Management of microbial contaminants in the In Vitro Gene Bank: a case study of taro [Colocasia esculenta (L.) Schott]

Ex situ conservation of vegetatively propagated crops by tissue culture is sometimes hampered by covert endophytic bacteria which become visible after repeated subculture over prolonged periods. In the present study, we identified bacterial contaminants and devised a strategy for their elimination from 20 in vitro conserved accessions of taro [Colocasia esculenta (L.) Schott] held in the In Vitro Gene Bank (IVGB) at ICAR-National Bureau of Plant Genetic Resources (NBPGR). Visually, the cultures were exhibiting white to dark brown exudation in tissue culture growth medium and undergoing slow degeneration as recorded by wilting and necrosis of leaves and shoots. On culturing the macerated pieces of shoots, corms, and leaf petioles from the contaminated taro cultures on bacterial indexing medium, six distinct bacterial colonies were observed. The identity of these bacteria was established through 16S rDNA sequencing as gram-negative strains (2) of Ralstonia spp. and gram-positive strains (4) of Paenibacillus spp. Thereafter, two strategies were adopted for elimination of contaminants from the cultures: (i) use of antibiotic supplemented media and (ii) hardening of micro-corms in field and re-establishment under in vitro conditions. Antibiotic supplemented media was not effective in eliminating contamination as reappearance of bacterial colonies was observed after subculturing in antibiotic-free growth medium. In contrast, re-establishment in vitro from hardened infected plants resulted in an average of 6.03% bacteria-free cultures in 14 accessions. The findings indicate that endophytic bacteria associated with the host (taro) plants under tissue culture conditions may efficiently be mitigated through periodic transfer of the tissue culture derived corms to ex vitro conditions and re-introduction of the bacteria-free shoot tip explants in vitro.