Found in 21 U.S. states, S. integrifolium integrifolium Michx. (rosinweed) is a native herbaceous perennial plant closely related to sunflower (Van Tassel et al., 2017). Because of its long-lived nature, high quality forage biomass yield comparable to corn, along with edible seeds with high quality oil akin to domesticated oilseed sunflower, this species has piqued interest in the plant science community for its potential to become a domesticated perennial oilseed/forage crop (Vilela et al., 2018). In addition, S. integrifolium possesses a complex and deep root system that allows it to improve soil structure and health as well as provide food to a wide range of native pollinating insects (Vilela et. al, 2018). Domestication efforts have been ongoing for longer than 15 years with experimental trials occurring primarily in the Midwest. Currently, S. integrifolium is propagated via seed, cuttings, and divisions. As an obligate outcrossing species, growing from seed cannot achieve true-to-type propagation; propagation through cuttings and divisions however is slow, and cannot ensure disease-free production. Previously, there did not exist a sterilization and micropropagation protocol suitable to achieve rapid true-to-type multiplication of S. integrifolium. Obtaining significant numbers of elite genotypes obtained from breeding and selection efforts thus has been difficult to obtain since clonal propagation for S. integrifolium has been limited to cuttings and division.
Micropropagation is a tissue culture-based technique for the in vitro regeneration of whole plants from organs, tissues, cells or protoplasts (Beversdorf, 1990) Micropropagation is dependent on the in vitro aseptic culture of cells, tissues, organs or whole plant under controlled nutritional and environmental conditions in order to produce true-to-type clones of pathogen-free plants (George, 2008; Loyola-Vargas & Ochoa-Alejo, 2018). Perhaps the most important step for culture establishment is the sterilization of the explants. An effective way of preventing bacterial contamination in vitro requires the elimination of contaminating microorganisms from the initial plant explants that are introduced into the culture (Mihaljević et al., 2013). The methods for reducing contaminations include the use of explants from donor plants maintained under a strictly sanitary regime, reduction of the size of the initial explants to just the apical meristem and eliminating microbial contaminants by pretreatment with one or more chemical antiseptics as well as using antibiotics when explants are cultured on a nutrient medium (George, 2008; Mihaljević et al., 2013).
Perennial crops can have endophytes in specific tissues or can be infected systemically. Endophytes are mainly bacteria entering through natural openings on the plant surface, or through wounds (Cassells, 2012) although fungal endophytes are also known (Waqas et al., 2015). Surface sterilization is not always sufficient for field-grown plants if endophytic contamination is detected, and antibiotics and/or fungicides may be added to the culture medium as an attempt to eliminate the contaminant (Cassells, 2012; Shields et al., 1984). Many factors can determine the success of sterilization, including the types of sterilizing agent(s) used and their concentrations, the period of exposure of the plant tissue to the sterilization agent(s), and the plant organ type used (crown buds, stems, leaves, etc.). Some sterilizing agents include ethanol, sodium hypochlorite (bleach), hydrogen peroxide, and Plant Preservative Mixture PPM, a proprietary mixture of two broad-spectrum isothiazolone biocides. Although it is difficult to eradicate endophytic bacteria with PPM because of the time required for uptake, greater absorption of PPM can be achieved through vacuum infiltration, which allows the sterilizing compound to penetrate deeper into tissues than occurs by simply soaking tissues in the sterilizing compound (Tague et al., 2006). PPM was also found to infiltrate younger axillary buds more effectively, potentially because younger plant tissues are both thinner and less dense (Miyazaki et al., 2010).
Because of the convenience and the potential of decreasing the breeding cycle, in vitro techniques are now applied extensively in many breeding programs; however, the lack of well-established in vitro techniques often limits more extensive practical uses especially with newer species (Zulkarnain et al., 2015). At present, another species in the Silphium genus, Silphium perfoliatum L. (cup plant), has been studied regarding the processes required for seed sterilization and micropropagation from apical explants of seedlings in in vitro cultures (Tomaszewska-Sowa & Figas, 2011). But, in contrast, there does not exist a S. integrifolium micropropagation protocol. This current research aims to establish a S. integrifolium tissue sterilization protocol for stems (for clonal propagation), seeds (for embryo rescue or callus culture), and anthers (for anther culture), which would be useful for future research on S. integrifolium micropropagation and related tissue culture methods.