Comparative Studies of Net Photosynthesis and Transpiration of Some Citrus Species and Relatives

Comparisqns were made between ‘Campbell Valencia’ orange (Citrus sinensis (L.) Osbeck), and ‘Gabon Cherry-orange’ (Citropsis gabunensis (Engl.) Swing.), ‘Frost Lisbon’ lemon (Citrus limon (L.) Burm. f.) and ‘Eremolemon’ (Eremocitrus glauca (Lindl.) Swing. ×Citrus limon (L.) Burm. f.) and between diploid and autotetraploid ‘Lisbon’ lemons with respect to the influences of temperature and humidity on net photosynthesis and transpiration. Net photosynthesis, leaf conductance to water vapor and water-use-efficiency of Citropsis gabunensis were lower than with Citrus sinensis.‘Eremolemon’ had higher net photosynthesis and higher water-use-efficiency than ‘Lisbon’ lemon, but only small differences were observed between the two species in leaf conductance to water vapor. Small, nonsignificant, differences were observed between diploid and tetraploid ‘Lisbon’ lemons in responses of net photosynthesis and leaf conductance to temperature and humidity. Temperatures above 30°C and increases in vapor pressure difference caused declines in net photosynthesis and increases in vapor pressure difference resulted in decreases in leaf conductance to water vapor by all of the species used in these studies.     

Evaluation of Genetic Diversity in Lemons and Some of Their Relatives Based on SRAP and SSR Markers

Genetic diversity was evaluated by sequence-related amplified polymorphism (SRAP) and simple sequence repeat (SSR) markers among 45 lemons (Citrus limon (L.) Burm. f.), five citrons (Citrus medica L.), four rough lemons (Citrus jambhiri Lush), and two Citrus volkameriana accessions. Twenty-one SRAP primer combinations produced a total of 141 (77%) polymorphic fragments with an average of 6.7 fragments per primer combinations whereas 13 SSR primers produced a total of 26 (76%) polymorphic fragments with an average of 2.0 per primer. The unweighted pair-group method arithmetic average analysis as assessed with combined SRAP and SSR data demonstrated that the accessions had a similarity range from 0.65 to 1.00. Rough lemons and C. volkameriana accessions were relatively closely related. In lemon group, accessions from hybrid origin were distant from the others. We also applied principal components analysis (PCA) for a better presentation of relation among the accessions studies. Using PCA, 88.7% of the total variation in the original dimensions could be represented by just the two dimensions defined by the first two PCs. Although nearly all accessions could be distinguished, there was a low level of genetic diversity detected among lemon cultivars.     

Aconitase activity and expression during the development of lemon fruit

Citrus fruits are characterized by the accumulation of high levels of citric acid in the juice sac cells and a decline in acid level toward maturation. It has been suggested that changes in mitochondrial aconitase (EC 4.2.1.3) activity affect fruit acidity. Recently, a cytosolic aconitase (cyt-Aco) homologous to mammalian iron-regulated proteins was identified in plants, leading us to re-evaluate the role of aconitase in acid accumulation. Aconitase activity was studied in 2 contrasting citrus varieties, sweet lime ( Citrus limettioides Tan., low acid) and sour lemon ( Citrus limon var. Eureka, high acid). Two aconitase isozymes were detected. One declined early in sour lemon fruit development, but was constant throughout sweet lime fruit development. Its reduction in sour lemon was associated with a decrease in aconitase activity in the mitochondrial fraction. Another isozyme was detected in sour lemon toward maturation, and was associated with an increase in aconitase activity in the soluble fraction, suggesting a cytosolic localization. The cyt-Aco was cloned from lemon juice sac cells, but in contrast to the changes in isozyme activity, its expression was constant during fruit development. We present a model, which suggests that reduction of the mitochondrial aconitase activity plays a role in acid accumulation, while an increase in the cyt-Aco activity reduces acid level toward fruit maturation.     

The Dry Storage of Citrus Seeds

The survival of seeds of lemon (Citrus limon L.), lime [C. arantifolia(Christm.) Swing.] and sour orange (C. aurantium L.) was examinedunder a wide range of constant moisture contents and temperatures.Seed longevity was increased by decreasing the moisture contentand temperature of the storage environment. Maximum viabilitywas maintained in the combination of storage conditions includingthe lowest moisture content (5 per cent) and lowest temperature(–20 °C) investigated. The practicality of dry storageof citrus seed for genetic conservation is discussed. Citrus limon L., lemon, Citrus aurantifolia (Christm.), Swing, lime, Citrus aurantium L., sour orange, dry storage, moisture content, temperature, seed viability, seed longevity     

Relationships of leaf Fatty acids to cold hardening of citrus seedlings

Three cultivars of citrus with different sensitivities to freezing temperatures (citron, Citrus medica L.; rough lemon, C. limon Burm. F; sour orange, C. aurantium L.) were cold hardened for 4 weeks. Lipids from leaves of hardened and control seedlings were fractionated and analyzed for fatty acids. The absolute amount of triglycerides and phospholipids increased in the leaves upon hardening. With hardening, total linoleic acid also increased 141% in citron, 210% in rough lemon, and 233% in sour orange. Specific increases in linoleic acid were found in triglycerides, in the four phospholipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol), and in neutral lipids more polar than triglycerides. Trans-3-hexadecenoic acid was found only in phosphatidylglycerol.     

Cytogenetical studies inCitrus. Part I

The chromosome number ofCitrus penvisculata, Mayer lemon, Italian lemons. Kagzi Kalan lemon, Hill lemon and Nepale oblong lemon has been found to be 2n=18. Bridge-fragment configurations were observed inC. penvisculata, Mayer lemon, Italian round and Kagzi Kalan lemon. Univalents and pollen sterility have been observed in varying proportions in all of the seven species and varieties. Pollen sterility is probably due to presence of univalents and inversion heterozygosity     

Improved Transformation Efficiency in Citrus by Plasmolysis Treatment

Procedures for high efficiency production of transgenic citrus plants using an Agrobacterium tumefaciens system with plasmolysis treatment were developed. Longitudinally cut epicotyl segments of eight different citrus species [’Milam’ Rough lemon (Citrus jambhiri Lush), ‘Volkamer’ lemon (Citrus volkameriana L), Rangpur lime (Citrus limonia L), ‘Hamlin’ sweet orange (Citrus sinensis L Osbeck), ‘Duncan’ grapefruit (’Citrus paradisi’ Macf), Sour orange (Citrus aurantium L), ‘Cleopatra’ mandarin (Citrus reticulata Blanco) and Carrizo citrange (Citrus sinensis L Osbeck x Poncirus trifoliata L Raf) ] were plasmolyzed in different concentrations of sucrose and maltose [0, 3, 6, 8, 9, 10, 12 % (w/v) ] prior to Agrobacterium inoculation. Plasmolyzed epicotyl explants were cocultivated with either the hypervirulent Agrobacterium tumefaciens strain, the EHA-101 (harboring a binary vector pGA482GG) or Agl-1 (carrying pCAMBIA1303 vector). Both binary vectors contained neomycin phosphotransferase II (NPT II) and β-glucuronidase (GUS) genes. The binary vector, pCAMBIA1303 also contained a fused mGFP5 gene at the 3’ end of GUS gene as a reporter. Epicotyl explants of Rangpur lime, Rough and ‘Volkamer’ lemons plasmolyzed in 9–12 % maltose showed transient GUS gene expression comprising up to 95 % of the cut surface of explants, while Carrizo citrange showed 80 % expression when they were plasmolyzed in 6–10 % sucrose. On the other hand, epicotyl explants of ‘Hamlin’ sweet orange, Grapefruit, Sour orange and ‘Cleopatra’ mandarin showed transient GUS expession in 80–90 % of explants with 6–10 % sucrose. Basal portions of the regenerated putative transgenic shoots harvested from the cut surface of epicotyl explants within 2–3 months, were assayed for GUS, and apical portions were shoot-tip grafted in vivo for the production of whole plants. The transformation efficiencies in different species obtained are the highest so far reported for citrus.     

Citrus somatic hybridization with potential for improved blight and CTV resistance

Summary The production of five new somatic hybrids with potential for improved disease resistance is reported herein. Protoplast isolation, fusion, and plant regeneration was achieved from Caipira sweet orange (Citrus sinensis L. Osbeck) as an embryogenic parental source and Volkamer lemon (C. volkameriana Pasquale), Cleopatra mandarin (C. reticulata Blanco), and Rough lemon (C. jambhiri Lushington) as non-embryogenic parental sources. Fusion involving Cleopatra mandarin and Rangpur lime (C. limonia L. Osbeck) as embryogenic parental sources with Sour orange (C. aurantium L.) also resulted in somatic hybrid plants. Somatic hybridization was confirmed by leaf morphology evaluation, chromosome counting, and randomly amplified polymorphic DNA (RAPD) analyses. Somatic hybrids may combine complementary characteristics from both parental sources and have potential for tolerance to blight and citrus tristeza virus (CTV).     

Periodic Nocturnal Stomatal Opening of Citrus in a Steady Environment

In a steady environment, leaf stomates of sour orange (Citrus aurantium L.) remained closed in the dark, whereas those of rough lemon (Citrus jambhiri Lush) underwent several 20-minute periods of pronounced opening at intervals of 60–85 minutes. Peak nocturnal opening occurred about 10 minutes after the cycle began, as shown by a change in leaf diffusion resistance from 60 s cm^?1 (closed) to 2 s cm^minus;1 (open). This minimal leaf resistance equals that obtainable in rough lemon in strong illumination (26 kilolux). The transitory stomatal opening detected by leaf resistance measurements was corroborated by measurements of leaf temperature and leaf thickness. During nocturnal opening, leaf temperature was 2°C below the value representing non-cycling periods. Also, minimal leaf thickness coincided with maximal stomatal opening. The triggering mechanism for nocturnal stomatal opening appears to originate within the plant, since the environmental factors of air temperature, humidity, carbon dioxide concentration, and substrate (aerated water culture) were held steady.     

The introduction of cultivated citrus to Europe via Northern Africa and the Iberian Peninsula

The circumstances concerning the diffusion of the main cultivated citrus from their places of origin in Asia are studied here, showing that the citron (’Citrus medica L.) was the only one knew in Ancient times in Europe, while the lemon (C. limon [L.] Osbeck), lime (C. aurantiifolia [Christm.] Swingle), pomelo (C. maxima [Burm.] Merr.) and sour orange (C. x aurantium L.) were introduced to Europe by the Muslims via the Iberian Peninsula and Sicily, and that the grapefruit (C. paradisi Macfad.), mandarin (C. reticulata Blanco) and sweet orange (C. x aurantium L.) arrived to the West between the fifteenth and nineteenth centuries as a result of the trade with the British and Portuguese colonies.     

Growth and root hydraulic conductivity of several citrus rootstocks under salt and polyethylene glycol stresses

The water relations responses to salt of several important citrus rootstocks such as Swingle citrumelo, sour orange, and Milam lemon have not been studied in detail before. Studies were set up to compare growth and root hydraulic properties of these rootstocks to other citrus rootstocks by exposing them to NaCl and polyethylene glycol (PEG) stresses. Seedlings of 7 citrus rootstocks were irrigated for 5 months with nutrient solutions containing NaCl or PEG that had been adjusted to osmotic potentials of -0.10, -0.20 or -0.35 MPa. The 7 rootstocks studied were sour orange (Citrus aurantium), Cleopatra mandarin (Citrus reticulata Blanco), Swingle citrumelo (C. paradisi x P. trifoliata), Carrizo citrange (C. sinensis x P. trifoliata), rough lemon (Citrus jambhiri Lush), Milam lemon (C. jambhiri hybrid), and trifoliate orange (Poncirus trifoliata [L.] Raf.). In both shoot and root growth, Cleopatra mandarin and sour orange were the least sensitive to salt, Milam and trifoliate orange were the most sensitive, and rough lemon, Swingle, and Carrizo were intermediate in sensitivity. Even though the roots were exposed to solutions of equal osmotic potentials, plant growth and root conductivity were reduced more by the PEG treatments than the corresponding NaCl treatments. At -0.10 and -0.20 MPa, shoot and root dry weights were reduced 16 to 55% by NaCl and 24 to 68% by PEG. Shoot root ratio was lowered at the higher concentrations, particularly by PEG. There was a major decrease in root conductivity caused by NaCl at -0.10 MPa (19 to 30% in sour orange and Cleopatra mandarin and 78 to 85% in trifoliate orange and Milam). Conductivity decreased more at -0.20 and -0.35 MPa, but not proportionally as much as at -0.10 MPa. Root weight per unit length increased at the higher salt levels, particularly in trifoliate orange. Water flow rate through root systems followed the same trend as root conductivity; salt affected sour orange and Cleopatra mandarin the least and trifoliate orange and Milam the most. However, reductions in fibrous root length by salt treatment differed. Root lengths of Swingle and Carrizo were least affected by salt while sour orange. Milam, and rough lemon were the most affected. Hence, even though sour orange and Cleopatra mandarin were more tolerant than the other rootstocks in terms of water flow rate or root conductivity, these 2 rootstocks showed a proportionally greater decrease in root length than Carrizo, Swingle, or trifoliate orange.     

Oviposition behaviour and larval development of Anastrepha fraterculus from Argentina in citrus

Citrus peel physicochemical attributes are considered the main components conferring partial or even total resistance to fruit fly (Diptera: Tephritidae) infestation. Fruit fly females adapt their ovipositional strategies to overcome such resistance. Here, we explored the effects of citrus species (Rutaceae) on the ovipositional behaviour of the South American fruit fly, Anastrepha fraterculus (Wiedemann), and on its immature development. Particularly, we investigated the effects of (1) citrus species on oviposition behaviour and immature development, (2) citrus species on oviposition preference and on the location of the eggs at different depth in the citrus peel, and (3) harvest season and post‐harvest storage time on oviposition behaviour and immature development in lemon. Citrus species influenced ovipositional behaviour and affected survival of immature stages. Females laid eggs in lemon [Citrus limon (L.) Burm.], orange [Citrus sinensis (L.) Osbeck], and grapefruit (Citrus paradisi Macfadyen). In orange and lemon, larvae were found dead close to the oviposition areas, suggesting chemically mediated resistance mechanisms. Under choice conditions, females preferred grapefruit over lemon and bigger clutches were found in the layers where embryonic development is favoured. Unsuitability of lemon as a medium to complete development was neither affected by harvest season nor by storage time of the fruit after harvest. The physical and chemical characteristics of the peel were distinctive to each citrus species and may have affected the specific levels of resistance of these citrus species to infestation by A. fraterculus.     

Effect of 2,4-D and 4-CPPU on somatic embryogenesis from stigma and style transverse thin cell layers of Citrus

Callus induction, somatic embryogenesis and plant regeneration were obtained in lemon [Citrus limon (L.) Burm. cv `Femminello'] and sweet orange [C. sinensis (L.) Osb. cv `Washington Navel GS'] from cultures of stigma and style transverse thin cell layer explants [(t)TCLs]. Explants were cultured on 16 different media, based on the nutrients and vitamins of Murashige and Tucker medium (MT) supplemented with different combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and N-(2-chloro-4-pyridyl)-N-phenylurea (4-CPPU). Sucrose (146 mM) was used as the sole carbon source. Somatic embryos arose from callus at the surface of stigma and style (t)TCLs 3–5 months after culture initiation of both sweet orange and lemon. The percentages of embryo formation from style (t)TCLs ranged from 0% (the media containing 2,4-D) to 16.0% (the medium supplemented with 4 M 4-CPPU) for C. limon. Better results were obtained when stigma (t)TCLs from C. limon were used; in fact, percentages ranged from 0% on the media containing 2,4-D, with the only exception for the medium supplemented with 0.4 M 2,4-D, to 24.8% on medium with 4 M 4-CPPU. The embryogenic response of lemon (t)TCLs was usually higher than for sweet orange (t)TCLs. After about 3 months, somatic embryos developed into plantlets at high frequencies ranging from 53% to 75% for sweet orange and lemon style derived embryos, respectively.     

Soil temperature and flooding effects on two species of citrus

Summary Rough lemon (Citrus jambhiri Lush.) and sour orange (C. aurantium L.) seedlings were grown at constant soil temperatures of 16, 24, and 33 C for 3 months. Shoot and root growth of rough lemon was greatest at 33 C while growth of sour orange was greatest at 24 C. There were no significant effects of soil temperature on shoot: root ratio, leaf water potential or stomatal conductance. The hydraulic conductivity of intact root systems of both species was highest when seedlings were grown at 16 C. Thus, acclimation through greater root conductivity at low soil temperature may have compensated for decreased root growth at 16 C and negated effects of soil temperature on plant water relations. Half the plants growing at each soil temperature were subsequently flooded. Within 1 week, the soil redox potential (Eh) dropped below zero mV, reaching a minimum Eh of –250mV after 3 weeks of flooded conditions. Flooded plants exhibited lower root conductivity, a cessation of shoot growth, lower leaf water potentials, lower stomatal conductances, and visual sloughing of fibrous roots. Decreases in root conductivity in response to flooding were large enough to account for the observed decreases in stomatal conductance.Florida Agricultural Experiment Stations Journal Series No. 4080.     

An alternative method for the genetic transformation of sweet organce

Summary An alternative method for transforming sweet organe [Citrus sinensis (L.) Osbeck] has been developed. Plasmid DNA encoding the non-destructive selectable marker enhanced green fluorescent protein gene was introduced using polyethylene glycol into protoplasts of ‘Itaborai’ sweet organe isolated from an embryogenic nucellar-derived suspension culture. Following protoplast culture in liquid medium and transfer to solid medium, transformed calluses were identified via expression of the green fluorescent protein, physically separated from non-transformed tissue, and cultured on somatic embryogenesis induction medium. Transgenic plantlets were recovered from germinating somatic embryos and by in vitro rooting of shoots. To expedite transgenic plant recovery, regenerated shoots were also micrografted onto sour orange seedling rootstocks. Presence of the transgene in calluses and regenerated sweet organe plants was verified by gene amplification and Southern analyses. Potential advantages of this transformation system over the commonly used Agrobacterium methods for citrus are discussed.     

Water status indicators of lemon trees in response to flooding and recovery

Potted 2-year-old lemon trees [Citrus limon (L.) Burm. fil, cv. Verna] grafted on sour orange (C. aurantium L.) rootstock were subjected to flooding for 3 d. Control plants were irrigated daily to field capacity. Continuously (sap flow, trunk diameter fluctuations) and discretely (predawn and midday leaf water potential, leaf conductance) measured plant-based water status indicators were compared. The sensitivity of the maximum daily trunk shrinkage signal intensity to flooding and its behaviour during the recovery period demonstrated that this indicator is more feasible than the others for use in automatic irrigation. The responses to flooding of continuously and discretely measured plant-based water status indicators were very similar to those observed in response to drought stress indicating that it necessary to use soil water measurement automatic sensors to detect the cause of the stress. The results underlined the robustness of the compensation heat-pulse technique for estimating instantaneous and daily transpiration rates on flooding stress and recovery.     

GROWTH RESPONSES FROM WHOLE FRUIT AND FRUIT HALVES OF LEMON CULTURED IN VITRO

A comparative growth study was conducted on juice vesicles cultured in the form of various fruit explant types (equatorially bissected fruit halves, longitudinally bissected fruit halves, one-eighth sections of fruit, one-quarter sections of fruit, whole carpel segments, 2 or 3 mm thick equatorial slices of fruit, and 1 cm² fruit endocarp pieces) from 15 mm diam Citrus limon (L.) Burm. f. cv. Eureka lemons. Juice vesicles within equatorial fruit halves produced the least amount of callus. Furthermore, these juice vesicles grew similarly to juice vesicles occurring in the tree grown fruit. A study of cultured equatorial fruit halves using 10–45 mm diam lemons was then conducted. Fruit half cultures containing juice vesicles could be readily established from 15–45 mm diam lemons. Vesicles from 10 mm diam fruit halves, however, invariably produced callus. Vesicles cultured within fruit halves produced proportionately less callus as their fruit diam increased. Juice vesicles cultured in 15–30 mm diam fruits lost their original green color and turned opaque as they matured (i.e., after 3–6 months in culture). A method is also presented, whereby whole lemon fruits can be established and maintained in vitro. Lemons, 35–45 mm in diam, were the best explant sources for establishing whole fruit cultures. Juice vesicles in whole fruit cultures may remain viable for up to 8 months in culture.     

Stem and leaf water potentials, gas exchange, sap flow, and trunk diameter fluctuations for detecting water stress in lemon trees

The sensitivity of continuous (on a whole-day basis) and discretely (at midday) measured indicators of the plant water status in adult lemon trees in response to a cycle of water deprivation and recovery, and the feasibility of obtaining baselines for tree water status indicators was investigated in 30-year-old Fino lemon trees (Citrus limon (L.) Burm. fil.) grafted on sour orange (C. aurantium L.) rootstocks. Control plants (T0) were irrigated daily above their crop water requirements in order to obtain non-limiting soil water conditions, while T1 plants were subjected to water stress by withholding irrigation for 50 days, after which time irrigation was restored and plant recovery was studied for 16 days. In T0 plants the water relations and the plant symptoms confirmed that they had not suffered waterlogging. In contrast, T1 plants showed a substantial degree of water stress, which developed very slowly. Maximum daily trunk shrinkage (MDS) increased in response to water stress during the first 15 days of the experiment, but when the stem water potential (Ψ_stem) fell below −1.8 MPa, the MDS signal intensity decreased. However, Ψ_stem and sap flow (SF) signal intensities progressively increased during the water stress period. The results showed that MDS is a very suitable plant-based indicator for precise irrigation scheduling in adult lemon trees. Reference or baseline relationships for MDS, Ψ_stem, and SF measurements as a function of several parameters related to the evaporative demand of the atmosphere were obtained. This fact open up the possibility of considering a plant-based indicator measurement at a given time relative to the expected value under non-limiting water conditions, which can be calculated from the reference relationships.     

The influence of the composition of essential lemon oils on their antioxidant properties and the stability of the components

The antioxidant properties of various essential oils of lemon (Citrus limon L.) were studied by capillary gas liquid chromatography and compared with those of individual limonene and citral and their mixture in model systems. Some differences in resistance towards oxidation of the major components of lemon oils were ascertained and the synergetic effects in the antioxidant activity of the components and their stability were found.