A histochemical method using a substrate of beta-glucuronidase for detection of genetically modified papaya.
A histochemical assay for detecting genetically modified (GM) papaya (derived from Line 55-1) is described. GM papaya, currently undergoing a safety assessment in Japan, was developed using a construct that included a beta-glucuronidase (GUS) reporter gene linked to a virus coat protein (CP) gene. Histochemical assay was used to visualize the blue GUS reaction product from transgenic seed embryos. Twelve embryos per fruit were extracted from the papaya seeds using a surgical knife. The embryos were incubated with the substrate 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide (X-Gluc) in a 96-well microtiter plate for 10-15 hours at 37 degrees C. Seventy-five percent of GM papaya embryos should turn blue theoretically. The histochemical assay results were completely consistent with those from a qualitative polymerase chain reaction (PCR) method developed by this laboratory. Furthermore, the method was validated in a five-laboratory study. The method for detection of GM papaya is rapid and simple, and does not require use of specialized equipment.
Wakui
Chiseko
C
National Institute of Health Sciences: 1-18-1, Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.
Akiyama
Hiroshi
H
Watanabe
Takahiro
T
Fitch
Maureen M
MM
Uchikawa
Seiji
S
Ki
Masami
M
Takahashi
Kunihiko
K
Chiba
Ryoko
R
Fujii
Akemi
A
Hino
Akihiro
A
Maitani
Tamio
T
D029441:Carica; D030841:Food, Genetically Modified; D017930:Genes, Reporter; D005966:Glucuronidase / Q000032:analysis* / Q000235:genetics; D020719:Glucuronides; D006651:Histocytochemistry; D016133:Polymerase Chain Reaction; D012639:Seeds
D020719:Glucuronides; D005966:Glucuronidase