Optimal growth conditions of Agrobacterium Rhizogenes for use in the transformation of banana (musa spp).

Abstract

In recent years, rapid procedures for obtaining transgenic roots have been developed using Agrobacterium rhizogenes, a soil pathogen which elicits adventitious and genetically transformed roots from a range of tissue types. The resultant plants are “composite” comprising a transgenic hairy root system attached to non-transformed shoot system. Hairy roots are important in the transformation process because of their first growth and high laterally branched nature. Agrobacterium rhizogenes strains vary in their virulence for induction of hairy roots in plants. The objective of this study was to 1) to determine the efficacy of A. rhizogenes ability to induce the hairy root trait in banana, 2) to determine optimal conditions (OD600nm and inoculation time) required for effective induction of banana hairy roots using A. rhizogenes, and 3) to determine response of selected local East African Highland banana to transformation by A. rhizogenes. Out of nine A. rhizogenes strains tested, only the agropine-producing strains, namely, LBA9402, 15835, A4 and PMP90 infected and induced varying amounts of hairy roots in banana corms, apical meristems and leaves of genotypes such as M. balbisiana, 9518S-12, 5610S-1, Calcutta 4, Kisansa, KM5 and 3165K-5, whereas the remaining 5 strains; LMG150, LMG151, PGR2260, NCPPB4042 and 5083, did not induce hairy roots when inoculated on the explants. An optical density (OD600nm) 0.8 and inoculation time of 30 minutes was found to be optimum to induce hairy roots in the majority of the genotypes and explants that were able to respond to A. rhizogenes infection. The most responsive explants were apical meristems, followed by corms and finally the leaves. Beyond an optimal inoculation time of 30 minutes, overgrowth of bacteria was observed on infected sites leading to death and decomposition of explants and no transformation was achieved. At lower optical densities, only traces of hairy roots were produced while at higher levels such as OD600nm 1.0 no hairy roots were produced. The explants (corms, apical meristems and leaves) used were obtained from cultivars that belong to 3 ploidy level groups; diploids (M. balbisiana, Calcutta 4, 5610S-1 and 9518S-12), triploids (Kisansa and KM5) and one tetraploid (3165K-5). Diploid Musa genotypes responded best with highest percentage hairy root induction frequencies observed in LBA9402 + M. balbisiana; A4 + M. balbisiana; LBA9402 + 9518S-12; PMP90 + M. balbisiana; 15835 + M. balbisiana; LBA9402 + 9518S-12; 15835 + M. balbisiana; 15835 + 5610S-1 with the highest %HRI as 12.5% (M. balbisiana) as compared to 2.08% (3162K-5). PCR analysis was performed on hairy root sets to x confirm presence and integration of rolA gene into the explants using Ri specific primers. Bands of similar expected size (300bp) were obtained from strains 15835, A4, PMP90 and LBA9402. This study demonstrated the susceptibility of banana to A. rhizogenes mediated transformation and hairy roots induction indicated that A. rhizogenes can be used to transform banana