Network


Latest external collaboration on country level. Dive into details by clicking on the dots.

Hotspot


Dive into the research topics where Caroline A. Sparks is active.

Publication


Featured researches published by Caroline A. Sparks.


Plant Cell Reports | 2003

Factors influencing successful Agrobacterium-mediated genetic transformation of wheat

Huixia Wu; Caroline A. Sparks; B. Amoah; Huw D. Jones

The development of a robust Agrobacterium-mediated transformation protocol for a recalcitrant species like bread wheat requires the identification and optimisation of the factors affecting T-DNA delivery and plant regeneration. We have used immature embryos from range of wheat varieties and the Agrobacterium strain AGL1 harbouring the pGreen-based plasmid pAL156, which contains a T-DNA incorporating the bar gene and a modified uidA (β-glucuronidase) gene, to investigate and optimise major T-DNA delivery and tissue culture variables. Factors that produced significant differences in T-DNA delivery and regeneration included embryo size, duration of pre-culture, inoculation and co-cultivation, and the presence of acetosyringone and Silwet-L77 in the media. We fully describe a protocol that allowed efficient T-DNA delivery and gave rise to 44 morphologically normal, and fully fertile, stable transgenic plants in two wheat varieties. The transformation frequency ranged from 0.3% to 3.3%. Marker-gene expression and molecular analysis demonstrated that transgenes were integrated into the wheat genome and subsequently transmitted into progeny at Mendelian ratios.


Plant Physiology | 2011

Molecular Characterization of Rht-1 Dwarfing Genes in Hexaploid Wheat

Stephen Pearce; Robert Saville; S. P. Vaughan; Peter M. Chandler; Edward P. Wilhelm; Caroline A. Sparks; Nadia Al-Kaff; Andrey V. Korolev; Margaret I. Boulton; Andrew Phillips; Peter Hedden; P. Nicholson; Stephen G. Thomas

The introduction of the Reduced height (Rht)-B1b and Rht-D1b semidwarfing genes led to impressive increases in wheat (Triticum aestivum) yields during the Green Revolution. The reduction in stem elongation in varieties containing these alleles is caused by a limited response to the phytohormone gibberellin (GA), resulting in improved resistance to stem lodging and yield benefits through an increase in grain number. Rht-B1 and Rht-D1 encode DELLA proteins, which act to repress GA-responsive growth, and their mutant alleles Rht-B1b and Rht-D1b are thought to confer dwarfism by producing more active forms of these growth repressors. While no semidwarfing alleles of Rht-A1 have been identified, we show that this gene is expressed at comparable levels to the other homeologs and represents a potential target for producing novel dwarfing alleles. In this study, we have characterized additional dwarfing mutations in Rht-B1 and Rht-D1. We show that the severe dwarfism conferred by Rht-B1c is caused by an intragenic insertion, which results in an in-frame 90-bp insertion in the transcript and a predicted 30-amino acid insertion within the highly conserved amino-terminal DELLA domain. In contrast, the extreme dwarfism of Rht-D1c is due to overexpression of the semidwarfing Rht-D1b allele, caused by an increase in gene copy number. We show also that the semidwarfing alleles Rht-B1d and Rht-B1e introduce premature stop codons within the amino-terminal coding region. Yeast two-hybrid assays indicate that these newly characterized mutations in Rht-B1 and Rht-D1 confer “GA-insensitive” dwarfism by producing DELLA proteins that do not bind the GA receptor GA INSENSITIVE DWARF1, potentially compromising their targeted degradation.


Molecular Genetics and Genomics | 1999

Creation of ecdysone receptor chimeras in plants for controlled regulation of gene expression.

Alberto Martinez; Caroline A. Sparks; P. Drayton; John Thompson; Andrew James Greenland; Ian Jepson

Abstract Transformation with a chimeric receptor containing the glucocorticoid transactivation and DNA-binding domains fused to an ecdysteroid receptor ligand-binding domain permits ecdysone agonist-inducible gene expression in monocotyledonous plant cells. The inducible system is based on the specific activation of a chimeric receptor containing the ligand-binding domain of the Heliothis virescens ecdysteroid receptor and the inducer RH5992 (a 20-hydroxyecdysone agonist). RH5992 is an non-steroidal agrochemical with a high specificity for lepidopteran ecdysone receptors. Addition of RH5992 to transformed cells results in high levels of inducible expression in a ligand-specific manner, particularly when the effector receptor is coupled to the strong transactivator VP16. A chimeric construct containing the Drosophila ecdysone ligand-binding domain failed to activate reporter gene activity with RH5992, while activation was observed in the presence of muristeroneA. The system described provides the basis for an inducible gene expression system that is compatible with agricultural use.


Plant Physiology | 2010

Down-Regulation of the CSLF6 Gene Results in Decreased (1,3;1,4)-β-d-Glucan in Endosperm of Wheat

Csilla Nemeth; Jackie Freeman; Huw D. Jones; Caroline A. Sparks; Till K. Pellny; Mark D. Wilkinson; Jim M. Dunwell; Annica A.M. Andersson; Per Åman; Fabienne Guillon; Luc Saulnier; Rowan A. C. Mitchell; Peter R. Shewry

(1,3;1,4)-β-d-Glucan (β-glucan) accounts for 20% of the total cell walls in the starchy endosperm of wheat (Triticum aestivum) and is an important source of dietary fiber for human nutrition with potential health benefits. Bioinformatic and array analyses of gene expression profiles in developing caryopses identified the CELLULOSE SYNTHASE-LIKE F6 (CSLF6) gene as encoding a putative β-glucan synthase. RNA interference constructs were therefore designed to down-regulate CSLF6 gene expression and expressed in transgenic wheat under the control of a starchy endosperm-specific HMW subunit gene promoter. Analysis of wholemeal flours using an enzyme-based kit and by high-performance anion-exchange chromatography after digestion with lichenase showed decreases in total β-glucan of between 30% and 52% and between 36% and 53%, respectively, in five transgenic lines compared to three control lines. The content of water-extractable β-glucan was also reduced by about 50% in the transgenic lines, and the Mr distribution of the fraction was decreased from an average of 79 to 85 × 104 g/mol in the controls and 36 to 57 × 104 g/mol in the transgenics. Immunolocalization of β-glucan in semithin sections of mature and developing grains confirmed that the impact of the transgene was confined to the starchy endosperm with little or no effect on the aleurone or outer layers of the grain. The results confirm that the CSLF6 gene of wheat encodes a β-glucan synthase and indicate that transgenic manipulation can be used to enhance the health benefits of wheat products.


Plant Molecular Biology | 1992

Construction and characterisation of a yeast artificial chromosome library containing three haploid maize genome equivalents.

Keith J. Edwards; Helen Thompson; David Edwards; Antoine de Saizieu; Caroline A. Sparks; John Thompson; Andrew James Greenland; Mark Eyers; Wolfgang Schuch

We have constructed a yeast artificial chromosome (YAC) library using high-molecular-weight DNA prepared from agarose-embedded leaf protoplasts of the maize inbred line UE95. This library contains 79 000 clones with an average insert size of 145 kb and should therefore represent approximately three haploid genome equivalents. The library is organised as an ordered array in duplicate microtitre plates. Forty-one pools of DNA from 1920 individual clones have been prepared for rapid screening of the library by the polymerase chain reaction (PCR). Using this approach, together with conventional colony hybridisation, we have been able to identify between one and eight positive clones for every probe used.


Scientific Reports | 2015

The first crop plant genetically engineered to release an insect pheromone for defence.

Toby J. A. Bruce; Gudbjorg I. Aradottir; Lesley E. Smart; Janet L. Martin; John C. Caulfield; Angela Doherty; Caroline A. Sparks; Christine M. Woodcock; Michael A. Birkett; Johnathan A. Napier; Huw D. Jones; John A. Pickett

Insect pheromones offer potential for managing pests of crop plants. Volatility and instability are problems for deployment in agriculture but could be solved by expressing genes for the biosynthesis of pheromones in the crop plants. This has now been achieved by genetically engineering a hexaploid variety of wheat to release (E)-β-farnesene (Eβf), the alarm pheromone for many pest aphids, using a synthetic gene based on a sequence from peppermint with a plastid targeting amino acid sequence, with or without a gene for biosynthesis of the precursor farnesyl diphosphate. Pure Eβf was produced in stably transformed wheat lines with no other detectable phenotype but requiring targeting of the gene produced to the plastid. In laboratory behavioural assays, three species of cereal aphids were repelled and foraging was increased for a parasitic natural enemy. Although these studies show considerable potential for aphid control, field trials employing the single and double constructs showed no reduction in aphids or increase in parasitism. Insect numbers were low and climatic conditions erratic suggesting the need for further trials or a closer imitation, in the plant, of alarm pheromone release.


Molecular Breeding | 2006

Characterisation of T-DNA loci and vector backbone sequences in transgenic wheat produced by Agrobacterium-mediated transformation

Huixia Wu; Caroline A. Sparks; Huw D. Jones

Detailed molecular characterisation of transgene loci is a requirement for gaining regulatory approval for environmental release of genetically modified crops. In cereals, it is generally accepted that Agrobacterium-mediated transformation generates cleaner transgene loci with lower copy number and fewer rearrangements than those generated by biolistics. However, in wheat there has been little detailed analysis of T-DNA insertions at genetic and molecular level. Wheat lines transformed using Agrobacterium tumefaciens with bar and gusA (GUS) genes were subjected to genetic and molecular analysis. Unlike previous studies of transgene loci in wheat, we used functional assays for PAT and GUS proteins, combined with PCR and Southern analysis to detect the presence, copy number, linkage and transmission of two transgenes inserted in the same T-DNA. Thirty-four independent transgenic lines were categorised into three types: type I events (38% of total) where the gusA and bar genes displayed complete genetic linkage, segregating together as a single functional locus at the expected ratio of 3:1; type II events (18%), which possessed two or more transgene loci each containing gusA and bar; and type III events (44%), containing an incomplete T-DNA in which either the gusA or bar gene was lost. Most lines in this last category had lost the bar gene situated near the left T-DNA border. Southern analysis indicated that 30% of all lines possessed a single T-DNA copy containing gusA and bar. However, when data on expression and molecular analysis are combined, only 23% of all lines have single copy T-DNAs in which both gene cassettes are functioning. We also report on the presence of plasmid backbone DNA sequence in transgene loci detected using primer pairs outside the left and right T-DNA borders and within the plasmid selectable marker (NptI) gene. Approximately two thirds of the lines contained some vector backbone DNA, more frequently adjacent to the left border. Taken together, these data imply unstable left border function causing premature T-strand termination or read-through into vector backbone. As far as we are aware, this is the first report revealing near border T-DNA truncation and vector backbone integration in wheat transgenic lines produced by Agrobacterium-mediated transformation.


Archive | 2004

Transformation of Wheat by Biolistics

Caroline A. Sparks; Huw D. Jones

Food products derived from wheat are one of the most important sources of calorific intake worldwide and have formed an important part of man’s diet since Neolithic times. In 2002, 573 million tonnes of wheat grain were produced worldwide, of which approx. three quarters was eaten by humans (1). Wheat grain is rich in carbohydrates, proteins and essential vitamins and minerals such as vitamins B and E, magnesium and phosphorous, as well as fibre. It is the only cereal with enough gluten to make leavened bread and is a major constituent of many other foods including biscuits, cakes, breakfast cereal and pasta. Low-grade wheat and industrial wheat by-products are used for animal feed. Wheat is highly adaptable and is grown throughout the world, from the Arctic Circle to south of the Tropic of Capricorn, although it is most suited to more temperate latitudes between 30°N-50°N and 25°S-30°S. The global area of land under wheat cultivation has fluctuated greatly over recent decades. In the early 1960s, approx. 210 million hectares were grown. By the early 1980s, this had increased to approx. 240 million hectares but by the year 2000 the area had fallen again to 213 million hectares. However, in the same time frame, the world wheat production has steadily increased from 200 million tonnes to nearly 600 million tonnes per year today. It is projected to increase further to 860 million tonnes by 2030 (2).


Methods of Molecular Biology | 2009

Biolistics transformation of wheat.

Caroline A. Sparks; Huw D. Jones

We present a complete, step-by-step guide to the production of transformed wheat plants using a particle bombardment device to deliver plasmid DNA into immature embryos and the regeneration of transgenic plants via somatic embryogenesis. Currently, this is the most commonly used method for transforming wheat and it offers some advantages. However, it will be interesting to see whether this position is challenged as facile methods are developed for delivering DNA by Agrobacterium tumefaciens or by the production of transformants via a germ-line process (see other chapters in this book).


Annals of Botany | 2008

Detailed Analysis of the Expression of an Alpha-gliadin Promoter and the Deposition of Alpha-gliadin Protein During Wheat Grain Development

T.W.J.M. van Herpen; M. Riley; Caroline A. Sparks; Huw D. Jones; Cristina S. Gritsch; E. H. Dekking; R.J. Hamer; Dirk Bosch; Elma M. J. Salentijn; M.J.M. Smulders; Peter R. Shewry; L.J.W.J. Gilissen

BACKGROUND AND AIMS Alpha-gliadin proteins are important for the industrial quality of bread wheat flour, but they also contain many epitopes that can trigger celiac (coeliac) disease (CD). The B-genome-encoded alpha-gliadin genes, however, contain very few epitopes. Controlling alpha-gliadin gene expression in wheat requires knowledge on the processes of expression and deposition of alpha-gliadin protein during wheat grain development. METHODS A 592-bp fragment of the promotor of a B-genome-encoded alpha-gliadin gene driving the expression of a GUS reporter gene was transformed into wheat. A large number of transgenic lines were used for data collection. GUS staining was used to determine GUS expression during wheat kernel development, and immunogold labelling and tissue printing followed by staining with an alpha-gliadin-specific antibody was used to detect alpha-gliadin protein deposited in developing wheat kernels. The promoter sequence was screened for regulatory motifs and compared to other available alpha-gliadin promoter sequences. KEY RESULTS GUS expression was detected primarily in the cells of the starchy endosperm, notably in the subaleurone layer but also in the aleurone layer. The alpha-gliadin promoter was active from 11 days after anthesis (DAA) until maturity, with an expression similar to that of a 326-bp low molecular weight (LMW) subunit gene promoter reported previously. An alpha-gliadin-specific antibody detected alpha-gliadin protein in protein bodies in the starchy endosperm and in the subaleurone layer but, in contrast to the promoter activity, no alpha-gliadin was detected in the aleurone cell layer. Sequence comparison showed differences in regulatory elements between the promoters of alpha-gliadin genes originating from different genomes (A and B) of bread wheat both in the region used here and upstream. CONCLUSIONS The results suggest that additional regulator elements upstream of the promoter region used may specifically repress expression in the aleurone cell layer. Observed differences in expression regulator motifs between the alpha-gliadin genes on the different genomes (A and B) of bread wheat leads to a better understanding how alpha-gliadin expression can be controlled.

Collaboration


Dive into the Caroline A. Sparks's collaboration.

Top Co-Authors

Avatar
Top Co-Authors

Avatar
Top Co-Authors

Avatar
Top Co-Authors

Avatar
Top Co-Authors

Avatar
Top Co-Authors

Avatar
Top Co-Authors

Avatar
Top Co-Authors

Avatar
Researchain Logo
Decentralizing Knowledge