Steve Millam

Potato Virus X-Induced Gene Silencing in Leaves and Tubers of Potato

Virus induced gene silencing (VIGS) is increasingly used to generate transient loss-of-function assays and has potential as a powerful reverse-genetics tool in functional genomic programs as a more rapid alternative to stable transformation. A previously described potato virus X (PVX) VIGS vector has been shown to trigger silencing in the permissive host Nicotiana benthamiana. This paper demonstrates that a PVX-based VIGS vector is also effective in triggering a VIGS response in both diploid and cultivated tetraploid Solanum species. We show that systemic silencing of a phytoene desaturase gene is observed and maintained throughout the foliar tissues of potato plants and was also observed in tubers. Here we report that VIGS can be triggered and sustained on in vitro micropropagated tetraploid potato for several cycles and on in vitro generated microtubers. This approach will facilitate large-scale functional analysis of potato expressed sequence tags and provide a noninvasive reverse-genetic approach to study mechanisms involved in tuber and microtuber development.

Stability of potato (Solanum tuberosum L.) plants regenerated via somatic embryos, axillary bud proliferated shoots, microtubers and true potato seeds: a comparative phenotypic, cytogenetic and molecular assessment

The stability, both genetic and phenotypic, of potato (Solanum tuberosum L.) cultivar Desiree plants derived from alternative propagation methodologies has been compared. Plants obtained through three clonal propagation routes—axillary-bud-proliferation, microtuberisation and a novel somatic embryogenesis system, and through true potato seeds (TPS) produced by selfing were evaluated at three levels: gross phenotype and minituber yield, changes in ploidy (measured by flow cytometry) and by molecular marker analysis [measured using AFLP (amplified fragment length polymorphism)]. The clonally propagated plants exhibited no phenotypic variation while the TPS-derived plants showed obvious phenotypic segregation. Significant differences were observed with respect to minituber yield while average plant height, at the time of harvesting, was not significantly different among plants propagated through four different routes. None of the plant types varied with respect to gross genome constitution as assessed by flow cytometry. However, a very low level of AFLP marker profile variation was seen amongst the somatic embryo (3 out of 451 bands) and microtuber (2 out of 451 bands) derived plants. Intriguingly, only AFLP markers generated using methylation sensitive restriction enzymes were found to show polymorphism. No polymorphism was observed in plants regenerated through axillary-bud-proliferation. The low level of molecular variation observed could be significant on a genome-wide scale, and is discussed in the context of possible methylation changes occurring during the process of somatic embryogenesis.

Cloning and molecular characterisation of a potato SERK gene transcriptionally induced during initiation of somatic embryogenesis

Somatic embryogenesis offers great potential in plant propagation, long-term germplasm conservation, and as a suitable model system for deciphering early events during embryogenesis. The up-regulation and ectopic expression of a SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) gene has been shown to mark and enhance embryogenic competence in somatic cells of model plant species. We have cloned and characterised a SERK gene (StSERK1) from potato (Solanum tuberosum L.), an important crop plant. Sequence analysis of StSERK1 revealed high levels of similarity to other plant SERKs, as well as a conserved intron/exon structure which is unique to members of the SERK family. Furthermore, StSERK clustered most closely with SERK gene family members such as MtSERK1, CuSERK1, AtSERK1, and DcSERK, implicated in evoking somatic embryogenesis. Monitoring of SERK expression during progression of potato somatic embryogenesis revealed increased StSERK expression during the induction phase. Subsequently, during the embryo transition phases, StSERK expression was unchanged and did not vary among embryo-forming and inhibitory conditions. However, in isolated somatic embryos StSERK expression was again up-regulated. In other plant parts (leaves, true potato seeds, microtubers and flower buds), StSERK showed different levels of expression. Expression analysis suggests that the isolated StSERK could be a functional SERK orthologue. The possible role of SERK as a marker of pluripotency, rather than embryogenesis alone, is discussed.

Plant cell and biotechnology studies in Linum usitatissimum – a review

The species Linum usitatissimum (flax/linseed) has been the focus of a great deal of both basic and applied research effort in plant cell and biotechnology studies in recent years. In this review we consider applications of the techniques of plant biotechnology in this species under several distinct headings. Plant cell and tissue regeneration strategies and applications are discussed, and the applications of the techniques of somatic embryogenesis, protoplast isolation, culture and fusion and cell suspension cultures in this species are described. A major area of study is the use of anther and microspore culture where clear advantages to breeding programmes could be applied. In addition, embryo and ovary culture studies have resulted in significant findings. The more recent technologies of gene transfer and expression by genetic transformation are reviewed, and a section on strategies for improvements in technological quality is also included. Finally we propose conclusions and future prospects for this ancient, but still highly relevant crop.

Genotype-specific regeneration from a number of Rubus cultivars

Eight Rubus genotypes were examined for their ability to regenerate on 55 media differing in their plant growth substance content. Regeneration was obtained from all cultivars examined, though efficiency varied greatly depending on genotype. A minimum threshold level of thidiazuron was found to be required for regeneration from leaf discs, and the optimum level for efficient regeneration varied among cultivars. Zeatin was not found to be as effective. Thidiazuron supplemented with a range of individual auxins was investigated, and it was found that NAA had a complementary effect, applicable across a wide range of cultivars at relatively low levels. Using five random primers, 28 polymorphic PCR amplification products (RAPD markers) were stably generated and used to discriminate all eight genotypes investigated. A similarity matrix and dendrogram was generated from the markers and this was related to their regenerative ability.

Genetic transformation of barley microspores using anther bombardment

Bombardment of intact anthers of commercial barley (Hordeum vulgare) varieties resulted in 0.5–1.0% of transformed microspores of which 20–40% continued in androgenic development (0.2% of all bombarded microspores). Using a system based on bombardment of anthers is therefore likely to be more technically efficient than the use of a microspore isolation, transformation and regeneration system. Bombardment of anthers has a number of technical and scientific advantages over existing systems for gene transfer and can be considered as a alternative method to existing methods for genetic transformation in barley.

The establishment and regeneration of a range ofCuphea germplasm t in vitro

The range of Cuphea species previously investigated in vitro has been extended, and the potential of a number of species for uptake into gene transfer systems evaluated. Eight species were successfully established in culture and assessments of relative subculturing potential rates after 4 weeks varied from 10.3 per initial shoot in C. wrightii to 2.3 in C. leptopoda. In tissue regeneration studies, callus formation was rapid and efficient from seven of the eight lines investigated and the species C. lutea, C. lanceolata, C. leptopoda, C. paucipetala and C. laminuligera (in addition to the previously reported C. wrightii) regenerated shootsin vitro from leaf explants after three to six weeks culture on a medium containing 8.8 μM BA, in a two-stage protocol. A high incidence of in vitro flowering was also recorded in C. lutea, C. leptopoda and C. paucipetala. Though initial growth rates were comparable, it was found that extended culture on a matrix of Phytagel rather than agar was detrimental to subsequent shoot development.

Potato (Solanum tuberosum L.).

Potato (Solanum tuberosum L.) is a globally important crop plant producing high yields of nutritionally valuable food in the form of tubers. It has been the focus of substantial study because of its use both as a staple food crop and as a potentially significant source of compounds of interest. This has included the development and application of transgenic technology for introducing novel traits of fundamental and applied interest. This chapter describes a rapid, efficient, and cost-effective system for the routine transformation of this crop plant at rates above 40% efficiency, calculated as the mean number of Southern blot- confirmed independent transgenics per number of internodal explants originally plated. Internodal sections are co-cultivated with Agrobacterium tumefaciens and subjected to a two-stage callus induction/shoot outgrowth system under kanamycin selection. Shoot regeneration rates are high using the described method, and excised independent shoots rooting from the cut end of the stem after two further subcultures on kanamycin are 95% certain to be transformed. The transgenic status can be confirmed by molecular analysis and the plants grown on for tuber production enabling a wide spectrum of further studies.

Developments in Transgenic Biology and the Genetic Engineering of Useful Traits

Publisher Summary This chapter focuses on the development of transgenic biology and the genetic engineering of useful traits. There has also been considerable public concern over GM technology in general, and to allay many of the perceived problems, substantial scientific advances have been made in such areas as developing “clean-gene” technology, studies on resistance breakdown, large-scale studies of gene flow in the environment and the actual composition and safety of introduced traits. The changing emphasis in target traits has been a key feature in the development of transgenic technology in potato, which has been at the forefront of progress in crop plants due to the relative ease of transformation of this species. The more complex traits have been facilitated by the development of increasingly efficient and sophisticated approaches, including the vastly increased knowledge of genomes and gene function. Furthermore, such output traits are considered to be more acceptable to consumers and have thus been the focus of commercially funded as well as academic projects. The potato has been a model crop for transformation, due to the relative ease of A. tumefaciens-mediated transformation, but developments in areas such as VIGS may lead to hitherto unconsidered possibilities for testing genes and host: pathogen interactions.

Factors influencing the utility of gametic microprotoplasts for partial genome transfer in potato

Abstract The potential of gametic microprotoplasts as a tool for partial genome transfer in potato (Solanum tuberosum L.) was investigated and a number of technical findings are reported. The phosphoric amide herbicide amiprophos-methyl was effective for microcell induction when applied at a concentration of 25 μm to excised anther material; however, neither whole buds nor isolated microspores responded to treatment. One selected clone responded well to spindle toxin treatment, and microcells were observed in 9% of tetrads, for which 4% (wt/vol) cellulase Onozuka R10 and 0.25% (wt/vol) driselase was found to be optimal for the release of protoplasts from the tetrads with conversion rates exceeding 40%. Technical problems identified at a number of crucial stages that may preclude the more widespread application of this technology are discussed.