Initial exploration of unreduced gamete (2n gamete) pollen development through nitrous oxide (N₂O) application in Limonium sinuatum : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Horticultural Science at Massey University, Palmerston North, New Zealand

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Hybridisation and polyploidisation are techniques that can rapidly generate proprietary diversity in plant breeding programs. Limonium sinuatum (2n = 16) is diploid, with polyploid plants being of potential value for breeding and commercial applications. Although somatic polyploidisation has been reported in Limonium, sexual polyploidisation potentially offers scope to speed up hybridisation and breeding. Sexual polyploidisation results in increased ploidy level via formation of unreduced (2n) gametes. Unreduced gametes are produced naturally but usually at levels too low to be of practical use, and production can be dependent on genotype and environmental conditions. Practically useful levels of unreduced gametes can be artificially induced by judicious timing of treatment with nitrous oxide (N₂O). Flower buds at the onset of meiosis are suggested as the best stage to treat with N₂O. However, there is no information available on when meiosis occurs in relation to flower development, nor what the optimum conditions of treatment might be in L. sinuatum. The aims of this study were to identify the stage of flower development when meiosis occurs and then to test the effect of N₂O treatment on 2n gamete formation before investigating the potential of 2n gametes in direct hybridisation. The results showed that meiosis across the raceme is asynchronous and occurs in very small flowers (0.8 mm in diameter) on each array of a spike in raceme. Additionally, meiotic division occurred from just after 6 AM., reaching its peak between 7 and 8 AM. (sunrise at ca 6.15 AM). N₂O treatments (600 Kpa) of 24 hours and 48 hours durations gave significant increases in formation of 2n gametes as indicated by a wider size range or bimodal pollen grain size distribution of pollen. Though legitimate combinations were used in crosses using 2n pollen to a diploid female plant did not result in polyploid progeny. The most likely reason for this is that a triploid block mechanism operates preventing normal embryo development.
Figures 1(a) and (b) (=Zhang et al., 2014 Fig 1 and Koppolu & Schnurbusch, 2019 Fig 7A respectively) and 3 (=Younis et al., 2014 Fig 1) have been removed for copyright reasons. Figure 2 is re-used under a Creative Commons Attribution 4.0 International (CC BY 4.0) license.