Evaluation of the efficacy of the inducible over-expression of 9-cis-epoxycarotenoid dioxygenase1 (NCED1) to confer improved water use efficiency in transgenic plants : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Plant Biology at Massey University, Palmerston North, New Zealand

Abstract

In order to trial the concept of inducible overproduction of the plant hormone abscisic acid (ABA) to confer increased water use efficiency, the forage legume Trifolium repens (L.) (white clover) and the model plant species Nicotiana tabaccum (L.) (tobacco) were transformed with the construct 9-cis-epoxycarotenoid dioxgenase 1 (NCED1) gene from Solanum lycopersicon (SlNCED1) driven by the RUBISCO small subunit promoter (SSUp). For white clover, a total of 18 putatively genetically-independent transgenic lines were obtained through selection in tissue culture, and these were further cloned by vegetative propagation to give 56 plants in total. Ten of these tested positive for NCED1 insertion using polymerase chain reaction (PCR) with genomic DNA. Establishment of transgenic clover on soil was problematic, but seven putatively transformed lines were established. Of these, only one line potentially expressed SlNCED1, but the transcriptional levels were too low, as determined by semi-quantitative reverse-transcriptase dependent PCR (sqRT-PCR), for any further analysis. This low expression, and the fact that only one line was identified, led to the decision to discontinue investigations with white clover. In parallel, the more amenable tobacco transformation system was also used with the SlSSUp::NCED1 construct to act as proof-of-concept. Fourteen putatively genetically independent transgenic lines of tobacco were obtained through tissue culture, six of which were successfully established onto soil. A range of integrated gene copy numbers and NCED1 expression levels were identified in the To lines using genomic PCR and sqRT-PCR. Self-fertilised seed was collected from each transgenic line, but the germination rate from all of the transgenic lines was significantly lower than wild-type. Those lines that did germinate often displayed a range of aberrant growth phenotypes. After trialing methods to evaluate water use efficiency, a total of 47 T1 seedlings displaying a normal seedling phenotype were established on soil. A range of water use efficiencies were observed as determined by analysis of plant growth rates against water use, followed by a transpiration assay of plants deemed „efficient‟ and „poor‟ users of available water. No correlation of SlNCED1 expression with the more efficient users of available water was observed, and so ultimately it was concluded that the transgene was ineffective at raising the water use efficiency of tobacco as determined by the parameters measured in thesis.