Abstract
We have developed a method for detecting a transgene and its protein product in maize endosperm that allows the kernel to be germinated after analysis. This technique could be highly useful for several monocots and dicots. Our method involves first sampling the endosperm with a hand-held rotary grinder so that the embryo is preserved and capable of germination. This tissue is then serially extracted, first with SDS-PAGE sample buffer to extract proteins, then with an aqueous buffer to extract DNA. The product of the transgene can be detected in the first extract by SDS-PAGE with visualization by total protein staining or immuno-blot detection. The second extract can be purified and used as template DNA in PCR reactions to detect the transgene. This method is particularly useful for screening transgenic kernels in breeding experiments and testing for gene silencing in kernels.
Similar content being viewed by others
References
Anderson OD, Greene FC, Yip RE, Halford NG, Shewry PR and Malpica-Romero JM (1989) Nucleotide sequences of the two high-molecular-weight glutenin genes from the D-genome of a hexaploid bread wheat,Triticum aestivum L. cv Cheyenne. Nucleic Acids Res 17: 461–462.
Bennetzen JL and Freeling M (1993) Grasses as a single genetic system: Genome composition, colinearity and compatibility. Trends Genet 9: 259–261.
Harlow E and Lane D (1999) Using Antibodies: A Laboratory Manual, pp 495, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
Kumpatla SP, Chandrasekharan MB, Iyer LM, Li G and Hall TC (1998) Genome intruder scanning and modulation systems and transgene silencing. Trends Plant Sci 3: 97–104.
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685.
Osborne TB (1924) The Vegetable Proteins, pp 154, Longmans Green and Co., New York.
Senior ML and Heun M (1993) Mapping maize microsatellites and polymerase chain reaction confirmation of the targeted repeats using a CT primer. Genome 36: 884–889.
Shewry PR (1995) Plant storage proteins. Biol Rev Camb Philos Soc 70: 375–426.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Sangtong, V., Mottl, E.C., Long, M.J. et al. Serial extraction of endosperm drillings (SEED)—A method for detecting transgenes and proteins in single viable maize kernels. Plant Mol Biol Rep 19, 151–158 (2001). https://doi.org/10.1007/BF02772157
Published:
Issue Date:
DOI: https://doi.org/10.1007/BF02772157