Limonium sinuatum (2n=16) is a diploid ornamental flowering plant. Polyploid plants have a potential value for breeding and commerce. Sexual polyploidisation via the formation of unreduced (2n) gamete is an approach to speed up hybridisation and breeding. It is possible to artificially induce unreduced gametes in high frequency by applying nitrous oxide (N2O) to flowers at the onset of meiosis. However there is no information available in Limonium sinuatum on whenmeiosis occurs in relation to flower development, nor what the optimum timing and duration of N2O treatment might be in L. sinuatum. The aims of this study were to determine the stage of flower development at which meiosis takes place, identify when each meiosis stage occurs, and then examine the effect of N2O treatment on 2n gamete formation. The results showed that meiosis across the inflorescence is typically asynchronous and is most of the time occurring in very small flowers (0.2 cm in diameter) located at 3 flowers behind the flower marker on each array of a sub-inflorescence. The most dominant cell meiotic division stage occurred from 4 am to 6 am was the pre-meiotic stage then progressed rapidly from just after 6 am marked by the observation of multiple meiotic cell stages and reaching its activity peak between 7 a.m. and 8 a.m. (sunrise at 6.15 a.m.). N2O treatments (600 Kpa) of 24 hours duration offered significant increases in the presence of formation of 2n gametes as indicated by the presence of large pollen in high frequency.

The production of statice (Limonium sp.) plants with higher ploidy through induction of whole-genome duplication (WGD) via the spindle disrupter nitrous oxide (N2O) was examined as a strategy to increase the germplasm diversity of the species. Furthermore, the impact of the resulting ploidy changes on the morphological features of the progeny was examined. Intraspecific crosses between diploid plants of Limonium sinuatum (L.) Mill and L. perezii (Stapf) Hubb. were conducted daily for seven consecutive days, with subsequent exposure to N2O. Within the resulting progeny, between 16% and 35% of plants were polyploid when N2O was applied between one and four days after pollination. A comparative analysis between diploid and tetraploid progeny was conducted, using a selection of 10 L. sinuatum (5 diploids and 5 tetraploids) and 7 L. perezii (4 diploids and 3 tetraploids) genotypes. The results revealed differences between tetraploids and their diploid counterparts for most of the evaluated characteristics. Tetraploid plants of L. sinuatum and L. perezii exhibited pollen grains 1.5 times larger in plan area; the leaves and main floral stem diameter were 1.2 and 1.5 times thicker for L. sinuatum and L. perezii, respectively, the guard cell length was 1.4 times greater for both species, while the stomatal density was 0.6 times lower for L. perezii and 0.8 for L. sinuatum. The leaf area and main floral stem wings were affected by the ploidy increase only for L. sinuatum. In this regard, tetraploid plants of L. sinuatum displayed leaves 1.8 times bigger and main floral steam wings 2.4 times wider in comparison to diploid plants. In conclusion, the production of tetraploid Limonium plants using N2O shortly after pollination creates new diversity for breeding. These findings underscore the potential for leveraging polyploidy as a strategy to enhance desirable traits in Limonium species.

We investigated the possibility of either exogenous ethylene or endogenous ethylene production having an association with the increase in shoot number when nodal explants of Gentiana spp. ‘Little Pinkie’ were cultured in an in vitro medium supplemented with ethephon (10 mg⋅L–1). For the first time within an in vitro system, we report the application of laser ethylene detector technology, and optimization of the methodology to quantify concentrations of ethylene (in the part-per-billion range) released from ethephon decomposition within the atmosphere of gas-exchangeable culture vessels including nodal explants. Compared with continuous (continuous measurements on the same replicate of vessels) and repeated (sampling same replicate of vessels every 48 hours) sampling methodologies, the nonrepeated (sampling fresh replicate of vessels every 48 hours) method of measurement of ethylene concentration was more representative of the actual condition within vessels. Although no prior published data exist showing the positive or negative effect of gaseous ethylene in the headspace of culture vessels on bud outgrowth in gentian, our study shows gaseous ethylene in the headspace of culture vessels was not effective in increasing shoot formation in gentian explants cultured in vitro, whereas ethephon supplementation in agar was effective. Plant material in culture vessels did not have a significant effect on ethylene production regardless of the presence or absence of ethephon. Therefore, although ethephon supplementation in the medium produced gaseous ethylene in the headspace, it was unlikely to cause endogenous ethylene production in explants, but it did trigger shoot formation in ‘Little Pinkie’, perhaps through decomposition to ethylene within the explant tissue, enhancing the internal ethylene level possibly at a locally high concentration.

Polyploidy is a key driver of significant evolutionary changes in plant species. The genus Actinidia (kiwifruit) exhibits multiple ploidy levels, which contribute to novel fruit traits, high yields and resistance to the canker-causing dieback disease incited by Pseudomonas syringae pv. actinidiae (Psa) biovar 3. However, the genetic mechanism for resistance to Psa observed in polyploid kiwifruit is not yet known. In this study we performed detailed genetic analysis of a tetraploid Actinidia chinensis var. chinensis population derived from a cross between a female parent that exhibits weak tolerance to Psa and a highly Psa-resistant male parent. We used the capture-sequencing approach across the whole kiwifruit genome and generated the first ultra-dense maps in a tetraploid kiwifruit population. We located quantitative trait loci (QTLs) for Psa resistance on these maps. Our approach to QTL mapping is based on the use of identity-by-descent trait mapping, which allowed us to relate the contribution of specific alleles from their respective homologues in the male and female parent, to the control of Psa resistance in the progeny. We identified genes in the diploid reference genome whose function is suggested to be involved in plant defense, which underly the QTLs, including receptor-like kinases. Our study is the first to cast light on the genetics of a polyploid kiwifruit and suggest a plausible mechanism for Psa resistance in this species.

Pseudomonas syringae pv. actinidiae (Psa) Biovar 3, a virulent, canker-inducing pathogen is an economic threat to the kiwifruit ( Actinidia spp.) industry worldwide. The commercially grown diploid (2 x ) A. chinensis var. chinensis is more susceptible to Psa than tetraploid and hexaploid kiwifruit. However information on the genetic loci modulating Psa resistance in kiwifruit is not available. Here we report mapping of quantitative trait loci (QTLs) regulating tolerance to Psa in a diploid kiwifruit population, derived from a cross between an elite Psa-susceptible ‘Hort16A’ and a tolerant male breeding parent P1. Using high-density genetic maps and intensive phenotyping, we identified a single QTL for Psa tolerance on Linkage Group (LG) 27 of ‘Hort16A’ revealing 16-19% phenotypic variance and candidate alleles for susceptibility and tolerance at this loci. In addition, six minor QTLs were identified in P1 on distinct LGs, exerting 4-9% variance. Complete tolerance in the F1 population is attained by additive effects from ‘Hort16A’ and P1 QTLs providing evidence that divergent genetic pathways fend-off virulent Psa strain. Two different bioassays further identified new QTLs for tissue-specific responses to Psa. Transcriptome analysis of Psa-tolerant and susceptible genotypes in field revealed hallmarks of basal defense and provided candidate RNA-biomarkers for screening Psa tolerance.

The international ornamentals industry is benefiting from perennial plants developed in New Zealand (NZ) such as begonia Bonfire™, limonium Sinzii™, and the gentian Showtime™ series. The 'Bonfire' series of begonia, with its distinctively new flower shape, was released in the year 2000. While flower and foliage colour were important; architectural form, vigour and floral productivity were key selection criteria. Understanding the underlying physiology of plant architecture (physiologically informed breeding) was key in the introduction of subsequent new cultivars to the series. These screening criteria allowed us to evaluate annually ten times more begonia seedlings per season. Planting of limonium Sinzii continues to expand exponentially: in 2018/19 plantings almost doubled compared with those of the previous year. The limonium Sinzii range resulted from successful hybridisation between Limonium sinuatum and L. perezii. Embryo culture, chromosome doubling, and backcrossing were some of the in vitro and in vivo tools and strategies applied to achieve the current range. The sterile gentian 'Little Pinkie' was another plant developed using wide crosses and embryo rescue. As with other gentians in the Showtime series, a physiologically informed breeding strategy was used. In the case of 'Little Pinkie', an understanding of how crown buds arise was used to generate at least double the number of shoots on propagules. This has underpinned the success of this crop for us, and our industry partners. The preceding examples illustrate how, for each crop species, our breeding programmes have diversified germplasm, through exploiting a diverse skill base within the team. Our conclusion is that our most successful examples of breeding programmes have been those in which plant breeders, plant physiologists, in vitro specialists, horticulturalists, and marketers are all actively engaged.

Branching has a major influence on the overall shape and productivity of a plant. Strigolactones (SLs) have been identified as plant hormones that have a key role in suppressing the outgrowth of axillary meristems. CAROTENOID CLEAVAGE DIOXYGENASE (CCD) genes are integral to the biosynthesis of SLs and are well characterized in annual plants, but their role in woody perennials is relatively unknown. We identified CCD7 and CCD8 orthologues from apple and demonstrated that MdCCD7 and MdCCD8 are able to complement the Arabidopsis branching mutants max3 and max4 respectively, indicating conserved function. RNAi lines of MdCCD7 show reduced gene expression and increased branching in apple. We performed reciprocal grafting experiments with combinations of MdCCD7 RNAi and wild-type 'Royal Gala' as rootstocks and scion. Unexpectedly, wild-type roots were unable to suppress branching in MdCCD7 RNAi scions. Another key finding was that MdCCD7 RNAi scions initiated phytomers at an increased rate relative to the wild type, resulting in a greater node number and primary shoot length. We suggest that localized SL biosynthesis in the shoot, rather than roots, controls axillary bud outgrowth and shoot growth rate in apple.

A successful in vitro propagation system for gentian (Gentiana sp.) 'Little Pinkie' as a potted plant depends on a rapid multiplication rate to produce shoots that, when excised (explants), result in highly branched plants. As a part of an ongoing research programme to improve our understanding of this gentian grown in vitro, the effect of growth duration, nodal position from which explants were taken, and inclusion of ethephon in the culture medium were investigated. In the absence of ethephon, increasing the duration of growth from 5 to 8 weeks did not improve branching. Furthermore, the multiplication rate did not change, as after 8 weeks most explants from the tip position became floral, resulting in the total number of usable explants generated being reduced by 30%. Not every explant taken from different nodal positions grew equally, however: fresh weight and number of new shoots per explant taken from the second node below the tip were respectively, 1.5 and 3 times more than the explants taken from the tip. In contrast, when ethephon was applied, after 8 weeks growth in vitro, explants produced twice as many shoots per explant as the control, and only 10% of the tip-derived explants flowered. The number of shoots produced per explant taken from the second node below the tip was 4.5 times more than tip-derived explants. In spite of the improvement in branching however, ethephon in the growing medium decreased the multiplication rate as a result of a decrease in the length of the longest shoots rendering fewer shoots as useable explants. We conclude that culture for 5 weeks in the absence of ethephon is preferable for in vitro propagation of 'Little Pinkie'. Higher multiplication rates may be achievable if ethephon can be used to stimulate the number of branches formed, but also subsequently release those branches into rapid growth.

When propagules of gentian 'Little Pinkie' were generated in vitro, each progressive subculture resulted in a reduction in the duration to flower production when deflasked plants were subsequently grown in vivo. The initiation of flowering in propagules while growing in vitro reduced the quality of potted plants produced, and reduced the overall efficiency of the micro propagation system by 40%. Hence determining factor(s) influencing the onset of flowering and developing strategies to maximise persistent vegetative growth of explants were considered to be important for commercialisation of this cultivar. The effect of position from which explants originated on flowering in vitro was investigated. A gradient of propensity for explants to flower was found to exist along the shoot from which explants were derived. Explants derived from the tip had the greatest propensity to flower (35%), and the percentage of flowering decreased with distance from the tip. Explants derived from the first node below the tip produced four times fewer flowering plants than those derived from the tip, while only 6% of explants flowered when derived from the second node. To optimise the efficiency of propagation of 'Little Pinkie' therefore, modifying the propagation protocol from mass propagation to selective propagation is suggested, wherein explants derived from the tip of original shoots are not recommended for inclusion. In contrast, lower nodes may be used as propagules for either subculture in vitro or production of potted plants.

Cut flower productivity and quality of gentian is associated with growth and development of crown buds. Experiments were carried out with the gentian cultivar Showtime Diva to identify the response to treatments that break dormancy [cold temperature (chilling), gibberellic acid (GA 3 )] applied at different stages of development of crown buds (plants with nonemerged crown buds, shoots recently emerged, or shoots emerged and elongated). The comparative growth potential of crown buds within the cluster was also investigated. At the stages of development examined, the application of GA 3 (100 ppm) increased emergence of crown buds as shoots, leading to development of more flowering shoots. A similar response was observed with exposure to cold, but only on plants with nonemerged crown buds. Shoot emergence increased in response to increased duration of cold from 0 to 42 days (5 °C). Both chilling and GA 3 could potentially be used to reduce the duration to, and spread of, harvest maturity if applied before shoot emergence. The hierarchical relationship of buds in crown bud clusters led to differential responses to application of GA 3 . Buds ontogenetically positioned at the proximal end of the bud cluster took a similar duration to reach shoot emergence or harvest maturity. For buds located at the distal end there was a positive correlation between ontogenetic bud position and the duration to reach shoot maturity. Shoot length and number of nodes at harvest maturity showed slight negative correlations with the position of the bud in the bud cluster. The results provide an explanation for possible sources of the variation in quality and quantity of floral shoots, and spread in time to harvest maturity within a single plant, and with development stage.

Gentians are herbaceous perennials that utilize preformed crown buds as part of their perennating structure. We used leaf removal at different times in the season to assess the importance of accumulating carbohydrate reserves for plant survival during winter and subsequent re-growth in spring. Defoliation of gentian 'Velvet Glove' during the growth season significantly influenced the development of crown buds, winter survival, re-growth in spring, and carbohydrate reserves in crowns (mainly because of reduction in gentianose and sucrose concentrations). Carbohydrate reserves stored in the storage roots of crowns were likely to influence the development of crown buds, winter survival and re-growth. Given the absence of starch in gentians, the predominant carbohydrate reserve in crowns, gentianose, may play a similar role to that of starch in other plant species. From the time of emergence, at least 2884 GDD (base 0 degrees C) retention of full leaf canopy on plants of 'Velvet Glove' was required for sufficient accumulation of carbohydrate reserves in crowns to result in similar winter survival and re-growth in spring, as with undefoliated control plants. (C) 2016 Elsevier B.V. All rights reserved.

To be economically valuable as a potted plant it is desirable for gentians to produce uniform plants with multiple branches (secondary shoots) at flowering. Not all propagules of the cultivar 'Little Pinkie' grew equally when propagated in vitro from either a single node explant, or just the apical region of the donor plant in vitro. Explants from the tip position produced a single primary shoot derived from the continued morphological development of the apex and no secondary shoots. In contrast, explants from lower positions produced between 0 and 8 secondary shoots. The number of shoots and total weight of the in vitro plantlets increased up to three and two-fold, respectively, with increased distance of the explant position from the tip. In addition, with increased distance from the tip, morphological non-uniformity increased for all growth variables. Scanning electron microscopy evidenced the formation of additional vegetative buds (adventitious), especially for explants taken toward the base of the original shoot. However, in spite of the increase in number of secondary shoots raised on the explants taken from lower down the stem, the total number of usable explants was the same for plantlets derived from all positions. The physiological mechanisms and underlying hormonal control will be discussed together with strategies to increase the number of usable explants.

During the development and senescence of florets in gentian 'Showtime Spotlight', there was a dramatic change in petal non-structural carbohydrates (NSCs), including accumulation, and hydrolysis. Gentianose concentration increased more than ten-fold with the development of florets, to a maximum of 26.1 mg g(-1) fresh weight (FW) just before floret opening. Subsequently, as florets began opening, the gentianose concentration sharply decreased to almost nothing as flowers progressed from fully open to naturally senesced. Gentiobiose concentration increased gradually during early development of florets, with the pattern of increase with each stage of development being slightly behind that for gentianose, reaching a maximum of 21.2 mg g(-1) FW as florets began opening. These stage-specific changes in concentrations in each NSC were paralleled by significant changes in activity of both gentianose and gentiobiose glycoside hydrolase. In a plant system devoid of starch, and where changes in sucrose and glucose concentration were comparatively small, the stage-specific and intensive fluctuation of the unique carbohydrates gentianose and gentiobiose imply an important role in controlling gentian floret development and opening. The significant positive correlation between the osmolality of soluble NSCs and pressure potential supports the hypothesis that this carbohydrate metabolism role is via osmotically driven cell expansion. (C) 2016 Elsevier B.V. All rights reserved.

This paper investigates the influence of postharvest science on the New Zealand floriculture industry and then considers how new drivers of the affluent flower consumers (i.e., environmentally sustainable production) might be addressed by the NZ industry in the future. Market demand trends have historically been achieved through delivering novelty (e.g., new genetics or new crops), 'massaging' harvest windows to meet demand (production technologies), understanding plant physiology to deliver quality products (postharvest technologies), and through marketing techniques (targeting specific demographics, social obligations, cultural influences, value perceptions). The role of postharvest science has been to support delivery of quality products, usually defined by having an optimal vase life. This research meets the needs of our 'direct' customers: the exporters, and wholesale and retail marketers. Rarely do we consider the purchase drivers for end-consumers. As the NZ floriculture industry strives to deliver into high-value niche markets around the world, there is a need to better understand the drivers of our end-consumers. To this end, we believe there is an opportunity for postharvest science working in conjunction with preharvest science to support more sustainable production of cut flowers.

Crown buds are an important determinant of yield and quality in the production of flowering shoots in gentians. While anecdotally it has been reported that the harvesting of flowering shoots can have a negative effect on the formation of crown buds, the current study endeavoured to quantify what, if any, relationship existed between shoots and crown-bud formation. Plants of genotype '03/04-114' and the cultivar 'Showtime Spotlight' were used. Clusters of crown buds mainly formed at the base of developing floral shoots. At the end of the growing season total crown-bud number (TCBN) was determined by both crown-bud cluster number (CN) and crown-bud number per cluster (CBNC). While there was no correlation between CN and CBNC for either genotype 03/04-114 or 'Showtime Spotlight', shoot number (SN) was positively correlated with CN (03/04-114: r(2)=0.48; P=0.004 and 'Showtime Spotlight': r(2)=0.52; P<0.001), but not with CBNC. As there was no evidence of a positive correlation between SN and CN following extended periods of defoliation of genotype 03/04-114 (r(2)=0.14; P=0.29), the likely implications of shoot removal and defoliation are discussed in terms of photo-assimilate acquisition and/or hormonal control of development of clusters of crown buds.

Branching is a key determinant of architectural quality of ornamental plants. Using plants trained to a single primary shoot, cultivars of Japanese maple (Acer sp.) 'Fireglow', 'Katsura', 'Orangeola', 'Red Emperor', 'Skeeter's Broom' and 'Sango Kaku' presented a diverse range of growth and sylleptic branching patterns. Shoot extension was more vigorous in 'Sango Kaku', which displayed uninterrupted extension to develop more than 30 metamers. In other cultivars, some primary shoots had a pause after extension of approximately six preformed metamers, forming shorter bicyclic shoots (e.g., 'Red Emperor'); and some of the shoots were monocyclic, comprising mostly preformed metamers (e.g., 'Fireglow'). Sylleptic branching in the six cultivars differed in both branching intensity and the location of branches along the primary shoot. 'Sango Kaku' and 'Katsura' featured two intensely branched zones with lower branching frequency between them. 'Orangeola' and 'Skeeter's Broom' had a single branching zone with lower branching intensity located at the basal and middle sections of the parent shoot, respectively. In addition, xylem sap of 'Red Emperor' contained more strigolactone than that of 'Sango Kaku', supporting the hypothesis that sylleptic branching appears to correlate inversely with the amount of strigolactone. The above results support the hypothesis that the occurrence of sylleptic branching and these branches' distribution along the primary shoot are subject to combined hormonal and developmental control. Possible relationships between the primary shoot organogenesis/extension and the time course of sylleptic branching are currently being investigated.