Epichloë sp. enhances micropropagation efficiency in grasses

PITTA-ALVAREZ, S. I.; REGALADO GONZÁLEZ JJ; IANNONE, L. J.; VIGANLE, M.V.; NOVAS, M.V.; BERDION, M.V.

Salamanca

Simposio; 10th International Symposium on Fungal Endophytes of Grasses; 2018

Fungi of the genus Epichloë (Tul) are a group of Ascomycota (Hypocreales, Clavicipitaceae) including both sexual and asexual species, which establish symbiotic associations as foliar endophytes. Asexual species are commonly called “endophytes of grasses” because they grow within the tissues of some C3 grasses (Poaceae, subfamily Pooideae)[1]. The association with Epichloë endophytes usually impacts significantly on the physiology and ecology of host plants[2]. Particularly, asexual Epichloë spp. promote biomass and seeds production, regrowth capacity and increasethe resistance to abiotic and biotic stresses) in the host plant. Thus, these endophytes have been used in grass breeding. However, biotechnological tools are difficult to use in this breeding because grasses are recalcitrant to in vitro culture[3]. The objective of our research is to study the effect of Epichloë sp. on the micropropagation of different grasses and develop micropropagation protocols to allow the application of different biotechnological tools in these grasses.Seeds of different grasses species (Lolium multiflorum, Bromus auleticus and Bromus Pictus) infected with Epichloë spp. (E+) or not (E-) were sterilized[4] and cultured in Petri dishes with MS medium supplemented with 30 gL-1sucrose or with CIM medium (Callus Induction Medium), MS supplemented with 30 gL1sucrose, 2 mgL1 2,4-D and 0.1 mgL1 BA. After 4 weeks of incubation in dark at 25±2°C, germination and callus formation percentage were calculated and compared between E+ and E- seeds in each species (Table 1). In all species, both in vitro germination and callus formation were significantly higher in E+ seeds. The calluses obtained were re-cultured in new plates with MIC medium every 4 weeks until reaching the size to start plant regeneration. In the case of L. multiflorum, callus growth was studied, being the growth of the callus E+ significantly faster than the callus E-[4].The calluses (E+ and E-) were cultured to induce shoot regeneration in plates with Regeneration Medium (RM), MS supplemented with 30 gL1 sucrose and 0.2 mgL1 Kinetine, and incubated for 8 weeks at 25±2°C under 16:8 h (L:D)photoperiod. Shoot regeneration was significantly higher in the L. multiflorum and B. auleticus callus E+ than E- (Table 1). However, regeneration was unsuccessful in B. pictus and we are performing additional tests to fine-tunethis regeneration. The L. multiflorum shoots rooted in the medium RM[4] and the B. auleticus shoots were ex vitro rooted during the acclimatization. Finally the B. auleticus plant size was studied, and in both in vitro and ex vitro the size of the plantlets regenerated from E+ seeds was significantly higher than E- seeds.All results obtained indicated that the presence of Epichloë sp. in the seeds of different grasses promotes substantially in vitro germination, the success in different micropropagation steps (callus formation or regeneration) and the growth of the regenerated plantlets. This interaction allows successful micropropagation of grasses considered recalcitrant, such as L. multiflorum or B. auleticus, allowing the use of biotechnological techniques in breeding grasses and the use of in vitro plants in different studies, as the interaction with other microorganisms.