Wednesday, November 15, 2006

Isolation and Identification of a Wheat Seed Endophyte.

Rima Thapa, Department of Plant and Soil Sciences, Oklahoma State University, 368 Ag Hall, Stillwater, OK 74078 and Michael Anderson, Dept of Plant and Soil Sciences, Oklahoma State University, 368 Ag Hall, Stillwater, OK 74074.

Endophytes are bacteria or fungi, which live inside plant tissues causing no harm. In fact, most endophytes are known to contribute towards plant growth, disease resistance, resistance to abiotic stresses, and crop productivity. Endophytes are present in roots, stems, leaves, fruits and tubers of a wide-range of agricultural, horticultural and forest species. The objective of this research was to isolate and characterize bacterial endophytes in wheat seeds. Seed-borne endophytes have the potential for vertical transmission across generations. Seeds were surface sterilized, germinated, homogenized, diluted, and plated on 1/10 Tryptic Soy Agar. Plates were incubated at room temperature for 48 hours and seven colonies were picked and reinoculated into Tryptic Soy Broth for a 24 hours incubation. Endophytic DNA from the culture media was extracted using a MoBio kit. All seven colonies were fingerprinted using RAPD-PCR to distinguish clonal isolates. All isolates produced identical fingerprint patterns indicating that they are most likely very similar if not identical. Consequently, a single isolate was used for sequencing and identification. Amplification by PCR of the near-full length16S rRNA gene was performed using universal primers. The PCR product was sequenced and compared with 16S rRNA sequences from the NCBI database yielding a very close match to Bacillus sp. related to Bacillus subtilis. The sequence was compared using cluster analysis to a wide-range of bacterial standard species from the Bacillus subtilis group, which showed an almost identical relationship to Bacillus subtilis. Further biochemical tests are necessary to confirm this identification. Further experiments will be performed to isolate more seed-borne endophytes and to confirm vertical transmission.