Chlorocholine chloride application effects on photosynthetic capacity and photoassimilates partitioning in potato (Solanum tuberosum L.)

Treatment of potato (Solanum tuberosum L.) with chlorocholine chloride (CCC) applied twice as a foliar spray 25 and 30 days after planting has shown to decrease shoot and stolon growth but increase tuber yield. However, the regulatory role of CCC on translocation of recently fixed photoassimilates into different parts of potato plants has not been fully illustrated. In this study, ¹⁴C-isotope labelling technique was used to estimate the photosynthetic capacity and photoassimilate partitioning among leaves, stems, roots + stolons, and tubers of potted potatoes treated with 1.5 g l⁻¹ CCC. CCC treatment significantly increased tuber dry mass but reduced leaf dry mass. CCC-treated leaves had significantly higher chlorophyll and carotenoid contents and assimilated 22.0% more ¹⁴CO₂ per leaf dry mass than the controls. Compared with the control, CCC treatment reduced the translocation of ¹⁴C-photoassimilates into leaves, stems and roots + stolons but increased that into tubers. CCC-treated leaves exported 14.6% more ¹⁴C-photoassimilates into other parts of the plants. In addition, CCC treatment reduced ¹⁴C-soluble sugar and ¹⁴C-starch accumulation in leaves and stems but enhanced them in tubers and roots + stolons. Collectively, the results indicate that CCC treatment significantly improves the photosynthetic capacity of potato leaves and promotes photoassimilates partitioning into tubers thereby enhancing tuber growth.     

Development of a plant regeneration system from seed-derived calluses of centipedegrass [Eremochloa ophiuroides (Munro.) Hack]

The objective of this study is to establish plant regeneration system with the seed of the new Chinese selection “E-126”of centipedegrass [Eremochloa ophiuroides (Munro.) Hack] as explant. In present study, the following results were obtained: (1) The medium formulation most suitable for calluses induction was identified to be MS with 1.0 mg l⁻¹ 2,4-D + 30 g l⁻¹ mannitol + 50 ml l⁻¹ coconut milk and the ratio of calluses induction was 96.0%, including 5.2% of yellow granule calluses induction. The above medium formulation was adopted for subculture. (2) The rate of shoot regeneration from yellow granular calluses was 98.0% by MS optimum medium formulation with 2.0 mg l⁻¹ KT + 50 ml l⁻¹ coconut milk. The differentiated rate retained as high as 88.0% even after 5 times of subculture and 18.6% after 15 times of subculture. The optimum medium formulation for shoot growth was identified to be MS medium plus 2.0 mg l⁻¹ BAP, 0.8 mg l⁻¹ NAA and 50 ml l⁻¹ coconut milk. (3) The optimum medium for shoot rooting was identified to be MS medium with 0.6 mg l⁻¹ NAA + 50 ml l⁻¹ coconut milk, and the rooting rate to be 98.0%. The survival rate of transplanted plantlets from tubes to basin with soil was 92.0%. In conclusion, the plant regeneration system was successfully developed in this study, which may provide basic reference for screening of somaclonal variants and genetic transformation of centipedegrass.     

Molecular characterization and analysis of somaclonal variation in chrysanthemum cultivars using RAPD markers

Molecular characterization using RAPD analysis was carried out in eight cut flowers and two pot plant cultivars of chrysanthemum. Three of them (‘Refocus’, ‘Red Reagan’, and ‘Sheena Select’) were established in vitro and the occurrence of somaclonal variation was studied using the same molecular technique. Two induction media (MS + 0.1 mg l⁻¹ NAA + 0.1 mg l⁻¹ BA, and MS + 2.0 mg l⁻¹ IAA + 0.5 mg l⁻¹ Kinetin), and two proliferation media (MS + 0.1 mg l⁻¹ NAA + 0.2 mg l⁻¹ BA, and MS + 4.0 mg l⁻¹ IAA + 2.0 mg l⁻¹ Kinetin) were employed in order to evaluate the effect of the medium composition in the shoots’ stability. Likewise, the effect of the culture age was considered in assessing genetic stability. Monthly subcultures were carried out, identifying the origin and history of the shoots, throughout a nine-month proliferation period followed by acclimatization. Molecular markers were obtained in every subculture cycle and from the acclimatized plants. Only one shoot from the 7th subculture of the cultivar ‘Refocus’ showed a different band pattern. The use of RAPD for chrysanthemum cultivar characterization and somaclonal variation detection is discussed.     

Effect of enhanced ultraviolet-B on allelopathic potential of Zanthoxylum bungeanum

The effect of enhanced ultraviolet-B on allelopathic potential of Zanthoxylum bungeanum was investigated. A significant inhibitory effect on germination rate of crop seeds under bioassay was observed at 25 g l⁻¹ and 50 g l⁻¹ by extracts from Zanthoxylum leaf both treated with enhanced ultraviolet-B radiation and untreated control. Medicago sativa and lettuce were more sensitive than radish to the extract from Zanthoxylum leaf treated with enhanced UV-B radiation, as the germination rates of M. sativa and lettuce were significantly reduced compared to control at 25 g l⁻¹ and 50 g l⁻¹, and so did alfalfa at 12.5 g l⁻¹. However, as for radish (Raphanus sativus) there was no significant reduction in germination rate at any concentration under bioassay compared to control. Content of UV-B absorbing compounds and total phenols in Zanthoxylum seedlings responded positively to enhanced UV-B radiation. The results suggest that the allelopathic potential of Z. bungeanum was generally improved under enhanced UV-B radiation.     

Evaluation of the effectiveness of soil-applied plant derivatives of Meliaceae species on nitrogen availability to peach trees

We assessed the effect of soil-applied derivatives of melia (Melia azedarach L.) and neem (Azadirachta indica A. Juss) on nitrogen (N) soil availability, root uptake and peach (Prunus persica L.) growth. First we evaluated the effectiveness of experimentally prepared amendments made with fresh ground melia leaves or commercial neem cake incorporated into the soil as nitrification inhibitors, then we evaluated the effect of fresh ground melia fruits and neem cake on growth and N root uptake of potted peach trees, and on soil microbial respiration. Soil-applied fresh ground melia leaves at 10 and 20 g kg⁻¹ of soil as well as commercial neem cake (10 g kg⁻¹) were ineffective in decreasing the level of mineral N after soil application of urea-N as a source of mineral N, rather they increased soil concentration of nitric N and ammonium N. The incorporation into the soil of fresh ground melia fruits (at 20 and 40 g kg⁻¹) and neem cake (at 10 and 20 g kg⁻¹) increased N concentration in leaves of GF677 peach × almond (Prunus amygdalus) hybrid rootstock alone or grafted with one-year-old variety Rome Star peach trees. An increase in microbial respiration, leaf green color and plant biomass compared to the control trees were also observed. The Meliaceae derivatives did not affect, in the short term (7 days), N root uptake efficiency, as demonstrated by the use of stable isotope ¹⁵N, rather they promoted in the long term an increase of soil N availability, N leaf concentration and plant growth.     

Production of inter-section hybrids between Primula filchnerae and P. sinensis through ovule culture

Inter-section hybrids were obtained in the reciprocal crosses between Primula filchnerae (2n = 2x = 24) of Sect. Pinnatae and P. sinensis ‘Fanfare’ (2n = 2x = 24) of Sect. Auganthus by rescuing ovules on half-strength (1/2) Murashige and Skoog's (MS) medium supplemented with 50 g l⁻¹ sucrose, 2.5 g l⁻¹ gellan gum, 0.1 mg l⁻¹ α-naphthaleneacetic acid (NAA), 0.1 mg l⁻¹ 6-benzyladenine (BA) and 50 mg l⁻¹ gibberellic acid (GA₃). In ovule culture, germination occurred with radicle elongation but no plumule was observed. The radicle kept on the initial medium showed root proliferation with callus formation. When the calluses were transferred to (1/2)MS media containing 30 g l⁻¹ sucrose and 3 g l⁻¹ gellan gum, without plant growth regulators (PGRs) or with 1 mg l⁻¹ zeatin and 0.1 mg l⁻¹ NAA, plantlets were regenerated. The plants thus obtained were confirmed to be hybrids through flow cytometry (FCM) and random amplified polymorphic DNA (RAPD) analyses. The hybrid obtained when P. filchnerae was used as the maternal parent was diploid, whereas hexaploid hybrid was obtained when using P. sinensis as the maternal parent. The hexaploid hybrid might be produced through chromosome doubling of a triploid originated from the fertilization of P. sinensis with unreduced pollen of P. filchnerae.     

Effects of the commercial product based on Trichoderma harzianum on plant,bulb and yield characteristics of onion

Effects of the commercial product TrichoFlow WP™ (Agrimm Technologies Ltd., New Zealand), based on the fungus Trichoderma harzianum, on quality characteristics and yield of bulb onion was investigated. Bulb sets of the local cultivar Kantartopu was planted in soil with in and between row distances of 0.15 m and 0.40 m, respectively. The product, at considerably high dosages of 5 g m⁻², 10 g m⁻² and 15 g m⁻², was mixed with water and sprinkled once to the plots at planting. Analyses of data at harvest did not show statistical significance for Trichoderma effect on total bulb yield, bulb diameter, leaf length, number of shoot apex, %titratable acidity, number of internal (fleshy) leaves, number of external (papery) leaves, %soluble solids and %bulbs with diameters of 20–39 mm, 40–69 mm and ≥70 mm. The yields obtained from the plots treated with the dosages of 5 g m⁻², 10 g m⁻² and 15 g m⁻² and the control plots were 1063.7 kg da⁻¹, 1051.0 kg da⁻¹, 1066.5 kg da⁻¹ and 985.0 kg da⁻¹, respectively. Our results showed that high dosages of the Trichoderma product were not effective in enhancing onion bulb and yield characteristics under the given conditions.     

Influence of yeast extract and casein hydrolysate on callus multiplication and somatic embryogenesis of date palm (Phoenix dactylifera L.)

Complex organic additives are known to improve growth and differentiation of in vitro plant cultures. The present investigation was conducted to determine the effect of various concentrations of yeast extract (YE) and casein hydrolysate (CH) on callus growth and somatic embryogenesis in date palm cultivar Nabout Saif. Callus induced from shoot tip explants was grown on callus multiplication medium supplemented with either YE or CH at 0.0, 0.1, 0.25, 0.5, and 1 g l⁻¹. To induce somatic embryogenesis, callus was transferred to a hormone-free medium containing the corresponding concentration of additives. The results have shown that callus weight and the number of somatic embryos were directly proportional to increases in the concentration of organic additives tested. Callus growth was best achieved when 1 g l⁻¹ of either YE or CH was added to the culture medium. At this concentration of YE, callus growth was double that of the control medium. On CH-containing media growth was 2.3 times that of the control. This indicates that CH is more effective in enhancing callus growth. However, YE was more effective in enhancing somatic embryogenesis. The data show that the best somatic embryo formation was obtained on either 1 g l⁻¹ YE or 0.5 g l⁻¹ CH which produced 45 and 30 embryos per culture, respectively, as compared to 20 embryos produced in the control treatment. Resultant somatic embryos successfully rooted and regenerated plantlets which exhibited normal growth in the greenhouse. Enhanced plant regeneration, an essential criterion for commercial micropropagation, was achieved.     

Coconut water and BAP successfully replaced zeatin in olive (Olea europaea L.) micropropagation

The data presented report on trials conducted during 24 months using the Portuguese olive cultivar ‘Galega vulgar’. The effectiveness of coconut water, BAP, or kinetin, as possible zeatin substitutes in olive micropropagation protocols, was investigated. In all stages of the micropropagation process, the mineral and vitamin formulation of olive medium (OM) was used. Regarding culture establishment the best results were achieved when 50 ml l⁻¹ coconut water and 2.22 μM BAP were used as medium supplements. For the in vitro multiplication stage, the highest proliferation rates with an average of 3.4 new explants on each 30 days were achieved maintaining the coconut water concentration at 50 ml l⁻¹ and increasing BAP up to 8.87 μM. The effects of IBA and activated charcoal on the in vitro root induction were also studied. Rooting rates of over 85% were obtained by basal immersion of the explants in IBA solution at 3 g l⁻¹ for 10 s, followed by inoculation in the OM culture medium, added with 2 g l⁻¹ of activated charcoal and without growth regulators. All in vitro rooted plants were transferred into Jiffy-Pots filled with vermiculite–perlite 3:1 (v/v) substrate. Those were subsequently wetted with the OM mineral solution, placed into polystyrene plates each one with 100 Jiffy-Pots capacity, which were transferred to traditional rooting mist benches, on a water-cooling equipped greenhouse. Such a simple acclimatization procedure allowed for 95% of plants survival.     

Seed germination and tissue culture of Cymbidium giganteum Wall. ex Lindl.

Efficient protocols were established for in vitro seed germination, neo-formation of secondary (2°) protocorms from primary (1°) protocorms and multiple shoot buds and protocorm-like body (PLB) induction from pseudo-stem segments of in vitro-raised seedlings of Cymbidium giganteum. Four nutrient media, namely Murashige and Skoog (MS), Phytamax (PM), Mitra et al. (M), and Knudson ‘C’ (KC) were evaluated for seed germination and early protocorm development. In addition, the effects of peptone, activated charcoal (AC) and two plant growth regulators [6-benzylaminopurine (BAP) and 2,4-dichlorophenoxyacetic acid (2,4-D)] were also studied. Both M and PM supplemented with 2.0 g l⁻¹ peptone or 1.0 mg l⁻¹ BAP resulted in ∼100% seed germination. Media supplemented with 2.0 g l⁻¹ AC could effectively induce large protocorms (1.6 ± 0.1 mm in diameter). Neo-formation of 2° protocorms from 1° protocorms was achieved in liquid and agar-solidified PM medium fortified with different concentrations and combinations of auxins (α-naphthalene acetic acid (NAA) and 2,4-D) and cytokinins [BAP and kinetin (KN)]. The highest number of 2° protocorms was obtained in liquid medium (10.7 ± 0.9/1° protocorm) supplemented with 2.0 mg l⁻¹ BAP + 1.0 mg l⁻¹ NAA. Although protocorms proliferated profusely in liquid medium, these did not develop further unless transferred to agar-solidified medium within 6–8 weeks. Multiple shoot buds and PLBs were induced from pseudo-stem segments on agar-solidified PM medium fortified with different concentrations and combinations of BAP and NAA and the maximum number of PLBs (6.00 ± 0.20) was recorded when BAP and NAA were applied at 2.0 mg l⁻¹ each. A solid root system was induced from PLBs and shoot buds when these were transferred to half-strength PM or M media fortified with 0.5 mg l⁻¹ indole-3-acetic acid. Well-rooted plants were transferred to the greenhouse with 95% survival.     

Effect of genotype,explant size,position, and culture medium on shoot generation of Gerbera jamesonii by receptacle transverse thin cell layer culture

An unique procedure for the mass shoot propagation of Gerbera using receptacle transverse thin cell layer (tTCL) culture procedure was developed. Genotype, flower bud age, explant size, position of receptacle tTCLs and culture media were found to affect the success of culture. Ten interspecific crosses of Gerbera showed different shoot regeneration rates and callus induction via receptacle tTCL culture, all of which had shoot regeneration rates higher than 57%. Flower buds collected on the 10th day resulted in 91% shoot regeneration after 6 weeks of culture on basal MS medium [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol. Plant. 15, 475–497] supplemented with 0.02 mg l⁻¹ thidiazuron (TDZ), 0.8 mg l⁻¹ adenine and 10% (v/v) coconut water (CW). This was significantly higher than those from flower buds on the 7th and 14th days (22% and 54%), respectively. Shoot regeneration rate was the highest (94–100%) in the middle layers of the receptacle. For mass shoot propagation, shoot clusters were subcultured on half-strength MS medium supplemented with 0.5 mg l⁻¹ indole-3-butyric acid (IBA), 0.5 mg l⁻¹ 6-benzyladenine (BA) and 2.0 mg l⁻¹ kinetin after every 4 weeks. Plantlets formed when single shoots were cultured on half-strength MS medium containing 1 mg l⁻¹ IBA. All plantlets acclimatized well in the greenhouse.     

The effects of foliar applied CaCl2·2H2O,Ca(OH)2 and K2CO3 combined with the surfactants Glucopon and Plantacare on gas exchange of 1 year old apple (Malus domestica BORKH.) and broad bean (Vicia faba L.) leaves

Calcium chloride, calcium hydroxide, potassium carbonate and the alkylpolyglycoside surfactants Glucopon 215 CSUP and Plantacare 12 UP are salts applied to leaves as foliar nutrients and fungicides. These chemicals were sprayed on apple (Malus domestica BORKH.) and broad bean (Vicia faba L.) leaves. Stomatal conductance and rates of net photosynthesis were measured continuously in the light and in the dark using a Portable Photosynthesis System CIRAS-1. All compounds with the exception of Ca(OH)₂ affected stomatal conductance and net photosynthesis, albeit to different degrees. In light, Plantacare either alone (0.2 g l⁻¹) or in combination with CaCl₂·2H₂O (5 g l⁻¹) or K₂CO₃ (5 g l⁻¹) caused a rapid initial increase in stomatal conductance during the first 1–3 h after spraying on the leaves, maximum conductances were observed about 6 h after application. A rather high stomatal conductance was observed during the dark period when Glucopon (0.2 g l⁻¹) was applied either alone or in combination with Ca(OH)₂. The combination CaCl₂·2H₂O + Glucopon did not cause this elevated stomatal conductance during the dark. CaCl₂·2H₂O reduced stomatal conductance in combination with both Glucopon and Plantacare. The surfactant Plantacare reduced net photosynthesis during the first light period (12 h), if applied alone or in combination with CaCl₂·2H₂O. Treatment of broad bean leaves with K₂CO₃ + Plantacare resulted in a rapid decrease in net photosynthesis during the first hour, and then the rates of net photosynthesis increased rapidly and approached to those of the water control. The effects of surfactants and salts on net photosynthesis had nearly disappeared by the beginning of the second light period. Non-specific glycosidases presumably cleaved the glycosidic bond between the alkyl and the sugar moieties during the preceding night. Our data showed that foliar applications of CaCl₂·2H₂O and K₂CO₃ together with alkyl polyglycoside surfactants can affect gas exchange. However, the effects of the chemicals at the concentrations used in our study were not very large and were transient. They practically vanished within 24 h and a detrimental effect on growth and development of crops was not likely.     

Direct regeneration of plants derived from in vitro cultured shoot tips and leaves of three Lysimachia species

The regenerability of three ornamental species—Lysimachia christinae, Lysimachia rubinervis and Lysimachia nummularia ‘Aurea’, were investigated using in vitro leaves and shoot tips. 6-Benzylaminopurine (BAP) and α-naphthalene acetic acid (NAA) added to Murashige and Skoog (MS) medium were tested for their effect on organogenesis. On the medium, shoot regeneration occurred directly without callus formation. In these species, L. christinae developed the highest regeneration rate and numbers of shoots/explant from shoot tips (100%, 12.25) and leaf bases (100%, 13.01) on the MS medium containing 3.0 mg l⁻¹ BAP and 0.1 mg l⁻¹ NAA. For L. rubinervis, the highest shoot induction rate and number of shoots/explant were obtained from shoot tip (100%, 16.87–17.20) on the MS medium with 0.1 mg l⁻¹ NAA and 3.0–5.0 mg l⁻¹ BAP. L. nummularia ‘Aurea’, however, showed the highest regeneration rate and number of shoots/explant (100%, 12.73) from leaf bases on MS medium supplemented with 1.0 mg l⁻¹ BAP and 0.1 mg l⁻¹ NAA. All in vitro shoots rooted well on half macronutrient MS medium containing 0.1 mg l⁻¹ NAA. After acclimatization, transplanted plantlets grew normally and flowered in the field.     

Priming abiotic factors for optimal hybrid Cymbidium (Orchidaceae) PLB and callus induction,plantlet formation,and their subsequent cytogenetic stability analysis

Abiotic factors affect the induction of PLBs and callus in hybrid Cymbidium Twilight Moon ‘Day Light’. The initiation and proliferation of new PLBs and callus could be achieved on NAA and kinetin, supplemented at 0.1 mg l⁻¹ each, respectively, both within 45–60 days. Bacto agar was found to be the most suitable solidifying agent for PLB induction, although a higher shoot fresh weight was obtained on Gelrite; a pH 5.3 was optimal while pH 4.5 caused 100% explant necrosis; coconut water, when supplied at 10–20% (v/v) resulted in a significant increase in the number of PLBs formed per PLB segment (23.1 versus 14.6 in controls) while a massive (almost four-fold) increase in fresh top weight occurred when PLB explants were placed in liquid culture, as a result of hyperhydricity; Fe-EDTA (1 mg l⁻¹) and activated charcoal (1 g l⁻¹) stimulated total fresh weight and PLB formation in the presence of PGRs; PLB formation decreased but total fresh shoot weight increased with the addition of niacin or myo-inositol, both vitamins. Dark-grown PLB-induced plants were etiolated and had longer internodes and higher fresh weight than light-grown control plants at 45 μmol m⁻² s⁻¹; at 15 μmol m⁻² s⁻¹ shoots were slightly etiolated, fragile, and PLB formation was scarce. RAPD and mtDNA analysis of all resultant PLBs, callus or plants showed them to be genetically identical, with comparable chlorophyll contents. Despite the detection of cytological variation between different plant parts, little variation resulted from abiotic factor treatment.     

Impact of iron stress on biomass,yield, metabolism and quality of potato (Solanum tuberosum L.)

In order to study the influence of variable iron on biomass, economic yield and role of iron in potato (Solanum tuberosum) cv. Chandramukhi metabolism, plants were grown in refined sand at variable iron ranging from 0.001 to 2.0 mM. Exposure of potato plants to Fe stress (i.e. a Fe concentration different from 0.1 mM) shows retarded growth, decreased chlorophyll concentration and Hill reaction activity, induced changes in enzyme activities and concentration of Fe and Mn. The visible symptoms of iron deficiency appeared on day 15 at 0.001 mM Fe as chlorosis of young leaves. The excess of iron (at >0.1 mM Fe) appeared later, after 25 days and the chlorosis was observed on older leaves. Both deficiency (0.001 mM) and excess (>0.1 mM) of iron reduced the tuber yield, deteriorating its quality by lowering the concentration of sugars, starch and protein nitrogen and increasing the accumulation of non-protein nitrogen and phenols in tubers.     

Effect of organic and inorganic fertilizers applied during successive crop seasons on growth and nitrate accumulation in lettuce

A romaine-type lettuce (Lactuca sativa L.) cv. Corsica was cultivated during three successive crop seasons (late-spring, late-autumn and late-winter) in the same soil of an experimental greenhouse in S.W. Peloponnese, Greece. Seven long-term fertilization treatments were tested for their effect on plant growth and nitrate concentration in the external lettuce leaves. Treatments included: three different doses of organic fertilization (composted sheep manure) applied at the start of each crop season, three different doses of inorganic N fertilization applied via fertigation during each crop season, and a control treatment in which no fertilizer was applied. A drip irrigation system was used to water all plants. The highest nitrate levels were observed in the medium and maximum inorganic fertilization treatments (572–664 mg kg⁻¹) in all crop seasons. They were significantly higher compared to the respective organic fertilization treatments (253–435 mg kg⁻¹) and all other fertilization treatments (148–435 mg kg⁻¹). Crop season affected lettuce growth more than nitrate accumulation in the lettuce leaves: lettuce biomass production was the smallest and most uniform in the late-autumn season and did not respond to the fertilization treatments tested (ranging from 409 to 439 g plant⁻¹), while in the late-spring season biomass production was the highest and most variable (561–841 g plant⁻¹), it correlated with nitrate concentration in the leaves and in the medium and maximum inorganic fertilizer doses it significantly exceeded production from all other fertilization treatments (827–841 g plant⁻¹). Following the three crop seasons the residual availability of N, P and K was clearly enhanced in the soil receiving the organic compared to the inorganic fertilization. Nitrate concentration in lettuce leaves was far below the upper limits set by the European Commission in all fertilization treatments throughout the three crop seasons, a result attributed mainly to the sufficient level of light intensity and duration throughout the year in Southern Greece.     

Factors affecting haploid induction through in vitro gynogenesis in summer squash (Cucurbita pepo L.)

Haploid production using in vitro ovule cultures has long been recognized as an important tool to produce haploid and homozygous double-haploid plants for genetic studies and plant breeding programs. In the present study, four experiments were carried out to study the influence of genotype, position of female flowers on plant stem, temperature and sucrose concentration on the in vitro gynogenesis induction of squash. (1) Ovules of 12 genotypes were excised from female flowers, 1 day before anthesis, and cultured onto MS medium containing 3% sucrose and 1 mg l⁻¹ from each of kinetin and 2,4-D (2,4- dichlorophenoxy acetic acid). Differences in response among genotypes were demonstrated. Raad F₁ showed the highest percentage of responding ovules and number of plantlets per dish with 48.8% and 15 plants, respectively. The results revealed that genotype is a key factor influencing the in vitro gynogenesis in squash. (2) Ovules were excised from first, second and third female flower of two hybrids (Giad and Raad) and cultured onto the mentioned above medium. The highest percentage of responding ovules and number of plantlets per dish were obtained from ovules excised from the second female flower on the plant stem. (3) Effect of temperature (4 and 32 °C) for 0, 4, 7 and 12 days on the ovule culture of Queen F₁ was studied. Ovules incubated at 4 or 32 °C for 4 days produced a better embryogenic response. (4) Three sucrose concentrations (30, 60 and 90 g l⁻¹) were tested with the ovule cultures of the local cultivar (Eskandrani). Differences among sucrose concentrations were statistically significant and ovules cultured on the MS medium containing 30 g l⁻¹ produced the best result. MS medium containing 90 g l⁻¹ did not produce gynogenic ovules.     

Plant regeneration from stem segment-derived friable callus of “Fonio” (Digitaria exilis (L.) Stapf.)

“Fonio” (Digitaria exilis (L.) Stapf.) is a member of the grass family with excellent culinary and nutritional properties. In spite of its economic values, hardly has any improvement work been done. To enhance genetic improvement of this grain, plant regeneration protocol was developed using 8 cultivars. Stem segments of 5 mm long excised from 1 month-old seedlings germinated in vitro were cultured on 6 types of media for friable callus induction. Best result was obtained on Murashige and Skoog (MS) medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1 g l⁻¹ casamino acid, where 91.3, 88.9 and 87.8% of the explants formed friable calli in cultivars ‘Kurelep’, ‘Churiwe’ and ‘Agyong’, respectively. Shoots appeared when friable calli were transferred to two regeneration media, i.e., MSBZ (MS medium + 0.022 mg l⁻¹ 2,4-D, 0 .22 mg l⁻¹ 6-benzylaminopurine (BA), 0.22 mg l⁻¹ zeatin) and MSBG (MS medium + 0.5 mg l⁻¹ BA, 0.1 mg l⁻¹ gibberellic acid). The highest frequency of plant regeneration was attained on MSBG, with 91.7% of the friable calli forming shoots in cultivar “Churiwe”. Regenerated plants were rooted on hormone free MS medium. Flow cytometric analysis revealed 100% of the regenerants to be diploid. The protocol developed here can be used in the transformation of “Fonio” to increase the yield potential of this crop by incorporating characteristics such as disease resistance and stress resistance.     

Effect of synthetic auxins on fruit size of five cultivars of Japanese plum (Prunus salicina Lindl.)

Most of the Japanese plum (Prunus salicina) cultivars grown in Israel produce relatively small fruit. Application of 2 l solution tree⁻¹ of 25 mg l⁻¹ 2,4-dichlorophenoxypropionic acid (2,4-DP) as butoxyethyl ester (Power™), 15 mg l⁻¹ 3,5,6-trichloro-2-pyridyloxyacetic acid (3,5,6-TPA) as free acid (Maxim^®), or 25 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) + 30 mg l⁻¹ naphthaleneacetic acid (NAA) (0.3% Amigo™) at the beginning of pit-hardening, when fruitlet diameter was ca. 22 mm, caused an appreciable and significant increase in fruit size. The yield of large fruit per cv.: ‘Kesselmen’ (100% increase), ‘Songold’ (100%), ‘Black Diamond’ (800%), ‘Royal Diamond’ (160%) and ‘Royal Zee’ (100%). As a result, the total yield of all five cultivars was also increased dramatically. Anatomical studies with ‘Songold’ revealed that the main effect of these synthetic auxins was via direct stimulation of fruit cell enlargement. The above auxins had no negative effect either on fruit quality at harvest (and after 1 week in shelf-life), or on return yield in the following year.     

Effects of exogenous application of plant growth regulators on the development of ovule and subsequent embryo rescue of stenospermic grape (Vitis vinifera L.)

Seedless grapevine cultivars (Vitis vinifera L.) are widely grown in Europe, America and Asia. Abortion of zygotic embryos in seedless grapes largely limits the efficiency of breeding of seedless cultivars through genetic crossing. The present study was designed to investigate effects of exogenously applied plant growth regulators (PGRs) to the grapevines in field condition on ovule and subsequent embryo rescue of seedless grapes of small seed traces. First experiment was performed by measuring ovules weight, proportion of each category ovules in maturity and embryo development in vitro of seedless grape cv. Centennial Seedless, Thompson Seedless and Crimson Seedless sprayed by chlormequat (CCC), benzyladenine (BA), ethephon (CEPA) and putrescine (Put). The effects of different application concentration and date of CCC were further evaluated in Centennial Seedless in later experiment. The results showed that exogenous application of all PGRs did not affect the total number of ovules per berries in maturity. CCC increased the ovules weight and proportion of ovules >4 mm in length of three varieties in maturity. The effects of two application times of PGRs on weight of berries and ovules and proportion of each category ovules in maturity were not significantly different. In the proceeding of embryo rescue, CCC at 100 and 1000 mg l⁻¹, BA at 100 mg l⁻¹ and Put at 20 mg l⁻¹ increased the percentage of developed embryos of Centennial Seedless and Thompson Seedless. The results showed that the size of ovules excised for embryo rescue significantly affected embryo formation and plant regeneration. The percentage of embryos formation in ovules >4 mm in length was significantly more than in ovules 2–4 mm in length, no embryo was found in ovules <2 mm in length. Exogenous application of CCC at 100–500 mg l⁻¹ significantly increased percentage of ovules >2 mm in length by 80.0–82.7% in Centennial Seedless, therefore improving embryo formation. The statistical correlation was found between the proportion of ovules >2 mm and embryo formation (r = 0.92) in Centennial Seedless. Among the different spraying time in Centennial Seedless, CCC applied 14 days before bloom produced significantly more ovules >2 mm in length and embryos formation.