TITLE:
In Vitro Introduction of Hardy Alcohol Resistant Bacillus spp. through Aseptically Grown Watermelon Seedlings
AUTHORS:
Pious Thomas, Chennareddy Aswath
KEYWORDS:
Alcohol Resistant Bacteria, Hardy Bacillus spp., Bacterial Endospores, Microbial Contamination, Micropropagation, Plant Tissue Culture
JOURNAL NAME:
Advances in Microbiology,
Vol.4 No.9,
July
11,
2014
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.