Indirect somatic embryogenesis in Coffea canephora and Coffea eugenioides: endogenous hormone levels and copy number of genes related to auxin biosynthetic pathway and morphogenic in vitro response

Abstract

Coffea canephora and Coffea eugenioides indirect somatic embryogenesis (ISE) has been established and compared during callus formation and somatic embryo regeneration, showing that these species exhibit the same response in vitro. However, intraspecific differences regarding ISE responses have also been reported. ISE is influenced by genetic and physiological aspects. We aimed to: (a) induce ISE in C. canephora and C. eugenioides; (b) identify and quantify the endogenous levels of indole-3-acetic acid (IAA), zeatin (Z) and gibberellic acid GA 3 and GA 4 during the callus induction; and (c) determine the number of gene copies of AUX/IAA33 and YUC4 that are involved in the auxin biosynthetic pathway, and of WOX4 and LEC1 that are involved in the morphogenic pathway. Using liquid chromatography coupled to mass spectrometry (LC-MS), the identification and quantification of IAA, GA 3 , GA 4 and Z was performed in both Coffea. From the fluorescence in situ hybridization, the copy number of the AUX/IAA33, YUC4, WOX4 and LEC1 genes was verified. ISE was established for C. canephora and C. eugenioides, which exhibited the same in vitro response. However, the two Coffea differed in relation to endogenous hormone levels, with the exception of IAA at 90 days. These species possess two gene copies of AUX/IAA33, YUC4, WOX4 and LEC1, corroborating to their diploidy. Therefore, our results show that C. canephora and C. eugenioides differ in the physiological aspect, but not in the genetic aspect investigated here. Thus, our data contribute as a basis to understand the in vitro response and for the improvement of the in vitro propagation procedures. Keywords: Coffee. Plant Tissue Culture. In vitro regeneration. Somatic Embryo. Genetic. Plant Physiology.