Tissue culture and plant regeneration of Spartina alterniflora: Implications for wetland restoration

Abstract

A tissue culture and plant regeneration protocol for the salt marsh grass, Spartina alterniflora, has been developed. Callus was efficiently induced on Murashige and Skoog (MS) medium supplemented with 1 mg L⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1 mg L⁻¹ indole-3-acetic acid (IAA). Callus initiation from 6-day-old seedlings was faster and occurred with a greater frequency than callus initiation from coleoptile-covered segments from the same age seedlings. However, only the coleoptile-covered segments produced regenerable callus, which was maintained on MS medium supplemented with 1 mg L⁻¹ 2,4-D and 1 mg L⁻¹ naphthaleneacetic acid (NAA). The regenerable callus differentiated into shoots upon transfer to shoot regeneration medium. A high frequency of shoot regeneration was obtained when the medium contained 3 mg L⁻¹ 6-benzylaminopurine (BA) or Thidiazuron (TDZ), with or without the addition of 0.2 mg L⁻¹ IAA. Regenerated shoots were transferred to root regeneration medium, the optimal of which was determined to be half-strength MS medium supplemented with 1 mg L⁻¹ indole-3-butyric acid (IBA). TDZ in the shoot regeneration medium inhibited root formation in the root regeneration medium, making BA rather than TDZ the optimal hormone for the shoot regeneration medium. The mode of plant regeneration was organogenesis. Upon transfer to soil, the most successful growth of plants occurred in a mixture of commercially available potting soil and natural marsh mud. The development of a tissue culture and regeneration protocol for S. alterniflora provides the possibility of selecting lines of this species, via somaclonal variation, with characteristics desirable for wetland creation and restoration.