Monika Krzewska

Current insights into hormonal regulation of microspore embryogenesis

Plant growth regulator (PGR) crosstalk and interaction with the plant’s genotype and environmental factors play a crucial role in microspore embryogenesis (ME), controlling microspore-derived embryo differentiation and development as well as haploid/doubled haploid plant regeneration. The complexity of the PGR network which could exist at the level of biosynthesis, distribution, gene expression or signaling pathways, renders the creation of an integrated model of ME-control crosstalk impossible at present. However, the analysis of the published data together with the results received recently with the use of modern analytical techniques brings new insights into hormonal regulation of this process. This review presents a short historical overview of the most important milestones in the recognition of hormonal requirements for effective ME in the most important crop plant species and complements it with new concepts that evolved over the last decade of ME studies.

Changes in gene expression patterns associated with microspore embryogenesis in hexaploid triticale (×Triticosecale Wittm.)

Abstract To gain a better understanding of the molecular mechanisms controlling microspore embryogenesis (ME) in triticale (×Triticosecale Wittm.), the expression patterns of 13 genes, previously identified in bread wheat to be associated with microspore-derived embryo development, were analysed. Four triticale doubled haploid (DH) lines, significantly different with respect to embryogenic potential, were studied. The gene expression profile was dissected at different points of the ME induction procedure up to the 8th day of in vitro culture (dc). RT-PCR revealed that these 13 genes were expressed during triticale ME. Variations in gene expression profiles were observed between the studied DH lines. DH28 (highly embryogenic) was the only one in which all analysed genes (Ta.TPD1-like, TAA1b, GSTF2, GSTA2, CHI3, Tad1, XIP-R1, TaAGL14, TaNF-YA7, SERK2, SERK1, TaEXPB4, TaME1) were up-regulated during the first 8dc. In the less embryogenic DH31, TAA1b, GSTA2 and TaEXPB4 were already induced on 4dc. In DH25, ME was initiated quite efficiently but soon inhibited, which coincided with the lack of gene expression (TaEXPB4, TaME1) or down-regulation (Tad1, XIP-R1, TaAGL14, TaNF-YA, SERK2, SERK1) on 8dc. In the recalcitrant DH50 line, the majority of genes were expressed at a lower level or not at all, indicating disturbances in ME initiation. In this study, the molecular mechanisms involved in triticale ME induction were analysed for the first time, laying the foundation for further characterisation of specific genes controlling microspore-derived embryo development.

Tissue Culture and Regeneration: A Prerequisite for Alien Gene Transfer

Introgression of genes from alien species into crop plants could be achieved through distant hybridisation aided by tissue culture-based embryo rescue techniques. Beside this, in vitro mutagenesis, gametoclonal/somaclonal variation and transgenesis are the other tools which can generate additional variability. However, all these tissue culture-based tools require totipotent tissues. The direct regeneration of plants from an explant without a callus stage via organogenesis or somatic embryogenesis is the quickest path for micropropagation. Because of their speed and low costs of culture phase and the fidelity of the genotype in the cloned progeny, systems with direct somatic embryogenesis or organogenesis are often recommended and subjected to transformation. On the other hand, most micropropagation procedures with a callus stage can be applied as a basis for transformation, and the fresh friable calli can be directly used as the transformation target. Cell and microspore suspension cultures have also been seen as the ideal targets for genetic transformation due to the large amount of homogenous material, easy selection of the targeted cells and less chances of chimeric regeneration, while protoplasts due to exposed plasma membrane can introduce foreign DNA very easily and therefore form the ideal targets for generating unique and novel plants. This chapter discusses various plant regeneration methods and the factors affecting them towards achieving alien gene transfer in crop plants.

Doubled Haploids in Triticale

Climate changes, a rapid pathogen evolution, and market change, put breeders under constant pressure to deliver new and better varieties in a shorter time while simultaneously reducing the costs of variety development. Substantial progress has been generated in triticale doubled haploid (DH) technologies during the last decade that widens their application in breeding and research at large. The progress in triticale androgenesis including the recent research on its physiological and genetic aspects is presented here. It is completed with the brief description of triticale x maize crosses to raise DH. Application of triticale DH populations to map QTL of triticale resistance to abiotic and biotic stresses, morphogenetic, and morphology traits is also reviewed. Albinism and problems of DH lines stability is discussed among the unsolved problems of triticale DH production.

Changes in protein abundance and activity involved in freezing tolerance acquisition in winter barley (Hordeum vulgare L.)

The changes in protein abundance induced by cold hardening were analysed by 2 DE in ten doubled haploid (DH) lines of winter barley, highly differentiated with respect to freezing tolerance level. Among 45 differential proteins identified by MALDI-TOF/TOF, the majority was classified as related to photosynthesis, carbohydrate metabolism, oxidation-reduction reactions and stress response. Among the detected proteins, higher abundance of RuBisCO large and small subunits, RuBisCO activase, two Oxygen-evolving enhancer proteins, Ferredoxin-NADP reductase, Cytochrome P450-dependent fatty acid hydroxylase and 14-3-3 protein was associated with higher freezing tolerance level. Lower relative level of hypothetical ATP synthase beta subunit, uncharacterized mitochondrial protein AtMg00810 and ribosomal RNA small subunit methyltransferase G also seems to be important. The results of proteomic studies were complemented by the evaluation of photosynthetic apparatus acclimation, showing distinctive differences between the studied genotypes in the number of active PSII reaction centres (RC/CSm). Additionally, the analysis of antioxidative enzyme activities suggests the importance of H2O2 as a signalling molecule possibly involved in the initiation of cold-induced plant acclimation. However, in DH lines with high freezing tolerance, H2O2 generation during cold hardening treatment was accompanied by more stable activity of catalase, H2O2-decomposing enzyme. SIGNIFICANCE In the study, the changes in protein abundance induced by cold hardening treatment were analysed by two-dimensional gel electrophoresis in ten doubled haploid (DH) lines of winter barley. Harnessing DH technology resulted in distinctive widening of genetic variation with respect to freezing tolerance level. Both the cold-hardening effect on the protein pattern in an individual winter barley DH line as well as the variation among the selected DH lines were investigated, which resulted in the identification of 45 differentiated proteins classified as involved in 14 metabolic pathways and cellular processes. Among them, eight proteins: (1) the precursor of RuBisCO large subunit, (2) RuBisCO small subunit (partial), (3) RuBisCO activase small isoform, (4) the precursor of Oxygen-evolving enhancer protein 1-like (predicted protein), (5) Oxygen-evolving enhancer protein 2, (6) the leaf isozyme of Ferredoxin-NADP reductase, (7) hypothetical protein M569_12509 Cytochrome P450-dependent fatty acid hydroxylase-like and (8) hypothetical protein BRADI_1g11290 (14-3-3 protein A-like) were accumulated to a higher level in leaves of cold-hardened seedlings of freezing tolerant winter barley DH lines in comparison with susceptible ones. Three others: (9) hypothetical protein BRADI_5g05668 F1 ATP synthase beta subunit-like, (10) predicted protein uncharacterized mitochondrial protein AtMg00810-like and (11) BnaA02g08010D Ribosomal RNA small subunit methyltransferase G-like were detected at lower level in freezing tolerant seedlings in comparison with susceptible genotypes. The last two were for the first time linked to cold acclimation. The results of complementary analyses indicate that PSII activity and stability of antioxidative enzymes under low temperature are also very important for freezing tolerance acquisition.

Changes in protein abundance and activity induced by drought during generative development of winter barley (Hordeum vulgare L.)

The present study investigated drought-induced changes in proteome profiles of ten DH lines of winter barley, relatively varied in water deficit tolerance level. Additionally, the parameters describing the functioning of the photosynthetic apparatus and the activity of the antioxidative system were analysed. Water deficit (3-week growth in soil with water content reduced to ca. 35%) induced significant changes in leaf water relations and reduced photosynthetic activity, probably due to decreased stomatal conductance. It was associated with changes in protein abundance and altered activity of antioxidative enzymes. From 47 MS-identified proteins discriminating more tolerant from drought-sensitive genotypes, only two revealed distinctly higher while seven revealed lower abundance in drought-treated plants of tolerant DH lines in comparison to sensitive ones. The majority were involved in the dark phase of photosynthesis. Another factor of great importance seems to be the ability to sustain, during drought stress, relatively high activity of enzymes (SOD and CAT) decomposing reactive oxygen species and protecting plant cell from oxidative damages. Low molecular weight antioxidants seem to play less important roles. Our findings also suggest that high tolerance to drought stress in barley is a constitutively controlled trait regulated by the rate of protein synthesis and their activity level. BIOLOGICAL SIGNIFICANCE According to our knowledge, this is the first comparative proteomic analysis of drought tolerance performed for the model set of several winter barley doubled haploid (DH) lines. We analysed both the drought impact on the protein pattern of individual winter barley DH lines as well as comparisons between them according to their level of drought tolerance. We have identified 47 proteins discriminating drought-tolerant from drought-sensitive genotypes. The majority was involved in the dark phase of photosynthesis. Another factor of great importance in our opinion seems to be the ability to sustain, during drought stress, relatively high activity of antioxidative enzymes (SOD and CAT) decomposing reactive oxygen species and protecting plant cell from oxidative damages. Our findings also suggest that high tolerance to drought stress in barley is a constitutively-controlled trait regulated by the rate of protein synthesis and their activity level.