Calogênese e embriogênese somática de cana-de-açúcar, variedade RB867515: otimização de protocolo

Abstract

The sugarcane is a crop of major importance for Brazil, which currently leads the ranking of production and export of sugar and ethanol. Research has been developed in order to obtain new cultivars with desirable agronomic characteristics such as resistance to biotic and abiotic stresses. In addition to traditional breeding methods, biotechnological tools such as genetic transformation have been developed in order to meet this demand for more productive and resistant varieties. The establishment of efficient protocols for somatic embryogenesis, thereby producing more appropriate target cells for this procedure, is a key point to the success of the biotechnological approach. The present study aimed to (1) test different disinfestations protocols for the in vitro introduction of explants; (2) evaluate the induction of somatic embryogenesis in different types of explants; and (3) analyse the influence of different culture media and growth regulators on the steps of somatic embryogenesis of sugar cane, cv. RB867515. Preliminary tests with three different explants (shoots, meristem and shoot tips) and different disinfection protocols were performed. The disinfestations protocols involved the immersion of explants in sodium hypochlorite (1.25%), 70% ethanol, mercuric chloride, or fungicide (Cercobin, with and without vacuuming). The frequency of callus induction from the shoot apex and base on MS medium with 1.0; 2.0; 3.0; 4.0 mg.L-1 of 2,4-D was evaluated. The frequency of callus induction from the apexes of micro propagated plants in media with and without supplementation of coconut water and the four aforementioned 2,4-D concentrations were evaluated as well. The calli formed in the induction media were transferred to multiplication media containing 3.0 mg.L-1 of 2,4-D with or without IAA (4 mg.L-1) and without the supplementation of coconut water, aiming to increase the embryogenic area. For the regeneration step, MS basal media with and without 1 mg.L-1 BAP and GA3 were evaluated. After plants obtaining, the efficiency of the activated charcoal in the roots induction was evaluated. In disinfestations treatments, the decontamination and explants induction were more efficient when sodium hypochlorite (1.25%) and 70% ethanol were applied in the disinfestation procedure. No difference was observed in the callus induction originated from either the apex or the base of shoots at all concentrations of 2,4-D. The use of 2,4-D at a concentration of 3 mg.L-1 provided the best callus induction from shoot apexes. The greater intensity multiplication of embryogenic areas was achieved with the medium without the supplementation of IAA. Satisfactory results for plant regeneration were observed in the medium without the supplementation of growth regulators. The activated charcoal was beneficial to rooting.