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Plant Molecular Biology Reporter

, Volume 24, Issue 1, pp 81–91 | Cite as

A rapid method for tissue collection and high-throughput isolation of genomic DNA from mature trees

  • Josquin F. G. Tibbits
  • Luke J. McManus
  • Antanas V. Spokevicius
  • Gerd Bossinger
Protocols

Abstract

Collection of tissue and subsequent isolation of genomic DNA from mature tree species often proves difficult. DNA extraction from needles, leaves, or buds is recommended in many protocols. Collecting these tissues from mature trees generally requires the use of firearms or climbing if sampling is to be nondestructive. As a result, sample collection is a major expense of many tree-based projects. Tree (and plant) tissues generally contain large amounts of polysaccharides and phenolic compounds that are difficult to separate from DNA. Many methods aim to overcom these problems, with most involving extraction in buffers containing the nonionic detergent cetyltrimethyl-ammonium bromide (CTAB), followed by numerous steps to clean contaminants from the DNA, using organic solvents and differential salt precipitation. These steps are time-consuming, such that isolation of DNA becomes the bottleneck in many molecular studies. This paper presents a new, efficient, cambium collection method for tree species and a DNA extraction protocol based on that of Doyle and Doyle (1987), with follow-up purification using the Wizard nuclei lysis and protein precipitation solutions (Promega). Results show a significant improvement in yield and DNA purity compared with other published methods, with consistently high yields of pure genomic DNA and high sample throughput. The relatively low cost per extraction, no requirement for use of liquid nitrogen, no requirement for freezer storage, and long-term sample stability after collection are important additional benefits.

Key words

DNA extraction cambial scrapings Eucalyptus Pinus tree sample collection 

Abbreviations

BSA

bovine serum albumin

CTAB

cetyltrimethylammonium bromide

PVP

poly vinylpyrrollidone

TE

tris-EDTA buffer

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Copyright information

© Springer 2006

Authors and Affiliations

  • Josquin F. G. Tibbits
    • 1
  • Luke J. McManus
    • 1
  • Antanas V. Spokevicius
    • 1
  • Gerd Bossinger
    • 1
  1. 1.School of Forest and Ecosystem ScienceThe University of MelbourneCreswickAustralia

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