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Vladimir Orbović, Manjul Dutt and Jude W. Grosser

The effects of fruit age on the seed quality and germination percentage of ‘Duncan’ and ‘Flame’ grapefruit and ‘Hamlin’ sweet orange were investigated. Our results suggested that seed germination varied from 98% to 100% for the two grapefruit cultivars and 85% to 100% for ‘Hamlin’ regardless of time of harvest. Within the first 5 months of the harvest season, chilling of ‘Duncan’ and ‘Hamlin’ seeds at 4 °C for 7 days after fruit sampling resulted in a lower germination percentage only with the ‘Hamlin’ seeds. Seed moisture content of all three cultivars varied slightly through the season and remained steady at 60% and 70% for batches of fresh seeds stored at room temperature or at 4 °C. Our results suggest that high seed viability and germination percentage can allow the use of these seeds for experimentation regardless of the time the fruit were picked during the harvest season.

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Vladimir Orbović, Diann Achor and James P. Syvertsen

Copper (Cu)-based fungicidal sprays are widely used on many crops although Cu sprays can be phytotoxic under some conditions. The mechanism of phytotoxicity is poorly understood but must involve toxic levels of Cu penetrating plant tissues. We studied the effect of different adjuvants on the deposition pattern of droplets and penetration of Cu (in Kocide fungicide) through isolated cuticles of ‘Marsh’ grapefruit leaves and ‘Valencia’ orange fruit. The addition of the silicone-based L-77 surfactant to the Kocide suspension markedly increased the spread of the droplets on cuticles and increased the penetration of Cu through fruit and abaxial leaf cuticles, both with stomatal pores, but not through astomatous adaxial leaf cuticles, which had much lower permeability. Urea and petroleum spray oil adjuvants had no effect on surface area of droplets or the penetration of Cu through leaf and fruit cuticles. Spray tank mixes of Cu fungicides with organosilicone surfactants should be avoided because these surfactants can enhance the penetration of Cu into citrus leaves and fruit thereby leading to phytotoxicity.

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Vladimir Orbovic, Diann Achor, Peter Petracek and James P. Syvertsen

We examined the effects of air temperature, relative humidity (RH), leaf age, and solution pH on penetration of urea through isolated cuticles of citrus leaves. Intact cuticles were obtained from adaxial surfaces of different aged grapefruit leaves. A finite dose diffusion system was used to follow movement of 14C-labeled-urea from solution droplets across cuticles throughout a 4-day period. The rate of urea penetration increased as temperature increased from 19 °C to 28 °C, but penetration was not further increased at 38 °C. Increasing RH increased droplet drying time and urea penetration at both 28 °C and 38 °C. Cuticle thickness, weight per area, and the contact angle of urea solution droplets increased as leaves aged. Cuticular permeability to urea decreased as leaf age increased from 3 weeks to 7 weeks, but permeability increased in cuticles from leaves older than 9 weeks. Contact angles decreased with increased urea solution concentration on six 7-week-old leaf surfaces, but solution concentration had no effect on contact angle on cuticles from younger and older leaves. Reducing pH of urea solution from pH 8 to pH 4 accelerated the loss of urea from breakdown, possibly due to hydrolysis.

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Vladimir Orbovic, John L. Jifon and James P. Syvertsen

Urea solutions, with or without non-ionic (X-77) and organosilicone (L-77) surfactant, were applied to Citrus leaves and isolated cuticles to examine adjuvant effects on urea uptake and leaf net gas exchange. When compared to X-77, L-77 exhibited superior features as a surfactant, resulting in smaller contact angles of droplets deposited on teflon slide. Both L-77 and X-77 had a strong effect on penetration rate of urea within first 20 min of experiment. Effect of L-77 on urea penetration rate decreased quickly within next 20 min, whereas the effect of X-77 was sustained over a 24-h period following application. When compared to solution of urea alone, addition of X-77 to urea resulted in significant increase of the total amount of urea that penetrated the cuticles. The effect of L-77 was smaller, although the total amount of urea that penetrated the cuticles within a 4-day period was similar for both surfactants. Solutions of either urea alone, urea+L-77 and urea+X-77, or L-77 alone, induced a negative effect on net CO2 assimilation (ACO2) for 4 to 24 h after they were sprayed onto leaves. X-77, when applied alone, had no effect on ACO2. Scanning electron microscopy revealed that 1 h after application, leaf surfaces treated with X-77 appeared to be heavily coated, as opposed to those treated with L-77, which appeared similar to untreated control leaves.

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Vladimir Orbovic, John L. Jifon and James P. Syvertsen

Although urea can be an effective adjuvant to foliar sprays, we examined effects of additional surfactants on urea penetration through leaf cuticles along with the effect of urea with and without surfactants on net gas exchange of leaves of `Marsh' grapefruit (Citrus paradisi Macf.) trees budded to Carrizo citrange (C. sinensis L. Osbeck × Poncirus trifoliata L. Raf.) rootstock. Various combinations of urea, a nonionic surfactant (X-77), and an organosilicone surfactant (L-77), were applied to grapefruit leaves and also to isolated adaxial cuticles. When compared to X-77, L-77 exhibited superior surfactant features with smaller contact angles of droplets deposited on a teflon slide. Both L-77 and X-77 initially increased penetration rate of urea through cuticles, but the effect of X-77 was sustained for a longer period of time. The total amount of urea which penetrated within a 4-day period, however, was similar after addition of either surfactant. Solutions of either urea, urea + L-77, urea + X-77, or L-77 alone decreased net assimilation of CO2 (ACO2) for 4 to 24 hours after spraying onto grapefruit leaves. A solution of X-77 alone had no effect on ACO2 over the 4-day period. Although reductions in ACO2 were similar following sprays of urea formulated with two different surfactants, the underlying mechanisms may not have been the same. For the urea + X-77 treatment, X-77 increased the inhibitory effects of urea on ACO2 indirectly by increasing penetration of urea into leaves. For the urea + L-77 formulation, effects of L-77 on ACO2 were 2-fold, direct by inhibiting ACO2 and indirect by increasing urea penetration. One hour after application, scanning electron microscopy (SEM) of leaf surfaces treated with X-77 revealed that they were heavily coated with the residue of the surfactant, whereas leaves treated with L-77 looked similar to nontreated leaves with no apparent residues on their surfaces. The amount of X-77 residue on the leaves was lower 24 hours after application than after 1 hour as observed by SEM.

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Vladimir Orbović, Diann Achor, Peter Petracek and James P. Syvertsen

Effects of air temperature, relative humidity (RH), and leaf age on penetration of urea through isolated leaf cuticles of `Marsh' grapefruit (Citrus×paradisi Macfad.) trees on `Carrizo' citrange (C. sinensis L. Osbeck × Poncirus trifoliata (L.) Raf. rootstock were examined. Intact cuticles were obtained from adaxial surfaces of `Marsh' grapefruit leaves of various ages. A finite dose diffusion system was used to follow movement of 14C-labeled urea from urea solution droplets across cuticles throughout a 4-day period. Within the first 4 to 6 hours after urea application, the rate of urea penetration increased as temperature increased from 19 to 28 °C, but there was no further increase at 38 °C. Increasing relative humidity increased urea penetration at 28 °C and 38 °C. Cuticle thickness, cuticle weight per area, and the contact angle of urea solution droplets increased as leaves aged. Cuticular permeability to urea decreased as leaf age increased from 3 to 7 weeks, but permeability increased in cuticles from leaves older than 9 weeks. Contact angles decreased with increased urea solution concentration on leaf surfaces that were 6 to 7 weeks old, but solution concentration had no effect on contact angle on cuticles from younger and older leaves. Changing urea solution pH from 8.0 to 4.0 could have an effect on the amount of urea penetrating the cuticle through the loss of urea from breakdown possibly due to hydrolysis. Results from this study define leaf age, environmental conditions, and formulation for maximum uptake of foliar-applied urea.

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Xiuli Shen, Vladimir Orbović, Manjul Dutt, William S. Castle and Frederick G. Gmitter Jr.

An efficient in vitro regeneration system through direct shoot organogenesis was established for Murraya paniculata (L.) Jack (Orange Jessamine). Epicotyls, leaves, roots, and cotyledons from in vitro-germinated seedlings and several plant growth regulators (PGRs) were evaluated for their effects on plant regeneration. Longitudinally cut epicotyl segments were observed to be the optimal explants followed by uncut epicotyls (not longitudinally cut). Roots, leaves, and cotyledons were not suitable as explants as a result of little or no shoot induction. Adventitious shoot induction was enhanced by the addition of 6-benzyladenine (BA). The highest percentage of shoot induction (87%) and the greatest number of shoots per explant (12.7) occurred on Murashige and Skoog (MS) medium supplemented with 15 μM BA from longitudinally cut epicotyls followed by 5.2 shoots per explant from uncut epicotyls. Optimal concentration of gibberellic acid (GA3) for shoot elongation was observed to be 15 μM. Eighty-five percent of the regenerated shoots produced roots with an average of three roots per shoot on MS medium supplemented with 5 μM indole-3-butyric acid (IBA). Our protocol for direct shoot organogenesis can potentially lead to the development of a robust method for production of transgenic plants of M. paniculata through Agrobacterium-mediated genetic transformation.

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Milica Ćalović, Chunxian Chen, Qibin Yu, Vladimir Orbović, Frederick G. Gmitter Jr and Jude W. Grosser

Six mandarin cultivars, Ponkan (Citrus reticulata), Willowleaf (Citrus deliciosa), Kinnow (Citrus nobilis × C. deliciosa), Murcott (purported C. reticulata × Citrus sinensis), W. Murcott [purported (C. reticulata × C. sinensis) × C. reticulata)], and Snack (purported C. reticulata hybrid), were used in protoplast fusion with different parental combinations to generate somatic hybrids. Sixty-five somatic regenerants were obtained using optimized formulation of enzymes and molecular weight of polyethylene glycol for improved protoplast yield and heterokaryon fusion rate, respectively. Flow cytometry was used to determine the ploidy level of somatic regenerants, and nuclear expressed sequence tag–simple sequence repeat (EST-SSR) markers to determine their parental source. Of the 65 somatic regenerants, 46 were identified as autotetraploids, 18 allotetraploids, and one undefined. The EST-SSR markers also revealed that some ‘W. Murcott’ embryogenic callus lines that were presumed to be of nucellar origin were actually derived unexpectedly from individual ovules of zygotic origin. These mandarin-derived tetraploids are valuable as potential breeding parents for interploid crosses with an aim at seedlessness and easy-peeling traits.