HORTSCIENCE 47(12):1710–1713. 2012. been met (e.g., a period of low temperature). We anticipate that a successful production protocol for V. californicum collected from Chilling Requirements to Break natural populations in temperate regions, sites above 2400 m in elevation, will include defined Dormancy of Veratrum californicum chilling treatments. In this study we report the influence of Youping Sun and Sarah A. White varied periods of chilling on shoot emergence School of Agricultural, Forest, and Environmental Sciences, Clemson and growth using mature, field-collected V. University, 167 Poole Ag. Center, Clemson, SC 29634 californicum plants. Our goal was to force V. californicum to emerge before a hypothetical David Mann natural period of snow cover as a means of Infinity Pharmaceuticals, Inc., Cambridge, MA 02139 shortening the production cycle. Thus, we de- termined the minimum length of cold treat- 1 Jeffrey Adelberg ment required to break dormancy and whether School of Agricultural, Forest, and Environmental Sciences, Clemson shortened dormancy affected shoot emergence University, 275 Poole Ag. Center, Clemson, SC 29634 and vigor. Growth of the shoot systems was also observed in both greenhouse and artifi- Additional index words. corn lily, shoot emergence, leaf expansion, photosynthesis cially illuminated growth rooms. Abstract. Veratrum californicum, a native of the western United States, has long been used in herbal medicine and now also has potential pharmaceutical uses. As a result of Materials and Methods a projected increasing demand for V. californicum biomass for pharmaceutical purposes, Chilling. A few hundred plants of Vera- the development of a chilling protocol for enhanced cultivation efficiency is needed. To trum californicum, rhizomes with attached study the effects of chilling on the growth of V. californicum, field-collected rhizomes with bulbs and roots, were selected for uniformly attached bulbs and roots were potted, stored at 10 8C for 2 weeks, and subsequently large size from a wild population of thousands chilled at 5 8C for 30 to 180 days before transfer to a greenhouse or growth room. Twenty dug in a meadow 1000 m2 (mechanically plants were transferred to the greenhouse every 30 days to observe growth. Ten plants harvested for the purpose of pharmacological were harvested at shoot emergence and the remaining 10 when leaves were fully research) in Boulger Canyon, UT (lat. 39°36# N, expanded. In addition, 10 plants were transferred from 5 8C to a growth room every long. 111°13# W, elevation 2671 m). This 30 days where net photosynthetic rates were measured. Longer chilling duration site was chosen because the planting stock correlated with a reduction in days to shoot emergence and leaf expansion. The net from which the plants were collected had high photosynthetic rates of V. californicum plants chilled for 120, 150, or 180 days were concentrations of cyclopamine and related higher than those of plants chilled for only 30, 60, or 90 days. Plants exposed to longer alkaloids (unpublished data). The next day, chilling durations were taller and had larger, more numerous leaves. Interestingly, 14 Sept. 2010, plants were shipped overnight V. californicum shoot emergence was also observed in the dark at 5 8C after the bulbs had to Clemson, SC, and on arrival, the rhizomes, been stored for 210 days. Growth of the root systems of plants was also observed during each with one bulb, were sorted into two size chilling. In conclusion, chilling was necessary at 5 8C for a minimum of 120 days to force groups based on bulb circumference [12.2 ± early emergence and vigorous growth of V. californicum. 1.0 cm (mean ± SD) and 10.2 ± 1.4 cm for large and small bulb sizes, respectively] and potted into 7.6-L (large) or 3.8-L (small) plastic Veratrum californicum Durand (corn lily) Extensive field surveys were conducted to containers filled with Fafard 3B mix [45% is an herbaceous perennial monocot native to select planting stock with high concentra- Canadian sphagnum peatmoss, 25% pro- wet meadows across much of western North tions of cyclopamine and related alkaloids. cessed pine bark, 15% perlite, 15% vermic- America [USDA NRCS (Natural Resources An ecotype of primary interest was identified ulite, starter nutrients (40 to 230 mg·L–1 Conservation Service), 2011]. Veratrum cal- in numerous high-elevation bogs and meadows nitrogen; 5 to 30 mg·L–1 phosphorus; 40 to ifornicum has long been used in herbal in Utah and Idaho where plants have had the 200 mg·L–1 potassium, calcium, and sulfur; medicine and now also has potential pharma- highest yields of alkaloids over several suc- 25 to 80 mg·L–1 magnesium), wetting agent, ceutical applications. In recent years, the V. cessive years of observation (unpublished dolomitic limestone; Conrad Fafard, Inc., californicum-derived phytochemical cyclop- data). In these higher (above 2400 m) regions Anderson, SC]. Each plant was drenched amine and its derivatives have been explored with native V. californicum populations, bulbs with 330 ppm SubdueÒ [25.1% Metalaxyl: N- as promising therapeutic agents for the treat- undergo prolonged (seven- to eight-month) (2, 6-dimethylphenyl)-N-(methoxyacetyl) al- ment of tumors arising from activation of the periods of winter dormancy under a deep anine methyl ester, 74.9% inert ingredients; Hedgehog signaling pathway (Berman et al., snow pack. Syngenta Crop Protection, Inc., Greensboro, 2002; Chen et al., 2002; James et al., 2004; Average daily soil-temperature data col- NC] to prevent root rot. Taipale and Beachy, 2001; Tremblay et al., lected between 2003 and 2010 at three depths Veratrum californicum plants were first 2009). To meet the projected pharmaceutical (5, 20, and 51 cm) from several independent stored in the dark at 10 °C and 70% relative demand for this alkaloid, a dependable culti- USDA weather station locations in Utah humidity for 2 weeks (pretreatment) and then vation system will be required. To that end, proximal to natural V. californicum popula- chilling treatments were initiated at 5 °C and we are developing cultivation protocols for tions of interest show that soil temperatures 65% relative humidity for 30, 60, 90, 120, 150, the greenhouse production of V. californicum. typically drop below 10 °C by 1 Oct. and do and 180 d in a controlled environment room not return above 10 °C until after 1 May. (Model# 120-208; Climate Technologies, Inc., Field observations report a rapid burst of Laytonsville, MD) in the Clemson University vegetative growth after the recession of the Biosystems Research Complex. Bulbs and Received for publication 18 July 2012. Accepted snow line. Taylor (1956) reported that a period rhizomes not included in these experiments for publication 5 Oct. 2012. of exposure to cold temperatures is neces- were handled in the same manner as experimen- We gratefully acknowledge the generous support from Infinity Pharmaceuticals, Inc. that allowed us to sary to permit successful vegetative growth tal units and were retained under experimental conduct the work. We also thank Kenny Krantz for of V. californicum and that this prolonged conditions for 220 d. All bulbs and rhizomes his help in potting, plant care, and data collection. exposure to cold is essential for successful were watered at 2-week intervals and sub- 1To whom reprint requests should be addressed; cultivation. Dormancy is defined as a lack of strate volumetric water content was main- e-mail [email protected]. growth because specific conditions have not tained above 44.2% ± 5.8% as measured 1710 HORTSCIENCE VOL. 47(12) DECEMBER 2012 using Decagon 10-HS soil moisture sensors upward from the second most mature leaf (Decagon Devices, Inc., Pullman, WA). at the base of the stalk. At the end of the Greenhouse and growth room forcing. experiment (24 May), dead plants were Nineteen plants from each bulb size treat- counted. ment were removed from the cold environ- Photosynthetic light response curves were ment room on a monthly basis. Four plants recorded for all plants surviving after 40 d in were destructively harvested for analysis the growth room by using a CIRAS-2 porta- (harvest described subsequently), 10 plants ble photosynthesis system with an integrated transferred into a greenhouse, and five plants Chlorophyll Fluorescence Module (CFM) transferred into a growth room. Both the unit (PP Systems International, Inc., Ames- greenhouse and growth room are located in bury, MA) mounted with an automatic uni- the Clemson University Biosystems Research versal PLC6 broad leaf cuvette. All plants Complex. were well watered before measurements. The Plants transferred to the greenhouse envi- third, fourth, or fifth fully expanded leaf, ronment were fertigated with 100 mg·L–1 counting from the top of the plant downward, nitrogen CalMag special fertilizer (15N– was chosen for the measurements. Two of the 2.2P–12.5K; Scotts Peters Excel, Marysville, three leaves per plant were recorded. The OH). Substrate volumetric water content was number of plants measured per chilling pe- measured by using Decagon10-HS soil mois- riod for which photosynthetic light response ture sensors and maintained at or above curves were recorded include one, five, nine, 48.5% ± 4.9%. An ECD DataWorker (ECD, 10, nine, and eight from 30, 60, 90, 120, 150, Inc., Milwaukie, OR) recorded canopy air and 180 d of chilling, respectively. The CO2 temperature on an hourly basis. Ambient air concentration within the leaf chamber was temperatures in the greenhouse were 20.2/ maintained at 375 mmol·mol–1, whereas the 17.0 ± 6.5/5.2 °C day/night during the exper- photosynthetic light response curve was mea- Fig. 1. Effect of chilling duration at 5 °Con imental period. Light intensity at the canopy sured. Constant temperature (25 °C) and rela- Veratrum californicum shoot emergence (A) level was monitored hourly with LI-190 tive humidity (75%) were maintained within and leaf expansion (B).