Citrange Seedlings Research Articles

Irrigating with high boron concentration improved the physiological response of ‘Carrizo’ citrange seedlings under drought stress and recovery conditions

Irrigating with high boron concentration improved the physiological response of ‘Carrizo’ citrange seedlings under drought stress and recovery conditions

A transcriptional analysis reveals an extensive range of genes responsible for increasing the tolerance of Carrizo citrange to oxygen deficiency

Little information is available on the Citrus genus and its relatives with regard to their ability to tolerate oxygen deficiency, establishing physiological and structural modifications. In order to gain insight into how citrus rootstocks respond to low-oxygen stress, a transcriptomic analysis (using a custom microarray) was performed on Carrizo citrange (CC) seedlings. These seedlings were transformed with OsMybleu transcription factor (TF), known for inducing tolerance to oxygen deficiency, and compared with CC wildtype. They were flushed for 24 h with N2 and microarray, carrying out expressed sequence tags of Citrus and relatives isolated from the roots, was hybridized with RNA of roots before and after hypoxia treatment. The genes involved in fermentation, Krebs cycle, sugar metabolism, cell wall metabolism, hormones, and TFs all resulted significantly altered in response to hypoxia in both samples. Quantitative expression analysis was performed on 42 selected genes to validate microarray results. The outcome was that most of them were confirmed. The main results lead to the conclusion that CC is naturally tolerant to oxygen limitation. Transformed CC responded to hypoxia by activating the main genes which are known in other plants to be responsible for this type of tolerance such as pyruvate decarboxylase and alcohol dehydrogenase. Among TFs, several were also induced, such as an HDZipIII homologous to AtHB15, target of mir166, itself overexpressed exclusively in transformed CC under hypoxia compared with all other samples. The present manuscript represents one of the very few investigative works focused on hypoxia-responsive transcriptional networks in citrus.

A real-time PCR assay for detection and absolute quantitation of Citrus exocortis viroid in two sensitive and tolerant rootstocks

Citrus exocortis viroid (CEVd) is the causal agent of exocortis disease, the economically important viroid disease of citrus. In this research, CEVd copy number was quantified in sour orange (Citrus aurantium L.) and citrange (Poncirus trifoliata L. Raf. × Citrus sinensis L.) seedlings by absolute real-time PCR. A full-length in vitro transcript of a single-sequence CEVd-S1 variant was synthesized and mechanically inoculated into sour orange and Troyer citrange seedlings as tolerant and sensitive hosts, respectively. CEVd copy number was subsequently monitored at different times after inoculation in the green bark of infected seedlings for up to 16 weeks post inoculation (wpi). CEVd was detected at 3 wpi and totally increased up to 16 wpi. The copy number of CEVd per milligram of bark tissue in both host species was in the range of 103 to 104 at the third and sixth weeks post inoculation, without any significant differences. Although the viroid titer was remarkably higher in citrange (sensitive host) than in sour orange (tolerant host) at 10–16 wpi, we demonstrated that CEVd titer differed in citrus species depending on the time of sampling and host plant species.

Pathogenicity of Phyllosticta citricarpa Ascospores on Citrus spp.

Citrus black spot, caused by Phyllosticta citricarpa, is one of the most important fungal diseases in many citrus-growing regions with hot and humid summers. Ascospores and conidia are known to contribute to epidemic development of the disease. However, pathogenicity testing has never been done for pure ascospores produced from fully characterized P. citricarpa isolates, due to the inability to induce the sexual state in vitro. Recently, an in vitro mating technique was developed to readily produce pure P. citricarpa ascospores for use in host inoculation studies. To test the pathogenicity of P. citricarpa ascospores, we inoculated Troyer citrange leaves and Murcott tangor fruit with ascospores produced in vitro from characterized P. citricarpa isolates. Typical symptoms of citrus black spot occurred. Recovery of P. citricarpa isolates from symptomatic lesions and their characterization using genetic markers enabled us to identify recombinant genotypes among the isolates recovered from ascospore inoculations and, as such, fulfill Koch's postulates for ascospores. We have also identified Troyer citrange seedlings as a potential model system for citrus black spot inoculation studies, because it allows typical symptoms of citrus black spot to be expressed with a much shorter latent period than on fruit. This will facilitate future studies of epidemiological aspects of P. citricarpa ascospores relative to conidia and improve our understanding of the citrus black spot pathosystem. The susceptibility of Troyer citrange seedlings will also facilitate experimenting with disease management methods, aimed at reducing the impact of citrus black spot.

Changes in chemical composition and structure of root cell wall of citrus rootstock seedlings in response to boron deficiency by FTIR spectroscopy

ABSTRACTBoron (B) is an essential microelement for higher plants, and plays a role in cell wall formation. Citrange seedlings with different amounts of B were studied through Fourier-transform infrared spectroscopy (FTIR) analyses. The results showed that the growth and development of new roots were evidently inhibited by B deficiency. Boron-deficiency significantly increased cell wall biomass (CWB) as a percentage of root fresh weight and the ratio of B concentration in cell wall to the total B in roots. The findings from CWB FTIR spectra showed the band at 3429 cm−1 under control condition was shifted to 3442 cm−1 after being B deprived, suggesting that the mode of hydrogen bonding was changed by B deficiency. Boron deficiency clearly decreased the peak height of carboxylic ester band around 1741 cm−1, but increased that of COO− stretching band around 1400 cm−1, suggesting that the relative amount and degree of esterification of carboxylic groups was decreased and pectin content and structure was altered. These results demonstrate that changes in amount, structure, and assembly of root cell wall polymers may be either specific or adaptive responses of citrange seedlings to B deficiency and FTIR can be an appropriate method to study changes in cell wall under B deficiency.

Tetraploidy enhances the ability to exclude chloride from leaves in carrizo citrange seedlings

Tetraploid citrus seedlings are more tolerant to salt stress than diploid genotypes. To provide insight into the causes of differences in salt tolerance due to ploidy and thus to better understand Cl− exclusion mechanisms in citrus, diploid and tetraploid seedlings of Carrizo citrange (CC) were grown at 0 (control) and 40mM NaCl (salt-treated) medium for 20 days. Chloride uptake and root-to-shoot translocation rates were on average 1.4-fold higher in diploid than in tetraploid salt-treated plants, which resulted in a greater (1.6-fold) Cl− build up in the leaves of the former. Root hydraulic conductance and leaf transpiration rate were 58% and 17% lower, respectively, in tetraploid than in diploid control plants. Differences remained after salt treatment which reduced these parameters by 30–40% in both genotypes. Morphology of the root system was significantly influenced by ploidy. Tetraploid roots were less branched and with lower number of root tips than those of diploid plants. The cross-section diameter and area were lower in the diploid, and consequently specific root length was higher (1.7-fold) than in tetraploid plants. The exodermis in sections close to the root apex was broader and with higher deposition of suberin in cell walls in the tetraploid than in the diploid genotype. Net CO2 assimilation rate in tetraploid salt-treated seedlings was 1.5-fold higher than in diploid salt-treated plants, likely due to the loss of photosynthetic capacity of diploid plants induced by Cl− toxicity. Leaf damage was much higher, in terms of burnt area and defoliation, in diploid than in tetraploid salt-treated plants (8- and 6-fold, respectively). Salt treatment significantly reduced (37%) the dry weight of the diploid plants, but did not affect the tetraploids. In conclusion, tetraploid CC plants appear more tolerant to salinization and this effect seems mainly due to differences in morphological and histological traits of roots affecting hydraulic conductance and transpiration rate. These results may suggest that tetraploid CC used as rootstock could improve salt tolerance in citrus trees.

The response of different genotypes of citrus and relatives to Citrus psorosis virus inoculation

Damage caused by Citrus psorosis virus (CPsV) could be avoided obtaining resistant cultivars by breeding, but the sensitivity of most citrus hosts to CPsV is unknown. To find potential sources of resistance we inoculated 63 cultivars and hybrids of Citrus and related genera [Citrus (36), Microcitrus (5), Fortunella (6), Eremocitrus (1), Pleiospermium (1), Atalantia (2), Severinia (1), Clausena (1), Swinglea (1), Afraegle (1), Poncirus (1) and hybrids (7)] with the CPsV isolate PB-143 and monitored symptoms and CPsV infection by ELISA. Microcitrus inodora and Fortunella hindsii were symptomless but gave high ELISA values, suggesting tolerance to CPsV, whereas Citrus depresa, Clausena excavata, Cleopatra mandarin, sour orange, Carrizo citrange and CPB4475 citrumelo were ELISA negative, suggesting resistance. Further examination of CPsV infection by reverse transcription quantitative real time PCR (RT-qPCR) in Cleopatra mandarin, Poncirus trifoliata and Carrizo citrange seedlings inoculated with CPsV isolates P-121, PB-102 and PB-143, revealed different infection rates and CPsV accumulation, depending on the isolate and citrus genotype. Bud propagation of these genotypes on CPsV-inoculated sweet orange plants incited a bud union disorder that hindered flushing and scion growth, suggesting that the high viral load in the rootstock induced a hypersensitive-like reaction in the partially resistant scion.

First Report of Armillaria Root Rot Caused by Armillaria mellea Infecting Carrizo Citrange and Sour Orange Rootstocks in Turkey.

Citrus rootstocks, Carrizo citrange (Citrus sinensis [L.] Osb. × Poncirus trifoliata [L.] Raf.) and sour orange (C. aurantium L.) grown in containers filled with 5 liters of potting mix of 40% peat and 60% volcanic tuff declined in a 0.2-ha commercial nursery in Adana, Turkey, between 2004 and 2007. Seedlings with symptoms of root rot were found with an average disease incidence of 20% among 1,000 Carrizo citrange seedlings and 10% among 15,000 sour orange seedlings. The potting mixture preparation unit was located next to an oak tree (Quercus sp.) showing symptoms of Armillaria root rot. Six- to 12-month-old seedlings of both rootstocks were stunted and the crowns were necrotic with the presence of white mycelium. Mycelial fans were observed beneath the bark of infected roots and they expanded into the crown. The root systems and nearby potting mix contained rhizomorphs. Thus, Armillaria spp. was suspected as a possible causal agent. Three diseased crowns and three rhizomorphs were surface-sterilized with 1% NaClO for 1 min and cultured on benomyl-dichloran-streptomycin containing selective medium (3) at 25°C in the dark for 1 week. Six isolates transferred to 1.5% malt extract agar at 33°C in the dark for 7 weeks consistently yielded abundant aerial hyphae and mean diameter growth range was 4 to 21 mm and the mycelium margin was regular (1). To confirm pathogen identity, total DNA was extracted using the PowerSoil DNA Isolation Kit (MO BIO Laboratories, Inc., CA) directly from 7-day-old cultures grown in potato dextrose broth (PDB). The ribosomal DNA internal transcribed spacer (ITS) region was amplified by PCR using the primer pair ITS1 and ITS4 (5) and sequenced. The sequences were 99% identical to that of Armillaria mellea isolates from Japan (AB510880) and China (KF032535). This confirmed the identity of the causal agent as A. mellea (Vahl.) P. Kumm. Ten 3-month-old seedlings of Carrizo citrange and sour orange were transplanted into steam-sterilized potting mix and inoculated with wood pieces of oak (Quercus sp.) colonized by the fungus (two pieces for each container) (2). The oak wood pieces were sterilized prior to the colonization by the pathogen. Plants were maintained in a greenhouse (23 to 25°C) until symptoms appeared. Ten non-inoculated seedlings from each rootstock served as controls and were maintained in the same environment. After 4 months, the crowns of the seedlings developed necrotic areas and root systems contained rhizomorphs on all inoculated seedlings and fungus was re-isolated from crowns and rhizomorphs. All control plants remained disease-free and no fungus was re-isolated. A. mellea was reported to infect citrus rootstocks in Spain in 1999 (4). To our knowledge, this is the first report of Armillaria root rot caused by A. mellea infecting Carrizo citrange and sour orange rootstocks in Turkey. This indicates that citrus rootstocks could be at risk for infection and sterilization of the potting mix and good sanitation practices in nurseries are very important.

Study of the Effects of Compost and Nitrogen on Concentrations of Nutrients in Citrange Seedlings

A one-year factorial experiment using the randomized complete block design with three replications was carried out to study the effects of compost and nitrogen on the concentrations of nutrients in citrange seedlings. The treatments included various levels of the organic matter compost (zero, 2.5, 5, and 7.5%) and different levels of pure nitrogen from ammonium sulfate (zero, 20, 40, and 80 mg/kg soil). In all, 48 treatments were applied on citrange stocks. Comparison of mean squares for the effects of compost and nitrogen fertilizer on micronutrients showed that these effects were significant on potassium and manganese at the five percent probability level (and on iron, zinc, and copper at the one percent probability level), but were not significant on elements such as nitrogen, phosphorous, or magnesium, in citrange rootstocks. The maximum nitrogen and potassium concentrations in citrange leaves (2.90 and 1.83%, respectively) were achieved in the treatment of applying 7.5% compost with no nitrogen fertilizer. The highest phosphorous concentration in citrange leaves (0.80%) was observed in the treatment of applying 2.5% compost with no nitrogen fertilizer. The maximum potassium concentration in citrange leaves in the treatment of applying 7.5% compost and 40 mg/kg nitrogen was 0.74%. The highest iron and zinc concentrations in citrange leaves (201.4 mg/kg and 16.32 mg/kg, respectively) were achieved by the application of 2.5% compost and 40 mg/kg of the nitrogen fertilizer. The maximum potassium concentration in citrange leaves by applying 7.5% compost and 80 mg/kg of the nitrogen fertilizer was 32.35 mg/kg.The highest copper concentration in citrange leaves by the application of 5% compost with no nitrogen fertilizer was 15.24 mg/kg.

FUSARIUM SPP. ASSOCIATED TO CITRUS DRY ROOT ROT: AN EMERGING ISSUE FOR MEDITERRANEAN CITRICULTURE

FUSARIUM SPP. ASSOCIATED TO CITRUS DRY ROOT ROT: AN EMERGING ISSUE FOR MEDITERRANEAN CITRICULTURE

Lack of Evidence for Seed Transmission of 'Candidatus Liberibacter africanus' Associated with Greening (Huanglongbing) in Citrus in South Africa.

'Candidatus Liberibacter africanus' is associated with citrus greening (huanglongbing [HLB]) in South Africa. Various unpublished reports have suggested that the related bacterium 'Ca. L. asiaticus' associated with HLB in citrus might be seed transmissible based on real-time PCR results. Seed transmission poses a risk of long distance disease spread, especially with the dissemination of rootstock seed. Therefore, it was essential to determine whether 'Ca. L. africanus' is seed transmitted in citrus. Fruit from 26 'Ca. L. africanus'-infected branches of six citrus cultivars showing greening symptoms were collected and the seed was harvested. Cultivars included were Minneola tangelo (Citrus reticulata × C. paradisi); sweet oranges (C. sinensis) Premier midseason, Clanor midseason, and Olinda Valencia; Eureka lemon (C. limon) and Troyer citrange (Poncirus trifoliata × C. sinensis) rootstock variety. Branches bearing each fruit were collected and confirmed to contain 'Ca. L. africanus' by real-time PCR testing using Taqman probe HLBp and HLBaf and HLBr primers as described by Li et al. (3). The seed of each sample was sorted into five categories ranging from healthy looking to totally aborted based on their appearance before planting. Germination was done in seed trays under vector-free conditions at 24 to 28°C. Thereafter the seedlings were planted in small, plastic bags and monitored for greening-like symptoms or other abnormalities for up to 2 years. A slow-release, balanced fertilizer was applied and supplemented with micro-nutrient sprays for plant maintenance. Plants showing abnormal symptoms were potted into larger pots and closely monitored. These samples and a number of other seedlings showing growth abnormalities were tested for 'Ca. L. africanus' by real-time PCR as described above. In total, 1,570 seedlings were obtained. Some abnormal symptoms such as small chlorotic leaves, interveinal chlorosis, yellow veins, and stunting were seen in some seedlings. Most symptoms resembled deficiencies, and no blotchy mottle typical of 'Ca. L. africanus' infection was noted on any of the seedlings. Abnormal seedlings arose from normal and abortive seed. One hundred and eighteen of these seedlings (8 Minneola tangelo; 24 Premier midseason, 42 Clanor midseason, 33 Olinda Valencia, and 11 Troyer citrange seedlings) were individually tested using real-time PCR for 'Ca. L. africanus' detection. These seedlings had germinated from essentially healthy-looking seed (category 1) to seeds with severe abnormalities (category 5) and 33, 24, 23, 30, and 8 seedlings, respectively, were tested from each of the five seed categories. No samples tested positive with real-time PCR based on a positive/negative threshold Cq value of 35. Buds of some seedlings that yielded the lowest Cq values above 35 were grafted onto healthy 'Madam vinous' sweet orange (C. sinensis) seedlings and monitored for symptom development for 3 months. No symptoms developed and all these indicators also tested negative for 'Ca. L. africanus', indicating the absence of a transmissible agent. Just as other researchers (1,2) have recently indicated a lack of evidence for seed transmission of 'Ca. L. asiaticus', no seed transmission of 'Ca. L. africanus' could be demonstrated in this experiment either.

Effect of exogenously applied spermidine on growth and physiology of citrus rootstock Troyer citrange under saline conditions

The aim of this study was to assess the effects of spermidine (Spd) on ameliorating adverse effects of salinity in Troyer citrange seedlings. For this purpose, 6-month-old, uniform-sized seedlings of Troyer citrange were transplanted to plastic containers containing Hoagland nutrient solution. Addition of 75 mM NaCl into the nutrient solution reduced the plant growth, leaf chlorophyll content, chlorophyll fluorescence yield (Fv/Fm), net photosynthetic rate, respiration rate, and total Spd, N, and Ca^{2+} + Mg^{2+} contents of the leaves. It increased the leaf proline, total putrescine (Put), total spermine (Spm), Na^+, and Cl^- contents of the leaves. Addition of Spd (0.1 or 0.5 mM) to the saline nutrient solution and its weekly sprays (1 or 5 mM) on NaCl-stressed plants improved leaf number, chlorophyll content, Fv/Fm, net photosynthetic rate, and N content; increased total Spd and Spm contents; and reduced Na+ contents of the plants. Leaf Ca^{2+} + Mg^{2+} contents were slightly improved only when Spd (0.5 mM) was added to the saline nutrient solution. Leaf P and K^+ contents were not significantly affected by the salinity or Spd treatments. Among the polyamines, Put content was least affected, while there was a sharp increase in Spm content due to the salinity and Spd treatments. These results suggest involvement of Spm in the salinity tolerance of citrus rootstock Troyer citrange.

Substrate Type and Salinity Affect Growth Allocation, Tissue Ion Concentrations, and Physiological Responses of Carrizo Citrange Seedlings

To gain insight into salinity tolerance of citrus, we studied growth, leaf, and root Cl– concentrations and physiological responses of 5-month-old seedlings of the citrus rootstock Carrizo citrange [Citrus sinensis (L.) Osb. × Poncirus trifoliate L.] grown in a greenhouse in three different substrates: Candler sand soil, Floridana sandy clay soil, or a commercial soilless peat/perlite/vermiculite potting media. Plants were kept well-watered with a complete nutrient solution plus either no salt (control) or 50 mM NaCl for 9 weeks. Without salinity, substrate type did not affect total plant growth although there were differences in shoot/root dry weight ratio and mineral nutrient relationships attributable to substrate. Predawn leaf water potential, midday CO2 assimilation, and leaf water use efficiency were highest in seedlings grown in the soilless peat. The salt treatment decreased leaf and root growth, reduced leaf Ca2+, and increased leaf K+ concentration in all the three substrates. Overall, plant growth was negatively related to leaf Cl–. Leaf growth reductions were least in Candler-grown seedlings and greatest in Floridana soil as Cl– concentrations were lowest in Candler sand and highest in Floridana soil. Leaf Na+ was also highest in Floridana seedlings. In contrast to salt ions in leaves, roots of salinized seedlings in Candler sand had the highest Na+ and Cl– concentration. Salinity reduced net gas exchange of leaves similarly in all three substrates. Salinity reduced both leaf water potential and osmotic potential such that leaf turgor was increased. Thus, salinity-induced reductions in growth and net gas exchange were not the result of loss of turgor but more likely resulting from toxic ion accumulation in leaves. Based on the relative rankings of leaf growth and leaf Cl– concentrations, Carrizo seedlings from Candler sand had the highest salt tolerance and those grown in Floridana soil had the lowest salt tolerance. Substrate type should be considered when characterizing plant growth and physiological responses to salinity.

Leaf water relations and net gas exchange responses of salinized Carrizo citrange seedlings during drought stress and recovery.

Since salinity and drought stress can occur together, an assessment was made of their interacting effects on leaf water relations, osmotic adjustment and net gas exchange in seedlings of the relatively chloride-sensitive Carrizo citrange, Citrus sinensis x Poncirus trifoliata. Plants were fertilized with nutrient solution with or without additional 100 mm NaCl (salt and no-salt treatments). After 7 d, half of the plants were drought stressed by withholding irrigation water for 10 d. Thus, there were four treatments: salinized and non-salinized plants under drought-stress or well-watered conditions. After the drought period, plants from all stressed treatments were re-watered with nutrient solution without salt for 8 d to study recovery. Leaf water relations, gas exchange parameters, chlorophyll fluorescence, proline, quaternary ammonium compounds and leaf and root concentrations of Cl(-) and Na(+) were measured. Salinity increased leaf Cl(-) and Na(+) concentrations and decreased osmotic potential (Psi(pi)) such that leaf relative water content (RWC) was maintained during drought stress. However, in non-salinized drought-stressed plants, osmotic adjustment did not occur and RWC decreased. The salinity-induced osmotic adjustment was not related to any accumulation of proline, quaternary ammonium compounds or soluble sugars. Net CO(2) assimilation rate (A(CO2)) was reduced in leaves from all stressed treatments but the mechanisms were different. In non-salinized drought-stressed plants, lower A(CO2) was related to low RWC, whereas in salinized plants decreased A(CO2) was related to high levels of leaf Cl(-) and Na(+). A(CO2) recovered after irrigation in all the treatments except in previously salinized drought-stressed leaves which had lower RWC and less chlorophyll but maintained high levels of Cl(-), Na(+) and quaternary ammonium compounds after recovery. High leaf levels of Cl(-) and Na(+) after recovery apparently came from the roots. Plants preconditioned by salinity stress maintained a better leaf water status during drought stress due to osmotic adjustment and the accumulation of Cl(-) and Na(+). However, high levels of salt ions impeded recovery of leaf water status and photosynthesis after re-irrigation with non-saline water.

Influence of Chloride and Transpiration on Net15NO3−Uptake Rate byCitrus Roots

Three-month-old Carrizo citrange (hybrid of Citrus sinensis L. Osbeck×Poncirus trifoliata Blanco) seedlings were grown in controlled environment chambers in pots of fine sand. Plants were irrigated with either non-saline or saline solutions over a 3-week period. After these treatments, plants were transferred to vessels containing a 5 m m15NO3K (96% atom excess15N) solution, and transpiration as well as concentration of15N and Cl−in roots, stem and leaves were measured after 24 h. Transpiration and15NO3−uptake rates were inhibited after exposure to NaCl and the concentration of salt pre-treatment determined the intensity of this inhibitory effect. To determine the effect of transpiration on NO3−absorption, net15NO3−uptake rate was measured in salt stressed and non-stressed plants exposed to different light intensities or relative humidities and also in detached roots. Reduction in NO3−uptake was more closely related to Cl−antagonism from salt stress than to reduced transpiration rate.

Influence of growing media on the auxin‐induced growth of containerized ‘Carrizo’ citrange seedlings

Three growing media consisting of Sphagnum peat mixed with either a siliceous sand, a sandy soil or a fen peat, were studied in relation to the effect of indolebutyric acid (IBA), applied at 2.0 g I‐1 as a 10 s root dip, on the growth of containerized ‘Carrizo’ citrange seedlings. Plants grown in the fen peat mixture showed the highest root development and vegetative growth, but were insensitive to IBA. This auxin improved the growth of plants grown in both sand‐and soil‐based mixes. Similar trends were observed for nitrogen (N) and phosphorus (P) foliar concentrations, but potassium (K) concentrations in leaves were only increased in plants grown in the soil‐based growing medium.

Genetic transformation of lime (Citrus aurantifolia Swing.): factors affecting transformation and regeneration.

We have previously developed procedures for the efficient production of sweet orange (Citrus sinensis L. Osbeck) and Carrizo citrange (C. sinensis L. Osbeck×Poncirus trifoliata L. Raf.) transgenic plants using an Agrobacterium tumefaciens-mediated transformation and shoot tip grafting in vitro regeneration system. We now report on the optimization of the cocultivation, regeneration and selection conditions for efficient and reliable production of transgenic lime (C. aurantifolia Swing.) plants. Improved transformation frequencies were obtained by cocultivating the explants with Agrobacterium on feeder plates. Optimum regeneration of transgenic shoots was obtained by exposing the explants to darkness for 2 weeks and by using kanamycin at 100 mg/l as selective agent. Attempts to use geneticin as selection antibiotic were not successful. Shoot tip grafting of regenerated shoots on Troyer citrange seedlings resulted in 100% successful production of transgenic plants. The presence and expression of the transferred genes in the regenerated plants was verified by β-glucuronidase histochemical and fluorimetric assays, neomycin phosphotransferase ELISA assays, PCR and Southern analyses.

Salinity and calcium effects on emergence, growth and mineral composition of seedlings of eight citrus rootstocks

Salt tolerance of several citrus rootstocks during emergence and the early seedling stage of development was studied under greenhouse conditions. Salinity delayed and depressed seedling emergence, reduced shoot and root biomasses, and altered the mineral concentration of young citrus seedlings, although the magnitude of these changes differed between rootstocks. The addition of 50 mol m'3 NaCl to a nutrient solution delayed seedling emergence by three to five days with the exception of Troyer citrange (TC). Final emergence was reduced by less than 30% in Carrizo citrange (CC), TC, and Swingle cit- rumelo (SC), and by more than 65% in Ridge pineapple (RP), Cleopatra mandarin (CM), and rough lemon (RL). In the 50 mol m~3 NaCl treatment, shoot and root biomass were reduced by over 50%. Even in the no-salt control treatment, emergence of the first seedling and time to 50% emergence varied between rootstocks. Troyer citrange seedlings emerged fastest beginning ten days after sowing. Carrizo citrange and SC eme...

In-vitrograft-enhanced nucellar plant development in the monoembryonicCitrus grandisL.

SummaryFailure to obtain plants or shoots of sufficient size for removal to greenhouse has been a problem with nucellus cultures of some monoembryonic Citrus cultivars. The problem has been solved for C. grandis L. ‘Ma Dou Wen Dan’ by grafting emerging shoots onto ‘Troyer’ citrange seedlings in vitro. Grafted stems elongated rapidly, produced healthy leaves and manifested no shoot-tip necrosis. The elongated shoots were severed after two months and regrafted onto nursery stock in the greenhouse.

Growth of Citrus Rootstock (Carrizo Citrange) Seedlings During and After Long-term CO2 Enrichment

Abstract Carrizo citrange seedlings [Citrus sinensis (L.) Osbeck × Poncirus trifoliata (L.) Raf.] were grown in field enclosures for 17 weeks during 1984 at 330, 660, or 990 µl·liter-1 CO2 and then transferred to ambient air for 10 weeks to examine growth during and after these treatments. Immediately after the CO2 treatment period, plants grown at the highest CO2 level had accumulated about 120% more total dry matter than those at 330 µl·liter-1 CO2, compared to a mean increase of 67% at 660 µl·liter-1. Shoot length, leaf area, and leaf number were also increased 71%, 55%, and 39%, respectively, at 990 µl·liter-1 CO2 and somewhat less at 660 µl·liter-1. Percentage gains from CO2 enrichment decreased during the posttreatment period, but absolute differences were maintained or increased for at least 10 more weeks. During the treatment period, dry matter partitioning among leaves, stems, and roots did not vary with CO2 level, but root growth after transplanting and transfer to ambient air occurred at the expense of shoot growth in plants that had been grown at ambient CO2 levels. Plant growth, in terms of dry weight, shoot length, leaf area, and leaf number, was advanced 1.0 to 3.5 weeks after 17 weeks at elevated CO2 levels, and fresh weight per centimeter of stem length was about 1 month ahead. This last parameter can be used as an approximate indicator of readiness for scion budding, suggesting that CO2 enrichment could represent a considerable value to the citrus nursery industry. Data are also consistent with the hypothesis that a greater response to elevated CO2 is likely to occur where a high sink demand and/or low levels of leaf starch are maintained. Both such demand and low leaf starch occur in citrus seedlings, which have a pronounced juvenility period and capacity for continuous flush-type growth.