Transgene copy number estimation and analysis of gene expression levels in Populus spp. transgenic lines

BackgroundThe genus Populus has certain important features, suchas a relatively small nuclear genome, it can be easilyregenerated easily in vitro and genetically transformedby Agrobacterium vector system, which make it ideal forgene transfer and molecular genetic studies in foresttrees [1]. Insect-tolerant poplars have been obtainedusing several types of insecticidal genes coding for Bacil-lus thuringiensis-toxins. Regenerated plants with insect-resistance were obtained in different studies. Agrobacter-ium-mediated transformation has been the favoredmethod for the introduction of foreign genes into plants.The effectiveness of insect-resistance in transgenicplants is related to the side effects of gene transfer (siteof gene insertion, copy number, gene silencing etc.).Moreover intransgenic plants, transgene copy numbercan greatly affect the expression level and genetic stabi-lity of the target gene, making estimation of transgenecopy numbers an important area of genetically modifiedplant research [2]. Thus molecular biological analysis oftransgenic plants, like real time PCR, widely used todetect and quantify DNA and cDNA [3], could repre-sent an useful tool to investigate the genetic stability oftransgenic forest trees having a long life cycleas well asfor determining copy number in transformed plants.Material and methodsThe present study was undertaken to investigatePopu-lus alba and P. tremula x P. tremuloides transgeniclines, obtained via Agrobacterium-mediated transforma-tion, carrying cry1Ab and nptII genes in the T-DNAregion. The plants were vegetatively propagated ingrowth chambers over 2 years. Ten individuals fromeach clone were planted in containers with “forest soil”,and grown in a climate chamber.Extraction of genomic DNA and RNA from leaves wasperformed for PCR and Real Time PCR (RT-PCR) ana-lysis to estimate the transgene copy number [4] as wellas expression of the inserted gene [5]in transgenicpoplar, respectively.Results and discussionAll lines contained one copy ofcry gene and two ofthem showed that the copy number was different forthe cry1Ab and nptII genes, suggesting rearrangementsor multiple but incomplete copies of the transferredDNA (Figure 1). The copy number was concordantamong the 3 individuals of each lines analysed and withthose determined from the same transgenic lines kept inmicropropagation for 2 years.The transcript levels from both genes were deter-mined in 3 individuals for each line growing in climaticchambers. High levels of mRNA expression weredetected with respect to the stable endogenousactingene for both transgenic lines (Figure 2). Comparing thetranscript level of inserted genes among lines, a signifi-cant low level of nptII gene (p = 0.005) in the line carry-ing 3 copies was observed.Preliminary results indicate a differential expression ofendogenous genes among transgenic lines and towardstheir isogenic form.ConclusionsThe evaluation of the copy number of the insertedgenes has indicated their stability after 2 years of


Background
The genus Populus has certain important features, such as a relatively small nuclear genome, it can be easily regenerated easily in vitro and genetically transformed by Agrobacterium vector system, which make it ideal for gene transfer and molecular genetic studies in forest trees [1]. Insect-tolerant poplars have been obtained using several types of insecticidal genes coding for Bacillus thuringiensis-toxins. Regenerated plants with insectresistance were obtained in different studies. Agrobacterium-mediated transformation has been the favored method for the introduction of foreign genes into plants. The effectiveness of insect-resistance in transgenic plants is related to the side effects of gene transfer (site of gene insertion, copy number, gene silencing etc.). Moreover intransgenic plants, transgene copy number can greatly affect the expression level and genetic stability of the target gene, making estimation of transgene copy numbers an important area of genetically modified plant research [2]. Thus molecular biological analysis of transgenic plants, like real time PCR, widely used to detect and quantify DNA and cDNA [3], could represent an useful tool to investigate the genetic stability of transgenic forest trees having a long life cycleas well as for determining copy number in transformed plants.

Material and methods
The present study was undertaken to investigate Populus alba and P. tremula x P. tremuloides transgenic lines, obtained via Agrobacterium-mediated transformation, carrying cry1Ab and nptII genes in the T-DNA region. The plants were vegetatively propagated in growth chambers over 2 years. Ten individuals from each clone were planted in containers with "forest soil", and grown in a climate chamber.
Extraction of genomic DNA and RNA from leaves was performed for PCR and Real Time PCR (RT-PCR) analysis to estimate the transgene copy number [4] as well as expression of the inserted gene [5]in transgenic poplar, respectively.

Results and discussion
All lines contained one copy of cry gene and two of them showed that the copy number was different for the cry1Ab and nptII genes, suggesting rearrangements or multiple but incomplete copies of the transferred DNA ( Figure 1). The copy number was concordant among the 3 individuals of each lines analysed and with those determined from the same transgenic lines kept in micropropagation for 2 years. The transcript levels from both genes were determined in 3 individuals for each line growing in climatic chambers. High levels of mRNA expression were detected with respect to the stable endogenous actin gene for both transgenic lines (Figure 2). Comparing the transcript level of inserted genes among lines, a significant low level of nptII gene (p = 0.005) in the line carrying 3 copies was observed.
Preliminary results indicate a differential expression of endogenous genes among transgenic lines and towards their isogenic form.

Conclusions
The evaluation of the copy number of the inserted genes has indicated their stability after 2 years of * Correspondence: cristina.vettori@cnr.it 1 Plant Genetics Institute, CNR, Italy Full list of author information is available at the end of the article micropropagation. The lower expression level of the nptII inserted gene in one line could suggest that factors like position effects or DNA rearrangements lead to differential expression.
The screening of the transcriptomic variations in transgenic plants carrying the cry gene and the comparison with position effects or DNA rearrangements is in course. The final aim is to unravel possible pleiotropic transcriptomic effects following cry gene expression in P. alba and P. tremula x P. tremuloides transgenic lines.
Author details Figure 1 Copy number values estimated by real-time PCR. Data are expressed as mean ± 95% confidence limit.

Figure 2
Relative expression obtained through qRT-PCR for the transgenes cry1Ab and nptII in transgenic Populus lines. The data are shown relative to the endogenous actin gene. Boxes: interquartile range, or the middle 50% of observations; dotted line: median gene expression; whiskers: minimum and maximum observations. Data are expressed as mean ± 95% confidence limit after 10000 permutations and have a p = 0.000 -0.002.