Improving Cold Tolerance through In Vitro Selection for Somaclonal Variations in Seashore Paspalum

Abstract

Cold stress is a major factor limiting the growth of warm-season turfgrass species. Cold tolerance in warm-season turfgrass species could be improved through in vitro selection for somaclonal variations. The objectives of this study were to establish an effective in vitro culture protocol for generating plants from calli using mature seeds of seashore paspalum (Paspalum vaginatum) and to determine whether in vitro cold selection of somaclonal variations would lead to improved cold tolerance in seashore paspalum. The optimal concentrations of supplemental compounds in the culture medium for callus induction, embryogenic callus formation, and plant regeneration were determined. The supplemental compounds included 2,4–dichlorophenoxy acetic acid (2,4-D), 6-benzylaminopurine (6-BA), kinetin (KT), naphthalene-1-acetic acid (NAA), CuSO4, and acidic hydrolysis casein (AHC). The highest rates of callus induction (97.50%), embryogenic callus formation (66.88%), and regeneration (55.94%) were obtained with the supplemental compounds of 3.0 mg·L−1 2,4-D and 10.0 mg·L−1 CuSO4 for callus induction; with 3.0 mg·L−1 2,4-D, 15 mg·L−1 CuSO4, and 1.0 g·L−1 AHC for embryogenic callus formation; and with 8.0 mg·L−1 6-BA, 0.2 mg·L−1 KT, 0.5 mg·L−1 NAA, and 10 mg·L−1 CuSO4 for plant regeneration. Embryogenic calli were subjected to 2 or 6 °C treatment for 90 days for in vitro cold selection of somaclonal variation. Plants regenerated from calli surviving cold treatment (cold-selected) for 45 or 60 days were then exposed to low temperatures [15/10 or 5/3 °C (day/night)]. Plant variants derived from cold-selected calli exhibited significant improvement in their tolerance to low temperature of either 15/10 or 5/3 °C (day/night), as manifested by higher turf quality, leaf chlorophyll content, and membrane stability as well as lower levels of lipid peroxidation compared with the control plants. This study demonstrated the feasibility of in vitro selection for cold tolerance in seashore paspalum. The cold-tolerant variants could be useful germplasm for breeding programs and further molecular characterization of cold tolerance mechanisms.