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

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

Callus induction and plant regeneration from embryonic axes of Kosteletzkya virginica

Kosteletzkya virginica, a perennial dicot halophytic species of the Malvaceae, is native to American salt marsh. It was introduced into China as a potential species to improve coastal wetlands and to develop ecologically sound saline agriculture. K. virginica adapts excellently to the tidal-flat habitats in China's east coast, with multiple eco-benefits; in particular, its seed oil could be used to produce biodiesel. The purpose of this study was thus to develop a standardized protocol to induce a high frequency of callus and subsequent plantlet regeneration system for a K. virginica breeding program with the final objective of applying transgenic techniques to improve seed oil yield. The embryonic axes of K. virginica were used as explants for callus induction, shoot induction from the callus and then adventitious root induction from the shoots on nine culture media with different hormone combinations. The best results were achieved on the following media: (1) 93.94% callus induction on MS medium supplemented with 1.0 mg L⁻¹ indole-3-acetic acid (IAA), 0.3 mg L⁻¹ kinetin, 30 g L⁻¹ sucrose and 8 g L⁻¹ agar; (2) 65.83% shoot induction on 1/2MS medium supplemented with 0.1 mg L⁻¹ IAA, 0.5 mg L⁻¹ zeatin, 30 g L⁻¹ sucrose and 8 g L⁻¹ agar; (3) 96.67% rooting on MS medium containing 30 g L⁻¹ sucrose and 8 g L⁻¹ agar. The survival rate of plantlets by organogenic regeneration was 85% after being transplanted into potting soil in flowerpots and placed in the greenhouse. This experiment indicates that we established successful callus induction and plant regeneration protocols for K. virginica.     

Factors affecting mango (Mangifera indica L.) protoplast isolation and culture

The major factors influencing protoplast isolation and culture of mango (Mangifera indica L.) cv. Kensington Pride were investigated. The resultant protocol was used to compare plating efficiency among 4 mango cultivars. Most responses differed between proembryonic masses (PEMs) and leaf sources. Protoplast yields of 15.22 × 10⁶ g⁻¹ from PEMs and 8.68 × 10⁶ g⁻¹ from greenhouse-derived leaves were obtained in a solution of 0.7 M mannitol CPW plus 1.5% cellulase, 1% hemicellulase and 0.75% macerozyme for PEMs or 0.5 M mannitol CPW plus 1.5% cellulose, 1% hemicellulase and 1.5% macerozyme for leaves. Culture in Ca-alginate beads with initial plating densities (IPD) of 2.5 × 10⁴ Pp mL⁻¹ for PEMs and 2.5 × 10⁵ for leaves gave the highest plating efficiencies (FPE). For PEMs 1 mg L⁻¹ 2,4-d and 3.5 mg L⁻¹ kinetin gave an FPE of 2.85% whereas lower kinetin (2 mg L⁻¹) plus 0.5 mg L⁻¹ 6-BAP was most effective for leaves (FPE of 2.12%). Most protoplast mortality occurred during the first week of culture and was more severe in liquid culture. In Ca-alginate beads, protoplast survival at 14 days was higher for PEMs (30%) than leaf (21%) as was the frequency of cell division (17.6% compared to 13.6%). PEMs protoplasts continued development through embryogenesis to in vitro plantlet regeneration whereas leaf protoplasts underwent cell division up to 40-cell colonies but failed to proceed further. For PEMs, polyembryonic cvs. Kensington Pride and Keow Savoey produced higher FPE (1.95%) than monoembryonic cvs. Tommy Atkins and Keitt (1.75%). There was no effect of cultivar for leaf protoplasts.     

Nutritional quality of greenhouse lettuce at harvest and after storage in relation to N application and cultivation season

The effect of five levels of nitrogen fertilization on the growth and nutritional quality of Cos lettuce (Lactuca sativa L. cv. Parris Island) at harvest and after storage was studied during autumn and winter in South-West Greece. Plants were cultivated hydroponically in a greenhouse and the nitrate, chlorophyll and ascorbic acid (vitamin C) concentrations within the plant tissues were measured at harvest and following storage at 5 or 10 °C for 10 days. Nitrate accumulated in the leaves with increasing amounts of N within the nutrient solution and was higher in the winter than in the autumn. At the lowest N level (20 mg L⁻¹), the inner leaves accumulated more nitrate than the outer leaves, whereas at higher N levels (140, 200 or 260 mg L⁻¹) nitrate accumulation was higher in the outer leaves. Overall, the highest nitrate concentrations were detected in the petiole and the proximal end of the leaf, but at the lowest N application rate (20 mg L⁻¹) nitrate accumulated in the distal region of the leaf too. Although the nitrate concentrations within the leaves did not change significantly during 10 days storage at 5 or 10 °C, the chlorophyll and vitamin C concentrations decreased. Chlorophyll loss was higher in lettuce that was grown under low N levels and was higher at 10 °C than at 5 °C, but was reduced by enclosure of the lettuce in polyethylene film. It is concluded that the optimum N application rate for Cos lettuce grown hydroponically under cover during autumn and winter in South-West Greece, and in other areas with a similar climate, is 200 mg N L⁻¹ because at this N rate yield is satisfactory and leaf nitrate concentrations are below the maximum acceptable level for human consumption. Nutritional value (vitamin C concentration) and market quality (chlorophyll content) are highest at harvest and decrease during storage, but quality in terms of nitrate concentration does not change.     

Growth and flowering of black iris (Iris nigricans Dinsm.) following treatment with plant growth regulators

The effects of application method and concentration of gibberellic acid (GA₃), paclobutrazol and chlormequat on black iris performance were assessed. Plants (10 cm high, 4 ± 1 leaves) were sprayed with 125, 250, 375 or 500 mg L⁻¹ or drenched with 0.25, 0.5, 1 or 2 mg L⁻¹ GA₃. In a second experiment, the plants were sprayed with 100, 250, 500 or 1000 mg L⁻¹ or drenched with 0.25, 0.5, 1 or 2 mg L⁻¹ paclobutrazol. Other plants were sprayed with 250, 500, 1000 or 1500 mg L⁻¹ or drenched with 100, 250, 375 or 550 mg L⁻¹ chlormequat. In each experiment, the control treatment consisted of untreated plants. Results indicated that the tallest plants (37.3 cm) in the GA₃ experiment were those sprayed with 250 mg L⁻¹. The most rapid flowering (160 days after planting) occurred when a 375 mg L⁻¹ GA₃ spray was used, whereas flowering was delayed to 200 days using 1 mg L⁻¹ GA₃ drench. Drenching with 1 mg L⁻¹ GA₃ increased height of the flower stalk by 7 cm compared to the control. Though relatively slow to flower, plants drenched with 1 mg L⁻¹ GA₃ had long and rigid stalks, which were suitable as cut flowers. Number and characteristics of the sprouts were not affected by GA₃. All paclobutrazol sprays resulted in leaf falcation. A 500 or 1000 mg L⁻¹ paclobutrazol spray resulted in severe and undesirable control of plant height, drastic reduction in stalk height and weight, and delayed flowering. Plants drenched with 0.25 or 1 mg L⁻¹ paclobutrazol were suitable as pot plants. Chlormequat reduced plant height only at the highest drench concentration, which also reduced flowering to 70%. No leaf falcation was observed with GA₃ or chlormequat. Chemical names: ( ± )-(R*,R*)-beta-((4-chlorophenyl)methyl)-alpha-(1,1,-dimethylethyl)-1H-1,2,4,-triazol-1-ethanol (paclobutrazol); (2-chloroethyl) trimethylammonium chloride (chlormequat).     

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.     

Rooting response of shoot cuttings from three peach growth habits

Current year shoot cuttings were collected in October and August from three growth habits of peach (Compact, Pillar, and Standard) and treated with one of four concentrations of indole butyric acid (0, 250, 1250, and 2500 mg L⁻¹ IBA). Rooting response was measured after 5 weeks in the greenhouse. Little or no rooting occurred with cuttings from any growth habit that was collected in October or in August when treated with 0 and 2500 mg L⁻¹ IBA. In August, the number of shoots that rooted was greater in cuttings from Pillar (79 and 45%) than Compact (13 and 3%) treated with 250 and 1250 mg L⁻¹ IBA, respectively. Cuttings from Standard trees had intermediate rooting of 56 and 6% at 250 and 1250 mg L⁻¹ IBA, respectively. Pillar trees consistently grew more roots with greater root length per cutting than the other growth habits. It is proposed that differences in rooting response among the growth habits may be associated with differences in endogenous auxin concentration that had been found in previous studies. Within peach and possibly other fruit trees, the capacity of shoot cuttings to develop adventitious roots can vary by cultivar and successful root induction with exogenous plant growth regulators may depend, in part, on endogenous hormone levels.     

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.     

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.     

An upper limit for elevated root zone dissolved oxygen concentration for tomato

It is well understood that insufficient oxygen within plant root zones can greatly diminish plant productivity. However, little is known about the effect of elevated root zone oxygen concentrations. Tomato (Lycopersicon lycopersicum Mill., cv. Trust) seedlings were grown in nutrient solutions containing dissolved oxygen (DO) concentration ranging from 5.3 to 40 mg L⁻¹ for 4 weeks. There were no visible symptoms observed on the leaves or stems in any of the treatments. Leaf chlorophyll content was higher in the 40 mg L⁻¹ treatment than with 20 and 30 mg L⁻¹ DO treatments. Two weeks from the start of the experiment, roots in the 40 mg L⁻¹ treatment exhibited stunted growth, became thicker, and had fewer side and fine roots compared to roots in the lower levels of DO treatment. Almost all the measured growth parameters (fresh and dry weights of root, stem, and leaf, leaf area, stem diameter) were significantly reduced in plants grown in the 40 mg L⁻¹ treatment compared to plants in the lower level of DO treatments, except that the plant height increased with the increasing DO concentration. Root respiration increased linearly with increasing DO concentration; however, there was no effect on leaf net CO₂ exchange rate. It is suggested that it was safe to enrich root zone DO to as high as 30 mg L⁻¹, although the growth benefit was minor by increasing DO from ambient air saturated level (∼8.5 mg L⁻¹) to 30 mg L⁻¹. Higher than 30 mg L⁻¹ could cause reduction in tomato plant growth.     

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.     

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

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

Ethylene influences development and flowering of Ptilotus spp. in vitro and ex vitro

The genus Ptilotus has immense potential for ornamental horticulture but its commercial development has been hindered by propagation limitations. Poor seed quality and germination are reported. Cutting propagation is limited by cutting supply as the juvenile phase of Ptilotus is short. Micropropagation has been used in an attempt to overcome these difficulties but explants become floral in vitro and this causes plantlets to elongate. Ethephon has been used to control flowering of stock plants of many ornamental species. This study investigated the effect of ethephon applied to young (3-week-old, deflasked from tissue culture) and mature (1-year-old) Ptilotus plants in a greenhouse. A system of applying gaseous ethylene at 0, 100, 200 and 300 mg l⁻¹ to the headspace of in vitro plantlets in glass jars was developed and the response of in vitro plantlets to ethylene studied. One-year-old Ptilotus plants were treated with 500 mg l⁻¹ ethephon 2 days before pruning or 1 or 2 weeks after pruning. Ethephon application 2 days before pruning decreased the number of inflorescences and increased the number of shoots (compared to the control) but was phytotoxic. Ethephon applications of 150 or 300 mg l⁻¹ applied weekly or fortnightly to 3-week-old plants deflasked from tissue culture reduced plant height and number of inflorescences and at low concentrations increased the number of new shoots. A fortnightly application at 150 mg l⁻¹ is recommended. Previous reports on the effects of ethylene on inflorescence production on plantlets in vitro are limited. Our study showed that exposure of in vitro plantlets of P. nobilis to ethylene gas at 100 mg l⁻¹ for 1 h significantly increased the number of shoots and plant height but this did not occur for plantlets of P. spicatus. Plantlets of P. spicatus exposed to transient ethylene at 200 and 300 mg l⁻¹ showed significantly greater rooting (52.4%) than the control (13.6%).     

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.     

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.     

Cercis siliquastrum L., Malus trilobata Schneid and Acer syriacum Boin and Gaill water use as affected by two fertilizer rates

There has been an increased demand for landscaping plants in Lebanon as a result of numerous reconstruction projects. Sustainable landscape regulations have created a need for regionally adapted taxa, especially those with low water requirements. Therefore, water use of container-grown plants and the impact of fertilization on water use were studied in the following native species: Cercis siliquastrum L. (six mother trees), Malus trilobata Schneid (two mother trees) and Acer syriacum Boin and Gaill (one tree). Two-year-old containerized seedlings were grown at The Ohio State University (Columbus, USA) under two fertilizer rates: 25 or 100 mg N L⁻¹ of 21 N–3.1 P–5.9 K water soluble fertilizer. Water use estimates were made by saturating the containers early in the morning, allowing them to drain for 1 h, weighing them and re-weighing approximately 5 h later. Although there were differences in seedling heights, those grown at 25 mg N L⁻¹ were taller than those at 100, there were few differences in water use per seedling. In August, Cercis seedlings grown under 100 mg N L⁻¹ had higher height adjusted water use (g water cm⁻¹ height h⁻¹, a method for standardizing water use among different sized plants) than those grown under 25 mg L⁻¹. However, there were no differences in height adjusted water use in September attributed to fertilizer rates. In September, Acer seedlings had higher water use cm⁻² leaf surface area under 25 than 100 mg N L⁻¹. There were no differences in water use among the progeny from the six Cercis mother trees. However, the seedlings from one Malus tree had higher water use cm⁻² leaf surface area than those from the other tree, even though the extant trees were separated by less than 20 m.     

Zinc influence and salt stress on photosynthesis,water relations,and carbonic anhydrase activity in pistachio

A greenhouse study was conducted to evaluate the ameliorative effects of zinc (0, 5, 10 and 20 mg Zn kg⁻¹ soil) under saline (800, 1600, 2400 and 3200 mg NaCl kg⁻¹ soil) conditions on pistachio (Pistacia vera L. cv. Badami) seedlings’ photosynthetic parameters, carbonic anhydrase activity, protein and chlorophyll contents, and water relations. Zn deficiency resulted in a reduction of net photosynthetic rate and stomatal conductance. The quantum yield of photosystem II was reduced at zinc deficiency and salt stress. Zinc improved plant growth under salt-affected soil conditions. Increasing salinity in soil under Zn-deficient conditions, generally decreased carbonic anhydrase activity, protein, chlorophyll a and b contents. However, these adverse effects of salinity alleviated by increasing Zn levels up to 10 mg kg⁻¹ soil. Under increasing salinity, chlorophyll a/b ratio significantly increased. Zinc treatment influenced the relationship between relative water content and stomatal conductance, and between leaf water potential and stomatal conductance. It concluded that Zn may act as a scavenger of ROS for mitigating the injury on biomembranes under salt stress. Adequate Zn also prevents uptake and accumulation of Na in shoot, by increasing membrane integrity of root cells.     

Regeneration and characterization of plants derived from leaf in vitro culture of two sweet orange (Citrus sinensis (L.) Osbeck) cultivars

In the current work attempts were made to investigate culture of leaf explants derived from in vitro seedlings of two sweet orange (Citrus sinensis (L.) Osbeck) cultivars, Bingtangcheng and Valencia. Effects of several factors, including culture medium, lighting condition, explant age and genotype on regeneration response were examined based on three parameters, percentage of explants producing shoots, mean number of shoots per explant and shoot forming capacity. Culture of the explants on shoot-inducing media (SIM) composed of MT salts supplemented with different growth regulators gave rise to disparate shoot regeneration, in which SIM₁ (MT + 0.5 mg L⁻¹ BA + 0.5 mg L⁻¹ Kinetin + 0.1 mg L⁻¹ NAA + 3% sucrose + 0.8% agar, pH 5.8) was shown to be the most effective medium for direct induction of shoots from leaf explants. Highly significant difference in the response of shoot bud regeneration was noted between the two cultivars, with Bingtangcheng being more responsive than Valencia. Culture of explants from fully developed leaves led to better shoot regeneration capacity in comparison to undeveloped ones. However, the two lighting conditions used herein did not cause significant difference in shoot regeneration. Phenotypic observation and randomly amplified polymorphic DNA (RAPD) analysis confirmed that all the regenerated plants from both genotypes were genetically identical to their donor plants, suggesting absence of detectable genetic variation in the regenerated plants. The data presented here demonstrated that direct initiation of plants from leaf explants has been successfully accomplished. To our knowledge, this is the first report on direct regeneration of shoots from leaf explants in Citrus, which will provide an alternative source for citrus genetic manipulation in the future.     

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 synthetic auxins on fruit development of ‘Bing’ cherry (Prunus avium L.)

The main cherry cultivar grown in the warm climate of Israel, ‘Bing’, produces relatively small fruit. Over three consecutive years (2003–2005), application of 50 mg l⁻¹ 2,4-dichlorophenoxypropionic acid [2,4-DP; as its butoxyethyl ester (Power™)], 10 mg l⁻¹ 3,5,6-trichloro-2-pyridyloxyacetic acid [3,5,6-TPA; as the free acid (Maxim^®)], or 25 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) plus 30 mg l⁻¹ naphthaleneacetic acid (NAA; 0.3% Amigo™), at the beginning of pit-hardening when fruitlet diameter was ca. 13 mm caused appreciable and significant increases in fruit size and total yield, except when the crop load was heavy. Anatomical studies revealed that the main effect of these synthetic auxins was via direct stimulation of fruit cell enlargement. The above auxins had no negative effect on fruit quality, either at harvest or after 1 month of storage at 0 °C, or on return yield in the following year.