João Carlos Bespalhok Filho

Nitrogen Starvation, Salt and Heat Stress in Coffee (Coffea arabica L.): Identification and Validation of New Genes for qPCR Normalization

Abiotic stresses are among the most important factors that affect food production. One important step to face these environmental challenges is the transcriptional modulation. Quantitative real-time PCR is a rapid, sensitive, and reliable method for the detection of mRNAs and it has become a powerful tool to mitigate plant stress tolerance; however, suitable reference genes are required for data normalization. Reference genes for coffee plants during nitrogen starvation, salinity and heat stress have not yet been reported. We evaluated the expression stability of ten candidate reference genes using geNorm PLUS, NormFinder, and BestKeeper softwares, in plants submitted to nitrogen starvation, salt and heat stress. EF1, EF1α, GAPDH, MDH, and UBQ10 were ranked as the most stable genes in all stresses and software analyses, while RPL39 and RPII were classified as the less reliable references. For reference gene validation, the transcriptional pattern of a Coffea non-symbiotic hemoglobin (CaHb1) was analyzed using the two new recommended and the most unstable gene references for normalization. The most unstable gene may lead to incorrect interpretation of CaHb1 transcriptional analysis. Here, we recommend two new reference genes in Coffea for use in data normalization in abiotic stresses: MDH and EF1.

Transient gene expression of beta-glucuronidase in citrus thin epicotyl transversal sections using particle bombardment

Nosso objetivo e desenvolver um protocolo para transformacao genetica de Citrus atraves do bombardeamento de particulas usando cortes finos de epicotilo como explantes alvo. Neste trabalho foram otimizadas condicoes para a expressao transiente usando citrange carrizo (Citrus sinensis x Poncirus trifoliata) como modelo. Epicotilos de plântulas de citrange carrizo germinadas in vitro foram cortadas transversalmente em secoes de 1 mm e cultivadas em meio MS com 5 mM no escuro. Depois de uma semana, explantes foram bombardeados com o plasmidio pE2113-GUS usando um aparelho de alta pressao de helio (PDS 1000/He Bio-Rad). As melhores condicoes para a expressao transiente foram: particulas de tungstenio M-25, pressao de helio de 1550 psi, distância de 9 cm entre os explantes e DNA/particle holder e cultivo dos explantes em meio com alta osmolaridade (0,2 M manitol + 0,2 M sorbitol) 4 h antes e 20 h depois do bombardeamento. Usando estes parâmetros, uma media de 102 pontos azuis por bombardeamento (20 explantes/placa) foi obtida. Este protocolo esta sendo usado atualmente para transformacao de citrange Carrizo e adaptado para transformacao de laranja doce (Citrus sinensis).

Organogenesis and transient genetic transformation of the hybrid Eucalyptus grandis × Eucalyptus urophylla

The hybrid Eucalyptus grandis x Eucalyptus urophylla presents high levels of productivity and potential for use in paper, cellulose and fiber industries. The bud organogenesis from leaf explants of two clones of E. grandis × E. urophylla was studied in order to verify the effect of several factors: subculture duration on multiplication medium, type of explants, entire and half leaves: basal and apical portions, and duration of the culture on a regeneration medium. Differences in organogenic capacity of the two clones tested were observed. The explant most recommended for organogenesis is the basal section of the leaf collected from shoot clusters subcultured every 17 days. Moreover, the leaf explants must be transferred to a fresh bud induction medium every five days. This study also aimed at evaluating factors affecting the genetic transformation of leaf explants with the uidA gene, via co-cultivation with Agrobacterium tumefaciens, such as the pre-culture of the explants on a specific medium, the duration of their co-culture with the bacteria and the addition of acetosyringone to the culture media. The best conditions for the expression of the uidA gene were two days of pre-culture of the leaf tissues, three days of co-culture with the bacteria and the addition of acetosyringone in pre- and co-culture media.

Proposta e validação de escala para a ferrugem alaranjada da cana-de-açúcar

The aim of this work was to elaborate and validate a diagrammatic scale to quantify the severity of orange rust of sugarcane and to compare its efficiency in assessing the disease with the scale of brown rust. For the design of the scale, 120 sugarcane leaves were collected from a susceptible cultivar and the highest and the lowest severity founded were selected as extremes of scale. The intermediate levels were calculated according to the law of Weber-Fechner. The scale was proposed with nine levels of severity: 0,06; 0,14; 0,36; 0,89; 2,17; 5,18; 11,87; 24,92 and 45,00%. The validation was accomplished by eight appraisers who estimated the severity of 120 leaves in three assessments: without scale, with scale of brown rust and with the proposed diagrammatic scale. The scale proposed was more efficient than the scale of brown rust to assess the severity of this disease, providing an average coefficient of determination (R2) of 0,91 and absence of constant and systematic errors. With the scale of brown rust, the average R2 was 0,84 and three appraisers committed constant errors. Moreover, with the scale proposed, approximately 92% of the estimates of the appraisers had been in the range of variation of 5% of the value of real severity, while in the assessments without scale and with scale of brown rust, approximately 78% and 84% of the estimates were in that range, respectively.

Influence of calcium content of tissue on hyperhydricity and shoot-tip necrosis of in vitro regenerated shoots of Lavandula angustifolia Mill.

In the present study, the effects of two CaCl2.2H2O levels (440 and 1320 mg L-1) and two subcultures were evaluated on in vitro shoots of Lavandula angustifolia cv. Provence Blue. Ca2+ content of the apical, middle and basal portion of shoots was determined. Increasing CaCl2.2H2O level in the culture medium increased tissue Ca2+ content and decreased hyperhydricity. Shoot-tip necrosis also decreased with 1320 mg L-1 CaCl2.2H2O, but it did not occur in the second subculture. The middle and basal portion had higher Ca2+ content than apical portion. In non-hyperhydric tissues, there were smaller and more juxtaposed cells. Scanning electron microscopy of the leaves demonstrated that trichomes from in vitro leaf surface occurred in smaller quantities.

Evaluation of sugarcane genotypes and production environments in Paraná by GGE biplot and AMMI analysis

The purpose of this study was to evaluate sugarcane genotypes for the trait tons of sugar per hectare (TSH), stratifying five production environments in the state of Parana. The performance of 20 genotypes and 2 standard cultivars was analyzed in three consecutive growing seasons by the statistical methods AMMI and GGE Biplot. The GGE Biplot grouped the locations into two mega-environments and indicated the best-performing genotypes for each one, facilitating the selection of superior genotypes. Another advantage of GGEBiplot is the definition of an ideal genotype (G) and environment (E), serving as reference for the evaluation of genotypes and choice of environments with greater GE interaction. Both models indicated RB006970, RB855156 and RB855453 as the genotypes with highest TSH and Sao Pedro do Ivai as the environment with the greatest GE interaction. Both approaches explained a high percentage of the sum of squares, with a slight advantage of AMMI over GGE Biplot analysis.

Methods for assessment of pre-harvest sprouting in wheat cultivars

The objective of this work was to test methods for pre-harvest sprouting assessment in wheat cultivars. Fourteen wheat cultivars were grown in Londrina and Ponta Grossa municipalities, Parana state, Brazil. They were sampled at 10 and 17 days after physiological maturity and evaluated using the methods of germination by rainfall simulation (in a greenhouse), in-ear grain sprouting, and grains removed from the ears. The in-ear grain sprouting method allowed the differentiation of cultivars, but showed different resistance levels from the available description of cultivars. The sprouting of grain removed from the ears did not allow a reliable distinction of data on germination in any harvest date or location. The method of rainfall simulation is the most suitable for the assessment of cultivars as to pre-harvest sprouting, regardless of the sampling date and evaluated location.

Physical purity and germination of sugarcane seeds (caryopses) (Saccharum spp.)

Plant breeding is generally done through sexual reproduction even when the species is propagated asexually for commercial exploitation, as for example, in sugarcane. Therefore, the development of procedures to evaluate sugarcane seed viability is important for plant breeding programs. The objective of this research was to develop a methodology for analyzing the viability of sugarcane seeds (Saccharum spp.). Three crosses were used, two biparental crosses and one polycross. For the germination test study, two substrates (paper and sand) and three constant incubation temperatures (25 oC, 30 oC and 35 °C), in the presence of constant light and also an alternating temperatures (20-30 oC), with 8 hours light (30 oC) and 16 hours darkness (20 oC), were studied. Seedlings were evaluated every five days. The results demonstrated that temperature affected sugarcane seed germination with the most favorable conditions being the alternating temperature (20-30 oC) and the constant temperature of 30 °C on a paper substrate.

Indirect organogenesis from leaf explants of Eucalyptus benthamii x Eucalyptus dunnii and shoot multiplication

Background In Brazil, especially in the Southern region, stresses caused by cold and eventual frost are those that exert the most negative effect on the productivity of Eucalyptus spp. The genetic transformation techniques may contribute to forestry improvement programs in order to obtain genotypes expressing new interesting characteristics. They allow shortening the long breeding cycles and avoiding manipulation of adult trees. Their efficiency depends on establishment of regeneration procedures that allow the development of shoots from the transformed tissues. E. benthamiix E. dunnii hybrids have shown superiority to their parents concerning growth and frost tolerance [1], but no information about their in vitro organogenesis has been reported in the literature. The objective of this study was to evaluate the effect of some factors of culture medium on indirect organogenesis and shoot multiplication of anE. benthammi x E. dunnii clone. Methods In vitroestablished shoots,provided by EMBRAPA-Florestas (Colombo, PR, Brazil), were used as explant source. Cultures were maintained under white fluorescent tubes providing a photon flux density (PFD) of approximately 20 μmol m -2 s -1 , a 16-h photoperiod and a temperature of 25±2 °C. The cultures were performed in glass flasks containing 25 mL MS [2] medium supplemented with 1.11µM BA and sealed with rigid polypropylene caps. For the indirect organogenesis, leaves were excised from shoots at the petiole base, split into two halves and inoculated in culture media. The cultures were done in Petri dishes kept in a growth chamber in the dark throughout the experiment. The statistical design was performed in a factorial scheme (2:2:2) and a comparison was done between two culture media (MS-N/2, with half concentration of potassium and ammonium nitrates, and JADS [3] with 0.1 µM NAA, with and without PVP-40 (250 mg L -1 ) and two TDZ concentrations (0.1 and 0.5 µM). After 70 days, the percentages of explants forming callus, oxidized explants, explants producing anthocyanin, explants forming buds and shoots, and the number of shoots per explant were evaluated. For the multiplication test, the statistical design was performed in a factorial scheme (3:2) with three culture media (MS, WPM [4] and JADS, with 1.11µM BA) and two subcultures (28 and 56 days after the initial culture period). The analyzed variables for each subculture were: percentage of oxidation, of explants showing chlorosis, fresh weight and number of shoots. Results and discussion Regarding the oxidation, the higher rates (100%) were observed on JADS medium in presence or absence of PVP-40 and on MS-N/2 medium (68.3%) in presence of PVP-40. However, the JADS medium showed the highest percentage of callus formation (83.3%). In MS-N/2 medium the highest percentage of callus formation (55%) was found in the presence of PVP-40 and 0.5µM

Breeding of Sugarcane

Sugarcane is the main source for sugar production and the most important crop for energy production, as well as for byproducts like ethanol and fibers in the world. With a complex genome, the plant has its species from crosses between species of the genus Saccharum, which were the basis for sugarcane breeding programs worldwide. The production of sugarcane has increased worldwide due to breeding programs that have developed more productive clones for specific uses and adapted to different climatic conditions. The future objective of breeding programs is to develop sugarcane with high productivity, high sucrose content, drought tolerance, and high production of ethanol and biomass, i.e., plants with high fiber content and with cell walls easily broken to favor the production of ethanol from bagasse, efficient plants with low nitrogen fertilizer use, and others, and consequently to reduce environmental impacts. Currently, the demand for products derived from sugarcane is consistently increasing; the ethanol byproduct has been pointed out as one of the important sources to feed the demand for renewable energy in fossil and nonrenewable fuel substitution programs in different countries around the world. This chapter describes the genetic improvement of sugarcane and its current goals.