Serial extraction of endosperm drillings (SEED)--a method for detecting transgenes and proteins in single viable maize kernels

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 grin...

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Bibliographic Details
Published in:Plant molecular biology reporter 2001-06, Vol.19 (2), p.151-158
Main Authors: Sangtong, V, Mottl, E.C, Long, M.J, Lee, M, Scott, M.P
Format: Article
Language:English
Subjects:
Buffers
Corn
Deoxyribonucleic acid
DNA
embryo (plant)
Endosperm
enzyme-linked immunosorbent assay
Gel electrophoresis
gene expression
Gene silencing
Germination
Kernels
methodology
polymerase chain reaction
protein composition
Proteins
seed germination
Sodium lauryl sulfate
Transgenes
transgenic plants
viability
Zea mays
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Summary: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.
ISSN:0735-9640
1572-9818
DOI:10.1007/BF02772157