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.     

Combining endophytic fungi and bacteria for the biocontrol of Radopholus similis (Cobb) Thorne and for effects on plant growth

Eight endophytic fungal and bacterial isolates with antagonistic activity against Radopholus similis were evaluated in vivo for their individual and combined effects on biocontrol of R. similis and on the growth of “Grand Naine” cultivar banana plantlets in the greenhouse. Penetration efficiency (PE) of R. similis was between 3 and 21% in 29 biological agents (BAs) treatments, less than the 29% of the nematode-alone control (p ≤ 0.0001); 24 of the BAs treatments did not differ from the PE of 5% for a nematicide control. Twenty nine BAs treatments exhibited antagonistic activity against nematodes which reduced final population levels between 18 and 93%, relative to those on nematode-alone control plants (p ≤ 0.0001), and 14 BAs treatments were statistically similar to the nematicide treatment (88% reduction). Twenty four BAs treatments had increments of plant root biomass ranging from 20 to 58%, greater than the control plants; 37% of the treatments with single and combined BAs inoculations had root length increments ranging from 29 to 54% compared with control and chemical treatment. The nematicide, Terbufos 10GR, did not affect plant growth.     

Phenological,morphological and functional indicators of genetic variability and their implication in the sexual reproductive system of Olea europaea L. (Oleaceae)

The floral biology of ‘Koroneiki’, ‘Kalamata’ and ‘Mastoidis’ was studied for three consecutive years to elucidate the potential physiological and genetic controls of inflorescence architecture and phenology and to identify potential genotype-distinguishing characters that could be employed for morphological cultivar discrimination. The first open flowers were mainly observed at position I for ‘Koroneiki’ (83%) while they were mainly located at position II for ‘Mastoidis’ (40%) and ‘Kalamata’ (63%). The last open flowers were principally located at the apical position for ‘Koroneiki’ (46%) and ‘Kalamata’ (68%) and at position II for ‘Mastoidis’ (75%). Most of the first open flowers were perfect for ‘Koroneiki’ (89%) and ‘Mastoidis’ (97%) while lower percentages were observed in the last open flowers (67% and 80%, respectively). Higher percentages of perfect flowers were observed in ‘Mastoidis’ compared to ‘Koroneiki’ in both cases. The average flowering shoots of ‘Kalamata’ produced significantly more flowers (1523/m) compared to ‘Koroneiki’ (1139/m) and ‘Mastoidis’ (1044/m). The half of the flowers in ‘Mastoidis’ were located at position I while the 38% was located at position II and only the 12% at the apex. By contrast, approximately the 80% of flowers in ‘Koroneiki’ and ‘Kalamata’ panicles was equally distributed at positions I and II, while the 18% and 19%, respectively, was located at the apex. A marked relationship was also observed between flower position in the panicle and flower gender.     

Micropropagation of Dendrobium draconis Rchb. f. from thin cross-section culture

An efficient procedure is outlined for rapid and mass in vitro propagation of an orchid, Dendrobium draconis Rchb. f. through in vitro culture of thin cross-sections (TCSs) derived from young stems. The TCS explants were excised along the stem from the base to shoot tip of 6-month-old plantlets and cultured on Murashige and Skoog (MS) medium supplemented with 20 g/l sucrose and different concentrations of N⁶-benzyladenine (BA), kinetin (Kn) and 1-naphthaleneacetic acid (NAA), either individually or in combination. Protocorm-like bodies (PLBs) were directly induced from the TCS explants and completely developed into shoots within 6–7 weeks. The optimal growth regulators combination for maximal PLB development was 2 mg/l BA and 1.0 mg/l NAA, giving rise to 68% of responding explants with an average 11 PLBs per explant. Shoot development was best achieved on MS medium containing sucrose and coconut water. Plantlets, 6–8 cm height were transplanted into coconut husk peat with 92% survival rate in a nursery.     

Fruit and seed diversity of domesticated carob (Ceratonia siliqua L.) in Morocco

The carob tree (Ceratonia siliqua L.) is an important economic resource for Morocco's rural populations. This species is used in reforestation actions and its cultivation in modern orchards is being undertaken to valorize marginal lands and substitute for drought sensitive species. However, little data is available on its intra-specific variability and its adaptability. Morphological characters of pods and seeds from 13 ecoregions of private-domesticated carob were used to assess phenotypic variation of this species. These stands extend from south-west to north-east and cover a wide range of Morocco's ecoregions. Pods length, width, thickness, seeds number, pulp weight, seeds yield and seeds length, width, thickness and weight were measured for 390 trees (30 trees per ecoregion). Statistically significant differences were found between ecoregions for all characters which were examined, what indicates a high phenotypic diversity. Principal component analysis (PCA) and hierarchical cluster of all ecoregions lead to identify two major and opposite groups (the northern ecoregions; and the central and south-western ecoregions). Ecoregions of the north of Morocco exhibited the largest and the thickest pods with the highest pulp weight while other ecoregions have relatively short pods but largest proportion of seed yield. Similarly, the northern ecoregions are characterized by the heaviest seeds. A correlation matrix between morphological characters, geographic parameters and precipitation exhibits a positive and a negative correlation of pods thickness and pulp weight with the latitude and the altitude, respectively. Seed yield and weight are negatively and positively correlated to pod width, pod thickness and pulp weight, respectively. In addition, seed weight is positively correlated with the latitude. The geographic pattern of the carob tree and its variability are discussed in this paper.     

Fruit weight is related to ovary weight in olive (Olea europaea L.)

Fruit size is an important parameter both for scientific understanding and for commercial purposes. In many species, mature fruit size is often related to floral ovary size, but no literature exists in olive that demonstrates such a relationship. Previous work suggests that olive cultivars with different fruit sizes have similar cell number and size in the ovary transectional area, but ovary and fruit dry weight was not measured. In the present study, ovary dry weight and fruit dry weight during the whole fruit development season until harvest were measured in olive cultivars with different fruit size, over three years. Flower dry weight was also measured. Fruit weight at harvest was strongly correlated to ovary weight at bloom, both in single-year data and when data from three years were pooled. Flower dry weight, excluding the ovary, was also correlated to ovary dry weight. Ovary dry weight was strongly correlated not only to the fruit dry weight at maturity, but also at any date during fruit development. The mature fruit/ovary dry weight ratio ranged between 1000 and 4000 among cultivars, but was not correlated to the fruit dry weight at maturity. These results suggest that, in olive, fruit weight is genetically controlled through the ovary weight at bloom. This knowledge may have implications in the understanding of fruit set and source-sink relationships in olive.     

Effects of thermoperiodicity and plant population density on stem and flower elongation, leaf development, and specific fresh weight in single stemmed rose (Rosa hybrida L.) plants

Thermoperiodicity, i.e. growth in the alternating temperature regime with the same diurnal mean compared with growth at the constant temperature at which optimal growth occurs, was studied at three plant population densities in four cultivars of Rosa hybrida L. Single-node cuttings with five-leaflet leaves were excised and grown as single-stemmed rose plants at an average photosynthetic photon flux density of about 260 μmol m⁻² s⁻¹ and supplied with carbon dioxide at about 1000 μmol mol⁻¹. The optimal constant temperature regime was 22 °C day (20 h)/22 °C night (4 h); alternating temperatures were 23 °C day (20 h)/18 °C night (4 h). The plant population densities were 100, 131 and 178 plants m⁻² of bench area. Thermoperiodicity was absent, or could not be detected, in the parameters related to the growth period, the formation of fresh biomass, the bloom quality, and most parameters related to shoot elongation. However, classic thermoperiodic effects of alternating regime were significant in the cultivars Red Velvet and Sonia, with shoot elongation promoted (7.1 and 10.5%, respectively) in the growth phase from onset of axillary bud growth until the flower bud became visible. Compared with the other two cultivars, plants of Red Velvet and Sonia tended to develop longer internodes. The results, obtained concurrently at three different plant population densities, suggest that thermoperiodicity can affect (single-stemmed) plant growth and development in R. hybrida. Increased plant population density also increased plant height at visible flower bud, but the bloom quality, expressed as specific fresh weight, and the flower height at anthesis was decreased at the highest density. Increased plant population density increased the number of five-leaflet leaves developed in Red Velvet, but had no effect on leaf number in Texas and Sonia, while, in Lambada the leaf number was decreased at the highest density.     

Establishment of in vitro tissue cultures from Echinacea angustifolia D.C. adult plants for the production of phytochemical compounds

The establishment of in vitro cultures of Echinacea angustifolia D.C. was obtained directly from sections of flower stalks of adult plants. The shoot formation was obtained from this plant material placed on a modified MS basal medium named CH supplemented with 0.5 mg L⁻¹ 6-benzylaminopurine (BA). The in vitro propagation procedure of E. angustifolia consisted of three distinct phases: an initial regeneration phase from stalk sections (IP shoots on basal medium with 0.25 mg L⁻¹ BA), an elongation phase on active charcoal and an axillary proliferation of the shoots (AP shoots on basal medium with 0.5 mg L⁻¹ BA).Regenerating calli were established from leaves of in vitro shoots cultured on CH medium supplemented with 3 mg L⁻¹ BA and 0.5 mg L⁻¹ indole-3-butyric acid (IBA). Developed shoots from the callus cultures were subcultured on the CH medium with 0.5 mg L⁻¹ BA (leaf regenerated shoots: LR shoots). The secondary metabolite content of the in vitro plant material was compared with that of the greenhouse growing plants. The quali-quantitative LC-DAD-ESI-MS analysis on the extracts from axillary proliferation shoots (AP shoots) showed significant production of caffeic acid derivatives while leaf callus and LR shoots, accumulated mainly alkamides. These results showed that the proper choice of the procedures for in vitro multiplication allowed us to obtain plant biomass able to produce the active compounds typical of E. angustifolia plants.     

Transformation of Brassica oleracea var. capitata with bacterial betA gene enhances tolerance to salt stress

The bacterial betA gene for biosynthesis of glycinebetaine was transferred to cabbage (Brassica oleracea var. capitata) cultivar ‘Golden Acre’ through Agrobacterium-mediated transformation of hypocotyl explants. Transgenic status was established through Southern hybridization and mRNA expression in the shoots. The transformants exhibited higher tolerance to NaCl stress compared to untransformed parent plants. In physiological assessment of salinity tolerance, transgenics showed better growth response and greater stability in maintaining plant water relations at increasing levels of salinity. These results demonstrate that engineering glycinebetaine biosynthetic pathway into cabbage can lead to enhanced salt tolerance.     

In vitro induction of the amphiploid in interspecific hybrid of blueberry (Vaccinium corymbosum × Vaccinium ashei) with colchicine treatment

Amphiploids were produced from pentaploid hybrids between Vaccinium corymbosum (4x) and Vaccinium ashei (6x) by colchicine treatment. Seeds of each parental species and those obtained by the interspecific crossing were treated with colchicine at 0, 500, 1000 and 2000 mg/l for 7 days, and the ploidy level of the seedlings was determined with flow cytometry. Either amphiploid (10x) or ploidy chimera (5x + 10x) was obtained from interspecific hybrid seeds treated with all colchicine concentrations, while no chromosome-doubled plants were obtained from both parents. Cross direction in interspecific hybridization affected the results of colchicine treatment and amphiploids were obtained only when V. corymbosum was used as the seed parent. In this cross, 17–25% of the seedlings turned to be amphidiploids or ploidy chimeras by treating with 500 mg/l colchicines for 7 days. These results indicate that susceptibility to colchicine may increase in the interspecific hybrid compared with the parental species but only when V. corymbosum was used as female parent.     

Role of bioinoculants in development of salt-resistant saplings of Morus alba (var. sujanpuri) in vivo

Pot experiments were conducted for the development of salt-resistant saplings of Morus alba (var. sujanpuri) involving bioinoculants, namely arbuscular mycorrhizal (AM) fungi, Azotobacter and indole butyric acid (IBA). The IBA and sodium chloride (NaCl) concentrations were optimized prior to the experiments. By using both low and high concentrations, 15 ppm IBA and 0.05% NaCl (w/v) were found to be optimum in acting synergistically with AM fungi and Azotobacter and also for increasing all the growth parameters and microbial count in the rhizosphere. For in vivo development of salt-resistant saplings, the optimal concentration of IBA, along with AM fungi and Azotobacter in different combinations, was applied. The saplings were irrigated regularly with 0.05% NaCl water. Although growth parameters such as AM infection percentage, AM spores per 100 g soil and Azotobacter cells/g soil were affected by NaCl watering, the inoculation of both bioinoculants significantly enhanced survival percentage of saplings from 25 to 50% under salt stress. Maximum survival (55%) of saplings was found with IBA (15 ppm)+AM fungi+Azotobacter.     

In vitro micropropagation of the ornamental prickly pear cactus Opuntia lanigera Salm–Dyck and effects of sprayed GA3 after transplantation to ex vitro conditions

We established the conditions to micropropagate the ornamental prickly pear cactus Opuntia lanigera Salm–Dyck through axillary shoot development from isolated areoles. For the shoot proliferation stage different explant orientation (vertical and horizontal), type of cytokinin (BA, DAP and K), and concentrations (0, 1.25, 2,5, 5.0 and 7.5 mg/L) were evaluated. Media [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco cultures. Phys. Plant. 15, 473–497: 50 and 100%], and carbohydrate concentration (0.25, 0.5, 0.75 and 1.0%) were studied to optimize individual shoot growth and elongation. Following micropropagation and plantlet acclimatization, the effects of GA₃ on plant growth were determined by spraying a series of increasing concentrations (0, 150, 300 and 450 ppm). A reliable and efficient protocol of micropropagation was established for this particular plant species. The greatest propagation ratio (shoot proliferation) was obtained when explants were cultured in vertical orientation (4.975 shoots per explant) as compared to horizontal position (3.692 shoots per explant). The addition of BA to the media resulted in increased shoot number per explant (8) in comparison to K and DA, which produced only 2 shoots in average. However, after 42 days of culture, significantly higher shoot length was obtained with DAP (14 mm) compared to K and BA (4 mm). After the shoot proliferation stage, an elongation subculture was performed prior rooting in which shoot growth was enhanced when crowns of shoots were cultured in 50% of basal salt formulation of Murashige and Skoog (1962) and low sucrose concentration (2.5 and 5%). Exogenous application of GA₃ after plantlet acclimatization on glasshouse conditions increased spine-hair (developed from areoles in young plants) length as part of short-term effects. However, significantly higher values were obtained in plantlets treated with 300 ppm of GA₃ when compared with the rest of the treatments. At the end of the study, the most important long-term effect produced by GA₃ was the suppression of total shoot growth. The micropropagation protocol described here and the conditions to grow the plants through fertigation plus the application of GA₃ that induced changes in the phenotype may be used in commercial exploitations to regenerate 12,500 plantelts in average after 12 months of culture and produce healthy plants with better ornamental characteristics and higher commercial value.     

Analysis of genetic diversity in Portuguese Ceratonia siliqua L. cultivars using RAPD and AFLP markers

Although carob (Ceratonia siliqua L.) is of great economic importance little is still known about the pattern of genetic variation within this species. Morphological characteristics based on 31 fruit and seeds of continuous characters determinant for agro-industrial uses, were compared with RAPD and AFLP markers for assessing genetic distances in 68 accessions of carob trees, from different cultivars, varieties and eco-geographic regions of Algarve. Eighteen selected RAPD primers applied to the 68 accessions produced a total of 235 fragments ranging from 200 to 2000 bp, of which 93 (40%) were polymorphic. Four AFLP selective primer combinations generated a total of 346 amplification fragments of which 110 were polymorphic. The average level of polymorphism based on four primer combinations was 31.8%. The phenetic trees based on RAPD and AFLP analyses gave high co-phenetic correlation values, and were found to be consistent in general with the analysis of morphological data, carried out on the same accessions. A number of RAPD and AFLP markers were found to be diagnostic for ‘Canela’ cultivar and 13 wild ungrafted trees.     

Interactions of photoperiod,temperature, duration of short-day treatment and plant age on flowering of Fragaria x ananassa Duch. cv. Korona

The effects of photoperiod (10, 12, 16, 20 or 24 h), day-temperature (12, 15, 18, 24 or 30 °C), the number of short days (14, 21 or 28 days), plant age (4, 8 or 12 weeks) and their interactions on flower and inflorescence emergence were investigated in strawberry cv. Korona. No flowers emerged in plants exposed to photoperiods of 16, 20 or 24 h or to a short-day treatment for 14 days. All plants exposed to short days at daily photoperiods of 10 or 12 h for 21 days or longer, emerged flowers at temperatures between 12 and 18 °C. A further increase in temperature led to a drastic decrease in the total number of flowers per plant. A short-day treatment (10 or 12 h photoperiod) of 28 days resulted in highest numbers of inflorescences and flowers per plant, while a short-day treatment of 21 days resulted in the highest numbers of flowers per inflorescence. Complete flower induction was observed in only 4-week-old runner plants. The number of inflorescences and the number of flowers per inflorescence increased with plant age. However, the start of flowering was delayed with increasing plant age.     

Potential hazard characteristics of Tilia, Betula, and Acer trees removed in the Helsinki City Area during 2001–2003

In order to study decay, and to improve the management and protection of old urban trees, a total of 256 felled urban trees were examined during 2001–2003: 95 Tilia spp., 74 Betula spp., and 87 Acer spp. Most of the trees (73%) were located in the main parks and along the main streets in the downtown area of Helsinki City, Finland. The mean age of the trees was over 60 years, and the majority (64%) were old park trees. Poor condition and increasing risk of failure were the main reasons for felling in 82% of the cases. Thirty three percent of these trees were degenerated or dead, but the amenity value of 14% of the risk trees was still high. The latter were old, big trees which posed a potential hazard, but had a vital and balanced crown.Some characteristic profiles for potential failure were identified for each of the tree species studied: Ganoderma lipsiense in the butts and hollows in the stems of Tilia spp., weak fork formations together with Rigidoporus populinus on Acer spp., and degeneration together with decay in the stem on Betula spp.Decay fungi most commonly identified were R. populinus, G. lipsiense, Inonotus obliquus and Piptoporus betulinus. In addition, Kretzschmaria deusta was very common in three of the parks, and on every one of the tree species investigated.     

Development of a male sterile eggplant by utilizing the cytoplasm of Solanum virginianum and a biparental transmission of chloroplast DNA in backcrossing

To develop a male sterile line of eggplant (Solanum melongena L.) utilizing the cytoplasm of S. virginianum, an interspecific F₁ hybrid between S. virginianum and S. melongena ‘Senryo Nigou’ was continuously backcrossed to S. melongena ‘Uttara’ using ‘Uttara’ as a recurrent pollen parent and four backcross generations, BC₁, BC₂, BC₃ and BC₄ were produced. All the plants in backcross generations were anther indehiscent although the F₁ hybrid, S. virginianum and S. melongena were dehiscent. Pollen stainability with acetic carmine and in vitro germination ability of pollen in all the backcross progenies were quite lower than those of the parental species. Fruit set percentage, number of seeds per fruit and seed germination rate were high in all the backcross progenies. The present results indicate that anther indehiscent type of functional male sterile line of eggplant could be developed by utilizing the cytoplasm of S. virginianum. PCR-RFLP analysis of chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA) were performed to identify the organelle inheritance. The F₁ hybrid and all the backcross progenies displayed maternal inheritance of mtDNA. The cpDNA of the examined single BC₁ plant exhibited the recombinant cpDNA pattern of the parental F₁ hybrid and ‘Uttara’, indicating occurrence of the biparental inheritance of cpDNA. As all the BC₂, BC₃ and BC₄ progenies showed the same recombinant cpDNA patterns of the BC₁ plant, the recombinant cpDNA might be stable and harmonize with the nuclear genome of S. melongena. The present male sterile line can contribute to expand the male sterility source of eggplant.     

Immunolocalization and quantification of IAA after self- and free-pollination in Olea europaea L.

In order to characterise the self-compatibility in ‘Pisa 9’, that is an improved clone of cultivar Leccino (Olea europaea L.), biological, histological and biochemical analyses were carried out. Observations were also focused on ‘Pendolino’, the main pollinator for Italian cultivars considered as being self-incompatible. After self-pollination, in ‘Pisa 9’ the majority of the pollen tubes reached the distal part of the style, entered the ovule leading to a high fruit-set percentage. These same processes were also noted in the Pendolino cultivar, though in a lesser degree. The indole acetic acid (IAA) concentration and its immunocytochemical localization were determined in pollinated flowers after self- and free-pollination and in unpollinated flowers. In both cultivars the highest IAA levels and signal intensity were observed in free-pollinated pistils, whereas after self-pollination a different behaviour emerged. In ‘Pendolino’ the immunostaining was not uniformly distributed and stopped at the style level. This finding sustained the low percentage of fruit-set obtained in the cultivar. In ‘Pisa 9’ the auxin distribution was similar to the free-pollinated pistils where the immunolocalization staining reached the ovary. The correspondence between the IAA signal in the ovary and the high percentage of fruit-set in ‘Pisa 9’ self-pollinated pistils, confirms the role of this hormone in the induction and regulation of the fertilisation process.     

Efficient production of transgenic plants using the bar gene for herbicide resistance in sweetpotato

Efficient production of transgenic sweetpotato (Ipomoea batatas (L.) Lam.) plants using the bar gene for herbicide resistance was achieved through the use of embryogenic suspension cultures and Agrobacterium tumefaciens-mediated transformation. Cell aggregates from embryogenic suspension cultures of sweetpotato cv. Lizixiang were cocultivated with A. tumefaciens strain EHA 105 harboring a binary vector pCAMBIA3300 with the bar gene and uidA gene. Selection culture was conducted using 0.5 mg/l PPT. A total of 1431 plants were produced from the inoculated 870 cell aggregates via somatic embryogenesis. GUS assay and PCR analysis of the regenerated plants randomly sampled showed that 86.5% of the regenerated plants were transgenic plants. Stable integration of the bar gene into the genome of transgenic plants was confirmed by Southern blot analysis and transgene expression was demonstrated by Northern blot analysis. The copy number of integrated bar gene ranged from 1 to 3. Transgenic plants exhibited functional expression of the bar gene by in vivo assay for herbicide resistance. This study also provides a simple and efficient transformation system of sweetpotato based on the use of bar gene as a selectable marker gene, which can be combined with other agronomically important genes for the improvement of sweetpotato.