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    Cytokinin, jasmonates and postharvest physiology of Asparagus officinalis L. : a thesis submitted for the degree of Master of Science in Plant Biology, Massey University,
    (Massey University, 1998) Gapper, Nigel Esteven
    The asparagus spear is a rapidly growing shoot, dependent on the crown and storage roots for substrate . Once harvested, spears have a very short shelf-life. Investigations to date point to a physiological cause of this deterioration rather than a pathogenic one. Since loss of membrane integrity is a notable feature of the postharvest deterioration, spears were treated immediately following harvest with cytokinin (which promotes membrane integrity), and jasmonic acid (produced by deteriorating membranes). Treated plant material was collected and monitored for physiological and compositional changes. Results show a reduction in postharvest elongation of spears treated with cytokinin, and a reduction of shelf-life of spears treated with jasmonic acid, when compared with control spears treated with water. Also an extension of shelf-life was observed for spears treated with cytokinin. We quantified jasmonates using ELISA in spears after harvest, and also in naturally senescing cladophylls. Jasmonate concentration increased in spears rapidly after harvest, which is most likely to be in response to wounding. Results also showed that jasmonates may be involved in desiccation stress and cessation of elongation in asparagus spears. Jasmonate production and metabolism appears to be more ordered during natural foliar senescence than during harvest induced senescence of the spear. Jasmonic acid and dihydrojasmonic acid are metabolised to cucurbic acid during the later stages of natural foliar senescence. The presence of jasmonates in asparagus spears was confirmed by electrospray ionisation mass spectrometry. This analysis also enabled to identify a novel jasmonate, tryptophan-dihydrojasmonic acid amino acid conjugate.
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    Strigolactones and hormonal interaction in control of branching in Zantedeschia and other horticultural species : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Plant Science at Massey University, Palmerston North, New Zealand
    (Massey University, 2016) Manandhar, Sarina
    Shoot branching that involves development of lateral buds into shoots is one of the important factors influencing crop productivity. Strigolactones have recently been found to be involved in the control of branching, but the actual bioactive compound/s that inhibits bud outgrowth is still unknown. A germination assay utilizing the seeds of a parasitic weed (Orobanche minor), detected strigolactones within the xylem exudates of different horticultural crop species; the strigolactone concentration negatively correlated with branching of cultivars or mutants. In Zantedeschia grown in vivo, the concentration of strigolactones was independent on the volume of guttation fluid (xylem exudates) suggesting the difference in concentration of strigolactones in high and low branched cultivars was due to the difference in potential of producing strigolactones between these cultivars and not due to differences in volume of guttation fluid. While identifying a bioactive compound using germination and branching assays in combination with liquid chromatography and mass spectrometry, compounds containing „N? were detected in the low branched wild-type Petunia, but not in the highly branched mutant, suggesting the possibility of such compounds being SL-conjugates which may be associated with bud outgrowth inhibition. In Zantedeshia (in vitro) and pea stems, strigolactone reduced the axillary shoot number stimulated by the cytokinin suggesting an antagonistic interaction between these two hormones on bud release. However, as cytokinin may stimulate subsequent growth of released buds by increasing the auxin transport out of the bud, strigolactone may have reduced subsequent growth by reducing auxin transport. Since GA3 enhanced subsequent growth of buds in pea stems, but not the release, an antagonistic interaction between strigolactone and gibberellins on subsequent growth is possible. Interestingly, strigolactone successfully reduced adventitious bud formation in Zantedeschia grown in vitro, adding a new role for strigolactones in plant development. Despite correlation between strigolactone and branching inhibition in different horticultural crops such as apple, kiwifruit, Zantedeschia and Acer, further studies relating to strigolactone and its interaction with other hormones on branching of these crops could be performed using in vitro techniques for a clear understanding of strigolactones? role on branching inhibition. More importantly, quantification of strigolactones using the germination assay may have significant implications in horticultural crop breeding for obtaining desired shoot branching.
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    Auxin action and cell elongation : a rational approach : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Plant Physiology at Massey University
    (Massey University, 1970) Penny, Pauline Elizabeth
    A method was developed to measure every minute the growth of a single segment excised from the elongating region of a plant. The method was used to determine the short term kinetics of growth in response to auxin addition. The method is not dependant on the use of hollow coleoptile tissue and the results are plotted as a growth rate against time. The technique has a resolution an order of magnitude higher than those in current use. The results show that there is a latent phase before auxin-induced increase in elongation rate occurs. After this latent phase, there is a rapid rise in rate to a maximum followed by a decrease and then usually a rise to a second maximum. Three hypotheses for explaining the growth rate curve are considered. It was found that neither RNA nor protein synthesis were required for the initial action of auxin but that protein synthesis became necessary within a few minutes after auxin addition. The apparent half-life of the protein whose synthesis is stimulated by auxin is about 12 min. This short half life suggests that, after the synthesis of the protein, there is a limited time during which it can act with auxin to increase elongation. A model which incorporated these results has been proposed and its relationship to the three hypothesis for explaining the growth rate curve is discussed.
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    Growth analysis and plant hormone studies in apple (Malus sylvestris Mill.) : a thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy
    (Massey University, 1975) Park, Heung Sub
    Previous data on gravitational effects on shoot growth and flowering have been inconsistent. Attempts have been made to investigate shoot growth and flowering on shoots with a 3/8 phyllotaxis in 90 cm and 150 cm laterals. These were bent at different times to the horizontal or to a pendulous position in apple varieties Red Delicious and Granny Smith on MM 106 rootstocks grown under a semi-intensive system on a commercial orchard in the major apple growing area of Hastings. The four treatments comprised: horizontal or pendulous bending during the dormant period, at petal fall, second cover stage and with normal vertical laterals as controls. Horizontal bending increased total shoot growth and flowering relative to the vertical controls in the 90 cm treatment in both varieties. There seemed to be a tendency to decrease total shoot growth when the time of bending was later in the season and no differences in flowering occurred among the horizontal bending treatment. On the other hand shoot growth was relatively constant in all treatments in the 150 cm treatment. A very significant increase in flowering, however, was found in the petel fall pendulous bending. In the dormant period pendulous bending there was a slight effect on the flower promotion relative to the verticals. The production of laterals and flower buds was always more pronounced on the upper side of the bent shoots, with an intermediate on the flanks, and greatly inhibited effects on the lower side, indicating a steep linear relationship from the lower to the upper during the dormant period treatment in all experiments. Generally, the percentages of shoot growth and flower production were increased from the dormant period bending to the petal fall, second cover and to the vertical control. The greatest increase in shoot length and increased percentage of flowering in all experiments were found in the apical whorl zone, and these further decreased from the, 1st to the 2nd, and to the 3rd whorl; this was the case for shoot growth and flowering in the first whorl was not increased due to the inherent properties in Red Delicious. The shoot growth and flowering at the different whorls in 150 cm length laterals bent pendulously in Red Delicious showed a quadratic relationship due to the longer shoots in the apical and the arch position on the shoot when bent at the 5th whorl in all treatments. But at the 5th whorl flowering was reduced considerably, because of substantial lateral growth. In order to describe the growth relationship between shoot volume and total leaf area an index based on the ratio of vegetative and reproductive responses was established e.g. vegetative 10.83 and reproductive 19.80-24.40. The relationship of shoot growth and flowering are discussed in terms of a hormone balance theory. Plant hormone studies In order to establish a ratio of different plant hormones for an understanding of physiological phenomena, appropriate extraction procedures are required for especially apple leaves which are rich in phenolic compounds and other inhibitors. Therefore extraction procedures and purification were examined using 14C-IAA and 3H-zeatin. Loss of 14C-IAA during extraction procedures was due to a high pH in the aqueous phase during solvent partitioning. The final recovery of 14C-IAA was 3.8% at pH 8.0 and 81.1% at pH 2.5 through solvent partition and column chromatography. 14C-IAA was chromatographed on a silica gel-celite column and a Sephadex LH-20 column, giving 80 recovery in 30 ml elution volume around the main peak and 90% recovery in 20 ml elution volume around the main peak respectively. Nearly 100% recovery from a Sephadex G-10 column was obtained. 50-57% recovery of 14C-IAA wes obtained in cellulose thin layer chromatography at the Rf of IAA, and no loss of 14C-IAA occurred during 3 days storage in a dark cabinet. The partition coefficient of 3H-zeatin at pH 8.3 was 13.12 with ethyl acetate and 0.488 with n-butanol; at pH 2.5, 108-89 with ethyl acetate and 16.73 with n-butanol. Backwashing can recover 3H-zeatin from ethyl acetate phase which was partitioned at pH 2.5. 80% recovery of 3H-zeatin in the first 1,000 ml was obtained from Sephadex G-10 and Dowex 50 W x 8. 88.6% recovery of 3H-zeatin could be obtained in a 20 ml peak using Sephadex LH-20 eluted with 95% EtOH containing 0.001 M HCl. The behaviour of 3H-zeatin was studied in paper chromatography and cellulose, DEAE cellulose and silica gel, thin layer chromatography, about 82-60% of 3H-zeatin the Rf of 3H-zeatin being recovered. Four series of plant hormones were determined from apple leaves by ethyl acetate partitioning, Sephadex G-10 column, silica gelcelite column and cellulose thin layer chromatography of acidic fractions containing auxin-, gibberellin-, and ABA-like substances, and by butanol partitioning, Sephadex G-10, Sephadex LH-20, and'DEAE cellulose thin layer chromatography for cytokinin-like substances from basic fractions. Possibly two kinds of auxin-like substances were found and possibly GA9, GA4, GA5, GA1, or GA3, and GA8-like substances were eluted from a silica gel-celite adsorption column. Several groups of cytokinin-like substances were obtained from Sephadex LH-20 column chromatography, possibly zeatin, zeatin-riboside and other cytokinins were found in apple leaves. Based on the estimation of each plant hormone from thin layer chromatography, a relative plant hormone index was established, i.e., Relative Auxin Activity Index, Relative Inhibitor Activity Index, Relative Gibberellin Activity Index, and Relative Cytokinin Activity Index, representing 6.59, 1.04, 2.64, and 8.16 respectively, the hormone giving the highest ratio being considered the dominant hormonal factor at that stage of development. GLC techniques were also studied for plant hormone analysis, using 3% OV-1 and NAA, IAA, TPA, GA1, GA3, GA4, GA5, GA7, GA9, GA13 and ABA markers to establish retention times and detector response at the 2.5 ng level. N.O.-bis(trimethylsilyl)trifluoroacetamide (BSTFA) together with Trimethylchlorosilane (TMCS) silyl reagents produced the best peak heights for IAA, IPA, GA3 and GA1 but reduced the ABA peak by half and the GA9 peak by 20%.
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    Ethylene biosynthesis during leaf maturation and senescence in white clover : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Massey University
    (Massey University, 1997) Butcher, Stephen Mark
    Aspects of leaf senescence in relation to ethylene biosynthesis in the plagiotropic herbaceous plant white clover (Trifolium repens L.) have been studied. Two stolons growing from clonally propagated plants were trained over a dry substratum that inhibits nodal root growth, and all axillary stolons and flowers were removed. White clover plants grown using this method produce leaves at all stages of development along a single stolon from initiation at the apex, through expansion, maturity, senescence and then necrosis. The study shows that modification of the stolon growth habit of white clover by the suppression of nodal roots provides a suitable system for the study of leaf development and senescence in relation to ethylene metabolism. The pattern of leaf development (the number of leaves at each stage of development present on the stolon) and senescence (as measured by changes in leaf chlorophyll content) along the white clover stolon is consistent between plants of the same genotype growing under the same environment, but varied greatly between the different cultivars and genotypes examined. The rate of change between the different stages of leaf development and senescence within the one genotype used in this study, AgResearch Grassland genotype 10F, differed when grown under two different environments. On mature stolons (stolons with 6 or more nodes with senesced leaves) of genotype 10F grown using the modified stolon system, the number of green leaves was maintained at a constant number as the leaf appearance rate was balanced by the senescence rate. However, the number of leaves maintained on the stolons differed between the two growing environments used in this study, from 9.85 +/- 0.23 for plants grown at Levin, to 14.57 +/- 1.99 for plants grown at Palmerston North. The total chlorophyll concentration in the leaves from plants grown at Levin increased from leaf one (the youngest opened leaf; 740 μg/g.fw) to a maximum in leaf five (mature green leaf; 2240 μg/g.fw), declined rapidly from leaf five to leaf seven (senescing leaves; 1500 μg/g.fw), and then remained constant from leaf seven to leaf ten. A similar pattern of change in chlorophyll concentration was measured in leaves from plants grown at Palmerston North, but the maximum concentration was found in leaf 4 (1750 μg/g.fw), remained relatively constant to leaf 8, before decreasing in leaf 9 (750 μg/g.fw) and declining to a minimum in leaf 15 (250 μg/g.fw). The chlorophyll a:b ratio in mature green leaves from plants grown at Palmerston North (1.46:1 to 2.63:1) was lower than the ratio in leaves from plants grown at Levin (3.72:1 to 4.98:1). Leaves of white clover produce ethylene. Ethylene evolution from attached leaves varied from 1 nL/g.fw/h (mature green leaves) to 3 nL/g.fw/h (senescing leaves). Ethylene evolution from detached leaves was initially high (12.6 nL/g.fw/h at 15 min) but declined to 3.8 nL/g.fw/h by 45 min before increasing again. Detached mature green leaves (leaves four to six) of white clover are sensitive (as measured by chlorophyll loss) to low concentrations (<1 ppm) of exogenous ethylene. The chlorophyll concentration in these leaves after four days of ethylene treatment (1, 10 or 100 ppm ethylene) was significantly lower than the chlorophyll concentration in freshly harvested leaves. However, the chlorophyll concentration in leaves two and three (early mature green) treated with ethylene was not significantly different from the concentration in freshly harvested leaves. 1-aminocyclopropane-1-carboxylic acid (ACC) concentration was low in leaves one to four (<1 nmoles/g.dw), increased to reach a maximum concentration of 11.4 nmoles/g.dw in leaf seven and declined to 2 nmoles/g.dw in leaf ten. 1-aminocyclopropane-1-carboxylic acid (MACC) concentration was highest in leaf one (11.3 nmoles/g.dw), declined to 6 nmoles/g.dw in leaf two, and remained constant for all other leaves. ACC synthase activity could not be determined in protein extracts from white clover leaf tissue. ACC oxidase activity in protein extracts varied in the different leaves examined. The activity versus substrate concentration curve for leaves one, three, five and six displayed saturation kinetics with respect to the substrate, ACC, whereas the data for leaves eight and ten did not show saturation kinetics over the range of ACC concentrations used. The ACC oxidase activity varied from 0.81 nL/mg.protein/h in extracts from leaf six, to 1.64 nL/mg.protein/h for leaf five. The apparent Km varied from 61 μM for leaf six to 138 μM for leaf five, while the Vmax varied from 0.92 for leaf six to 2.06 for leaf five. Degenerate oligonucleotide primers corresponding to conserved regions found among diverse ACC synthases were used for reverse transcriptase-polymerase chain reaction (RT-PCR) to amplify DNA fragments from RNA extracted from white clover leaf tissue. A DNA clone, ACS7, showed 88% homology at the nucleotide level to ACC synthase from Glycine max. The ACS7 sequence contained the three conserved domains (including the reaction centre, and the three residues known to bind the pyridoxal phosphate coenzyme) identified in published ACC synthase sequences. The derived amino acid sequence for the conserved domains are identical with other published sequences. Southern analysis indicates ACC synthase is represented by a multigene family in white clover. Northern analysis of the expression of ACC synthase using ACS7 as a hybridisation probe was unsucessful. Preliminary screening of a white clover leaf cDNA library produced a clone with 72.5% homology to a putative cysteine proteinase from Pisum sativum, and 63.4% homology to a cysteine proteinase from Vicia sativa.
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    Molecular characterisation of ethylene biosynthesis during leaf ontogeny in white clover (Trifolium repens L.) : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Massey University
    (Massey University, 1999) Yoo, Sang Dong
    Ethylene (C2H4) biosynthesis has been investigated during leaf ontogeny in white clover (Triforium repens L. cv. Glassland challenge, genotype 10F) with a particular emphasis on the production of the hormone in the apex and newly initiated leaves (designated as leaves 1 and 2). In these developing tissues, a relatively higher rate (5 to 6-fold) of ethylene production (0.5 to 0.9 nL C2H4 gFwt-1 hr-1) was associated with a higher accumulation (1.5-fold) of 1-aminocyclopropane-1-carboxylate (ACC), when compared with mature green leaves. Genes encoding the ethylene biosynthetic enzymes, ACC synthase (ACS) and ACC oxidase (ACO) have been cloned to characterise ethylene biosynthesis at the molecular level. The partial protein-coding regions of ACS genes have been cloned using reverse transcriptase-dependent polymerase chain reaction (RT-PCR) with degenerate nested primers using cDNA templates from RNA isolated either the apex or leaf 2. These ACS genes were identified and designated as TRACS1, TRACS2 and TRACS3. TRACS1 (680 bp) is 72% and 64% homologous to TRACS2 (674 bp) and TRACS3 (704 bp), respectively, and TRACS2 and TRACS3 are 63% homologous, in terms of nucleotide sequence. TRACS1 shows highest homology with PS-ACS2, an ACS cloned from etiolated pea (Pisum sativum) seedlings which is induced by IAA and wounding. TRACS2 shows highest homology with MI-ACS1, an ACS cloned from mature mango (Mangifera indica) fruit. TRACO3 shows highest homology with VR-ACS7, an ACS cloned from etiolated hypocotyls of mung bean (Vigna radiata) and also PS-ACS1, an ACS cloned from etiolated pea seedlings which is induced by IAA, but not by wounding. The TRACS3 gene was expressed as a 1.95 kb transcript mainly in the apex and newly initiated leaves as determined by northern analysis. TRACS1 has not been detected in the tissues examined. The expression of TRACS2 has not been determined. Three ACO genes, comprising ca. 1100 bp of the protein-coding region and 3'-untranslated region (3'-UTR) have been cloned using a combination of RT-PCR and 3'-rapid amplification of cDNA ends (3'-RACE), and designated as TRACO1, TRACO2 and TRACO3. Comparison of a 813 bp protein-coding region of TRACO1, generated by RT-PCR using degenerate primers on cDNA templates from RNA isolated from the apex shows 77% and 75% homology to the protein-coding region sequences of TRACO2 (804 bp) and TRACO3 (816 bp), respectively, generated by RT-PCR using degenerate primers on cDNA templates from RNA isolated from leaf 2. The TRACO2 and TRACO3 protein-coding region sequences show 84% homology. The technique of 3'-RACE generated 3'-UTR sequences of 301 bp (TRACO1), 250 bp (TRACO2), and 92 bp (TRACO3) each amplified using mRNA extracted from the apex. The 92 bp 3'-UTR of TRACO3 has been shown to be a truncated version of a 324 bp sequence amplified from TRACO3 expressed in senescent leaf tissue of white clover (Dr. D. Hunter, IMBS, Massey University, personal communication), and it is this full-length version that was used in further experiments. The 3'-UTR sequences of the three ACO genes are more divergent, when compared with the protein-coding regions, showing 61%, 55%, and 59% homology between TRACO1 and TRACO2, TRACO1 and full-length TRACO3, and TRACO2 and full-length TRACO3, respectively. Using these 3'-UTR sequences as gene-specific probes, Southern analysis revealed that the three ACO genes are encoded by distinct genes in the white clover genome. Northern analysis, using either protein-coding regions or 3'-UTRs as probes, determined that the TRACO1 gene was expressed as a 1.35 kb transcript almost exclusively in the apex and the TRACO2 gene was expressed as a 1.35 kb transcript mainly in newly initiated leaves and mature green leaves, with maximum expression in newly initiated leaves. This pattern of gene expression coincides with the high rate of ethylene production from the apex and newly initiated leaves in white clover. The apex tissue-specific TRACO1 gene was also detected in axillary buds, and the mature leaf-associated TRACO2 gene was expressed in other mature vegetative tissues, including internodes, nodes and petioles. Both TRACO1 and TRACO2 genes are highly expressed in roots. TRACO3 gene expression was not detected in apex and mature green leaf tissues examined using the 3'-UTR gene-specific probe, but two transcripts (1.17 kb and 1.35 kb) were visualised using the protein-coding region probe and with an extended exposure time. ACO enzyme activity, in vitro, was highest in just fully expanded mature green leaves (leaf 3 and leaf 4) during leaf ontogeny in white clover. Apex, axillary bud and floral bud tissues show a relatively higher enzyme activity, when compared with mature nodes and internode tissues, but lower than that measured in petiole tissue. Highest activity of ACO, in vitro, has been detected in root extracts. Polyclonal antibodies, raised against the TRACO1 gene product expressed in E. coli, recognised a high molecular weight (ca. 205 kD) protein complex with highest accumulation in the apex. This complex was also detected in axillary bud, floral bud, and leaf 1 tissue. Immunoaffinity-based purification of the ca. 205 kD protein was carried out to obtain sufficient protein for amino acid sequencing. However, no sequence was obtained. Polyclonal antibodies, raised against the TRACO2 gene product in E. coil, recognised ACO protein (ca. 36 kD) in newly initiated leaves and mature green leaves as well as petioles and roots. This recognition pattern coincides with ACO enzyme activity, in vitro, as well as TRACO2 gene expression in the tissue. Expression of the TRACO2 and TRACO3 genes has been characterised in response to a combination of wounding, ethylene, indole-3-acetic acid (IAA), aminoethoxyvinylglycine (AVG), and 1 -methylcyclopropene (1-MCP) treatments using mature green leaves. Expression of TRACO2 gene is enhanced in response to ethylene and IAA. Ethylene-induced TRACO2 gene expression was not blocked by 1-MCP. Expression of TRACO3 was induced in response to wounding in mature green leaves. Wound-induced TRACO3 gene expression was not induced by the ethylene produced in the leaf tissue, indicating ethylene-independent ACO expression in the wounded leaves. Induction of either TRACO2 by IAA treatment or TRACO3 by wounding was delayed with AVG treatment of mature green tissue, suggesting that changes in ACS activity in the tissue is associated with induction of ACO gene expression.
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    Role of cytokinin and ethylene during senescence in broccoli (Brassica oleracea var. Italica) : a thesis submitted for the degree of Doctor of Philosophy at Massey University
    (Massey University, 2003) Gapper, Nigel Esteven
    Broccoli (Brassica oleracea var. italica) deteriorates rapidly following harvest. The two plant hormones ethylene and cytokinin are known to act antagonistically on harvest-induced senescence in broccoli: ethylene acts by accelerating the process, whereas additional cytokinin delays it. The overall aim of this thesis was to gain a better understanding of how these two hormones control postharvest senescence. The effects of exogenous cytokinin (6-benzyl aminopurine, 6-BAP), 1-aminocyclopropane-1-carboxylic acid (ACC) and sucrose on senescence-associated gene expression were measured in both wild-type plants and transgenic plants harbouring an antisense tomato ACC oxidase gene (pTOM13). Exogenous cytokinin caused both a reduction (BoACO) and an increase (BoACS) in ethylene biosynthetic gene expression as well as reduced expression of genes encoding sucrose transporters and carbohydrate metabolising enzymes, indicating a significant role for cytokinin in the delay of senescence. Transgenic broccoli was produced using Agrobacterium tumefaciens-mediated transformation. Ethylene biosynthesis was targeted via an antisense BoACO2 gene fused to the harvest-induced asparagine synthetase (AS) promoter from asparagus. In addition, broccoli was transformed with constructs harbouring the Agrobacterium tumefaciens isopentenyl transferase (ipt) gene using the senescence-associated SAG12 and floral-associated MYB305 gene promoters to enhance the levels of cytokinin either during senescence or in floral tissue, respectively. The presence of the antisense AS-ACO construct was associated with an increased rate of transformation when compared to control constructs. Physiological analyses of mature plants showed that the antisense AS-ACO gene construct caused delayed senescence in both detached leaves and detached heads. Gene expression analyses of harvested floret tissue from AS-ACO lines showed decreases in transcript levels of senescence marker genes compared to wild-type and transgenic control lines, as well as a reduction in expression of sucrose transporter and carbohydrate metabolising genes, confirming the key role of ethylene in the promotion of senescence. In addition, genes involved with cytokinin biosynthesis and metabolism were isolated by PCR using primers based on Arabidopsis clones. The four broccoli ipt sequences aligned closely to four of the Arabidopsis sequences and were subsequently named BoIPT4, BoIPT5, BoIPT6 and BoIPT7. A cytokinin oxidase clone (BoCKX) was also isolated from broccoli. The four BoIPT genes were expressed in a number of different tissues, suggesting that the different genes may be involved in different biological processes in the plant. BoIPT4 was expressed early and BoCKX expressed late in florets during senescence. A model depicting the regulation of senescence in broccoli through the expression of cytokinin biosynthesis and metabolism genes, and their interaction with ethylene and carbohydrate metabolism is presented and discussed.