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

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.

Expression and inheritance of the wheat Glu-1DX5 gene in transgenic maize

“…Endosperm material from individual kernels was collected using a hand-held rotary grinder (Dremel) as described (Sangtong et al 2001). Extracted proteins were separated by electrophoresis on a 12% SDS-PAGE (Acrylamide/bis 37.5:1) gel (Laemmli 1970) in the presence of β-mercaptoethanol using a Mini-Protean II Electrophoresis Cell from BIO-RAD with a 20-well comb.…”

“…Polymerase chain reaction DNA was extracted and purified from the endosperm pellet remaining in the 96-well plate after extraction of the proteins with SDS-PAGE (Sangtong et al 2001). Leaf DNA was prepared using the Puregene DNA isolation kit (Gentra Systems, Minneapolis, Minn.).…”

Maize as production and delivery vehicle of edible vaccines against the enterotoxigenic Escherichia coli and the swine transmissible gastroenteritis (TGE)

“…Abnormalities in foreign gene transmission have been reported. In two extreme cases, Hood et al (1997) observed that transgenic plants expressing avidin were male sterile as a result of the effect of the transgene, while Sangtong et al (2001) observed that transgenic maize plants expressing the wheat glutenin 1DX5 gene did not transmit the gene through the pollen.…”

“…To identify kernels expressing LT-B for planting for the next generation, individual kernels from each ear (two ears per event) were analyzed by a partial destruction method (Sangtong et al, 2001). With the use of a fine drill and without damaging the embryo, part of the endosperm from each kernel was removed for ELIS A analysis.…”

“…To identify transgenic Ro events expressing LT-B, individual kernels from each ear (two ears per event) were analyzed by a partial destruction method, where kernels were partially drilled to remove part of the endosperm without damaging the embryo as described by Sangtong et al (2001). Protein was extracted from endosperm as described for whole kernels.…”