The M4 insulator, the TM2 matrix attachment region, and the double copy of the heavy chain gene contribute to the enhanced accumulation of the PHB-01 antibody in tobacco plants.

The expression of recombinant proteins in plants is a valuable alternative to bioreactors using mammalian cell systems. Ease of scaling, and their inability to host human pathogens, enhance the use of plants to generate complex therapeutic products such as monoclonal antibodies. However, stably transformed plants expressing antibodies normally have a poor accumulation of these proteins that probably arise from the negative positional effects of their flanking chromatin. The induction of boundaries between the transgenes and the surrounding DNA using matrix attachment regions (MAR) and insulator elements may minimize these effects. With the PHB-01 antibody as a model, we demonstrated that the insertion of DNA elements, the TM2 (MAR) and M4 insulator, flanking the transcriptional cassettes that encode the light and heavy chains of the PHB-01 antibody, increased the protein accumulation that remained stable in the first plant progeny. The M4 insulator had a stronger effect than the TM2, with over a twofold increase compared to the standard construction. This effect was probably associated with an enhancer-promoter interference. Moreover, transgenic plants harboring two transcriptional units encoding for the PHB-01 heavy chain combined with both TM2 and M4 elements enhanced the accumulation of the antibody. In summary, the M4 combined with a double transcriptional unit of the heavy chain may be a suitable strategy for potentiating PHB-01 production in tobacco plants.

Field Trial and Molecular Characterization of RNAi-Transgenic Tomato Plants That Exhibit Resistance to Tomato Yellow Leaf Curl Geminivirus.

RNA interference (RNAi) is a widely used approach to generate virus-resistant transgenic crops. However, issues of agricultural importance like the long-term durability of RNAi-mediated resistance under field conditions and the potential side effects provoked in the plant by the stable RNAi expression remain poorly investigated. Here, we performed field trials and molecular characterization studies of two homozygous transgenic tomato lines, with different selection markers, expressing an intron-hairpin RNA cognate to the Tomato yellow leaf curl virus (TYLCV) C1 gene. The tested F6 and F4 progenies of the respective kanamycin- and basta-resistant plants exhibited unchanged field resistance to TYLCV and stably expressed the transgene-derived short interfering RNA (siRNAs) to represent 6 to 8% of the total plant small RNAs. This value outnumbered the average percentage of viral siRNAs in the nontransformed plants exposed to TYLCV-infested whiteflies. As a result of the RNAi transgene expression, a common set of up- and downregulated genes was revealed in the transcriptome profile of the plants selected from either of the two transgenic events. A previously unidentified geminivirus causing no symptoms of viral disease was detected in some of the transgenic plants. The novel virus acquired V1 and V2 genes from TYLCV and C1, C2, C3, and C4 genes from a distantly related geminivirus and, thereby, it could evade the repressive sequence-specific action of transgene-derived siRNAs. Our findings shed light on the mechanisms of siRNA-directed antiviral silencing in transgenic plants and highlight the applicability limitations of this technology as it may alter the transcriptional pattern of nontarget genes.

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%.

Intron-hairpin RNA derived from replication associated protein C1 gene confers immunity to tomato yellow leaf curl virus infection in transgenic tomato plants.

The whitefly-transmitted Tomato Yellow Leaf Curl Virus (TYLCV) is the major pathogen of tomato crop in Cuba and one of the most outstanding viral diseases of plants worldwide. In this work, we have developed transgenic tomato plants, transformed with an intron-hairpin genetic construction to induce post- transcriptional gene silencing against the early TYLCV replication associated protein gene (C1). The intron-hairpin RNA produced involves 726 nts of the 3' end of the TYLCV C1 gene as the arms of the hairpin, and the castor bean catalase intron. Transgenic tomato plants belonging to line 126, which harbor a single transgene copy, showed immunity to TYLCV, even in extreme conditions of infection (4-leaf-stage plants and 300 to many hundreds viruliferous whiteflies per plant during 60 days). Dot blot hybridization of these plants showed no TYLCV DNA presence 60 days after inoculation. Small interfering RNA molecules were detected in both inoculated and non-inoculated plants from line 126. These transgenic tomato plants of the otherwise very TYLCV-susceptible Campbell-28 tomato cultivar, are the first report of resistance to a plant DNA virus obtained by the use of the intron-hairpin RNA approach.