Organelles isolated from castor bean (Ricinus communis L.) endosperm by sucrose density gradient centrifugation were assessed for purity by mixing isolated radiolabelled organelles with unlabelled homogenates and recentrifuging. The glyoxysomal fraction was found to be very pure but glyoxysomal membrane fragments contaminated the mitochondria and microsomes. Integral membrane proteins of the three major organelle fractions were isolated using the Triton X-114 method. SDS-PAGE analysis of Triton X-114 extracts revealed that the microsomes and glyoxysomes contained proteins of similar molecular weights. The slight glyoxysomal membrane contamination of the microsomes was dismissed as the cause of these similarities. The e.r.-derived microsomal fraction was shown to possess lipid glucosyltransferase activity. The microsomal fraction was subfractionated on flotation gradients. Two major subfractions were obtained and appeared to represent microsomes of rough- and smooth- e.r. origin, the lighter subfraction being enriched in N-acetyl glucosaminyl and mannosyl transferases while the heavier subfraction was slightly enriched in protein fucosyltransferase. Glycoproteins of the glyoxysomal membrane were shown by in vivo [3H] and [14c] sugar incorporation to possess oligosaccharide moieties containing glucosamine (GlcNAc), fucose and galactose residues. Mannose residues were detected by overlaying SDS-PAGE separated polypeptides with [125i] concanavalin A. The presence of GlcNAc and mannose indicated the presence of e.r. synthesized asparagine-linked oligosaccharide chains and this was confirmed by demonstrating (a) their susceptibility to endo-H and (b) inhibition of glycosylation by tunicamycin. Glyoxysomal membrane protein synthesis was studied in vivo and in vitro. Structural similarities between shared microsomal and glyoxysomal membrane polypeptides were shown using antiserum to glyoxysomal Triton X-114 extracts. Castor bean mRNA translation in vitro demonstrated that the major glyoxysomal membrane proteins did not undergo co-translational insertion into canine microsomes and were possibly synthesized on free, cytosolic ribosomes. The results are discussed with reference to recent models for microbody formation in other seeds and mammalian liver.

A collection of heritage variety accessions were characterised using Amplified Fragment length Polymorphisms (AFLPs) (200 accessions ) and multivariate analysis of morphological characters (366 accessions); key features of interest for the conservation of Plant Genetic Resources were the identification of diversity within and between accessions. Motivations and practices of heritage variety growers were explored using questionnaires. Heritage varieties are herein defined as traditional crop varieties that have a historical origin of over 40 years, are non-hybrid and non-GMO and are of cultural/heritage value to their users; they are part of the suite of plant genetic resources currently utilised by growers and of potential use to plant breeders in the future. A large range of morphological and genetic diversity was present between accessions in all crops; in addition, diversity was found within accessions, particularly in Vicia faba, Daucus carota and Cucumis sativum. Comparisons between data sets were made for diversity, relationships, comparisons with commercial standards and identifying potential duplicates. The synthesis of both data sets highlighted the three potential duplicates for further investigation by HSL (all in Pisum sativum). The findings highlight the importance of heritage varieties and the Heritage Seed Library, both culturally and in terms of conservation for present and future use.

There is an extensive diversity of crops and their wild relatives in Mexico, which are distributed throughout the country. Crop wild relatives (CWR) play a special role for present and future food security strategies: they represent a potential source of variation for the domesticated species, contributing to the genetic improvement of these crops. However, the effects of climate change, among other threats, are reducing significantly this biodiversity. The purpose of this study was to analyse the diversity of wild relatives of the most important crops in Mexico as a basis for the development and implementation of a national conservation strategy for these genetic resources. The methods involved the identification of priorities and creation of a national CWR inventory, in situ and ex situ gap analyses at taxon and ecogeographic levels, the evaluation of the impacts of climate change, threat assessment and predictive characterisation. Applying these methods, 310 CWR taxa were identified as priorities and recommendations for immediate in situ and ex situ conservation actions were made to ensure their representativeness under current and future climatic conditions. All these components contribute to the systematic active long-term conservation of priority CWR diversity in the country and enhance their sustainable utilisation thus helping mitigate the threats to Mexican agrobiodiversity and global food security.

The study was aimed at applying molecular marker techniques to locate QTL and determine the efficiency of the marker-assisted selection. The research was done using Brassica oleracea and Arabidopsis thaliana. The Brassica DH lines represented a population of homozygous individuals while the F\(_2\) and F\(_3\) generations of Arabidopsis represented a segregating population. Marker-assisted selection was applied after the detection of QTL which allowed the identification of markers linked to the QTL and hence the selection for such markers. In Brassica, 40 QTL were detected using the marker regression method. Between 1 and 6 QTL were located per trait, which individually explained 2-49% of the additive genetic variance. In Arabidopsis the marker regression method detected 23 QTL in the F\(_2\), whereas 40 QTL were detected by the interval mapping method in the F\(_3\) generation. 1 7 QTL mapping to similar positions and showing similar modes of action were detected by both methods. Alleles for various QTL were dispersed between parents in both crosses. The efficiency of MAS was determined using various approaches, based on the number of top ranks, number of lines in a group, phenotypic value and as the ratio between response based on MAS and response obtained in the F\(_3\) by applying phenotypic selection to the F\(_2\) generation. The MAS gave generally better response compared to phenotypic selection, particularly when heritability was low. MAS for single QTL was always more effective while multiple QTL and QTL showing linkage posed some practical problems in MAS applications. Overall, MAS has to be applied in conjunction with phenotypic selection to get best results as QTL of minor effect cannot be tackled through marker/QTL associations.

Perennial ryegrass (Lolium perenne L.) is one of the most economically and environmentally important grass species for the temperate zone. It maintains effective self-incompatibility (SI), which promotes outbreeding as well as limits the efficient production of inbred lines and hybrids. SI in L. perenne is controlled by the S and Z loci, mapping to linkage groups 1 and 2, respectively. None of the gene products has been identified so far. Comparative mapping has identified regions on rice chromosomes 5 (R5) and 4 with synteny to regions of L. perenne genome containing the S and Z loci, respectively. Markers were developed from the syntenic rice genomic region to refine the S and Z maps. The closest flanking markers had a map distance of 2 cM from S and 0.2 cM from Z. SI cDNA libraries were developed from in-vitro pollinated stigma subtracted with unpollinated stigma to identify SI components and SI response related genes. Through a BLAST search, candidates identified from the SI libraries that were orthologous to sequences on the S and Z flanking regions on rice R4 and R5 were the prime candidate SI genes. Altogether ten SI candidate genes were identified with incompatible response associated differentially expression pattern: a rapid increase in expression within two minutes after pollen-stigma contact and reaching a maximum between 2-10 minutes, implying their roles in the SI response. Attempts were carried out to determine the linkage relationships between the identified candidates and the S or Z loci. Large fine scale mapping populations were developed individually for the S and Z loci to generate high resolution maps of S and Z towards map-based cloning. Tightly linked markers were identified mapping at a distance of 1.4 cM from S and 0.9 cM from Z. The studies performed in this project have implications on both the underlying genetic control and the associated biochemical responses involved in L. perenne SI. The closely linked markers for S or Z could be applied in future marker assisted selection breeding programmes and map-based cloning.

Oilseed rape (Brassica napus) is a commercially important agricultural crop susceptible to damage from invertebrate herbivores, such as caterpillars, aphids and slugs. Plants can detect the presence of invertebrates via physical contact, tissue consumption, and on recognition of compounds in saliva. Plant retaliation includes the production of proteinase inhibitors to impair gut function and the accumulation of phenylpropanoids and potentially toxic glucosinolates to decrease plant palatability. Previous work has shown that a component of sunlight, ultraviolet-B (UV-B) radiation, can regulate defence related responses in a manner similar to that of pests and the plant wound-response hormone, Jasmonic acid (JA). The molecular basis behind UV-B- enhanced plant defence against invertebrates, however, remains elusive. This project aims to better understand invertebrate resistance in oilseed rape along with the genetic and metabolic basis of UV-B-enhanced defence against two agricultural pests, the grey field slug and caterpillars of the Diamondback moth (Plutella xylostella). UV-B treatment of B.napus and Arabidopsis thaliana has been found to enhance their resistance to these pests, and gene expression analysis of B.napus identified several genes similarly regulated by UV-B radiation, JA application, and/or slug or Plutella grazing. It is thought that these genes are important in UV-B enhanced plant resistance. Transgenic Arabidopsis lines over-expressing three of these oilseed rape genes have been generated to evaluate their role in UV-B-mediated defence. If found to be more resistant to pests, these lines will serve as ‘proof of concept’ that manipulation of the UV-B response pathway in members of the Brassica family could be used to develop new invertebrate resistant varieties.

Phosphorus (P) is one of the essential nutrients for plant growth; however, it is usually in low availability to plants in most soils. P deficiency/low-P in the early growth stages of wheat can cause reductions in tiller and head formation, which poses a threat to wheat productivity and global food security. Genetic variation of phosphate use efficiency (PUE) has been documented in wheat. PUE can be improved under P deficiency/low-P by P-stress-responsive adaptation mechanisms that increase P acquisition and/or utilisation. Major regulatory components involved in PUE include Pi itself, microRNAs, hormones and sugars. In addition, several transcriptional factors (TFs) appear to play crucial roles in the regulatory complexity controlling the expression of P-stress-responsive genes. A better understanding of their roles may help to achieve favourable expression patterns of downstream genes and hence potentials to develop wheat cultivars with improved PUE in future crop improvement programme. Using bioinformatic approaches, this study identified two TFs, TaPtf1 and TaMyb67, which may act as key components in PUE in wheat. Their roles in regulating PUE were investigated through molecular and transgenic approaches. Overexpression constructs for TaPtf1 and TaMyb67 were created and subsequently transformed into wheat by Agrobacterium-mediated transformation. Selected transgenic lines were studied for overexpression of these transgenes and their effects on growth-, harvest- and PUE-related plant traits in a soil-pot experiment under different P supply. The phenotypic effects of TaPtf1 in the transgenic lines were implicated to be P-stress responsive and likely associated with plant height, biomass, grain filling and P accumulation in shoots. The results appeared to be consistent with previous studies of TaPtf1 in wild-type wheat suggesting that TaPtf1 has a functional divergence from OsPtf1/ZmPtf1. On the other hand, TaMyb67 was shown to be a likely ecotypic variation of TaPhr1-B1. TaMyb67 transgenic lines gave no clear evidence of phenotypic differences, presumably due to the downstream P-stress-responsive genes regulated by TaMyb67 being unresponsive to high-P and the low level of (or no) overexpression of TaMyb67 under low-P in these lines.

Tomato (Solanum lycopersicum L.) is one of the most important vegetable crops cultivated worldwide. Rab GTPases are involved in the processes of intracellular membrane traffic, protein secretion and signal responses for targeting of molecules for secretion into the cell wall. Silencing these genes is likely to affect fruit softening by blocking the export of a range of cell wall modifying factors. A cDNA clone from the tomato fruit encoding a protein homologous to rab11/YPT3 was developmentally regulated during fruit ripening. Antisense fruit changed colour but failed to soften normally (Lu et al., 2001). The aim of the project is to investigate the role of Rabs and other elements of intracellular membrane trafficking in secretion of cell wall modifying factors associated with fruit softening. Sixty genes encoding Rab GTPases in tomato were identified by in silico analysis by screening the EST databases and were classified as Rab GTPases according to Release 12 of TIGR Tomato Gene Index. The genes were grouped into 8 different functional classes ranging from Rab1, 2, 5, 6, 7, 8, 11 and Rab18. A phyletic tree was constructed which indicated all the genes were falling into 8 different clades belonging to each class. A comprehensive in silico analysis of other genes involved in intracellular membrane trafficking like Rab GEF, GAP, GDI, and other genes like ARF, ARL, RAN, ROP, SAR (Vernoud et al., 2003)was done by using the same databases. The results showed that there are thirteen ARF GTPases, eighteen ARL GTPases, seven SAR GTPases, eight ROP GTPases, and a total of nine RAN GTPases in tomato. The Rab GTPase interacting partners like GAP, GDI and GEF were also identified. The results revealed that there are twenty three Rab GAPs, two Rab GEFs and a total of six Rab GDIs were found. The information about the genes was updated throughout the course of the present investigation right from the TIGR DFCI TGI release11,12 and 13, SGN Unigene Build 1 and 2 as well the latest release of tomato whole genome sequence database at SGN from SGN ITAG-1 to ITAG-2.4. After the final analysis, a total of 55 Rab GTPases were grouped into eight different sub classes with five Rab1/D, five Rab2/B, four Rab5/F, three Rab6/H, four Rab7/G, five Rab8/E, twenty five Rab11/A and four Rab18/C. Solyc Rab11/A was further classified into 6 sub groups from A1 to A6. The results of classification led to identification of seven RabA1, six RabA2, one RabA3, five RabA4, five RabA5 and one RabA6. The phylogenetic analysis of the tomato Rab GTPases and a comparative analysis with Arabidopsis Rab GTPase confirmed the groups and classes. Similarly four RAN, ten ROP, twelve ARF, four SAR, six ARL GTPases were identified. In case of ARF GTPases seven ARFA, four ARFB, and one ARF C was found. The regulators of small GTPases identified in tomato had one RAB GEF, two ROP GEF, four RABGDI and five ROP GDI, twenty two RabGAP, 2 RAN GAP, nine ROP GAP and nineteen ARF GAP. Comparative phyletic analysis of all the above with Arabidopsis genes confirmed the classification. The molecular and phenotypic characterization confirmed the presence of antisense LeRab11a transgene and the phenotypes were heritable. The textural analysis to characterize the pattern of fruit softening of the wild type and transgenic plants by compression and penetration test from different fruit developmental stages was done and the results showed a sequential pattern of delayed softening and the transgenic fruits were firmer than wild type at the later stages of fruit ripening and had enhanced shelf life and reduced susceptibility to fungal diseases. The relative expression profiles generated by qPCR also showed the pattern of differential expression of Rab11 group of GTPases in wild type and transgenic fruits. Along with this the latest freely available expression database were also used to generated the expression profiles of Rab GTPases during different developmental stages not only in wild type Ailsa Craig (AC++) but also in Heinz cultivar as well as in Solanum pimpinellifolium.

Radial patterning of root hair bearing cells (trichoblasts) in Arabidopsis thaliana (arabidopsis) follows the type 3 root hair patterning mechanism whereby the radial positioning of trichoblasts is coordinated according to the position of underlying cortex cells (Pemberton et al. 2001). Epidermal cells which are positioned over the cleft between two underlying cortex cell files adopt trichoblast identity. Low phosphate stress in arabidopsis roots has been show to result in an increase in the number of cortex cell files from 8 to 12-16, which in turn results in an increase in the number of trichoblast position cells (Zhang et al. 2003, Cederholm and Benfey, 2015). However, little is known about what mechanisms control this proliferation in cortex tissue. Zhang et al. (2003) demonstrate that eir1 (an allele of pinformed2, pin2) mutants are deficient in this response to low P. This finding suggests that PIN2 (an efflux facilitator of directional auxin transport) is required to orchestrate proliferation of cortex cell files under low P. This implies that directional auxin transport and, ultimately, auxin response are required to enable proliferative divisions in cortex tissues under low P. First of all, a deeper anatomical understanding of the cortex cell file number response to low P was developed. That showed that the divisions which lead to the increase in cortex cell file number occur in the first cortex cell next to the quiescent centre. It was also found that the anatomical changes only significantly affect the number of cortex cell and trichoblast cell files, suggesting that it is a cortex specific response to increase radial root hair density. It was further discovered that the root is sensitive to up to 400μM P and responds with increased cortex cell file number within 24 hours. I also showed that this response is independent of iron concentration. It was next hypothesised that the role of directional auxin transport implied that other PIN and AUX/LAX genes may be required as well as activating AUXIN RESPONSE FACTORs (ARFs). These experiments revealed that no other PINs or ARFs play a crucial role in this response. However it was found that PIN2:GFP protein changes its subcellular localisation in response to low P, suggesting that changes in auxin flux direction is required. Based on this, it was hypothesised that PIN2 complementation in the cortex in pin2 would rescue the phenotype. This was indeed the case, but also for PIN2 under the AUX1 promoter, suggesting an additional role for PIN2 in the epidermis and root cap during the low P response. Mathematical modelling suggested that auxin flux out of the cortex, mediated by PIN2, would be required for the response. However, results testing this in vivo with the DII:VENUS auxin reporter were inconclusive. It was suspected that ground tissues patterning regulators, BIRD genes, may also play a role in tissue patterning under low P. jkd (jackdaw) and mgp (magpie) mutants did not show any strong phenotype on low P, but expression analysis using reporter lines and reverse transcription qPCR did suggest changes in regulation. It was then hypothesised that BIRD genes and PIN2 may interact, by crossing pin2 and jkd I found that the two interact to a small degree but it is not evident that they interact directly within the same pathway. It was concluded that this response to low P was controlled by a number of regulatory networks which definitely involves PIN2 mediated auxin transport with a contribution from BIRD genes.

Wheat is a crop of global importance accounting for 20% of global calorie consumption and a similar percentage of the daily protein for 2.5 billion people in less developed countries. To meet the food production demands of a predicted global population of 9 billion people by 2050, wheat yields need to increase by 1.7% per annum, whilst facing the added pressures of reduced fertilizer inputs and potentially reduced land availability. Plant root systems are key for efficient water and nutrient uptake and thus have a direct impact on yield, and yet there has been competitively little research into root system improvement. A high-throughput root phenotyping pipeline for wheat seedlings was designed consisting of a germination paper-based growth system combined with image segmentation and analysis software. A number of lines from the A.E. Watkins landrace population were characterised to test the final pipeline design. The pipeline was then utilized to phenotype a population of 94 lines from a doubled haploid population for quantitative trait loci (QTL) discovery. In total, 29 root QTL were discovered, with 2 loci co-localising with QTL for grain yield and nitrogen uptake efficiency discovered in field trials. Modern wheat varieties may have limited genetic diversity, due to changes in ploidy throughout evolution and subsequent domestication. With this in mind, thirty five ancient wheat relatives and eighteen amphidiploid hybrids were phenotyped for seedling root architectural traits, to determine the amount of phenotypic variation within ancient wheat species, and whether this variation can be transferred to modern varieties. The utilization of seedling root trait phenotyping is discussed and future research directions are identified.