Plant tissue culture and artificial seed production techniques for cauliflower and their use to study molecular analysis of abiotic stress tolerance

A protocol for cauliflower micro-propagule production was developed and optimised for both micropropagation and artificial seed production techniques using meristematic tissues from cauliflower curd. All steps in the protocol were empirically optimised including: blending, sieving, culture methods, liquid culture media composition and plant growth regulator combinations and concentrations. The cost of the micro-propagules could be reduced by as much as 50% on the initial costings reported previously since treatments doubled the number of microshoots produced per culture unit. The research confirmed the suitability of cauliflower microshoots to be encapsulated as artificial seeds and an effective protocol for microshoot encapsulation was designed through the optimization of 1) the production of cauliflower microshoots suitable for encapsulation, 2) encapsulation procedures, 3) artificial seed artificial endosperm structure, 4) conversion materials. The possibility of culturing cauliflower artificial seeds in commercial substrates such as perlite, sand, vermiculite and compost was confirmed. The use of plant preservative mixture (PPM) for the control of contamination in cauliflower culture media and artificial seeds was optimised and the effect of this material on the development of plant material was assessed. It was confirmed that cauliflower artificial seed could be stored in a domestic refrigerator for up to 6 months which could have a great impact in cauliflower breeding programmes. The huge number of cauliflower microshoots that could be produced using this protocol and the homogeneity of the culture system, provided a tool for the molecular analysis of cauliflower microshoots (and artificial seed) abiotic stress tolerance analysis. Various treatments were conducted to improve microshoot cold tolerance and the up-regulation of the CBF/DREB1 transcription factor including low temperature acclimation, mannitol, ABA (abscisic acid) and Mo (molybdenum). Microshoots were confirmed to acclimate successfully using low temperature. Mo was shown to improve the cold tolerance of cauliflower microshoots and to up-regulate CBF/DREB1 in the absence of low temperature acclimation. Acclimation did not increase the accumulation of dehydrin proteins and it is concluded that dehydrins do not play a significant role in the cold tolerance of cauliflower microshoots. Since cauliflower breeding and seed multiplication protocols make extensive use of micropropagation, the studies reported in this research could make a significant impact by decreasing the cost of micropropagation and increasing its reliability. It also opens new perspectives for further research for cauliflower artificial seed production and the possibility of sowing these seeds directly in the field. Furthermore, this research helps to facilitate cauliflower breeding programmes by improving the understanding of abiotic stress tolerance mechanisms and the relationship between different types of abiotic stresses such as cold and drought.Damascus University, Syri

Genetic analysis of developmental traits associated with enhanced winter survival in autumn-seeded rye (Secale cereale L.).

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Ice activities in New Zealand Chionochloa species

Overwintering plants produce ice active agents with low thermal hysteresis but a higher ability to inhibit the recrystallization of ice crystals already formed. Many plants also minimize the damage from freezing by triggering ice formation at high sub-zero temperatures. These processes in plants differ from those in fishes and insects, which prevent intracellular ice formation and maintain a supercooled liquid state by secreting low molecular weight cryoprotectants and thermal hysteresis proteins (THP) to depress the freezing point of body fluid. Chionochloa tussocks are mainly endemic to New Zealand and dominate the alpine grasslands. Alpine areas in the South Island can experience snow and freezing at any time of the year due to the incursion of polar air masses. Alpine plants here have developed various mechanisms to overcome the cold weather. A previous study showed significantly high ice nucleation activity (INA) in two alpine Chionochloa species but provided no more information about other ice activities in this genus. Moreover, there are only a few studies on the phylogeny of the Chionochloa group based on ribosomal DNA and/or mitochondrial markers and the lack of genetic information has hampered further research in this genus. Ice activities were investigated in Chionochloa species collected at the Dunedin Botanic Garden and significant seasonal variations were reported with winter collections showing the highest and summer collections showing the lowest ice activities. Non-bacterial intrinsic proteins were essential in maintaining all three types of ice activities, which showed different responses to physical and chemical treatments including heat, pH, high salt, proteinase K, lysosome, reducing and oxidizing agents. Ice-shell enriched ice active proteins (IAP) were isolated from C. macra and C. spiralis respectively. This technique showed better purification efficiency than the traditional ice-finger method. Recrystallization inhibition proteins (RIP), class II endochitinase antifreeze proteins (EAF) and other potential ice-binding proteins were identified in the ice fraction (IF). Interestingly, ice nucleation activity (INA) was not present in the ice fraction (IF) suggesting weak ice-binding ability of ice nucleation protein (INP) or the requirement of other compounds such as metal ions to maintain the INA. INA was separated by a protocol of 100 kDa MWCO centrifugal device, size exclusion chromatography and ion exchange chromatography. MALDI-TOF mass spectrometry failed to identify any potential INP as fewer peptide fragments were present. The transcriptome of winter C. macra was annotated for the better understanding of essential proteins and metabolic pathways involved in the species-specific features including high polysaccharide contents, triterpenoid expression, water conservation and cold tolerance in this genus. In addition, alternative splicing events, SSRs markers, SNPs, miRNAs were reported and gene expression profiles were provided for further genetic research in C. macra and other species in this genus. The C. macra transcriptome also served as a personalized MASCOT database in assist with the search for ice-binding proteins in Chionochloa species assessed by mass spectrometry. Finally, potential ice active protein (IAP) genes in the C. macra transcriptome were investigated by Blast search against current known IAPs. Two putative IAP genes, IRI2 and EAF2, which showed high similarity to the mentioned RIP and EAF respectively, were expressed in the prokaryotic E. coli system and corresponding proteins were purified with ice activities confirmed. Phylogenetic analysis of these IAPs and Pooideae specific cold stress-related genes (C-repeat binding factors and fructosyltransferase) in C. macra indicated these genes from C. macra showed different phylogenetic relationships with core Pooideae and Brachypodium distachyon. Taken together, these findings confirmed that utilizing transcriptome combined with putative gene expression, and protein isolation combined with ice activity assays, were useful to study IAPs in New Zealand Chionochloa species. The application of a set of improved methods including DSC, ice-shell and transcriptome based MASCOT database not only saved time and labor but also gave accurate results in studying ice activities. Moreover, the first annotated transcriptome of winter C. macra provided a clear genetic map for the further genetic research in Chionochloa specific features and cold tolerance in this genus

Identification and characterisation of genes controlling the resistance response to ascochyta blight (Ascochyta rabiei (Pass.) Labrousse) in chickpea (Cicer arietinum L.)

Ascochyta blight, caused by Ascochyta rabiei (Pass.) Labrousse, is one of the most destructive diseases of chickpea (Cicer arietinum L.) worldwide. Despite the existence of highly resistant uncultivated genotypes, attempts to develop cultivars with a high level of durable resistance have been unsuccessful. This study investigated the chickpea defence response to A. rabiei using a functional genomics approach, which has the capacity to improve the overall understanding of the coordinated defence response at a molecular level. An existing cDNA library was used to generate a resource of Expressed Sequence Tags (ESTs) that, after clustering, comprised 516 unigenes. The unigenes were functionally annotated resulting in the identification of 20 specific defence-related unigenes, as well as numerous transcripts with possible involvement in the coordination of defence responses. To explore the expression patterns of the defence-related unigenes in an A. rabiei resistant and susceptible genotype, the unigenes were employed as probes in microarrays. Resulting expression data was analysed to identify differentially expressed unigenes over a time-course after infection. Comparison of the expression profiles from the resistant and susceptible genotype identified three putative genes that were exclusively up-regulated in the resistant genotype, thus may be involved in an effective defence response. Considering that a defence response can involve hundreds of genes, the entire set of chickpea unigenes were used to construct large-scale microarrays. To supplement the chickpea probes, 156 putative defence-related grasspea (Lathyrus sativus L.) ESTs and 41 lentil (Lens culinaris Med.) Resistance Gene Analogs (RGAs) were also included. Expression profiles for three chickpeas and one wild relative were generated over a time course. 97 differentially expressed ESTs were identified using a robust experimental system that included confirmation by quantitative RT-PCR. The results indicated that genes involved in the active defence response were similar to those governed by R-gene mediated resistance, including the production of reactive oxygen species and the hypersensitive response, down-regulation of 'housekeeping' gene expression, and expression of pathogenesis-related proteins. The comparison between resistant and susceptible genotypes identified certain gene expression 'signatures' that may be predictiv e of resistance. To further characterise the regulation of potential defence-related genes, the microarray was used to study expression profiles of the three chickpea genotypes (excluding the wild relative) after treatment with the defence signalling compounds, ethylene (E), salicylic acid (SA), and jasmonate (JA). 425 ESTs were differentially expressed, and comparison between genotypes revealed the presence of a wider range of inducible defence responses in resistant genotypes. Linking the results with the previous microarray results indicated the presence of other pathogen-specific signalling mechanisms in addition to E, SA and JA. The lower arsenal of defence-related gene expression observed in the susceptible genotype may be a result of 'breaks' in the pathways of defence-related gene activation. To draw together the findings of all experiments, a model was constructed for a hypothetical mechanism of chickpea resistance to A. rabiei. The model was synthesised based on the evidence gathered in this study and previously documented defence mechanisms in chickpea, and identified signal transduction as a key to resistance

Identification and Characterization of Cold-Tolerance Associated Genes in Wheat

The low temperature remains as one of the major limiting factors of crop productivity in the temperate region, and identification of cold tolerance related genes is crucial for developing cold tolerant crop plants to increase agricultural productivity. The objective of my thesis is to identify cold tolerance related candidate genes in wheat, one of the major crops in the temperate region. In Chapter 2, I have reviewed the literature pertaining to the mechanisms of cold tolerance in plants with specific emphasis on Wheat. In Chapter 3, forty candidate genes with increased expression under cold exposure based on published microarray data were selected and further characterized. These genes belonging to four categories namely defense-related regulators; transcriptional and epigenetic regulators; post-transcriptional and post-translational regulators; and genes of unknown functions revealed many differentially expressed genes including Remorin – upregulated in response to cold; a novel gene in wheat homologous to RD29B of Arabidopsis-upregulated in response to cold and ABA; and another novel gene regulated by both ABA and MetJA. In chapter 4, the results of genome-wide identification and characterization of the wheat remorin family and its association with cold tolerance are presented. A search of the wheat database revealed the existence of twenty different remorin genes that we classified into six groups sharing a common structure and phylogenetic origin. Promoter analysis of TaREM genes revealed the presence of putative cis-elements related to diverse functions like development, hormonal regulation, biotic and abiotic stress responsiveness. Expression levels of TaREM genes were measured in plants grown under in field and laboratory conditions and in response to hormone treatment. Our analyses revealed twelve members of the remorin family that are regulated during cold acclimation of wheat in four different tissues (root, crown, stem and leaves), with the highest expression in roots. Differential gene expression was found between wheat cultivars with contrasting degree of cold tolerance suggesting the implication of TaREM genes in cold response and tolerance. Additionally, eight genes were induced in response to ABA and MetJA treatment. This genome-wide analysis of TaREM genes provides valuable resources for functional analysis aimed at understanding their role in stress adaptation. The chapter 5 is focused on gaining insights into the evolutionary history and in-silico functional characterization of a novel cold-responsive gene in wheat. This gene in wheat has distant homology to known abiotic stress-related genes in other plants including CAP160 in Spinacia oleracea, RD29B in Arabidopsis and CDeT11-24 in Craterostigma plantagineum. The results show that these genes are homologous and may have evolved from a common ancestor. The Bayesian phylogenetic analyses of the protein sequences of this gene from various plant species revealed three distinctive clades. Further analyses revealed that this gene has predominantly evolved through neutral processes with some regions experiencing signatures of negative selections and some regions showing signatures of episodic positive selections. These genes contained common K-like segments and function predictions revealed that these protein-coding genes may share at least two functions related to abiotic stress conditions. One function is similar to the cryoprotective function of LEA protein, and the second function as a signalling molecule by binding specifically to phosphatidic acid