A rapid and non‐destructive screenable marker, FAST, for identifying transformed seeds of Arabidopsis thaliana

• Published in: The Plant journal : for cell and molecular biology Vol. 61; no. 3; pp. 519 - 528
• Main Authors: Shimada, Takashi L., Shimada, Tomoo, Hara‐Nishimura, Ikuko
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01-02-2010; Blackwell
• Subjects: Arabidopsis - chemistry; Arabidopsis - genetics; Arabidopsis - ultrastructure; Arabidopsis thaliana; Biological and medical sciences; Biomarkers - analysis; Biotechnology; Fundamental and applied biological sciences. Psychology; Genetic markers; green fluorescent protein; Green Fluorescent Proteins - analysis; Green Fluorescent Proteins - genetics; Luminescent Measurements - methods; Microscopy, Electron; Microscopy, Fluorescence - methods; oleosin; Plant biology; Plant physiology and development; Plants, Genetically Modified - chemistry; Proteins; screenable marker; seed; Seeds; Seeds - chemistry; Seeds - genetics; Seeds - ultrastructure; Time Factors; transformation; Transgenic plants; Transformation; Seeds; Identification; Marker; Arabidopsis thaliana; oleosin; Cruciferae; Dicotyledones; Seed; screenable marker; Angiospermae; Green fluorescent protein; Spermatophyta
• ISSN: 0960-7412; 1365-313X
• DOI: 10.1111/j.1365-313X.2009.04060.x

Summary
The creation of transgenic plants has contributed extensively to the advancement of plant science. Establishing homozygous transgenic lines is time‐consuming and laborious, and using antibiotics or herbicides to select transformed plants may adversely affect the growth of some transgenic plants. Here we describe a novel technology, which we have named FAST (fluorescence‐accumulating seed technology), that overcomes these difficulties. Although this technology was designed for use in Arabidopsis thaliana, it may be adapted for use in other plants. The technology is based on the expression of a fluorescent co‐dominant screenable marker FAST, under the control of a seed‐specific promoter, on the oil body membrane. The FAST marker harbors a fusion gene encoding either GFP or RFP with an oil body membrane protein that is prominent in seeds. The marker protein was only expressed in a specific organ (i.e. in dry seeds) and at a specific time (i.e. during dormancy), which are desirable features of selectable and/or screenable markers. This technique provides an immediate and non‐destructive method for identifying transformed dry seeds. It identified the heterozygous transformed seeds among the T1 population and the homozygous seeds among the T2 population with a false‐discovery rate of <1%. The FAST marker reduces the length of time required to produce homozygous transgenic lines from 7.5 to 4 months. Furthermore, it does not require sterilization, clean‐bench protocols or the handling of large numbers of plants. This technology should greatly facilitate the generation of transgenic Arabidopsis plants.