Consistency of yield and quality of seed are traits not yet optimised by the brassica seed industry in New Zealand. As of 2008, seed producers in Canterbury, New Zealand, exported approximately $18m of brassica seed. However, there is a need to increase both seed quantity and/or quality. The plant hormone group, the cytokinins, regulates many stages of plant growth and development, including cell division and enhancement of sink strength, both of which are important processes in seed development and embryonic growth. The two gene families targeted in this project play a key role in maintaining cytokinin homeostasis. Isopentenyl transferase (IPT) catalyzes the rate limiting step in the formation of cytokinins, and cytokinin oxidase/dehydrogenase (CKX) irreversibly inactivates cytokinins. The aim of this project was to identify those cytokinin gene family members expressing specifically during the early phases of pod and seed development. Initially this study used a rapid-cycling Brassica rapa (RCBr) because of its rapid life cycle, then, as the project developed, a commercial crop of forage brassica (B. napus) was studied. Reverse transcriptase PCR (RT-PCR) and BLAST analysis was used to identify putative IPT and CKX genes from RCBr and B. napus. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was used to measure the expression of individual gene family members during leaf, flower, pod and seed development. BrIPT1, -3, and -5, and BrCKX1, -2, -3, and -5/7 and were shown to express differentially both temporally and spatially within RCBr root, stem, leaf, seed, and pod tissues. BnIPT1, 3 and 7 and BnCKX1, 2, 5 and 7 were also differentially expressed. Particularly strong expression was shown by BrIPT3, BrIPT5 and BrCKX2 in developing seeds. Both Brand BnIPT3 expressed strongly in maturing leaves. In normal plant growth and development, biosynthesis and metabolism of cytokinin is tightly regulated by the plant. Increasing the levels of cytokinins during seed development, either by over expressing IPT3 or IPT5, or decreasing the expression of CKX2, or both, could potentially increase both seed yield and seed vigour.

The factors influencing senescence in white clover (Trifolium repens L.) are of considerable importance to the pastoral sector of New Zealand’s economy. The plant hormones, ethylene and the cytokinins, have been implicated as having opposing influences on senescence. This project focused on the cytokinins. The rate limiting step in cytokinin biosynthesis is catalysed by isopentenyl transferase (IPT) and the primary enzyme in the degradation of cytokinins is cytokinin oxidase/dehydrogenase (CKX). Both IPT and CKX genes are present as multi-gene families. A reduction in the level of active cytokinins either via a decrease in IPT expression, or an increase in CKX expression, or both, would implicate the cytokinins in developmental leaf senescence in white clover. White clover grows in a sequential pattern with leaves at all stages of development making it a good model for studying leaf development and senescence. A decrease in leaf chlorophyll is used as a marker for the onset of senescence. A micro-scale chlorophyll analysis was developed using the NanoDrop™ thus allowing tissue from the same leaflet to be used for gene expression and chlorophyll measurements. The pattern of chlorophyll changes was similar to that shown by Hunter et al.(1999) and Yoo et al.(2003) in white clover stolons used for ethylene research. Reverse transcriptase PCR (RT-PCR) and BLAST analysis was used to identify five putative IPT genes and seven putative CKX genes from white clover. RT-PCR demonstrated the expression of seven of these genes (TrIPT1. TrIPT13, TrIPT15 TrCKX1, TrCKX2, TrCKX6). Analysis with quantitative real-time PCR showed expression of TrCKX2 increased markedly during leaf expansion and was consistently high during senescence, suggesting a potential role for CKX in facilitating the progression of senescence.