Morphological and molecular analyses of Rosa damascena × R. bourboniana interspecific hybrids

Rosa damascena Mill is the most important scented rose species cultivated for rose oil production. Rosa bourboniana L. (Edward rose), a related species, is popular on account of its longer blooming period and ease of propagation. With an aim to combine the oil quality of R. damascena and recurrent flowering habit of R. bourboniana, two cultivars (Jwala and Himroz) of R. damascena were crossed with R. bourboniana. The F1 hybrids obtained were evaluated using morphological, random amplified polymorphic DNA (RAPD) and microsatellite (SSR) markers. Twenty-two selected RAPD and three SSR primer pairs were utilized for hybrid identification. According to presence or absence of bands RAPD and SSR markers were classified into seven types of markers. The bands specific for the pollen parent and occurring in the hybrids were good markers to confirm the hybridity. The non-parental bands expressing uniquely in hybrids were effective in distinguishing the hybrids from each other. Cluster analysis, based on Jaccard's similarity coefficient using unweighted pair group method based on arithmetic mean (UPGMA), reliably discriminated the hybrids into two main clusters. These results indicate the practical usefulness of RAPD and SSR markers in hybrid identification in scented roses. The approach is advantageous for its rapidity and simplicity, for identification of hybrids at the juvenile stage. One of the studied morphological traits – prickle density, can also complement in the identification of interspecific hybrids between R. damscena (♀) and R. bourboniana (♂).     

Agrobacterium × plant factors influencing transformation of ‘Joseph's coat’ (Amaranthus tricolor L.)

A protocol is developed for Agrobacterium-mediated genetic transformation of Amaranthus tricolor via explant co-cultivation with Agrobacterium rhizogenes. Bacteria-plant specific factors which influenced transformation were optimized. Of the two Agrobacterium strains employed, LBA9402 was more infectious compared to A4. Bacterial suspensions grown overnight with 100 μM acetosyringone and experiencing O.D.₆₆₀ = 0.6 followed by dilution to a density of 10⁹ cells ml⁻¹ were the most effective. Explants from garden-grown plants were more responsive than those from in vitro cultures; stem internodes being better than leaves. Immersion of the pre-pricked explants in bacterial suspension resulted in a markedly higher transformation frequency compared to the direct injection method. The infection of internode explants with the LBA9402 strain followed by co-cultivation on growth regulator-free MS medium (MS0) for 5 days resulted in emergence of hairy roots up to a maximum frequency of 97.22%. Roots were individually cultured in MS0, but fortified with bactericidal antibiotic (500 μg ml⁻¹ cefotaxime). Rhizoclones showing prolific growth were renewed through successive subcultures in MS0. Opine gene expression was revealed by positive agropine and mannopine synthesis in all selected transformed rhizoclones. Shoot regeneration from root clones, capable of auxin-independent growth and opine proficiency, was stimulated in MS augmented with 2.0 mg l⁻¹ zeatin. pRi TL–DNA rolB and pRi TR–DNA man2 ORF were detected in leaf tissues of regenerated plants from selected hairy root clones through PCR amplification. The implication of such findings is discussed on the possibility of conferring protection to crop amaranths against biotic stress challenges, particularly due to insects, viruses or fungal pathogens.     

Aneuploid strawberry (2n = 8x + 2 = 58) was developed from homozygous unreduced gamete (8x) produced by second division restitution in pollen

Unreduced gamete formation is significant in the evolutionary development of complex polyploidy series found in wild strawberry, genus Fragaria (Rosaceae). Also, it is important for genetics and breeding in strawberry plants to elucidate the mechanism of unreduced gamete formation. The objective of this study was to search for ploidy anomalies resulting from artificial diploid × octoploid crosses, and examine the mechanism through which these unreduced gametes were produced. Five everbearing cultivars of Fragaria vesca L. diploid (2n = 2x = 14) were crossed with pollen from six June-bearing cultivars of Fragaria × ananassa Duch., octoploid (2n = 8x = 56). A total of 3000 mature seeds, 100 from each of the 30 parental combinations were sown at 23 °C/20 °C (day/night) under artificial lighting with a 16 h day. The seedlings were transplanted to pots and grown in a greenhouse. Reproductive and morphological observations, flow cytometry analyses, chromosome counts and DNA analyses using CAPS markers were performed to identify the genetic background of the offspring. Most of the seed (79%) did not germinate or died soon after germination. Of the seedlings produced, 7% seemed to be pure F. vesca based on morphological characteristics, flow cytometry analyses and chromosome counts; 14% were pentaploids (2n = 5x = 35), 0.1% were hexaploids (2n = 6x = 42), and 0.03% (one individual) was aneuploid (2n = 8x + 2 = 58). Electrophoresis banding patterns obtained by CAPS marker analysis were heterozygotic in the 8x pollen parent but homozygotic in the aneuploid progeny. Judging from the chromosome counts and the CAPS marker analysis, the aneuploid was the result of a homozygous unreduced pollen grain (8x) crossed with an incomplete chromosome compliment from the egg. Because of the homozygosity, the unreduced male gamete must have been derived from second division restitution (SDR) in the octoploid pollen parent.     

In vitro proliferation from axillary buds and ex vitro protocol for effective propagation of Syringa × hyacinthiflora ‘Luo Lan Zi’

As a precondition for lilac mass propagation, the optimal shoot-multiplication medium for Syringa × hyacinthiflora ‘Luo Lan Zi’ was ascertained mainly based on clustered microshoot inducement and large leaf area establishment in 6-benzyladenine (BAP) (1.00 mg L⁻¹) and zeatin (Z) (0.10 mg L⁻¹) combination. Medium supplied with lower level of BAP (0.50 mg L⁻¹) and auxin (IAA) (0.25 mg L⁻¹) was not suitable for lilac shoot proliferation, but it could be competent for long-term preservation of the un-rooted shoots so that subsequent proliferation culture could be carried out at anytime. In addition, excess height growth which resulted in low transplanting survival rate was effectively controlled by decrease in node number when paclobutrazol (PBZ) was applied in rooting medium at a concentration of 1.00 mg L⁻¹ after taking into account the effects on shoot height, rooting, persistent leaf area and PBZ carry-over. An important overwintering treatment was to use a plastic chamber covering for plants in the greenhouse prior to field planting to ensure adequate biomass of stem and underground parts not only in the current growing season but also in the subsequent years.     

Effect of plant growth temperature on membrane lipids in strawberry (Fragaria × ananassa Duch.)

Changes in membrane lipid composition are important in the acclimation of plants. The influence of four day/night growing temperature combinations (18/12, 25/12, 25/22, and 30/22 °C) on membrane lipids of ‘Earliglow’ and ‘Kent’ strawberry (Fragaria × ananassa Duch.) were studied. The monogalactosyl diglyceride (MGDG) and digalactosyl diglyceride (DGDG) were the major galactolipids in the strawberry leaves. ‘Earliglow’ contained a higher amount of galactolipids in the leaves than ‘Kent’. The major phospholipids in the strawberry leaves, roots, and fruit were phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylglycerol (PG), and phosphatidylethanolamine (PE). PC and PE were the two predominant phospholipids in the strawberry. The leaves and fruit of ‘Earliglow’ contained higher amounts of phospholipids compared to those of ‘Kent’, whereas ‘Kent’ strawberry roots had higher phospholipids. Palmitic (C16:0), stearic (C18:0), oleic (C18:1), linoleic (C18:2), and α-linolenic (C18:3) acids were major fatty acids in galacto- and phospholipids of the ‘Earliglow’ and ‘Kent’ strawberry. PC is very rich in linolenic acid in leaves compared to the fruit and root tissues. PC had the highest ratio of unsaturated to saturated fatty acids among all phospholipids. There was a significant increase in the content of galactolipids (MGDG, DGDG) and phospholipids (PC, PI, PG and PE) and unsaturation of their fatty acids in the cool day/night growth temperature. Increasing day/night growth temperatures decreased MGDG/DGDG ratios. The shifts in saturation and composition of fatty acids observed with strawberry may be an adaptation response of plants to the temperature changes.     

Auxin-dependent development and habituation of highbush blueberry (Vaccinium × covilleanum But. et Pl.) ‘Herbert’ in vitro shoot cultures

The influence of IAA (1.0 mg dm⁻³), and IBA (1.16 mg dm⁻³), on the development of highbush blueberry (Vaccinium × covilleanum But. et Pl.) ‘Herbert’ in vitro shoot cultures was examined. Depending on the kind of auxin and 2iP concentration in vitro cultures consisted of various number of axillary (AX) and adventitious (AD) shoots. Three different categories of AD shoots were found: leaf shoots (AD-L), node-adjoin shoots (AD-P), and base-adjoin shoots (AD-M, madshoots). The AX shoots were the least habituated (towards auxins, cytokinins and vitamins) whereas the AD-M shoots (madshoots) the most. In comparison to IAA, IBA caused dying or callusing higher number of initial explants. However, IBA generally suppressed development of AD shoots, especially madshoots whereas slightly weakened multiplication of AX shoots. IBA significantly enhanced elongation of AX shoots also. Axillary shoots obtained on IBA-media had relative long internodes and rigid, well-developed leaves. The adventitious shoots, especially base-adjoin (AD-M) ones, were easily distinguishable as were more thin and fragile, more or less vitrified, and had short internodes and smaller, sometimes unfolded leaves. Development of blueberry in vitro cultures on auxin-free and IAA-supplemented media was similar. AX shoots grown on such media resembled AD shoots. 2iP applied in higher doses along with IAA promoted much proliferation of AD than AX shoots. In contrast, 2iP applied in higher doses together with IBA stimulated significantly only growth of AX shoots whereas in general, development of adventitious shoots was not affected. Micropropagation carried out through routine method based on subculturing of shoot explants or shoot clumps on the medium supplemented with IAA (4 mg dm⁻³) and 2iP (10–15 mg dm⁻³) as well as stimulation of shoot elongation on the blank medium causes in fact the propagation of highbush blueberry through highly habituated adventitious madshoots. Replacement of IAA by IBA facilitates micropropagation of highbush blueberry cv. Herbert through axillary shoots.     

Rootstocks influence fruit oleocellosis in ‘Hamlin’ sweet orange (Citrus sinensis L. Osbeck)

Oil spotting or oleocellosis, is a major problem in citrus crops. As the rootstock and fertilization play important roles in citrus growth and fruit development, we investigated the influence of different rootstocks on the growth, mineral nutrition metabolism, water relations, and fruit oleocellosis of eight-year-old field ‘Hamlin’ sweet orange trees. Trees grafted onto Lichi16-6 trifoliata (Poncircus trifoliate) had the greatest rate of oleocellosis (RO), and trees grafted onto Goutou orange (Citrus aurantium) had the greatest degree of oleocellosis (DO). In contrast, trees grafted onto Rangpur lime (Citrus limonia Osbeck) had the lowest RO and DO. Trees were the most vigorous on Rangpur lime rootstocks, followed by Lichi16-6 trifoliata, and then Goutou orange. In addition, because the scion/stock girth ratio showed significant correlations with the RO and DO, oleocellosis parameters can be a good indicator of scion/stock affinity. The total N, total P, Ca²⁺, and Mg²⁺ in leaves from trees on Rangpur lime were significantly lower than in leaves from trees on Goutou orange or Lichi16-6 trifoliata. In addition, the RO showed a significant correlation with the leaf Ca²⁺ and S concentrations and with the peel Mg²⁺ concentration. The DO was significantly correlated with the total peel N and S concentrations. In addition, the RO showed a significant correlation with the net assimilation of CO₂ (ACO₂), stomatal conductance (GS), transpiration rate (ET), and water-use efficiency (WUE). However, the DO showed a significant correlation with the GS, ACO₂, and WUE. Taken together, these results indicate that rootstocks affect the development of oleocellosis in ‘Hamlin’ sweet orange due to their effects on the mineral nutrition balance and water relations.     

Changes in morphological phenotypes and essential oil components in lavandin (Lavandula × intermedia Emeric ex Loisel.) transformed with wild-type strains of Agrobacterium rhizogenes

Hairy roots were induced from leaf-derived calli of lavandin (Lavandula × intermedia Emeric ex Loisel.) by infection with wild-type strains of Agrobacterium rhizogenes, A-5 (MAFF 02-10265) and A-13 (MAFF 02-10266). A-5-inoculated calli formed hairy roots more efficiently than A-13 ones. The transgenic shoots could be obtained from hairy root segments mediated by each Agrobacterium strain. However, different plant growth regulators were required for efficient adventitious shoot formation in each strain. In A-5, the most efficient adventitious shoot formation rate of 23.8% was observed in a medium with 4.4 × 10⁻⁶ M of 6-benzylaminopurine. On the other hand, a significantly higher rate of 13.2% was detected in a medium with 4.0 × 10⁻⁷ M of N-(2-chloro-4-pyridyl)-N′-phenylurea in A-13. Most of the regenerated plants showed dwarfism with closed internodes and extensive lateral branching, which were typical characteristics of ‘hairy root syndrome’. On the other hand, only nine of the 45 regenerated plants formed flower buds in early June, a delay of about one month compared with nontransgenic regenerated plants. The floral stalks and spikes of these plants were very short, resulting in a compacted form. Many regenerants showed a significantly lower productivity of essential oil than nontransgenic regenerants. Moreover, the relative percentage of the linalyl-cation-derived compounds, linalool and linalyl acetate, decreased in most of the regenerated plants. Compact plants with the ability of flower bud formation are assumed to be valuable not only for lavandin breeding, but also for clarifying the interaction between rol genes expression and essential oil production.     

Interaction of short day and timing of nitrogen fertilization on growth and flowering of ‘Korona’ strawberry (Fragaria × ananassa Duch.)

The effects of timing of nitrogen (N) fertilization relative to the beginning of a 4-week floral-inducing short-day (SD) period have been studied in ‘Korona’ strawberry plants under controlled environment conditions. Groups of low fertility plants were fertilized with 100 ml of calcium nitrate solution for 3 days a week for a period of 3 weeks starting at various times before and at the beginning of the SD period, as well as at different times during the SD period. All plants, including SD and long day (LD) control plants, received a weekly fertilization with a low concentration complete fertilizer solution throughout the experiment. Leaf area, fresh and dry matter increments of leaves, crowns and roots, as well as leaf chlorophyll concentration (SPAD values) were monitored during the experimental period. A general enhancement of growth took place at all times of N fertilization. This was paralleled by an increase in leaf chlorophyll concentration, indicating that the control plants were in a mild state of N deficiency. When N fertilization was started 2 weeks before beginning of the SD period, flowering was delayed by 7 days, and this was gradually changed to an advancement of 8 days when the same treatment was started 3 weeks after the first SD. The amount of flowering was generally increased by N fertilization although the effect varied greatly with the time of N application. The greatest flowering enhancement occurred when N fertilization started 1 week after the first SD when the number of flowering crowns and the number of inflorescences per plant were more than doubled compared with the SD control, while fertilization 2 weeks before SD had no significant effect on these parameters. Importantly, the total number of crowns per plant was not affected by N fertilization at any time, indicating that enhancement of flowering was not due to an increase in potential inflorescence sites. No flowering took place in the control plants in LD. Possible physiological mechanisms involved and practical applications of the findings are discussed.     

Fruit nutrient content and lipoxygenase activity in relation to the production of malformed and button berries in strawberry (Fragaria × ananassa Duch.)

Fruit nutrient content and lipoxygenase (LOX) activity were determined in strawberry fruit to establish a relationship, if it exists, between nutrients, and LOX activity with the fruit malformation and nubbins or button berry disorders. Nearly 17% fruit were affected by malformation and 10% by nubbins in open-field-grown strawberries. ‘Etna’ produced higher proportion of malformed (22.7%) as well as button berries (16.9%) and ‘Sweet Charlie’ the lowest (8.9% and 3.3%, respectively). Dry matter content (%) was lower in malformed (5.2%) and button berries (3.23%) than normal berries (7.41%). The concentration of P and Mg did not differ significantly, but that of N and K was notably higher and of Ca and B was lower in malformed and button berries than normal berries. Consequently, the N/Ca and K/Ca ratios were higher in malformed and button berries. LOX activity was significantly higher in malformed as well as button berries than normal berries, with significant differences among cultivars. The correlations between N, K and malformed and button berries were positive and between Ca and B, and malformed and button berries were negative. Similarly, the correlation between LOX activity and malformed, and button berries were also positive, indicating that excess of N and K, and deficiency of Ca and B are related to the production of malformed and buttons or nubbins in strawberry.     

Germination of Passiflora mollissima (Kunth) L.H.Bailey, Passiflora tricuspis Mast. and Passiflora nov sp. seeds

Passiflora mollissima, Passiflora tricuspis and Passiflora nov sp. are three passion fruit species occurring in Bolivia. Germination percentages and rates were determined for 11 different treatments. Per species, germination of 100 seeds was monitored every 3 days, during 90 days. Germination started after 9 days and 50% of final germination was reached within a month or less. Successful, recommended methods for P. mollissima are removing the basal point of seeds (27% germination) or removing the basal point in combination with pre-soaking seeds for 48 h in 50 ppm GA₃ (18%). Pre-soaking seeds for 24 h in 400 ppm GA₃ (42%) and removal of the basal point in combination with pre-soaking seeds for 48 h in 50 ppm GA₃ (36%) are suggested methods to improve germination of P. nov sp. Removing the apical point of P. tricuspis seeds resulted in maximal germination (57%). No unique treatment gave satisfactory results for the three species tested. Exogenous dormancy, probably a combination of mechanical and chemical dormancy is present in the three species studied. Presence of physical dormancy was found in P. mollissima.     

H2O2 metabolism during sweet orange (Citrus sinensis L. Osb.) ‘Hamlin’ Xanthomonas axonopodis pv. citri interaction

Sweet orange (Citrus sinensis L. Osb.) ‘Hamlin’ is a canker (Xanthomonas axonopodis pv. citri: Xac) susceptible citrus genotype grown commercially worldwide. Canker causes severe economic losses and restricts the marketability of crop for export. Little is known about the role of oxidative stress in canker development. In the present investigation, sweet orange ‘Hamlin’ leaves were artificially inoculated with Xac to determine the impact of Xac infection on hydrogen peroxide (H₂O₂) metabolism. Characteristic symptoms following artificial inoculation were water soaking of the infiltrated zone between 2 and 8 days after inoculation (dai); raised epidermis accompanying tiny yellow colored bacterial colonies at 8 dai; and yellowing and necrosis of the infected zone by 12–16 dai. In planta Xac population increased 1000 fold by 14 dai from an initial population of 7.3 × 10⁶ cfu cm⁻² (0 dai). Peak concentrations of H₂O₂ were observed at 24 h and between 8 and 10 dai and coincided with higher activity of total superoxide dismutase (SOD). Lower levels of H₂O₂ in infected leaves were maintained by Xac induced higher activities of catalase (CAT), ascorbate peroxidase (APOD), and guaiacol peroxidase (POD). It appears Xac altered H₂O₂ metabolism in C. sinensis L. Osb. ‘Hamlin’ to enhance survival and growth.     

Quantitative trait loci controlling flowering time and related traits in a Solanum lycopersicum × S. pimpinellifolium cross

Flowering time is an important factor determining early yield in tomato. However, the quantitative trait loci (QTLs) controlling flowering time and their relation to other QTLs for morphological and physiological traits have not been well studied. The aim of this study was to map the chromosomal regions controlling days to flowering (DTF) concurrently with other traits, such as the number of leaves preceding the first inflorescence (LN), length of the largest leaf (LL), number of lateral shoots (LS), fresh weight (FRW) and plant height (PH). This was undertaken using an inbred backcross population derived from a cross between the commercial cultivar Solanum lycopersicum cv. ‘M570018’ and its close wild relative S. pimpinellifolium (PI124039). S. pimpinellifolium flowers earlier than the cultivated tomato. Plants were grown in spring and summer. Composite interval mapping detected 16 QTLs for the six traits evaluated. These QTLs explained 10–42% of the individual phenotypic variation. QTLs detected in spring generally did not differ from those detected in summer. In chromosome 1, the DTF QTL was co-located with the QTLs for LL, LS and FRW, while in chromosome 3 it was co-located with the QTLs for LN, FRW (summer) and PH. One DTF QTL that was detected in chromosome 3 and conferred by the S. pimpinellifolium allele hastens flowering. The co-location of the DTF QTL with the LN QTL suggested that the DTF QTL in chromosome 3 controls the period from the vegetative to reproductive phase. Co-locations of DTF QTLs with the other traits might be pleiotropic effects of a single gene or cluster of genes via physiological relationships among traits because they were found to be highly significantly correlated.     

Changes in carbohydrate contents in shoot tips,leaves and roots of strawberry (Fragaria × ananassa Duch.) during flower-bud differentiation

Flowering is an important step in crop production. Under flower-inducing conditions, biochemical or physiological changes can be recognized. Changes in carbohydrates have an important role in flower development in plants; however, carbohydrate changes during flower bud differentiation in strawberry have not been thoroughly investigated. In this study, runner plants potted in 18 cm diameter pots and grown under non-inducing conditions (28 ± 3/22 ± 3 °C day/night; 16 h day length). When the plants were established, half of the plants then were put under inducing conditions (23 ± 3/13 ± 3 °C day/night; 8 h day length). After the induction period of 21 short-day cycles, plants were brought to non-inductive conditions again. In order to evaluate the carbohydrate changes during flower differentiation, shoot tips, leaves and roots were sampled from four replicated plants collected weekly for the period of 7 weeks. Sucrose, glucose and fructose contents were determined by HPLC, and starch by the anthrone method. The results obtained indicated that the most abundant soluble sugar in all organs tested was sucrose. Sucrose in the shoot tips of induced plants at 42, 56 and 70 days after the start of the short-day treatment were significantly higher than corresponding time in non-induced plants. However, the glucose, fructose and starch contents in shoot tips, leaves and roots of non-induced plants in most sampling dates were greater than those of induced plants. In other words, the shoot tips (bud) of induced plants acted as strong ‘utilizing sink’ and preferentially metabolized carbohydrates rather than storing them. It seems that non-structural carbohydrate contents in shoot tips, leaves and roots of strawberry may have an important role in flower-bud differentiation.     

Rapid seedlings regeneration from seeds and vegetative propagation with sucker and rhizome of Eremospatha macrocarpa (Mann & Wendl.) Wendl and Laccosperma secundiflorum (P. Beauv.) Kuntze

To develop efficient seedling production methods for Laccosperma secundiflorum and Eremospatha macrocarpa, a study was conducted to examine regeneration using offsets combined with several physical and chemical treatments of seeds. Offsets categorized into small, medium and large diameters, were planted in three conditions: shaded and open nursery, and greenhouse. We tested sucker from E. macrocarpa, and sucker and rhizome from L. secundiflorum. For both species, high viability percentage (ranging from 55% to 100%) were observed for small and medium suckers planted in shaded nursery and greenhouse, against less than 49% for sucker planted in open nursery. The mean seedling emergence times were estimated to 84, 77 and 75 days after planting (DAP) for small, medium and large sucker of L. secundiflorum, respectively under open nursery condition, and 76, 75, 95 DAP for small, medium and large suckers of the same species, respectively in shaded condition. Greenhouse has a significant positive effect on E. macrocarpa seedlings emergence time. For this species, the mean seedling emergence times were estimated to 43 DAP for small sucker and 76, 93 DAP for medium and large suckers. No seedling was obtained from rhizome planted in all the growing conditions tested. Concerning seed dormancy breaking, germination percentages and rates were determined for 13 treatments. The best treatments were pre-soaking unscarified seeds for 4 days in 1.01 g l⁻¹ and 0.10 g l⁻¹ KNO₃, with 79% and 68% of germination, respectively and in 3.46 × 10⁻³ g l⁻¹ GA₃ for 68% of germination. These methods are suggested to improve germination of L. secundiflorum seeds. Successful and recommended methods for E. macrocarpa are pre-soaking scarified seeds in 3.46 × 10⁻³ g l⁻¹ and 3.46 × 10⁻⁴ g l⁻¹ GA₃, 96% and 94% of germination, respectively. Dormancy, probably a combination of mechanical and chemical dormancy, is present in the two species.     

Dormancy and germination in Rosa multibracteata Hemsl. & E. H. Wilson

Most of the achenes produced by Rosa multibracteata Hemsl. & E. H Wilson are dormant on maturity and require pretreatment to stimulate germination. To investigate the mechanism of dormancy and to develop effective methods of improving germination, roles of the pericarp, testa, and embryo of R. multibracteata in regulating dormancy were studied by investigating the effect of different pretreatments on germination. The effects of temperature and water stress were also tested with achenes treated by warm plus cold stratification. In freshly harvested achenes, pericarps are permeable and the embryo fully developed, which eliminates the possibility of physical, morphological, or morphophysiological dormancy. Germination percentage remained low (<5%) despite softening the pericarp or even removing it fully. However, fully removing the testa improved germination significantly (39%), indicating the possible presence of germination inhibitors in the testa. Dry storage, scarification with sulphuric acid (H₂SO₄), and warm stratification proved ineffective by themselves but when combined with cold stratification, improved germination and shortened the cold stratification period needed to break dormancy. Dry storage for 68 weeks followed by cold stratification for 16 or 24 weeks resulted in maximum germination (72–79%) among all the treatments. In achenes scarified with H₂SO₄, germination increased with an increase in the duration of cold stratification. Neither gibberellic acid (GA₃) nor ‘smoke water’ (water through which smoke had been bubbled for 2 h) had any positive effect on germination even on seeds that had been mechanically scarified or stratified. Both high temperature and water stress lowered germination in achenes treated with warm plus cold stratification. Our results suggest that R. multibracteata achenes have an intermediate physiological dormancy, and that dry storage for 68 weeks followed by cold stratification for 16 or 24 weeks is the best method for propagating R. multibracteata from seed.     

Relation of carbohydrate reserves with the forthcoming crop,flower formation and photosynthetic rate,in the alternate bearing ‘Salustiana’ sweet orange (Citrus sinensis L.)

The aim of this work was to assess the relation between carbohydrate levels and flower and fruit production, as well as the role of carbohydrates on CO₂ fixation activity, by analysis of leaves, twigs and roots from the alternate bearing ‘Salustiana’ cultivar of sweet orange (Citrus sinensis [L.] Osbeck). A heavy crop load (on year) did not affect photosynthesis activity when compared to non-fruiting trees (off year). Fruiting trees accumulated most of the fixed carbon in mature fruits, whilst no accumulation was observed in roots before harvest. Non-fruiting trees transported part of the fixed carbon to the roots and mobilize it for growth processes and, at the end of the season (December), store it as reserves. Reserve carbohydrates accumulation in leaves started by early December for both tree types, showing the same levels in on and off trees until spring bud sprouting. A heavy flowering after an off year caused the rapid mobilization of the stored reserves, which were exhausted at full bloom. We found no evidence on carbon fixation regulation by either fruit demand or carbohydrate levels in leaves. Carbohydrate reserves played little or no role over fruit set, which actually relied on current photosynthesis.     

Comparative analysis of laboratory freezing methods to establish cold tolerance of detached rhizomes and intact crowns in garden chrysanthemums (Dendranthema × grandiflora Tzvelv.)

Since 1926, the University of Minnesota herbaceous perennial breeding program has released N = 84 garden chrysanthemum cultivars (Dendranthema × grandiflora) with important traits for northern temperate climates, such as winter hardiness. Recent breeding objectives have identified the need for development of non-destructive phenotypic markers and destructive laboratory freezing tests for co-selection of cold tolerance in Dendranthema, Gaura, and other herbaceous perennial flower crops. Such methods have become critical to flower breeding programs in northern temperate regions during periods of above-average winter temperatures and minimal snow cover due to the ‘el Niño’ effect. Two different, destructive laboratory freezing tests were evaluated for their effectiveness in determining cold tolerance. Acclimated crowns of n = 6 garden chrysanthemum genotypes, ranging from hardy to non-hardy in USDA Z3-4, were used in Omega Block (using detached, emergent rhizomes) and chamber (using entire, intact crowns with emergent, non-emergent rhizomes) freezing test methods. Comparative winter survival in the field was monitored over locations and years. Cold tolerance was assessed at 0 to −12 °C with varying ramp and soak time periods. LT₅₀ temperatures and number of living emergent rhizomes were also determined. Rhizome quality at 1, 3, and 5 cm depths was rated for regrowth on a 0 (dead) to 5 (undamaged) scale. The chamber freezing method was the most powerful to discern accurate LT₅₀ values. Cold tolerant genotypes included ‘Duluth’ and Mn. Selection 98-89-7 (LT₅₀ = −12 °C). Four genotypes were rated as non-hardy (LT₅₀ = ≤−10 °C). Cold-tolerant genotypes also had significantly higher regrowth ratings for rhizomes at 1 and 3 cm depths. Future research will implement the chamber freezing method to assay the inheritance of winter hardiness in intact crowns of segregating populations.     

The effect of gibberellic acid (GA3) on some phenolic substances in globe artichoke (Cynara cardunculus var. scolymus (L.) Fiori)

Globe artichoke has important nutritional values related to its high content of phenolic compounds such as cynarin and chlorogenic acid. Cynarin (1,5-dicaffeoylquinic acid) is a derivative of caffeic acid and has effect on hepatobiliary diseases, hyperlipidaemia, dropsy, rheumatism and cholesterol metabolism. The effect of gibberellic acid (GA₃) on the yield of the phenolics, chlorogenic acid and cynarin, both in leaves and in the edible part of the head of globe artichoke, were studied. Plant samples were collected for chemical analyses at the laboratories of the Chair of Vegetable Science, Department for Plant Sciences, Technische Universität München in Freising-Weihenstephan, Germany. GA₃ at 60 ppm was applied either at 4, 6 or 8 weeks after transplanting date (ATD). When transplant were used, application of GA₃ at 4 weeks ATD significantly increased the content of chlorogenic acid in leaves while the level of cynarin was similar to the untreated control. In contrast when plants were direct seeded into the field, GA₃ treatments had no affect on phenolics in leaves compared to the untreated control. Whereas GA₃ reduced the time to flowering, it did not increase the content of chlorogenic acid or cynarin in leaves or in most of the flower receptacles, except when GA₃ was applied at 6 or 8 weeks. On the other hand, cynarin yield in the 3rd–5th harvested flower heads was higher than controls. The timing of GA₃ application is, thus, critical for decreasing time to flower as well as altering the content of cynarin and chlorogenic acid in globe artichoke.