A novel method based on combination of semi-in vitro and in vivo conditions in Agrobacterium rhizogenes-mediated hairy root transformation of Glycine species

Despite numerous advantages of the many tissue culture-independent hairy root transformation protocols, the process is often compromised in the initial in vitro culture stage where inability to maintain high humidity and the delivery of nourishing culture medium decrease cellular morphogenesis and organ formation efficiency. Ultimately, this influences the effective transfer of produced plantlets during transfer from in vitro to in vivo conditions, where low survival rates occur during the acclimation period. We have developed an intermediate protocol for Agrobacterium rhizogenes transformation in Glycine species by combining a two-step in vitro and in vivo process that greatly enhances the efficiency of hairy root formation and which simplifies the maintenance of the transformed roots. In this protocol, cotyledonary nodes of Glycine max and Glycine canescens seedlings were infected by A. rhizogenes K599 carrying a reporter gene construct constitutively expressing green fluorescent protein (GFP). Glass containers containing sand and nutrient solution were employed to provide a moist clean microenvironment for the generation of hairy roots from inoculated seedlings. Transgenic roots were then noninvasively identified from nontransgenic roots based on the detection of GFP. Main roots and nontransgenic roots were removed leaving transgenic hairy roots to support seedling development, all within 1 mo of beginning the experiment. Overall, this protocol increased the transformation efficiency by more than twofold over traditional methods. Approximately 88% and 100% of infected plants developed hairy roots from G. max and G. canescens, respectively. On average, each infected plant produced 10.9 transformed hairy roots in G. max and 13-20 in G. canescens. Introduction of this simple protocol is a significant advance that eliminates the long and genotype-dependent tissue culture procedure while taking advantage of its optimum in vitro qualities to enhance the micropropagation rate. This research will support the increasing use of transient transgenic hairy roots for the study of plant root biology and symbiotic interactions with Rhizobium spp.