Antonio Carlos Torres

Garlic viral complex: identification of Potyviruses and Carlavirus in Central Brazil

Garlic viruses often occur in complex infections in nature. In this study, a garlic virus complex, collected in fields in Brazil, was purified. RT-PCR was performed using specific primers designed from the consensus regions of the coat protein genes of Onion yellow dwarf virus, a garlic strain (OYDV-G) and Leek yellow stripe virus (LYSV). cDNA of Garlic common latent virus (GCLV) was synthesized using oligo-dT and random primers. By these procedures individual garlic virus genomes were isolated and sequenced. The nucleotide sequence analysis associated with serological data reveals the presence of two Potyvirus OYDV-G and LYSV, and GCLV, a Carlavirus, simultaneously infecting garlic plants. Deduced amino acid sequences of the Brazilian isolates were compared with related viruses reported in different geographical regions of the world. The analysis showed closed relations considering the Brazilian isolates of OYDV-G and GCLV, and large divergence considering LYSV isolate. The detection of these virus species was confirmed by specific reactions observed when coat protein genes of the Brazilian isolates were used as probes in dot-blot and Southern blot hybridization assays. In field natural viral re-infection of virusfree garlic was evaluated.

Detection of three Allexivirus species infecting garlic in Brazil

Garlic viruses often occur in mixed infections under field conditions. In this study, garlic samples collected in three geographical areas of Brazil were tested by Dot-ELISA for the detection of allexiviruses using monoclonal specific antibodies to detect Garlic virus A (GarV-A), Garlic virus B (GarV-B), Garlic virus C (GarV-C) and a polyclonal antiserum able to detect the three virus species mentioned plus Garlic virus D (GarV-D). The detected viruses were biologically isolated by successive passages through Chenopodium quinoa. Reverse Transcriptase Polimerase Chain Reaction (RT-PCR) was performed using primers designed from specific regions of the coat protein genes of Japanese allexiviruses available in the Genetic Bank of National Center of Biotechnology Information (NCBI). By these procedures, individual garlic virus genomes were isolated and sequenced. The nucleotide and amino acid sequence analysis and the one with serological data revealed the presence of three distinct allexiviruses GarV-C, GarV-D and a recently described allexivirus, named Garlic mite-borne filamentous virus (GarMbFV), in Brazil.

Bioensaio para detecção de sementes de soja transgênicas tolerantes ao glifosato

Abstract – Glyphosate is a systemic, nonselective, postemergence herbicide that inhibits growth ofboth weeds and crop plants. Once inside the plant, glyphosate interferes with biosynthesis of aromaticamino acids phenylalanine, tyrosine, and tryptophan, by inhibiting the activity of5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), a key enzyme of the shikimate pathway.The objective of this work was to develop a simple, effective and inexpensible method for identificationof transgenic soybean tolerant to glyphosate. This technique consisted in germinating soybean seeds infilter paper moistened with 100 to 200 µM of glyphosate. Transgenic soybean seeds tolerant toglyphosate germinated normally in this solution and, between 7 and 10 days, started to develop aprimary root system. However non-transgenic seeds stopped primary root growth and emission ofsecondary roots.Index terms: Glycine max , herbicide resistance, transgenic plants, biosynthesis. Bioensaio para deteccao de sementes de soja transgenicas tolerantes ao glifosato

Absorção de macronutrientes por explantes de bananeira in vitro

The absorption of the nutrients (N, P, K, Ca, Mg and S) by banana (Musa sp.) cv. Prata Ana explants on the basic medium of Murashige & Skoog (1962) supplemented with sucrose (30 g/L) and BAP (3.5 mg/L) were evaluated at 0, 10, 20, 30, 40, 50 and 60 days after inoculation. The seven treatments were arranged on a completely randomized design with three replicates. The sequence of nutrient absorption by the explants was K > N > Ca ³ P > Mg @ S. The P was the nutrient with the fastest absorption rate and at the 30th day the explants had already absorbed 75% of the P from the medium. The P absorption stopped by the 50th day. The S absorption stopped at the 50th day with 66% of it remaining in the medium. The results suggested a close relationship between these two nutrients. The highest rates of nutrient absorption were observed during the first 20 days of culture. The rhizome, pseudostem and leaves behaved differently to the extraction and accumulation of nutrients.

Viral reinfection affecting bulb production in garlic after seven years of cultivation under field conditions

Six sequential field experiments were conducted from 1999 to 2002 to evaluate virus reinfection in garlic cv. Amarante and its effect on bulb production. The treatments in the year 1999 were: T1 – virus-free garlic-seed obtained by thermotherapy and meristem-tip culture and indexed for virus by immuno-sorbent electron microscopy (ISEM) under its first field cycle; T2, T3 and T4 – garlic-seed in the second, third and fourth field cycles, respectively; and C – standard garlic-seed from the grower (with no control of virus infection) as a control. In the years 2000 to 2002, a new plot of virus-free seed was added to the experiment and cloves from the previous treatments were again grown under field conditions. In the fourth year of experiments, the treatments comprised T1 (virus-free garlic seeds under the first field cycle) to T7 (garlic-seed in the sixth field cycle) and C. Two experiments were conducted in the years 1999 and 2000, at two locations and in the years 2001 and 2002 only one experiment per year at one location. The combined analysis of variance for all experiments indicated a significant difference between the treatments for plant height and yield. The bulbs were classified into commercial classes according the Brazilian regulation and the commercial classes 4–7 were 72 %, 60%, 59%, 53% and 35% of the total number of bulbs harvested, from T1 to T5, respectively. Virus reinfection at the end of the second field cycle, determined by serology using antisera against the most common viruses of garlic in Brazil reached 83%. Treatments T1–T7 yielded 118%, 79%, 57%, 51%, 39%, 33% and 31% higher than the control.

Shoot tip culture and thermotherapy for recovering virus-free plants of garlic.

Garlic shoot tip culture associated with dry heat thermotherapy (cloves exposed to 37°C for 35 days) were essential for recovering virus free plants of the cv Amarante. In this condition 70% of the explants developed in vitro and produced plants. A total of 77% of those plants was virus free when indexed by ISEM, which resulted in a final index of 54% of virus free plants from treated cloves. The percentage of regeneration decreased to 20% as the temperature increased up to 40°C. However 90% of those plants were virus free, leading to a final index of 18% virus free plants out of treated cloves.

Indução de embriogênese somática em genótipos de batata-doce

Hortalicas (‘92’, ‘94’, ‘184’, ‘188’, ‘202’, ‘319’, ‘370’, ‘374’, ‘442’, ‘449’, ‘549’, ‘594’, ‘605’, ‘633’, ‘643’, ‘645’, ‘PI3138463’ e as cultivares Jewel e White Star) foram utilizados como fonte de explantes para inducao da embriogenese somatica. Inicialmente foi testada, como referencia, a metodologia proposta por Cantliffe et al. (1987) para inducao de calo embriogenico na cultivar White Star de batata-doce. Explantes constituidos do meristema apical com dois primordios foliares e porcao subadjacente do caule foram excisados de plantas desenvolvidas em casa-devegetacao, e cultivados no meio basico contendo sais minerais de Murashige & Skoog (1962), com 3% sacarose, 0,2% de phytagel e, em mg/L: tiamina.HCl, 1,0; piridoxina.HCl, 0,5; acido nicotinico, 0,5; e glicina, 2,0. Esse meio foi suplementado com 2,0 mg/L de 2,4D. O pH do meio foi ajustado a 5,7 ± RESUMO A diferenciacao e o desenvolvimento de embrioes somaticos foram observados em 15 genotipos de batata-doce a partir de explantes de apices caulinares com um ou dois primordios foliares. A embriogenese foi induzida utilizando o meio composto de sais minerais MS e vitaminas, 3% de sacarose, 0,2% de phytagel, suplementado com 2,4-D (0,0; 0,5; 1,0 e 2,0 mg/L). As culturas foram mantidas no escuro a temperatura de 27°C. A adicao de 2,4-D foi necessaria para a inducao de calo embriogenico nos genotipos ‘92’, ‘94’, ‘184’, ‘188’, ‘319’, ‘370’, ‘374’, ‘442’, ‘449’, ‘5491, ‘594’, ‘645’, ‘PI3138463’, ‘White Star’ e ‘Jewel’. Os clones ‘169’, ‘202’, ‘605’, ‘633’ e ‘643’ nao responderam a inducao de calo embriogenico. Os calos embriogenicos apresentaram a coloracao amarelo claro, textura compacta e granular, enquanto que os calos nao embriogenicos apresentaram coloracao hialina e consistencia friavel. Os calos formaram-se na regiao periferica dos explantes. Nas condicoes estabelecidas, melhor producao de calo embriogenico foi obtida com 2,0 mg/L de 2,4-D. A transferencia desses calos para meio sem 2,4-D favoreceu o desenvolvimento dos embrioes para os estadios cordiforme, torpedo e cotiledonar.

Efeito da sacarose, cinetina, isopentenil adenina e zeatina no desenvolvimento de embriões de Heliconia rostrata in vitro

Embrioes provenientes de frutos maturos de Heliconia rostrata Ruiz & Pavon foram excisados e inoculados em meio de cultura contendo os sais basicos MS, vitaminas e sacarose. A adicao de sacarose foi essencial para o desenvolvimento dos embrioes. Em meio desprovido de sacarose os embrioes morreram em cultura. Concentracoes de 1%, 2% e 3% (p/v) de sacarose favoreceram o desenvolvimento dos embrioes. Concentracoes de 6%, 9% e 12% (p/v) de sacarose inibiram o crescimento dos embrioes. A adicao de cinetina, isopentenil adenina e zeatina nao favoreceram o crescimento e o desenvolvimento dos embrioes.

Genetic transformation of potato cultivar Achat by Agrobacterium tumefaciens

Nodal segment explants of potato (Solanum tuberosum L.) cv Achat were excised from in vitro growing plants, and transformed with Agrobacterium tumefaciens LBA4404 carrying the binary vector pBI121 with npt II and gus-intron genes. The regeneration of putative transformants was done in medium supplemented with 50 mg.L-1 of kanamycin. Putative transformants expressing kanamycin resistance and b-glucuronidase activity were identified. PCR analysis of these plants showed amplification of the npt II in gus+ plants. Southern blotting hybridization revealed the insertion of at least one copy of the npt II gene.

Características fotossintéticas de batata cv. Baronesa e seu genótipo transformado geneticamente para resistência ao PVY

Potato breeding is difficult and requires a great deal of time and energy. The use of recombinant DNA technology, with its potential capacity of isolating and transferring genes from any organism, allows incorporating in plants new characters of agricultural interest. However, consequences of the incorporation of determined genes on physiological characteristics are sometimes unknown. In this study we evaluated photosynthetic characteristics of potato plants genetically modified with resistance genes to PVY. Potato tubers of cv. Baronesa and its respective transformed genotype were planted in pots and kept in greenhouse. During the plant life cycle, parameters of chlorophyll fluorescence, net photosynthesis and potential photosynthesis were evaluated. Plants transformed with PVY resistant genes showed higher maximum photochemical efficiency and higher oxygen evolution rate, however the parameters of chlorophyll fluorescence and net photosynthesis were the same.