Lorenzo Burgos

Using quantitative real-time PCR to detect chimeras in transgenic tobacco and apricot and to monitor their dissociation

BackgroundThe routine generation of transgenic plants involves analysis of transgene integration into the host genome by means of Southern blotting. However, this technique cannot distinguish between uniformly transformed tissues and the presence of a mixture of transgenic and non-transgenic cells in the same tissue. On the other hand, the use of reporter genes often fails to accurately detect chimerical tissues because their expression can be affected by several factors, including gene silencing and plant development. So, new approaches based on the quantification of the amount of the transgene are needed urgently.ResultsWe show here that chimeras are a very frequent phenomenon observed after regenerating transgenic plants. Spatial and temporal analyses of transformed tobacco and apricot plants with a quantitative, real-time PCR amplification of the neomycin phosphotransferase (nptII) transgene as well as of an internal control (β-actin), used to normalise the amount of target DNA at each reaction, allowed detection of chimeras at unexpected rates. The amount of the nptII transgene differed greatly along with the sub-cultivation period of these plants and was dependent on the localisation of the analysed leaves; being higher in roots and basal leaves, while in the apical leaves it remained at lower levels. These data demonstrate that, unlike the use of the gus marker gene, real-time PCR is a powerful tool for detection of chimeras. Although some authors have proposed a consistent, positive Southern analysis as an alternative methodology for monitoring the dissociation of chimeras, our data show that it does not provide enough proof of uniform transformation. In this work, however, real-time PCR was applied successfully to monitor the dissociation of chimeras in tobacco plants and apricot callus.ConclusionsWe have developed a rapid and reliable method to detect and estimate the level of chimeras in transgenic tobacco and apricot plants. This method can be extended to monitor the dissociation of chimeras and the recovery of uniformly-transformed plants.

Improving knowledge of plant tissue culture and media formulation by neurofuzzy logic: a practical case of data mining using apricot databases.

Plant tissue growth can be regulated and controlled via culture media composition. A number of different laborious and time-consuming approaches have been used to attempt development of optimized media for a wide range of species and applications. However, plant tissue culture is a very complex task, and the identification of the influences of process factors such as mineral nutrients or plant growth regulators on a wide spectrum of growth responses cannot always well comprehended. This study employs a new approach, data mining, to uncover and integrate knowledge hidden in multiple data from plant tissue culture media formulations using apricot micropropagation databases as an example. Neurofuzzy logic technology made it possible to identify relationships among several factors (cultivars, mineral nutrients and plant growth regulators) and growth parameters (shoots number, shoots length and productivity), extracting biologically useful information from each database and combining them to create a model. The IF-THEN rule sets generated by neurofuzzy logic were completely in agreement with previous findings based on statistical analysis, but advantageously generated understandable and reusable knowledge that can be applied in future plant tissue culture media optimization.

Agrobacterium‐mediated transformation of Vitis Cv. Monastrell suspension‐cultured cells: Determination of critical parameters

Although some works have explored the transformation of differentiated, embryogenic suspension‐cultured cells (SCC) to produce transgenic grapevine plants, to our knowledge this is one of the first reports on the efficient transformation of dedifferentiated Vitis vinifera cv Monastrell SCC. This protocol has been developed using the sonication‐assisted Agrobacterium‐mediated transformation (SAAT) method. A construct harboring the selectable nptII and the eyfp/IV2 marker genes was used in the study and transformation efficiencies reached over 50 independent transformed SCC per gram of infected cells. Best results were obtained when cells were infected at the exponential phase. A high density plating (500 mg/dish) gave significantly better results. As selective agent, kanamycin was inefficient for the selection of Monastrell transformed SCC since wild type cells were almost insensitive to this antibiotic whereas application of paromomycin resulted in very effective selection. Selected eyfp‐expressing microcalli were grown until enough tissue was available to scale up a new transgenic SCC. These transgenic SCC lines were evaluated molecularly and phenotypically demonstrating the presence and integration of both transgenes, the absence of Agrobacterium contamination and the ability of the transformed SCC to grow in highly selective liquid medium. The methodology described here opens the possibility of improving the production of valuable metabolites.

Silencing of Agrobacterium tumefaciens oncogenes ipt and iaaM induces resistance to crown gall disease in plum but not in apricot

BACKGROUND In this study, two vectors with short-length chimeric transgenes were used to produce Prunus rootstocks resistant to crown gall disease through RNA-interference-mediated gene silencing of the Agrobacterium tumefaciens oncogenes ipt and iaaM. RESULTS Transgenic plum and apricot lines were produced with efficiencies of up to 7.7 and 1.1% respectively. An in vitro evaluation method allowed identification of susceptible lines and reduction in the number of lines to be evaluated in the greenhouse. Five transgenic plum lines, expressing transgene-derived small interfering RNA (siRNA) and low levels of transgene hairpin RNA (hpRNA), showed a significant reduction in the development of the disease after infection with Agrobacterium strains C58 and A281 under greenhouse conditions. However, unexpectedly, all transgenic apricot lines were gall susceptible. The infection of apricot plants with a binary vector containing only the 6b oncogene demonstrated that the expression of this gene is involved in the induction of tumours in the apricot species. CONCLUSION RNAi-mediated gene silencing can be used for inducing crown gall resistance in plum rootstocks. These could be used to graft non-genetically modified commercial fruit cultivars reducing, or eliminating, the disease symptoms.