Efficient DNA Minipreparation by Modified Benzyl Chloride Method from Hairy Roots of Ajuga reptans to Detect rolB Gene from Ri Plasmid

Ajuga reptans, a member of the family Labiatae, is not only a horticultural plant which is used as ground cover, but also a medicinal herb which has been used for the treatment of jaundice and rheumatism in European countries for centuries. The roots of A. reptans contain several types of phytoecdysteroid and mainly 20-hydroxyecdysone (20-HE) known as a principal physiological inducer of molting and metamorphosis in arthropoda. For production of 20-HE, some A. reptans hairy root lines with high productivity of 20 HE and their culture methods have been established [see review 1]. Hairy roots are adventitious roots induced by infection with Agrobacterium rhizogenes. There are variations in the characteristics, such as morphology, the capacity for growth and the productivity of secondary metabolites, of hairy roots lines and, moreover, the characteristics of each hairy root line are very stable [2]. Since hairy roots of A. reptans are relatively thick (1-2mm in diameter) and firm, the difference of characteristics such as morphology, elongation, branching, and gravity response among the independently isolated root lines can be easily distinguishable [3,4]. This trait of A, reptans hairy roots prompts us to use the hairy roots for analysis of genes, in particular, rooting locus (rol) genes, responsible for exhibiting such characteristics [4]. At present, we have isolated more than 15 hairy root lines with distinctive features (unpublished result) . During the selection of hairy root lines, we need to develop a small scale DNA isolation method (minipreparation) from the hairy roots to confirm integrated T-DNA of Ri plasmid, in particular, rol genes. In minipreparation of DNA from plant materials, a method using cetyltrimethylammonium bromide (CTAB) is well devised [5], and the freezing of plant materials in liquid nitrogen is also indispensable for disruption of plant materials. The major problem with this method is that part of the disrupted plant materials are lost because the fine powder of the plant materials adheres to the surface of the grinding apparatus. Recently, Kikuchi et al. introduced some excellent methods including the Benzyl chloride method for the isolation of DNA from plant materials