Transformation of Nicotiana tabacum L. root by Agrobacterium rhizogenes for in vitro cultivation and symbiotic association with Rhizophagus clarus

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Abstract

Rhizophagus clarus is an arbuscular mycorrhizal fungus and an obligatory symbiont of plant roots, capable of improving plant growth and development, as it increases root area and facilitates the absorption, mainly, of water and phosphate compounds. R. clarus in vitro cultivation associated with carrot roots (Daucus carota) transformed by the Ti plasmid of Agrobacterium rhizogenes is promising in obtaining propagules for agricultural inoculation, already tested in crops of soybean and cotton. Aiming to a large-scale production, tobacco roots (Nicotiana tabacum L.) were transformed, due to the high mycotrophy evidenced in greenhouse experiments, to evaluate colonization to be the basis for an inoculant. The transformation of tobacco roots was performed from leaf disks and disarmed strains of A. rhizogenes. The disks were submerged for 10 seconds in Luria Bertani broth containing A. rhizogenes, and then transferred to Petri dishes with Murashige and Skoog (MS) medium for 3 days/25 °C. Subsequently, they were transferred to MS medium added with 22 mg/L of cefotaxime, to inhibit bacterial growth. After three weeks, the grown roots were transferred to fresh MS medium with antibiotic, and it was repeated until reaching good root growth. All cultures were maintained at 25 °C in the dark. After transformation, roots were multiplied every 30 days in minimal mineral medium (M). The fungus R. clarus was cultivated associated with carrot roots in M medium that was sectioned into 2.2 cm² squares to be used to inoculate the transformed roots after 10 days of growth. Mycelial growth was estimated by the Root Length formula (RL= π.N.A/2H), where N = intersections; A = plate area; H = sum of the length of the lines, and π = 3.14. The intersections of hyphae were counted in lines spaced 0.5 cm apart and for root growth was used the same method, with 1 cm apart spacing. Spores were quantified by direct counting in an optical stereoscope. Root growth started 10 days after reaching the peak and intensified up to 45 days reaching 394 cm in length and 487 spores after 150 days. The fungus showed high mycelial growth and sporulation reaching 1545 cm in length and 1325 spores after 150 days. This study shows the viability of using transformed tobacco roots for in vitro multiplication of R. clarus propagules and it can be the basis of a future inoculant, therefore further research is needed.

Institutions
  • 1 Departamento de Microbiologia / Centro de Ciências Biológicas / Universidade Estadual de Londrina
  • 2 Centro de Ciências Biológicas / Universidade Estadual de Londrina
  • 3 Centro de Ciências Agrárias / Universidade Estadual de Londrina
  • 4 Centro de Ciências Exatas / Universidade Estadual de Londrina
Track
  • Inovação e Biotecnologia
Keywords
transformed roots
Tobacco
mycorrhiza
arbuscolar mycorrhizal fungi