Effect of supernatant of associative bacteria of the genus Pseudomonas on germination, morphogenesis and growth of Dactylorhiza incarnata (L.) Soo (Orchidaceae) in vitro

No studies on the influence of supernatant of associative bacteria on the growth and development of orchid temperate climate of the northern hemisphere in vitro have been conducted to date. However, it is necessary to develop technologies for the cultivation of rare and protected species in vitro using biologically active components of culture media. The aim of this research was to assess the effect of two supernatants of associative bacteria of the genus Pseudomonas from the rhizosphere of generative individuals of Dactylorhiza incarnata on seed germination, morphogenesis and seedling growth of this species in vitro, a representative of orchids in the middle part of the European part of Russia. We isolated bacteria of the genus Pseudomonas (P chlororaphis subsp. aurantiaca GRP225 and P. brassicacearum GRT221) from the rhizosphere of generative individuals of D. incarnata, growing in a sedge-herb community in the middle zone of the European part of Russia (Yaroslavl region). Molecular genetic analysis of the nucleotide sequences of 16S rRNA genes was used to identify the strains. Bacteria were grown on LB medium with constant stirring for 5-7 days at 30 °C. Supernatant was obtained by separation of the bacterial mass by centrifugation (5000 rpm, 20 min, at 4 °C). The content of IAA in supernatant was determined by the Salkovsky method [Tsavkelova EA et al, 2007]. For supernatant strain P. chlororaphis subsp. aurantiaca GRP225, this parameter was 18.2±1.30 mg/l, for P. brassicacearum GRT221 it was 31.1±1.94 mg/l. We carried out the study according to the following scheme: 1) a modified Knudson nutrient medium (control); 2) the same with IAA 1.75 mg/l (10 pM); 3) the same with supernatant P. chlororaphis subsp. aurantiaca GRP225 (10% of volume); 4) the same with supernatant P. brassicacearum GRT221 (10% of the volume). Crops of seeds were cultivated for 2 months in the dark and 16 months in the light (2000 lux, 16 h). Seed germination was judged by the amount of proto-feeds formed. The number of germinated seeds was determined at the 3rd, 6th, 12th and 18th months of cultivation by the number of formed protocorms. Full germination of seeds was determined by the number of seedlings 18 months after sowing. We distinguished a protosome and a juvenile plant when describing morphogenesis [Kolomeytseva GL et al, 2012]. The morphophysiological parameters of the seedlings (linear dimensions of protosomes, shoots and adventitious roots, the wet weight of seedlings) were determined at the 3rd, 6th, 12th and 18th months of cultivation. We revealed a stimulating effect of supernatant ofP chlororaphis subsp. aurantiaca GRP225 and P. brassicacearum GRT221 for the germination of mature seeds of D. incarnata in vitro (See Fig. 2). Moreover, the effect of the first supernatant is 27% more effective compared to the second supernatant and is expressed in an increase in the number of sprouted seeds in relation to the control 5 times and in the test with IAA 1.8 times. D. incarnata seedlings on nutrient media with a supernatant are characterized by accelerated rates of development (See Fig. 3). This is confirmed by the early and active formation of protorizomes, orthotropic shoots and accessory roots. The effect of the supernatant of the strain P. brassicacearum GRT221 coincides with the action of IAA. On a medium with supernatant P chlororaphis subsp. aurantiaca GRP225, more seedlings with photosynthetic leaf - juvenile plants are formed by the 18^th month (See Fig. 1f). The growth parameters of D. incarnata seedlings on nutrient media with a supernatant are high (See Table 1 and 2). For linear sizes of protorizomes, this was noted from the 3rd month and amounts to 20-30% compared with the control. Orthotropic shoot growth is most pronounced in seedlings on a medium with supernatant P. chlororaphis subsp. aurantiaca GRP225. Its length is 30% more than the corresponding values in other tests. On the same medium, seedlings have the maximum wet weight. Thus, the use of supernatant of associative bacteria is effective for accelerating the germination of mature seeds, increasing their germination, the rate of development of seedlings, the size of the orthotropic shoot and adventitious roots, as well as the accumulation of fresh mass. The maximum stimulating effect is shown for the supernatant of P. chlororaphis subsp. aurantiaca GRP225. This opens up prospects for its use in vitro cultivation of rare and protected orchid species of the middle zone of the European part of Russia for their conservation and reproduction. The paper contains 3 Figures, 2 Tables and 32 References.

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