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

PERERA M.F; BUDEGUER F.; CASTAGNARO, ATILIO P.; OVEJERO S.N; WELIN, BJÖRN; RACEDO J.; CUENYA M.I.; NOGUERA A.S.

Istambul

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

Genetic transformation in sugarcane is a valuable tool that allows overcoming the limitationsassociated with conventional breeding. This is mainly carried out by bombardingembryogenic callus (Indirect Somatic Embryogenesis, ISE) with genes of interest. However,an alternative technique with several advantages is direct somatic embryogenesis (DSE),which allows faster regeneration, fewer subcultures and less probability of the occurrence ofsomaclonal variation. In this sense, molecular markers can be used to detect the possiblesomaclonal variation generated during the genetic transformation process. The objective ofthe present study was to evaluate and compare the somaclonal variation, through the use ofTRAP markers, generated by both regeneration techniques (ISE and DSE). The embryogeniccalli and leaf roll discs of TUC 95-10 variety were bombarded using a plasmid containing theepsps and nptII genes, which confer resistance to glyphosate herbicide and kanamycin,respectively. Stable transformation and integration of both genes were determined by PCRwith specific primers and the transgenic events were characterized by functional TRAPmarkers. The seven transgenic lines obtained by bombardment of calli showed 67% similarityto the parental variety; however, they shared more than 98% similarity among them, whilethe five lines obtained by bombardment of leaf roll discs showed more than 98% similarity tothe parental genotype. This last technique has proven to be a better alternative to the currentlyapplied methodology since the required cultivation time was shortened from 14 to 22 weeksto produce a transformed plant versus 24 to 36 weeks for the conventionally ISE technique.Furthermore, the molecular characterization of the TUC 95-10 transgenic lines confirmed thatthe genetic changes produced by ISE are greater than the changes induced by DSE. Inaddition, TRAP markers are a rapid and recommendable first approach to identify prominenttransgenic lines (with more than 95% similarity to the parental genotype) before carrying outany other greenhouse tests