Springer 2005
Plant Cell, Tissue and Organ Culture (2005) 82: 207–211 DOI 10.1007/s11240-004-7836-6
Research note
Tissue culture propagation of Mongolian cherry (Prunus fruticosa) and Nanking cherry (Prunus tomentosa) Kris Pruski1,*, Tess Astatkie2 & Jerzy Nowak3 1
Department of Plant and Animal Sciences, Nova Scotia Agricultural College (NSAC), P.O. Box 550, Truro, NS B2N 5E3, Canada; 2Department of Engineering, NSAC, P.O. Box 550, Truro, NS B2N 5E3, Canada; 3 Department of Horticulture, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA (*requests for offprints; Fax: +1-902-897-9762; E-mail:
[email protected]) Received 16 January 2003; accepted in revised form 18 December 2004
Key words: culture initiation, ex vitro rooting, growth regulators, in vitro culture, shoot proliferation
Abstract In vitro culture establishment, shoot proliferation and ex vitro rooting responses of Mongolian cherry (Prunus fruticosa L.), and Nanking cherry (Prunus tomentosa L.), were examined using various combinations of growth regulators. Dormant buds, taken during winter months, were used as explants. In both species, Murashige and Skoog Minimal Organic (MSMO) solid medium supplemented with 0.49 lM indole-3-butyric acid (IBA) and either 4.44 or 8.88 lM 6-benzylaminopurine (BA), was the best for culture initiation, and with 8.88–15.16 lM BA for shoot proliferation. Good rooting responses were also obtained with shoots produced on media containing 0.91 lM thidiazuron (TDZ). Auxin treatments were required for ex vitro rooting of approximately 20 mm long shoots in peat/perlite (1:1 v/v) mixture, at 25 C, under mist. The best rooting (79%) was obtained with IBA/NAA (naphthaleneacetic acid) (9.80/2.69 lM) combination. A commercial rooting powder, Rootone F, containing IBA/NAA (0.057/0.067%), was also effective (73%). The ex vitro rooted plantlets did not require any additional acclimatization prior to transplanting to the regular greenhouse conditions. Abbreviations: BA – 6-benzylaminopurine; cv(s) – cultivar(s); IBA – indole-3-butyric acid; MSMO – Murashige and Skoog’s minimal organic medium; NAA – naphthaleneacetic acid; TDZ – thidiazuron
Mongolian (Prunus fruticosa L.) and Nanking (Prunus tomentosa L.) cherries are transcontinentally distributed, fruit–bearing, ornamental shrubs, well adapted to severe winter conditions of Canadian Prairie provinces (Knowles, 1975). Since valuable new selections, bearing relatively large (4–5 g), good quality fruits, suitable for processing were made, the fruit growers in Western Canada are currently considering these two species as new crops that could be commercially grown under prairie conditions. To make a large scale cultivation and an effective breeding viable, an efficient clonal multiplication method needs to be established. In Western Canada, in vitro propagation has been success-
fully utilized in the production of other fruitbearing, native prairie shrubs, such as: Amelanchier alnifolia Nutt., the Saskatoon berry (Pruski et al., 1991), Prunus pensylvanica L., the pincherry (Pruski et al., 2000), and. P.virginiana, the chokecherry (Pruski et al., 2000; Zhang et al., 2000). Although tissue culture propagation has been used for cherry species for over 20 years, there are no published protocols or micropropagation of P fruticosa and P. tomentosa fruit lines. This study was undertaken to determine requirements for in vitro establishment, multiplication and rooting of cultures, to develop an efficient micropropagation protocol for transplant production of these two species.
208 Explants of Mongolian and Nanking cherry were taken from 10-year-old plants grown in a shelterbelt row at the Crop Diversification Centre North, Edmonton, Alberta, Canada. Terminal (dormant) buds were dissected from the current year growth, in December. December collection was chosen to avoid winter injury to buds that occur on the Canadian prairies between January and March due to frequent freezing and thawing cycles (Pruski et al., 2000). Bud explants were rinsed in a stream of tap water for 60 min, outer scales pealed off, and the buds surface-sterilized by agitation (150 rpm) in 1% sodium hypochlorite (10· diluted commercial bleach) containing 0.1% Tween-20(surfactant), for 10 min, rinsed three times in sterile distilled water, and trimmed to approximately 3 mm length. The explants were then placed on 12 ml solid medium (0.6% agar) in culture tubes (25 · 150 mm) on Murashige and Skoog Minimal Organic (MSMO) media (Murashige and Skoog, 1962) supplemented with sucrose (30 g l)1) and either 0.49 lM indole-3butyric acid (IBA) or 0.54 lM naphthalene acetic acid (NAA), and 4.44 or 8.87 lM 6-benzylaminopurine (BA) (Table 1). Cultures were incubated for 4 weeks in an environmental chamber (Conviron S10h, Winnipeg, Manitoba, Canada) at 24/ 22 C day/night temperature, 16-h photoperiod, 150 lmol m)2 s)1 mixed fluorescent (F40T12 tubes, General Electric (GE), USA) and incandescent illumination (40W, GE, USA). Percentage
survival and the number of rosettes were recorded. The individual rosettes were then subcultured onto MSMO shoot proliferation media containing various concentrations of BA (0.00, 2.22, 4.44, 8.87, 22.19 lM) and thidiazuron (TDZ) (0.00, 0.45, 0.91, 1.82 lM). Two rosettes per Magenta GA7 vessel (Magenta Corp., Chicago, IL, USA) filled with 50 ml of solid medium, were grown for 4 weeks. Other media components and the growth conditions were as described above. Approximately 15 to 25-mm long shoots derived from proliferating cultures were rooted ex vitro, in peat moss/perlite (1:1 v/v) mixture, in Hillson-type plastic rootrainers with 38 mm2 · 140 mm cells, 32 cell stray1 (Spencer-Lemaire Ind. Ltd., Edmonton, Alberta, Canada). One shoot was transplanted into each cavity. Trays were then placed on the bottom heated (25 C) greenhouse bench equipped with an intermittent mist system (2 s, every 25 min). The following treatments were evaluated: no auxin (control), 9.80 lM IBA and 9.80 lM IBA + 2.70 lM NAA applied with daily watering for the first week of rooting period, and a commercial rooting powder Rootone F (Amchem Prod.Inc., Ambler, PA, USA) containing 0.057% IBA + 0.067% NAA mixture, applied as a basal dip on microcuttings prior to planting. The number of rooted plantlets wat recorded after 5 weeks from planting. The culture establishment experiment that had four treatments (Table 1) was conducted as a
Table 1. Effects of growth regulators on the initiation, performance (survival and rosette production) and rooting of Nanking cherry and Mongolian cherry cultures Establishment of culture Treatments for establishment
Rooting of plantlets in both species Species Nanking cherry
NAA 0.54 + BA 4.44 lM NAA 0.54 + BA 8.88 lM IBA 0.49 + BA 4.44 lM IBA 0.49 + BA 8.88 lM
0.97 1.20 0.93 1.23
1
Treatments for rooting
Rooted plantlets (mean)
Control IBA IBA+NAA Rootone F
18.7 20.3 25.3 23.3
Mongolian cherry 2
b (28 , 90% ) a (37,96%) b (27,80%) a (36, 86%)
1.00 1.40 1.30 1.50
b (39,90%) a (45,93%) a (30,96%) a (42, 100%)
c (59%3) bc (64%) a (79%) ab (73%)
The results are given as the mean number of rosettes per explant. In brackets are the total number of produced rosettes and the percentage of culture survival. n = 30 per each treatment and species; within each species, values followed by the same letter are not significantly different at the 5% level according to Kruskal–Wallis and the Mann–Whitney non-parametric tests. 1 Total number of rosettes produced out of 30 explants. 2 Percentage culture survival. n = 32 for last two columns; means followed by the same letter are not significantly different at the 5% level of significance using Duncan’s multiple range test. 3 Percentage of rooted plantlets.
209 completely randomized design, with three replicates of 10 explants. The response variable, number of rosettes, had either 0 (no growth) or 1 or 2 or 3 value. Examination of residuals, as described in Montgomery (2001), to verify the normal distribution and constant variance assumptions needed for the validity of the ANOVA results suggested that the distribution was not normal and no transformation was able to induce normality. As a result, the Kruskal–Wallis and the Mann-Whitney non-parametric tests were used for each of the two species and Median groupings were made. In the multiplication experiment, each treatment had three replications of 16 cultures. For each species, the relationship between BA concentration and shoot number (SN) and shoot length (SL in mm) was best described by a second-order polynomial. For each species, the value of BA concentration that maximizes shoot number was calculated, and the values of SN and SL at this BA concentration were calculated and summarized in Figure 2. The effect of TDZ on shoot number and length was determined using average of 30 plantlets within each replicate (three replicates). The data were analyzed as a 2-factor factorial design, species and TDZ concentration. In the rooting experiment, a 2-factor factorial ANOVA was performed (species and treatment) to analyze the results. The number of rooted plantlets (out of 32) was the response. Duncan’s multiple range test was used to separate the means. All statistical analyses were done using SAS. Combination of 0.49 lM IBA or 0.54 lM NAA with 8.87 lM BA,were the most effective in the initiation of Nanking chery cultures. The number of rosettes produced on media with 8.87 lM BA was significantly higher than on media with 4.44 lM BA (Table 1). Following 4 weeks in culture with 8.87 lM BA at least 86% explants survived. Most of them produced a single rosette and a few two rosettes, with a mean number per explant of 1.2 (Table 1). In Mongolian cherry the number of rosettes was also the highest on media with 8.87 lM BA (Table 1). NAA combination with 4.44 lM BA gave siginificantly lower average number of rosettes per culture. Nanking cherry were more difficult to establish than Monogolian cherry. The percentage survival varied from 80 to 96%. Mongolian Cherry Percentage survival varied from 90 to 100%, being the heighest on media with IBA 0.49 lM BA and
8.87 lM. Similar results were obtained in establishment of chokecherry (Pruski et al., 2000; Zhang et al., 2000) and pincherry cultures(Purski et al., 2000). Both Monogolian and Nanking cherry cultures benefitted from the higher concentration of BA (8.87 lM) in culture establishment (Table 1). Proliferating cultures of Nanking cherry and their rosettes are shown in Figure 1a. Summary of the regression analysis for shoot proliferation responses as affected by BA concentration is presented in Figure 2. For each species tested, the BA concentrations that maximize SN, and the SN and SL for the optimal BA concentration were calculated. Controls (zero BA) produced about two shoots, approx. 25 mm long, in Mongolian cherry, and 1.5 shoots, approx. 27 m long, in Nanking cherry per rosette (Figure 2). Compared to the
Figure 1. (a) Proliferating cultures of Nanking cherry and (b) rooted microcutting of Mongolian cherry.
210 controls, the 4.44 lM BA treatment increased the number of shoots in both species. With increasing BA concentration in the medium, more, but shorter shoots were produced. Above 15 lM BA concentration, the shoot number in both species decreased. Length of shoots in both Mongolian and Nanking cherry cultures decreased with increasing BA concentration in the medium (Figure 2). At 22.19 lM BA the shoots became dark green showing symptoms of hyperhydricity. Shoot deformation in cultures of related species, grown on media with high (22.5 lM) BA content, were also reported by Linebeger (1983) and Pruski et al. (1991, 2000). The BA concentrations for the maximal SN, calculated from the fitted regression equations, were: 15.16 lM for Nanking cherry and 17.94 lM for Mongolian cherry (Figure 2). BA concentrations 8.87–15.16 lM were optimal for shoot proliferation in both species. Approximately 3–4 good quality, 16 to 20-mm long shoots, with well-developed leaves (15–25 mm), were produced per rosette in Nanking cherry and 4–5, 5–18 mm long, in Mongolian cherry, after 4 weeks in culture. BA concentration of 17.94 lM determining a maximal SN in Mongolian cherry was slightly too high for obtaining long, easy to handle, shoots for ex vitro rooting. The 8.87-lM BA level has been also reported as optimal for proliferation of Prunus cerasifera · Prunus
munsoniana rootstocks (Dalzoto and Docampo, 1997) and 8.87–12.82 lM BA for P. virginiana (Pruski et al., 2000). The effect of TDZ on shoot proliferation was significant for the number of shoots only (data not shown). Compared to controls the number of shoots almost doubled on media with 0.45 lM TDZ in both species after 4 weeks in culture. Four good quality shoots, 15– 20 mm long with well-developed leaves were produced in Mongolian cherry cultures and 2–3 shoots (18–24 mm long) in Nanking cherry cultures at lower concentrations of TDZ (0.45–0.91 lM). Higher TDZ concentration of 1.8 lM induced more shoots (close to 5) but considerably shorter in both species and a mass of stunted, dark green, shoots that were difficult to handle. Similar responses have been reported by a number of researchers, with several woody plant species (Sarwar and Skirvin, 1997; Thengane et al., 2001). From our study, 0.91lM TDZ appeared to be optimal for the production of quality shoots in both species. TDZ has been reported to be a potent growth stimulant on in vitro plant morphogenesis and shoot orgnogenesis in several woody species, including Malus (Sarwar and Skirvin, 1997) and Prunus (Mante et al., 1989). Similarly to other fruit bearing woody species (Maene and Debergh, 1983: Pruski et al., 1991, 2000; Murai and Harada, 1997), in vitro produced shoot rosettes of both Mongolian and Nanking cherry rooted readily ex vitro when treated with
Figure 2. BA concentration effect on SL and SN in Mongolian cherry and Nanking cherry. Actual values (solid circles), the fitted by the second order polynomial (solid lines), and the fitted regression model of SL and SN for each species.
211 auxins. Shoots of Mongolian cherry were easier to root (average 73% rooting) than shoots of Nanking cherry (64%). Rooted Mongolian cherry shoots are illustrated in Figure 1b. The control yielded significantly fewer rooted plants (59%) than all other treatments (Table 1). The best rooting, 79%, was observed in IBA/NAA combination. A commercial rooting powder, Rootone F, was almost equally effective (73%) for both species and did not differ significantly from the IBA/NAA treatment(Table 1). Similar rooting benefits of the IBA/NAA auxin combination were reported by Struve and Lineberger (1985) and Pruski et al. (1991, 2000). Poor adventitious root formapon is a major obstacle in micropropagation systems (de Klerk, 2002). The conditions during in vitro rooting treatment(s) may have a profound effect on performance of plantlets after transfer ex vitro. Particularly, accumulation of ethylene during in vitro rooting can have a devastating effect (de Klerk, 2002). This problem could be avoided when rooting is performed ex vitro. Auxin concentrations optimal for root initiation during rooting in vitro can be inhibitory for root elongation (Maene and Debergh, 1983). Rooting ex vitro under intermittent mist creates leaching conditions, there the initial high concentration of auxin decreases with each application of mist. The 79% rooting rate (Table 1) reported in our study can also be taken as a transplant survival rate since ex vitro rooted plantlets do. not require acclimatization prior to transfer to greenhouse conditions.
Acknowledgements Authors thank Ms Tina Lewis (Crop Diversification Centre North, Edmonton, Alberta, Canada) for technical help and data collection. The financial assistance of the Plant Industry Division (Alberta Agriculture Food and Rural Development (AAFRD), Edmonton, Alberta, Canada) is gratefully acknowledged.
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