effect was not ovary-specific and that for optimal efficiency the co-culture should be applied for at least 5 days, the first application taking place no later than 5 days after microspore culture initiation.

Efficient microspore embryogenesis in wheat ( Triticum aestivum L.) induced by starvation at high temperature

Efficient microspore embryogenesis in wheat ( Triticum aestivum L.) induced by starvation at high temperature

Efficient microspore embryogenesis in wheat (Triticum aestivum L.) induced by starvation at high temperature

We have established an efficient method to induce embryo formation from isolated wheat (Triticum aestivum L.) microspores. Culture of excised anthers under starvation and heat shock conditions induced the formation of embryogenic microspores at high frequency in nine Austrian winter wheat genotypes, including cultivars that had been considered as recalcitrant in anther culture. Percoll gradient centrifugation of the mechanically isolated microspores allowed us to obtain homogeneous populations of embryogenic microspores in all genotypes which, after transfer to a rich medium containing immature ovaries for conditioning, divided and produced globular embryos. Thousands of embryos were produced in one petri dish. Many of these embryos developed into plantlets after transfer to a solid medium without ovaries.

Wheat microspore embryogenesis during in vitro anther culture

Cytological analysis of microspore embryogenesis during in vitro culture reveals a high mortality in the first week and a latency phase of about one week before the first embryogénic mitosis. Genotypic differences observed during our wheat anther culture do not seem to originate at the induction level but are linked to the different abortion rates.