An efficient and rapid Rhizobium rhizogenes root transformation protocol for Lemna minor
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Duckweeds belong to the smallest aquatic flowering plant family, Lemnaceae, and have a rapid doubling time, making this group an excellent system to study reduced morphology and wide environmental adaptability at the molecular level. Despite the availability of genomic and transcriptomic data for duckweed member, Lemna minor, lack of an efficient genetic transformation system has limited its use in plant molecular biology research. The present study reports an efficient and rapid Rhizobium rhizogenes-mediated root transformation system for L. minor. Two different factorial experiments were designed to test the effect of explant type, age, culture media and inoculation methods on transformation efficiency. Leaf and root tip cut explants were inoculated with R. rhizogenes strain MSU 440 harboring pBIN-YFP vector using yellow fluorescent protein (YFP) as a reporter gene for identification of transgenic roots. In addition, two different culture media, full MS and 0.25X Hoagland, and four different infection methods, solid culture, centrifugation, liquid culture and sonication, were compared. After 8 weeks, about 17% of the root tip-cut explants infected via the solid culture method and maintained in 0.25X Hoagland medium had YFP-expressing roots. These transgenic L. minor roots were morphologically similar to normal roots and PCR analysis demonstrated that the YFP-expressing roots were positive for the integration-expected rol genes. The described optimized root transformation procedure is a valuable tool for pursuing high-throughput gene characterization studies in L. minor.
KeywordsLemna Plant transformation Rhizobium rhizogenes Agrobacterium rhizogenes Duckweed Hairy roots
We thank Dr. Denneal Jamison-McClung, Director of UC Davis Biotechnology Program for helpful comments provided for improving the manuscript. We would like to thank Dr. Bhagya Chandrasekara for her support on the molecular analysis and staff members of the Agricultural Biotechnology Center, University of Peradeniya for their support and encouragement throughout the research period.
Compliance with ethical standards
Conflict of Interest
Both authors, R.W.M.K. Kanchanamala and P.C.G. Bandaranayake declare that no conflicts of interest exist regarding the materials included in the manuscript.
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