1. A general introduction to the theory, operation and biological activity of oxidation ponds has been given as well as specific introductions to the two areas of research studied, namely the role of fungi and some aspects of the nitrogen cycle in the Mangere oxidation ponds. 2. The enumeration and identification of fungi present in the bottom layers of the pond showed that the numbers were not sufficiently large to make a valuable contribution to the degradation of organic material in that particular environment. The dominant fungi present were species of the genera Penicillium, Mucor, Trichoderma and Geotrichum. 3. Isolates of some of the fungi most frequently isolated from these lower regions were grown under conditions of low redox potential. It was found that although the fungi did grow over the one month test period, they did not show great metabolic activity. 4. Direct examination of pond water containing a whitish glassy scum showed only the presence of clumps of heterogenous biomass but no dominant microorganism. Filamentous yeasts isolated from these scum samples formed a surface layer when grown in liquid medium. When inoculated into pond water it appeared that these filamentous yeasts formed a matrix to which the biomass adhered producing a condition similar to that seen in the original samples. 5. Use of ultra think layers of cetyl alcohol prevented scum formation by these filamentous yeasts but not growth in the medium underneath. Larger scale trials would be required to determine if this method is feasible for reducing scum formation in oxidation ponds. 6. Investigation of the proteolytic activity of pond sludge showed that the part of the nitrogen cycle which concerns the degradation of protein was present and active in the oxidation ponds. Proteolytic activity was found to increase with increasing depth of pond water up to the maximum (220 cm) tested. This suggests that increasing the depth of the ponds to approximately 185 cm (6 ft.) would improve pond efficiency in relation to degradation of protein. 7. The bacteria responsible for nitrification in the oxidation pond surface water were isolated and identified. Nitrosomonas was responsible for the oxidation of ammonia to nitrite and Nitrobacter for the oxidation of nitrite to nitrate. A twelve month survey of these bacteria showed Nitrite accumulated only when large numbers of Nitrosomonas were present. Statistical analysis of the data obtained indicated that populations of these bacteria were influenced by pH, alkalinity and ammonia. 8. Enumeration of denitrifying bacteria at different pond depths showed that greatest numbers were present in the sludge and interface although they were well distributed through the aqueous phase. 9. Identification of the isolated denitrifying bacteria showed that they were strains of Pseudomonas denitrificans, Micrococcus denitrificans and Bacillus lichenformis. Their identity was confirmed by comparison with standard cultures. 10. The denitrifying properties of these bacteria were studied to compare their ability to remove nitrate, as well as their reaction to additives. All three bacteria were able to remove low levels of nitrate but high nitrate concentrations were shown to be inhibitory. The use of additives to promote denitrification appeared to depend on the additive and the bacterium and not on the carbon nitrogen ration. Of the materials tested glucose was the most satisfactory and methanol the most inhibitory. Further work is necessary to clarify the role of methanol because in small scale studies with sewage effluents in other laboratories it has been found to be most efficient in promoting denitrification. 11. Continous measurements of the redox potential interface of a sludge, and pond top liquid (10-15cm. from the surface) revealed that the electronegative potential of the sludge remained remarkably steady. When the electrode was at either the interface or near the surface it recorded a distinct diurnal pattern. In pond top liquid the length of the positive potential period was dependent on day length. A change from the normal redox potential pattern of pond top liquid preceded deterioration of pond stability. The pattern returned to normal after a change in environmental conditions. It appears that continuous redox potential measurements could be valuable in showing changes in pond performance before they can be detected by other methods.

Actinidia chinensis, a diploid relative of kiwifruit, has valuable fruit characteristics, and varieties with superior flavour and marketable size have recently been selected in a classical breeding programme. However, the marketability of the fruit of A. chinensis and many other species of Actinidia is limited by poor fruit storage properties. Pioneering work in tomato has demonstrated that fruit ripening and senescence can be very effectively delayed by down-regulating genes required for biosynthesis of the phytohormone ethylene. The goal of this work was to isolate genes for ethylene biosynthesis in A. chinensis, characterise their expression, and to generate transgenic plants containing T-DNA constructs designed for ethylene downregulation. A small cDNA library was constructed from RNA isolated from the ripe fruit of A. chinensis. The library was screened for genes encoding each of the enzymes in the ethylene biosynthetic pathway, by probing with PCR products amplified from kiwifruit cDNA and with a cDNA clone previously isolated from kiwifruit. Three distinct cDNA clones encoding S-adenosyl-L-methionine (SAM) synthetase (ACSAM1, ACSAM2 and ACSAM3) were isolated from the library, together with two distinct 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase cDNA clones (ACAO1 and ACAO2). No ACC synthase cDNAs were detected in the library, indicating low transcript abundance. However, a partial ACC synthase cDNA (ACAS1) was amplified from ripe fruit using PCR techniques, and subsequently cloned in a plasmid vector. Phylogenetic analysis of SAM synthetase protein sequences from A. chinensis and other plant species indicates bifurcation of angiosperm SAM synthetase sequences into two main branches; ACSAM3 was assigned to a different branch from ACSAM1 and ACSAM2. The peptide sequence of ACAS1 shows higher homology to several auxin-inducible ACC synthase peptides than the product of the ethylene-inducible ACC synthase gene which is predominantly transcribed in ripening tomato fruit. RNAse protection assays were employed to estimate the relative transcript levels of each of the ethylene biosynthetic genes isolated from A. chinensis during ethylene-induced, post-harvest fruit ripening, and in immature fruit and floral samples. The response of the mature fruit to exogenous ethylene indicated a clear separation of ethylene sensitivity and ethylene production in A. chinensis. The application of exogenous ethylene correlated with increased transcript levels for all three SAM synthetase genes (ACSAM1, ACSAM2 and ACSAM3) and for the ACC oxidase gene family. Transcription of the ACC synthase gene ACAS1 was not affected by exogenous ethylene, but transcript levels increased during subsequent ethylene biosynthesis, consistent with this being a controlling step for the onset of ethylene production. One or more ACC oxidase transcripts increased significantly both prior to and during ethylene production. Only one of the SAM synthetase transcripts (ACSAM3) was induced during the late ethylene burst, and these transcripts were also abundant in floral tissues and young fruit. A role for SAM synthetase genes in the methionine salvage pathway is discussed. The expression patterns for ACAS1 and the ACC oxidase gene family arc consistent with the consensus view that the rate of ethylene biosynthesis in plant tissues is dependent on both ACC synthase and ACC oxidase activity levels. Therefore, with the aim of down-regulating ethylene biosynthesis in A. chinensis, expression cassettes containing ACAS1 and ACAO1 cDNAs, each controlled by a d35S promoter, were inserted in tandem into the Agrobacterium binary vector pCGN1549, in both the sense and antisense orientations. Leaf tissue from the ‘Earligold’ variety of A. chinensis was transformed with the resulting binary vectors, and transgenic plants were regenerated. PCR and Southern analysis indicated intact T-DNAs were integrated in at least half of the transformed plants, and Northern analysis detected mRNAs from one of the transgenes transcribed from both the sense and antisense constructs. No decrease in wound-induced ethylene biosynthesis was detected in the leaves of a small sample of these transgenic plants, and a larger number of transformants are now being grown for phenotypic screening. Down-regulation of ethylene biosynthesis may improve the storage properties and/or the shelf life of transgenic A. chinensis plants and may provide insights into the roles of ethylene in fruit ripening. / Appendix 1 restricted at the request of the author

The export of tamarillo is an important component of the New Zealand, exotic fruit industry. However, the quality of tamarillo fruit is severely decreased by tamarillo mosaic potyvirus (TaMV) which detrimentally affects fruit colour resulting in the fruit being unacceptable on the international marketplace. Virus incidence was surveyed in four tamarillo growing regions. Viruses were detected by indicator plant symptomology, and the incidence confirmed by dot blot analysis or ELISA. TaMV was present in 100% of tamarillo trees analyzed in ten of the twelve orchards surveyed. Incidence of potato aucuba mosaic potexvirus, cucumber mosaic cucumovirus, alfalfa mosaic virus and arabis mosaic nepovirus, which had all been previously reported in tamarillo was also determined. Tomato spotted wilt tospovirus was identified for the first time in tamarillo plants in New Zealand. TaMV RNA was purified from infected tamarillo leaves and a 1600 base pair cDNA clone generated to the 3'-terminus. The clone was sequenced and the location of the gene for the coat protein was identified by direct amino-terminal sequencing of purified TaMV coat protein. Comparison with other potyvirus coat protein sequences established that TaMV represents a new member of the potyvirus group. Seven chimeric transgenes containing TaMV sequences were constructed in three different binary vectors suitable for Agrobacterium-mediated transformation of plants. Of the twenty-six independent Nicotiana benthamiana plant lines generated expressing either TaMV coat protein or TaMV RNA sequences modified to block translation, eight plant lines demonstrated resistance to virus infection (more than 10% of resistant plants/line). All plants of one line, PT#25, were resistant to TaMV infection using dilute inocula; 40% were also resistant after inoculation with more concentrated inocula. The level of accumulation of CP in transgenic plant lines did not correspond with the degree of resistance to TaMV infection. In eight of the twenty-six transgenic lines a proportion of plants demonstrated recovery from systemic infection. Recovery was manifested as an absence or significant reduction of virus symptoms in newly developed leaves of plants that had previously shown symptoms of systemic TaMV infection. The mechanism of recovery from systemic infection is discussed. The protocol for the regeneration of transgenic tamarillo by Agrobacterium-mediated transformation was improved. The efficiency of transformation was optimized and the rate of transgenic shoot elongation was increased. Two tamarillo plants transformed with a transgene designed to express the TaMV coat protein were produced and were demonstrated to express the neomycin phosphotransferase gene and TaMV coat protein gene. However, following challenge with a low concentration of inoculum, micropropagants of these tamarillo plants failed to demonstrate resistance to TaMV infection. Three in vitro enzymatic activities of the virus encoded cytoplasmic inclusion protein were studied. The cytoplasmic inclusion protein was purified to near homogeneity using differential centrifugation and sucrose gradient purification. RNA-stimulated ATPase activity was demonstrated and the Km and Vmax determined. RNA binding and energy-dependent dissociation were characterized. RNA helicase activity of the cytoplasmic inclusion protein was demonstrated in the presence of NTP using RNA duplexes with single-stranded overhangs. These results have confirmed and extended the previous findings of the likely RNA helicase function of the potyvirus cytoplasmic inclusion protein, and suggest possible future mechanisms for obtaining resistance to potyviruses.

The intertidal red alga, Gracilaria chilensis Bird, McLachlan et Oliveira (Rhodophyta, Gracilariales), lives in an environment in which light is highly variable in terms of both amplitude and duration. A laboratory investigation of the photophysiology of G. chilensis was conducted to assess the response of the photosynthetic apparatus to light variability characteristic of the natural environment. Freshly collected Gracilaria chilensis was found to exhibit an endogenous rhythm of photosynthesis in conditions of constant limiting light and temperature. However, such a phenomenon was not observed in saturating light. Rhythms of phycobiliprotein concentration and dark respiration were also observed but were not as well-defined and could nor account for the photosynthetic rhythm. The photosynthetic response of G. chilensis to light fluctuations of various durations (0.25 to 900 seconds) and light levels was compared to that in static light. G. chilensis was able to utilise rapidly fluctuating light (< 1 second) more efficiently than fluctuations of longer duration (60-900 second). Mean photosynthetic rates were enhanced by up to l50% in fluctuating light of less than 60 sec duration over that predicted from steady-state. The photosynthetic apparatus of freshly collected G. chilensis was found to have many low-light "shade" acclimation characteristics. These included a low compensation point (5 µmolm-2 s-l) and onset of saturation (80 µmolm-2 s-l) suggesting sensitivity to photoinhibition. However, (laboratory) low-light acclimated G. chilensis was able to tolerate periods of constant high light (2000 µmolm-2 s-l)for periods of six hours without detectable detrimental effect on photosynthetic capacity, although photosynthetic efficiency was significantly inhibited after two hours of this treatment. The time course and characteristics of photoacclimation were determined by culturing G. chilensis in low- (15 µmol m-2 s-l) and high- (180 µmol m-2 s-l) light and high- and low-nitrogen regimes. The observed change in photosynthetic characteristics and pigment concentration indicated that acclimation began after a time lag of l-2 days, was complete after approximately a week and was reversible. Acclimation to growth light included changes in growth rate, P-I response curves, pigment concentration and composition and other biochemical components (e.g. carbon/nitrogen ratio). The nitrogen regime significantly affected pigment concentration in the high-light grown plants and the response suggests pigments play a role in nitrogen storage as well as light harvesting. These various physiological characteristics described above were interpreted as important mechanisms that enable G. chilensis to optimise photosynthetic response in the highly dynamic and stressful zone of the intertidal environment.

Persistence of Colletotrichum acutatum Simmonds f.sp. pinea Dingley & Gilmour in pine nursery soils and its infectivity of Pinus radiata D. Don seedlings were studied. Sampling procedures were compared in the search of soils for fungal propagules. Those most frequently used were soil dilution plate method, surface-soil dilution plate technique, and plating of debris picked from soil. A selective medium for direct isolation from soil was prepared comprising potato dextrose agar as the basal medium and incorporating benomyl (50 ppm), quintozene (7.5 or 75 ppm), streptomycin sulfate (100 ppm), chloramphenicol (100 ppm) and chlortetracycline HCl (100 ppm). Studies in plots at Auckland, Tokoroa and Rotorua indicated that fungal structures within P. radiata debris persisted for long periods; up to 25 months in the Auckland plots. In vitro, the viability of conidia in natural soil declined very rapidly; more than 50% of conidia were non-viable within 4 weeks of their introduction into soil incubated at 15 and 25 C. Similar experiments conducted at different matric potentials indicated that drier soils (-0.3 or -0.4 bar) had higher levels of viable conidia over periods of time compared to wetter soils of -0.1 bar or at saturation. Segments of pine seedlings introduced to soils into which various concentrations of conidia were previously incorporated showed increasing colonization with increasing conidial concentration at both 15 and 25 C after 4 days. Maximum colonization of segments occurred at conidial concentrations 106 ml-1 to 107 ml-1. Artificially-inoculated segments of pine seedlings were introduced into soil and then these were recovered at different time periods. After two months in soil 100% recovery of the fungus was obtained from all leaf, stem and root segments plated. Recovery levels from all the plated stem and root segments after 8.5 months were never lower than 68%. Recovery levels from leaf segments were lower because many of these had decomposed leaving only the central xylem strands. Conidia-laden membrane filters were introduced into soil. A method of microscopic study used after removal of these from soil enabled easy observation of resultant structures. Appressoria were formed while the fungus was in soil. Other potential survival propagules were thick-walled, darkly-pigmented, short hyphae within pine debris. It is considered that the short individual cells of such hyphae could survive as chlamydospores, and these are believed to be the prime means of long-term survival in soil. Several fumigants were tested for effectiveness in eradication of the fungus from soil. All chemicals tested, namely chloropicrin, methyl bromide, "Di-Trapex" and dazomet significantly reduced viability of conidia and the fungus in pine debris. Weeds from a pine nursery were inoculated with conidia; of these, only Epilobium ciliatum Raf. became infected. Infection of healthy parts was infrequent but saprophytic growth in older parts frequently killed the plant. This species is therefore a potential alternative host, although its importance as an inoculum source for infection of pine seedlings is unknown. Experimentally it was shown that infection could occur by rain splash of conidia onto healthy plants. Infection of P. radiata seedlings was studied at several constant and alternating temperatures. Initial infection was greatest at higher temperatures (ca 24 C); while full symptoms of the disease were best expressed at lower temperatures (12 to 18 C). Inoculated seedlings had significantly reduced shoot and root development at 12, 15, 18 C, but not at 6, 9, 23, 24, 27 C, after 53 days. Increase in height of healthy seedlings was significantly greater than that of inoculated seedlings, after 52 days at 18 C. Pathogenicity on P. radiata seedlings of an isolate of C. acutatum f.sp. pinea isolated from soil, "whitish isolate", was comparable to the normal "coloured isolate". / Whole document restricted, but available by request, use the feedback form to request access.

1. A tobacco cell-suspension tissue culture system derived from pith cells of Nicotiana tabacum L. var Wisconsin 38 was established. This system was used to study methods of purifying nucleic acids, phosphate ester and nucleic acid metabolism in cells subjected to nutritional shifts, and problems relating to the control of cell division in plant cells. 2. Cell cultures consisting of single cells, groups of cells and small aggregates were maintained in liquid medium on rotary shakers. Cultures exhibited an exponential growth period during which the cell generation time was 2, 3 days. 3. Cells removed from culture medium and resuspended in dilute inorganic salt solutions (step-down nutritional shift) exhibited marked changes in their phosphate and sulphate accumulation rates and in phosphate ester metabolism, but not in the type and rate of respiration. 4. Such metabolic responses to culture shifts were not related to cell damage or shock effects, but appeared to be a feature of changes in the nutritional environment of cells. The response of cells to a step-down nutritional shift was maximal at exponential- and late-stationary phases of culture growth. Step-up nutritional shifts (resuspending cells in enriched culture medium) produced some effects similar to those found for step-down cells. Actinomycin D did not prevent the phosphate accumulation response of cells to culture shifts, and was shown to inhibit only 50 per cent of the synthesis of rapidly-labelled ribonucleic acid. 5. RNA prepared from tobacco cells or rat liver by conventional phenol extraction was contaminated with other materials which composed as much as sixty per cent of the weight of the total preparation. RNA prepared from (32P) tobacco cells contained minor radioactivity in RNA but major radioactivity in a wide range of contaminating phosphate esters of high specific radioactivity. 6. Purification of RNA by the Kirby two-phase partition procedure removed most major contaminants but failed to remove (32P) phosphate esters. The procedure also resulted in degraded RNA. 7. A new method was developed for the purification of RNA prepared by the phenol procedure. It involved recovery of RNA from the upper phase of the two-phase Kirby extraction mixture by precipitation as the cetyltrimethylammonium (CETA) salt instead of by dialysis. This procedure reduced manipulation times and much more effectively removed RNAase and (32P) phosphate esters from the RNA. 8. TMV-RNA prepared by the new procedure was fully infectious. RNA prepared and purified from a mixture of whole rat liver and TMV was almost equally as infectious as RNA prepared from TMV alone. Infectivity of purified RNA, prepared from a mixture of tobacco cells and TMV, was stable in solution at 30°C for four hours. TMV-RNA prepared in this way and stored at -12°C in vacuo over P2O5 retained infectivity for at least 6 months. 9. Extraction of (32P) exponential-phase cells by aqueous phenol plus detergent released only RNA with the ribosomal type of base composition. Similar extractions in the presence of high salt concentrations released this RNA together with DNA and rapidly-labelled RNA. 10. During short treatments with (32P) orthophosphate, exponential-phase cells synthesised RNA with a base composition intermediate between that of ribosomal RNA and DNA. Cells subjected to a step-down nutritional shift prior to treatment with (32P) orthophosphate, synthesised RNA with a base composition close to that of tobacco DNA. 11. Following treatment times with (32P orthophosphate, or (32P) plus (3H) uridine, and following a step-down or steady-state nutritional shift to non-radioactive medium, tobacco cells exhibited the following changes in phosphate ester and RNA metabolism:- a. Changes in the levels of radioactivity present in the nucleoside triphosphate RNA precursors and other phosphate esters. b. A reduction in the rate of increase of specific radioactivity of RNA of step-down cells, followed by a subsequent recovery. c. Changes in the (32P) base composition of the RNA synthesised following the culture shift. d. Changes in the radioactive sedimentation profiles of RNA. 12. Extracts prepared from exponential-phase tobacco cells contained cytokinin (cell division stimulant) activity. Fractionation of cells, and bioassay of levels of cytokinins present in the various fractions, demonstrated the presence of soluble compounds with cytokinin activity in cell debris and cytoplasmic fractions, but not in the nuclear fraction. 13. Unhydrolysed (polymeric) tobacco nucleic acids (RNA plus DNA), prepared from exponential-phase tobacco cells, showed no cytokinin activity. A ribonucleotide mixture prepared by alkaline hydrolysis of these tobacco-cell nucleic acid preparations, mixtures of purified ribonucleotides, or a mixture of tobacco deoxyribo- and ribonucleosides resulting from snake-venom digestion, contained no activity. Deoxyribonucleotides were inhibitory. 14. A ribonucleotide mixture prepared by alkaline hydrolysis of RNA isolated from rat liver and sheep liver, contained cytokinin activity. Deoxyribo- and ribonucleoside mixtures resulting from snake-venom digestion of sheep-liver RNA and DNA contained similar levels of cytokinin activity to that found in extracts prepared by alkaline hydrolysis.

Ecophysiological attributes could be causes for rarity in plants. We tested the hypothesis that a species’ ability to regulate photosynthesis and growth in response to environmental factors is indicative of its environmental resilience and that this is linked to its conservation status. In this study, the ecophysiology of Graptophyllum reticulatum, an Australian endangered endemic species, was compared with that of its three closely related and more common congeners G. ilicifolium, G. excelsum and G. spinigerum. Ecophysiological attributes were measured on the four species in their natural habitats and under artificially imposed environmental stresses, including changed soil conditions, excess light and low water availability, in a glasshouse experiment. Photosynthesis was determined at the photosystem II and leaf level using chlorophyll a fluorescence and gas exchange techniques. Applied to the chlorophyll fluorescence transient of leaves, the JIP test provides a Performance Index which quantifies the main steps in PSII photochemistry including light energy absorption, excitation energy trapping, and conversion of excitation energy into electron flow. At the leaf level, gas exchange measurements allow determination of maximum CO2 assimilation rates, intercellular CO2 concentrations, stomatal conductance for water vapour and instantaneous water use efficiency. Growth analysis was performed to assess relative growth rates and physiological and morphological responses. Analysis of physiological differences and responses indicated that, compared to its more common relatives, the endangered G. reticulatum was an intrinsically slow growing species, exhibited the lowest fitness when growing in favorable environments and was most sensitive to excess light stress. Photoinhibition is therefore likely to restrict the endangered species to shade habitats. Compared with the endangered G. reticulatum, the vulnerable G. ilicifolium and common G. spinigerum species were better adapted to high light and changed nutrient levels, but were more susceptible to water stress. The rare G. excelsum had the fastest growth rate and the highest fitness in favorable environments. Based on the ecophysiological attributes examined here, it is proposed that excess light is likely to be the most critical abiotic factor restricting distribution of the endangered species in a fragmented landscape. The survival of the species may be most dependent on the intactness of the habitat over-storey. In contrast, the vulnerable G. ilicifolium showed strong susceptibility to water limitation, and survival might be threatened if climate change alters habitat water relations to cause, for example, more pronounced dry periods. The rare G. excelsum which had highest carbon gain and growth in the experiments carried out in this study, may become the most successful adaptation out of the rainforest environment due to its tolerance to higher light and limited water availability. To examine the generality of the link between rarity and ecophysiology with Graptophyllum species, two dipterocarp species, narrowly endemic Dipterocarpus condorensis and local common Shorea roxburghii that are actually co-located in South-eastern Vietnam were studied. Findings in this case study confirmed the usefulness of the comparative approach based on physiological measurements, either in situ or ex situ, to explain plant rarity. The results of this study indicate ecophysiological research is a tool for examining causes of rarity and possible abiotic threats. The information gained allows assessment of environmental resilience of species and contributes essential knowledge for management and conservation of threatened plants. Such knowledge is also useful for ex situ conservation including propagation, translocation and re-introduction in restoration programs.

Four New Zealand species are recognised: (1) P. tremula R.Br., (also in Australia, Lord Howe Is., Norfolk Is., the Kermadecs Is. and the Chatham Is.). (2) P. carsei sp. nov. (previously ‘P. comans’) (also in Australia and the Kermadec Is.). (3) P. macilenta A. Rich. (previously P. macilenta var. saxatilis Carse) endemic. (4) P. pendula Col. (previously P. macilenta Auct. non. Rich. and P. macilenta var. pendula (Col.) Cheeseman) endemic. The taxonomy and nomenclature of these species is discussed in detail and the nomenclature is also discussed for P. kingiana Endl. (previously sometimes treated as P. tremula) and P. zahlbruckneriana Endl. (previously treated under ‘P. comans’ or P.endlicheriana Agardh) both Norfolk Is. endemics, and the taxonomy of P. sp.aff.comans LHI (previously ‘P. comans’) endemic to Lord Howe Island is also discussed. Detailed study of the spores and paleae using conventional light microscopy and SEM was made for these species and also P. comans Forst. f. (from the New Hebrides) and P. novae-caledoniae from New Caledonia. Comparisons of the distribution, fronds, stipes, venation, rhizomes, paleae, indumentum, apices, sori, sporangia and spores have been made and where appropriate material of P. dentata ssp. flabellata, P. pacifica and P. vittata, has also been compared. Further comparisons have been made with material of ‘P. comans’ from other Pacific Islands including Fiji (three species ), Rarotonga, Samoa, Tahiti (each one species). P. tremula, P. kingiana and P. novae-caledoniae are exceptional in the genus because they lack paraphyses in the sori. P. kingiana and P. novae-caledoniae have a copious waxy deposit around and among the sporangial stalks but P. tremula has no accessory sporangial features at all. Germination and gametophyte growth follow the normal pattern for the genus. Some gametophytes can be kept alive and growing for considerable periods (up to three years) and become elongate, ribbon-like and unisexual (female). Hybridisation was achieved between P. carsei and P. macilenta. The progeny resemble the natural hybrid swarms suspected of being the product of the same parents.

Actinidia chinensis, a diploid relative of kiwifruit, has valuable fruit characteristics, and varieties with superior flavour and marketable size have recently been selected in a classical breeding programme. However, the marketability of the fruit of A. chinensis and many other species of Actinidia is limited by poor fruit storage properties. Pioneering work in tomato has demonstrated that fruit ripening and senescence can be very effectively delayed by down-regulating genes required for biosynthesis of the phytohormone ethylene. The goal of this work was to isolate genes for ethylene biosynthesis in A. chinensis, characterise their expression, and to generate transgenic plants containing T-DNA constructs designed for ethylene downregulation. A small cDNA library was constructed from RNA isolated from the ripe fruit of A. chinensis. The library was screened for genes encoding each of the enzymes in the ethylene biosynthetic pathway, by probing with PCR products amplified from kiwifruit cDNA and with a cDNA clone previously isolated from kiwifruit. Three distinct cDNA clones encoding S-adenosyl-L-methionine (SAM) synthetase (ACSAM1, ACSAM2 and ACSAM3) were isolated from the library, together with two distinct 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase cDNA clones (ACAO1 and ACAO2). No ACC synthase cDNAs were detected in the library, indicating low transcript abundance. However, a partial ACC synthase cDNA (ACAS1) was amplified from ripe fruit using PCR techniques, and subsequently cloned in a plasmid vector. Phylogenetic analysis of SAM synthetase protein sequences from A. chinensis and other plant species indicates bifurcation of angiosperm SAM synthetase sequences into two main branches; ACSAM3 was assigned to a different branch from ACSAM1 and ACSAM2. The peptide sequence of ACAS1 shows higher homology to several auxin-inducible ACC synthase peptides than the product of the ethylene-inducible ACC synthase gene which is predominantly transcribed in ripening tomato fruit. RNAse protection assays were employed to estimate the relative transcript levels of each of the ethylene biosynthetic genes isolated from A. chinensis during ethylene-induced, post-harvest fruit ripening, and in immature fruit and floral samples. The response of the mature fruit to exogenous ethylene indicated a clear separation of ethylene sensitivity and ethylene production in A. chinensis. The application of exogenous ethylene correlated with increased transcript levels for all three SAM synthetase genes (ACSAM1, ACSAM2 and ACSAM3) and for the ACC oxidase gene family. Transcription of the ACC synthase gene ACAS1 was not affected by exogenous ethylene, but transcript levels increased during subsequent ethylene biosynthesis, consistent with this being a controlling step for the onset of ethylene production. One or more ACC oxidase transcripts increased significantly both prior to and during ethylene production. Only one of the SAM synthetase transcripts (ACSAM3) was induced during the late ethylene burst, and these transcripts were also abundant in floral tissues and young fruit. A role for SAM synthetase genes in the methionine salvage pathway is discussed. The expression patterns for ACAS1 and the ACC oxidase gene family arc consistent with the consensus view that the rate of ethylene biosynthesis in plant tissues is dependent on both ACC synthase and ACC oxidase activity levels. Therefore, with the aim of down-regulating ethylene biosynthesis in A. chinensis, expression cassettes containing ACAS1 and ACAO1 cDNAs, each controlled by a d35S promoter, were inserted in tandem into the Agrobacterium binary vector pCGN1549, in both the sense and antisense orientations. Leaf tissue from the ‘Earligold’ variety of A. chinensis was transformed with the resulting binary vectors, and transgenic plants were regenerated. PCR and Southern analysis indicated intact T-DNAs were integrated in at least half of the transformed plants, and Northern analysis detected mRNAs from one of the transgenes transcribed from both the sense and antisense constructs. No decrease in wound-induced ethylene biosynthesis was detected in the leaves of a small sample of these transgenic plants, and a larger number of transformants are now being grown for phenotypic screening. Down-regulation of ethylene biosynthesis may improve the storage properties and/or the shelf life of transgenic A. chinensis plants and may provide insights into the roles of ethylene in fruit ripening. / Appendix 1 restricted at the request of the author

The export of tamarillo is an important component of the New Zealand, exotic fruit industry. However, the quality of tamarillo fruit is severely decreased by tamarillo mosaic potyvirus (TaMV) which detrimentally affects fruit colour resulting in the fruit being unacceptable on the international marketplace. Virus incidence was surveyed in four tamarillo growing regions. Viruses were detected by indicator plant symptomology, and the incidence confirmed by dot blot analysis or ELISA. TaMV was present in 100% of tamarillo trees analyzed in ten of the twelve orchards surveyed. Incidence of potato aucuba mosaic potexvirus, cucumber mosaic cucumovirus, alfalfa mosaic virus and arabis mosaic nepovirus, which had all been previously reported in tamarillo was also determined. Tomato spotted wilt tospovirus was identified for the first time in tamarillo plants in New Zealand. TaMV RNA was purified from infected tamarillo leaves and a 1600 base pair cDNA clone generated to the 3'-terminus. The clone was sequenced and the location of the gene for the coat protein was identified by direct amino-terminal sequencing of purified TaMV coat protein. Comparison with other potyvirus coat protein sequences established that TaMV represents a new member of the potyvirus group. Seven chimeric transgenes containing TaMV sequences were constructed in three different binary vectors suitable for Agrobacterium-mediated transformation of plants. Of the twenty-six independent Nicotiana benthamiana plant lines generated expressing either TaMV coat protein or TaMV RNA sequences modified to block translation, eight plant lines demonstrated resistance to virus infection (more than 10% of resistant plants/line). All plants of one line, PT#25, were resistant to TaMV infection using dilute inocula; 40% were also resistant after inoculation with more concentrated inocula. The level of accumulation of CP in transgenic plant lines did not correspond with the degree of resistance to TaMV infection. In eight of the twenty-six transgenic lines a proportion of plants demonstrated recovery from systemic infection. Recovery was manifested as an absence or significant reduction of virus symptoms in newly developed leaves of plants that had previously shown symptoms of systemic TaMV infection. The mechanism of recovery from systemic infection is discussed. The protocol for the regeneration of transgenic tamarillo by Agrobacterium-mediated transformation was improved. The efficiency of transformation was optimized and the rate of transgenic shoot elongation was increased. Two tamarillo plants transformed with a transgene designed to express the TaMV coat protein were produced and were demonstrated to express the neomycin phosphotransferase gene and TaMV coat protein gene. However, following challenge with a low concentration of inoculum, micropropagants of these tamarillo plants failed to demonstrate resistance to TaMV infection. Three in vitro enzymatic activities of the virus encoded cytoplasmic inclusion protein were studied. The cytoplasmic inclusion protein was purified to near homogeneity using differential centrifugation and sucrose gradient purification. RNA-stimulated ATPase activity was demonstrated and the Km and Vmax determined. RNA binding and energy-dependent dissociation were characterized. RNA helicase activity of the cytoplasmic inclusion protein was demonstrated in the presence of NTP using RNA duplexes with single-stranded overhangs. These results have confirmed and extended the previous findings of the likely RNA helicase function of the potyvirus cytoplasmic inclusion protein, and suggest possible future mechanisms for obtaining resistance to potyviruses.