HORTSCIENCE 53(1):62–66. 2018. https://doi.org/10.21273/HORTSCI12396-17 into October. Plants bloom the first year and increase in width and number of flowering stems as they age. Plants bloom when their Micropropagation of Epilobium stems are 30–45 cm tall (Love et al., 2009) and grow best in full sun but can tolerate part canum garretti (Firechalice) by Axillary shade. This herbaceous perennial plant dies back in fall and regrows from rhizomes each Shoot Culture spring (Winger, 1996). Firechalice is a good choice for rock Areej A. Alosaimi1 and Robert R. Tripepi2 gardens and is drought tolerant, although it Department of Plant Sciences, University of Idaho, 875 Perimeter Drive requires some supplemental irrigation to be MS 2333, Moscow, ID 83844-2333 attractive throughout the season and may grow better with light fertilization (Robson Stephen L. Love et al., 2008). The species tolerates most types Department of Plant Sciences, University of Idaho, Aberdeen Research and of soil over a wide pH range (5.5–8.5). Firechalice is an outstanding xeric landscape Extension Center, 1693 S 2700 W, Aberdeen, ID 83210 plant that deserves more attention in the Additional index words. meta-topolin, benzyladenine, single-node stem explants, axillary nursery trade. shoot proliferation Firechalice can be propagated by using various techniques. Seeds of this species are Abstract. Epilobium canum subsp. garrettii (firechalice) is an herbaceous wildflower with difficult to harvest, and seeds from some landscape potential, but its seeds are difficult to germinate because of dormancy ecotypes germinate poorly, meaning asexual requirements. The objective of this study was to develop a complete micropropagation propagation would be an appropriate alterna- procedure for a selected accession of firechalice. Single-node stem explants from the plant tive. Close to 100% of the stem cuttings taken were examined for their ability to establish on Murashige and Skoog (MS) medium or from E. canum can root if the stock plants are Woody Plant Medium (WPM). Shoot explants on MS medium supplemented with 4.4 mM held at 4 to 10 C (Anderson and Rupp, 2012). benzyladenine (BA) produced more than double the number of axillary shoots compared Root cuttings can also be successful (Love to explants on WPM (12.6 vs. 4.9 shoots, P = 0.0001). Benzyladenine, kinetin (kin), 6- et al., 2009). With firechalice, stem cutting (g,g-dimethlyallylamino)purine (2iP), thidiazuron (TDZ), and meta-topolin (mT) at techniques are somewhat limited with respect concentrations of 0, 1.1, 2.2, 4.4, or 8.8 mM were evaluated for shoot proliferation efficacy. to number of cuttings obtained from each Stem explants treated with 8.8 mM of BA or mT produced the most shoots, 11 or 15, parent plant, rooting success rate, and overall respectively. Benzyladenine, 2iP, and kin failed to affect shoot height even at the highest propagation efficiency. concentrations used, but 4.4 or 8.8 mM TDZ reduced shoot height to less than half of the Tissue culture propagation could provide heights of control shoots (3.1 vs. 1.2 cm, P = 0.0001). Firechalice shoots formed three to a viable alternative for rapid propagation of four roots easily without auxin added to the medium, but four to six roots formed per firechalice with a limited amount of stems shoot when using up to 9 mM of indole-3-butyric acid (IBA). In contrast, 9 mM available to propagate. For plants in general, naphthaleneacetic acid (NAA) prevented root formation. When using 0–9 mM IBA for axillary shoot proliferation is the best tissue rooting, 82.5% of the rooted shoots survived transplanting. Based on these results, culture technique for true-to-type reproduc- optimum micropropagation of firechalice may be achieved with shoots established on MS tion (Kane, 2011). Several related species in medium plus 4.4 mM BA, a concentration of 4.4 or 8.8 mM BA or mT used for shoot Onagraceae, including Ludwigia repens € proliferation, and use of up to 6 mM IBA during root induction should result in >80% (Ozturk et al., 2004), Lopezia racemosa shoot survival after transplanting. (Salinas et al., 2014), Fuchsia magellanica (Parveen and Rasheed, 2013), and several Oenothera species (Hafez et al., 2015; Many species of native plants have unique useful native plants have been used spar- Skrzypczak et al., 1994; Taniguchi et al., traits, including adaptability and drought ingly in the landscape trade because of seed- 2006), have been reproduced by tissue cul- tolerance, and provide value in managed based propagation difficulties. Vegetative ture. Most of these species have been micro- home and commercial landscapes. Potentially propagation could provide an alternate tool propagated via adventitious shoot regeneration for commercial production of these plants. from excised seedling tissues (roots, stems, Asexual plant propagation may be a viable leaves, etc.), with the exception of L. repens Received for publication 10 Aug. 2017. Accepted option for producing plants with low seed and F. magellanica that can be reproduced for publication 14 Nov. 2017. germination rates and slow seedling growth by axillary buds. Three Epilobium species— This research was supported, in part, by the USDA (Unander et al., 1995) or for obtaining Epilobium parviflorum (Deliu et al., 2007), National Institute of Food and Agriculture Hatch genetic uniformity. Epilobium hirsutum (Tamasx et al., 2009), and project IDA01418 and the Idaho Agricultural Epilobium canum (Greene) P.H. Raven gar- Epilobium angustifolium (syn. Chamaenerion Experiment Station. rettii (A. Nelson) P.H. Raven (syn. Zauschneria angustifolium) (Deliu et al., 2013; Dreger et al., This paper is a part of a dissertation submitted by garrettii), common name firechalice or hum- 2016) have been micropropagated mainly by A.A. Alosaimi in fulfilling Ph.D. requirements. mingbird flower, is in the Onagraceae family. We thank the Nursery Advisory Committee of the adventitious shoot regeneration from seed- Idaho State Department of Agriculture for their This species is native to the Intermountain ling tissues. Dreger et al. (2016) used shoot grant support NAC/ISDA 2014-6. We also thank West, and it grows naturally in all western tips to reproduce E. angustifolium but found William Price for statistical advice. U.S. states except Washington, Montana, that they were less successful for producing Mention of a trademark or proprietary product does and Colorado. Firechalice spreads as a ground- new shoots compared with using adventi- not constitute a guarantee or warranty of the cover, and the flowers are bright orange-red tious shoot regeneration. product by the College of Agricultural and Life and attractive to hummingbirds (Love et al., The goal of this research was to develop Sciences at the University of Idaho and does not 2009). Plants are relatively small, usually 30– a micropropagation procedure that used ax- imply its approval to the exclusion of other 45 cm tall and 30–60 cm wide with some older illary shoot proliferation for rapid production products that may be suitable. plants being much wider than tall. Leaves are 1Current address: Biology Department, Imam of a selected firechalice plant. We demon- Abdulrahman Bin Faisal University, 18th Street, dark green, and lance-shaped foliage is strate that firechalice can multiply quickly in Al Rayyan, Dammam 32256, Saudi Arabia. slightly pubescent. Stems grow upright and the first two stages of micropropagation 2Corresponding author. E-mail: btripepi@uidaho. are weakly spreading with thin branches that along with high percentages of rooting edu. tend to arch. Bloom starts in June and extends and transplant survival resulting in high

62 HORTSCIENCE VOL. 53(1) JANUARY 2018 throughput propagation of a selected acces- on the number of axillary shoots produced on used at various concentrations for axillary shoot sion. Having a rapid method to multiply plant stem explants. These cytokinins were used at proliferation and rooting studies. The type of numbers will allow a superior selection of concentrations of 0, 1.1, 2.2, 4.4, or 8.8 mM cytokinin and their concentrations were used as a native plant to be introduced more quickly and were added to the culture medium before independent variables for analyses of axillary into the landscape trade than by using stem autoclaving. After 4 weeks, shoot growth shoot proliferation, whereas auxin type and cuttings. parameters were determined (described in concentrations were used as independent vari- the following paragraphs). ables for analyses of rooting experiments. For Material and Methods Several treatments were used to root shoot all analyses, if the interaction between the explants. Combinations of BA and IBA were growth regulators and their concentrations A selected firechalice accession (E. evaluated for their ability to maintain or was significant, then effects of the growth canum garrettii) used for micropropagation improve shoot quality while trying to induce regulator concentrations were tested for each was obtained from Dr. Stephen Love, Uni- root formation. Firechalice shoots 1 cm tall individual growth regulator. Significant differ- versity of Idaho. The selection was made were placed on MS medium with various ences between treatment means were deter- from a wild collection in 2008 near Tony concentrations of BA (0, 1.1, 2.2, or 4.4 mM) mined by least-square means at the 5% level Grove Lake, Cache County, UT. This plant combined with various concentrations of IBA when comparing growth differences of ex- was selected for upright growth habit, long at (0, 1, or 2 mM). In a second study, fire- plants placed on different media. Plantlet bloom period, intense red flower color, and chalice shoots were placed on MS medium survival data were analyzed by ANOVA limited rhizomatous spreading compared with different concentrations of IBA or NAA assuming a Poisson distribution (Proc Glim- with the species norm. Single-node stem (0, 3, 6, or 9 mM). After 4 weeks, shoot mix, SAS, 2012) because count data were explants were taken from rooted stem cut- growth and rooting parameters were deter- evaluated. Single degree-of-freedom contrasts tings of the selected plant. Leaves were mined (described in the following para- were analyzed to determine the difference removed from stems, and 2-cm stem ex- graphs). In a third study, 1-cm-tall shoots in survival between IBA- or NAA-treated plants that contained a single node were taken directly from in vitro cultures had the plantlets. surface sterilized for 20 min in 0.6% (v/v) bottom 3 mm of stem dipped into distilled sodium hypochlorite containing two drops of water. The shoots were treated with 0.2% Results TweenÒ 20. naphthaleneacetamide (w/w) (Rootone; Smith Two media were evaluated for establish- Industries, Kansas City, MO), 0.3% IBA Single-node stem sections placed on ei- ing firechalice shoots in culture. Single-node (w/w) (Hormex #3; Maia Products, Inc., ther MS medium or WPM readily established explants were placed on MS medium Simi Valley, CA), or distilled water (con- in culture with <10% contamination. By the (Murashige and Skoog, 1962) or WPM trol) and then placed in Sunshine #1 potting third subculture, firechalice shoots on MS (Lloyd and McCown, 1980). Murashige mix(SunGroHorticulture, Vancouver, medium produced 2.6 times more shoots and Skoog medium (product M524; Phyto- Canada). Treated shoots were placed in (P = 0.0001), 2.9 times more shoot dry weight Technology Laboratories, Shawnee Mission, three clamshell containers arranged in a ran- (P = 0.0001), and grew three times taller (P = KS) contained 4.3 g·L–1 mineral salts, vita- domized complete block design to determine 0.0001) than shoots grown on WPM (Table 1). mins, glycine, and myo-inositol concentra- the efficacy of an ex vitro rooting protocol. Leaf color of shoots grown on MS medium tions used by Murashige and Skoog (1962), Ten shoots in one row were placed in each was also greener than that of leaves on shoots 2mg·L–1 thiamin, and 30 g·L–1 sucrose, clamshell container (20 cm · 20 cm · 9cm) grown on WPM. If a stem was damaged or solidified with 7 g·L–1 agar (product A111; with 30 shoots used per treatment. crushed rather than cut cleanly or if leaves PhytoTechnology Laboratories), and had a pH Plantlets were placed in clamshell con- touched the medium, these tissues produced 5.7. The medium included 3.5 mM BA in the tainers to acclimate in vitro–rooted shoots to a black substance, most likely a phenolic first subculture, which was increased to 4.4 mM conditions outside of the tissue culture ves- compound. Keeping leaves above the me- in the second subculture. Woody plant me- sels. In vitro–rooted shoots were used from dium and cutting the stem without damaging dium (product L449; PhytoTechnology Lab- the IBA and NAA rooting study. The clam- it usually prevented most phenolic produc- oratories) contained 2.3 g·L–1 salts; the same shell containers were placed in the growth tion. Firechalice shoots became stabilized concentrations of vitamins, glycine, and chamber under the same environmental con- after three subcultures (judged by good shoot myo-inositol as the MS medium (described ditions described previously. The percentage color and consistent, regular growth of the previously); and 20 g·L–1 sucrose. It was of plantlets that survived was determined shoots) and axillary shoot multiplication ex- solidified with 7 g·L–1 agar and the pH was 6 weeks after transplanting. periments were started. adjusted to 5.2 and included 3.5 mM BA. The Data analyses. Four experimental units Different cytokinins were tested for their media were dispensed into GA7 vessels (vessels) with six subsamples (six shoots) ability to induce axillary shoot proliferation. (Magenta Corp. Ltd., Chicago, IL) and were used in each treatment. Vessels were Benzyladenine (from 0 to 8.8 mM) was con- autoclaved at 120 C for 20 min. After arranged on shelves in the growth chamber in sidered the standard cytokinin to use as inoculation, stem cultures were incubated in a randomized complete block design with a comparison with other cytokinins. The a SG 30S germinator (Hoffman Manufactur- one vessel from each treatment in each block two-way analyses revealed significant inter- ing Inc., Albany, OR) at 25 ± 1 C under a 16-h (one block per shelf). Data taken in each actions (P < 0.001) between the type of photoperiod (cool-white fluorescent lamps), experiment included the number of shoots cytokinin used and the concentrations used with 38 mmol·m–2·s–1 photosynthetic photon formed (from axillary buds) per stem explant, for the number of shoots formed, shoot flux (PPF). After 4 weeks, shoot cultures were height of the tallest shoot on the explant, and heights, and shoot dry weights. Therefore, subcultured and placed on fresh MS medium shoot dry weight. Shoot dry weights were concentration effects on all three shoot or WPM two times before axillary shoot determined by drying the shoots in a 70 C growth parameters were analyzed separately proliferation was started. Because stem ex- oven for at least 3 days. The number of roots for each cytokinin. plants grew best on the MS salt formulation, per responding shoot, percentage of explants The two most effective cytokinins for this medium supplemented with 4.4 mM BA forming roots, and length of the longest root promoting axillary shoot proliferation were was used in subsequent experiments for on each rooted shoot were additional data BA and mT (Table 2). A concentration of producing shoots used for optimizing shoot recorded for the rooting experiments. Growth 8.8 mM BA induced 11 shoots to form per proliferation and root formation. parameters for the shoots in each vessel were explant, whereas 4.4 mM mT was needed to Studies were conducted to optimize axil- averaged, and the means were analyzed by induce 14 shoots per explant (Table 2). As the lary shoot proliferation in preparation for analysis of variance (ANOVA) (mixed model concentration of mT increased from 0 to later stages of micropropagation. The se- procedure) (Proc Mixed, SAS, 2012) for shoot 8.8 mM, shoot height in culture increased lected cytokinins, BA, kin, 2iP, TDZ, and establishment data and two-way ANOVA when significantly by 1cm(P = 0.0059), but mT, were examined mainly for their effects comparing different plant growth regulators shoot height seemed unaffected by BA

HORTSCIENCE VOL. 53(1) JANUARY 2018 63 Table 1. Effects of using two different media, Murashige and Skoog (MS) or Woody Plant Medium concentrations of IBA was 2.6 vs. 1.8 across (WPM), on the mean number of shoots, mean shoot height, and mean shoot dry weight during NAA concentrations. establishment of Epilobium canum garretti shoots in vitro. Plant growth data were from the third Two-way ANOVA for shoot growth and subculture of firechalice shoots, and these shoots were grown on MS medium or WPM for 4 weeks for rooting responses showed a significant in- each subculture. Four vessels were used per treatment, and within each vessel, six shoots were teraction between the type of auxin used and averaged before analysis. auxin concentrations on shoot height, shoot Medium Number of shoots Shoot ht (cm) Shoot dry wt (g) dry weight, number of roots formed per z MS 12.6 b 2.4 b 0.038 b responding shoot, mean length of the longest WPM 4.9 a 0.8 a 0.013 a root, and percentage of rooted shoots. For zDifferent letters within a column indicate significant differences between means as determined by least- # shoot height during rooting, the type of auxin squares means tests at P 0.05 level (n = 4). (main effect) was significant (P = 0.0001), but auxin concentration lacked significance. Table 2. Effects of benzyladenine (BA), meta-topolin (mT), thidiazuron (TDZ), kinetin (kin), or 6- Shoot height was unaffected by different (g,g-dimethlyallylamino)purine (2iP) concentrations on the mean number of shoots, mean shoot concentrations of IBA (P = 0.2413), but with heights, and mean shoot dry weights of Epilobium canum garretti shoots grown on Murashige and NAA in the medium shoot heights decreased Skoog medium for 4 weeks. Four vessels were used per treatment, and within each vessel, six shoots with higher concentrations (e.g., control were averaged before analysis. shoots were 2.8-fold taller than shoots treated Type of cytokinin Concn (mM) Number of shoots Shoot ht (cm) Shoot dry wt (g) with 9 mM NAA, Table 3, P = 0.0001). BA 0 1.8 az 2.5 a 0.017 a The type and concentration of auxin 1.1 6.6 b 2.7 a 0.022 ab affected shoot dry weights differently. Naph- 2.2 7.1 bc 2.7 a 0.022 ab thaleneacetic acid in the medium failed to 4.4 9.0 cd 2.7 a 0.030 bc affect shoot dry weight (P = 0.1169). The 8.8 11.1 d 2.7 a 0.037 c highest shoot dry weights were obtained mT 0 2.0 a 2.3 a 0.025 a when using the highest concentration of 1.1 10.0 b 2.6 ab 0.048 b IBA (9 mM), and the mean dry weight of 2.2 10.5 b 2.8 bc 0.060 bc these shoots was 2.4 times heavier than that 4.4 14.4 c 3.1 c 0.071 cd 8.8 15.0 c 3.2 c 0.088 d of control shoots (Table 3, P = 0.0017). TDZ 0 1.9 a 3.1 c 0.027 a The number of roots formed per respond- 1.1 6.0 b 2.5 bc 0.064 b ing shoot decreased by at least 36%, and the 2.2 6.2 b 1.8 ab 0.072 b length of the longest root on rooted shoots 4.4 6.3 b 1.5 a 0.073 b decreased at least 3-fold as NAA concentra- 8.8 7.2 b 1.2 a 0.126 c tion increased from 0 to 6 mM. Root formation kin 0 1.4 a 2.7 a 0.016 a was completely inhibited by 9 mM NAA 1.1 2.9 a 3.0 a 0.023 a (Table 3). 2.2 3.1 a 3.0 a 0.028 a The highest IBA concentrations (6 or 9 mM) 4.4 6.2 b 3.1 a 0.057 b 8.8 8.9 b 3.1 a 0.058 b significantly increased the number of roots per 2iP 0 2.6 a 2.9 a 0.025 a responding shoot (P = 0.0468), yet the length 1.1 3.0 a 2.8 a 0.024 a of the longest root was unaffected. Indolebu- 2.2 5.1 b 2.8 a 0.036 ab tyric acid concentration failed to affect the 4.4 5.1 b 3.0 a 0.041 ab percentages of shoots that rooted (P = 0.1828). 8.8 6.0 b 3.0 a 0.051 b In contrast, using NAA strongly inhibited zDifferent letters within a column for each separate cytokinin indicate significant differences between rooting percentage as the concentration in- # means as determined by least-squares means tests at P 0.05 level (n = 4). creased up to 9 mM (Table 3). Because in vitro firechalice shoots rooted easily on MS medium without auxin, rooting concentration (Table 2). The highest BA 2iP increased shoot dry weights by 3.5-fold powders (Rootone and Hormex #3) were concentration (8.8 mM) increased shoot dry or 2-fold compared with controls (Table 2). used in an attempt to bypass in vitro rooting. weight 2-fold compared with the stems Firechalice shoots rooted easily without Analysis of treated shoots showed that fire- grown without added cytokinin (controls) any phytohormones in the culture medium. chalice shoots rooted and survived at similar (Table 2). In contrast, 8.8 mM mT increased Roots failed to form on shoots placed on levels (P = 0.1714) regardless of the treat- shoot dry weight by more than 3-fold com- media with any concentration of BA even if ments used. The mean rooting percentages pared with control shoots (Table 2). this growth regulator was combined with (and subsequent survival) were 47% for Thidiazuron promoted shoot growth yet various concentrations of IBA. Roots formed Hormex #3-treated shoots, 40% for Rootone- had detrimental effects on firechalice shoots. on 88% of the shoot explants when using IBA treated shoots, and 20% for control shoots For instance, even though 8.8 mM TDZ in- without BA, and similar numbers of roots (treated with distilled water). creased the number of axillary shoots by 3.7- formed on the stem explants regardless of the In a preliminary experiment, rooted fold and shoot dry weight by 4.6-fold over the IBA concentration used (P = 0.5567). The shoots acclimated well and survived trans- control treatment, shoots on medium supple- mean number of roots formed per responding planting, but nonrooted shoots (treated as mented with the highest concentration of stem explant was 2.3 for the control, 2.8 for microcuttings) failed to form roots and died. TDZ were 2.5 times shorter than control 1 mM IBA, and 2.4 for 2 mM IBA treatments. Half of the shoots from the IBA and NAA shoots (Table 2, P = 0.0005). The highest When testing IBA or NAA concentrations rooting experiment were used in the acclima- concentration of TDZ also produced the for their effects on rooting, an interaction tization study. Although plantlets from the heaviest shoots (0.126 g, Table 2). between the type of auxin and auxin concen- 6 mM IBA treatment survived acclimatiza- The highest level of kin increased the tration was absent for the number of shoots tion the best (93%), shoots treated with 0, 3, shoot multiplication rate by more than 6-fold formed. Only the main effect for type of or 9 mM of IBA survived at similar levels compared with the control shoots (Table 2), auxin was significant (P = 0.0111) for the (72%, 88%, or 77%, respectively, P = 0.153). whereas 8.8 mM 2iP increased shoot multipli- number of shoots formed, and IBA induced Only a few (11.1%) rooted shoots treated with cation rate by only 2.3-fold over control 32% more shoots to form than NAA, 3 mM NAA survived transplanting, and all shoots (Table 2). Neither kin nor 2iP concen- regardless of the concentration used for shoots treated with highest concentra- trations affected shoot heights (P = 0.8382 or either auxin. The average number of tions of NAA (6 or 9 mM)failedtosurvive P = 0.8122, respectively), yet 8.8 mM kin or shoots formed per responding explant across transplanting.

64 HORTSCIENCE VOL. 53(1) JANUARY 2018 Table 3. Effects of different concentrations of indole-3-butyric acid (IBA) and naphthaleneacetic acid (NAA) on the mean number of shoots, mean shoot heights, mean shoot dry weights, mean number of roots per responding shoot, mean length of the longest root per shoot, and mean rooting percentages of Epilobium canum garretti shoots grown on Murashige and Skoog medium for 4 weeks. The same control treatment was used for IBA and NAA analyses. Six vessels were used per treatment, and within each vessel, three shoots were averaged before analysis. Number of roots per Type of auxin Auxin concn (mM) Shoot ht (cm) Shoot dry wt (g) responding shoot Length of the longest root (cm) Percentage rooting IBA 0 3.4 az 0.018 a 3.8 a 1.8 a 99 a 3 3.4 a 0.020 a 3.8 a 2.2 a 88 a 6 4.6 a 0.028 ab 5.5 b 2.3 a 75 a 9 4.6 a 0.044 b 5.9 b 2.7 a 83 a NAA 0 3.4 c 0.018 a 3.8 b 1.8 c 99 c 3 2.6 bc 0.019 a 4.6 b 1.1 b 65 b 6 1.8 ab 0.014 a 2.4 b 0.5 ab 43 b 9 1.2 a 0.014 a 0 a 0 a 0 a zDifferent letters within a column for each separate auxin indicate significant differences between means as determined by least-squares means tests at P # 0.05 level (n = 6).

Discussion The cytokinins 2iP and kin, had only when half-strength MS medium was used moderate effects on axillary shoot multipli- without auxin (unpublished data). Firechalice stem explants established well cation by firechalice stem explants, which Using Rootone or Hormex #3 to bypass in on MS medium compared with stem explants differs with results from Pavingerova et al. vitro rooting failed to produce acceptable on WPM. This result was anticipated because (1996) who found that 2iP stimulated adven- percentages of rooted shoots. Although twice MS medium is formulated for herbaceous titious shoot regeneration on leaf and root as many firechalice microcuttings treated plants (Pierik, 1987) compared with WPM, explants from five cultivars of Oenothera with RootoneÒ or HormexÒ #3 survived trans- which is formulated for woody plants. Fire- biennis, an Onagraceae species. In contrast, planting compared with water-treated con- chalice stem explants exuded a black sub- Hafez et al. (2015) found that kin-supplemented trols, less than 50% of the hormone-treated stance, most likely a phenolic compound, but media induced the highest shoot multiplication cuttings survived the ex vitro rooting process. an antioxidant compound was unnecessary in rates from apical bud and petiole explants These results were unexpected because the medium if leaves were kept above the from O. biennis compared with BA-amended Anderson and Rupp (2012) showed that medium and explant stems were cut without media. Leaf, petiole, and root explants from almost 100% of E. canum stem cuttings crushing. Leaf, stem, and root cultures of E. E. angustifolium on BA-supplemented media formed roots when the cuttings were treated angustifolium (Dreger et al., 2016) and nodal produced more adventitious shoots than those with 0.1% IBA. Rooting the selected fire- cultures of E. parviflorum and E. hirsutum explants on kin-supplemented media (Turker chalice accession in vitro appeared to (Deliu et al., 2013) readily established on MS et al., 2008). improve survival of this clone because of medium. Dreger et al. (2016) used 0.1 g·L–1 Thidiazuron is a potent cytokinin-like at least 72% of the shoots that rooted in vitro ascorbic acid in the culture medium for stem plant growth regulator that can promote or without auxin in the rooting medium sur- segments and leaf explants of E. angustifo- inhibit the growth of shoot cultures. In this vived transplanting. Using ex vitro auxin lium because of browning and tissue necrosis study with firechalice, shoot height reduc- treatmentstobypassinvitrorootingwasof of the explants. Nodal explants of E. parvi- tion associated with increased TDZ concen- limited value in this study, but perhaps florum and E. hirsutum also produced phenolic tration indicated that this compound manipulation of the shoot cultures [e.g., compounds in culture, but the intensity was inhibited shoot elongation, similar to results exposing in vitro shoots to a cold treatment low enough to avoid the use of antioxidant (Anderson and Rupp, 2012) before attempt- compounds in the media (Deliu et al., 2013). describedbyHuettemanandPreece(1993) with woody plant tissue culture. Turker et al. ing ex vitro rooting] will improve the In shoot proliferation studies with fire- percentage of ex vitro rooting and survival. chalice, mT promoted shoot multiplication (2008) found that 2.3 or 4.5 mM TDZ more than all other cytokinin treatments. combined with 1.2, 2.5, or 4.9 mM IBA failed to induce adventitious shoot regeneration Besides considering the best plant responses Conclusion in culture, propagators must also consider the for E. angustifolium. In contrast, TDZ can costs of the biochemicals used. The costs of induce high numbers of adventitious shoots Axillary shoot proliferation is a viable 1 g of mT from two tissue culture supply to form on cotyledon explants of three option for rapid propagation of the selected companies was $219 from one and $265 commercial cultivars of evening primrose, firechalice accession with limited foliage from the other in June 2017, whereas the cost a related Onagraceae species (DeGyves available for propagation. Murashige and of BA from both companies was $5 per et al., 2001). Skoog medium was the best medium for gram. The higher cost of mT may not be In this study, firechalice rooted easily establishing firechalice stem explants in cul- justified for commercial propagation of fire- without auxin added to the medium, indicat- ture. Shoot explants multiplied the best when chalice given that BA, which is at least 44 ing that its shoots naturally produced enough using BA or mT at 4.4 or 8.8 mM. Firechalice times cheaper, promoted shoot multiplication auxin to promote root formation. Of the shoots rooted easily without auxin added to almost as well as mT. Additional plant auxins tested, IBA induced more roots to the medium, yet adding up to 9 mM IBA species observed by other researchers also form than NAA for firechalice stem explants increased the number of roots per responding responded well to mT in Stage II. Aloe (Table 3). Because the higher concentrations shoot although the percentages of rooted polyphylla (an endangered medicinal and of NAA (6 or 9 mM) decreased shoot height shoots were similar to the control treatment. ornamental aloe) was grown on full-strength and inhibited root formation, these NAA Transplant survival percentages were similar basal medium with different concentrations concentrations appeared to have toxic effects if the shoots were rooted with up to 9 mM IBA of cytokinins (Bairu et al., 2007). Meta- on firechalice, and this auxin should probably or without auxin in the medium. Using NAA topolin induced the best rate of shoot multi- be excluded from any firechalice-rooting in the rooting medium should be avoided plication, and the optimum concentration was medium. Turker et al. (2008) found that because it inhibited root formation and de- 5 mM (Bairu et al., 2007). These results were rooting of E. angustifolium shoots decreased creased transplant survival. The firechalice similar to the firechalice ecotype used in this from 100% for shoots treated with 2.7 mM micropropagation procedures developed in study. To our knowledge, this study is the NAA to 30% for shoots treated with 16.1 mM this study can be used by the nursery industry first to use mT in an Epilobium micropropa- NAA. In our subsequent studies, firechalice to produce large numbers of tissue culture gation protocol. shoots rooted well (at least >80%) in vitro plantlets quickly.

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