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The Ascomycete Ophiostoma novo-ulmi threatens elm populations worldwide. The molecular mechanisms underlying its pathogenicity and virulence are still largely uncharacterized. As part of a collaborative study of the O. novo-ulmi-elm interactome, we analyzed the O. novo-ulmi ssp. americana transcriptomes obtained by deep sequencing of messenger RNAs recovered from Ulmus americana saplings from one resistant (Valley Forge, VF) and one susceptible (S) elm genotypes at 0 and 96 h post-inoculation (hpi). Transcripts were identified for 6424 of the 8640 protein-coding genes annotated in the O. novo-ulmi nuclear genome. A total of 1439 genes expressed in planta had orthologs in the PHI-base curated database of genes involved in host-pathogen interactions, whereas 472 genes were considered differentially expressed (DEG) in S elms (370 genes) and VF elms (102 genes) at 96 hpi. Gene ontology (GO) terms for processes and activities associated with transport and transmembrane transport accounted for half (27/55) of GO terms that were significantly enriched in fungal genes upregulated in S elms, whereas the 22 GO terms enriched in genes overexpressed in VF elms included nine GO terms associated with metabolism, catabolism and transport of carbohydrates. Weighted gene co-expression network analysis identified three modules that were significantly associated with higher gene expression in S elms. The three modules accounted for 727 genes expressed in planta and included 103 DEGs upregulated in S elms. Knockdown- and knockout mutants were obtained for eight O. novo-ulmi genes. Although mutants remained virulent towards U. americana saplings, we identified a large repertoire of additional candidate O. novo-ulmi pathogenicity genes for functional validation by loss-of-function approaches. Published version

Salinity is a major environmental stress in agriculture with significantly detrimental effects on crop productivity. The development of strategies to enhance salinity stress tolerance in plants is essential to ensure crop production in saline environments. Melatonin (Mel) and serotonin (Ser) accumulate in response to environmental stresses and are presumed to play protective roles and improve growth of tissues during recovery. In this study, the effects of Mel and Ser were investigated in Arabidopsis under NaCl stress. Exogenous Mel (10 µM) and Ser (10 µM) treatment significantly increased fresh weight, lateral root number, and shoot height in A. thaliana seedlings exposed to NaCl stress (25 mM and 50 mM) compared to the non-treated control seedlings. In order to understand the role of these indoleamines in alleviating salt stress, we investigated the effects of Mel and Ser treatments on the expression of salt stress responsive genes including, transcription factors involved in abscisic acid (ABA) signaling pathway, ABA-INSENSITIVE 3 (ABI3)and ABA-INSENSITIVE 5 (ABI5); ABA responsive gene, RESPONSIVE TO DESSICATION 29B (RD29B), ABA-independent gene, RESPONSIVE TO DESSICATION 29A (RD29A) and Arabidopsis trithorax-like gene (ATX1) which function in stress responses via ABA-dependent and ABA-independent manner. Other genes included, ROS-signaling transcription factor ZAT10 and ZAT12, and the genes encoding ion transporters crucial for maintaining ion homeostasis, HIGH AFFINITY K+ TRANSPORTER 5 (HAK5) and SALT OVERLY SENSITIVE 1 (SOS1). Mel (10 µM) pre-treatment for 24 hrs followed by 50 mM salt treatment up-regulated ABI3, RD29B, ZAT12 and HAK5. The Ser (10 µM) pre-treatment significantly up-regulated ZAT12.These results indicate that indoleamine pre-treatment improved plant growth under salt stress with Mel facilitating salt tolerance via upregulation of ABA responsive genes, mediation of antioxidant defense systems to counteract the salt-induced ROS overproduction as well as controlling ion homeostasis. Although Ser displayed no significant effects on ABA signaling, it was found to increase the expression of antioxidant defense gene, ZAT12. This study demonstrates the importance of indoleamine pathway in mediation of salt stress response and provides the first indication of the involvement of Ser in salt stress tolerance.

Commercial micropropagation of hybrid hazelnuts (Corylus avellana L. × C. americana Marshall) has been limited, owing to their poor rooting ability in vitro as well as ex vitro, leading to high mortality of plantlets transplanted in the greenhouse. The objective of this study was to develop an efficient and cost-effective protocol for rooting and plantlet acclimation of in vitro grown hazelnut shoots. Efficient in vitro rooting was accomplished in a rocker-based temporary immersion bioreactor system. The use of a temporary immersion system (TIS) in combination with the inert substrate Oasis® In Vitro Express (IVE) significantly improved the in vitro rooting efficiency (100%) compared with semi-solid medium (27%) after four weeks of culture. A higher density (36 explants/vessel) of shoot explants in the TIS was found to support a significantly greater shoot height, chlorophyll content, and longest root length, compared with the lowest density treatment (12 explants/vessel). Efficiency of rooting and the number of roots formed were similar for both the high and low density of explants in the culture vessels, and the resulting plantlets exhibited > 80% survival in the greenhouse. These results demonstrate the usefulness of rocker-based TIS for commercial micropropagation of hazelnuts and, potentially, other tree species.

Cryopreservation in liquid nitrogen (− 196 °C) ensures safe and cost-effective conservation of plant genetic resources using diverse materials, including roots. To date, successful cryopreservation of root explants isolated from in vitro grown plants as well as adventitious and hairy root cultures has been reported in large numbers of plant species. In particular, significant progress has been made in cryopreserving in vitro cultures of adventitious and hairy roots that attract both scientific and commercial interest as an alternative source of plant-derived bioactive compounds with potential application in pharmaceutical, cosmetic, and natural health product industries. The process of cryopreservation has been shown to have little or no impact on growth, and biosynthetic and genetic stability of roots following recovery in an ambient growth environment. However, structural integrity of roots is critical for successful cryopreservation as thin and fragile root tips are very sensitive to stressful conditions associated with various steps of a cryopreservation protocol and a thorough optimization of each step, particularly the treatment with vitrification solution, is essential. This review summarizes the results of recent studies on cryopreservation of plant roots and highlights the importance of continued research in this area. Roots are uniquely suited for long term conservation of plant genetic resources due to their ease of excision, in vitro maintenance, and the ability to undergo morphogenesis. The root based regeneration and cryopreservation systems offer excellent opportunities to investigate a range of regulatory signalling and adaptation mechanisms that are activated in response to physical and chemical stresses. This review summarizes the results of recent studies on cryopreservation of plant roots, including hairy and adventitious root cultures used in biotechnology, and underpins their significance in biobanking of economically important food, ornamental, and medicinal crops, as well as, endangered plant species.

Plant tissue culture techniques have been used to propagate horticultural crops at a commercial scale for more than three decades. However, due to the high cost it is generally only used for high value crops. To increase production efficiency and make micropropagation viable for a wider range of species, new approaches to address key steps of the process with high labor inputs need to be evaluated. For this study, a two‐piece scaffold system was designed, prototyped using 3D printing, and tested to physically hold plants upright thereby facilitating liquid based rooting. This system was evaluated with Malus domestica, Betula lenta, and Musa sp. using static liquid culture as well as rocker based temporary immersion system and compared to rooting in semi‐solid based medium as is commonly practiced. Significantly, earlier rooting was observed in all three species in liquid when compared to semi‐solid culture system, and plants cultured in liquid on the rocker generally performed better than those in static liquid. In addition to quicker, more uniform rooting, reducing labor requirements, and preventing root damage. This newly designed system is simple, easy to use, will help to improve efficiency, and reduce the cost of micropropagation.

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Yukon Draba (Draba yukonensis) is a small, short-lived perennial mustard species that is endemic to southwestern Yukon in Canada. This plant has been categorized as a species of Special Concern. It faces the threat of habitat loss due to natural and man-made causes and a population that is unevenly distributed to a few large and several small subpopulations in the area. It will therefore be judicious to undertake investigations on the conservation of this species to save it from further deterioration which may lead to its extinction. In this study, a protocol was developed for in vitro propagation and cryopreservation of Yukon Draba. The micropropagation protocol was optimized using shoot tips which enabled clonal propagation and in vitro storage of the species. Shoots grew best in the medium containing MS basal salts and had the highest multiplication with the addition of 2 µM 6-benzylaminopurine or 5 µM Kinetin with 3% sucrose. The addition of 10 µM Indole Butyric Acid (IBA) produced the highest number of adventitious roots on the shoots and the longest root length was observed at 2 µM IBA. The rooted plantlets were transferred to greenhouse and the highest survival (87.5%) was observed for the plantlets treated with a lower concentration of IBA (2 µM). Cryopreservation protocol was developed using the droplet-vitrification method for in vitro shoot tips. Two-week-old shoots had the highest survival and regrowth following exposure to plant vitrification solution 3 (PVS3) for 30 min, prior to direct immersion of the droplets into the liquid nitrogen. The optimized protocols for the micropropagation and cryopreservation may be useful for the long-term germplasm conservation and reintroduction of this species in its natural habitat.

Apples (Malus&nbsp, domestica Borkh) are prone to preharvest fruit drop, which is more pronounced in ‘Honeycrisp’. Hexanal is known to improve fruit retention in several economically important crops. The effects of hexanal on the fruit retention of ‘Honeycrisp’ apples were assessed using physiological, biochemical, and transcriptomic approaches. Fruit retention and fruit firmness were significantly improved by hexanal, while sugars and fresh weight did not show a significant change in response to hexanal treatment. At commercial maturity, abscisic acid and melatonin levels were significantly lower in the treated fruit abscission zone (FAZ) compared to control. At this stage, a total of 726 differentially expressed genes (DEGs) were identified between treated and control FAZ. Functional classification of the DEGs showed that hexanal downregulated ethylene biosynthesis genes, such as S-adenosylmethionine synthase (SAM2) and 1-aminocyclopropane-1-carboxylic acid oxidases (ACO3, ACO4, and ACO4-like), while it upregulated the receptor genes ETR2 and ERS1. Genes related to ABA biosynthesis (FDPS and CLE25) were also downregulated. On the contrary, key genes involved in gibberellic acid biosynthesis (GA20OX-like and KO) were upregulated. Further, hexanal downregulated the expression of genes related to cell wall degrading enzymes, such as polygalacturonase (PG1), glucanases (endo-β-1,4-glucanase), and expansins (EXPA1-like, EXPA6, EXPA8, EXPA10-like, EXPA16-like). Our findings reveal that hexanal reduced the sensitivity of FAZ cells to ethylene and ABA. Simultaneously, hexanal maintained the cell wall integrity of FAZ cells by regulating genes involved in cell wall modifications. Thus, delayed fruit abscission by hexanal is most likely achieved by minimizing ABA through an ethylene-dependent mechanism.

In grafted plants, the movement of long-distance signals from rootstocks can modulate the development and function of the scion. To understand the mechanisms by which tolerant rootstocks improve scion responses to osmotic stress (OS) conditions, mRNA transport of osmotic responsive genes (ORGs) was evaluated in a tomato/potato heterograft system. In this system, Solanum tuberosum was used as a rootstock and Solanum lycopersicum as a scion. We detected changes in the gene expression levels of 13 out of the 21 ORGs tested in the osmotically stressed plants, of these, only NPR1 transcripts were transported across the graft union under both normal and OS conditions. Importantly, OS increased the abundance of StNPR1 transcripts in the tomato scion. To examine mRNA mobility in transgrafted plants, StNPR1 and StDREB1 genes representing the mobile and non-mobile transcripts, respectively, were overexpressed in tobacco (Nicotiana tabacum). The evaluation of transgenic tobacco plants indicated that overexpression of these genes enhanced the growth and improved the physiological status of transgenic plants growing under OS conditions induced by NaCl, mannitol and polyethylene glycol (PEG). We also found that transgenic tobacco rootstocks increased the OS tolerance of the WT-scion. Indeed, WT scions on transgenic rootstocks had higher ORGs transcript levels than their counterparts on non-transgenic rootstocks. However, neither StNPR1 nor StDREB1 transcripts were transported from the transgenic rootstock to the wild-type (WT) tobacco scion, suggesting that other long-distance signals downstream these transgenes could have moved across the graft union leading to OS tolerance. Overall, our results signify the importance of StNPR1 and StDREB1 as two anticipated candidates for the development of stress-resilient crops through transgrafting technology.

The growth and productivity of several apple rootstocks have been evaluated in various previous studies. However, limited information is available on their tolerance to osmotic stress. In the present study, the physiological and molecular responses as well as abscisic acid (ABA) levels were assessed in six apple rootstocks (M26, V3, G41, G935, B9 and B118) osmotically stressed with polyethylene glycol (PEG, 30%) application under greenhouse conditions. Our results showed that V3, G41, G935 and B9 had higher relative water content (RWC), and lower electrolyte leakage (EL) under stress conditions compared to M26 and B118. Additionally, water use efficiency (WUE) was higher in V3, G41 and B9 than M26, which might be partially due to the lower transpiration rate in these tolerant rootstocks. V3, G41 and B9 rootstocks also displayed high endogenous ABA levels which was combined with a reduction in stomatal conductance and decreased water loss. At the transcriptional level, genes involved in ABA-dependent and ABA-independent pathways, e.g., SnRK, DREB, ERD and MYC2, showed higher expression in V3, G41, G935 and B9 rootstocks compared to M26 in response to stress. In contrast, WRKY29 was down-regulated in response to stress in the tolerant rootstocks, and its expression was negatively correlated with ABA content and stomatal closure. Overall, the findings of this study showed that B9, V3 and G41 displayed better osmotic stress tolerance followed by G935 then M26 and B118 rootstocks. Published version

In grafted plants, the movement of long-distance signals from rootstocks can modulate the development and function of the scion. To understand the mechanisms by which tolerant rootstocks improve scion responses to osmotic stress (OS) conditions, mRNA transport of osmotic responsive genes (ORGs) was evaluated in a tomato/potato heterograft system. In this system, Solanum tuberosum was used as a rootstock and Solanum lycopersicum as a scion. We detected changes in the gene expression levels of 13 out of the 21 ORGs tested in the osmotically stressed plants; of these, only NPR1 transcripts were transported across the graft union under both normal and OS conditions. Importantly, OS increased the abundance of StNPR1 transcripts in the tomato scion. To examine mRNA mobility in transgrafted plants, StNPR1 and StDREB1 genes representing the mobile and non-mobile transcripts, respectively, were overexpressed in tobacco (Nicotiana tabacum). The evaluation of transgenic tobacco plants indicated that overexpression of these genes enhanced the growth and improved the physiological status of transgenic plants growing under OS conditions induced by NaCl, mannitol and polyethylene glycol (PEG). We also found that transgenic tobacco rootstocks increased the OS tolerance of the WT-scion. Indeed, WT scions on transgenic rootstocks had higher ORGs transcript levels than their counterparts on non-transgenic rootstocks. However, neither StNPR1 nor StDREB1 transcripts were transported from the transgenic rootstock to the wild-type (WT) tobacco scion, suggesting that other long-distance signals downstream these transgenes could have moved across the graft union leading to OS tolerance. Overall, our results signify the importance of StNPR1 and StDREB1 as two anticipated candidates for the development of stress-resilient crops through transgrafting technology.

In this study, an effective root-based cryopreservation method was developed for Hypericum perforatum L., an important medicinal species, using in vitro plants. A systematic approach was applied to determine effective combinations of protocol steps such as preculture, osmoprotection, vitrification solution treatment, and unloading, followed by protocol optimization using a single-factor approach. The effects of root section type (root tips, middle sections, or basal sections), duration of root section culture after excision, and donor plant age were also investigated. In a wild genotype, middle and basal root sections excised from 8-wk-old plants and cryopreserved at the age of 10 d after excision showed the highest plant regrowth after cryopreservation. In the optimized protocol, root sections were precultured in 10% (w/v) sucrose for 17 h, osmoprotected with a solution composed of 17.5% (w/v) glycerol and 17.5% (w/v) sucrose for 20 min, followed by a vitrification solution of 40% (w/v) glycerol and 40% (w/v) sucrose for 30 min, and cryopreserved using aluminum foil strips (droplet-vitrification). After rewarming in preheated 25% (w/v) sucrose solution and 30-min unloading, root segments were recovered on medium supplemented with 1.0 mg L−1 gibberellic acid and showed 78% plant regrowth. This cryopreservation method was successfully adapted for five elite lines of H. perforatum with a 45 to 87% regrowth rate after cryopreservation. These results suggest that root cryopreservation may be an effective method for medicinal plant conservation and should be tested with a broader range of species.

This DNA fingerprinting test confirmed 195 unique Corylus sp. accessions that were used to build a reference database for identity verification of unknown hazelnut trees from three locations in Ontario. Hazelnut is one of the most profitable tree nuts worldwide. Development of a hazelnut industry in Ontario is urgently required, but economically important cultivars must be genetically verified first in order to meet industry standards. Traditional methods for cultivar identification are largely trait-based and unreliable. In this study, a multiplexed fingerprinting test was modified to allow for hazelnut cultivar discrimination at the DNA level. Fourteen highly polymorphic SSR markers covering the 11 linkage groups of Corylus genome were PCR amplified in multiplex using fluorescent-labelled primers. PCR conditions and primer physical properties were optimized to generate a clear signal for each locus. The 14 SSRs were used to fingerprint 195 unique Corylus accessions collected from the USDA-NCGR. Fragment sizes were subjected to a UPGMA clustering analysis which separated Corylus accessions based on species and geographic origin. For validation purposes, hazelnut leaves from three locations in Ontario were collected for identity verification using this DNA fingerprinting test. As a result, 33.3% of the unknown trees were duplicates of seven distinct genotypes and a small percentage (8.3%) of these were identical to reference Corylus hybrids. These results reflect common mislabelling issues and genotype duplications that can prevent a uniform plant propagation system. Implementation of this test together with the addition of more unique accessions to the reference database will help verification of trueness-to-type of economically important cultivars for the hazelnut industry.

Apples (Malus domesticaBorkh) are prone to pre-harvest fruit drop which is more pronounced in ‘Honeycrisp’. Using a transcriptomic approach, we analyzed the molecular mechanisms of fruit retention in ‘Honeycrisp’. A total of 726 differentially expressed genes (DEGs) were identified in the abscission zone of hexanal-treated and untreated fruit (FAZ). Hexanal down-regulated the genes involved in ethylene biosynthesis, such as S-adenosylmethionine synthase (SAM2) and 1-aminocyclopropane-1carboxylic acid oxidases (ACO3,ACO4andACO4-like). Genes related to ABA biosynthesis (FDPSandCLE25) were also down-regulated. On the contrary, gibberellic acid (GA) biosynthesis genes, gibberellin 20 oxidase1-like (GA20OX-like) and ent-kaurene oxidase (KO) were up-regulated. Further, hexanal down-regulated the expression of genes related to cell-wall remodelling enzymes such as polygalacturonase (PG1), glucanases (endo-β-1,4-glucanase; EG) and expansins (EXPA1-like, EXPA6, EXPA8, EXPA10-like, EXPA16-like). Hexanal also reduced ethylene, and abscisic acid (ABA) production at commercial harvest stage. Hexanal reduced ethylene production in fruits and thus reduced the sensitivity of FAZ cells to ethylene and ABA. Simultaneously, hexanal maintained the cell-wall integrity of FAZ cells by regulating genes involved in cell-wall modifications. Our findings show that fruit abscission is delayed by hexanal, by down regulating ABA through an ethylene-dependent mechanism.HighlightHexanal, a naturally occurring plant compound, increased fruit retention in apples by decreasing ethylene and ABA production and maintaining the cell-wall integrity in the fruit abscission zone.

Tulsi (Ocimum sanctum L.) is a sacred plant of medicinal and spiritual significance in many cultures. Medicinal properties of Tulsi are ascribed to its phytochemicals with antioxidant capabilities. The current study was undertaken to screen a large seed population of Tulsi to select germplasm lines with high antioxidant potential and to standardize protocols for micropropagation and biomass production to produce a phytochemically consistent crop. A total of 80 germplasm lines were established under in vitro conditions and screened for their antioxidant potential determined with the 2,2-diphenyl-1-picrylhydrazyl (DPPH) bioassay. The micropropagation of a selected line, named Vrinda, was established using nodal cultures grown on Murashige and Skoog medium containing benzylaminopurine (1.1 µM), gibberellic acid (0.3 µM), and activated charcoal (0.6%). The antioxidant phytohormones melatonin and serotonin were quantified in the field and greenhouse grown tissues of Vrinda and melatonin levels were found to be consistent in both conditions with higher serotonin levels under field conditions. This integrated approach combining the in vitro selection and propagation offers potential applications in the development of safe, effective, and novel natural health products of Tulsi, and many other medicinal plant species.

Conservation of threatened flora in genetic collections ex situ using in vitro culture and cryopreservation is receiving an increasing recognition as a complementary strategy to in situ conservation in natural habitats. The present study is focused on an integrated approach which involves conservation and propagation, emphasizing the usefulness of cryopreservation techniques for germplasm conservation of streambank lupine (Lupinus rivularis Douglas ex Lindl.), an endangered species in Canada. This included in vitro seed germination on Murashige and Skoog basal medium supplemented with 1 µM thidiazuron to induce multiple shoot formation, micropropagation on a medium with 5 µM benzylaminopurine, and in vitro rooting on medium with 20.0 µM indole-3-butyric acid. Cryopreservation of seeds and shoot tips of in vitro grown plants was successful with over 60% seed germination and 62% regrowth of cryopreserved shoot tips, respectively. Plants developed from cryopreserved seeds had chlorophyll contents in leaves and the growth characteristics including the development of inflorescence, similar to plants raised from non-cryopreserved seeds. These results provide further evidence that the combination of micropropagation with cryopreservation of seeds and vegetative parts may effectively facilitate long-term preservation of L. rivularis and other endangered species.

Hill's thistle (Cirsium hillii (Canby) Fernald) is a perennial plant endemic to the Great Lakes region of North America. Hill's thistle is listed as threatened in Ontario and Canada where it is found in globally rare alvar habitats. The main objective of this study was ex-situ conservation of Hill's thistle using in vitro culture techniques and reintroduction of micropropagated plants back to their natural habitat in Bruce Peninsula National Park, Ontario, Canada. Two out of twenty-nine available seeds were successfully germinated under in vitro condition. An efficient micropropagation protocol was optimized with 100% survival during acclimatization of plantlets in the greenhouse. Three hundred micropropagated plants were reintroduced to twelve different sites within Bruce Peninsula National Park in June and July 2017. Plants were monitored for survival, rosette growth, and flowering on all sites from 2017-2019. After four months of planting, 67 to 99% of the plants were alive in different sites and 90 to 99% of them survived over winter. In the following years, shoot regeneration and flowering were observed on most sites. This study further confirms the benefit of plant tissue culture techniques to ensure revival of Hill's thistle ecological biodiversity through the reintroduction of micropropagated plants. This approach consisting of the components of conservation, propagation, and reintroduction (CPR) may potentially serve as a model for saving and enriching other species at risk.

Plants respond to changes in their environments through hormonal activation of a physiological cascade that redirects metabolic resources and growth. In filberts (Corylus sp.), chelated iron promotes the growth of new shoots but the mechanism(s) are not understood. To use untargeted metabolomics and hormonomics approaches to generate novel hypotheses for the morphoregulatory role of ferric ethylenediamine-N,N'-di-(ortho-hydroxyphenyl) acetic acid (Fe-EDDHA) in filbert shoot organogenesis in vitro. Data were generated using previously optimized standardized untargeted metabolomics protocols with time of flight mass spectrometry. Multivariate statistical tools (principal component and partial least squares discriminant analysis) did not detect significant differences. Discovery tools Significance Analysis of Microarrays (SAM), multiple linear regression analysis, Bayesian analysis, logical algorithms, machine learning, synthetic biotransformations, targeted hormonomics, and online resources including MetaboAnalyst were used. Starch/sucrose metabolism and shikimate pathway metabolites were increased. Dose dependent decreases were found in polyphenol metabolism, specifically ellagic acid and its methylated derivative 3,4,3′-tri-O-methylellagic acid. Hormonomics analysis revealed significant differences in phytohormones and their conjugates. FeEDDHA treatment reduced indole-3-acetic acid, abscisic acid, salicylic acid, jasmonic acid conjugates (JA-Trp, JA-Ile, OH-JA) and dihydrozeatinglucoside in regenerating explants. Serotonin (5HT) was decreased in FeEDDHA-treated regenerating tissues while the related metabolite melatonin was increased. Eight phenolic conjugates of 5HT and eight catabolites were affected by FeEDDHA indicating that metabolism to sequester, deactivate and metabolize 5HT was induced by Fe(III). Tryptophan was metabolized through kynurenine but not anthranilate. Seven novel hypotheses were generated to guide future studies to understand the regulatory control(s) of shoot organogenesis.

An efficient method for in vitro propagation of the bryophyte moss Plagiomnium cuspidatum (Hedw.) T.J. Kop is presented. Protocol optimization investigated media salt strength (quarter, third, or half-strength Murashige and Skoog; MS), sugar concentration (1 to 3% (w/v) sucrose), growth regulator content (presence of benzylaminopurine (BA) at 0–5 μM and napthaleneacetic acid (NAA) at 0–5 μM), and the addition of the phenylpropanoid biosynthesis inhibitor 2-aminoindan-2-phosphonic acid (AIP). Optimal media composition was determined to be half-strength MS with 2% (w/v) sucrose and 0.1 μM NAA. This method was then utilized to examine the effects of modified phenylpropanoid metabolism via application of AIP, an inhibitor of the first dedicated enzyme in phenylpropanoid biosynthesis, phenylalanine ammonia lyase (PAL). Treatment with AIP greatly reduced tissue browning and initiation of branching in P. cuspidatum and resulted in prolific production of straw to pale green-colored rhizoids. Treatment of plants with AIP in combination with the biosynthetic product of PAL, trans-cinnamic acid, was not able to fully recover the branching or browning phenotype, but several other phenolics, including p-coumaric acid and kaempferol, produced farther downstream in the biosynthetic pathway, were capable of partial recovery of the phenotype. Additionally, treatment with two indoleamines, melatonin and its biosynthetic precursor N-acetylserotonin, was also capable of partial recovery of the phenotype, showing greatly increased branching and increased browning of rhizoids. These results suggest that cross talk between phenylpropanoid and indoleamine metabolism is involved in bryophyte growth and development, beyond their traditional roles, leading to modified developmental outcomes.

In this study conservation of Castilleja levisecta Greenm., a globally endangered species was addressed through in vitro cryopreservation of shoot tips. In vitro cultures were successfully established using seedlings received from British Columbia, Canada. Shoot tips excised from in vitro propagated plants were cryopreserved using a droplet-vitrification method following optimization of individual protocol steps such as pre-culture, treatment with vitrification solutions, and unloading. The highest plant regrowth after cryopreservation (66%) was achieved when shoot tips were pre-cultured in 0.3 M sucrose for 17 h followed by 0.5 M sucrose for 4 h, incubated in an osmo-protectant solution (17.5% [v/v] glycerol and 17.5% [w/v] sucrose) for 20 min, exposed to vitrification solution A3 (37.5% [v/v] glycerol plus 15% [v/v] dimethylsulfoxide (DMSO) plus 15% [v/v] ethylene glycol (EG) plus 22.5% [w/v] sucrose) on ice for 40 min, and unloaded in 0.8 M sucrose solution for 30 min. Healthy plants were developed from cryopreserved shoot tips and propagated in vitro using nodal segments. Plants derived from in vitro culture and from cryopreserved tissues were successfully rooted and acclimated in a greenhouse with 100% survival rate. Acclimatized plants were reintroduced in a naturalized propagation area at the Conservation Nursery at Fort Rodd Hill, Canada. Twenty of 94 reintroduced plants (21%) survived the transit from lab to field and some had started to flower. This is the first report for cryopreservation of C. levisecta, an important step in conserving and re-introducing this critically imperiled species in nature.

Melatonin is a ubiquitously present indoleamine with a vast capacity for modulating the growth and behavior of plants, animals, and microbes. Though melatonin was discovered in plants decades after its discovery in mammals, its presence has now been confirmed in almost all plant families. Despite this, the in vitro and in vivo mechanisms of action of melatonin are still poorly defined. Although there are an increasingly large number of investigations into the roles of melatonin in plants, few take advantage of in vitro culture systems. Melatonin has been found to possess several important roles in plant growth and development, including functions in rhythmic and cyclic processes, such as chronoregulation, seasonal and senescence processes, as well as modulation of reproductive development, control of root and shoot organogenesis, maintenance of plant tissues, and responses to biotic and abiotic stresses. This review highlights the potential for use of melatonin in several in vitro systems, the roles it plays in plant morphogenesis, and the importance of melatonin in communication within and between plants, and how in vitro systems can be exploited to better understand these understudied functions of melatonin.

‘Honeycrisp’ (Malus domestica Borkh.), a premium applecultivar, is highly susceptible to bitter pit and decline in quality during long-term storage. In order to enhance the quality, an aqueous composition containing hexanal was applied as a preharvest spray. The effects of hexanal were assessed on the treated fruit and compared with HarvistaTM (a sprayable 1-Methylcyclopropene based commercial formulation) applied and control fruit under both cold (2.5 °C, four months) and cold after room temperature storage (20 °C, 14 days) conditions. Color, firmness, and total soluble solids (TSS) did not show a significant change in response to any treatment at harvest, while abscisic acid (ABA) significantly reduced and tryptophan increased in response to hexanal, compared to HarvistaTM and control. The treatment effects on quality traits were observed during storage. Both hexanal and HarvistaTM sprayed apples had higher TSS under both cold and room temperature storage. In addition, both sprays enhanced firmness at room temperature storage. However, the effects of sprays on other quality traits showed a different pattern. Apples sprayed with hexanal had lower phospholipase D enzyme (PLD) activity, lower incidence of bitter pit, and decreased expression of MdPLDα1 compared to HarvistaTM and control. On the other hand, HarvistaTM treated fruit produced lower ethylene. Both sprays decreased the expression of MdPLDα4, MdCaM2, MdCaM4 and MdCML18 genes. Generally, PLD alpha has a direct role in promoting fruit senescence, whereas the calcium senor proteins (CaM/CMLs) may involve in fruit ripening process via calcium and ethylene interactions. Therefore, improved postharvest qualities, including the lower incidence of bitter pit in hexanal treated ‘Honeycrisp’, may be associated with lower membrane damage due to lower PLD enzyme activity and decreased expression of MdPLDα1 and MdPLDα4 genes throughout the storage period.

Cryopreservation is considered an ideal strategy for the long-term preservation of plant genetic resources. Significant progress was achieved over the past several decades, resulting in the successful cryopreservation of the genetic resources of diverse plant species. Cryopreservation procedures often employ in vitro culture techniques and require the precise control of several steps, such as the excision of explants, preculture, osmo- and cryoprotection, dehydration, freeze-thaw cycle, unloading, and post-culture for the recovery of plants. These processes create a stressful environment and cause reactive oxygen species (ROS)-induced oxidative stress, which is detrimental to the growth and regeneration of tissues and plants from cryopreserved tissues. ROS-induced oxidative stresses were documented to induce (epi)genetic and somatic variations. Therefore, the development of true-to-type regenerants of the source germplasm is of primary concern in the application of plant cryopreservation technology. The present article provides a comprehensive assessment of epigenetic and genetic integrity, metabolic stability, and field performance of cryopreserved plants developed in the past decade. Potential areas and the directions of future research in plant cryopreservation are also proposed.

A simple and efficient system was developed for the rapid in vitro multiplication of Baptisia australis using nodal explants derived from axenic cultures. Among the four cytokinins [benzylaminopurine (BA), kinetin, N6-(2-isopentenyl)adenine (2-iP) and zeatin] tested for axillary bud induction, BA at 4.4 µM proved to be the most effective treatment for induction of multiple shoots from nodal explants after six weeks. Supplementation of Murashige and Skoog (MS) and Driver and Kuniyuki walnut (DKW) media with a chelated iron source, ethylenediamine Bis (2-hydroxyphenylacetic acid) (FeEDDHA) and BA had a favorable effect on axillary bud proliferation from explants (apical buds and nodal segments) further improving the regeneration response as well as number of shoots induced. DKW media proved to be superior to MS for multiple shoot induction of B. australis. A combination of 4.4 µM BA and 23 µM of FeEDDHA in DKW produced a positive response resulting in 100% of nodal segments responding and regenerating 7.2 shoots per explant. The highest rooting percentage was observed on MS media containing 1 µM IBA. In vitro-rooted plants were successfully acclimated in a greenhouse with a survival rate of 97%. The micropropagation method described in this study has a potential for commercial multiplication of this horticultural plant.

Attempts of clonal propagation of sugar maple through conventional and micropropagation techniques have been largely unsuccessful and the basis for this longstanding problem has remained unknown. Typical symptoms of stress in in vitro cultures of sugar maple and its intrinsic tolerance to highly shaded growing conditions indicated that optimization of light intensity might overcome its recalcitrance. To test this hypothesis we evaluated the growth, morphology, chlorophyll content and various chlorophyll fluorescence parameters of in vitro plantlets cultured under three intensities (4, 16 and 40 µmol m−2 s−1) using two spectra of light (red-blue and full spectrum white). Higher light intensities increased fresh and dry mass, but had no significant effect on the number of nodes or plant height. Anthocyanin accumulation in tissues, reduction in chlorophyll content, and significantly increased physiological stress quantified by chlorophyll fluorescence parameters, indicated increasing levels of light induced damage to the tissues with increasing light intensities. The critical factor limiting sugar maple’s response to in vitro propagation was light intensity and the levels commonly used for in vitro propagation are not suitable for sugar maple. The current study demonstrates the unique low light requirement, between 4 and 16 µmol m−2 s−1, of this species to overcome the recalcitrance to in vitro propagation. Spectral differences in quality of light and its effect on in vitro growth and stress in cultures are also discussed. In general light induced stress symptoms begin to appear at lower intensities with RB spectrum as compared to the full spectrum lights. While this study is limited to maples, requirement of specific light intensity is likely the cause of recalcitrance in other woody species with similar ecological roles.

cv. Anthos) is presented. Isotope tracer experiments were performed on plantlets regenerated from thidiazuron-induced stem explants and grown on MS basal medium for 2 months. Radiolabel from 14C-tryptophan was recovered as 14C-indoleacetic acid, 14C-tryptamine, 14C-5-hydroxytryptophan, 14C-serotonin and 14C-melatonin in the treated St. John's wort plantlets. Chromatographic peak identity was confirmed by high performance liquid chromatography-mass spectrometry-mass spectrometry and quantification of melatonin by radioimmunoassay. Significantly more radiolabel was recovered in serotonin relative to melatonin under low light conditions with this ratio being reversed under increased lighting, indicating that the rate of flow through this biosynthetic pathway is regulated, at least in part, by light.

In spite of their lack of central organized nervous system, plants possess many of the same signaling compounds which are employed in the mammalian nervous system and commonly referred to as neurotransmitters or neuromodulators. These include classes such as the indoleamines, melatonin and serotonin, and the catecholamines, dopamine, epinephrine (adrenaline), and norepinephrine (noradrenaline) and acetylcholine. These compounds, since their discoveries in plants, have been found to play important and diverse roles in plant life, including organogenesis, growth and development, flowering and reproduction, sensing environmental cues, and survival against a myriad of environmental stresses. This chapter will provide an overview of the roles these compounds play in plant life, and the mechanisms by which these compounds serve to mediate and direct growth, reproduction, and morphogenesis in plants and the as yet unidentified receptors for these compounds.

Hazelnut (Corylus spp.) flowering occurs during late winters and early spring which coincides with erratic fluctuating weather patterns. Irregular and reduced precipitation, especially in the form of snow, is assumed to dictate poor flowering and enhanced injury to catkins, especially in the sourced cultivars of interest to growers. We investigated the effect of water (precipitation received as rain and or snow) percolation during snowmelt period using phenological endpoints such as female flower ratio, bud position of female flower occurrence, and catkin dehiscence in sourced and locally adapted cultivars. These reproductive events were also analyzed for biochemical endpoints such as the concentrations of tryptophan, serotonin and melatonin in temporally collected bud or catkin samples. In addition, the yield responses of the tagged twigs were tracked and analyzed. When snow was consistently removed from the root zone (NS) or when a prior placed plastic sheet (P) disallowed water percolation, the percentage of female flowers and apical dominance declined in the sourced cultivars suggesting an erratic gradient between aerial and belowground temperature. The presence of snow cover resulted in increased probability of female flowers from dormant buds in sourced cultivars. Tryptophan was found to accumulate in the dormant buds of sourced cultivars where snow was not disturbed, while serotonin concentrations were significantly higher in NS and P treatments. Melatonin concentrations were notably high in dormant flower and vegetative buds of locally adapted cultivars especially subjected to P treatment. The biochemical markers may not only assist in identifying cold tolerance but also reveal abiotic stress negating responses modulated by relative concentrations of these molecules. Our results demonstrate that ideal conditions of good precipitation, especially as snow, would not only blanket to provide thermoregulation but also maintain the sensitive gradient on which temperate trees rely to break dormancy and initiate flower development. These findings also suggest that the use of horticultural alternatives such as thermal mulch could be an option for mitigating adverse conditions of reduced water percolation which affect reproductive physiology of hazelnut.

Cherry birch (Betula lenta L.) is an endangered species in Canada, with only a single natural population of 18 trees found in the Niagara region of Ontario. The tree was reportedly used for medicinal purposes by First Nations and Native American peoples. The current study describes a cryopreservation method for seeds and in vitro shoot tips of B. lenta in hopes of conserving available germplasm. Cryopreservation of mature seeds was successful, with 24% of seeds germinating in the greenhouse and 12% of seeds germinating in vitro on medium supplemented with 10 μmol·L–1 thidiazuron or 6-benzylaminopurine. Postcryopreservation regrowth of in vitro shoot tips was achieved using a droplet-vitrification protocol after preculture in 0.3 mol·L–1 sucrose for 24 h followed by osmoprotection in loading solution for 20 min and treatment with vitrification solution A3 composed of glycerol, sucrose, ethylene glycol, and dimethyl sulfoxide at 0 °C for 60 min. The highest plant regeneration (52%) was obtained after unload...

An efficient procedure for the micropropagation and conservation of cherry birch (Betula lenta L.), an endangered species in Canada, is reported. The model utilizes in vitro proliferation of fresh and dormant buds from both greenhouse and mature trees. Various factors were evaluated to optimize the protocol, including plant growth regulators, type and concentration of carbohydrates, and the composition of basal salts. 6-benzylaminopurine was the most effective cytokinin for shoot multiplication, with an optimal concentration of 5 µM. Sucrose was a more effective carbohydrate source than glucose, with the highest multiplication rate occurring at 3% sucrose (w/v). Shoot multiplication was similar on Murashige and Skoog (MS) and Driver and Kuniyuki Walnut (DKW) basal salts, but significantly lower on Woody Plant medium (WPM). Culture media with half-strength DKW basal salts and 20 µM indole-3-butyric acid was found to be the best for rooting (80%). Rooted plantlets were acclimatized to the greenhouse environ...

An efficient protocol for the micropropagation of hybrid hazelnut (Corylus L.) was developed using a temporary immersion system (TIS) for the proliferation of shoots in a liquid medium. Growth and proliferation of shoots as well as the length and number of nodes in developing shoots were significantly improved by combining two forms of chelated iron: ethylenediamine-N,N′-di-(ortho-hydroxyphenyl) acetic acid (FeEDDHA), and FeSO4/EDTA (FeEDTA). The use of TIS with optimized concentrations of FeEDTA and FeEDDHA significantly increased the number of shoots, shoot height, leaf area, and chlorophyll content in all of the four cultivars tested, compared with a semi-solid medium. Proliferated shoots developed roots on a semi-solid medium containing 2.5 μmol/L indole-3-butyric acid and rooted plantlets acclimatized in greenhouse conditions with 80% survival.

Microtubers are an important source of tissues for conservation and exchange of germplasm. An efficient method for cryopreservation of whole microtubers that will ensure a safe and long-term storage was developed in this study. Potato cultivars (Climax, Avon and Ceza) were evaluated for responses to in vitro tuberization and cryopreservation. Treatments include pretreatment on medium with sucrose (30–100 g l−1) and desiccation in silica gel (0, 3 and 6 h) followed by rapid plunging into liquid nitrogen (LN). There were significantly higher number of microtubers produced on the medium with 80–100 g l−1 sucrose (8–11.33) compared to 30 g l−1 sucrose (0.7–1.7). The microtuber fresh weights varied among genotypes on sucrose (80–100 g l−1) with ‘Climax’ showing the highest value followed by ‘Avon’ and ‘Ceza’. The microtuber moisture content ranged from 26 to 36 % after 3 h of desiccation and from 17 to 28 % after 6 h of desiccation for all three cultivars. After cryopreservation, the percentage of surviving (green) microtubers of ‘Climax’ increased with an increase in sucrose concentration, and was significantly higher than the control. For ‘Avon’, the percentage of green microtubers produced on sucrose (80–100 g l−1) was significantly higher than other treatments. Also, ‘Ceza’ produced significantly more green microtubers with 100 g l−1 sucrose compared to other sucrose treatments, similar to the control. Cryopreserved microtubers produced the most shoots on medium with 100 g l−1 sucrose. This study demonstrates the feasibility of long term storage of microtubers in LN for conservation purposes.

Serotonin (5-hydroxytryptamine) is an indoleamine with important physiological functions in plants, including developmental processes and stress responses. In this study, a simple and rapid bioassay was developed to investigate the inhibition of the fruit browning enzyme, polyphenol oxidase (PPO) via serotonin, employing cut apple discs as well as apple tissue homogenate treated with serotonin. The present study demonstrated that serotonin is significantly effective in inhibiting browning compared to ascorbic acid. Serotonin reduced fruit browning in tissues as well as suppressed the activity of PPO enzyme in the browning model solutions containing epicatechin (Epi) and caffeoyl tartrate (CT) as polyphenol substrates. The determination of enzyme kinetic parameters also suggested that serotonin may act as a putative, non-competitive inhibitor of PPO. Apart from its anti-browning effect, the application of serotonin significantly increased total phenolics content and antioxidant capacity when applied to the apple homogenate. Furthermore, the analysis of MdPPOs showed that five (MdPPO2, MdPPO4, MdPPO6, MdPPO8 and MdPPO10) out of the nine MdPPOs expressed in apple fruits were down-regulated in apple tissues treated with serotonin. To the best of our knowledge, this is the first report documenting a role for serotonin in the biochemical and transcriptional regulation of PPO enzymes and a simple system to study the biochemical control of browning in pome fruits.

Melatonin and serotonin are important phytochemicals enabling plants to redirect growth in response to environmental stresses. Despite much research on their biosynthetic routes, localization of their biosynthetic enzymes and recent identification of a phytomelatonin receptor, localization of the molecules themselves has to date not been possible. Elucidation of their locations in living tissues can provide an effective tool to facilitate indolamine research across systems including both plants and animals. In this study, we employed a novel technique, quantum dot nanoparticles, to directly visualize melatonin and serotonin in axenic roots. Melatonin was absorbed through epidermal cells, travelled laterally, and accumulated in endodermal and rapidly dividing pericycle cells. Serotonin was absorbed by cells proximal to the crown with rapid polar movement toward the root tip. Thermal stress disrupted localization and dispersed melatonin and serotonin across cells. These data demonstrate the natural movement of melatonin and serotonin in roots directing cell growth and suggest that plants have a mechanism to disperse the indolamines throughout tissues as antioxidants in response to environmental stresses.