This study developed quantitative PCR (qPCR) methods to estimate transgene copy number and quantify mRNA levels in transgenic cassava plants obtained through Agrobacterium-mediated transformation. qPCR analysis of 15 lines found most contained 1-2 copies of the GUSPlus and hptII genes. Comparison to Southern blot analysis showed 83.3% agreement between the two methods. qPCR also grouped lines into high, medium, and low expression categories based on GUSPlus and hptII mRNA levels, with up to 380-fold and 3,000-fold differences between highest and lowest expressors. Results validated qPCR as a more efficient technique than conventional methods for characterizing transgene integration and expression in cassava.

1. Quantitative real time PCR assessment of cassava transgenic plants: Copy number
estimate and quantification of gene expression
Beltrán J1., Chavarriaga P1., Echeverry M2., Jaimes H1., Ladino J1., López D 1., Duque M1 and Tohme J1.
1. Conservation and Use of Tropical Genetic Resources, CIAT, AA 6713 , Cali, Colombia 2. Universidad de Puerto Rico
INTRODUCTION • Southern blot and qPCR, for copy number estimation
To improve the conventional molecular analysis of cassava transgenic The analysis revealed different integration patterns, confirming
plants, we developed qPCR methods to estimate copy number and that the lines arose from independent transformation events.
M 11 27 42 43 53 54 90 NT P M 11 27 42 43 53 54 90 NT P
quantify mRNA levels of transgenes in cassava lines obtained through hptII GUSPlus
Agrobacterium-mediated transformation.
Six lines were also analyzed by Southern blot. The copy number was
concordant in most cases with those estimated by qPCR. Most of the
transgenic events (86.6%) had low copy number (1 or 2), thus 0.5 kb
0.5 kb
corroborating that Agrobacterium generally inserts a low copy number
of transgenes into plants. Of the six lines evaluated, five coincided exactly with the qPCR
estimates indicating an 83.3% agreement between the two
Quantitative mRNA expression data grouped the transgenic lines into methodologies:
three expresser categories —high, medium, and low— for hygromycin Table 2. Comparison of copy numbers for two transgenes, as estimated by Southern
phosphotransferase (hptII) and ß-glucuronidase (GUSPlus) genes. blot and qPCR for six transgenic cassava lines
GUSPlus mRNA data agreed with results from histochemical GUS Copy number for gene:
staining. GUSPlus hptII
Line qPCR Southern blot qPCR Southern blot
11 2 2 2 2
METHODS 27
42
2
2
2
3
2
2
2
3
43 2 2 2 2
53 3 3 3 3
90 1 1 1 1
Transformed cassava lines
• Quantifying transgene expression using qRT-PCR
DNA RNA By comparing the highest and lowest expresser lines, we calculated
differences of up to 380 times for GUSPlus and 3000 times for hptII:
Transgene Detection by melting cDNA synthesis
curve analysis
3.5
Transgene mRNA detection by
Relative amount of mRNA
3
melting curve analysis
Copy number estimation by 2.5
real-time PCR Quantification of mRNA levels 2
Gusplus
by real-time RT-PCR 1.5
hpt II
1
Copy number by Southern blot Gus test
0.5
0
11 29 42 43 52 54 60 90 131
RESULTS AND DISCUSSION Transgenic lines
• Copy number estimation by real-time PCR RT-PCR results agreed with the qRT-PCR data for most samples
and for both transgenes:
M 11 29 42 43 52 54 60 90 131 NT H2O
Copy numbers for the genes GUSPlus and hptII were estimated in 15 181 bp GUSPlus
transgenic lines 182 bp hptII
169 bp 18S
Table 1. Copy number for the GUSPlus and hptII genes, estimated by qPCR for 15
transgenic cassava lines
• GUS test
Copy number for gene:
Line GUSPlus hptII
A differential pattern of intensities for GUS can be seen. These
2 1 1 intensities mirrored the data obtained with qRT-PCR:
11 2 2
27 2 2 29 90 43 11 52 54 42 60 131 NT
29 1 2
42 2 2
43 2 2
46 1-2 1-2
50 2 2
51 2 1 • Summarizing, qPCR was more efficient than the laborious techniques
52 1 1
53 3 3 conventionally used to detect transgene copy number and expression.
55 3 3
60 1 1
• Results suggest that, in 3-year-old transgenic cassava plants, high and
90 1 1
131 1 1 stable expression of transgenes can be found.
Most lines contained one or two copies of each gene; in some, the copy
PERSPECTIVES
number was different for the two genes, suggesting rearrangements of •Use qPCR for future characterization of transgenic events in cassava,
the T-DNA which could facilitate the selection of the most promising events.