In vitro establishment of nitrogen-fixing strawberry (Fragaria × ananassa) via artificial symbiosis with Azomonas insignis

Abstract

Artificial symbiosis was established between diazotrophic Azomonas insignia and strawbeH'y (Fragaria × ananassa). The partnership was created by in vitro techniques through callus induction and organogenesis. Suitable micropropagation [M3 = Murashige and Skoog (1962) (MS) basal medium supplemented with 2.5 btM N"-benzyladenine (BA), 0.3 gM gibberellic acid (GA3) , 2.2 gM indole-3-butyric acid (IBA), and 3% sucrose] and plant regeneration [R3 = MS mineral salts + 555 gM myo-inositol, 1.2 I.tM thiamine HC1, 4.4 gM BA, 0.5 gM IBA, 0.3 gM c~-naphthaleneacetic acid (NAA), 0.5 gM 2,4-dichlorophenoxyacetic acid (2,4-D)] media were developed for the test cultivar Fert(;di F5. New shoots containing bacteria were rooted, acclimatized, and planted outdoors. The basis of the partnership during the in vitro phase is the bacterial dependence on the plant metabolic activity, using maltose in the medium as carbon and energy source that can be utilized by the plant cells only. The presence of bacteria in the intercellular spaces of the callus tissues and regenerated plants was proved by re-isolation and microscopic techniques. Nitrogenase activity was also detected in the plant tissues.

BIOCHEMICAL PROFILE OF BACTERISED CALLI AND EMBRYOGENIC CALLI OF OIL PALM (Elaeis guineensis Jacq.)

“…As the result revealed, Z78 bacterial cells could possibly create an artificial symbiotic relation with the calli and embryogenic calli tissues and without conferring pathogenesis. Our results are in agreement with an earlier finding by Preininger et al (1997) wherein the fundamental of natural symbiosis interaction is able to create interdependency between the partners. Practically, the interdependency regulation of carbon and energy sources for both macro and micro-symbiont was able to create a most suitable artificial association condition.…”

“…As a consequence of the interactions, diazotroph could probably initiate cell proliferation and differentiation of oil palm calli and embryogenic calli in response to nitrogen supplied via nitrogen (N 2 ) fixation process and hormonal stimuli such as IAA (Lim et al, 2016b). Due to the potential application of diazotrophs with in vitro plant, various morphological analyses have been done to understand the interactions of bacteria and in vitro plant culture materials such as carrot, strawberry, rice, cormlet and oil palm (Varga et al, 1994;Preininger et al, 1997;Oliveira et al, 2002;Parray et al, 2015;Lim et al, 2016a, b). In spite of the potentials and current understanding of diazotrophs and in vitro plant interactions, research data could not fully explain the mechanism of interactions at biochemical level.…”

Trials to create artificial nitrogen-fixing symbioses and associations using in vitro methods: An outlook

“…Although callus tissues developed from the mixed suspension culture contained the introduced bacteria, no plants could be regenerated. A stable association, however, was obtained when injured leaves of micropropagated strawberries were inoculated with bacteria (Preininger et al, 1997b). Azomonas cells entered into the injured leaves, and persisted after callus formation.…”

“…The continued growth of bacteria in the associated cultures shows that they are able to utilize metabolites from the callus tissues and regenerated plants (Varga et al, 1994;Preininger et al, 1997b).…”

“…The main advantage of the methods detailed in this section is that bacterium cells are present in all parts of the entire plant (Preininger et al, 1997b). Aerobic bacteria, like Azotobacter, can also function in the photosynthetically active leaves or stems in addition to the root systems.…”