Influential factors in nectar composition and yield in Leptospermum scoparium : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Science, Institute of Agriculture and the Environment, College of Sciences, Massey University, Palmerston North, New Zealand

Abstract

Material omitted from digital version of thesis: Nickless, E. M., Anderson, C. W. N., Hamilton, G., Stephens, J. M., & Wargent, J. (2016). Soil influences on plant growth, floral density and nectar yield in three cultivars of manuka (Leptospermum scoparium). New Zealand Journal of Botany, 55(2), 100-117. doi:10.1080/0028825X.2016.1247732 ; Nickless, E. M., Holroyd, S. E., Stephens, J. M., Gordon, K. C., & Wargent, J. J. (2014). Analytical FT-Raman spectroscopy to chemotype Leptospermum scoparium and generate predictive models for screening for dihydroxyacetone levels in floral nectar. Journal of Raman Spectroscopy, 45(10), 890-894. doi:10.1002/jrs.4576 ; Nickless, E. M., Holroyd, S. E., Hamilton, G., Gordon, K. C., & Wargent, J. J. (2016). Analytical method development using FTIR-ATR and FT-Raman spectroscopy to assay fructose, sucrose, glucose and dihydroxyacetone, in Leptospermum scoparium nectar. Vibrational Spectroscopy, 84 (2016), 38-43. doi:10.1016/j.vibspec.2016.02.011Leptospermum scoparium (Mānuka) is the plant nectar source for medically bioactive honey, commercially marketed in New Zealand as Unique Mānuka Factor honey (UMF-honey). Methylglyoxal (MGO) is the unique bioactive component of UMF honey with Mānuka nectar containing significant amounts of the carbohydrate dihydroxyacetone (DHA), the chemical precursor for MGO. Anecdotal evidence and recently published data from nectar samples collected from various cultivars in natural sites or botanical gardens has indicated that the DHA and overall composition of L. scoparium nectar varies according to cultivar. The source of this variation is not clearly understood and although there is considerable literature on climatic and genetic influences on nectar composition and yield within various other plant species, there is little published work available on the influence of genetic and environmental factors on the composition and yield of nectar in L. scoparium. Of value to the commercial UMF honey industry in New Zealand is the ability to assess cultivars from breeding programs for the best potential to increase overall UMF honey yield. Predictive modelling of yields is invaluable to the developing honey industry to allow assessment of environmental influences that may affect overall yield along with seasonal influences on nectar production in Mānuka. The research in this thesis establishes the effect of various parameters on overall DHA yield from Mānuka and the beginnings of modelling influencing environmental factors. To determine influences on dihydroxyacetone (DHA) concentration and yield in the nectar of L. scoparium a number of studies were carried out. Methodologies for the collection and analysis of nectar were established. Ten different cultivars of L. scoparium with a range of genetic parentage were studied in controlled glasshouse conditions to assess phenotypic variability in terms of nectar composition and yield as well as plant growth and flowering amongst these cultivars. Significant differences in plant growth and flowering habits were observed amongst the ten cultivars, significant differences in nectar yield and nectar composition with regard to DHA yield were also observed. DHA yields ranged from 2714-7459 mg of DHA/kilogram normalised to 80 oBRIX, with total nectar sugar yields ranging between 0.7 and 4.8 mg amongst the ten cultivars studied. Preliminary research into the effect of temperature, radiation and humidity on nectar composition and yield were also undertaken. Effects of soil composition on these same parameters were researched with a subset of three of the ten cultivars grown on ten different soil types. Plant relative growth rates, dry weights and total plant height were measured throughout a 15 month glasshouse trial. Plant growth, flowering phenology, floral density, nectar yield and DHA composition data was gathered. Soils were analysed for various macronutrient and micronutrient levels and these parameters were modelled against plant data to determine which soil components were influencing plant parameters of interest. Soil type was shown to have no significant effect on DHA concentrations in nectar but results did show that soil type had a significant effect on flowering density amongst the three L. scoparium cultivars studied in the trial. Results from regression analysis of soil chemistry against measured plant parameters indicate that a fertiliser regime has the potential to increase nectar yields due to increased flower numbers. Multivariate analysis using partial least squares regression of soil composition data against plant parameters of value showed that soil components; phosphorus, sulphate, ferric and chloride were commonly shown to influence plant parameters measured. Analytical spectroscopy was investigated as a method to chemotype L. scoparium cultivars and also as a method for quantifying nectar components sucrose, glucose, fructose and DHA. Nectar composition was analysed using high pressure liquid chromatography (HPLC) and compared with fourier transform Raman spectroscopy (FT-Raman) and attenuated total reflectance infrared spectroscopy (ATR-FTIR) analytical spectroscopy methods. FT-Raman spectroscopy was shown to be useful in chemotyping cultivars and in addition proved to be a useful analytical method to predict DHA yield using leaf material from L. scoparium plants from the ten cultivars. FT-Raman and ATR-FTIR proved to be relatively accurate techniques to quantify L. scoparium nectar components DHA, fructose, glucose and sucrose, compared with HPLC methods which use extensive preparation techniques. R-squared values were very good for all nectar components measured excepting the sucrose model at R2 = 0.77. The R2 for the FT-Raman predictions of DHA against HPLC data are very good at 0.85. FTIR prediction data against HPLC data was also good at 0.86 R2. Overall an accurate model is possible for quantifying DHA concentrations in nectar using both FTIR-ATR and FT-Raman spectroscopy. Overall results show that various factors need to be considered when assessing plants for commercial use in the (UMF) Mānuka honey industry within New Zealand. Due to their large impact on overall nectar yield; floral density and plant growth rate parameters are the two key factors of value for commercial assessment of Mānuka cultivars. This research also highlights the importance of assessing not just DHA concentration in deducing cultivar value, but overall nectar yield. These key features must be explored when assessing L. scoparium plants within breeding programs, prior to selection for large-scale field production of high UMF Mānuka honey