Use of molecular markers to compare regeneration routes for developing sugarcane transgenic plants

PERERA, M. F.; RACEDO, J; OVEJERO, N; BUDEGUER, F; CUENYA, MI; WELIN, B; CASTAGNARO, AP; NOGUERA, AS

Estambul

Congreso; International Conference on Agricultural and Food Science (4th ICAFS 2020); 2020

Genetic transformation in sugarcane is an invaluable tool to overcome limitations associated with conventional breeding. It has been mainly focused on bombardment of genes into embryogenic callus (Indirect Somatic Embryogenesis, ISE); however, an alternative with several advantages, including faster regeneration, fewer subcultures and lower probability of occurrence of somaclonal variation is Direct Somatic Embryogenesis (DSE). In that sense, molecular markers could be used to screen for somaclonal variation during the process of genetic transformation. The aim of the present work was comparing ISE and DSE regeneration routes and assessing somaclonal variation by using TRAP markers. Embryogenic calli and leaf roll disc of TUC 95-10 variety were bombardment by using a plasmid harbouring epsps and nptII genes that confer resistance to glyphosate herbicide and kanamycin, respectively. The stable transformation and integration of both genes were determined by using PCR with specific primers and transgenic events were characterized by functional TRAP markers. The seven transgenic lines obtained by bombardment of calli, showed 67% similarity to the parental variety; however, they shared more than 98% similarity among them, whereas the five lines obtained by bombardment of disc leaf rolls showed more than 98% similarity to the parental genotype. The bombardment of leaf roll discs followed by DSE regeneration has been demonstrated as an improvement on the current methodology applied since the time required in culture is shortened, 14?22 weeks to produce a transformed plant versus 24?36 weeks for the conventionally used ISE route. Even more, the molecular characterization of transgenic lines of TUC 95-10 obtained confirmed that genetic changes induced by ISE were higher than those produced by DSE. In addition, TRAP markers is a rapid and recommendable first approach to identify most valuable transgenic lines (with more than 95% similarity to the parental genotype) before conducting any greenhouse tests.