Abstract
The present study was taken up with a view to ascertain the possibility of introduction of alcohol resistant bacteria in vitro through the aseptically raised watermelon (Citrullus lanatus) seedlings in the backdrop of isolating such organisms from micropropagated watermelon stocks. Watermelon cv. Arka Manik seedlings grown in vitro from surface-sterilized seeds with the intact seed coat on MS medium appeared visibly clean largely, but upon subjecting them to tissue-indexing, the segments from the collar or root tissue showed bacterial colony growth on Nutrient Agar (NA) from 72% of such healthy seedlings and the cotyledon and hypocotyl tissue of 44% seedlings. The pooled colony growth from NA upon challenge with 90% alcohol yielded 10 distinct colony types, identified as B. pumilus (4×), B. subtilis (4×), B. cereus (1×) or B. safensis (1×) based on partial 16S rRNA sequence analysis. The shoot-tip tissue from the healthy index-negative seedlings cultured on watermelon proliferation medium partly turned index-positive within 2 - 4 sub-culture cycles while being apparently clean. On the other hand, those from the previously index-positive cultures tended to show obvious bacterial growth during subsequent in vitro culturing. The observations suggested the possibility of introduction of spore-forming Bacillus spp. through surface-sterilized seeds, their gradual emergence in vitro in visibly clean seedlings, possible transmittal of spores to the alcohol through tissue-culturing tools and the survival therein with the chances of unsuspected lateral spread. Seed coat removal followed by surface sterilization with sodium hypochlorite facilitated the raising of clean seedlings with no detectable bacterial association.
Highlights
Microbial contamination is a serious limiting factor with plant tissue culture research and commercial production units [1]-[3]
Recent studies have indicated that alcohol once got contaminated with the hardy spores of bacteria like Bacillus, Brevibacillus, Lysinibacillus or Paenibacillus spp., the spores could survive therein for long periods, and the adulterated alcohol could serve as a source of inoculum leading to the unsuspected lateral spread of contaminants [6] [7]
The seeds that were imprinted on Nutrient Agar (NA) following surface sterilization as well as the last wash solution did not show any bacterial growth which prima facie suggested efficient surface sterilization
Summary
Microbial contamination is a serious limiting factor with plant tissue culture research and commercial production units [1]-[3]. Recent studies have indicated that alcohol once got contaminated with the hardy spores of bacteria like Bacillus, Brevibacillus, Lysinibacillus or Paenibacillus spp., the spores could survive therein for long periods, and the adulterated alcohol could serve as a source of inoculum leading to the unsuspected lateral spread of contaminants [6] [7]. At this laboratory, two alcohol surviving bacteria were initially isolated from the micropropagated cultures of grapes as covertly associated endophytic colonizers, namely B. pumilus [8] and Brevibacillus sp., the latter an uncommon Gram-negative spore-former [9]. The spores of the above organisms showed survival in ethanol for varying periods to the tune of over one year depending on the concentration of ethanol, the organism involved, stage of growth of source culture, spore content and the growing conditions [7]-[10]
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