Pepper, Sweet (Capsicum annuum)

Capsicum (pepper) species are economically important crops that are recalcitrant to genetic transformation by Agrobacterium ( Agrobacterium tumefaciens ). A number of protocols for pepper transformation have been described but are not routinely applicable. The main bottleneck in pepper transformation is the low frequency of cells that are both susceptible for Agrobacterium infection and have the ability to regenerate. Here, we describe a protocol for the effi cient regeneration of transgenic sweet pepper ( C. annuum ) through inducible activation of the BABY BOOM (BBM) AP2/ERF transcription factor. Using this approach, we can routinely achieve a transformation effi ciency of at least 0.6 %. The main improvements in this protocol are the reproducibility in transforming different genotypes and the ability to produce fertile shoots. An added advantage of this protocol is that BBM activity can be induced subsequently in stable transgenic lines, providing a novel regeneration system for clonal propagation through somatic embryogenesis

A 28 nt long synthetic 5^′UTR (synJ) as an enhancer of transgene expression in dicotyledonous plants

<p>Abstract</p> <p>Background</p> <p>A high level of transgene expression is required, in several applications of transgenic technology. While use of strong promoters has been the main focus in such instances, 5^′UTRs have also been shown to enhance transgene expression. Here, we present a 28 nt long synthetic 5^′UTR (synJ), which enhances gene expression in tobacco and cotton.</p> <p>Results</p> <p>The influence of synJ on transgene expression was studied in callus cultures of cotton and different tissues of transgenic tobacco plants. The study was based on comparing the expression of reporter gene <it>gus</it> and <it>gfp</it>, with and without synJ as its 5^′UTR. Mutations in synJ were also analyzed to identify the region important for enhancement. synJ, enhances gene expression by 10 to 50 fold in tobacco and cotton depending upon the tissue studied. This finding is based on the experiments comparing the expression of <it>gus</it> gene, encoding the synJ as 5^′UTR under the control of 35S promoter with expression cassettes based on vectors like pBI121 or pRT100. Further, the enhancement was in most cases equivalent to that observed with the viral leader sequences known to enhance translation like Ω and AMV. In case of transformed cotton callus as well as in the roots of tobacco transgenic plants, the up-regulation mediated by synJ was much higher than that observed in the presence of both Ω as well as AMV. The enhancement mediated by synJ was found to be at the post-transcriptional level. The study also demonstrates the importance of a 5^′UTR in realizing the full potential of the promoter strength. synJ has been utilized to design four cloning vectors: pGEN01, pBGEN02, pBGEN02-<it>hpt</it> and pBGEN02-<it>ALS</it>^<it>dm</it> each of which can be used for cloning the desired transgene and achieving high level of expression in the resulting transgenic plants.</p> <p>Conclusions</p> <p>synJ, a synthetic 5^′UTR, can enhance transgene expression under a strong promoter like 35S as well as under a weak promoter like nos in dicotyledonous plants. synJ can be incorporated as the 5^′UTR of transgenes, especially in cases where high levels of expression is required. A set of vectors has also been designed to facilitate this process.</p