engleski

Induction of organogenesis and influence of selective treatments and antimicrobial substances on adventitious roots in Coleus blumei Benth.

Introduction The genetic transformation of plants requires selection and regeneration of transformed tissues. The purpose of this study was to determine the suitable conditions for shoots and roots regeneration and to examine effect of selective agents and antimicrobial substances on Coleus blumei organogenesis. Nodal, internodal and leaf explants were analyzed for the regeneration potential. Multiple shoots were induced on nodal explants cultivated on MS medium with different concentrations of indoleacetic acid (IAA) and 6-benzylaminopurine (BAP). Adventitious roots were induced on leaf explants cultivated on MS media with α -naphthaleneacetic acid (NAA). The organs formed directly from the explants without an intermediate callus phase. The sensitivity of non-transformed adventitious roots of Coleus blumei to selective agents (kanamycin, neomycin, geneticin, hygromycin and glufosinate ammonium) and antimicrobial substances commonly used to eliminate Agrobacterium from tissue culture (plant preservative mixture: PPM, carbenicillin: carb, cefotaxime: cef and vancomycin: van) was measured. Materials and Methods The leaves excised from in vitro growing Coleus plants, were cut into 1 cm2 squares. Leaf explants were inoculated upside-down on MS medium supplemented with different NAA concentrations (0.01, 0.1, 0.5, 1 mg/l) and/or antimicrobial substances (500 mg/l carb, 250 mg/l cef, 100 mg/l van, 1ml/l PPM) and selective substances kanamycin (2 to 50 mg/l), neomycin (10 to 100 mg/l), geneticin (0.1 to 20 mg/l), hygromycin (1 to 20 mg/l) and glufosinate ammonium (0.01 to 5 mg/l). The plant stem was cut on internodal and nodal explants that were cultured on MS medium supplemented with different IAA and BAP concentrations. Leaf, internodal and nodal explants were cultured at 24 °C under fluorescent tube light (16 h photoperiod). Influence of different supplements on adventious organ induction was tested. Results were scored after 21 day. For each treatment 12 explants were analyzed. Results and Discussion Root induction NAA induced root development on leaf explants. All explants cultured on MS medium supplemented with 0.5 mg/l NAA developed roots but number of root origin was higher (up to 30) on medium with 1 mg/l NAA (Fig. 1a). NAA concentrations higher then 1 mg/l blocked root elongation. Media with different concentrations of IAA and/or BAP, thidiazuron and N-(2-chloro-4-pyridyl)-N′ -phenylurea (CPPU) did not induced shoots on leaf and internodal explants. BAP alone, or in combination with IAA induced multiple shoots formation on nodal explants of Coleus blumei (Fig. 1b). The influence of antimicrobial substances used for Agrobacterium elimination after transformation was examinated. Leaf explants were cultured up side-down on MS medium supplemented with 1 mg/l NAA and appropriate antimicrobial or selective substance. Carbenicillin reduced root induction and caused sward root growth. Cefotaxime, vancomycin and PPM alone or in combination were suitable for bacteria elimination since they did not derange root induction and growth (Fig. 2). For the selection of transgenic tissue different selective agents are commonly in use. Their effect on root induction on leaf explant of Coleus blumei cultivated on MS medium with 1 mg/l NAA was examinated. Concentrations higher than 20 mg/l kanamycin, 70 mg/l neomycin, 2 mg/l geneticin, 10 mg/l hygromycin and 1 mg/l glufosinate ammonium completely stopped normal root induction on leaf explants of Coleus blumei. Figure 1. a) Effect of NAA on root induction on leaf explants of Coleus blumei. b) Effect of IAA and BAP on shoot induction on nodal explants of Coleus blumei. Figure 2. Root induction on leaf explants of Coleus blumei. Influence of antimicrobial substances (0: without substance ; cef: 250 mg/l cefotaxime ; van: 100 mg/l vancomycin ; carb: 500 mg/l carbenicillin ; PPM: 1 ml/l PPM) added to MS medium supplemented with 1 mg/l NAA. Conclusions Organogenesis of Coleus blumei leaf, nodal and internodal explants was analyzed. The highest number of roots, up to 30 per explant, developed on leaf explants on MS medium supplemented with 1 mg/l NAA. The highest number of multiple shoots was induced on nodal explants cultured on MS medium supplemented with 0.3 mg/l BAP. Leaf explants might be the best for incorporation into a transformation system where transgenic roots are expected. Antibacterial substances cefotaxime, vancomycine and plant preservative mixture did not influence root development and are good for bacteria elimination after transformation. Susceptibility of normal roots to different selective treatments was analyzed. Transgenic roots could be regenerated on media with 20 mg/l kanamycin, 70 mg/l neomycin, 2 mg/l geneticin, 10 mg/l hygromycin or 1 mg/l glufosinate ammonium.

Coleus blumei; organogenesis; selective treatment; antimicrobial substances

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