A comparison of strategies for multiple-gene co-transformation via hairy root induction

Abstract

Hairy root is a transformed root tissue in which transfer DNA (T-DNA) is inserted in the genome by Agrobacterium rhizogenes. To establish a system for multiple-gene co-transformation in hairy roots, we evaluated four different strategies using A. rhizogenes. The genes gusA and mgfp5 were located in separate plasmids, which were transformed into two different batches of A. rhizogenes (strategy 2AR) or a single batch (strategy 2BV). The two reporter genes were also inserted in one T-DNA (strategy 1TD) or two different T-DNAs (strategy 2TD) in a binary vector. Over 90 % of infected Nicotiana tabacum leaf discs formed hairy roots in all four groups, which was not significantly different from the infection efficiency of wild-type A. rhizogenes. Proportions of co-transformed hairy roots with strategies 2AR, 2BV, 1TD, and 2TD were 65.4, 40.0, 78.6, and 82.1 %, respectively, which indicated that all of the strategies were suitable for co-transformation of multiple genes. High variation in growth rate and heterologous protein expression indicated that further screening is required to identify the clone with the highest productivity. Our results indicated that strategies 1TD and 2TD achieved the highest co-transformation efficiency. Combination with strategy 2AR or 2BV provides additional options for co-transformation of multiple transgenes.

Production of a tumour‐targeting antibody with a human‐compatible glycosylation profile in N. benthamiana hairy root cultures

“…Starting from an inoculum size of ≈0.2 g in a 40 mL culture, the FW biomass value of DXTFT N. benthamiana HRs was 11 g (275 g/L) after 25 days, while in the case of N. tabacum it was lower (100 g/L), but comparable to that already described by other groups [11]. It has been previously demonstrated that transgene expression in HRs is strongly influenced by positional effects (genes are randomly inserted in different regions of the genomic DNA), affecting not only protein expression but also hairy root growth and morphology [4,28]. This accounts for the strong variation in protein expression and growth that we and other groups observed among individual transgenic HRs expressing monoclonal antibodies in different plant species [8,11].…”

“…transgenes located in different plasmids and transformed in different batches of A. rhizogenes , was used to co‐express GUS and GFP in N. tabacum hairy roots. In this case a co‐transformation efficiency of 65% was obtained, while a higher value (82%) was obtained when the reporter genes were inserted in two different T‐DNAs within the same binary vector .…”

“…Along with cell suspension cultures, hairy root (HR) cultures represent a valid alternative thanks to their time efficiency in establishing transgenic HR lines, intrinsic genetic stability, fast biomass accumulation and manufacturing scalability . HR cultures are normally generated by infecting transgenic plants expressing the recombinant protein with Agrobacterium rhizogenes or by transforming plant tissues with A. rhizogenes strains bearing a plant expression vector encoding the protein of interest . These cultures lead to the formation of extensive secondary roots that can be cultivated under contained sterile conditions in hormone free media.…”

In Vitro Plant Cultures as Biofactories

“…Multiple-gene transformation of hairy roots has also been used to overexpress or suppress genes (Peebles et al 2011 ;Huang et al 2013b ). An example is the culture of transgenic C. roseus hairy roots; the approaches employed were two A, rhizogenes strains carrying each one a binary vector with the marker genes, an A. rhizogenes strain with both binary vectors with the marker gene, or an A. rhizogenes with the transgenes in the same or different T-DNA region of a single binary vector.…”

“…While, when the transgenes are in different binary vectors, the co-transformation effi ciency was lower, perhaps for plasmid incompatibility. However, it can be an alternative tool when trying to introduce a large number of transgene in the plant genome (Huang et al 2013b ).…”