Genetic characterisation of oak seedlings, epicormic, crown and micropropagated shoots from mature trees by RAPD and microsatellite PCR

DNA was isolated from seedlings of Quercus robur, collected from a single provenance, and from epicormic, crown shoots and in vitro shoots from a single tree of Q. petraea using a CTAB method of extraction. DNA was obtained in sufficient quantity and purity, from 13 out of 30 seedlings, and from all isolations from epicormic and in vitro shoots (2.5–10.0 μg/g fresh/ weight). Smearing was minimised at a primer concentration of 0.12 μM with Taq polymerase at 0.5 unit/reaction. Nine primers produced 142 bands, 28 of which were polymorphic. A similarity index showed that 11 seedlings were closely related with high coefficients (0.85–0.90), but each could be identified from another using only 9 primers (OPA-02 and -05, OPG-04 and -05, OPE-01, -02, -03, -08, -09). DNA was isolated from crown, epicormic and in vitro leaves originating from a single 150-yr old tree of Q. petraea and analysed by randomly amplified polymorphic DNA (RAPD) and microsatellites. With each primer, a characteristic RAPD pattern was obtained, and it was common to all six epicormic shoots derived from different parts of a single branch of this tree; also to the shoots from the crown of the same tree with OPE1 OPA-05, OPA-08, OPA-01, OPA-02, OPA-04, OPA-05, OPG-02, OPG-10, OPE-12. Similarly, the RAPD pattern obtained from shoot cultures in vitro, derived from individual nodes of epicormic shoots produced by six different branch segments, were uniform for each of 15 primers. This work was repeated using microsatellite PCR. Three microsatellite loci AG16, AG 1/2 and AG 1/5 were amplified by PCR. It showed a uniformity of these microsatellite loci in shoots from the crown of the tree, and from epicormic shoots cultures derived from six different sections of branch.     

Effect of explants source on shoot proliferation and adventitious regeneration in 10 Buddleia cultivars

Viable shoot cultures and weaned plants were obtained from cultured apical meristems with 10 Buddleia cultivars giving viabilities of 32–72%. The number of shoots produced, the micropropagation rate and the root number produced in vitro was higher in meristem derived shoots compared to those derived from shoot-tips. The subsequent growth rate of meristem derived plants, in the greenhouse, was also greater. The number of roots produced by conventional cuttings collected from meristem derived plants was significantly higher than in cuttings which were collected from plants derived from shoot-tips or from the original stock plants.Endogenous bacteria were not detected in either shoot cultures derived from meristems or in 10-week-old weaned plants derived from meristems whereas those derived from shoot-tips showed the presence of endogenous bacteria when sterilized explants were cultured on nutrient agar or on tryptic soy broth.Factors affecting adventitious bud and shoot production in leaf and internode explants was determined for ‘Lochinch’, ‘Border Beauty’, ‘Ile de France’ and ‘Pink Delight’ using meristem derived shoot cultures. Adventitious shoots appeared after 4 weeks of culture, in both types of explant when cultured on MS supplemented with 0.5–5.0 μM TDZ. The highest percentage regeneration was achieved from bisected internode explants cultured on 0.5 μM TDZ, with 93–100% regeneration among the cultivars whereas BA was less effective. The best response was obtained using 5.0 μM TDZ which gave over 10–11 shoots per explant in all bisected explants for all cultivars.     

Production of virus-free plantlets by anther culture of LiliumבEnchantment’

Anthers of the LiliumבEnchantment’, excised at the uninucleate microspore stage, were cultured on MS media containing 6% sucrose with auxin and cytokinin. When anthers were cultured on the medium with 2 mg l⁻¹ picloram and zeatin, 12–86% of them formed nodular calli. Anthers excised from greenhouse- and field-grown plants showed different responses: anthers of greenhouse-grown plants had a significantly higher capability to form callus and regenerate bulblets than those of field-grown plants. In anthers from greenhouse-grown plants, bulblet formation was dependent on the time at which anthers were excised from donor plants: anthers collected from early forced mother-plants had a higher capability of forming bulblet than others. All regenerated plantlets were diploid, which was substantiated by histological observation showing that the anther-derived calli originated from anther wall tissues. Virus tests by ELISA were made for 49 plantlets selected randomly at transplanting: 20 plantlets (41%) were virus-free, and the rest showed positive reactions for lily symptomless virus, cucumber mosaic virus and/or tulip breaking virus.     

Plant regeneration from protoplasts of calamondin (Citrus madurensis Lour.)

Diploid callus cells arose from the hypocotyl region of plantlets differentiated by anther culture of Calamondin (Citrus madurensis Lour.). The callus grew vigorously on Murashige and Tucker's medium containing 10 mg l⁻¹ benzyladenine, and differentiated embryoids on a medium with 5% lactose and no plant growth regulators. This embryogenic potential was transferred to protoplasts which regenerated plantlets.

Preculture of callus in liquid medium containing 5% lactose, for 4–7 days prior to protoplast isolation, effected a high yield of protoplasts. Plating efficiency was increased in a medium solidified with agarose and/or layered with liquid medium.     

The growth and flowering of Hymenocallis × festalis

Bulbs of Hymenocallis (including Ismene) have showy, fragrant flowers. Little is known of the horticultural potential of these plants, and observations and trials on a stock of Hymenocallis × festalis are described.

In a stock of glasshouse-raised bulbs, bulb grade exerted a marked effect of the number of florets produced, which increased from 2 in 9–10-cm-circumference bulbs, to 7 in 18–19-cm-grade bulbs. Field-raised bulbs of the same grades produced fewer florets. Bulbs were usually planted for flower production in the glasshouse in April; earlier planting (February) leading to greater floret size but a longer period in the glasshouse before anthesis. Planting could be delayed at least until June, or later flowering could be achieved by storing the dry bulbs over winter at a low temperature (5°C), but the latter treatment reduced the percentage of bulbs which flowered. The long scapes could be dwarfed by ethephon (as Ethrel). Hot-water treatment, as a pest and disease control measure, did not result in damage to the flowers provided it was delayed until after the staminal cup initial had been formed.

In the field, growth of the main bulb was more vigorous than in the glasshouse, but pot culture in the glasshouse led to copious offset production. Data are presented for bulb increases for various grades of bulbs planted in outdoor beds at rates of 350–1050 g per metre row.     

Apical shoot tip culture of tomato

We studied the effect of phytohormones on growth and development of apical shoot tip of tomato (Lycopersicon esculentum Mill.). Whole plants rarely developed on basal medium containing NAA (-naphthylacetic acid). K (kinetin) and BAP (6-benzylamino purine) stimulated shoot development within the range of 0.1–10 μM. BAP at 5 and 10 μM induced shoot multiplication. The clonal progenies were phenotypically uniform and without chromosomal changes or meiotic irregularities. The potential uses for propagating genetically valuable tomato lines under in vitro conditions are discussed.     

Physico-chemical and antioxidant properties of cornelian cherry fruits (Cornus mas L.) grown in Turkey

Cornus mas L. is a naturally growing dogwood species in Anatolia. In present study, physical, chemical and antioxidant properties of cornelian cherry fruits were studied. The fruit weight was in the range of 0.39–1.03 g, fruit length 14.24–22.20 mm, fruit width 9.59–13.21 mm, flesh/seed ratio 1.34 to 6.72. Hunter L values of the samples ranged between 10.82 and 19.69, and a value was between +6.25 and +15.59, and b value was between +3.46 and +6.64. In addition to the levels of dry matter, soluble solids, pH, total acidity, total sugar content, reduced sugar content, unreduced sugar content, ascorbic acid, total anthocyanin and total phenolics were within the range of 15.88–28.19%, 12.50–21.00%, 3.11–3.53, 1.10–2.53%, 76.80–154.00 g kg⁻¹, 52.80–120.00 g kg⁻¹, 0.00–32.30 g kg⁻¹, 0.16–0.88 mg g⁻¹, 1.12–2.92 mg g⁻¹ and 2.81–5.79 mg g⁻¹, respectively. On the other hand, ferric reducing antioxidant power (FRAP) and EC₅₀ values were between 16.21 mmol g⁻¹ and 94.43 mmol g⁻¹, 0.29–0.69 mg mL⁻¹. Anthocyanin extracts of the fruits were analysed by high-performance liquid chromatography (HPLC) with UV–vis detection. Pelargonidin 3-glucoside was the main pigment found in cornelian cherry fruits.     

Flowering influences adventitious root formation in chrysanthemum cuttings

Root formation in chrysanthemum (Dendranthemum grandiflora (Ramat.) Kitamura) cuttings was reduced as flowers developed on stock plants. This effect was shown for all ten cultivars evaluated in this study. Not all cultivars were affected equally by the presence of flower buds on cuttings. There was no relationship (r² = 0.06) between root formation in vegetative cuttings and the ability for a cultivar to root from flowering cuttings. IBA (1 mM) could partially overcome the negative effect of flowering on root formation, but cuttings taken after the flower buds had fully opened failed to root even after auxin treatment. Removing buds from cuttings or continually removing flower buds during stock plant growth reduced rooting compared to cuttings with flower buds intact. Furthermore, cuttings taken from the top three nodes of the stock plant containing flower buds rooted comparably to cuttings taken from the lower stem section that contained only vegetative buds. The negative influence of flowering on root formation appears to be due to the photoperiodic induction of the flowering stimulus rather than a direct competition for resources between flowers and developing roots.     

In vitro propagation of Symphytum species

Methods are described for the in vitro propagation of Symphytum × uplandicum Nyman from bud, root and stem explants. Highest shoot numbers were produced from root explants > 4 mm in diameter, cultured vertically with their distal cut surface on the medium. The most suitable medium for shoot production was Murashige and Skoog's (MS) with 0.3 mg l⁻¹ 6-benzylaminopurine (BAP). These shoots developed roots on MS medium without hormones and were successfully transplanted into pots. Subculturing shoots onto MS medium containing BAP, kinetin (K), 6-λ,λ-(dimethylallylamino)-purine or gibberellic acid (GA₃) at 0.5, 1.0, 5.0, 10.0 or 30.0 mg l⁻¹ failed to stimulate outgrowth of axillary buds in culture. In vitro propagation from root explants was also achieved with S. asperum Lepech., S. officinale L. and S. × uplandicum cultivars ‘Bocking 1’, ‘Bocking 2’, ‘Bocking 4’ and ‘Bocking 17’, but not with S. bohemicum, S. grandiflorum DC, S. tuberosum L. or S. × uplandicum ‘Bocking 7’ and ‘Variegatum’.     

Optimizing the lighting regime for Alstroemeria with respect to photoperiod and fluence rates

The Alstroemeria cultivars Diamond, King Cardinal and Libelle were grown for 18 months under five lighting regimes with, and without, soil cooling. The aim was to optimize the daily investment of light energy from artificial sources with respect to photoperiod and photosynthetic fluence rates and to elucidate possible links between reactions to photoperiod and root-zone temperature. The more photons (photosynthetically active radiation, PAR) that were supplied to the plants per day (8, 11 and 13 mol m⁻²), the higher was the total production of flowering stems. The total yield from regimes with 13 mol m⁻² day⁻¹ was higher when the light was spread over 20 and 16 h compared to 12 h. In treatments with soil cooling, the plants flowered continuously under all combinations of photoperiods and photosynthetic fluence rates, and the summer and autumn recession in flower production that occurred for non-cooled ‘King Cardinal' and ‘Diamond' was the same under all lighting regimes. It is concluded that it might be more cost-effective to spread the daily investment of light over 20 rather than 16 or 12 h when the total energy budget and CO₂ costs are taken into consideration.     

Effects of CO2, temperature and their interaction on the growth, development and yield of cauliflower (Brassica oleracea L. botrytis)

Stands of summer cauliflower were grown within polyethylene-covered tunnels along which a temperature gradient was imposed. Two tunnels were maintained at either normal or elevated CO₂ concentrations. At the last harvest (88 days from transplanting) no interaction between CO₂ and temperature on total biomass was detected. The total dry weight of plants grown at 531 μmol mol⁻¹ CO₂ was 34% greater than those grown at 328 μmol mol⁻¹ CO₂, whereas a 1 °C rise reduced dry weight by 6%. From serial harvests the radiation conversion coefficient was 2.01 g MJ⁻¹ and 1.42 g MJ⁻¹ at 531 μmol mol⁻¹ CO₂and 328 μmol mol⁻¹ CO₂, respectively, but was not greatly affected by differences in temperature. No effect of either CO₂ or temperature on the canopy light extinction coefficient was detected. The rate of progress towards curd initiation increased to a maximum at 15.5 °C, and declined thereafter. Provided the effect of temperature was accounted for, CO₂ enrichment did not affect the time of curd initiation. From serial harvests after curd initiation, the logarithm of curd weight or diameter were negative linear functions of mean temperature from initiation. Increases in curd weight and diameter at 531 compared with 328 μmol mol⁻¹ CO₂ were greater at warmer temperatures (27% at 13 °C compared with 47% at 15 °C, 57 days after initiation). Effects of CO₂ on curd diameter were less than those on curd dry weight because the curd dry matter content was greater at 531 compared with 328 μmol mol⁻¹ CO₂. Thus, the effects of elevated CO₂ concentrations on fresh weight based yield parameters of cauliflower were less than the increase in total dry matter production.     

Leaf structure and function of sweet cherry tree (Prunus avium L.) cultivars with open and dense canopies

Variations in morpho-anatomy, chemical composition, gas exchange and stem water potential (Ψ_stem) were studied in leaves of Prunus avium L. cultivars with different growth habit: Burlat and Summit (upright, dense canopies), and Van (spreading, open canopy). Light transmittance was highest in Van canopies. Therefore, Van allowed an easier penetration of the visible radiation through the canopy, which improves photosynthetic rates, especially at harvest. Conversely, no differences in Ψ_stem were found among cultivars. Van showed the highest leaf mass per unit area and variations in this parameter were mainly associated with alterations in leaf thickness than in leaf density. Leaves of Summit and Van were thicker than those of Burlat, mainly due to increased palisade and spongy parenchyma thickness. However, Burlat leaves had the highest palisade/spongy ratio. Total chlorophyll concentration per area and the chlorophyll a/b ratio were always higher in Van leaves than in the other two cultivars. In contrast, Van leaves had the lowest concentrations of soluble carbohydrates and starch. In dense canopies of Summit and Burlat, light reduction caused a notable increase in the relative concentration of total phenols. The above results provide useful information about the biological behaviour of sweet cherry cultivars with open and dense canopies in order to help the agronomical decisions regarding the local environmental conditions and the consumers’ preference. In particular, it is important to manipulate factors such as orchard exposure, row orientation, plant spacing, training system and pruning, which are able to reduce shading in the canopy.     

Response to drought of two olive tree cultivars (cv Koroneki and Meski)

The effect of water deprivation on plant water status, photosynthetic gas exchange and fluorescence parameters in two different olive tree varieties (Olea europaea L. var. ‘koroneiki’, ‘Meski’) was studied. Two-year-old olive trees, grown in pots in greenhouse, were subjected to one of three drought treatments (i.e., mild, moderate and severe drought stress) and compared to control trees. Both the leaf water potential (Ψ_w) and the relative water content (RWC) of the two varieties decreased with increasing levels of drought stress. Koroneiki showed higher (less negative) values of Ψ_w and lower values of RWC than the Meski, particularly during severe drought stress.

Net photosynthetic assimilation and stomatal conductance declined with drought. This inactivation of photosynthetic activity was accompanied by changes in the fluorescence characteristics. The PSII maximal photochemical efficiency (F_v/F_m), the intrinsic efficiency of open PSII reaction centres (F_v′/F_m′), the photochemical efficiency of PSII (Φ_PSII) and the total electron flow (J_t) decreased during the development of drought stress.

The non-quenching photochimique (q_N) showed an increase in the response to water deficit. These observations are discussed in relation to the strategies developed to grow drought-resistant olive trees in arid areas.     

Effect of sucrose concentrations on somatic embryogenesis in carnation (Dianthus caryophyllus L.)

The effect of sucrose concentration on callus induction followed by differentiation of embryogenic callus derived from petal explants of four carnation cultivars (Nelson, Sagres, Spirit and Impulse) was investigated. Embryogenic calli were produced on Murashige and Skoog [Murashige, T., Skoog, F.A., 1962. Revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 154, 73–479] basal medium (MS) culture medium containing six concentrations of sucrose (3, 6, 9, 12, 15 and 18%, w/v) all supplemented with 9 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.8 μM 6-benzyladenine (BA). Maximum frequency of embryogenic callus was obtained from the media containing 9 and 12% sucrose. Somatic embryos were induced on a hormone-free MS media containing the seven concentrations of sucrose. Development of somatic embryos was enhanced by increasing sucrose concentration from 1.5 to 12%, while it was reduced in higher concentrations of 15 and 18%. However, normal embryos were not developed in the media containing 1.5 and 3% sucrose. Ninety-five percent of somatic embryos were regenerated to form the entire plantlets when they transferred onto the half-strength hormone-free MS culture medium containing 3% sucrose. Plantlets were also continued to grow normally under greenhouse condition.     

Phenolic compounds in the phloem of Prunus trees, section Eucerasus

The flavans were the predominant compounds in the phloem of mature Prunus shoots (section Eucerasus), constituting about 5–12% of the dry substance. Chlorogenic acids were less concentrated and varied between 1.2 and 0.2% of the dry weight. The flavans were higher in P. avium than in P. cerasus. The reverse was true with chlorogenic acids. This pattern did not change remarkably near the union when cultivars of P. avium were grafted on clones of P. cerasus or P. fruticosa.     

Breeding of disease-resistant seedless grapes using Chinese wild Vitis spp.: I. In vitro embryo rescue and plant development

Seedless grape cultivars (Vitis vinifera L.) are widely grown in Europe, America and Asia. Fungal diseases are a great threat of them. Several wild Chinese Vitis species showed high resistance to many fungal diseases. Therefore, an investigation was conducted to assess the potential to incorporate these species in a breeding project for development of the disease-resistant seedless cultivars. Hybridization was conducted using V. vinifera as female parents and the wild Chinese Vitis spp. as male parents. In-ovulo embryo rescue was used to develop hybrid plants from the seedless females. An efficient protocol is reported here for the in vitro embryo rescue and plant development from the cross Emerald Seedless × Beichun. Ovules were excised from immature fruits 7 weeks after pollination (WAP) and cultured in the double-phase ER medium supplemented with 6.0% sucrose and 0.3% activated charcoal (AC). Following 8–12 weeks of culture, embryos were removed from the ovules and transferred onto WPM supplemented with 1.0 μM 6-benzyladenine (6-BA), 2.0% sucrose and 0.2% AC and solidified with 0.6% agar. After 8 weeks of culture, the embryos germinated and subsequently grew into whole plantlets. With the optimized parameters developed in the present study, about 34.0%, 91.2% and 77.4% of embryo formation, embryo germination and plant development were obtained, respectively. When this protocol was applied to 11 other cross combinations, genotype was found to significantly influence embryo formation, embryo germination and plant development, with different frequencies of hybrid plants successfully obtained in all crosses.     

Storage conditions and ripening of chiku fruits Achras sapota L.

Chiku fruits (Achras sapota L.) were found to be climacteric with the respiratory peak occurring at the same time, or 1–2 days after peak ethylene production. The optimum storage temperature was near 20° C. Storage life at this temperature could be increased by removing ethylene (C₂H₄) and adding 5–10% (v/v) carbon dioxide (CO₂) to the storage atmosphere. Extreme relative humidities or high concentrations of CO₂ impaired the quality of stored fruits. Ripening was not affected by treatment with oxygen, C₂H₄ or indoleacetic acid. Ascorbic acid and glucose levels increased with ripening but ascorbic acid decreased when the fruit became over ripe. Recommended storage conditions are about 20° C with 5–10% CO₂ coupled with the complete removal of C₂H₄ from the storage atmosphere. Short-term holding of the fruit at lower temperatures is also possible.     

Development of a regeneration protocol through indirect organogenesis in prickly pear cactus (Opuntia ficus-indica (L.) Mill)

An indirect organogenesis regeneration protocol for Opuntia ficus-indica (L.) Mill var “Blanco sin Espinas” is described. One centimeter square cladode explants sections from previously micropropagated prickly pear plants were cultured in Murashige and Skoog (MS) basal medium supplemented with 20 different combinations of 2,4-dichlorophenoxy acetic acid (2,4-D) and benzyladenine (BA). The best calli induction and regeneration response were observed when 2.26 μM 2,4-D and 2.21 μM BA combination was applied to the nopal explants. Regenerating calli was capable of forming new buds when transferred to MS basal medium supplemented with 0.5 μM BA (proliferation medium). Shoot elongation and rooting were achieved on MS medium without plant growth regulators. Excellent acclimatization to greenhouse conditions was observed for all transferred plantlets. By this procedure no morphological differences were observed between the regenerated and mother plants. This protocol may be also utilized to carry out plant regeneration after genetic transformation, in order to develop transformed plants without the presence of chimeric zones.     

Effect of night humidity on the vegetative growth and the mineral composition of tomato and strawberry plants

Conditions of low night humidity (50–55%) and high night humidity (90–95%) were imposed on tomato plants (Lycopersicon esculentum Mill. cv. Young-Kwang) for 30 days, and on strawberry plants (Fragaria grandiflora Ehrh. cv. Bogyo) for 40 days. Shoot dry weight and leaf area in tomato plants increased with high night humidity whereas the strawberry plants did not show any such increases. The concentrations of five major nutrients (N, P, K, Ca and Mg) were reduced in tomato plants under high night humidity, with the calcium content in younger leaves being the most severely affected. For the strawberry plants, however, high night humidity tended to increase the concentrations of major nutrients, including calcium in the younger leaves. Possible reasons for the differences in response of these two species to night humidity are discussed.     

Changes during the respiratory climacteric in ripening plantain fruits

Fruits of a Ghanaian plantain (Musa, AAB group) of the “French” type, cultivar ‘Apem’, were used to study changes before and after commencement of the climacteric at 25°C. Fruits exhibited an extended preclimacteric period during which C₂H₄ production was undetectable, then a well defined maximum in C₂H₄ production prior to a 10-fold increase in CO₂ production. Maximum C₂H₄ production was much higher than in dessert banana cultivars (Musa, AAA and AAAA groups). During ripening, the high initial content of alcohol-insoluble solids was almost completely converted to sugars, accompanied by rapid peel colouration and pulp softening.