In vitro Decline in Plant Cultures: Detection of a Legion of Covert Bacteria as the Cause for Degeneration of Long-term Micropropagated Triploid Watermelon Cultures

Abstract

In vitro cultures of triploid seedless watermelon cv. Arka Manik displayed a decline in shoot and root growth after 4–5 years of active culturing. Visibly clean cultures upon indexing on enriched media showed covert bacteria, and a significant improvement in proliferation and rooting in response to surface sterilization. The bacteria however survived endophytically. Low pH and reduced clarity of agar gelled medium were found to mask the expression of bacteria in the tissue culture medium. Gentamycin, streptomycin or broad-spectrum bactericide cefazolin provided as a 2 ml overlay in the medium in factorial combination at 0 or 50 mg l⁻¹ resulted in selective suppression of some bacteria depending on the treatment and eight bacterial clones comprising of four Gram-positive (Bacillus spp.) and four-Gram negative (3 × Pseudomonas spp. and 1 × Aeromonas sp.) strains were isolated from the cultures. Provision of 50 mg l⁻¹ gentamycin in 2 ml overlay in the multiplication or rooting medium coupled with occasional decontamination of cultures helped in circumventing the decline problem. The plants established in the field after 6 years of active in vitro culturing appeared normal and fertile suggesting the feasibility of keeping cultures for long periods, thus saving time and other resources. Freeing the cultures from covert bacteria was complicated by the presence of different bacterial types and this will be addressed later.