Plant regeneration from protoplast of Brazilian citrus cultivars

A procedure is described to regenerate plants from protoplasts of Brazilian citrus cultivars, after isolation, fusion and culture. Protoplasts were isolated from embryogenic cell suspension cultures and from leaf mesophyll of seedlings germinated in vitro. The enzyme solution for protoplast isolation was composed of mannitol (0.7 M), CaCl2 (24.5 mM), NaH2PO4 (0.92 mM), MES (6.15 mM), cellulase (Onozuka RS - Yakult, 1%), macerase (Onozuka R10 - Yakult, 1%) and pectolyase Y-23 (Seishin, 0.2%). Protoplast culture in liquid medium after chemical fusion lead to the formation of callus colonies further adapted to solid medium. Somatic embryo formation occurred spontaneously after two subcultures, on modified MT medium supplemented with 500 mg/L of malt extract. Well defined embryos were germinated in modified MT medium with addition of GA3 (2.0 muM) and malt extract (500 mg/L). Plant regeneration was also achieved by adventitious shoots obtained through direct organogenesis of not well defined embryos in modified MT medium with addition of malt extract (500 mg/L), BAP (1.32 muM), NAA (1.07 muM) and coconut water (10 mL/L). Plantlets were transferred to root medium. Rooted plants were transferred to a greenhouse for further adaptation and development.

Polyembryony and identification of Volkamerian lemon zygotic and nucellar seedlings using RAPD

The objectives of this work were to evaluate the frequency of polyembryony, and to identify zygotic and nucellar seedlings of Citrus volkameriana using RAPD. Twenty-five polyembryonic and eight monoembryonic seeds were cultivated in vitrofor six months. DNA from seedlings was extracted and used in combination with five RAPD primers to identify zygotic or nucellar origin of the seedlings. Environmental conditions of the year affected significantly (P<0.05) the morphological characteristics of fruitsand the number ofembryos per seed. Polyembryonic seeds ranged from 30.9%, 44.8% to 54.4% over three years. Morphological characteristic was not correlated with polyembryony. In vitro culture enable all embryos of each seed to grow, favoring the percentage of seedlings identified as zygotic. In polyembryonic and monoembryonic seeds, 25.9% and 87.5% of the seedlings, respectively, were sexually originated. In polyembryonic seeds, not all zygotic seedlings were produced by small embryos located at the micropyle.

Plant growth and phosphorus uptake in mycorrhizal rangpur lime seedlings under different levels of phosphorus

The objective of this work was to evaluate the response of rangpur lime (Citrus limonia) to arbuscular mycorrhiza (Glomus intraradices), under P levels ranging from low to excessive. Plants were grown in three levels of soluble P (25, 200 and 1,000 mg kg-1), either inoculated with Glomus intraradices or left noninoculated, evaluated at 30, 60, 90, 120 and 150 days after transplanting (DAT). Total dry weight, shoot P concentration and specific P uptake by roots increased in mycorrhizal plants with the doses of 25 and 200 mg kg-1 P at 90 DAT. With 1,000 mg kg-1 P, mycorrhizal plants had a transient growth depression at 90 and 120 DAT, and nonmycorrhizal effects on P uptake at any harvesting period. Root colonization and total external mycelium correlated positively with shoot P concentration and total dry weight at the two lowest P levels. Although the highest P level decreased root colonization, it did not affect total external mycelium to the same extent. As a result, a P availability imbalance affected negatively the mycorrhizal symbiosis and, consequently, the plant growth.

Citrus asymmetric somatic hybrids produced via fusion of gamma-irradiated and iodoacetamide-treated protoplasts

The objective of this study was to produce citrus somatic asymmetric hybrids by fusing gamma-irradiated protoplasts with iodoacetamide-treated protoplasts. Protoplasts were isolated from embryogenic suspension cells of grapefruit (Citrus paradisi Macfad.) cultivars Ruby Red and Flame, sweet oranges (C. sinensis Osbeck) 'Itaboraí', 'Natal', Valencia', and 'Succari', from 'Satsuma' (C. unshiu Marcow.) and 'Changsha' mandarin (C. reticulata Blanco) and 'Murcott' tangor (C. reticulata x C. sinensis). Donor protoplasts were exposed to gamma rays and receptor protoplasts were treated with 3 mmol L-1 iodoacetamide (IOA), and then they were fused for asymmetric hybridization. Asymmetric embryos were germinated, and the resulting shoots were either grafted onto sour orange, rough lemon or 'Swingle' (C. paradisi x Poncirus trifoliata) x 'Sunki' mandarin rootstock seedlings, or rooted after dipping their bases in indol-butyric acid (IBA) solution. The products were later acclimatized to greenhouse conditions. Ploidy was analyzed by flow cytometry, and hybridity was confirmed by amplified fragment length polymorphism (AFLP) analysis of plantlet DNAsamples. The best treatment was the donor-recipient fusion combination of 80 Gy-irradiated 'Ruby Red' protoplasts with 20 min IOA-treated 'Succari' protoplasts. Tetraploid and aneuploid plants were produced. Rooting recalcitrance was solved by dipping shoots' stems in 3,000 mg L-1 IBA solution for 10 min.

Evaluation of substrates on the emergence of "araticum-de-terra-fria" (Annona emarginata (Schltdl.) H. Rainer) Seedlings

Choosing a substrate is the determinant factor for the seedling producer; thus, the aim of this study was to evaluate the effect of different types of substrates on the emergence of "araticum-de-terra-fria" (Annona emarginata (Schltdl.) H. Rainer) seedlings. The experiment was carried out in a greenhouse and the experimental design was in randomized blocks, with three treatments and five replicates of 72 seeds per plot. The treatments consisted of the following substrates: coconut fiber, vermiculite and Plantmax® Citrus. The number of emerged seedlings was weekly counted for 105 days. Data regarding seedling height were obtained, and the emergence velocity index and mean time, besides total emergence percentage and that over time were calculated. Results from total mean emergence percentage, seedling height, emergence velocity index (EVI), and mean emergence time (MET) were subjected to analysis of variance and means were compared by the Tukey's test at 5% significance. The curves concerning the emergence percentage over time were fit by the logistic growth equation for each treatment and the means of each parameter (A, B, C) were compared by the Duncan's test at 5% significance. The substrates vermiculite led to the highest values of emergence percentage differing from the PlantMax® Citrus, but not of the coconut fiber, however the vermiculite promoted seedling height in a shorter time; therefore, this substrate is recommended for the initial development of "araticum-de-terra-fria" (Annona emarginata (Schltdl.) H. Rainer) seedlings.

Breeding, genetic and genomic of citrus for disease resistance

Although the citriculture is one of the most important economic activities in Brazil, it is based on a small number of varieties. This fact has contributed for the vulnerability of the culture regarding the phytosanitary problems. A higher number of varieties/genotypes with potential for commercial growing, either for the industry or fresh market, has been one of the main objectives of citrus breeding programs. The genetic breeding of citrus has improved, in the last decades, due to the possibility of an association between biotechnological tools and classical methods of breeding. The use of molecular markers for early selection of zygotic seedlings from controlled crosses resulted in the possibility of selection of a high number of new combination and, as a consequence, the establishment of a great number of hybrids in field experiments. The faster new tools are incorporated in the program, the faster is possibility to reach new genotypes that can be tested as a new variety. Good traits should be kept or incorporate, whereas bad traits have to be excluded or minimized in the new genotype. Scion and rootstock can not be considered separately, and graft compatibility, fruit quality and productivity are essential traits to be evaluated in the last stages of the program. The mapping of QTLs has favored breeding programs of several perennial species and in citrus it was possible to map several characteristics with qualitative and quantitative inheritance. The existence of linkage maps and QTLs already mapped, the development of EST and BAC library and the sequencing of the Citrus complete genome altogether make very demanding and urgent the exploration of such data to launch a wider genetic study of citrus. The rising of information on genome of several organisms has opened new approaches looking for integration between breeding, genetic and genome. Genome assisted selection (GAS) involves more than gene or complete genome sequencing and is becoming an import support in breeding programs of annual and perennial species. An huge information amount can be derivate from genome analysis. The use and benefit of such informations will depend on the genetic basis of the breeding program.

Woody Gall symptoms in different citrus species and varieties artificially colonized by the citrus brown aphid Toxoptera citricidus

Seedlings of 41 different citrus species and varieties were massively colonized with the citrus brown aphid Toxoptera citricidus, obtained from Pêra sweet orange (Citrus sinensis) trees, presenting symptoms of the "Capão Bonito" complex of the Citrus tristeza virus (CTV). The objective was to evaluate resistance or tolerance of the varieties to that virus complex, but even after eight months of inoculation no stem pitting was observed in the plants. Otherwise, the presence of galls similar to those induced by the vein enation-woody gall disease was observed in 73% of the plants of Volkamer Palermo (Citrus volkameriana), 60% of the Volkamer Catania 2, 2% of the Rangpur Lime D.22.30 (Citrus limonia), 13% of the Volkamer Australian Red, the Rangpur Lime hybrid, the Orlando tangelo (Citrus reticulata x Citrus paradisi) and the Florida Rough lemon (C. jambhiri), and 7% of the Carrizo citrange (Poncirus trifoliata x Citrus sinensis). The highest incidence and the largest gall size were observed in the Palermo Volkamer showing that this clone would be the most suitable to be used as an indicator plant in biological indexing tests for the disease. There are no previous reports in the literature about the occurrence of woody galls in Orlando tangelo and Carrizo citrange.

Energy demand in citrus production under varied operational efficiency values

Citrus orchards are very important in Brazil, especially in São Paulo State, where occupy an area of 600,000 ha approximately. To identify sustainability degree of citrus production system, an energy analysis allows evaluating efficiency of direct and indirect applied inputs. Thus, this study aimed to evaluate citrus production system under energetic point of view, in which invested energy is paid back with citrus production; being compared within three scenarios for operational field efficiency. As result, by sensitivity analysis was determined that fuel was the main energy demander, followed by pesticides and fertilizers. In operational work capacity analysis, all combinations between efficiency (minimum, typical and maximum) and yield levels became positive in the seventh year, except for the combination minimum efficiency and 10 % less yield, positive in the eighth year. The best combination (maximum efficiency and 10 % more yield) has promoted investment payoff around the sixth and seventh year. By this study, it is possible to determine the total energy demand to produce citrus and indentify the applied inputs that need more attention by the decision-makers. Labor and seedlings can be ommited for further studies with citrus, since they were irrelevant. Management of agricultural machinery may pose an important role on decreasing environmental impact of citrus production.

Poda e raleio manual de tangerineira (Citrus deliciosa Tenore) cv. Montenegrina no Sudoeste do Paraná

O bom calibre e a coloração adequada dos frutos cítricos para consumo in natura são importantes fatores de qualidade que elevam o seu consumo e melhoram os preços do produto. Este trabalho teve como objetivo avaliar o efeito de diferentes intensidades de raleio manual dos frutos e da poda sobre a produção e qualidade dos frutos da tangerina Montenegrina. O experimento foi realizado em pomar comercial no município de Dois Vizinhos, no Sudoeste do Paraná. O experimento bifatorial (raleio x poda) constituiu-se dos seguintes tratamentos. 1) sem raleio e sem poda (testemunha); 2) sem raleio, com poda; 3) 33% de raleio, sem poda; 4) 33% de raleio, com poda; 5) 66% de raleio, sem poda; e 6) 66% de raleio, com poda. O delineamento experimental foi de blocos completamente casualizados, com quatro repetições, sendo cada unidade experimental constituída por uma planta. Nas condições em que o experimento foi realizado, concluiu-se que o raleio nas intensidades de 33 e 66% promove aumento da massa fresca, melhora a cor da epiderme, aumenta o diâmetro e melhora a classificação comercial dos frutos da tangerina Montenegrina. A poda leve, de abertura e levantamento da base da copa, não contribui para a melhoria da qualidade dos frutos.

Growth and nutrient uptake of coffee seedlings cultivated in nutrient solution with and without silicon addition

In recent years, the application of silicon (Si) in crops, including coffee, has become a common practice. The objective of this study was to assess the silicon uptake by coffee seedlings and its effects on plant growth, water and macro and micronutrient uptake. The research was conducted using nutrient solution in a greenhouse at the Departamento de Fitotecnia da Universidade Federal de Viçosa, in a completely randomized design with two treatments (with and without silicon) and three replications. Each plot consisted of three plants grown in a 800 mL vessel containing the treatment solutions. At every three days, water consumption, the concentration of OH - and the depletion of Si and K were assessed in the nutrient solutions. After 33 days, the plants were assessed with regard to their fresh and dry weight of leaves, roots and stem, shoot height and total length of the plant (shoot and root). Number of leaves and internodes, and the content and accumulation of silicon, macro, and micronutrients were also determined. The consumption of water, the amount of potassium uptake and, biomass accumulation were greater in plants grown in solution without silicon addition. However, the concentration of OH- in the solution and the amount of silicon uptake were greater in plants grown in solution with added silicon. Silicon accumulation was greater in leaves than in stem and roots. Silicon decreased coffee plant accumulation of phosphorus, potassium, calcium, zinc, copper and iron.

Identification of 'Ubá' mango tree zygotic and nucellar seedlings using ISSR markers

Polyembryonic seeds are characterized by the development of over one embryo in the same seed, which can be zygotic and nucellar. The objective of this work was to identify the genetic origin, whether zygotic or nucellar, of seedlings of polyembryonic seeds of 'Ubá' mango tree using ISSR markers, and relating them with the vigor of the seedlings. Thus, mangos were harvested in Visconde do Rio Branco (accession 102) and Ubá (accessions 112, 138, 152 and 159), whose seeds were germinated in plastic trays filled with washed sand. Fifty days after sowing, seedlings from five seeds of each one of the accessions 102, 112, 138, 159 and from 10 seeds of the accession 152, were analyzed. These sseedlings were characterized and evaluated for plant height, stem circumference and mass of fresh aerial part and the most vigorous seedling was the one displaying at least two of these traits higher than the other seedlings from seed. Leaves were collected for genomic DNA extraction, which was amplified using seven ISSR primers previously selected based on the amplification profile and considering the number and resolution of fragments. Zygotic seedlings were found in 18 seeds, which were the most vigorous in six seeds. The results evidenced the existence of genetic variability in orchards using seedlings grown from seeds, because the farmer usually uses the most vigorous ones, assuming that this is of nucellar origin. These results also indicate that the most vigorous seedling are not always nucellar, inasmuch as of 20% of the total seeds evaluated, the zygotic seedling was the most vigorous.

Acclimation of croton and hibiscus seedlings in response to the application of indobultiric acid and humic acid for rooting

The vegetative propagation of ornamental plants can be accelerated by applying plant growth regulators. Amongst them, the use of auxins, plant hormones with physiological effects on cell elongation and rooting have stood out. Alternatively, the application of humic acids, bioactive fraction of soil organic matter, also results in increases in rooting cuttings of ornamental plants. The objective of this work was to study the growth characteristics and the nutritional contents of croton and hibiscus plants during acclimation of seedlings in response to different concentrations of indolebutyric acid (IBA) and humic acid (HA) applied to cuttings for rooting. The experiment was conducted in greenhouse, and the apical stem cuttings were treated with solutions with concentrations of 0, 250, 500, 1000 and 2000 mg L-1of IBA and 0, 10, 20, 30 and 40 mg L-1 of C from HA. At 45 days of rooting in carbonized rice husk, they were individually transferred to plastic bags of 2.0 dm3 containing a mixture of soil: sand: manure (2: 1: 1) as substrate. At 90 days of acclimation, the plants were collected for measurement of growth and nutritional variables. The results showed that the application of the IBA stimulates the absorption of nutrients and growth of croton cuttings and transplanted hibiscus, contributing to formation of vigorous seedlings. A similar response occurred with the application of HA in hibiscus cuttings

Growth and nutrient accumulation in mycorrhized papaya seedlings cultivated in a phosphorus-fertilized substrate

ABSTRACT The indiscriminate use of mineral fertilizers in papaya orchards has increased production costs, and the use of arbuscular mycorrhizal fungi is a promising alternative to reduce such expenses. Therefore, the present research aimed at studying the efficiency of arbuscular mycorrhizal fungi (AMF) on dry matter and nutrient accumulation in Sunrise Solo papaya seedlings, by applying doses of P2O5 (triple superphosphate) that are harmful to the symbiosis. The experiment was carried out in a protected environment and was set up in a randomized block design with four replications, and consisted of four P2O5 doses (0, 672, 1386 and 2100 mg dm-3), three mycorrhizal fungi species (Gigaspora margarita, Entrophospora colombiana and Scutellospora heterogama) and the control treatment (mycorrhiza-free). Shoot and root dry matter as well as nitrogen, phosphorus and potassium contents in leaf and root tissues were assessed. Mycorrhizal inoculation promoted a 30% increase in shoot dry matter in relation to the control treatment. Mycorrhizal fungi promoted increases in leaf and root nitrogen content up to 672 mg dm-3 P2O5. Inoculation of E. colombiana favored the highest gains in root and shoot dry matter. P2O5 fertilization increased foliar and root phosphorus content.

DISTINGUISHING Campa guianensis Aubl. FROM Campa procera D.C. (MELIACEAE) BY MORPHOLOGY OF YOUNG SEEDLINGS

Seedling morphology was studied in Campa guianensisAubl. and Carapa proceraD. C. from germination to 90 days age. In both species germination is hypogeal and cryptocotylar. Both have rare albino seedlings. Though both species have compound leaves when adult, C, proceraputs out an average total of six simple leaves at germination, while leaves of C. guianensisare compound at all stages. This is the best diagnostic character for separation of the two species at the young seedling stage. Not diagnostic, but none the less useful, is the fact, that polyembryonic seeds are often found in C. procera,but not observed in C guianensis.

Mites (Arachnida, Acari) on Citrus sinensis L. Osbeck orange trees in the state of Amazonas, Northern Brazil

Despite the importance of citriculture in Brazil, very little is known about mite populations in citrus crops in the Northern Region. In the municipality of Manaus, 12 sprayed sweet orange orchards were surveyed every two weeks during seven months to record mite species amount, and to describe the abundance and distribution of the most important species. The size and age of the orchards varied from 3,360 to 88,080 m² and seven to 25 years, respectively. In the fourteen sampling period, leaves, twigs and fruits were collected from 12 trees, one per orchard. In total, 3,360 leaves, 672 twigs and 1,344 fruits were sampled from 168 trees. Mites were manually extracted from the fruits, and by the washing method on leaves and twigs. We identified pests with the potential to cause economic loss. Fourteen species of phytophagous and mycophagous mites from Eriophyidae, Tarsonemidae, Tenuipalpidae, and Tetranychidae were recorded. Brevipalpus phoenicis (Geijskes 1939) and Phyllocoptruta oleivora (Ashm., 1879), the two commonest phytophagous mites in other Brazilian regions were dominant, showing that local orchards are susceptible to their infestation. Eleven predatory mites were recorded, comprising 10% of the mite population, belonging to Phytoseiidae and Ascidae. Phytoseiidae was the richest family, with ten species. The results are discussed in relation to the temporal variation aspects and habitat use of the most important species. Long-term research encompassing chemical applications followed by evaluations of the mite community are necessary for a better management of the orchards, taking into consideration the seasonal phenology of key pests.