Effect of promoter driving selectable marker on corn transformation.
Identification of an appropriate selection agent and its corresponding selectable marker gene is one of the first steps in establishing a transformation protocol for a given plant species. As the promoter controls expression level of the genes, the promoter driving the selectable marker gene can affect transformation. However, investigations into the direct effect of promoters driving selectable marker on transformation are lacking in the literature though many reports of relative strengths of promoters driving reporter genes like GUS or CAT or GFP are available. In the present study, we have compared rice Actin1 and CaMV.35S (commonly used promoters in monocotyledonous plant transformation) promoters driving nptII for their effectiveness in paromomycin selection of transgenic corn events. To enable statistically meaningful analysis of the results, a large sample size of nearly 5,000 immature embryos (explants) was employed producing approximately 1,250 independent events from each of the two constructs in four independent experiments. The rate of appearance of resistant calli and percentage of resistant calli recovered was higher with P-Os.Actin1/nptII/nos3' as compared to P-CaMV.35S/nptII/nos3' in all four experiments. There was no appreciable difference either in the frequency of plant regeneration or in the morphological characteristics of plants recovered from the two constructs. Although the escape rate trended lower with P-Os.Actin1 as compared to P-CaMV.35S, the recovery of low copy events was significantly higher with P-CaMV.35S. The higher transformation frequency with P-Os.Actin1 could be related to the strength of this promoter as compared to P-CaMV.35S in the explants and/or calli. Based on these results, we infer that the promoter driving the selectable marker is an important factor to be considered while establishing a high throughput transformation protocol as it could not only influence the transformation frequency but also the copy number of the transgene in the recovered transgenics.
Prakash
N Shiva
NS
Monsanto Research Centre, #44/2A, Vasanths' Business Park, Bellary Road, NH:7, Hebbal, Bangalore, India.
Prasad
V
V
Chidambram
Thillai P
TP
Cherian
Shoba
S
Jayaprakash
T L
TL
Dasgupta
Santanu
S
Wang
Qi
Q
Mann
Michael T
MT
Spencer
T Michael
TM
Boddupalli
Raghava S
RS
D000199:Actins / Q000235:genetics; D017796:Caulimovirus / Q000235:genetics; D018628:Gene Dosage; D018506:Gene Expression Regulation, Plant; D019868:Kanamycin Kinase / Q000235:genetics / Q000378:metabolism*; D012275:Oryza / Q000235:genetics / Q000378:metabolism; D018520:Plant Shoots / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D014170:Transformation, Genetic / Q000235:genetics*; D003313:Zea mays / Q000235:genetics* / Q000378:metabolism
D000199:Actins; D019868:Kanamycin Kinase