Establishing clones of Veratrum californicum, a native medicinal species, for micropropagation

Abstract

Factors affecting the micropropagation of Veratrum californicum, a slow-growing species that is a potentially valuable source of cyclopamine, were investigated. Sterile cultures were initiated on modified Murashige and Skoog medium, and clones from individual donor plants were assigned to experimental conditions when approximately 100 shoots of each clone were available. The effects of temperature, light quality, and plant growth regulators on multiplication and survival were assessed. Four clones from which large greenhouse populations were obtained were selected for in-depth analysis. When shoots were cultured at 10°C and 16°C, multiplication ratios consistently >1 were observed from three of four clones and two of four clones, respectively, during the five-subculture cycles. None of the clones stably increased when cultured at 24°C, and plants from this treatment did not survive acclimatization in the greenhouse. Only one clone showed increased multiplication ratios in response to plant growth regulator treatments, with maximum multiplication when shoots were cultured with 9 μM benzyladenine and 0.5 μM naphthaleneacetic acid. Light quality in the laboratory did not affect multiplication ratio but did affect subsequent greenhouse survival. The size of plants derived from culture was most often equivalent (65% of 1,271) to 3-yr-old seed-derived plants. Although the growth of clones during acclimatization differed, plants derived from cultures incubated at 16°C had the best rates of overall greenhouse survival. Temperature and light treatments in vitro critical to long-term plant survival were demonstrated and will assist the establishment of a mass propagation system for V. californicum.

Introduction

Veratrum californicum Durand (Melanthiaceae family) has been identified as the best source of cyclopamine, a hedgehog antagonist and starting material for promising anticancer therapeutics (McFerren 2006). A renewable supply of cyclopamine for a potential pharmaceutical market would require robust propagation and cultivation techniques. This bulbous perennial species is a monocotyledon and grows in high mountain ranges of the western USA. It is often found in open alpine meadows, woodlands, marshes, and swamps (Knight and Walter 2001). For successful container production of V. californicum, bulbs and rhizomes must be chilled at 5°C for a minimum of 120 d to break the dormancy (Sun et al. 2012). V. californicum is not readily propagated by seed or division (Sun et al. 2013). It grows slowly from seeds and requires more than 10 yr to establish mature plants (Taylor 1956; Williams and Kreps 1970). The growing season for V. californicum is approximately 2 mo in duration, and plants spend the remainder of the year dormant. Crown divisions of mature plants are not viable because, even if immediately replanted in native soils, mature divisions do not re-establish colonies. Plant tissue culture techniques may offer improved methods for propagation of V. californicum.

In vitro propagation of the Melanthiaceae family has been the subject of limited study. Ma et al. (2006) reported a culture system for somatic embryogenesis with limited whole plant propagation. With Fritillaria and Erythronium, two genera within the Liliaceae family (closely related to the Melanthiaceae family), in vitro culture at lowered temperatures increased both plant growth and quality (Mondoni et al. 2011; Carasso et al. 2012). In general, the success of micropropagation of bulbous plants depends upon the plant growth regulators (PGRs) naphthaleneacetic acid (NAA) and benzyladenine (BA), which increase multiplication efficiency (Bisht et al. 2012). V. californicum may also benefit from lowered culture temperatures and PGRs during micropropagation.

A V. californicum ecotype capable of producing high levels of cyclopamine alkaloids was identified in the high elevation (>8,000 ft.) bogs of southern Idaho and northern Utah. Variations in cyclopamine content exist among mature individuals within this ecotype (unpublished data, Infinity Pharmaceutical Co, Cambridge, MA). Because of this variability in cyclopamine yield, propagation of multiple clones is necessary to maximize potential for selection of clones with high cyclopamine yield. To that end, a micropropagation protocol broadly applicable to genotypes within this selected ecotype should be developed. Establishing consistent shoot formation from stage I (initiating sterile culture) propagative material may require repeated subculture cycles (McCown and McCown 1986). Once stage I material consistently forms shoots (become stable), efforts then turn to increasing the multiplication ratio (rate at which distinct shoots form) on the stage II propagative material. Once shoot multiplication rates are consistent, the clonal materials are placed in a medium which encourages rooting (stage III), before transfer and acclimatization (stage IV). Losses during any of the four stages may affect genotypes differently and inadvertently accelerate the in vitro loss of diversity.

The objectives of this study were to establish propagation methods for several clones of V. californicum by varying temperature, light quality, and PGRs during establishment of stable stage II culture and to quantify the impact of these factors on greenhouse survival. We also compared the growth of acclimatized tissue culture-derived plants with seed-derived plants of this slow-growing species.

Materials and Methods

Plant materials.

Rhizomatous bulbs and roots of senescing V. californicum plants were unearthed from a native population, in Boulger Canyon, UT (39° 36′ N, 111° 13′ W, latitude 2,671 m). Plant materials were selected from large, mature colonies with relatively uniform appearance on September 12, 2010 and shipped overnight on September 13, 2010 to Clemson, SC, for stage I initiation. Upon arrival, bulbs and roots were potted into 7.6 L plastic containers filled with Fafard 3B mix [45% Canadian sphagnum peat moss, 25% processed pine bark, 15% perlite, 15% vermiculite, starter nutrients (40–230 mg L⁻¹ N; 5–30 mg L⁻¹ P; 40–200 mg L⁻¹ K, Ca, and S; 25–80 mg L⁻¹ Mg), wetting agent, and dolomitic limestone (Conrad Fafard, Inc., Anderson, SC] and stored at 5°C for up to 6 mo prior to separating the rhizome and roots from the bulb. The sheath and outer layers of each bulb were removed, leaving bulbs that were roughly 2 to 5 cm in diameter. Bulbs were rinsed in running tap water, washed in detergent water (40 μL Tween 80 in 1 L deionized water), rinsed with deionized water, and surface-sterilized by placing them in a 95% (v/v) ethanol solution for 2 min and then in a 50% commercial bleach (Clorox™, 5.25% sodium hypochlorite) for 2 min. The bulbs were then rinsed with sterilized deionized water. The remaining outer layers of the bulb were removed, crosscut into wedge-shaped pieces, and the bulb wedges were immersed a second time in a 10% commercial bleach for 10 min and then rinsed with sterilized deionized water. The bulb wedges were then further split at the basal plate with at least three scales in an explanted tri-scale (as described by Ulrich et al. 1999). The tri-scale explants, hereafter referred to as clones, were placed in 50 mL of Murashige and Skoog (MS) medium (Murashige and Skoog 1962), containing 23 μM BA with 3% (w/v) sucrose and solidified with 0.7% (w/v) agar (Micropropagation-Type II, Caisson Labs, North Logan, UT). All culture medium used were adjusted to pH 5.7 using NaOH prior to autoclaving. Shoots from the clones were subcultured and multiplied. When approximately 100 shoots were available per clone, they were assigned to experimental conditions, and the treatments described below were assessed for their influence on multiplication in vitro and greenhouse survival.

Treatment temperatures.

Six vessels with five shoots of each clone were placed in each chamber at 10°C, 16°C, and 24°C for five successive subculture periods on agar-solidified MS medium containing 3 μM BA and were sorted by growth rate: 312, 327, and 328 (4-wk subculture); 315 and 317 (5-wk subculture); and 318, 329, and 330 (6-wk subculture). Plant materials were cultured under 20 μmol m⁻² s⁻¹ photosynthetic photon flux density (PPFD) light provided by cool white fluorescent tubes (treatment = “20fluor”) with a 16-h d⁻¹ photoperiod and maintained at 10°C or 16°C in low temperature incubators or at 24°C in a culture room. The first subculture cycle facilitated adaptation to culture conditions, and data were not recorded. Data were collected from the subsequent subculture cycles, and a multiplication ratio was calculated for each clone from subculture cycles 2 to 5. Multiplication ratios were calculated by counting the number of shoots per vessel at end of the culture cycle and dividing by the number of shoots used to initiate that vessel.

LED light treatments.

Shoots from five clones (315, 318, 327, 328, and 329) were cultured on MS medium supplemented with 3 μM BA and 0.7% (w/v) agar and cultured at 16°C. Plants were grown with a 16-h d⁻¹ photoperiod and four light treatments: 20 μmol m⁻² s⁻¹ monochromatic red (peak at 660 nm, “20red”), 20 μmol m⁻² s⁻¹ blue (peak at 480 nm, “20blue”), 20 μmol m⁻² s⁻¹ red/blue (1:1, “20red:blue”), and 40 μmol m⁻² s⁻¹ red/blue (3:1, “40red/blue”) light emitting diodes (LEDs). Each light treatment consisted of five vessels of three shoots of each clone. The photon energy distribution profile for each light source was measured with a spectroradiometer (LI-1800, LI-COR® Biosciences, Lincoln, NE). The impact of light quality treatments on the multiplication ratio of the clones was recorded for subculture cycles 2 through 5.

PGR effects.

Shoots from three clones (318, 328, and 329) were placed in the chamber at 16°C with a 16-h d⁻¹ photoperiod under 40red/blue light and were cultured on MS medium supplemented with 0.7% (w/v) agar and 3, 6, or 9 μM BA alone or with 0.5 μM NAA. Each PGR treatment consisted of three vessels of two shoots of each clone. The impact of PGR treatments was recorded for subculture cycles 3 through 6.

Rooting and acclimatization.

All shoots derived from the various treatments were transferred onto a root-inducing medium (MS medium supplemented with 5 μM NAA and 0.7% [w/v] agar) for 5 wk at 16°C under 40red/blue LED light. After 5 wk, roots had formed and rooted plants were removed from the culture medium, and agar was washed away under running tap water. These plants were transferred into 36-cell trays (53.3 × 27 × 5.7 cm) filled with Fafard 3B mix, placed in a mist bed, and covered with a plastic dome for 10 d. The plastic domes were then removed, and the trays remained in the mist bed for an additional 20 d. Well-acclimatized plants were then transferred to a non-misted bench in the greenhouse for 30 d, and survival ratios (the number of plants surviving divided by the number planted) were calculated. These experiments were conducted during the fall and winter season when cool greenhouse temperatures could be maintained. Temperature was recorded at 15-min intervals using an ECD dataworker (ECD, Inc., Milwaukie, OR). The mean air temperature during the experimental period in the greenhouse was 18.1 ± 4.4°C, and the mean low and high temperatures were 14.7 ± 2.6°C and 20.7 ± 3.7°C, respectively. The light intensity at the canopy level was monitored with LI-190 Quantum sensors (LI-COR® Biosciences). Supplemental light was provided when sunlight was <100 W m⁻² (as described by Sun et al. 2013).

Seedling culture.

One- and 2-yr-old seed-derived plants were shipped to Clemson from Boehringer Ingelheim Pharma GmbH & Co (Mainz, Germany) on March 21, 2012. Seedlings were grown according to greenhouse methods of Sun et al. (2013), held in dormancy using the methods of Sun et al. (2012), and grown for a second season. The morphology and mass of tissue culture-derived plants was compared with that of 2- to 4-yr-old seed-derived plants grown in the greenhouse.

Statistical analysis.

Shoots from a given clone were assigned randomly to treatments and analyzed using a completely randomized design with a minimum of three replicated vessels. Data were analyzed using JMP version 9.0 (Statistical Analysis System, Cary, NC). The main effects and interactions among clone, subculture cycle, and temperature, light, or PGRs were analyzed utilizing a three-way analysis of variance (ANOVA). Each vessel was considered an individual experimental unit. Hypothesis testing was conducted at a significance level of p = 0.01.

To streamline the “Results and Discussion” section, only data from four clones (315, 318, 328, and 329) will be discussed. These four clones were selected for detailed presentation because large numbers of tissue-cultured shoots were acclimatized to greenhouse conditions and due to the differential responses of these clones to temperature, light quality, and PGR treatments in vitro. Data from all clones included in the experiments and pertinent statistical results are presented in supplemental Tables 1 and 2.

Results and Discussion

Effects of temperature, light quality, and PGRs on multiplication ratio.

Clone multiplication ratios varied in response to temperature treatments over repeated subculture cycles (Table 1, p < 0.0001). Clones were considered to be stably increasing when multiplication ratios were greater than 1, and a decrease in multiplication was not predicted after five subculture cycles (with 95% confidence). Clones 315, 328, and 329 stably increased over the five subculture cycles at 10°C. When cultured at 16°C, clones 315 and 328 stably increased. When cultured at 24°C, none of the clones stably increased and the multiplication ratio of clones 318 and 328 declined to <1 by the fourth and fifth subculture cycle. Symptoms of decline included leaf yellowing and senescence (Fig. 1). For example, clone 328 plantlets often started with a rapid flush of elongated foliage that began to senesce before the crown and basal plate developed proper divisions.

Table 1. Effect of temperature and subculture cycle on multiplication ratio and predicted multiplication ratio during stabilization of selected clones of Veratrum californicum

Figure 1.

Shoots of clone 328 grown at (A) 10, (B) 16, and (C) 24°C. Cooler temperature favored growth of bulb and basal plate, while warmer temperature promoted extension leaf blades. Leaves at 24°C were more prone to turn yellow, brown, and then senesce.

Typical plant culture temperatures range from 20°C to 27°C, but optimum temperatures for in vitro propagation vary widely and depend upon the plant species cultured (Read and Preece 2003). V. californicum clones do not stably increase under the more typical culture room temperatures, which would be expected, as stands of our ecotype of V. californicum in nature are in high mountain regions with cool soil.

Four clones (315, 318, 328, and 329) cultured at 16°C were evaluated for their responses to five different light treatments (Fig. 2). Light quality treatments did not affect clone multiplication ratios (p = 0.40).

Figure 2.

Spectral distribution in relative energy of light treatments: (A) 20 μmol m⁻² s⁻¹ fluorescent (20fluor), (B) 40 μmol m⁻² s⁻¹ red/blue (3:1, 40red/blue) (peaks at 660 and 440 nm, respectively), (C) 20 μmol m⁻² s⁻¹ blue (20blue) (peak at 440 nm), (D) 20 μmol m⁻² s⁻¹ red (20red) (peak at 660 nm), and (E) 20 μmol m⁻² s⁻¹ red/blue (1:1, 20red/blue) (peaks at 440 and 660 nm, respectively) lights.

Three clones (318, 328, and 329) were cultured at 16°C under 40red/blue LED and evaluated for responses to different combinations of BA and NAA. There were not enough shoots of 315 to include in this experiment. The multiplication ratio for clone 329 increased from 1.1 (3 μM BA) to 1.5 (9 μM BA and 0.5 μM NAA) (Fig. 3, p = 0.003). Higher concentrations of NAA and BA might further improve performance of clone 329 if further trials were conducted. The multiplication ratios of clones 318 and 328 were not influenced by PGR treatments (p = 0.08, p = 0.34, respectively). For bulbous plants, a combination of BA and NAA typically promotes successful shoot multiplication (Hong and Lee 2012). However, in this study, only one of three clones of V. californicum had better multiplication after combined treatments with BA and NAA in the ranges we had selected.

Figure 3.

Effect of BA (3, 6, 9 μM) alone and in combination with NAA (0.5 μM) on multiplication ratio in clone 329 of Veratrum californicum. Vertical bars represent the standard error of the mean.

Effect of temperatures and light quality during acclimatization.

Plants from all stage II cultures were placed in root-inducing medium for 5 wk at 16°C under 40red/blue LED. Despite this common treatment, prior culture conditions influenced plant acclimatization in the greenhouse. When stage II clones were cultured under 20fluor light at 24°C, none of the plants survived acclimatization in the greenhouse. The best overall survival for the 15 original clones was from 16°C laboratory stage II culture conditions (p < 0.02), which resulted in survival ratios of 20% to 50% (Fig. 4). When taken from the 10°C treatment, survival of the clones ranged from 30% to 40%.

Figure 4.

The effects of laboratory culture temperature on the subsequent survival ratio during acclimatization in greenhouse January to March in Clemson, SC, with various clones of Veratrum californicum. Vertical bars represent the standard error of the mean.

Because the survival ratios varied greatly among clones cultured under the 20fluor at 16°C, additional stage II material were grown under LED lights with different spectral qualities at 16°C during three subculture cycles (Fig. 2). The 40red/blue light treatment in the laboratory enhanced survival in the greenhouse of clones 315 and 328, when compared with survival under the 20fluor treatment (Fig. 5, p < 0.0001). However, survival of clone 318 was the greatest when grown under 20fluor light, and the survival of clone 329 was enhanced when grown under 20blue or 20red/blue LED light.

Figure 5.

The effects of light quality treatments on the survival ratio of clones of Veratrum californicum during acclimatization. Vertical bars represent the standard error of the mean.

Even though the multiplication rates of in vitro clonal material were relatively low after 2 yr in culture, the 15 best clones yielded approximately 1,000 plants that survived greenhouse transfer (Supplemental Table 2). By altering light quality during stage II culture, the average survival rates of stage IV material in the greenhouse increased from 30% to 50% to 50% to 80%. Selecting clones and integrating light quality and temperature information with stage III rooting environment would be a next logical step toward improved greenhouse performance.

Evaluation of micropropagated V. californicum plants.

The value of a tissue culture-derived plant is in part determined by the stage at which it leaves the nursery. V. californicum is a slow-growing species that may not divide in nature until plants are over 10 yr of age, and seedlings are rarely, if ever, observed (Infinity Pharmaceutical Co., unpublished data). We grew 1- and 2-yr-old seedlings in the greenhouse for an additional two seasons to quantify growth in an effort to better assign value to tissue culture-derived plants. The sizes of tissue culture-derived plants acclimatized in the greenhouse tracked with that of 2- to 4-yr-old seed-derived plants, with most (65%) being the size of 3-yr-old seed-derived plants (Fig. 6, Table 2, and Supplemental Table 1). Plant sizes varied widely depending on the clone and experimental treatments. For example, clone 328 grew larger from shoots cultured at relatively low (10°C) temperatures, whereas plants of clone 329 were larger from shoots cultured at middle and high temperatures (16°C and 24°C). Plants of clone 329 grew larger from shoots cultured under the 40red/blue LED light treatment. Overall, plants of clone 328 were most adaptable to culture under multiple temperature and light conditions.

Figure 6.

Comparison of seed- and tissue culture-derived plants. (A) Typical 4-yr-old seed-derived plant grown with subirrigation in the greenhouse, (B) tissue culture-derived plant acclimatized to and grown in the greenhouse for 4 mo, and (C) tissue culture-derived plants, including some with multiple shoots (clone 329), acclimatized to and grown in the greenhouse for 4 mo.

Table 2. Mass of selected tissue culture-derived plants transferred to the greenhouse, compared with seed-derived plants that were grown in the greenhouse

Formation of multiple shoots on tissue culture-derived plants was common, but was never observed in the 2,000+ seed-derived plants (Table 2, Fig. 5, and Supplemental Table 1). Formation of multiple shoots in the tissue-cultured plantlets may have been an effect of residual BA from micropropagation (Werbrouk et al. 1995), but did not appear to interfere with rooting. The potential for production of multiple shoots from tissue culture-derived plants can serve an ancillary benefit during nursery production, as additional divisions of V. californicum may be possible during outdoor nursery production. The value of plants largely depends on the number of years required to grow a harvestable plant, which for V. californicum in the field, was at least 10 yr. Micropropagated plants enter the production chain with similar mass to 3-yr-old seedlings, which helps assign value to tissue cultures.

Conclusions

Although the donor plants were similar in appearance before harvest from a native stand, the physiological responses of clonal material to culture conditions and their resulting morphology under controlled environments were quite different. Clones responded differentially to varied temperatures during successive subculture cycles. With high temperature (24°C) culture conditions, some clones started off well but deteriorated over time. Most clones multiplied well at 10°C to 16°C (Supplemental Table 1). We suggest multiplying V. californicum tissue cultures at 16°C because this temperature also enhances subsequent greenhouse survival. Light quality in the cool growth chamber also influenced subsequent survival, but this could not be generalized for the clones in this study. After acclimatization in the greenhouse, some of the plants continued to divide. Nursery divisions subsequent to tissue culture also increase the numbers of plants produced. The laboratory environment had a long-lasting influence on subsequent greenhouse growth. Although we have not described a rapid method for large-scale micropropagation, stable increase of selected materials in this uncultivated, slow-growing species marks significant progress.