Universität Tübingen Botanisches Institut
Lehrstuhl Physiologische Ökologie der Pflanzen
De Filippis L, Hoffmann E, Hampp R (1996) Identification of somatic hybrids of tobacco generated by electrofusion and culture of protoplasts using RAPD-PCR. Plant Science 121: 39-46. Positive identification of somatic hybrids generated through protoplast fusion is often difficult or impossible based primarily on morphological or biochemical analysis. The use of molecular markers potentially offers a reliable and repeatable technique, allowing early assessment of polymorphic variation of hybrids directly at the DNA level. In this study we have applied random amplification of polymorphic DNA (RAPD) by polymerase chain reaction (PCR) to analyse the relationship between two parental species of tobacco, and six somatic hybrids produced as a result of fusion of vacuolated and evacuolated protoplasts, and subsequent culture. It was necessary to test a small number of commercially available ten-base synthetic oligonucleotide primers before arriving at a number which clearly distinguished the species and hybrids. The use of micro polyacrylamide gel electrophoresis coupled to silver staining for DNA provided a quick, reliable and very sensitive method of detecting polymorphisms. We demonstrated that the procedures and protocols developed were applicable to parental species as well as to all hybrids tested, and using just four primers provided enough polymorphic bands to statistically distinguish all plants. Our results show that RAPD technology is a versatile, precise, sensitive and cost effective method for genomic analysis of hybrids.
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