PCR-based RAPD technique to determine induced salinity tolerance in vitro in Acacia auriculiformis

Abstract

Acacia auriculiformis is an agro-forestry species in India with application in paper industry. The species is saline intolerant and fails to establish. Experiments were conducted to induce saline tolerance in callus cultures of A. auriculiformis on Murashige & Skoogs medium supplemented with different concentrations of NaCl+CaCl_2––– (2:1; 50, 100, 150, 200, 250 or 300 mM). Calli were trained for eight sub-culture generations. The genome from saline-tolerant calli was extracted by modified cetyl trimethyl ammonium bromide method and subjected to polymerase chain reaction-based random amplified polymorphic DNA technique. The polymorphic DNA bands were eluted and cloned to Escherichia coli with the help of pTZ57R/T. The transformed plasmid DNA was sequenced and subjected to BLAST at National Center for Biotechnology Information. Results indicated that saline training of calli resulted in the expression of tolerance to 100 and 150 mM saline concentration after eight sub-cultures. Both the saline concentrations produced conspicuous bands in trained calli. Three nucleotide sequences were similar to those of Bacillus amyloliquefaciens sub sp. plantarum and BLAST search result indicated high homologies to ThiT thiamine transporter, YuaG flottilin type band 7, and U32 class peptidase protein genes in bacterial genome. The annotation of these protein genes in saline tolerance was discussed. The study suggested that the endophytic B. amyloliquefaciens sub sp. plantarum might be inducing saline tolerance in A. auriculiformis.

Keywords::

• Acacia
• Bacillus amyloliquefaciens
• calli
• PCR-based RAPD
• salinity tolerance
• ThiT thiamine transporter
• YuaG band 7 protein

Acknowledgements

The first author thanks Kuvempu University for financial support during the course of study and Department of Crop Physiology, University of Agricultural Science, Bangalore for providing laboratory facilities. The authors thank Mr R.S. Sajeevan, Department of Crop physiology, University of Agricultural Science, Bangalore for his help during RAPD analysis.