Microclonal propagation in vitro is being actively used in the production of healthy planting material of food and ornamental plants. However, it needs further improvement to increase the growth rates of microclones in vitro and enhance regenerant survivability ex vitro. A promising approach to this end could be inoculating in vitro-micropropagated plants with plant growth-promoting rhizobacteria, specifically Azospirillum. However, the influence of Azospirillum inoculation on microclone behavior throughout the production process, including plant adaptation ex vitro and food crop productivity, has been underinvestigated. In this study, in vitrogrowing potato (Solanum tuberosum L.) microclones were inoculated with Azospirillum brasilense strain Sp245. The microclones were then grown on in soil in the greenhouse and field, with the experiment lasting for 120 days. Rootassociated bacteria were identified immunochemically, and the mitotic index of root meristematic cells was determined by a cytological method. The plant morphological parameters determined were shoot length, number of nodes per shoot, number of roots per plant, maximal root length, leaf area, percentage of surviving plants in the soil, and tuber yield and weight. Our results show that bacterial inoculation of potato microclones in vitro enhances plant adaptive capacity ex vitro and increases minituber yield. The percent survival index of field-grown inoculated plants was 1.5-fold greater than that of uninoculated plants. The overall tuber weight per plant was more than 30 % greater in the inoculated plants than it was in the control ones. For all cultivars on average, tuber yield per square meter increased by more than 45 % as a result of inoculation in vitro. This study is the first to report that Azospirillum inoculation of potato microclones not only improves the quality of planting material produced in vitro but also significantly increases minituber yield through enhancing plant adaptive capacity in the field.
Water deficits inhibit plant growth and decrease crop productivity. Remedies are needed to counter this increasingly urgent problem in practical farming. One possible approach is to utilize rhizobacteria known to increase plant resistance to abiotic and other stresses. We therefore studied the effects of inoculating the culture medium of potato microplants grown in vitro with Azospirillum brasilense Sp245 or Ochrobactrum cytisi IPA7.2. Growth and hormone content of the plants were evaluated under stress-free conditions and under a water deficit imposed with polyethylene glycol (PEG 6000). Inoculation with either bacterium promoted the growth in terms of leaf mass accumulation. The effects were associated with increased concentrations of auxin and cytokinin hormones in the leaves and stems and with suppression of an increase in the leaf abscisic acid that PEG treatment otherwise promoted in the potato microplants. O. cytisi IPA7.2 had a greater growth-stimulating effect than A. brasilense Sp245 on stressed plants, while A. brasilense Sp245 was more effective in unstressed plants. The effects were likely to be the result of changes to the plant’s hormonal balance brought about by the bacteria.
The authors declare no conflict of interests Acknowledgements: We thank S.N. Dedysh for assistance in molecular genetic analysis and Yu.M. Serekbaeva for her help in 16S rRNA gene sequencing. We are also sincerely grateful to Prof. Dr Frank Oliver Glöckner for his valuable comments on the use of the resource https://www.arb-silva.de. Immunofluorescence microscopy was performed at the Symbiosis Center for collective use of scientific equipment (Institute of Biochemistry and Physiology of Plants and Microorganisms RAS, Saratov). DNA preparation, sequencing and assembly of the fragments were made in the Laboratory of microbiology of wetland ecosystems (Institute of Microbiology RAS, Moscow) and ZAO «Evrogen» (Moscow).
Received November 14, 2016
A b s t r a c tWe present the results of molecular, genetic, physiological and biochemical investigations of a bacterial isolate from the rhizosphere of potato (Solanum tuberosum L.) as an object to study plant-microbial associativity, used in particular to improve the existing technologies for the production of high-quality planting material by the method of plant culture micropropagation in vitro. To correctly identify the isolate at the species level, we took into account the results of analysis of the current status of prokaryote identification and systematics, reflected in a number of recent reviews. Phylogenetic constructs with strains of the genera Ochrobactrum, Brucella, Ensifer, Mesorhizobium, Rhizobium, and more (closely related to the isolate under study), which were generated by using DNA sequences of 16S rRNA genes, revealed the isolate in the immediate surroundings of members of the genus Ochrobactrum. The isolate turned out to be part of the taxonomic group Ochrobactrum anthropi -one of the 1912 taxonomic groups recorded to date, which comprise 6193 prokaryotic species and each include species with coinciding (or almost coinciding) sequences of 16S rRNA genes (http://www.ezbiocloud.net/identify). In accordance with the conceptual propositions formulated in the above-mentioned publications, the species differences within these groups are determined at the level of other molecular genetic (and biochemical and physiological) properties and, with a high probability, by horizontal gene transfer. With account taken of the resulting set of elements of the polyphasic approach, the strain isolated by us was found to be closest to the known type strain O. lupini LUP21, which is capable of reinfecting leguminous plants of the genus Lupinus and which carries nodulation and nitrogen fixation genes (nodD and nifH), transferred horizontally into it from rhizobial species. This gave us grounds to identify the isolate being examined as Ochrobactrum lupini IPA7.2.
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