In Vitro Sterilization Procedures For Micropropagating Silphium Integrifolium Michx.

Perennial crops are constantly exposed to fungi and bacteria in their environment, and thus explants from eld-grown plants are dicult to disinfect for micropropagation because of both endophytic and epiphytic microbes. Field grown S. integrifolium plants were potted and grown in the greenhouse for ve weeks; new stems and anthers along with seeds were tested for in vitro sterilization. We report successful sterilization protocols that involved disinfecting seeds with isothiazolone biocides (PPM) and NaClO and sterilizing stems and anthers through vacuum inltration of PPM. The establishing of the protocols is useful for subsequent micropropagation and would in this way facilitate Silphium breeding and domestication processes.


Introduction
Found in 21 U.S. states, S. integrifolium integrifolium Michx. (rosinweed) is a native herbaceous perennial plant closely related to sun ower (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 sun ower, 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 signi cant numbers of elite genotypes obtained from breeding and selection efforts thus has been di cult 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 Perennial crops can have endophytes in speci c 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 su cient for eld-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 di cult to eradicate endophytic bacteria with PPM because of the time required for uptake, greater absorption of PPM can be achieved through vacuum in ltration, 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 in ltrate 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.

Materials And Methods
S. integrifolium's stems, seed, and anthers were utilized in this work. In July 2021, three-year-old eldgrown plants were dug out of the soil from eld plots located in the St. Paul campus of the University of Minnesota, MN and immediately, washed, the top 25 percent of their stems were trimmed off, and they were planted in 12-in. pots with Sungrow professional sterile potting mix and set to grow in the greenhouse at 90F. After ve weeks, the new stems that were produced were cut into 4 cm long pieces with two nodes and utilized for sterilization protocols. Seed and anther tissues were also collected from other greenhouse-grown plants grown in the same conditions. MS medium (Murashige and Skoog, 1962), with 15 g of sucrose and 2 ml of PPM per liter was used as the growth media. Solutions for sterilization contained plant preservative media (PPM) which is composed of 5-chloro-2-methyl-3(2H)-isothiazolone and 2-methyl-3(2H)-isothiazolone (Plant Cell Technology, Washington, DC) ethanol, Softsoap antibacterial soap (Colgate-Palmolive, New York City, NY), commercial bleach (6% NaClO, Clorox, Oakland, CA) 20%, hydrogen peroxide (H 2 O 2 ), and 10%, Polysorbate 80 (Tween 80, a nonionic surfactant wetting agent to decrease surface tension). All plant tissues were grown in petri dishes with sterile MS medium after sterilization for one month in growth chambers at 25°C with a 12-hour light photoperiod at 28.5 µmol.m. 2 s 1 of light intensity supplied by cool white orescent tubes to promote growth and development.
Procedure for sterilizing stems 1. Removed all leaves from stems except leaf primordia near the meristem of each stem. 2. Washed tissues with antibacterial soap and 10% alcohol for 5 minutes. . Rinsed with 2 ml/L PPM. 9. [Alternative step] Placed into 10% H2O2 for 10 minutes followed with a nal wash with 2 ml/L PPM.
This step can be skipped if step 3 (vacuum in ltration) was performed.
10. Placed onto sterile MS culture medium.

Procedure for sterilizing seeds
This procedure was essentially the same as the above procedure for sterilizing stems but without the vacuum in ltration nor H 2 O 2 treatments. The seed coat was removed before sterilization.
Procedure for sterilizing anthers 1. Using sterile forceps, removed anthers from an unopened or slightly opened S. integrifolium ower bud.
4. Washed with 2 ml/L PPM for 10 seconds, dipped into 70% ethanol for 5 seconds, and followed with a nal wash with 2ml/L PPM for 10 seconds.
5. Placed onto sterile MS culture medium.

Results
After being grown in the growth chamber for 5 weeks, all stems sterilized with this protocol were still clean and showed no visible signs of microbial contamination. Many had begun growing primordial leaves with the stems displaying phototropic bending and rooted in the media, indicating that the sterilization procedure was effective (see Fig. 1). However, a small amount of necrosis was present on some stems which possibly occurred as a result of the 10% H 2 O 2 treatment. To test this possibility, the stem sterilization experiment was repeated using the stem materials from the same plants, and the H 2 O 2 was replaced with vacuum in ltration of 2% PPM in the beginning of the protocol; stem tissues resulting from vacuum in ltration exhibited a 100% success (no colonies of bacteria/fungi visible) without tissue damage. Seeds and anthers sterilized with the described protocols remained without observable contamination, with seeds germinating on their plates after strati cation for 4 weeks (see Fig. 2) and anthers remaining viable (not desiccated nor necrotic) for transfer onto growth media containing hormones such as auxins and cytokinins.

Discussion
The protocols to obtain microbe-free stem, seed, and anther tissue in S. integrifolium will facilitate future research on micropropagation and in vitro mutagenesis using these tissues. They are not dissimilar from the micropropagation protocol proposed for a related species, S. perfoliatum, which achieved good results with 30% bleach and 70% ethanol as the main agents in the seed sterilization process (Tomaszewska-Sowa & Figas, 2011); however, that procedure was limited to seeds which are of limited use in the development of elite germplasm lines, whereas this current protocol was shown to be effective with stems and anther tissues as explants, which would thus allow for clonal propagation as opposed to the seed-based protocol used in the S. perfoliatum experiments. Any sterilization protocol potentially may need to be adapted for plants grown in other environments or for genotypes with quite different morphologies. Such difference can in uence physical behavior during sterilization or, for plants grown under quite different conditions; it may affect external or endophytic microbial populations. Plant tissues used in these experiments were obtained from plants grown initially in elds in the upper Midwest in North America and are thus from a more or less uniform source. Although genetic and extrinsic difference can vary (i.e. characteristics such as stem size, presence, and size of trichomes on the stem), these protocols provide useful procedures that can be optimized for individual differences should changes prove necessary.
The stem sterilization protocol we utilized included taking plants from the eld and greenhouse acclimating the plants that produced shoots in the greenhouse over a ve-week period. However, the eldgrown plants in this experiment produce new shoots within a week in the greenhouse. Thus, it is possible that the shoots produced within the period one week of transplant or longer may be suitable should a more rapid eld to culture time be desired. Similarly, although the sterilized seeds required strati cation for germination after being sterilized, the dormancy requirement may be overcome by incorporating a growth regulator such as Ethephon (an ethylene-release agent) into the sterile growing media (Reinert et al., 2018). By combining methods such as reducing seed dormancy periods as well as tissue culture micropropagation utilizing this protocol, the breeding process of Silphium may be shortened considerably.

Figure 2
Sterilized seeds germinating in vitro