БАКТЕРИАЛЬНЫЕ МИКРООРГАНИЗМЫ, АССОЦИИРОВАННЫЕ С ТКАНЯМИ РАСТЕНИЙ В КУЛЬТУРЕ in vitro: ИДЕНТИФИКАЦИЯ И ВОЗМОЖНАЯ РОЛЬ

Abstract

Effective sterilization of plant explants and antiseptics rules compliance do not exclude the presence of so-called covert (endophytic) bacteria in in vitro cultures. But the role of these bacteria in tissues cultures has been not enough studied whereas it was related to the explants regeneration capacity and the possibility of animal and human cells transformation under in vitro cultivation. Bacterial strains pathogenic to humans can be stably maintained in cultivated tissues and ex vitro plants. The broadening of bacterial environments creates ecological and genetic risks leading to necessity of careful monitoring of endophytic communities in plants used as raw food and at use of in vitro technologies in practical plant growing and food production. Identification of bacterial microorganisms colonizing in vitro plant cultures allows studying the bacteria effect on the host, realizing special chemotherapy and developing the microorganisms’ databases. Two methods of identification are the most widespread: more available traditional one that does not allow detecting non-cultured forms (its base is the use of cultural and morphological characteristics as well as chemical and biochemical reactions) and molecular-genetic one. At the second approach different 16S-rRNA sequences are studied using metagenomic DNA and appropriate specific primers; these sequences have conserved sites identical for all prokaryotes and variable ones suitable for species specific regions identification. Internal transcribed spacers (ITS) are being mainly used to distinguish the microorganisms at the species level and even at strains one. Taxonomy of in vitro cultures’ bacterial endophytes indicates to their diversity and absence of specific composition as for cultures of plants belonging to different taxa as for different plant organs explants. Among identified endophytic bacteria potentially useful for intact plants Streptomycete, Pantoea agglomerans and others were found as well as those pathogenic for humans, e.g. Ralstonia mannitolytica, Staphylococcus epidermidis, Corynebacterium amycolatum, Bacillus neonatiensis, Salmonella and Nocaridia spp. At in vitro plant cultivation durable symptomless bacterial presence is caused on the one hand by bacterial growth repression with factors accompanying plant explants cultivation (PH, temperature below bacterial optimum, activation of the defense mechanisms), and on the other hand by simultaneous bacteria support due to exudates secreted by plant explants. The rapid bacterial cells proliferation can begin even at small changes in initial conditions, at increase in plant exudates concentrations and per se in consequence of in vitro cultivation as a stress at the absence of whole organism regulatory role. As the number of subcultivations increases a portion of plant cultures with latent bacterial contamination increases too; no-cultured endophytes have been reported to acquire the status of cultured ones. Covert bacterial contamination could depress regeneration, micropropagation, cause death of in vitro cultivated objects, restrict the protocols repeatability and concern induction of epigenetic somaclonal variability. For instance Acinetobacter and Lactobacillus plantarum filtrates extracted from degrading calluses strongly reduced shoot regeneration at inoculation in explants or addition into a medium; bacteria Mycobacterium obuense and M. aichiense repressed seeds development in in vitro cultures. The article accents the problem of gnotobiological plant cultures (specifically in in vitro collections of plants genetic banks) development caused by difficulties in identification and elimination of bacterial microorganisms.