Abstract
SummaryFor non‐model plants, functional characterization of genes is still hampered by lack of efficient stable transformation procedures. Here, we report a simple, fast and efficient transformation technique with Agrobacterium rhizogenes for generating stable transgenic roots in living plants to facilitate functional studies in vivo. We showed that injection of A. rhizogenes into stems of various plant species lead to stable transgenic root generation, which can sustain plant growth after the original, non‐transgenic roots were cut off. A transformation system was established for pigeon pea, a major woody food crop, after optimizing the selection of A. rhizogenes strains, bacterium concentration, injection position and seedling age. RT‐PCR and fluorescence observation indicated a transgenic root induction efficiency of about 39% in pigeon pea. Furthermore, induction of hairy roots was achieved in nine out of twelve tested economically important plants at an efficiency of 15–39%. As proof of concept, bimolecular fluorescence complementation (BiFC) assay was applied to test the interaction between CcCIPK14 and CcCBL1/2 in pigeon pea. Additionally, ectopic expression of the bZIP transcription factor MdHY5 from apple confirmed the utility of the transformation technique for engineering anthocyanin synthesis in roots. Taken together, we show that this method allows fast in vivo studies of gene function in a wide range of plant species.
Highlights
Agrobacterium-mediated plant transformation system has been the most widespread and successful method for plant genetic engineering in recent decades (Gelvin, 2003; Matveeva and Lutova, 2014; Vain, 2007)
Since the first widely accepted demonstration of successful transfer of exogenous genes using the Agrobacterium-mediated plant transformation method in tobacco in 1983, efficient transformation systems have been established for a wide range of model plants and crops facilitating gene functional research and plant genetic modifications (Zhang et al, 2006; Wenzler et al, 1989; Ishida et al, 1996)
The constructed vector was introduced into the Agrobacterium rhizogenes strains and injected into 15-day-old pigeon pea seedlings (Figure 1a)
Summary
Agrobacterium-mediated plant transformation system has been the most widespread and successful method for plant genetic engineering in recent decades (Gelvin, 2003; Matveeva and Lutova, 2014; Vain, 2007). Agrobacterium tumefaciens invades plants at wounds where it can cause tumours by transferring a tumour-inducing (Ti) plasmid to the plant cell nucleus Based on this mechanism, the Ti plasmid was modified and tumourinducing genes were replaced by genes of interest to be incorporated into the chromosomes of the plant cell. A target gene can be transferred and incorporated into the genome of the host plant by infection of R. rhizogenes harbouring a modified Ri plasmid, resulting in transgenic hairy roots. Based on this mechanism, a valuable biotechnological application was exploited, known as hairy root culture (HRC). The hairy root system has proven to be very valuable to investigate gene functions, there are very few reports on medicinal and woody plants
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