Nitrogen best management practice for citrus trees: II. Nitrogen fate,transport, and components of N budget

Soils in central Florida citrus production region are very sandy, hence are vulnerable to leaching of soluble nutrients and chemicals. The objective of this study was to develop nitrogen (N) and irrigation best management practices for citrus in sandy soils to maintain optimal crop yield and quality, and to minimize N leaching below the rootzone. A replicated plot experiment was conducted in a highly productive 20+ year-old ‘Hamlin’ orange [Citrus sinensis (L.) Osbeck] trees on ‘Cleopatra mandarin’ [(Citrus reticulata Blanco)] rootstock grove located on a well drained Tavares fine sand (hyperthermic, uncoated, Typic Quartzipsamments) in Highland County, FL. Nitrogen rates (112–280 kg ha⁻¹ year⁻¹) were applied as fertigation (FRT), water soluble granular (WSG), a combination of 50% FRT and 50% WSG, and controlled release fertilizer (CRF). Tensiometers were used to monitor the soil moisture content at various depths in the soil profile as basis to optimize irrigation scheduling. Fruit yield and quality and nutritional status of the trees were reported in a companion paper. Soil solution was sampled at 60, 120, and 240 cm depths under the tree canopy using suction lysimeters. Concentrations of NO₃-N in the soil solution at 240 cm deep, which is an indicator of NO₃-N leaching below the tree rootzone, generally remained below the maximum contaminant limit (MCL) for drinking water quality (10 mg L⁻¹) in most samples across all N sources and rates, but for few exceptions. Total N in the fruit was strongly correlated with fruit load, thus, at a given N rate N removal by the fruit was lower during years of low fruit yield as compared to that during the years of high fruit yield. Furthermore, there was a strong linear relation between N and K in the fruit. This supports the need to maintain 1:1 ratio between the rates of N and K applications. In a high fruit production condition, the N in the fruit accounted for about 45% of the total N input on an annual basis. Fifteen percent of the total N input at 280 kg N ha⁻¹ year⁻¹ was not accounted for in the citrus N budget, which could be due to leaching loss. This estimate of potential leaching was very close to that predicted by LEACHM simulation model. The improved N and irrigation management practices developed in this study contributed to an improved N uptake efficiency and a reduction in N losses.     

Hybridization and in vitro culture of an orchid hybrid Ascocenda ‘Kangla’

The present study focuses on hybridization program involving two species belonging to two different vandaceous genera, viz., Ascocentrum ampullaceum (Roxb.) Schltr. var. auranticum, a narrow endemic orchid of Manipur and Vanda coerulea Griff., an endangered orchid of Appendix I of CITES (Convention on International Trade in Endangered Species of Wild Fauna and Flora), to synthesize the primary hybrid genus with intermediate and improved characters in the F₁ generation. Observations on the crossability in the present bigeneric cross between V. coerulea and A. auranticum had been achieved with 60% success when V. coerulea was taken as female parent. Murashige and Skoog (MS) basal medium at half-strength was effective for the development of the hybrid seedlings of V. coerulea × A. auranticum followed by Vacin and Went (VW) and Knudson C (KC) media. The best response of seedling growth was observed on MS medium at half-strength supplemented with 2.3 μM kinetin + 0.5 μM α-naphthalene acetic acid with maximum shoot height (2.7 cm), leaf number (4.6) and root number (4.1) after 150 days of inoculation. The survival percentage and growth performance of the seedlings were found to be higher (80% survival) in potting substrate consisting of brick:charcoal in the ratio 2:1 mulched with moss (Sphagnum sp.) than in potting substrate consisting of brick:charcoal:tree fern in the ratio 2:1:1. The first flowering was observed in the hybrid seedlings of V. coerulea × A. auranticum after 2 years of transfer to the ex vitro environment. Morphologically, the flowers differed from that of the parents clearly showing the success of the hybridization experiment. Registration of the hybrid has been made with the Royal Horticultural Society with the nomenclature Ascocenda ‘Kangla’ (No. T128725).     

Effects of light,macronutrients, and sucrose on germination and development of the endangered fern Adiantum reniforme var. sinense (Adiantaceae)

Effects of light, macronutrients strength of Murashige and Skoog (MS) (1962), and sucrose in the culture medium on spore germination and gametophyte development of the endangered fern Adiantum reniforme var. sinense were investigated. The presence of light was found to be essential for both spore germination and gametophyte growth. Moreover, a medium consisting of 1/4 MS with 15 g/l sucrose was optimal for spore germination and early gametophyte development; whereas, MS medium with 30 g/l sucrose was optimal for further gametophyte development. Increasing amounts of sucrose (45–60 g/l) in the medium delayed gametophyte growth and development. Additionally, sporophyte formation and early growth of gametophytes in a medium consisting of clay and peat (v/v = 1:2) was higher than those in a medium consisting of pure river sand. These findings indicated that requirements for nutrients for spore germination and early gametophyte development of A. reniforme var. sinese were relatively low, but these increased with further gametophyte development, formation and growth of sporophytes.     

Modeling plant morphology and development of petunia in response to temperature and photosynthetic daily light integral

The effects of mean daily temperature (MDT) and mean photosynthetic daily light integral (MDLI) on flowering during the finish stage of two petunia (Petunia × hybrida) cultivars were quantified. Petunia ‘Easy Wave Coral Reef’ and ‘Wave Purple’ were grown in glass-glazed greenhouses at 14–23 °C or 14–26 °C and under 4–19 mol m⁻² d⁻¹ with a 16-h photoperiod. The flower developmental rate was predicted using a model that included a linear MDT function with a base temperature multiplied by an exponential MDLI saturation function. The flower developmental rate increased and time to flower decreased as MDT increased within the temperature range studied. For example, under a MDLI of 12 mol m⁻² d⁻¹, as MDT increased from 14 to 23 °C, time to flower of ‘Easy Wave Coral Reef’ and ‘Wave Purple’ decreased from 51 to 22 d and 62 to 30 d, respectively. Flower developmental rate increased as MDLI increased until saturation at 14.1–14.4 mol m⁻² d⁻¹. Nonlinear models were generated for effects of MDT and MDLI on flower bud number and plant height at flowering. The number of flower buds at flowering increased as MDT decreased and MDLI increased. For example, at an MDT of 14 °C with 18 mol m⁻² d⁻¹, plants had 2.5–2.9 times more flower buds than those grown at 23 °C and 4 mol m⁻² d⁻¹. Models were validated with an independent data set, and the predicted time to flower, flower bud number, and plant height were within ±7 d, ±20 flowers, and ±4 cm, respectively, for 96–100%, 62–87%, and 93–100% of the observations, respectively. The models could be used during greenhouse crop production to improve scheduling and predict plant quality of these petunia cultivars.     

Cucumber fruit quality at harvest affected by soilless system,crop age and preharvest climatic conditions during two consecutive seasons

Cucumber fruits (Cucumis sativus L., cv. Trópico F1) grown on perlite substrate and NFT (Nutrient Film Technique) were harvested during two seasons (winter and spring) to monitor the effects of climatic conditions and hydroponic growth systems on fruit quality at harvest. The best fruit quality at harvest, as measured by lightness and hue angle parameters, was obtained during the 3 weeks following the first winter picking. When values of a parameter of preharvest climate value called G^* were lower than 0.4 MJ m⁻² °C day⁻¹ interval⁻¹, the cucumber achieved its optimum quality at harvest, as measured by the dark green color of the skin. NFT-grown fruits showed darker and greener skin color (higher hue angle and lower lightness) compared with perlite-grown fruit, irrespective of the season considered. During the winter season, the plant transpiration rate was 30% higher in perlite than in NFT culture, which correlates with higher differences in G^* and 40% additional yield in perlite. However, in spring G^* was not sensitive enough to discriminate between the two hydroponic systems. In general, fruit quality at harvest in spring was lower than during the winter, due to flesh whitening, higher longitudinal and equatorial calibers, and slightly higher pH, as well as worse epidermal color coordinates (ranging from the dark and dull green color typical of winter fruit to light and a vivid green-yellow color). During the spring season, NFT-grown fruit were less acid than perlite-grown fruit with no apparent correlation with the climatic conditions or fruit nutrition.     

In vitro germination and plantlet establishment of Labisia pumila (Bl.) F. Vill.

In vitro seeds germination and plantlet establishment of Labisia pumila were studied in this report. The seeds obtained from the mature fruits of L. pumila were sterilized and cultured on Murashige and Skoog (MS) solid media supplemented with 1–3 μM of 6-benzylaminopurine (BAP) and 3% (w/v) sucrose. The presence of BAP in the medium significantly affects seeds germination. High percentage of seeds germination (up to 90%) was successfully achieved after 2 weeks of culture on medium supplemented with 2 μM BAP. Up to 70% of explants produced shoots through direct regeneration from newly emerged epicotyls after 5 weeks of culture. The average of 8.1 ± 1.0 shoots per explant obtained on media treated with 2 μM BAP. Seedlings were further transferred to growth media fortified with different types of cytokinin. Result observed after 12 weeks showed that medium supplemented with 1 μM zeatin (ZEA) promote the highest growth with an average of 2.9 ± 1.0 cm shoot length and 7.7 ± 3.2 leaves per explant after 12 weeks. In addition, medium added with 2 μM BAP and supplemented with 3–4% (w/v) of sucrose promote the best growth i.e., 3.0 ± 0.6 shoots per explant, 2.27 ± 0.2 cm length and 4.3 ± 0.5 leaves per explant.     

High frequency early flowering from in vitro seedlings of Dendrobium nobile

Dendrobium nobile Lindl. is a popular temperate Chinese orchid commonly marketed as a traditional medicinal plant. Seedlings of Dendrobium nobile Lindl. produced floral buds (33.3–34.8%) precociously on a defined basal medium (1/2 MS) containing paclobutrazol (PP₃₃₃) at 0.5 mg L⁻¹ or thidiazuron (TDZ) at 0.1 mg L⁻¹ within 4 months of culturing. The frequency of floral buds formation can be further increased to 95.6% by growing seedlings in a PN (PP₃₃₃ 0.3 mg L⁻¹ + NAA 0.5 mg L⁻¹)-containing medium followed by transfer onto 1/2 MS medium with PP₃₃₃ and TDZ (PP₃₃₃ + TDZ). However, flower developed was deformed under 25 °C but it developed fully when grown in a lower temperature regime (23 °C/18 °C, light/dark) for 45 days. Under optimal condition, in vitro flowering was observed about 6 months after seed sowing.     

Effects of greenhouse cooling method on growth,fruit yield and quality of tomato (Solanum lycopersicum L.) in a tropical climate

A tomato (Solanum lycopersicum L.) crop was grown in four greenhouses during the dry season 2005/06 in Central Thailand. Sidewalls and roof vents of two greenhouses were covered with nets and these greenhouses were mechanically ventilated when air temperature exceeded 30 °C (NET). The other two greenhouses were covered with polyethylene film and equipped with a fan and pad cooling system (EVAP). Overall mean air temperature was significantly reduced by 2.6 and 3.2 °C (day) and 1.2 and 2.3 °C (night) in EVAP as compared to NET and outside air, respectively. Temperature maxima in EVAP averaged about 4 °C lower than in NET and outside. The relative humidity was around 20 and 30% (day) and 10 and 15% (night) higher in EVAP than in NET or outside, respectively. Vapour pressure deficit averaged 0.25 kPa in EVAP, 1.03 kPa in NET and 1.48 kPa outside. The crop water-consumption was significantly lower in EVAP (1.2) than in NET (1.8 L plant⁻¹ day⁻¹), which is ascribed to reduced transpiration in EVAP. Total fruit yield was similar in NET (6.4 kg plant⁻¹) and EVAP (6.3 kg plant⁻¹). The quantity of undersized (mostly parthenocarpic) and blossom-end rot (BER)-affected fruits was reduced in EVAP. However, the proportion of marketable yield was significantly higher in NET (4.5 kg plant⁻¹) than in EVAP (3.8 kg plant⁻¹), owing largely to an increased incidence of fruit cracking (FC) in EVAP. Higher FC but lower BER incidence coincided with higher fresh weight and Ca concentration in the fruits in EVAP. It is concluded that in regions with high atmospheric relative humidity evaporative cooling without technical modifications allowing dehumidification will not improve protected tomato production.     

Effects of plant growth promoting bacteria (PGPB) on yield,growth and nutrient contents of organically grown strawberry

The effects of plant growth promoting bacteria (PGPB) on the fruit yield, growth and nutrient element content of strawberry cv. Fern were investigated under organic growing conditions between 2006 and 2008. The experimental plot was a completely randomized design with 3 replicates. Three PGPB strains (Pseudomonas BA-8, Bacillus OSU-142 and Bacillus M-3) were used alone or in combination as bio-fertilizer agent in the experiment. Data through 3 years showed that the use of PGPB significantly increased fruit yield, plant growth and leaf P and Zn contents. Root inoculation of M3 and floral and foliar spraying of OSU-142 and BA-8 bacteria stimulated plant growth resulting in significant yield increases. M3 + BA-8, BA-8 + OSU-142, M3, M3 + OSU-142 and BA-8 applications increased cumulative yield by 33.2%, 18.4%, 18.2%, 15.3% and 10.5%, respectively. Number of fruits per plant significantly increased by the applications of M3 + BA-8 (91.73) and M3 (81.58) compared with the control (68.66). In addition, P and Zn contents of strawberry leaves with bacterial inoculation significantly increased under organic growing conditions. Available P contents in soil were increased from 0.35 kg P₂O₅/da at the beginning of the study to 2.00, 1.97 and 1.82 kg P₂O₅/da by M3 + OSU-142, M3 + BA-8 and M3 + BA-8 + OSU-142 applications, respectively. Overall, the results of this study suggest that root inoculation of Bacillus M3 alone or in combination with spraying Bacillus OSU-142 or Pseudomonas BA-8 have the potential to increase the yield, growth and nutrition content of strawberry plant under organic growing conditions.     

Effects of diurnal temperature alternations and light quality on growth,morphogenesis and carbohydrate content of Cucumis sativus L.

The effects of the difference (DIF) between day/night temperature (DT/NT) and end-of-day (EOD) light quality on growth, morphology, dry matter (DM) content and carbohydrate status in cucumber (Cucumis sativus L.) were examined. Plants were grown under a 12 h high light intensity period and DT/NT of 25/19 °C (positive DIF) or 19/25 °C (negative DIF) in combination with an exposure of 30 min EOD-red (EOD-R) or far-red (EOD-FR) light. A significant interaction between DIF and EOD light quality was found on morphology, DM and carbohydrate content in axial plant organs like stems and petioles, but not in leaf blades and roots. Positive DIF induced taller stems, and higher DM and carbohydrate content than negative DIF when the plants were grown under EOD-R. The stems developed under EOD-FR were tall and accumulated the highest content of DM and carbohydrates and only small differences were found between positive DIF and negative DIF. Under EOD-R a higher content of glucose and fructose was found under positive than negative DIF, while EOD-FR light resulted in a high glucose and fructose content under both positive and negative DIF and thereby equalized the effect of the two temperature treatments. The results show that positive DIF can induce similar responses in elongation growth, DM and carbohydrate accumulation as EOD-FR, and further that phytochrome status interact with the responses to alternating DT and NT.     

Plant regeneration of Carica papaya L. through somatic embryogenesis in response to light quality,gelling agent and phloridzin

Difficulties to develop an easy and reproducible protocol to get healthy and well formed plants from somatic embryos of papaya (Carica papaya L.) had included low germination, callus production at the base of the embryo radicle and the occurrence of hyperhydric plantlets among others, and by consequence unsuccessful transfer to the field. With the aim of improving a propagation method, the effects of light quality, gelling agent and phloridzin concentration on the germination of somatic embryos of hermaphrodite C. papaya L. var. Maradol were studied. Somatic embryos were grown on half strength MS medium, with the addition of Chen vitamins [Chen, M.H., Wang, P.J., Maeda, E., 1987. Somatic embryogenesis and plant regeneration in Carica papaya L. tissue culture derived from root explants. Plant Cell Rep. 6, 348–351], solidified with three distinct gelling agents: Sigma^® Agar–Agar, Difco^® Bacto agar and Phytagel^®; supplemented with phloridzin and exposed to different light qualities: blue (54 μmol m⁻² s⁻¹), red (65 μmol m⁻² s⁻¹), gro-lux (68 μmol m⁻² s⁻¹), red + blue, white (32 μmol m⁻² s⁻¹) and wide spectrum (49 μmol m⁻² s⁻¹) during a period of 4 weeks. Results show that light quality and gelling agent had important effects on germination and plant growth, while 3.0 mg L⁻¹ phloridzin had an important role on germination as well as in root development. Somatic embryos exposed to white light, culture medium solidified with 3.0 mg L⁻¹ phytagel and 3.0 mg L⁻¹ phloridzin showed longer roots. Meanwhile, germination and plant length were promoted on an improved culture medium solidified with 7.5 g L⁻¹ Difco^® Bacto agar, 3.0 mg L⁻¹ phloridzin and exposed to gro-lux lamps. Under these conditions, 70% of somatic embryos germinated and developed normal roots without hyperhydricity. The regenerated plantlets with well developed roots and shoots were successfully transferred to a greenhouse with a survival rate of 95%.     

Inoculation of growing media with the rhizobacterium Variovorax paradoxus 5C-2 reduces unwanted stress responses in hardy ornamental species

Inoculation of growing media with plant growth promoting rhizobacteria (PGPR) has a number of potential benefits for the production of ornamental plants. Certain rhizobacteria synthesise the enzyme ACC deaminase, which cleaves ACC, the precursor of the plant hormone ethylene. Bacterial metabolism is now known to lead to a reduction in [ACC] in the plant transpiration stream and bacteria are hypothesised to act on ACC exuded from roots. This in turn reduces the ethylene generated in plants growing in growing media inoculated with these bacteria. Here we tested if applications of the ACC deaminase containing rhizobacteria Variovorax paradoxus⁵C–²could be of benefit to ornamental growers by reducing ethylene generation in stressed plants. Ethylene is produced at levels that are inhibitory to growth and development under a number of abiotic stresses. The propagation and production of hardy ornamentals is deleteriously affected by abiotic stresses that involve ethylene signalling, including drought and physical wounding. Inoculation of growing media with V. paradoxus 5C-2 lowered ethylene emission from mature leaves of Cytisus × praecox experiencing drought stress. In addition, bacterial inoculation of the growing media resulted in significantly reduced abscission of the mature leaves under drought treatment. Beneficial effects of inoculation where also found in the wounding response of Fargesia murielae following divisional propagation and late season senescence in Aquilegia × hybrida in response to drought stress. Together these results demonstrate that V. paradoxus⁵C–²has real potential for use on ornamental nurseries in situations where plant stresses are unavoidable.     

Effects of plant density in broccoli on yield and radiation use efficiency

The effects of plant density on broccoli (Brassica oleracea L. var. italica Plenck) commercial characteristics are well determined. However, it is not completely clear how the broccoli plant respond to changes in plant shading as a result of different plant densities. The objective of this experiment was to determine the effect of plant density on intercepted photosynthetically active radiation (PAR), plant architecture, and plant growth and production. “Legacy” broccoli plants were grown in pots in a greenhouse in the seasons of 2002 and 2003 at 2, 4, 6 or 8 plants m⁻² (temperatures: between 10.0 and 16.1 °C, average incident PAR: 12 mol m⁻² day⁻¹). Plant density affected the intercepted and accumulated PAR. There were not effects on the length of the vegetative and reproductive periods, the total and final number of leaves, and the spear diameter and fresh weight. The magnitude and evolution of leaf area (LA) was independent of plant density up to 70 days after transplant (dat). Since then on, LA increased linearly with plant density. The highest intercepted PAR was 70–72% with 6–8 plants m⁻². With the increase in plant density: the erectness of the upper leaves and stem length increased, the extinction coefficient decreased and commercial spear (inflorescence plus a portion of stem 10 cm long) weight decreased (but it was due to the stem portion of the spear and not to the edible portion). On an area basis, the decrease in commercial spear weight with plant density was more than compensated by the higher number of plants. The radiation use efficiency (RUE) increased proportionally with the leaf area index (LAI) up to a LAI of about 3, and then stabilized. The only effect of plant density on dry weight partitioning was to decrease the dry weight allocated to the stem portion of the spear. As plant density increased, and consequently the degree of shading increased, the net assimilation rate (NAR) decreased and the leaf area ratio (LAR) increased. This compensatory change between NAR and LAR, kept the relative growth rate (RGR) for individual plants almost constant.     

Factors affecting in vitro adventitious shoot formation on internode explants of Citrus aurantium L. cv. Brazilian

The in vitro formation of newly formed adventitious buds and shoots from internodal branch segments was studied on 12-month-old plants of Citrus aurantium L. cv. Brazilian. The effects of 6-Benzyladenine (BA) and α-Naphthalene acetic acid (NAA) treatments were evaluated on adventitious bud and shoot regeneration. High rates of bud initiation and shoot development were obtained both with BA supplemented medium, in the range from 1 mg L⁻¹ to 3 mg L⁻¹, and with 0.1 mg L⁻¹ NAA supplemented medium. NAA concentrations above 1 mg L⁻¹ significantly reduced bud initiation and shoot elongation. The results obtained using different in vitro culture vessels such as Petri dishes, tubes and glass culture jars were compared. The highest adventitious bud induction was observed in Petri dishes for internodes cultured in 2 mg L⁻¹ BA supplemented medium, with 95% responsive explants forming 9.0 ± 2.4 adventitious buds. The adventitious buds observed in Petri dishes reached a maximum height of 1 mm, with no further development, while some of the adventitious shoots cultured in tubes and glass culture jars grew over 1 cm in height. A shoot regeneration gradient of the internodes collected along the branch axis was noticed, with basal ones exhibiting higher regeneration frequency.     

The influence of irrigation system and nutrient solution concentration on potted geranium production under various conditions of radiation and temperature

Zonal geranium (Pelargonium × hortorum ‘Real Mintaka’) were grown in closed soilless systems to evaluate the effects of irrigation system (drip and subirrigation) and nutrient solution concentration (half and full) under various conditions of radiation and temperature (winter and summer) in terms of substrate electrical conductivity (EC_s), growth, quality, crop evapotranspiration (ET_c) and growth index water use efficiency (WUE_GI) and nutrient uptake. At he end of the cultural cycle the highest EC_s in the upper and lower layers were recorded in the spring season on plants grown in subirrigation using a full nutrient solution concentration. The highest shoot biomass, leaf area, plant growth index, and quality index were recorded in the winter season on plants grown in both drip-irrigation and subirrigation using half and full nutrient solution concentration, whereas the lowest value was observed in the spring season on plants grown with subirrigation using the full nutrient solution concentration. The highest maximum air temperature recorded during the first 20 days after transplanting in the spring growing season was presumably responsible for the reduction in shoot biomass production, growth and quality index, and in time of geranium flowering compared to the plants grown in the winter season. The ET_c was 44% higher in spring than in winter season treatment, while the effect of the irrigation system was less pronounced with an increase in 11% in the subirrigation treatment compared with the drip-irrigation system. WUE_GI was not stable and showed a seasonal variability. Solar radiaton (R_s), air temperature (T_a) and vapour pressure deficit (VPD) were greatly higher in the spring season, which influenced WUE_GI negatively. The WUE_GI improved especially when R_s, T_a and VPD were below 12 MJ m², 20 °C and 0.6 kPa, respectively. The highest N, and Mg uptake were recorded in the winter season, especially on plants grown with subirrigation at 2 dS m⁻¹. The highest P, K, and Ca uptake values were measured during winter season using subirrigation system, and on plants grown under full strength nutrient solution. The variation of the nitrate concentration and EC in the nutrient solution during the spring growing cycle was less pronounced in the subirrigation than with a drip-irrigation system which represents an important aspect for the simplification of the closed loop management of the nutrient solution.     

Chromosome doubling of Lychnis spp. by in vitro spindle toxin treatment of nodal segments

Lychnis (Caryophyllaceae) consists of about 30 species distributed throughout the temperate regions of the Northern Hemisphere, from East Asia to Europe. Many Lychnis spp. have high ornamental value and cultivated as pot or garden plants. In the present study, in vitro chromosome doubling of several Lychnis spp. was examined in order to widen their variability in horticultural traits. Initially effect of various spindle toxin treatments [100, 500 or 1000 mg l⁻¹ colchicine (COL), 10, 20 or 50 mg l⁻¹ oryzalin (ORY), or 1, 5, 10 mg l⁻¹ amiprophos-methyl (APM)] of nodal segments of a triploid genotype of L. senno (3x) was investigated on survival of nodal segments and chromosome doubling in nodal segment-derived plantlets. Significantly higher percentage (75.0%) of surviving segments after spindle toxin treatment was obtained in 10 mg l⁻¹ ORY treatment. Flow cytometry (FCM) analysis of leaf tissues showed that 9.4–13.8% of plantlets, which were derived from 10 to 20 mg l⁻¹ ORY, or 5 mg l⁻¹ APM treatments, were hexaploid (6x) or ploidy chimera (3x + 6x, 4x + 6x, 5x + 6x, 3x + 4x + 6x). The results obtained by FCM analysis were confirmed by chromosome observation in root tip cells. Thus 10 mg l⁻¹ ORY treatment of nodal segments is suitable for in vitro chromosome doubling of triploid L. senno. Efficient chromosome doubling was also achieved in diploid L. fulgens (2x) and L. sieboldii (2x) by treating nodal segments with 10 mg l⁻¹ ORY: 68.9–88.7% of nodal segments survived after ORY treatment, and 24.7–26.5% of plantlets derived from ORY-treated nodal segments were tetraploid (4x) or ploidy chimera (2x + 4x) in both species. These results indicate that the in vitro chromosome doubling method established for triploid L. senno may be applicable to a wide range of Lychnis spp. Tetraploid L. fulgens and L. sieboldii showed a compact plant form, and had thick stems and deep green leaves compared with the diploid mother plants. On the other hand, hexaploid L. senno showed very poor growth and died before flowering.     

Effect of different drip irrigation methods and fertilization on growth,physiology and water use of young apple tree

A pot experiment was conducted to investigate the effect of three drip irrigation methods (i.e. conventional drip irrigation (CDI), both sides of the root-zone irrigated with full watering, alternate drip irrigation (ADI), both sides of the root-zone irrigated alternatively with half of the full watering, and fixed drip irrigation (FDI), only one side of the root-zone irrigated with half of the full watering) on growth, physiology, root hydraulic conductance and water use of young apple tree under different nitrogen (N) or phosphorus (P) fertilization (i.e. CK (no fertilization), N₁ (0.2 g N/kg), N₂ (0.4 g N/kg), P₁ (0.2 g P₂O₅/kg) and P₂ (0.4 g P₂O₅/kg)). Results show that compared to CDI, ADI and FDI reduced mean root dry mass, daily transpiration, root hydraulic conductance (K_r), leaf photosynthesis rate, transpiration rate and stomatal conductance of young apple tree by 6.9 and 27.7, 29.3 and 45.0, 6.8 and 37.9, 2.5 and 4.8, 32.6 and 33.0, 22.1 and 22.3%, but increased leaf water use efficiency (WUE) by 31.3 and 29.8%, respectively when they saved irrigation water by 50%. Compared to the CK, N or P fertilization significantly increased K_r, and K_r was increased with the increased N or P fertilization level. There were parabolic correlations between K_r and root dry mass, daily transpiration and stomatal conductance. Our results indicate that ADI reduced transpiration rate significantly, but it did not reduce photosynthesis rate and K_r significantly, thus alternate drip irrigation improved WUE and the regulation ability of water balance in plants.     

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.     

Improvement of staining method for observation of mitotic chromosomes in octoploid strawberry plants

An important strategy to conduct intentional breeding of octoploid strawberry plants is to recognize the functions of every chromosome. To do so, a methodology must be developed to distinguish chromosomes one by one. We reported the possibility of distinguishing chromosomes using light microscopy when somatic cells of octoploid strawberry plants were stained using ordinary methods with lacto-propionic orcein (LPO). However, karyotype analysis of octoploid strawberry plants required clearer chromosome images. This study obtained clearer chromosome images of octoploid Fragaria × ananassa and F. chiloensis plants. Three staining methods were examined: 60% acetic acid (AA) alone, 1.5% LPO alone, and two-step treatments with 60% AA and 1.5% LPO. Collected root tips of the plants were placed in 2 mM 8-hydroxyquinoline solution for 1 h and were subsequently stored at 4 °C for 15 h. The samples were then fixed in a 3:1 absolute alcohol: glacial acetic acid solution for 40 min, followed by mixture with 1N HCl solutions at room temperature for 2 h and then at 60 °C for 10 min. For separate staining using 60% AA and 1.5% LPO, the root tip was expelled on the glass slide with a drop of each solution for a few minutes to stain the chromosomes. For the two-step staining method, the samples stained with 60% AA were frozen at −80 °C for at least 5 min. The cover slip was removed using a razor blade. Subsequently, the specimens were air-dried and stained with the 1.5% LPO for 3 min. Digital images of chromosomes were obtained using light microscopy. Samples of the two-step staining method produced the clearest chromosome images in both F. × ananassa and F. chiloensis. Furthermore, the greatest color difference between the chromosomes and the cytoplasm was obtained from images of the two-step staining method among the three staining methods. These results demonstrate that the two-step staining method is useful for chromosome counting and karyotype analysis in strawberry plants.