Iwona Żur

Progress in Doubled Haploid Technology in Higher Plants

In the early 1990s, many basic protocols were developed for haploidy and doubled haploidy, but most were inefficient. During the last decade, progress in technology has been achieved mainly by empirical, time and cost consuming testing of protocols; as a consequence success was proportional to the number of laboratories involved. In the most frequently studied crops (barley, wheat, triticale, maize, rice and rapeseed) improved protocols are now used routinely in breeding and although several problems remain the benefits make doubled haploidy well worthwhile. Significant advances have also been achieved in vegetable, fruit, ornamental, woody and medicinal species, though responses in many remain low with legumes being particularly recalcitrant. There has been resurgence in doubled haploids over the last few years with protocols published for almost 200 plant species. The present review aims to show the recent progress in haploid and doubled haploid technology of higher plants.

Stress-related variation in antioxidative enzymes activity and cell metabolism efficiency associated with embryogenesis induction in isolated microspore culture of triticale (x Triticosecale Wittm.)

Isolated microspore cultures of two spring triticale (x Triticosecale Wittm.) cultivars were used to examine the effect of various stress treatments (either high—32°C or low—5°C temperature with or without nitrogen/carbohydrate starvation) applied to excised anthers on the effectiveness of microspore embryogenesis induction. To quantify the effects of pretreatment conditions, the activity of antioxidative enzymes (catalase, peroxidase and superoxide dismutase) together with respiration rate and heat emission were measured. It was observed that heat shock treatment applied as the only one stress factor increased the activity of antioxidative enzymes which suggests intensive generation of reactive oxygen species. Such pretreatment effectively triggered microspore reprogramming but drastically decreased microspore viability. After low temperature treatment, the activity of antioxidative enzymes was similar to the control subjected only with the stress originated from the transfer to in vitro culture conditions. This pretreatment decreased the number of microspores entering embryogenesis but sustained cell viability and this effect prevailed in the final estimation of microspore embryogenesis effectiveness. For both, low- and high-temperature treatments, interaction with starvation stress was beneficial increasing microspore viability (at 5°C) or efficiency of embryogenesis induction (at 32°C). The latter treatment significantly reduced cell metabolic activity. Physiological background of these effects seems to be different and some hypothetical explanations have been discussed. Received data indicate that in triticale, anther preculture conditions could generate oxidative stress and change the cell metabolic activity which could next be reflected in the cell viability and the efficiency of microspore embryogenesis.

Stress-induced changes important for effective androgenic induction in isolated microspore culture of triticale (× Triticosecale Wittm.)

The accumulation of abscisic acid (ABA) and the activities of antioxidative enzymes along with cell metabolic activity were monitored during androgenesis induction in triticale (×Triticosecale Wittm.). Tested cultivars ‘Mieszko’ and ‘Wanad’ were selected due to their significantly different responses to androgenic induction. Significant variation was observed in respect of superoxide dismutase activity and endogenous ABA content in anthers isolated from freshly cut tillers. For both cultivars, tillers pretreatment with low temperature decreased peroxidase activity by 36%, highly accelerated respiration rate and reduced heat production. At the same time, the level of ABA in ‘Mieszko’ was increased to the level measured in ‘Wanad’. This effect was associated with higher microspore culture viability and increased stress tolerance in ‘Mieszko’. Low temperature and metabolic starvation during 4-day anther preculture did not influence activities of antioxidative enzymes, while it resulted in slight decrease in respiration rate and heat emission. The importance of these changes for effective androgenesis induction is discussed.

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.

β-1,3-glucanase and chitinase activities in winter triticales during cold hardening and subsequent infection by Microdochium nivale

The accumulation of pathogenesis-related proteins such as β-1,3-glucanases and chitinases was studied in cold induced snow mould resistance in two Polish cultivars of winter triticale, cv. Hewo and cv. Magnat that substantially differ in resistance to Microdochium nivale. The plants were pre-hardened at 12°C for 10 days and hardened at 4°C for 28 days. Subsequently, cold hardened plants were inoculated with fungal mycelium (M. nivale) and incubated at 4°C for 7 days in dark. Cold acclimatisation resulted in suppression of the total glucanase and chitinases activities in the resistant Hewo as well as sensitive Magnat cultivars that possibly coincides with altered metabolism. However, upon infection with M. nivale the chitinases were markedly induced in the cv. Hewo. At the same time, total β-1,3 glucanases activities did not seem to be affected by fungus in any of the tested triticale cultivars. The pattern and/or the activity of chitinases in plants might be indicative for the resistance/susceptibility against M. nivale.

Reaction of winter oilseed rape callus to different concentrations of elicitors: pectinase or chitosan

The aim of the presented methodical experiments was 1) the evaluation if callus of winter oilseed rape (Brassica napus var. oleifera L.) initiates a defence reaction to fungal elicitors: pectinase (polygalacturonase) or chitosan, and 2) the choice of the elicitor doses, which evoke the strongest tissue reaction. The results obtained will be used in the next experiments relating the studies of pathogenesis mechanisms proceeding in rape plants infected by necrotrophic fungi. The defence response was estimated on the basis of changes in electrolyte leakage from cells, metabolic efficiency, phenolic content and catalase activity. In the experiment pectinase was used at concentration of 3, 8, 16, 133 and 166 µl per 1 cm3 of culture medium while chitosan at: 25, 50, 75 and 100 µg·cm−3. Both elicitors increased cell membrane permeability: pectinase at the doses equal or greater to 16 µl·cm−3 while chitosan of 25 µg·cm−3. The greatest metabolic inefficiency was observed in calli elicited with 16 µl·cm−3 pectinase and with chitosan of 100 µg·cm−3. The decrease in phenolic content was noted under influence of most doses of both elicitors. The highest catalase activity was evoked by pectinase of 8 µl·cm−3 and chitosan of 75 and 100 µg·cm−3. The results indicated that 8–16 µl·cm−3 of pectinase and 100 µg·cm−3 of chitosan caused the strongest defence reaction of oilseed rape tissue.

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.

Photosynthesis-dependent physiological and genetic crosstalk between cold acclimation and cold-induced resistance to fungal pathogens in triticale (Triticosecale Wittm.)

The breeding for resistance against fungal pathogens in winter triticale (Triticosecale Wittm.) continues to be hindered by a complexity of the resistance mechanisms, strong interaction with environmental conditions, and dependence on the plant genotype. We showed, that temperature below 4 °C induced the plant genotype-dependent resistance against the fungal pathogen Microdochium nivale. The mechanism involved, at least, the adjustment of the reactions in the PSII proximity and photoprotection, followed by an improvement of the growth and development. The genotypes capable to develop the cold-induced resistance, showed a higher maximum quantum yield of PSII and a more efficient integration of the primary photochemistry of light reactions with the dark reactions. Moreover, induction of the photoprotective mechanism, involving at least the peroxidases scavenging hydrogen peroxide, was observed for such genotypes. Adjustment of the photosynthesis and stress acclimation has enabled fast plant growth and avoidance of the developmental stages sensitive to fungal infection. The same mechanisms allowed the quick regrow of plants during the post-disease period. In contrast, genotypes that were unable to develop resistance despite cold hardening had less flexible balancing of the photoprotection and photoinhibition processes. Traits related to: photosynthesis-dependent cold-acclimation and cold-induced resistance; biomass accumulation and growth; as well as protection system involving peroxidases; were integrated also at a genetic level. Analysing 95 lines of the mapping population SaKa3006×Modus we determined region on chromosomes 5B and 7R shared within all tested traits. Moreover, similar expression pattern of a set of the genes related to PSII was determined with the metaanalysis of the multiple microarray experiments. Comparable results for peroxidases, involving APXs and GPXs and followed by PRXs, indicated a similar function during cold acclimation and defense responses. These data provide a new insight into the cross talk between cold acclimation and cold-induced resistance in triticale, indicating a key role of photosynthesis-related processes.

Progress of snow mould infection in crowns of winter rye (Secale cereale L.) is related to photosynthetic activity during cold acclimation.

Resistance to snow mould is a feature determined by multiple genes. Therefore, determining the phenotype of resistant plants is difficult as it requires an investigation over a long period of time from cold acclimation through pathogenesis. The aim of the present study was (i) to determine the characteristics of the resistant genotype and (ii) to clarify the connections between photosynthesis during cold acclimation and then pathogenesis caused by Microdochium nivale. Two inbred lines of winter rye (Secale cereale L.) differing in their susceptibility to snow mould were used in the study. After cold acclimation snow mould resistant (SMR) line was characterised by higher values of CO2 assimilation and electron transport efficiency but did not differ from snow mould susceptible (SMS) line in carboxylation rate of RuBisCO (Vcmax). Higher soluble carbohydrate accumulation, due to higher photosynthesis intensity, as well as an ABA increase at 5 days post infection (DPI) in leaves and crowns were found in SMR line during the pathogenesis period. Callose deposition was found around non-infected bundle sheets and in cortex cells at 5 DPI (at the same time point as ABA peak) only in SMR line, which probably prevented the infection of leaf initials. Early leaf initials infection in SMS line may be responsible for inhibiting leaf growth and plant regeneration after stress cessation. The results show different physiological and biochemical characteristics of the investigated lines, which can be applied in the selection of resistant genotypes and identifying genomic regions responsible for metabolic pathways increasing pathogen resistance.

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.