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    Water-use efficiency in perennial ryegrass (Lolium perenne L.) : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Science at Massey University, Manawatu, New Zealand
    (Massey University, 2021) Weerarathne, Lekamalage Visna Yahani
    Knowledge of genetic and physiological bases of drought responses and stress tolerance properties of pasture plants is an integral part of designing efficient pasture improvement programs to combat the consequences of climate change. However, experimental evidence or theoretical analyses on that aspect is sparse in the literature. Therefore, considering wider applications, high economic importance, and acknowledged poor tolerance of (Lolium perenne L.; PRG) to drought, the main aim of this research was to evaluate morpho-physiological trait responses that are linked to water-use efficiency (WUE) in different PRG populations from different sources in simulated summer drought cycles under a controlled environment. This study also included a quantitative genetic analysis conducted on the key traits to ascertain which traits are under genetic control for future breeding purposes. Experiment 1 screened single potted PRG genotypes from three commercial cultivars with industry reputation for persistence for natural differences in morpho-physiological traits related to water use (WU) under simulated drought. Large within-population variation was observed for the measured traits which included, among others, WUE (g WU/g plant dry matter); shoot dry weight, SDW; leaf osmotic potential, OP; leaf relative water content; predawn leaf water potential; root dry weight at 20–50 cm depth, RDWD; gravimetric soil moisture at 30–40 cm depth, SMCD; post-cutting regrowth. Principal component analysis (PCA) identified important trait associations contributing to high WUE (i.e. WUE-OP-RDWD trait association) and one related to higher SDW together with ‘SMCD conservation’ indicative of ‘true WUE’ was used to make a divergent selection of 20 high- and 15 low-WUE genotypes (HWUE and LWUE, respectively). Experiments 2 and 3 were conducted simultaneously using the same methodology as Experiment 1 and inter-randomised in the same glasshouse space. Experiment 2 retested clones of HWUE and LWUE plants selected in Experiment 1 for consistency of trait expression across the two consecutive growing seasons, and also collected data for additional traits. Results confirmed that the key trait associations identified in Experiment 1 were almost identically expressed in Experiment 2. From data on additional traits, it was established that the accumulation of high molecular weight sugars in the shoots significantly contribute to ‘true WUE’ of a subset of PRG genotypes but, with the less involvement of gas exchange data under the conditions tested. It is speculated that enhanced mesophyll conductance of CO2 might underlie this important trait association. However, the large majority of genotypes exhibited a ‘SMCD-depleting’ trait association of WUE with improved gas exchange and maximum quantum efficiency of PSII, demonstrating late A.R. Blum’s theory of WUE. Thus, selection of PRG for drought tolerance should consider yield and soil moisture data together to establish the most appropriate category of WUE trait association in improved cultivars. Experiment 3 investigated drought response trait associations in two further populations: a group of elite plants from a commercial plant breeder’s breeding program (CBL) and a PRG germplasm line derived from crossing Mediterranean and Middle Eastern seed accessions (MMEL) compared with those from the HWUE selection. PCA results showed that the major trait associations found in the yield and water relations data of elite subsets of CBL closely followed those of the HWUE selection, but elite MMEL plants exhibited typical summer dormancy characteristics where the average SDW of MMEL was 40% of lower than that of the CBL plants. Results further suggested that the company field evaluation system could benefit from the consideration of water relations traits, including WUE and associated traits like OP, as externally-measured selection traits for PRG drought tolerance. Experiment 4 evaluated quantitative genetic parameters of the key traits using the same methodology from Experiment 1 in a breeding population of 36 ‘half-sib (HS) families’ under both stressed and non-stressed conditions. There were significant estimates of among- and within-HS family genetic variances, narrow-sense heritability, and predicted genetic gain estimates for the key traits, indicating high genetic potential of each trait for breeding purposes under the conditions tested. However, the correlated response to selection of each trait pair comprising highly genetically correlated morpho-physiological traits with WUE was lower than that gain from the single-trait selection, accommodating further research questions on the efficacy of indirect and multi-trait selection of key traits. Based on the current results, it was found that the direct selection of PRG for WUE or concurrent selection for OP, RDWD, SMCD, and SDW or RGS traits under drought is advisable. Furthermore, significant quantitative genetic parameters estimated for WUE under non-stressed conditions together with high genetic correlation observed for WUE between stressed and non-stressed conditions suggested that a PRG population can potentially be selected for this trait at early growth stages or before imposition of water deficit.
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    Exploring the genetic potential of locally adapted germplasm for drought tolerance : a case for cowpea (Vigna unguiculata (L.)) Walp) from Malawi : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Science, Institute of Agriculture and Environment, Massey University, Palmerston North, New Zealand
    (Massey University, 2014) Pungulani, Lawrent Lino Michael
    The shortage of improved cowpea (Vigna unguiculata (L.) Walp) varieties and increased frequency of droughts in Malawi have created a need to identify drought tolerant genotypes with desirable agronomic and utility characteristics. This research evaluated local germplasm maintained by the Malawi Plant Genetic Resources Centre (MPGRC), as an initial step towards the identification of genotypes with drought tolerance. Eco-geographic characterisation revealed diverse ecologies among the different germplasm collected. These genotypes were subsequently assessed for drought tolerance in a glasshouse study. All genotypes which tolerated low moisture conditions in the glasshouse originated from areas with high rainfall and low temperatures suggesting that extreme environmental conditions and/or human mediated actions interfered with adaptation processes. Furthermore, the eco-geographic characterisation identified germplasm gaps which need to be filled by either collection or repatriation of germplasm from international genebanks. The establishment of on-farm conservation in areas with low rainfall and high temperature such as Chikwawa and Nsanje districts may enhance adaptation of cowpea to drought conditions. Genotypes 479, 601, 645, 2226 and 3254 fully recovered from moisture stress, while 2232 started wilting within one week of drought stress initiation in the first glasshouse experiment. The genotypes which recovered from moisture stress showed low scores for wilting scales, low leaf wilting index (LWI), high relative water content, high scores for stem greenness and high levels for re-growth. In addition, the first glasshouse experiment resulted in the development of a leaf wilting index, which has been identified as an easily used method for scoring wilting, compared to common wilting scales. In a subsequent glasshouse experiment, all the genotypes which fully recovered from moisture stress showed high relative water content during the period of stress, but showed differences in other physiological traits. For example, genotypes 479, 601, 645 and 2226 had reduced stomatal conductance, transpiration rate and net photosynthesis, while 3254 maintained high scores for the three traits from the initial stage of moisture stress. Although 2232 showed a high transpiration rate and stomatal conductance, its net photosynthesis was significantly reduced, compared to all the other genotypes, after the third week of stress. The differences in physiological traits among genotypes indicated that 3254 has drought tolerance; 479, 601, 645 and 2226 avoid drought while 2232 is drought susceptible. The field performance of these six genotypes and two released varieties (Sudan 1 and IT82E16) was assessed in field trials in Malawi at Baka, Bvumbwe, Chitala, Chitedze and Kasinthula. Results from field trials revealed significant variation for reproductive, yield and seed characteristics. Sudan 1, IT82E16, 409 and 601 matured in less than 65 days after planting; 3254 took 70 days and 645, 2226 and 2232 took more than 85 days. Genotype 3254 consistently gave high yields at sites with low rainfall and high temperatures compared to 2232 which yielded poorly at the same sites. The eight genotypes showed variation in seed size with genotype 2226 producing large seeds (>20g/100 seeds) at all sites. The seed size of 2232 was significantly lower than 2226 at sites with low rainfall and high temperatures. The field performance of these genotypes reflects the physiological responses observed in the glasshouse, confirming the drought response categories of the genotypes. The agreement between glasshouse experiments and the field trial suggests there is intrinsic value in the locally adapted germplasm maintained by the Malawi Plant Genetic Resources Centre. Among the genotypes tested in the field, farmers selected 479 for early maturity; 2226 and 2232 for high leaf biomass; 3254 for high pod load; 2226 and 2232 for large seeds; Sudan 1 for small seeds; and 601, IT82E16 and Sudan 1 for smooth seed texture. Genotype 3254 was ranked poorly at all the sites due to rough seed texture. Genotypes for potential use in improving production of cowpea in drought prone areas were identified. In the absence of released drought tolerant varieties, it is recommended that genotypes with drought avoidance characteristics be promoted in areas with mild droughts, while 3254 with its typical drought tolerance may be suitable for areas with intense droughts. However, the rough seed texture of 3254 may limit its usefulness due to its poor ranking by farmers at all sites. Priorities for future cowpea in Malawi include investigating inheritance of drought tolerance in cowpea.
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    Breeding and transgenic approaches to improving water use efficiency in white clover (Trifolium repens L.) : 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
    (Massey University, 2014) Raynes, Julia Sarah Margaret
    This thesis investigates the potential role of the ABA biosynthetic gene 9-cis epoxycarotenoid dioxygenase 1(NCED1) as a determinant of water-use-efficiency (WUE) in plants as part of a longer term aim to confer improved WUE to the forage legume white clover (Trifolium repens L.). Two experimental approaches have been used. The first looked at the expression of NCED1 in a range of Trifolium species that display anatomical and morphological traits that confer some adaptations to growth and survival in dryland habitats. The second approach involved over-expression of NCED1 in the model species tobacco (Nicotiana tabacum) in proof-of-concept experiments to directly determine if any changes in plant water relations can be measured. Initially the constitutive expression of TrNCED1 was assessed, using the quantitative real-time polymerase chain reaction (q-RT-PCR), in two varieties of white clover. The first is an agronomically elite cultivar with a higher water requirement for optimal growth, cv. Grasslands Kopu II, and the second a dryland-adapted ecotype, Tien Shan. It was found that for both varieties, TrNCED1 expression was highest in the younger (first-fully-expanded) leaf (FFEL) tissue, followed by root tissue, then lastly apical tissue. When comparing the two varieties under well-watered conditions, the relative expression of TrNCED1 was higher in aerial parts of the cv. Kopu, but in the roots of the Tien Shan ecotype with respect to the reference genes, TrActin and TrGAPDH. Further, in the high biomass cv. Grasslands Kopu II, expression of TrNCED1 decreased in the apical and FFEL tissues of plants experiencing a water deficit, and decreased while for the dryland ecotype Tien Shan, TrNCED1 expression did not change in the apex reguardless of plant water status. Expression in the FFEL increased in tissue experiencing water deficit, and decreased in roots, suggesting different mechanisms for drought tolerance and response in the two varieties. For the range of Trifolium species assessed, constitutive expression of the TrNCED1 homologue was measured under well watered conditions in the FFEL and apical tissues. Essentially no significant difference in expression in either tissue between species was detected, with respect to the reference genes, TrActin and TrGAPDH. model species. In other studies, over-expression of NCED1 has been found to confer some characteristics associated with increased WUE, although abnormal growth associated with high levels of ABA at key developmental stages has proved problematic. For this thesis, the two senescence associated promoters, Senescence-Activated-Gene (SAG13) and Senescence-Associated-Receptor-Kinase (SARK), were selected to drive over-expression of NCED1 from Solanum lycopersicum in tobacco. Of those plants that came through tissue culture and were successfully established in soil, a single line transformed with SAG13p::SlNCED1, and three plants transformed with SARKp::SlNCED1, were shown to be positive for transgene insertion using a polymerase chain reaction (PCR) with genomic DNA. Of these, two plants, both transformed with SARKp::SlNCED1, were found to express SlNCED1 when tested using PCR with cDNA from isolated RNA. Water relations measurements performed on all four plants that were positive for the transgene, an empty vector control, a selection of plants that were negative for transgene insertion, and some wild-type controls, found that one of the lines confirmed as expressing the transgene, line 751-1, had a very low transpiration rate and low level of stomatal conductance. To extend these measurements to determine and increase in water-use-efficiency, a comparison must be made between growth rate and water uptake, and many more transgenic plant lines must be analysed. Finally, to determine whether the eventual transformation of white clover with SAG13p::SlNCED1 and SARKp::SlNCED1 would result in co-suppression of both types of transgene and the constitutive NCED1, expression of TrNCED1 in seven tissue types, from two varieties was measured. Highest expression was determined in the root tissue and in the younger leaf tissue, but was lower in the mature tissue examined. This suggests that transformation with SlNCED1 and expression in the mature tissues is not likely to be influenced by the constitutively expressed TrNCED1.
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    Neotyphodium lolii endophyte improves drought tolerance in perennial ryegrass (Lolium perenne. L) through broadly adjusting its metabolism : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy (PhD) in Microbiology and Genetics at Massey University, Manawatu, New Zealand
    (Massey University, 2014) Zhou, Yanfei
    Perennial ryegrass (Lolium perenne) is a widely used pasture grass that is frequently infected by Neotyphodium lolii endophyte. The presence of N. lolii enhances grass resistance to several biotic and abiotic stresses such as insect, herbivory and drought. Recent studies suggest the effect of N. lolii on ryegrass drought tolerance varies between grass genotypes. However, little is known about the molecular basis of how endophytes improve grass drought tolerance, why this effect varies among grass genotypes, or how the endophytes themselves respond to drought stress. This knowledge will not only increase our knowledge of beneficial plant-microbe interactions, but will also guide better use of endophytes, such as selection of specific endophyte - cultivar combinations for growth in arid areas. In this study, a real time PCR method that can accurately quantify N. lolii DNA concentration in grass tissue was developed for monitoring endophyte growth under drought. The effect of N. lolii on growth of 16 perennial ryegrass cultivars under drought was assessed, and a pair of endophyte-infected grasses showing distinct survival ability and performance under severe drought stress was selected. The transcriptome profiles of these two endophyte-infected grasses, as well as their clonal endophyte-free grasses, were analyzed using high-throughput RNA sequencing. The expression of endophyte and grass genes responsive to drought was analyzed simultaneously using different bioinformatic tools. The results demonstrated that N. lolii enhanced the growth of perennial ryegrass under drought, but the effect varied between cultivars. On the molecular level, endophytes living in both drought-tolerant and drought-sensitive grasses responded to drought in similar ways, including increased expression of endophyte genes related to scavenging of reactive oxygen species (ROS), DNA replication and the cell cycle, and also reduced expression of genes involved in alkaloids biosynthesis. The presence of endophytes also led to enhanced grass tolerance that was associated with broad adjustments in the plant metabolism. This included up regulation of grass genes involved in chloroplast maintenance and protection, osmotic adjustment and ROS scavenging capability. The extent of these endophyte-associated effects was greater in the drought tolerant grass genotype than in the drought sensitive genotype. This work highlights the role of fungal endophytes in grass drought stress tolerance and provides a comprehensive insight into the mechanisms involved.
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    Response to water deficit stress in the native New Zealand tussock, Festuca novae-zealandiae : submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy, Department of Plant Biology and Biotechnology, Massey University, Palmerston North, New Zealand
    (Massey University, 1996) Abernethy, Grant Andrew
    Responses to water-deficit stress by a xerophytic tussock native to sub-alpine grasslands of New Zealand have been investigated. Festuca novae-zealandiae possesses some structural adaptions such as curled laminae, limited stomatal distribution, and dense trichomes characteristic of dry-land adapted species. These features were compared with those of a mesophyte, F. arundinacea. In addition to structural adaptions, evidence that metabolic changes contribute to the drought tolerance of F. novae-zealandiae was sought. Plants of F. arundinacea and F. novae-zealandiae were grown in glasshouse pots and were subjected to water-deficit. Concentrations of the phytohormone ABA and the osmoprotectants proline and glycine-betaine increased in mature laminae of F. arundinacea as SWC declined to 8%. In F. novac-zealandiae, ABA concentration increased from 25-50 ng/gDW in two steps. At the first step (at 9-10% SWC), the increase was greatest in leaf bases (to 150 ng/gDW), and the second increase (at 6% SWC) was greatest in laminae (to 320 ng/gDW) and non-existent in leaf bases. In water-sufficient tillers, proline concentration was elevated in leaf bases (5-8 mg/ g DW) compared to laminae (2 mg/ g DW) and increased to 28 mg/ g DW in leaf bases, and to 14 mg/ g DW in laminae as SWC declined to 8-9%. Glycine-betaine concentration (40-100 µmol/ gDW) was unchanged by water-deficit stress. Leaf and tiller counts, and estimates of leaf tip scorching indicated that leaf death progressed with increasing water-deficit. Although leaf elongation declined, it did not cease until the last tillers had died. SDS-PAGE and Western analysis of soluble proteins using antisera to ubiquitin indicated that protein turnover increased in lamina, but not in leaf bases in response to water-deficit. It was concluded that in response to water-deficit stress, leaf bases of F. novae-zealandiae retained turgor and remained viable at the expense of existing leaf laminae, which died back. Southern analysis suggested that the F. novae-zealandiae genome contains sequence(s) with homology to group 2 LEA proteins. Western analysis using antisera raised against the dehydrin consensus sequence KIKEKLPG revealed several related proteins in seeds of F. novae-zealandiae, but these were not induced by water-deficit stress in leaves.
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    Tissue-specific responses to water deficit in the New Zealand xerophytic tussock species Festuca novae-zelandiae : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Biology at Massey University, Palmerston North, New Zealand
    (Massey University, 2005) Clark, Gregory Thomas
    Festuca novae-zelandiae (Hack.) Cockayne is an endemic New Zealand perennial tussock forming grass of the family Poaceae. Morphologically F. novae-zelandiae exhibits a number of leaf adaptations associated with dehydration postponement as reflected in the climatic distribution of this species and its occurrence as a physiognomic dominant grass in semi arid short-tussock grasslands. Biochemical studies into the drought tolerance of this species have indicated the occurrence of tissue specific responses with respect to abscisic acid (ABA) and proline accumulation and protein turnover suggestive of a preferential protection of the tiller base and associated meristematic zones at the expense of lamina tissues. Further tissue specific biochemical responses to water-deficit stress in F. novae-zelandiae have been investigated. Changes in water-soluble carbohydrates (WSC) were monitoured over a 49-day dry-down period (decline in soil water content from 30% to 4%) in consecutive leaf segments comprising the leaf base (meristem region), elongation zone, the enclosed and exposed lamina, as well as basal sheath segments from the two next oldest leaves. In fully hydrated leaf tissues polymers of fructose (fructans) were the main WSC present and were mainly low molecular weight fructans of the inulin and neokestose series with the average degree of polymerization (DP) of fructan pools from 6 to 9. The highest fructan concentrations were present towards the leaf base. Fructan concentrations decreased over the course of the dry-down, although remained significantly higher in the meristem region of the tiller base with respect to any other tissue, until tissue water content fell below 45%. By day 49 of the dry-down period, the average DP of the fructan pool in tissues was from 3 to 5. Sucrose content increased in each tissue during the course of the dry-down, and was highest at the leaf base, where a concentration of 200 µmol g-1 dry weight was measured after 49 days of dry-down. The negative correlation between fructan and sucrose content, which indicates an inter-conversion dependent on tissue water content, suggests that, in this specises, fructans serve as a carbohydrate storage pool, while sucrose stabilises the meristem during extreme water deficit.