Abstract
Casuarina equisetifolia is widely used in agroforestry plantations for soil stabilization, ecosystem rehabilitation, reclamation, and coastal protection. Moreover, C. equisetifolia has remarkable resistance to typhoons, desert, low soil fertility, drought, and salinity, but not cold. Therefore, it is significant to breed high-quality Casuarina varieties to improve the tolerance and adaptability to cold weather by molecular techniques. The establishment of a rapid and efficient callus induction and regeneration system via tissue culture is pre-requisite for the genetic transformation of C. equisetifolia, which is so far lacking. In this study, we reported an efficient and rapid regeneration system using stem segment explants, in which callus induction was found to be optimal in a basal medium supplemented with 0.1 mg⋅L–1 TDZ and 0.1 mg⋅L–1 NAA, and proliferation in a basal medium containing 0.1 mg⋅L–1 TDZ and 0.5 mg⋅L–1 6-BA. For bud regeneration and rooting, the preferred plant growth regulator (PGR) in basal medium was 0.5 mg⋅L–1 6-BA, and a combination of 0.02 mg⋅L–1 IBA and 0.4 mg⋅L–1 IAA, respectively. We also optimized genetic a transformation protocol using Agrobacterium tumefaciens harboring the binary vector pCAMBIA1301 with β-glucuronidase (GUS) as a reporter gene. Consequently, 5 mg L–1 hygromycin, 20 mg L–1 acetosyringone (As), and 2 days of co-cultivation duration were optimized to improve the transformation efficiency. With these optimized parameters, transgenic plants were obtained in about 4 months. Besides that, Agrobacterium rhizogenes-mediated transformation involving adventitious root induction was also optimized. Our findings will not only increase the transformation efficiency but also shorten the time for developing transgenic C. equisetifolia plants. Taken together, this pioneer study on tissue culturing and genetic transformation of C. equisetifolia will pave the way for further genetic manipulation and functional genomics of C. equisetifolia.
Highlights
Casuarina equisetifolia, a symbiotic nitrogen-fixing tree, plays an important role in economic and environmental improvements in tropical and subtropical littoral zones of Asia, the Pacific, and Africa (Diem and Dommergues, 1990; Zhang et al, 2008; Zhong et al, 2010)
A symbiotic nitrogen-fixing tree, plays an important role in economic and environmental improvements in tropical and subtropical littoral zones of Asia, the Pacific, and Africa (Diem and Dommergues, 1990; Zhang et al, 2008; Zhong et al, 2010). It is widely used in agroforestry plantations for several purposes, such as soil stabilization, ecosystem rehabilitation, Casuarina equisetifolia Transformation Protocol reclamation, and coastal protection (Pinyopusarerk and Williams, 2000)
The statistical results showed that the rate of callus induction was highest in the C4 medium, but that the frequency of callus browning was higher in the C4
Summary
A symbiotic nitrogen-fixing tree, plays an important role in economic and environmental improvements in tropical and subtropical littoral zones of Asia, the Pacific, and Africa (Diem and Dommergues, 1990; Zhang et al, 2008; Zhong et al, 2010). It is reported that C. equisetifolia establishes symbiotic relationships with soil microorganisms like Frankia for nitrogen fixation in Casuarina root nodules (Benson and Silvester, 1993; Diagne et al, 2013a) and mycorrhizas for increasing phosphorus uptake (Santi et al, 2003) These symbiotic relationships enhance its adaptability to harsh environments such as salinity, barrenness, and drought
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