Estabelecimento de protocolo de cultivo in vitro de calos embriogênicos da cana-de-açúcar RB034045 voltado à transformação genética, via biobalística, com o gene cp4-epsps

Abstract

Somatic embryogenesis is a key tool in the genetic improvement of sugarcane (Saccharum spp.), particularly in protocols involving genetic transformation. However, limitations such as endophytic contamination and the highly variable in vitro response among genotypes still hinder its efficiency. This study aimed to establish and optimize an indirect somatic embryogenesis protocol for the RB034045 variety, as well as to perform genetic transformation experiments via biolistics as a validation step, using the cp4-epsps gene, associated with glyphosate resistance, as a selectable marker. The initial protocol was developed using leaf roll explants from the shoot apical region, cultivated in medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) for induction and multiplication of embryogenic calli, followed by regeneration and plant development steps. Based on this system, optimization experiments were conducted focusing on three critical aspects: endophytic contamination, efficiency of the regeneration medium composition, and explant region suitability. Strategies involving 2.5% sodium hypochlorite combined with reduced plant tissue per flask effectively decreased contamination levels. Regarding regeneration, the best results were obtained with medium supplemented with coconut water (77.7%) and 6-benzylaminopurine (BAP) (75.8%). The basal region of the cylindrical leaf stalk stood out for its higher callogenesis efficiency and embryogenic yield. Additionally, the minimum inhibitory concentration of kanamycin for effective selection was determined to be 100 mg L−1. For validation, embryogenic calli were subjected to biolistic transformation using a construct carrying the cp4-epsps gene and subsequently cultured under selective pressure. Regenerated plantlets exhibited an albino phenotype and low vegetative vigor. PCR analysis did not detect the cp4-epsps gene, although amplification of the nptII gene was observed, confirming partial integration of the transgene. The results demonstrate the feasibility of the somatic embryogenesis protocol developed for the RB034045 variety and its potential application in genetic transformation strategies, with prospects for optimization toward the generation of stable transgenic events.