Crown Buds Research Articles

Deep-sequencing transcriptome analysis of field-grown Medicago sativa L. crown buds acclimated to freezing stress

Medicago sativa L. (alfalfa) 'Zhaodong' is an important forage legume that can safely survive in northern China where winter temperatures reach as low as -30°C. Survival of alfalfa following freezing stress depends on the amount and revival ability of crown buds. In order to investigate the molecular mechanisms of frost tolerance in alfalfa, we used transcriptome sequencing technology and bioinformatics strategies to analyze crown buds of field-grown alfalfa during winter. We statistically identified a total of 5605 differentially expressed genes (DEGs) involved in freezing stress including 1900 upregulated and 3705 downregulated DEGs. We validated 36 candidate DEGs using qPCR to confirm the accuracy of the RNA-seq data. Unlike other recent studies, this study employed alfalfa plants grown in the natural environment. Our results indicate that not only the CBF orthologs but also membrane proteins, hormone signal transduction pathways, and ubiquitin-mediated proteolysis pathways indicate the presence of a special freezing adaptation mechanism in alfalfa. The antioxidant defense system may rapidly confer freezing tolerance to alfalfa. Importantly, biosynthesis of secondary metabolites and phenylalanine metabolism, which is of potential importance in coordinating freezing tolerance with growth and development, were downregulated in subzero temperatures. The adaptive mechanism for frost tolerance is a complex multigenic process that is not well understood. This systematic analysis provided an in-depth view of stress tolerance mechanisms in alfalfa.

Defoliation affects carbohydrate reserves, crown bud development, and spring re-growth in gentian ‘Velvet Glove’

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°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.

Tolerance of Swallowworts (Vincetoxicumspp.) to Multiple Years of Artificial Defoliation and Clipping

AbstractThe European vines pale swallowwort and black swallowwort are invading various habitats in northeastern North America. It is unclear how these plants might respond to potential biological control agents, as they experience little herbivore damage in North America, or longer durations of mowing given the reported lack of efficacy of mechanical control. We evaluated the effect of six seasons of artificial defoliation (50 or 100% defoliation once or twice per season) and clipping (once, twice, or four times at 8 cm above the soil level) on the survival, growth, and reproduction of mature plants of the two species grown in a common garden field experiment. No plants died from damage after 6 yr. Black swallowwort produced more aboveground biomass, whereas pale swallowwort produced more root biomass and root crown buds, compared with its congener species. For most damage treatments, root biomass and the number of crown buds and stems increased over time, whereas aboveground biomass and viable seeds per plant generally did not change. Substantial overlap in plant size and seed production occurred among damage treatments and species. The most severe defoliation treatment did not substantially limit growth and reproduction compared with undamaged plants. While two clippings per season sometimes prevented seed production, four clippings per season was the only type of damage that consistently prevented plant growth and eliminated seed production. Pale and black swallowwort display a high tolerance to aboveground tissue loss in high-light environments without plant competition. The annual increase in plant size calls into question the potential efficacy of a defoliating insect against field populations of swallowworts, and it seems likely the only benefits of a long-term mowing regime will be to eliminate seed production.

The relevance of shoots in the formation of crown buds in gentian

The relevance of shoots in the formation of crown buds in gentian

Glyphosate's impact on vegetative growth in leafy spurge identifies molecular processes and hormone cross-talk associated with increased branching.

BackgroundLeafy spurge (Euphorbia esula) is a perennial weed that is considered glyphosate tolerant, which is partially attributed to escape through establishment of new vegetative shoots from an abundance of underground adventitious buds. Leafy spurge plants treated with sub-lethal concentrations of foliar-applied glyphosate produce new vegetative shoots with reduced main stem elongation and increased branching. Processes associated with the glyphosate-induced phenotype were determined by RNAseq using aerial shoots derived from crown buds of glyphosate-treated and -untreated plants. Comparison between transcript abundance and accumulation of shikimate or phytohormones (abscisic acid, auxin, cytokinins, and gibberellins) from these same samples was also done to reveal correlations.ResultsTranscriptome assembly and analyses confirmed differential abundance among 12,918 transcripts (FDR ≤ 0.05) and highlighted numerous processes associated with shoot apical meristem maintenance and stem growth, which is consistent with the increased number of actively growing meristems in response to glyphosate. Foliar applied glyphosate increased shikimate abundance in crown buds prior to decapitation of aboveground shoots, which induces growth from these buds, indicating that 5-enolpyruvylshikimate 3-phosphate (EPSPS) the target site of glyphosate was inhibited. However, abundance of shikimate was similar in a subsequent generation of aerial shoots derived from crown buds of treated and untreated plants, suggesting EPSPS is no longer inhibited or abundance of shikimate initially observed in crown buds dissipated over time. Overall, auxins, gibberellins (precursors and catabolites of bioactive gibberellins), and cytokinins (precursors and bioactive cytokinins) were more abundant in the aboveground shoots derived from glyphosate-treated plants.ConclusionBased on the overall data, we propose that the glyphosate-induced phenotype resulted from complex interactions involving shoot apical meristem maintenance, hormone biosynthesis and signaling (auxin, cytokinins, gibberellins, and strigolactones), cellular transport, and detoxification mechanisms.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1627-9) contains supplementary material, which is available to authorized users.

Coordinated Expression of FLOWERING LOCUS T and DORMANCY ASSOCIATED MADS-BOX-Like Genes in Leafy Spurge.

Leafy spurge (Euphorbia esula L.) is a noxious perennial weed that produces underground adventitious buds, which are crucial for generating new vegetative shoots following periods of freezing temperatures or exposure to various control measures. It is also capable of flowering and producing seeds, but requires vernalization in some cases. DORMANCY ASSOCIATED MADS-BOX (DAM) genes have been proposed to play a direct role in the transition to winter-induced dormancy and maintenance through regulation of the FLOWERING LOCUS T (FT) gene, which also is likely involved in the vernalization process. To explore the regulation of FT and DAM during dormancy transitions in leafy spurge, the transcript accumulation of two previously cloned DAM splice variants and two different previously cloned FT genes was characterized. Under long-photoperiods (16 h light), both DAM and FT transcripts accumulate in a diurnal manner. Tissue specific expression patterns indicated the tissues with high DAM expression had low FT expression and vice versa. DAM expression is detected in leaves, stems, shoot tips, and crown buds. FT transcripts were detected mainly in leaves and flowers. Under dormancy inducing conditions, DAM and FT genes had an inverse expression pattern. Additionally, chromatin immunoprecipitation assays were performed using DAM-like protein specific antibodies to demonstrate that DAM or related proteins likely bind to cryptic and/or conserved CArG boxes in the promoter regions of FT genes isolated from endodormant crown buds. These results are consistent with the hypothesis that DAM proteins play a crucial role in leafy spurge dormancy transition and maintenance, potentially by negatively regulating the expression of FT.

The possibility of crop cultivation and utilization of edible gum from herb (Dorema ammoniacum D. Don) in dryland farming

Gum ammoniacum (Dorema ammoniacum D. Don) is one of the most important food, industrial and medicinal plants of Iran which is mainly distributed in semi-arid and desert areas. It is endangered due to superfluous and unsustainable harvesting methods. The objective of this study was to evaluate the possibility of crop cultivation in terms of dryland farming and the best operation method for collecting gum resin. For this purpose, the effects of different scarification methods (traditional, concave and staircase) and cut-off frequencies (5, 10 and 13 times) on gum yield of the five-year-old plants were studied at Toroq farm in Mashhad. The treatments were studied under a split plot structure in time experiment which was arranged in a completely randomized design with three replications. The rate of the root gum production and the rate of plant survival in the years after the withdrawal were measured, and finally the data were analyzed. Results showed no significant differences among cutting methods at p<0.05. The yield of gum was affected by times of harvesting and the highest yield was observed (p<0.01) in 13 cut-off times with 31.67 g/plant. The lowest gum yields were seen in 5 cut-off times with 5.84 g/plant. The traditional cutting method destroyed crown buds and caused the death of the plant (p<0.01). Reasonable gum yields and plant regeneration rates were obtained with the use of stairs method which is an easy method to be applied by the farmers or beneficiaries.

Influence of photoperiod regime and exogenous plant growth regulators on crown bud formation in gentian

Following vegetative propagation of interspecific hybrids of Gentiana such as ‘Showtime Diva’ there can be a delay or failure in formation of crown buds, sometimes resulting in the death of the plant. The influence of photoperiod and exogenous applications of plant growth regulators were investigated in order to identify factors that could induce the formation of crown buds earlier in the season. The transfer of plant material to a naturally occurring short photoperiod increased the number of crown buds 5-fold compared to a long photoperiod. Application of gibberellic acid inhibited bud formation under all photoperiod regimes investigated. When applied under the normally inhibiting long photoperiod regime, ethephon tripled the number of crown buds and shoots compared to the control, with the promotional effects doubled when paclobutrazol or thidiazuron was also applied. When exogenous growth regulators, that promoted crown bud formation under a long photoperiod regime were applied under the naturally occurring short photoperiod regime, buds formed on storage roots instead of the normal position on the transition zone between the shoot and roots. The change in location of crown buds with growth regulator application and photoperiod regime is discussed in terms of changes in sink strength and carbohydrate accumulation.

Dehydration-induced endodormancy in crown buds of leafy spurge highlights involvement of MAF3- and RVE1-like homologs, and hormone signaling cross-talk.

Vegetative shoot growth from underground adventitious buds of leafy spurge is critical for survival of this invasive perennial weed after episodes of severe abiotic stress. To determine the impact that dehydration-stress has on molecular mechanisms associated with vegetative reproduction of leafy spurge, greenhouse plants were exposed to mild- (3-day), intermediate- (7-day), severe- (14-day) and extended- (21-day) dehydration treatments. Aerial tissues of treated plants were then decapitated and soil was rehydrated to determine the growth potential of underground adventitious buds. Compared to well-watered plants, mild-dehydration accelerated new vegetative shoot growth, whereas intermediate- through extended-dehydration treatments both delayed and reduced shoot growth. Results of vegetative regrowth further confirmed that 14 days of dehydration induced a full-state of endodormancy in crown buds, which was correlated with a significant (P < 0.05) change in abundance of 2,124 transcripts. Sub-network enrichment analyses of transcriptome data obtained from the various levels of dehydration treatment also identified central hubs of over-represented genes involved in processes such as hormone signaling (i.e., ABA, auxin, ethylene, GA, and JA), response to abiotic stress (DREB1A/2A, RD22) and light (PIF3), phosphorylation (MPK4/6), circadian regulation (CRY2, PHYA), and flowering (AGL20, AP2, FLC). Further, results from this and previous studies highlight homologs most similar to Arabidopsis HY5, MAF3, RVE1 and RD22 as potential molecular markers for endodormancy in crown buds of leafy spurge. Early response to mild dehydration also highlighted involvement of upstream ethylene and JA-signaling, whereas severe dehydration impacted ABA-signaling. The identification of conserved ABRE- and MYC-consensus, cis-acting elements in the promoter of leafy spurge genomic clones similar to Arabidopsis RVE1 (AT5G17300) implicates a potential role for ABA-signaling in its dehydration-induced expression. Response of these molecular mechanisms to dehydration-stress provides insights on the ability of invasive perennial weeds to adapt and survive under harsh environments, which will be beneficial for addressing future management practices.

The resemblance and disparity of gene expression in dormant and non-dormant seeds and crown buds of leafy spurge (Euphorbia esula).

BackgroundLeafy spurge (Euphorbia esula L.) is a herbaceous perennial weed and dormancy in both buds and seeds is an important survival mechanism. Bud dormancy in leafy spurge exhibits three well-defined phases of para-, endo- and ecodormancy; however, seed dormancy for leafy spurge is classified as physiological dormancy that requires after-ripening and alternating temperature for maximal germination. Overlaps in transcriptome profiles between different phases of bud and seed dormancy have not been determined. Thus, we compared various phases of dormancy between seeds and buds to identify common genes and molecular processes, which should provide new insights about common regulators of dormancy.ResultsCluster analysis of expression profiles for 201 selected genes indicated bud and seed samples clustered separately. Direct comparisons between buds and seeds are additionally complicated since seeds incubated at a constant temperature of 20°C for 21 days (21d C) could be considered paradormant (Para) because seeds may be inhibited by endosperm-generated signals, or ecodormant (Eco) because seeds germinate after being subjected to alternating temperature of 20:30°C. Since direct comparisons in gene expression between buds and seeds were problematic, we instead examined commonalities in differentially-expressed genes associated with different phases of dormancy. Comparison between buds and seeds (‘Para to Endo buds’ and ‘21d C to 1d C seeds’), using endodormant buds (Endo) and dormant seeds (1d C) as common baselines, identified transcripts associated with cell cycle (HisH4), stress response/transcription factors (ICE2, ERFB4/ABR1), ABA and auxin response (ABA1, ARF1, IAA7, TFL1), carbohydrate/protein degradation (GAPDH_1), and transport (ABCB2). Comparison of transcript abundance for the ‘Eco to Endo buds’ and ‘21d C to 1d C seeds’ identified transcripts associated with ABA response (ATEM6), auxin response (ARF1), and cell cycle (HisH4). These results indicate that the physiological state of 21d C seeds is more analogous to paradormant buds than that of ecodormant buds.ConclusionCombined results indicate that common molecular mechanisms associated with dormancy transitions of buds and seeds involve processes associated with ABA and auxin signaling and transport, cell cycle, and AP2/ERF transcription factors or their up-stream regulators.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-014-0216-4) contains supplementary material, which is available to authorized users.

Foliar Application of Glyphosate Affects Molecular Mechanisms in Underground Adventitious Buds of Leafy Spurge (Euphorbia esula) and Alters Their Vegetative Growth Patterns

Long-term control of leafy spurge with glyphosate requires multiple applications because the plant reproduces vegetatively from abundant underground adventitious buds, referred to as crown and root buds. Determining the molecular mechanisms involved in controlling vegetative reproduction in leafy spurge following foliar glyphosate treatment could identify limiting factors or new targets for manipulation of plant growth and development in invasive perennial species. Thus, we treated leafy spurge plants with 0 or 2.24 kg ai ha−1glyphosate to determine its impact on selected molecular processes in crown buds derived from intact plants and plants decapitated at the soil surface 7 d after glyphosate treatment. New shoot growth from crown buds of foliar glyphosate-treated plants was significantly reduced compared with controls after growth-inducing decapitation, and had a stunted or bushy phenotype. Quantification of a selected set of transcripts involved in hormone biosynthesis and signaling pathways indicated that glyphosate had the most significant impact on abundance ofENT-COPALYL DIPHOSPHATE SYNTHETASE 1, which is involved in a committed step for gibberellin biosynthesis, and auxin transporters includingPINs, PIN-LIKES, andABC TRANSPORTERS. Foliar glyphosate treatment also reduced the abundance of transcripts involved in cell cycle processes, which would be consistent with altered growth patterns observed in this study. Overall, these results suggest that interplay among phytohormones such as auxin, ethylene, and gibberellins affect vegetative growth patterns from crown buds of leafy spurge in response to foliar glyphosate treatment.

The Architectural Complexity of Crown Bud Clusters in Gentian: Anatomy, Ontogeny, and Origin

Shoot productivity and overwintering survival of gentians (Gentiana sp.) are determined by the initiation and subsequent development of crown bud clusters. Understanding of the anatomical features and origins of crown buds and bud clusters, and plant ontogeny, the morphological features of crown buds, and their associated development is required to achieve manipulation of bud initiation, emergence, and development. Anatomical features of the crown bud clusters were examined using both light and confocal microscopy using hybrids of Gentiana triflora × G. scabra. The initiation of bud clusters presented characteristics typical of adventitious buds in terms of their origin and presence of external vascular connection to the parental tissue. In contrast, crown buds forming subsequently within the cluster developed as axillary buds within that initial bud, collectively forming on a compact stem with minimal internode elongation. Stem elongation within the cluster after application of gibberellic acid enabled identification of a hierarchical arrangement of buds within the cluster with one bud at each node and arranged spirally at 90°. Arrangements of buds within the cluster were different from the opposite decussate phyllotaxis in floral shoots with two axillary buds at each node. Based on the current study, a crown bud cluster originated from a first bud initial, which was adventitious followed by development of subsequent crown buds within the cluster as axillary buds from this first bud initially with a single bud developing at each node.

Effects of different cutting methods and times of cutting on growth performance and gum resin production of ferula assa-foetida

Ferula assa-foetida L. is an important industrial and medicinal plant of Iran which is mainly distributed in arid and semi-arid areas of Khorasan province in north east of the country. Gum of this plant is widely used in pharmacological industry. This plant is endangered because of more harvesting and unsustainable harvesting methods. The objective of this study was to evaluate the effects of different cutting methods (conventional, cut of crown part, concave and surface) and times of cutting (5, 10 and 13 times) on gum yield of this species in its wild habitat in Mazar region in Bajistan. The treatments were studied under a factorial experiment which was arranged in a completely randomized design with three replications. Results showed significant differences among cutting methods and times of harvesting. Yield of gum was affected by times of harvesting and the highest yield was obtained in 10 cutting times. The highest and lowest gum yields were obtained in concave method with 10 cutting times (25.24 g/plant) and superficial method with 5 cutting times (2.02 g/plant), respectively. Results showed that conventional cutting method destroys crown buds, which causes death of the plant. The highest gum yields and the best plant regeneration rates were obtained by using of concave method with 10 cutting times.

Anatomical investigations determining the origin of crown buds on the transition zone of gentians

Gentians are herbaceous perennials of ornamental and medicinal value, which utilize preformed crown buds as part of the perennating structure. Understanding the morphology and associated development of these crown buds is required if manipulation of bud initiation, emergence and development are to be achieved. The point of origin of crown buds, or whether crown buds originate from axillary or adventitious meristems, has not previously been published. In the current study, therefore, anatomical features of the shoot/stem, storage root, transition zone between shoot and root, axillary buds and crown bud clusters, were examined using light microscopy. In seedlings of Gentiana lutea, G. straminea, G. triflora and G. scabra, the transition zone was determined to be the area of initiation of crown buds, which differed morphologically in vascular arrangement from both the true shoot/stem and storage root. Crown buds that first developed on the transition zone were considered to be adventitious in origin.

BAC Library Development and Clone Characterization for Dormancy-Responsive DREB4A, DAM, and FT from Leafy Spurge (Euphorbia esula) Identifies Differential Splicing and Conserved Promoter Motifs

We developed two leafy spurge bacterial artificial chromosome (BAC) libraries that together represent approximately 5× coverage of the leafy spurge genome. The BAC libraries have an average insert size of approximately 143 kb, and copies of the library and filters for hybridization-based screening are publicly available through the Arizona Genomics Institute. These libraries were used to clone full-length genomic copies of an AP2/ERF transcription factor of the A4 subfamily of DEHYDRATION-RESPONSIVE ELEMENT-BINDING PROTEINS (DREB) known to be differentially expressed in crown buds of leafy spurge during endodormancy, a DORMANCY ASSOCIATED MADS-BOX (DAM) gene, and several FLOWERING LOCUS T (FT) genes. Sequencing of these BAC clones revealed the presence of multiple FT genes in leafy spurge. Sequencing also provided evidence that two different DAM transcripts expressed in crown buds of leafy spurge during endo- and eco-dormancy result from alternate splicing of a single DAM gene. Sequence data from the FT promoters was used to identify several conserved elements previously recognized in Arabidopsis, as well as potential novel transcription factor binding sites that may regulate FT. These leafy spurge BAC libraries represent a new genomics-based tool that complements existing genomics resources for the study of plant growth and development in this model perennial weed. Furthermore, phylogenetic footprinting using genes identified with this resource demonstrate the usefulness of studying weedy species to further our general knowledge of agriculturally important genes.

Increase in ACC oxidase levels and activities during paradormancy release of leafy spurge (Euphorbia esula) buds

The plant hormone ethylene is known to affect various developmental processes including dormancy and growth. Yet, little information is available about the role of ethylene during paradormancy release in underground adventitious buds of leafy spurge. In this study, we examined changes in ethylene evolution and the ethylene biosynthetic enzyme ACC oxidase following paradormancy release (growth induction). Our results did not show an obvious increase in ethylene during bud growth. However, when buds were incubated with 1mM ACC, ethylene levels were higher in growing than non-growing buds, suggesting that the levels of ACC oxidase increased in growing buds. Real-time qPCR indicated that the transcript of a Euphorbia esula ACC oxidase (Ee-ACO) increased up to threefold following growth induction. In addition, a 2.5- to 4-fold increase in ACO activity was observed 4days after decapitation, and the Ee-ACO accounted for 40% of the total ACO activity. Immunoblot analyses identified a 36-kD Ee-ACO protein that increased in expression during bud growth. This protein was highly expressed in leaves, moderately expressed in crown buds, stems and meristems, and weakly expressed in roots and flowers. Immunolocalization of Ee-ACO on growing bud sections revealed strong labeling of the nucleus and cytoplasm in cells at the shoot apical meristem and leaf primordia. An exception to this pattern occurred in cells undergoing mitosis, where labeling of Ee-ACO was negligible. Taken together, our results indicated an increase in the levels of Ee-ACO during paradormancy release of leafy spurge that was not correlated with an increase in ethylene synthesis.

Induction of endodormancy in crown buds of leafy spurge (Euphorbia esula L.) implicates a role for ethylene and cross-talk between photoperiod and temperature

Leafy spurge is a model for studying well-defined phases of dormancy in underground adventitious buds (UABs) of herbaceous perennial weeds, which is a primary factor facilitating their escape from conventional control measures. A 12-week ramp down in both temperature (27→10°C) and photoperiod (16→8h light) is required to induce a transition from para- to endo-dormancy in UABs of leafy spurge. To evaluate the effects of photoperiod and temperature on molecular networks of UABs during this transition, we compared global transcriptome data-sets obtained from leafy spurge exposed to a ramp down in both temperature and photoperiod (RDtp) versus a ramp down in temperature (RDt) alone. Analysis of data-sets indicated that transcript abundance for genes associated with circadian clock, photoperiodism, flowering, and hormone responses (CCA1, COP1, HY5, MAF3, MAX2) preferentially increased in endodormant UABs. Gene-set enrichment analyses also highlighted metabolic pathways involved in endodormancy induction that were associated with ethylene, auxin, flavonoids, and carbohydrate metabolism; whereas, sub-network enrichment analyses identified hubs (CCA1, CO, FRI, miR172A, EINs, DREBs) of molecular networks associated with carbohydrate metabolism, circadian clock, flowering, and stress and hormone responses. These results helped refine existing models for the transition to endodormancy in UABs of leafy spurge, which strengthened the roles of circadian clock associated genes, DREBs, COP1-HY5, carbohydrate metabolism, and involvement of hormones (ABA, ethylene, and strigolactones). We further examined the effects of ethylene by application of 1-aminocyclopropane-1-carboxylate (ACC) to paradormant plants without a ramp down treatment. New vegetative growth from UABs of ACC-treated plants resulted in a dwarfed phenotype that mimicked the growth response in RDtp-induced endodormant UABs. The results of this study provide new insights into dormancy regulation suggesting a short-photoperiod treatment provides an additive cross-talk effect with temperature signals that may impact ethylene's effect on AP2/ERF family members.

Memory of prolonged Winter cold inhibits flowering and increases long-day leaf number in the chrysanthemum cultivar ‘Nagano Queen’

SummaryChrysanthemum (Chrysanthemum morifolium Ramat.) is a short-day plant; however, flowering is eventually initiated under non-inductive long-day or night-break conditions. When using Summer-to-Autumn-flowering cultivars, such flowering is undesirable for stable horticultural production, particularly for Summer cropping, because of the short length of the cut flower, the formation of a crown bud, and the development of a branched spray of flowers. Under non-inductive conditions, flowering depends on the number of leaves that form below the terminal flower bud (i.e., the long-day leaf number). Long-day leaf numbers varied among cultivars in this study. ‘Nagano Queen’ showed a seasonal change in long-day leaf number, being high in Spring and decreasing in Summer. In addition, rooted cuttings under night-break conditions initiated very early flowering. Here, we show how exposure to prolonged Winter cold influenced long-day leaf number, and its inhibitory effect on flowering. Cold pre-treatments inhibited flower initiation under subsequent short-day conditions, with noticeable genetic variation among cultivars. For instance, flower initiation in ‘Nagano Queen’ was strongly inhibited in Spring, but the inhibition decreased in Summer. This result indicated that the inhibitory effect of Winter cold caused a delay in Spring flowering, which gradually declined from Spring to Summer. Exposure to prolonged Winter cold also inhibited flower initiation under long-day and night-break conditions, and increased long-day leaf number in Spring. These results indicate that this seasonal change in the inhibitory effect of Winter cold influences long-day leaf number in ‘Nagano Queen’.

Temperature impacts changes in crown buds and flowering of gentian ‘Spotlight’

A better understanding of the origin and development of crown buds in gentian may help explain the observed spread in timing of flower harvest maturity. The types and numbers of buds, the time of their macroscopic appearance, emergence and development to flowering were tracked over time, in two growing environments for two growth cycles of the gentian cultivar ‘Spotlight’. Although different types of buds were present, only pre-formed primary-crown buds produced flowering shoots. In the first growth cycle, only 73% of these pre-formed buds emerged, leaving the rest of the buds to senesce and degenerate. Hence, the observed spread in time of flower harvest maturity was not due to either bud type or buds that appeared during the current growth cycle developing into floral shoots. The appearance of new crown buds for the following growth cycle's flower production occurred over an 8-month period, from summer through to winter. The time of harvest maturity for a shoot was not strongly correlated with the time of bud appearance or shoot emergence, as originally hypothesised. Compared with those grown in the cooler environment outside, cultivation in a protected environment resulted in 50% longer shoots at harvest, did not influence the time to flower harvest, but increased the rate of appearance of buds by an additional 0.18 per month. With no chilling however, within the protected environment the crown buds failed to emerge as shoots during the second growth cycle. In contrast, with chilling received by outdoor-grown plants, crown buds emerged and flowered. The lack of any correlation between the time of appearance of buds and/or their emergence with that of stem harvest maturity, as well as the chilling requirement for shoot emergence, highlights that factors associated with growth and development of a crown bud after its appearance may have a greater influence on the time of harvest maturity in ‘Spotlight’ and, therefore, the spread in timing.

Effects of soil water deficit on seedlings of different Medicago falcata L. populations

Medicago falcata L. survives in desiccation conditions and is used to provide the genetic material for improved drought survival of this otherwise productive forage species (crosses referred to as M. media). Seedlings were grown in one of three soil water contents (80, 50 and 25% of field capacity) to compare physiological and morphological response under soil water stress. After eight weeks, populations differed in drought responses, and populations from Russia displayed superior characteristics such as more survival than others; maintaining stomatal more open, high water use efficiency (WUEi) and photosynthesis rate; less leakage of electrolytes from the plasma membrane, large leaf area, more crown buds, big crown diameter, high root length and relative root length, and an approximately 1.5 fold root: shoot ratio than others, etc. We recommended that the Russian M. falcate L. populations with excellent drought tolerance are suitable to grow in arid and semi-arid regions in China, and also a potential selection for breeding new drought tolerant forage. Key words: Medicago falcata L., soil water deficit, photosynthesis, water use efficiency, leaf area, survival, biomass allocation.