A three-step screening procedure for detection of covert and endophytic bacteria in plant tissue cultures

摘要

A sequential three-step screening procedure to detect any covert or endophytic bacteria in plant tissue cultures was developed consequent to the observation that conventional detection methods were inconsistent and undependable. This procedure involved diligent visual examination of cultures for any inconspicuous growth (step-1), indexing the medium of visually clean cultures using bacteriological media (step-2), and subsequent tissue-indexing (step-3) using split segments from different plant parts. Step-2 indexing of grape, watermelon, papaya, capsicum, eggplant and gerbera cultures revealed bacteria in 0-100% cultures in different batches. Varying proportions of cultures that passed step-2 indexing turned positive during step-3 indexing, suggesting the essentiality of tissue-indexing. Use of two bacteriological indexing media (BIM), namely nutrient agar (BIM1) and 523 medium (BIM2) differing in nutrient constituents, pH (6.4 and 7.0 respectively) and gel strength (10 and 20 g l(-1) agar), pre-incubation of nutrient plates at 30-37degreesC to ensure freedom from incidental contaminants, post-indexing incubation at two different temperatures (25-30degreesC and 37degreesC respectively) and sterility testing of tools prior to use were other considerations during indexing. This screening procedure practised for two-four cycles allowed reliable scrutiny of plant tissue cultures for freedom from cultivable bacteria at culture initiation or while sanitizing contaminated cultures; this would find application for certification of in vitro cultures and gene banks.