Carlos Henrique Siqueira de Carvalho

Agrobacterium-mediated transformation of sorghum: factors that affect transformation efficiency

The results presented in this work support the hypothesis that Agrobacterium-mediated transformation of sorghum is feasible, analogous to what has been demonstrated for other cereals such as rice, maize, barley and wheat. The four factors that we found most influenced transformation were: the sensitivity of immature sorghum embryos to Agrobacterium infection, the growth conditions of the donor plant, type of explant and co-cultivation medium. A major problem during the development of our protocol was a necrotic response which developed in explants after co-cultivation. Immature sorghum embryos proved to be very sensitive to Agrobacterium infection and we found that the level of embryo death after co-cultivation was the limiting step in improving transformation efficiency. The addition of coconut water to the co-cultivation medium, the use of vigorous and actively growing immature embryos and the removal of excess bacteria significantly improved the survival rate of sorghum embryos and was critical for successful transformation. Hygromycin phosphotransferase (hpt) proved to be a good selectable marker for sorghum. We also found that β-glucuronidase (GUS) activity was low in most of the transgenic plant tissues tested, although it was very high in immature inflorescences. Although promising, the overall transformation efficiency of the protocol is still low and further optimization will require particular attention to be given to the number of Agrobacterium in the inoculum and the selection of sorghum genotypes and explants less sensitive to Agrobacterium infection.

Otimização dos parâmetros de bombardeamento de partículas para a transformação genética de linhagens brasileiras de milho

The objective of this work was to develop a genetic transformation system for tropical maize genotypes via particle bombardment of immature zygotic embryos. Particle bombardment was carried out using a genetic construct with bar and uidA genes under control of CaMV35S promoter. The best conditions to transform maize tropical inbred lines L3 and L1345 were obtained when immature embryos were cultivated, prior to the bombardment, in higher osmolarity during 4 hours and bombarded at an acceleration helium gas pressure of 1,100 psi, two shots per plate, and a microcarrier flying distance of 6.6 cm. Transformation frequencies obtained using these conditions ranged from 0.9 to 2.31%. Integration of foreign genes into the genome of maize plants was confirmed by Southern blot analysis as well as bar and uidA gene expressions. The maize genetic transformation protocol developed in this work will possibly improve the efficiency to produce new transgenic tropical maize lines expressing desirable agronomic characteristics.

A Phosphate Transporter Promoter from Arabidopsis thaliana AtPHT1;4 Gene Drives Preferential Gene Expression in Transgenic Maize Roots Under Phosphorus Starvation

Phosphorus (P) stress responsive genes have been identified and characterized, including the high-affinity phosphate transporter AtPHT1;4 from Arabidopsis thaliana. This gene encodes a membrane protein that is primarily expressed in roots under phosphorus deficiency. A 2.3-kb promoter region from AtPHT1;4 has been fused with the β-glucuronidase (GUS) encoding gene and introduced into maize via biolistic bombardment to evaluate its spatiotemporal activity in a heterologous system. AtPHT1;4::GUS expression is detected preferentially in transgenic maize roots under P deficiency. Further analysis of transgenic plants has also revealed that GUS activity is higher in roots than in leaves by about sixfold. These results demonstrate the ability of AtPHT1;4 promoter to direct expression of the reporter gene in a monocot root system under P stress. This property of AtPHT1;4 promoter makes it useful to engineer maize plants to modify the soil’s rhizosphere and increase efficiency of P acquisition under P stress conditions.

Resposta de eixos embrionários de cupuaçu (Theobroma grandiflorum Schum.) à concentração de sais, doses de sacarose e renovação do meio de cultivo

The effects of concentration of salts and sucrose and culture medium renewal frequency on cupuassu (Theobroma grandiflorum Schum.) embryonic axes development were studied. Two salts concentrations of MS (100 and 50%), two sucrose levels (1.5 and 3%) and three explants subcultives (without medium renewal, renewal at 30 and 60 days) were tested, with NAA (0.5 mg/L) and BAP (0.3 mg/L) in the MS medium. The use of MS at full concentration, with addition of 3% of sucrose, and renewals of the medium at 30 day intervals promoted better cupuassu embryonic axes development.

Genetic and molecular characterization of Candystripe1 transposition events in sorghum

In sorghum, the Candystripe1 (Cs1) transposable element causes a variegated pericarp phenotype due to its excision activity from the y1 (yellow seed1) locus. The Y1 is a transcription regulator which is required for the biosynthesis of red 3-deoxyflavonoid pigments. Somatic variability in the transposition behavior of Cs1 was observed via biochemical analysis of 3-deoxyflavonoids in the leaf tissues of the Y1-cs alleles. Using somatic excisions of Cs1 as a tool, we establish that the Cs1 is active in young seedlings and the y1 locus is also functional in these tissues. Several somatic and germinal excision events were characterized and sequence analysis of independent events predominantly showed 2-bp footprints. Further, with the goal of understanding the properties of Cs1 that would facilitate the development of a transposon tagging system in sorghum, germinal excisions of Cs1 from y1 were used as a marker. Transposition of Cs1 was followed by characterization of putative insertion events. Genetic linkage between mutant phenotypes and the co-segregating restriction fragments of Cs1 provided additional evidence that Cs1 is an active transposable element in sorghum.

Chemical, Technological and Sensory Properties of Wheat Grains (Triticum aestivum L) as Affected by Gaseous Ozonation

Gaseous ozonation, an emerging technology, is applied to microbiological decontamination and to the degradation of residues and contaminants in wheat grains. However, due to its high oxidizing capacity, ozone may cause undesirable effects on the quality of grains or in their derivatives. This study aimed to evaluate the influence of ozone gas on wheat grain quality when exposed to different levels of ozone concentration, exposure time, and grain mass, using a full 23 factorial design. After the milling of ozonized grains, two fractions were evaluated: flour (A) and bran plus germ (B). In fraction A, the quality of the wheat flour was analyzed by both alveography and farinography. The flour extraction rate, falling number and gluten contents were also measured. The chemical and mineral profiles were determined in both fractions. A sensory evaluation, using the difference-from-control test, was applied to investigate the possible differences in aroma or in overall appearance of the flour obtained from the ozonized grains. The results showed that ozone concentration (60 mg/L) positively affected (p < 0.05) the toughness and the falling number of the flour. The other parameters of alveography, as well as farinography, gluten content, chemical composition, mineral, and sensory profiles were not affected (p > 0.05) by ozonation. This study demonstrated that gaseous ozonation, when applied under the conditions of 10 to 60 mg/L, from 2 to 5 h of exposure, grain mass from 2 to 5 kg, does not cause any negative impact on the wheat quality.

Indução de embriogênese somática em cupuaçu (Theobroma grandiflorum Schum.)

Young leaves of cupuassu (Theobroma grandiflorum Schum.) were used to induce somatic embryogenesis, which were cultivated initially in MS basal medium suplemented with 6.0 mg/L BAP and 0.5 mg/L IAA (established medium ) followed by MS basal medium supplemented with 2,20 mg/L TDZ (induction medium). Vascular region of explants showed intumescement three days after inoculation in established medium, which was followed by the formation of calli clusters. Calli mass were transferred to induction medium, and the structures with proembryogenic characteristics could be observed after one week. Scanning electron microscopy studies revealed structures with somatic embryos characteristics in the medium with TDZ.

Indução de calos embriogênicos em explantes de cupuaçuzeiro

It was studied the induction of embryogenics calli in cupuassu, in function of kind of explant and culture medium. Cotyledons segments and embryonic axes were tested and divided in three parts: region of plumule, radicule and hypocotile. The explants were cultivated in two different culture medium: 1) MS supplemented with 2,4-D (1 mg L-1) and Kinetin (0,25 mg L-1); 2) MS supplemented with NAA (5 mg L-1) and Kinetin (0,25 mg L-1). The hypocotile region demonstrated to be more responsive segment of the embryonic axe, forming callus with white and friable aspect. No somatic embryogenesis was evidenced in callus of cupuassu with auxines testeds in the medium.

DESENVOLVIMENTO DE CALOS EM EXPLANTES DE CUPUAÇUZEIRO (Theobroma grandiflorum Schum.) EM FUNÇÃO DA CONCENTRAÇÃO DE AUXINAS E DO MEIO LÍQUIDO

Objetivou-se estudar o efeito da concentracao de auxina e do meio liquido sobre o desenvolvimento de calos de cupuacuzeiro. Segmentos de eixos embrionarios e cotiledones, obtidos de frutos de cupuacu dos tipos Mamorana e Redondo, foram cultivados em 4 meios de cultura diferentes: 1) meio MS (50%), suplementado com 2,4-D (1; 2; 4; 8 mg/L); 2) sais N6 (SIGMA) (4 g/L), acrescidos de 2,4-D (0; 2; 4 mg/L) e ANA (0; 3; 5 mg/L); 3) igual ao anterior, suplementado apenas com ANA (3 mg/L); e 4) meio MS, acrescido com ANA (1 mM). Calos com aspecto branco e brilhante foram observados em segmentos de eixos embrionarios e cotiledones, cultivados nas menores concentracoes de meio 1 (1 e 2 mg/L), enquanto nas maiores concentracoes (4 e 8 mg/L) se observou a formacao de calos e massa calosa branco-opaca, em eixos embrionarios e em segmentos cotiledonares, estas estruturas tornaram-se escuras dentro de oito semanas. Usando o meio 2, um grande numero de raizes foram formadas, enquanto o mesmo meio suplementado apenas com ANA (3; 5 mg/L) originou uma massa calosa. A combinacao de ANA e 2,4-D, 3 e 2 mg/L, respectivamente, promoveu a formacao de calos brancos e raizes. A transferencia das culturas para meio liquido, sem regulador de crescimento, promoveu aumento de tamanho dos explantes e escurecimento dos mesmos. O cultivo desses explantes no meio 3 resultou no aparecimento de calos amarelos, com aspecto friavel, que permaneceram com a mesma aparencia no meio 4.

A panel of the most suitable reference genes for RT-qPCR expression studies of coffee: screening their stability under different conditions

The reliability of analyses using real-time quantitative polymerase chain reaction (RT-qPCR) depends on the selection of appropriate reference genes to correct for sample-to-sample and run-to-run variations. The aim of the present study was to select the most suitable reference genes for gene expression analyses in tissue samples from coffee, Coffea arabica L. (Arabica) grown under well-watered (WW) and water-deficit (WD) conditions and C. canephora Pierre ex A. Froehner (Robusta) grown under WW conditions. Expression profiles and stabilities were evaluated for 12 reference genes in different tissues from C. arabica and for 8 genes in tissues from C. canephora. The web-based RefFinder tool, which combines the geNorm, NormFinder, Bestkeeper, and Delta-Ct algorithms, was employed to assess the stability of the tested genes. The most stable reference genes identified for all tissues grouped (WW/WD) of C. arabica were clathrin adaptor protein medium subunit (AP47), ubiquitin (UBQ), 60S ribosomal protein L39 (RPL39), and elongation factor 1α (EF1α), while class III alcohol dehydrogenase (ADH2), β-actin (ACT), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and ubiquitin (UBQ) genes were the most stable for all tissues grouped (WW) of C. canephora tissues. Validation by the expression level analysis of CaACO-like demonstrated that the use of the best and the worst set of reference genes produced different expression results. The results reinforce the general assumption that there is no universal reference gene and that it is essential to select the most appropriate gene for each individual experiment to apply adequate normalization procedures of RT-qPCR data.