Abstract
BackgroundIce plant (Mesembryanthemum crystallinum L.) is a model plant for studying salt-tolerant mechanisms in higher plants. Many salt stress-responsive ice plant genes have been identified with molecular and biochemical approaches. However, no further functional characterization of these genes in host plant due to lack of easy and effective transformation protocols.ResultsTo establish efficient transformation system of ice plants, three types of ice plant materials, hypocotyl-derived callus, aseptically-grown seedlings and pot-grown juvenile plants, were used to develop Agrobacterium-mediated transformation protocols. The highest transient transformation efficiency was with 5-day-old ice plant callus co-incubated with an Agrobacterium tumefaciens at 2.5 × 109 cells mL−1 for 48 h. The 3-day-old ice plant seedlings with root tip removed were successfully infected with A. tumefaciens or A. rhizogenes, and obtained 85% and 33–100% transient transformation rates, respectively. The transient transformation assays in ice plant callus and seedlings demonstrated that the concentrations of Agrobacteria, the durations of co-incubation time, and the plant growth stages were three important factors affecting the transient transformation efficiencies. Additionally, pot-grown juvenile plants were syringe-injected with two A. rhizogenes strains A8196 and NCPPB 1855, to establish transformed roots. After infections, ice plants were grown hydroponically and showed GUS expressions in transformed roots for 8 consecutive weeks.ConclusionsOur Agrobacterium-mediated transformation protocols utilized hypocotyl-derived callus and seedlings as plant materials, which can be easily obtained in large quantity. The average successful transient transformation rates were about 2.4–3.0% with callus and 33.3–100.0% with seedlings. We also developed a rapid and efficient protocol to generate transgenic roots by A. rhizogenes infections without laborious and challenging tissue culture techniques. This protocol to establish composite ice plant system demonstrates excellent improvements in efficiency, efficacy, and ease of use over previous ice plant transformation protocols. These Agrobacterium-mediated transformation protocols can be versatile and efficient tools for exploring gene functions at cellular and organ levels of ice plants.
Highlights
Ice plant (Mesembryanthemum crystallinum L.) is a model plant for studying salt-tolerant mechanisms in higher plants
Mesembryanthemum crystallinum is a facultative halophyte with distinctive ability to change from C3 photosynthesis to Crassulacean acid metabolism (CAM) under stress, tolerate high salinity by transporting sodium into vacuoles of specialized epidermal bladder cells (EBCs) and accumulate osmolytes in the cytosol during water deficit and salt stress (Bohnert and Cushman 2000; Cushman and Borland 2002)
Plasmids, and culture conditions Agrobacterium tumefaciens and A. rhizogenes strains were grown in 523 media (Kado and Heskett 1970) or on 523 agar supplemented with appropriate antibiotics at 28 °C
Summary
Ice plant (Mesembryanthemum crystallinum L.) is a model plant for studying salt-tolerant mechanisms in higher plants. Many salt stress-responsive ice plant genes have been identified with molecular and biochemical approaches. Mesembryanthemum crystallinum is a facultative halophyte with distinctive ability to change from C3 photosynthesis to Crassulacean acid metabolism (CAM) under stress, tolerate high salinity by transporting sodium into vacuoles of specialized epidermal bladder cells (EBCs) and accumulate osmolytes in the cytosol during water deficit and salt stress (Bohnert and Cushman 2000; Cushman and Borland 2002). CAM induction in M. crystallinum provides a prototype for plant scientists to study various gene and enzyme functions associated with the CAM pathway, such as CAMspecific isoform of phosphoenolpyruvate carboxylase (PEPC) (Cushman et al 1989; Winter and Holtum 2014)
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