Removal of bacterial dextran in sugarcane juice by Talaromyces minioluteus dextranase expressed constitutively in Pichia pastoris.

A gene construct encoding the mature region of Talaromyces minioluteus dextranase (EC 3.2.1.11) fused to the Saccharomyces cerevisiae SUC2 signal sequence was expressed in Pichia pastoris under the constitutive glyceraldehyde 3-phosphate dehydrogenase promoter (pGAP). The increase of the transgene dosage from one to two and four copies enhanced proportionally the extracellular yield of the recombinant enzyme (r-TmDEX) without inhibiting cell growth. The volumetric productivity of the four-copy clone in fed batch fermentation (51 h) using molasses as carbon source was 1706 U/L/h. The secreted N-glycosylated r-TmDEX was optimally active at pH 4.5-5.5 and temperature 50-60 °C. The addition of sucrose (600 g/L) as a stabilizer retained intact the r-TmDEX activity after 1-h incubation at 50-60 °C and pH 5.5. Bacterial dextran in deteriorated sugarcane juice was completely removed by applying a crude preparation of secreted r-TmDEX. The high yield of r-TmDEX in methanol-free cultures and the low cost of the fed batch fermentation make the P. pastoris pGAP-based expression system appropriate for the large scale production of dextranase and its use for dextran removal at sugar mills.

A transformation procedure for recalcitrant tomato by addressing transgenic plant-recovery limiting factors.

Agrobacterium tumefaciens technology is the battle horse for tomato genetic transformation. However, tomato varieties with low regeneration capacity are very difficult to transform. In the past, tomato transformation through Agrobacterium infection was focused on varieties capable of high regeneration yield, while successful transformation of low regenerable cultivars has not been reported. The genotype response to tissue culture conditions is believed to drive the frequency of regeneration of transgenic plant, whereas the capacity for cell proliferation could determine the transformation efficiency through this technology. The Campbell-28 cultivar is an example of constraints arising from a high morphogenetic potential with low conversion compared to normal plants. In the present work the roles that contribute to improved transgenic plant recovery from this recalcitrant variety were explored for factors like Agrobacterium concentration and antibiotics for bacterial removal and transformant selection. Analysis of the efficiency from independent transformation experiments revealed a more than twofold increase of transformant regeneration after selection on ammonium glufosinate compared to kanamycin selection, showing a transformation efficiency of 21.5%.

Efecto de diferentes métodos de cultivo sobre la regeneración in vitro de papa (Solanum tuberosum L.) de la vaeriedad comerciall Chieftain

Se evaluó la capacidad de regeneración de explantes de hoja, tubérculos y segmentos de tallo de la variedad comercial Chieftan, utilizando dos métodos: A, el mismo medio de cultivo desde la iniciación de los callos hasta la formación de los brotes y B, transferencia de los callos a un medio de cultivo libre de auxinas antes de la formación de los brotes. La mayor frecuencia de regeneración se obtuvo de discos de tubérculo en el medio (MRT-6) (método A) con 3,28 brotes por explante, seguido por 2,5 brotes en segmentos de tallo (medios MRE-4 y MRE-6, método B) y 0,8 brotes a partir de discos de hoja (medio MRH-3, método A). La combinación de 1 mg/L de nitrato de plata con 5 g/L de carbón activado, durante la propagación in vitro, mostró diferencias significativas tanto en el número de hojas por planta como en su calidad (AU)