The transformation of banana with potential virus resistance genes

One approach to reducing the yield losses caused by banana viral diseases is the use of genetic engineering and pathogen-derived resistance strategies to generate resistant cultivars. The development of transgenic virus resistance requires an efficient banana transformation method, particularly for commercially important 'Cavendish' type cultivars such as 'Grand Nain'. Prior to this study, only two examples of the stable transformation of banana had been reported, both of which demonstrated the principle of transformation but did not characterise transgenic plants in terms of the efficiency at which individual transgenic lines were generated, relative activities of promoters in stably transformed plants, and the stability of transgene expression. The aim of this study was to develop more efficient transformation methods for banana, assess the activity of some commonly used and also novel promoters in stably transformed plants, and transform banana with genes that could potentially confer resistance to banana bunchy top nanovirus (BBTV) and banana bract mosaic potyvirus (BBrMV). A regeneration system using immature male flowers as the explant was established. The frequency of somatic embryogenesis in male flower explants was influenced by the season in which the inflorescences were harvested. Further, the media requirements of various banana cultivars in respect to the 2,4-D concentration in the initiation media also differed. Following the optimisation of these and other parameters, embryogenic cell suspensions of several banana (Musa spp.) cultivars including 'Grand Nain' (AAA), 'Williams' (AAA), 'SH-3362' (AA), 'Goldfinger' (AAAB) and 'Bluggoe' (ABB) were successfully generated. Highly efficient transformation methods were developed for both 'Bluggoe' and 'Grand Nain'; this is the first report of microprojectile bombardment transformation of the commercially important 'Grand Nain' cultivar. Following bombardment of embryogenic suspension cells, regeneration was monitored from single transfom1ed cells to whole plants using a reporter gene encoding the green fluorescent protein (gfp). Selection with kanamycin enabled the regeneration of a greater number of plants than with geneticin, while still preventing the regeneration of non-transformed plants. Southern hybridisation confirmed the neomycin phosphotransferase gene (npt II) was stably integrated into the banana genome and that multiple transgenic lines were derived from single bombardments. The activity, stability and tissue specificity of the cauliflower mosaic virus 358 (CaMV 35S) and maize polyubiquitin-1 (Ubi-1) promoters were examined. In stably transformed banana, the Ubi-1 promoter provided approximately six-fold higher p-glucuronidase (GUS) activity than the CaMV 35S promoter, and both promoters remained active in glasshouse grown plants for the six months they were observed. The intergenic regions ofBBTV DNA-I to -6 were isolated and fused to either the uidA (GUS) or gfjJ reporter genes to assess their promoter activities. BBTV promoter activity was detected in banana embryogenic cells using the gfp reporter gene. Promoters derived from BBTV DNA-4 and -5 generated the highest levels of transient activity, which were greater than that generated by the maize Ubi-1 promoter. In transgenic banana plants, the activity of the BBTV DNA-6 promoter (BT6.1) was restricted to the phloem of leaves and roots, stomata and root meristems. The activity of the BT6.1 promoter was enhanced by the inclusion of intron-containing fragments derived from the maize Ubi-1, rice Act-1, and sugarcane rbcS 5' untranslated regions in GUS reporter gene constructs. In transient assays in banana, the rice Act-1 and maize Ubi-1 introns provided the most significant enhancement, increasing expression levels 300-fold and 100-fold, respectively. The sugarcane rbcS intron increased expression about 10-fold. In stably transformed banana plants, the maize Ubi-1 intron enhanced BT6.1 promoter activity to levels similar to that of the CaMV 35S promoter, but did not appear to alter the tissue specificity of the promoter. Both 'Grand Nain' and 'Bluggoe' were transformed with constructs that could potentially confer resistance to BBTV and BBrMV, including constructs containing BBTV DNA-1 major and internal genes, BBTV DNA-5 gene, and the BBrMV coat protein-coding region all under the control of the Ubi-1 promoter, while the BT6 promoter was used to drive the npt II selectable marker gene. At least 30 transgenic lines containing each construct were identified and replicates of each line are currently being generated by micropropagation in preparation for virus challenge.

Discovering, applying and integrating self-knowledge : a grounded theory study of learning in life coaching

Professional coaching is a rapidly expanding field with interdisciplinary roots and broad application. However, despite abundant prescriptive literature, research into the process of coaching, and especially life coaching, is minimal. Similarly, although learning is inherently recognised in the process of coaching, and coaching is increasingly being recognised as a means of enhancing teaching and learning, the process of learning in coaching is little understood, and learning theory makes up only a small part of the evidence-based coaching literature. In this grounded theory study of life coaches and their clients, the process of learning in life coaching across a range of coaching models is examined and explained. The findings demonstrate how learning in life coaching emerged as a process of discovering, applying and integrating self-knowledge, which culminated in the development of self. This process occurred through eight key coaching processes shared between coaches and clients and combined a multitude of learning theory.

Generalised self-efficacy in relation to the life transitions of adult learners in a university setting : towards a narrative constructivist model of self-regulation

This paper demonstrates a model of self-regulation based on a qualitative research project with adult learners undertaking an undergraduate degree. The narrative about the participant’s life transitions, co-constructed with the researcher, yielded data about their generalised self-efficacy and resulted in a unique self-efficacy narrative for each participant. A model of self-regulation is proposed with potential applications for coaching, counselling and psychotherapy. A narrative method was employed to construct narratives about an individual’s self-efficacy in relation to their experience of learning and life transitions. The method involved a cyclical and iterative process using qualitative interviews to collect life history data from participants. In addition, research participants completed reflective homework tasks, and this data was included in the participant’s narratives. A highly collaborative method entailed narratives being co-constructed by researcher and research participants as the participants were guided in reflecting on their experience in relation to learning and life transitions; the reflection focused on behaviour, cognitions and emotions that constitute a sense of self-efficacy. The analytic process used was narrative analysis, in which life is viewed as constructed and experienced through the telling and retelling of stories and hence the analysis is the creation of a coherent and resonant story. The method of constructing self-efficacy narratives was applied to a sample of mature aged students starting an undergraduate degree. The research outcomes confirmed a three-factor model of self-efficacy, comprising three interrelated stages: initiating action, applying effort, and persistence in overcoming difficulties. Evaluation of the research process by participants suggested that they had gained an enhanced understanding of self-efficacy from their participation in the research process, and would be able to apply this understanding to their studies and other endeavours in the future. A model of self-regulation is proposed as a means for coaches, counsellors and psychotherapists working from a narrative constructivist perspective to assist clients facing life transitions by helping them generate selfefficacious cognitions, emotions and behaviour.