Agrobacterium rhizogenes-transformed roots of coffee (Coffea arabica): conditions for long-term proliferation, and morphological and molecular characterization

Abstract

Background and aims: The aims of this study were to set up proliferation conditions for hairy roots of Coffea arabica regenerated after transformation by Agrobacterium rhizogenes strain A4-RS, and to carry out the morphological and molecular characterization of hairy root clones maintained over the long term.

Methods: Auxin supply, light conditions and sucrose concentration were modified with the aim of establishing efficient root proliferation conditions. The morphological variability among 62 established hairy root clones was phenotyped by scanning the roots and analysing the images using 'whinRHIZO' software procedures. PCR analysis of integration in transformed root cells of rol and aux oncogenes from the T-DNA of the Ri plasmid was used to study the molecular variability among clones.

Key results: Auxin supply was necessary to obtain and stimulate growth and branching, and IBA applied at 0.5 microm was the most efficient auxin. Significant differences were shown among the 62 clones for total root length and for the percentage of fine roots. These variables were stable across subcultures and could hence be used for efficient characterization of hairy root clones. The majority of hairy root clones (86 %) exhibited non-significant phenotype differences with non-transformed roots. Eight clones were significantly different from the non-transformed controls in that they possessed a low proportion of fine roots. Two other hairy root clones grew significantly faster than the other clones. The PCR analysis revealed a low variability in the integration of rol and aux oncogenes in transformed root cells. The T(R)-DNA was never integrated as aux1 and aux2 genes were not found, although rolB and rolC genes from the T(L)-DNA were always present.

Conclusions: The discovery of low morphological variability among coffee hairy roots together with the identification of morphological variables allowing easy identification of phenotypically altered clones represent two important results. They make hairy roots a possible, and efficient, tool for functional-genomic studies of coffee root genes.

Fig. 1.

Different phenotypes of coffee hairy root clones in comparison with non-transformed roots (control). (A) X16 hairy root clone showing a high growth rate and a large percentage of branched fine roots; (B) non-transformed root clone (C1, control); (C) X43 hairy root clone with low growth rate and small percentage of branched fine roots; (D) X22 hairy root clone exhibiting a normal phenotype with a large percentage of branched fine roots and an intermediate growth rate. Scale bar = 10 mm.

Fig. 2.

Correlations between growth variables characterizing the development of 62 coffee hairy root clones. The three variables were assessed at the end of the first subculture (21 d) and at the end of the following subculture (42 d). A coefficient of determination R² was calculated and is indicated on the graphs.

Fig. 3.

Relation between total root length (cm) after a 3-week subculture period and the percentage of fine roots for 62 hairy root clones and three non-transformed clones (controls) of Coffea arabica. Data from the control clones are shown in red and underlined (C1, C2 and C3). Hairy root clones significantly different from the controls for the percentage of fine roots (i.e. possessing thick roots) are shown in green and those differing for the total root length are shown in blue (i.e. fast growing). The hairy root clones within the ellipse were not significantly different from the controls in terms of their morphology.

Fig. 4.

Histological cross-sections of normal, non-transformed roots and A. rhizogenes-transformed roots. (A) Normal root; (B) transformed root showing a non-altered phenotype (clone X13); (C) transformed root exhibiting the altered ‘fast growing’ phenotype (clone X19); and (D) transformed root with the altered phenotype characterized by slow growth and low percentage of fine roots (diameter <0·5 mm; clone X39). cc, central cylinder; rh, rhizoderm; pc, cortical parenchyma. Scale bar = 100 µm.