Protocols for Mass Micropropagation of Antelope and Desert Bitterbrush

1 1 1 2 Annette Leege -Brusven , John L. Edson , David L. Wenny , and Min Hironaka

Leege -Brusven, Annette; Edson, John L.; Wenny, David L.; Hironaka, Min. 1994. Protocols for Mass Micropropagation of Antelope and Desert Bitterbrush. In Landis, T.D.; Dumroese, R.K., technical coordinators. Proceedings, Forest and Conservation Nursery Associations. 1994, July 11-14; Williamsburg, VA. Gen. Tech. Rep. RM-GTR-257. Fort Collins, CO: U.S. Department of Agriculture, Forest Service, Rocky Mountain Forest and Range Experiment Station: 239-245. Available at: http://www.fcnanet.org/proceedings/1994/leege-brusven.pdf

Abstract—Vegetative propagation of Purshia tridentata and P. glandulosa could potentially capture and multiply valuable genetic traits such as fire resistance and produce plants which reproduce in a shorter time than seedlings. The goal of this research was to develop mass propagation protocols for antelope and desert bitterbrush using micropropagation techniques. Microshoot tips were incubated in vitro on alternative nutrient media supplemented with different levels of cytokinin and auxin growth regulators. An average of 7.9 shoots/explant of P. tridentata were produced after 1 month on Woody Plant Medium (WPM) supplemented with 0.1 mg/l benzlyadenine (BA). P. glandulosa multiplied 5-fold over 1 month on the same media. Best rooting occurred ex vitro for both species. P. tridentate microshoots rooted at 88.6% after treatment with Hormex® #1 (0.1% IBA) and P. glandulosa microshoots rooted up to 78% after treatment with Hormex® #3 (0.3% IBA). Flowering and fruiting of micropropagated plantlets from both species occurred after one season’s growth in the greenhouse. These successful mass propagatio n systems for bitterbrush could play an important role in range restoration.

INTRODUCTION rial for revegetation projects This paper describes mass- have been hindered by seed propagation protocols for ante- Antelope and desert bitter- dormancy and seed predation lope and desert bitterbrush and brush (Purshia tridentata (Young and Evans, 1981), evaluates the effects of culture (Pursh) DC. and P. glandulosa vegetatively propagated plants media and plant growth regulators Curran, respectively) are could provide an alternative and on multiplication and rooting. roseaceous, nutritive forage more reliable source of planting shrubs in the semi-arid range- stock. Micropropagated antelope GENERAL METHODS lands of Utah, Nevada, Idaho and desert bitterbrush plants, and California (Welch et al., produced in a relatively short Culture initiation 1982). Wild-fires and livestock time, could potentially restore Our source of P. tridentata overgrazing have degraded range damaged habitat with fire- and P. glandulosa material was habitat and reduced the popula - resistant, palatable, nutritious wild seed, collected from range- tion of both bitterbrush species. and drought-hardy selections. land shrubs in southern Idaho Since efforts to propagate mate -

1 University of Idaho Forest Research Nursery, Moscow, Idaho 83844-1137.

2 Range Resources, Department of Range Resources, University of Idaho, Moscow, Idaho. and Nevada, respectively. Seed- Selecting a culture medium Hormex® #3 (0.3% IBA); lings were grown in pots at the Although MS medium has Hormex® #8 (0.8% IBA); or University of Idaho Plant Sci- commonly been used as a sub- Rootone® (0.2% naphthalen- ence greenhouse. Shoot tips of strate for micropropagation, eacetic acid (NAA) plus 4.04% new growth were collected from many woody plants grow better Thiram®); or a 5-sec dip in a 2-yr-old containerized seedlings on other defined media with 1:10 solution of Dip ‘n Grow® and placed in sterile culture for lower concentrations of mineral (0.1% IBA and 0.05% NAA). propagation. nutrients (Bonga and von All microshoots were immersed Aderkas, 1992) such as Woody in 1 g/l of the fungicide benomyl The cuttings were washed in Plant Medium (WPM) (Lloyd for 20 sec before being treated running tap water for 30 min. and McCown, 1980). Hence, we and planted. The treated Leaves were excised from the cultured microshoots on MS and microshoots were placed on stems, and the defoliated shoots WPM media to detect differ- propagation benches under 60% were agitated for 10 min in an ences in shoot elongation and the shade and natural photoperiod. aerated aqueous solution con- number of axillary shoots pro- Root zone temperatures varied taining 5 drops of Tween® 80 per duced. from 20° to 22°C, relative liter. The stems were then humidity from 86 to 92%, and surface sterilized for 15 min in a Shoot multiplication diurnal air temperatures from continuously stirred 20% Microplants exposed to potent 15° to 25°C. Clorox® bleach solution (1% and high levels of cytokinins in NaClO) and rinsed 3X in sterile, culture media often produce distilled, deionized water. The adventitious as well as axillary stem segments were given fresh shoots (Huetteman and Preece, PROPAGATION OF PURSHIA TRIDENTATA basal cuts and placed individu- 1993), and adventitious shoot ally in 25 X 150 mm test tubes production has been linked to Materials and methods containing 10 ml of Murashige increased rates of somaclonal Culture medium: 90 and Skoog (MS) basal medium variation (Pierik, 1987). To microshoots were equally as- (Murashige and Skoog, 1962). decrease the possibility of signed to hormone-free MS, These and all subsequent cul- adventitious shoot formation, WPM and ½ MS. To compare tures grew on open shelves thus, maintaining the genotypes microshoot elongation and new under an 18-h photoperiod of the bitterbrush shoots, the shoot production on the same 3 provided by cool-white fluores- cytokinin benzyladenine (BA) media containing BA, 450 shoot cent lights (40 W) which pro- was used at low to moderate tips were randomly assigned to duced approximately 20 umoles levels for shoot multiplication. WPM, MS or ½ MS, each -2 -1 m s PAR on the leaf surfaces. supplemented with 0.1 mg/l BA. Diurnal temperatures ranged Ex vitro rooting After 4 weeks, microshoot from 22° to 27°C. The effect of several commer- elongation, the number of new cial auxin preparations on the shoots and health scores were To increase the number of rooting of microshoots was recorded for each explant. microshoots available for experi- assessed after 6 weeks growth ments, stems were cut into 2- under fog humidification on a Shoot multiplication: 270, node segments and transferred to greenhouse propagation bench. 1.5-cm-long microshoot tips fresh media monthly until The auxin treatments were either were assigned in equal numbers ® sufficient numbers of uniform basal talc dips in: Hormex #1 to 9 concentrations of BA (0, healthy microshoot tips were (0.1% indolebutyric acid (IBA)); 0.025, 0.05, 0.1, 0.2, 0.4, 0.8, produced. # SHOOTS/EXPLANT 10 Putr Pugl 8 6 The new shoots on WPM 4 developed normal healthy foliage (mean health score 2.3) 2 versus slightly more chlorotic 0 growth on MS (2.1), and ½ MS WPM MS (1.4) media (p = 0.005). Be- MEDIA cause the bitterbrush microshoots grew more vigor- ously on WPM than on either 1.6, and 3.2 mg/l) on WPM, rooted microshoots with normal MS or ½ MS, we selected WPM arranged in a CR design. After 4 root development were trans- as the medium for subsequent in weeks, shoot elongation and planted to 215 cc capacity cells vitro experiments. counts of all new shoots were filled with a 1:1:1 mix of perlite, recorded. vermiculite, and peat (v/v). The Shoot Multiplication: New plants grew under natural light shoots developed from axillary In vitro rooting: 200 with greenhouse temperatures nodes and the bases of explants microshoots were assigned to 2 varying from 15° to 25°C during treated with varying levels of replicates of 4 auxin treatments the growing season. Survival, BA. In contrast, the explants on arranged in a RCB design. The shoot growth, number of stems, BA-free medium were stunted treatments included a 5-sec NAA and flowering habit were re- and failed to release axillary (1,000 mg/l) basal dip and corded 1 year later on a random shoots. The number of new culture on hormone-free WPM, sample of 30 survivors. shoots increased from 0 on and placement on WPM contain- hormone-free medium, to a ing 0, 0.1 and 1.0 mg/l NAA. Results and discussion maximum average of 15, after Rooted microshoots were tallied Culture Medium: After 1 treatment with 0.8 mg/l BA, and after 4, 6 and 8 weeks. In a month in culture, the then declined at higher concen- second experiment, 432 1-cm- microshoots on BA-free MS, ½ trations of BA. Maximum stem long microshoots were either MS and WPM elongated an growth (1.8 cm) occurred with dipped or not dipped in 1,000 average of only 4 mm and treatment of 0.1 mg/l BA, and mg/l NAA and placed on ½ MS produced no new shoots. In decreased significantly after or WPM. contrast, microshoots on the treatments > 0.2 mg/l BA (p = same 3 media supplemented with 0.0001). The difficulty of Ex vitro rooting: 480, 1-cm- 0.1 mg/l BA lengthened rapidly clearly identifying axillary long microshoots were removed and produced vigorous new shoots increased when BA from test tubes and assigned to 3 shoots. Greatest stem elongation concentrations rose above 0.2 replicates of a control (no hor- and new shoot production mg/l, due to shorter stem intern- mone), Hormex® #1, Rootone® occurred on WPM. The odes and increased basal shoot and Dip ‘n Grow® arranged in a microshoots grew an average of proliferation. Therefore, the RCB design. The treated shoots 0.8 and 1.1 cm taller on WPM maximum shoot count of 15 may were struck into 5-ml capacity versus MS and ½ MS, respec- include adventitious shoots less miniplug cells containing a 2:1:1 tively (p = 0.0008), and devel- desirable for clonal production. mix of perlite, peat and vermicu- oped an average of 5 more new The BA level of 0.1 mg/l re- lite (v/v). shoots per explant on WPM sulted in optimal elongation, a Acclimatization: 50-in vitro- versus MS (p = 0.0002). moderate number of new shoots produced, and reasonable assur- ance of the axillary nature of the new shoots.

In vitro rooting: NAA seemed to inhibit normal rooting. Microshoots on NAA-free WPM (control) developed white “normal” roots which penetrated to the bottom of the test tube. Explants on the NAA-supplemented media, however, developed “aerial” horizontal fibrous roots on the surface of the medium and produced few if any normal roots. In the second Figure 1. Ex vitro rooted Purshia tridentata plantlet 4 weeks after experiment, 11% of the NAA- treatment with 0.1% IBA. treated microshoots rooted versus 53% of the non-treated explants (p < 0.0001) after 6 weeks in culture. Rooting was similar on both WPM and ½ MS incubation media.

Ex vitro rooting: Microshoots rooted more successfully ex vitro than in vitro (Fig. 1). After 6 weeks, 62% of the untreated microshoots (control) had rooted versus 88.6% and 79.0% of the microshoots treated with Rootone® and Hormex® #1, respectively. The microshoots treated with Dip ‘n Grow® rooted at 65.3%. We infer that a low rate of auxin, applied as Figure 2. Flowering desert bitterbrush (left) and antelope either IBA or NAA powder bitterbrush (right) after 1 year in the greenhouse. treatment, will likely result in ex vitro rooting at a level sufficient for commercial production. Acclimatization: Four months after transplant, 85% of the plantlets remained alive with no subsequent death. By the end of the first year’s growth in the greenhouse, the plantlets had produced leaders with an average extension of 16.4 cm. The plantlets bloomed heavily within 1 month of budbreak with an average of 20 flower buds on each leader (Fig. 2). The preco- cious and prolific flowering of Figure 3. Effect of Woody Plant Medium (WPM) and Murashige the plantlets suggests that regen- and Skoog medium (MS) on shoot elongation of P. eration could occur the first year tridentata (Putr) and P. glandulosa (Pugl) microshoots after 4 weeks in culture. after outplanting this type of planting stock if conditions are favorable for establishment.

PROPAGATION OF PURSHIA GLANDULOSA

Materials and methods

Culture medium: 360, 2-cm- long shoot tips were excised and placed randomly on MS or WPM containing 0.1 mg/l BA. After 5 weeks, we calculated stem elongation and counted the number of axillary shoots produced. Figure 4. Effect of Woody Plant Medium (WPM) and Murashige and Skoog medium (MS) on shoot production of P. Shoot multiplication: 160 tridentata (Putr) and P. glandulosa (Pugl) shoot tips were randomly as- microshoots after 4 weeks in culture. signed to 24 treatment combina- tions of MS + 0, 0.05, 0.1, 0.2, In vitro rooting: 216, 2-cm- Ex vitro rooting: Non- 0.4, 0.9, 1.8 or 3.6 mg/l BA. long microshoots were divided lignified microshoots were cut Twenty microshoots were used among 6 media (MS, ½ MS and into 600, 3-cm-long shoot tips per treatment. After 4 weeks, WPM supplemented with or and 600 stem segments. Forty shoot elongation and the number without 1 mg/l NAA). Rooting tips and 40 segments were of axillary bud breaks were was recorded after 4, 6, 8 and 10 randomly assigned to each of 5 recorded for each shoot. weeks. hormone treatments: a control (no hormone), Hormex® #3, Hormex® #8, Rootone®, and Dip ‘n Grow®. The experiment was replicated twice in a ran- domized split-plot design. The explants were struck in 5-ml capacity miniplug cells containing a 1:1 mix of Sunshine® #3 and perlite.

Acclimatization: 40 rooted microshoots were transferred from agar medium to 164 cc capacity Ray Leach® containers containing Sunshine® #2. Observations were made a year later on shoot growth, number and location of branches, and incidence of flowering and fruiting.

Results and discussion Culture medium: Stem Figure 5. Purshia glandulosa microshoots after 1 elongation and axillary shoot month on hormone-free WPM. Note tall, production were enhanced single stem growth. significantly on the WPM (p<0.001) versus MS medium (3.85 vs. 2.75 cm and 2.75 vs. 2.06 shoots, respectively). P. the stunted growth of P. rooting on NAA-supplemented glandulosa microshoots devel- tridentata on BA-free media. MS and ½ MS, respectively. oped taller stems but fewer Explants achieved maximum None of the microshoots on MS axillary shoots than P. tridentata growth with 0.1 mg/l BA (3.5 produced roots, regardless of explants on both media (Figs. 3 cm) and were shorter with more whether NAA was present or not & 4). Using a low level of BA axillary bud break after treat- in the medium. This data sug- (0.1 mg/l) would avoid the foliar ments >0.2 mg/l BA. The gests the most important factor chlorosis and adventitious shoot number of new shoots increased in rooting is the basal medium. production associated with the from 0 on BA-free medium, to a In contrast to the response of P. higher levels of BA in the maximum average of 5.0 shoots tridentata, NAA did not inhibit medium, a distinct advantage per explant after treatment with rooting. when propagating selected 0.4 mg/l BA, and then declined genetic traits. at higher concentrations of BA. Ex vitro rooting: Microshoot tips rooted most successfully Shoot multiplication: In vitro rooting: The best after they were treated with a Microshoots developed tall, rooting (44%) occurred after 10 low concentration of auxin. single stems on hormone-free weeks on WPM + 1 mg/l NAA After 6 weeks, 28% of the medium (Fig. 5), in contrast to as compared to 0% and 19% untreated microshoots had rooted versus 74%, 72%, and 71% of the microshoots treated and Organ Cult. 33:105-119. with Hormex® #3, Hormex® #1 3. Plantlets of both species, and Dip ‘n Grow®, respectively. transferred to greenhouse Lloyd, G. and B. McCown. Microshoots treated with conditions, can provide 1980. Commercially-feasible Hormex® #8 rooted at 52%. flowering and fruiting plants micropropagation of mountain Explant tips also rooted better within 1 year. laurel, Kalmia latifolia, by use than stem segments (60% versus of shoot-tip culture. Comb. 36%, respectively). We have demonstrated that Proc. Int. Plant Prop. Soc. antelope and desert bitterbrush 30:421-427. Acclimatization: Four months can successfully be propagated after transplant, 85% of the by micropropagation techniques, Murashige, T. and R. Skoog. plantlets remained alive with no achieving numbers suitable for 1962. A revised medium for subsequent death. After 1 year commercial production. rapid growth and bioassays in the greenhouse, the leading with tobacco tissue cultures. shoots had extended an average With micropropagated ante- Physiol. Plant. 15:473-97. of 18.0 cm. Eighty-five percent lope and desert bitterbrush, of the plantlets flowered, with range managers and scientists Pierik, R.L.M. 1987. In Vitro the axillary bloom generally can potentially: Culture of Higher Plants. extending to the full length of all Martinus Nijhoff the stems (Fig. 2). 1. revegetate areas with locally Publ.,Dordrecht, The Nether- selected genetic stock lands.

CONCLUSIONS: 2. shorten the time for breeding Welch, C.L., S.B. Monsen, and THE PROPAGATION programs with selected clones N.L. Shaw. 1982. Nutritive PROTOCOL value of antelope and desert 1. Initiating and multiplying both 3. outplant material which could bitterbrush, Stansbury bitterbrush species on Woody rapidly produce seed cliffrose, and Apachi-plume, Plant Medium, supplemented p. 173-185 In: A.R. Tiedman with 0.1 mg/l BA, provides 4. lower the fire frequency, and R.L. Johnson (eds.), Proc. numerous elongated healthy enhance forage, and increase Research and Management of shoots suitable for multiplica- cover of damaged habitat. Bitterbrush and Cliffrose in tion in subculture. Western North America Symposium. Intermtn. Forest REFERENCES 2. Rooting the microshoots in ex and Range Exp. Sta. General vitro conditions with low Bonga, J.M. and P. von Aderkas. Tech. Rep. INT-152. levels of auxin would lower 1992. In Vitro Culture of propagation costs, although Trees. Kluwer Academic Young, J.A. and R.A. Evans. untreated microshoots of P. Publ., Dordrecht, The Nether- 1981. Germination of seeds of glandulosa and P. tridentata lands. antelope bitterbrush, desert rooted with moderate success bitterbrush, and cliff rose. under in vitro conditions (44% Huetteman, C.A. and J.E. USDA Science and Education and 61%, respectively) Preece. 1993. Thidiazuron: a Administration Agricultural potent cytokinin for woody Research Results, ARR-W-17. plant tissue. Plant Cell, Tissue