Abiotic stress is the predominant factor limiting turfgrass growth in many climatic areas. One of the major abiotic stresses in cool temperate regions is low temperature, which can be detrimental for growth of warm-season grass species. Warm-season turfgrasses grown in cool climatic regions enter dormancy early in the fall and green up later in the spring compared with cool-season species (Beard, 1973). Seashore paspalum is a warm-season turfgrass with many desirable traits such as superior tolerance to heat stress, salinity stress, and drought stress; however, this species is sensitive to low temperature stress and often losses turf quality during late fall or early winter in cool climatic regions, which limits its use to warm climatic areas (Duncan and Carrow, 1999). Improving cold tolerance of warm-season turfgrasses such as seashore paspalum is important to expand the range of use and extend the growing season throughout cooler climates.
Limited progress has been made in the improvement of cold tolerance in seashore paspalum through traditional breeding efforts as a result of insufficient genetic variability in cold tolerance or the lack of cold-tolerant cultivated germplasm (Duncan and Carrow, 1999). Somaclonal selection is a powerful tool to increase genetic variability with regard to plant stress tolerance (Karp, 1995). Previous studies reported improved cold tolerance through the selection of somaclonal variants in agronomic crops (Bertin and Bouharmont, 1997; Kendall et al., 1990; Lazar et al., 1988). For example, Kendall et al. (1990) showed that plants of winter wheat (Triticum aestivum) regenerated from callus and subsequently exposed to below-freezing temperature exhibited significant improvement in cold tolerance. Some plants that survived this cryoselection process were genetic variants, which suggest selection at the callus level can contribute to heritable improvement of stress tolerance, although some variations may be epigenetic (Kendall et al., 1990). Additionally, Bertin and Bouharmont (1997) reported enhanced chilling tolerance in rice (Oryza sativa) using somaclonal variations and in vitro selection. Cold tolerance of warm-season turfgrass could be improved through in vitro selection for somaclonal variations under cold treatment. However, in vitro selection for somaclonal variations of cold tolerance is not well documented in turfgrass species.
An effective and efficient tissue culture and regeneration system is required for the in vitro selection of somaclonal variations. Callus induction, embryogenic callus formation, and plant regeneration require specific concentrations of plant growth regulators such as auxin and cytokinins or their synthetic analogs such as 2,4-D and 6-BA in the culture medium (Murashige and Skoog, 1962; Neibaur et al., 2008). In addition to plant growth regulators, the use of CuSO4 in culture medium was found to stimulate callus induction, proliferation, somatic embryogenesis, and regeneration in some monocotyledonous plants (Dahleen, 1995; Sahrawat and Chand, 1999; Tahiliani and Kothari, 2004). Specific ingredients for culture media likely vary depending on explant type such as seed vs. vegetative organs or inflorescence or differences in genetic composition (Neibaur et al., 2008; Olesen et al., 1995; Zeng et al., 2009). A system for callus induction and plant regeneration has been developed for several turfgrass species, including seashore paspalum (Cardona and Duncan, 1997; Neibaur et al., 2008), perennial ryegrass [Lolium perenne (Olesen et al., 1995; Zeng et al., 2009)], zoysiagrass [Zoysia matrella (Chai et al., 2011)], centipede grass [Eremochloa ophiuroides (Barampuram et al., 2009)], kentucky bluegrass [Poa pratensis (Van Ark et al., 1991)], and creeping bentgrass [Agrostis stolonifera (Zhong et al., 1991)]. Mature seeds and immature inflorescence or embryos are used as explants in in vitro culture systems. The selection of somaclonal variations using seeds as explants offers several advantages over using immature inflorescence or embryos in grass species (Bai and Qu, 2001; Ha et al., 2001). For seashore paspalum seeds produced from outcrossing, use of seeds as explants may generate a wider range of somaclonal variation as compared with using vegetative organs. In addition, mature seeds are likely to be readily available as the source of explants in all seasons and are therefore considered one of the most efficient sources of explants for in vitro culture or somatic embryogenesis in grass species (Bai and Qu, 2001). However, an effective method for callus induction, embryogenic callus formation, and plant regeneration from mature seeds that can be successfully used in in vitro culture selection for seashore paspalum, particularly for in vitro selection of cold tolerance, is not yet available. The establishment of an efficient in vitro selection system is a prerequisite for increasing genetic variability of stress tolerance through somaclonal selection or genetic transformation.
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 and to determine whether in vitro cold selection of somaclonal variations would lead to improved cold tolerance in seashore paspalum.
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