Abstract
The effects of genotype, basal medium, plant growth regulator (PGR), dark treatment, and antibiotics on shoot regeneration of two Buddleia cultivars, B. davidii ‘Potters Purple’ and Buddleia ‘Lochinch’, were investigated. In vitro shoots were regenerated from leaf tissues in either Murashige and Skoog (MS) or woody plant medium (WPM) media supplemented with benzyladenine (BA). In general, more shoots were regenerated in WPM medium than in MS medium. Dark treatment for 3 to 5 weeks dramatically increased shoot regeneration. Addition of indole-3-butyric acid (IBA) or naphthalene acetic acid (NAA) significantly enhanced the regeneration rate and shoots of each explant. The maximum regeneration rate (100%) of B. davidii ‘Potters Purple’ was achieved when cultured in WPM containing 5 μm BA plus 5 μm IBA. The maximum regeneration rate (98.4%) of Buddleia ‘Lochinch’ was found in WPM supplemented with 20 μm BA plus 4 μm IBA. Carbenicillin at 250 to 500 mg·L−1 and cefotaxime at 125 to 250 mg·L−1, individually or combined, promoted shoot regeneration. Interactions between genotype and medium or PGRs were found. In vitro shoots were easily rooted in half-strength MS medium with or without NAA. Rooted plants were transferred to potting mix and grown in the greenhouse. This research will facilitate genetic improvement and fast propagation of Buddleia species using biotechnology.
The genus Buddleia consists of more than 100 species of which many are woody shrubs providing colorful and fragrant flowers for landscaping and gardening (Dirr, 1998; Hogan, 2003). Native to China, buddleia plants are well adapted to various environmental conditions. Some species are salt-tolerant and others are winter-hardy. Naturally, buddleia plants are seed-propagated. In nursery production, soft wood cutting propagation is the most common method to propagate buddleia species.
Buddleia davidii, known as butterfly bush, is commonly used for landscaping. Butterfly bush was imported to North America from China around the 1900s as a garden shrub. It is a fast-growing deciduous shrub reaching 1 to 5 m tall and is often lanky and wide-spreading. Its heavy flowers and long bloom period (from early summer to late fall) are attractive to gardeners. This species is also characterized as winter-hardy (U.S. Department of Agriculture zone 5) and tolerant of a high soil pH and can be grown in most states of the United States (Dirr, 1998; Hogan, 2003). However, this species has a few concerns such as lack of flower color, excessive growth, and virus (Podaras, 2005). Recently, invasiveness of this species has become an issue because Buddleia davidii is a prolific seed producer and its seeds can be dispersed by wind and water and may remain dormant in the soil for many years (Sheppard et al., 2006).
A colorful, compact, and sterile Buddleia davidii cultivar would be highly desirable for landscaping purposes. Attempts to improve Buddleia davidii using conventional breeding methods have resulted in limited success (Tobutt, 1993). Different ploidy levels of species in the genus Buddleia may impede the interspecific crosses; therefore, elite traits cannot be efficiently integrated into new plants (Tobutt, 1993). In vitro mutation and gene transformation are being explored (Rose et al., 2000). The objective of this study was to determine factors influencing regeneration of two Buddleia cultivars, B. davidii ‘Potters Purple’ and Buddleia ‘Lochinch’, from in vitro leaf tissues, which will facilitate development of a regeneration protocol for Buddleia species.
Materials and Methods
Plant materials
Two cultivars, Buddleia davidii ‘Potters Purple’ and Buddleia ‘Lochinch’, were used in this study. In vitro cultures were provided by Peter Podaras at Cornell University and subcultured every 4 weeks in Murashige and Skoog (MS) medium (Murashige and Skoog, 1962) supplemented with 2.67 μm benzyladenine (BA), 2% sucrose, and 0.35% phytagel (Sigma Chemical Co., St. Louis) and adjusted to pH 5.8 before autoclaving. These cultures were maintained at 25 °C under cool-white light at ≈36.4 μmol·m−2·s−1 with a 16-h photoperiod.
Plant regeneration
Experiments were conducted to determine factors affecting plant regeneration of Buddleia. Unless otherwise stated, all regeneration media are woody plant medium (WPM) (Lloyd and McCown, 1980) supplemented with MS vitamins, 2.0% sucrose, and 0.65% agar (#0140-01-0; Difco Co., Detroit, MI), and pH was adjusted to 5.2 before autoclaving. In vitro leaves were cut into two or three segments across the main vein and placed in a petri plate (100 mm × 15 mm) containing 25 mL medium. Each treatment had three plates (samples) with 10 leaf segments per plate. In vitro cultures were subcultured in the fresh medium every 4 weeks. Experiments were repeated two or three times.
Effects of basal medium, benzyladenine, and dark treatment on shoot regeneration.
Two basal media (MS and WPM) and six concentrations of BA (0, 2.5, 5, 10, 20, and 40 μM) under 0 or 4 weeks dark treatment were evaluated for their effects on shoot regeneration. After being in the dark for 0 or 4 weeks, plates were moved to the 16-h light/8-h dark photoperiod condition with a light intensity of 36.4 μmol·m−2·s−1. This experiment was conducted as a completely randomized design (CRD) consisting of two replications of a factorial arrangement with a 2 × 6 × 2 of basal medium, BA concentration, and weeks of dark treatment, respectively.
Effects of auxin on shoot regeneration.
Two auxins, indole-3-butyric acid (IBA) and naphthalene acetic acid (NAA), at four concentrations (0, 0.5, 1, and 5 μm for ‘Potters Purple’ or 0, 2, 4, and 20 μm for ‘Lochinch’) each, were added into the regeneration medium (5 μm BA WPM medium for ‘Potters Purple’ and 40 μm BA WPM medium for ‘Lochinch’). All cultures were maintained in the dark for the first 4 weeks and then moved to the 16-h photoperiod condition for shoot regeneration. The experiment was conducted as a CRD consisting of two replications of a 2 × 4 factorial arrangement of cultivar and auxin, respectively.
Effects of antibiotics on shoot regeneration.
Carbenicillin (Cb) (Caisson Laboratory, North Logan, UT) at 250 or 500 mg·L−1 and cefotaxime (Cf) (Caisson Laboratory) at 125 or 250 mg·L−1, individually or combined, was added to the regeneration medium. All cultures were maintained in the dark for 4 weeks and then moved to the 16-h photoperiod condition for shoot regeneration. This experiment was also designed as a CRD consisting of two replications of a 2 × 7 factorial arrangement of cultivar and antibiotics, respectively.
The regeneration rate was calculated as the percentage of leaf explants forming shoots after 8 weeks culture (two subcultures) for all experiments. The number of shoot regenerated from each leaf explant was counted in Expt. 2 (auxin effect). In other experiments, the shoot number was only estimated.
Rooting and acclimatization
Regenerated shoots were proliferated in MS medium with 2.67 μm BA, 2% sucrose, and 0.35% phytagel. In vitro shoots (greater than 1.5 cm) were rooted in half-strength MS medium containing 0.65% agar with four different auxin treatments (0, 0.1, 1, and 10 μm NAA) under regular tissue culture conditions (described in “Plant Materials”). Rooting percentage and root number were recorded after 4 weeks of root development. Each treatment had 27 cuttings and was replicated twice. Rooted plants were then transferred to a flat filled with Jiffy Mix (Jiffy Mix, Shippagan, Canada) and covered with a clear plastic top for 1 week. The cover was gradually removed during the next 1-week period. Survival plants were potted into Sunshine Mix #1 (Fisons Western Corp., Vancouver, Canada) and grown in the greenhouse.
Statistical analysis
Data from all experiments were subject to analysis of variation and mean comparison using the GLM procedure of SAS software Version 9.1 (SAS Institute, 2004).
Results and Discussion
Effects of basal medium, benzyladenine, dark treatment, and genotype on shoot regeneration.
Basic medium significantly affected shoot regeneration (Table 1). In vitro shoots were regenerated from both cultivars when leaf explants were cultured in either MS or WPM media containing BA (Fig. 1A). However, more shoots were regenerated from leaf explants in WPM medium than in MS medium. Addition of cytokinin (BA) was necessary for shoot regeneration of both cultivars. No shoots were produced and the original leaf explants were dead after 6 to 8 weeks culture if no BA was present in the medium. Significant interaction was found between cultivar and BA concentration. However, interactions between cultivar and other factors such as medium and dark treatment were not significant. Although interactions between dark treatment and BA or cultivar × BA were significant, no significant interactions were determined between dark treatment and other factors. Overall, an average of 20.6% leaf explants produced shoots in BA-containing medium after dark treatment for 4 weeks, whereas only 7.1% of leaf explants regenerated shoots without dark treatment. In vitro regeneration also showed genotype-dependence. With a 4-week dark treatment, an average of 18.6% and 25.3% of leaf explants of ‘Potters Purple’ regenerated shoots in MS and WPM medium, both containing 2.5 to 40 μm BA, whereas 23.0% and 32.0% of leaf explants of ‘Lochinch’ produced shoots in the same medium. Maximum regeneration rate for ‘Potter Purple’ and ‘Lochinch’ were 53.3% and 48.4%, respectively, which was obtained from 5 μm WPM medium for ‘Potters Purple’ and 40 μm BA medium for ‘Lochinch’. It was also found that the bigger the leaf explant, the more the shoots developed within the same species (estimated data, not shown).
Effects of basal medium, dark treatment, and benzyladenine (BA) on shoot regeneration of two Buddleia cultivars.
(A) In vitro shoots were induced from leaf explants in woody plant medium (WPM) containing 5 μm benzyladenine and 1 μm nmaphthalene acetic acid. (B) Roots were induced from microcuttings in half-strength Murashige and Skoog medium with or without auxin; (C) acclimated plants were potted and grown in the greenhouse.
Citation: HortScience horts 42, 7; 10.21273/HORTSCI.42.7.1670
The large F value of dark treatment in this study indicated that darkness was one of the most important factors determining shoot regeneration of buddleia species. Limited and controversial information is available regarding the effect of dark treatment on plant regeneration. Although there are reports that dark treatment significantly increased plant regeneration of apple, purslane, persimmon, castors, and so on (Ahn et al., 2007; Choi et al., 2001; Fasolo et al., 1989; Fiore et al., 1997; Gu and Zhang, 2005; Rossi-Hassani and Zryd, 1995), other researches showed no stimulatory effect of dark treatment on plant regeneration of Rubus and Juncus accuminatus (Fiola et al., 1990; Sarma and Rogers, 1998). Our other research results on several species, including aspen, birch, chokecherry, periwinkle, and viburnum, showed that the effect of dark was not only species- but also genotype-dependent (unpublished data). Little is known about why a dark treatment influences in vitro regeneration. It is known that indoleacetic acid (IAA) can be degraded by light. Thus, the inclusion of a dark condition may help keep the optimal ratio of endogenous auxin to cytokinin in in vitro tissues for callus/shoot regeneration. Dark treatment (etiolating plant tissues) may also speed up the process of organogenesis by stimulating dedifferentiation of plant cells (Herman and Hess, 1963). Shortening the photoperiod, decreasing light intensity, and dark treatment will result in thinner cell walls and lower cell wall deposits, facilitating penetration of plant growth regulators into cells (Herman and Hess, 1963).
Although shoots were regenerated in both MS and WPM media, high ammonia and other salts in the MS medium may inhibit shoot initiation. A low ammonium medium has performed better than high ammonium media for Prunus tissue culture (Dai et al., 2004; Perez-Tornero and Burgos, 2000). In addition, higher shoot regeneration rates with the WPM medium in this study indicated that buddleia may be sensitive to high ammonia and other salts during the in vitro regeneration. Thus, determination of medium type should always be one of the first important steps in establishing a regeneration system for different species and genotypes.
Effect of auxin on shoot regeneration.
In vitro shoot regeneration of Buddleia was enhanced by the addition of auxin into the regeneration medium (Table 2). Addition of IBA at 0.5 to 10 μm or NAA at 0.5 to 1 μm and IBA at 2 to 4 μm or NAA at 2 μm significantly increased regeneration rate for ‘Potters Purple’ and ‘Lochinch’, respectively. High concentration of auxin (except IBA for ‘Potters Purple’), especially NAA, showed significant inhibition of shoot regeneration. The number of shoots regenerated from each leaf explant was significantly affected by auxin type (Table 2). Significant interactions between cultivar and auxin were found in the regeneration rate rather than shoots per explant, indicating that the number of shoots regenerated per explant was largely dependent on the genotype and auxin type. An average of 8.7 shoots per responding explant was developed when IBA was added, whereas only 3.5 shoots were produced when NAA was added. More shoots were regenerated from ‘Potters Purple’ than ‘Lochinch’.
Effects of auxin on shoot regeneration of two Buddleia cultivars.z
In general, the direction of organogenesis of plants in tissue culture is largely determined by the ratio of auxin to cytokinin provided. Endogenous hormones in plants play an important role in balancing the ratio to a certain level for either shoot or root formation. In some species, the ratio of auxin to cytokinin in in vitro tissues needs to be adjusted by providing exogenous cytokinin or auxin to the culture medium. Auxin (NAA and IBA) promotes shoot regeneration for a few plant species such as Dianthus chinensis (Kantia and Kothari, 2002), Rhodiola spp. (Dimitrov et al., 2003; Liu, et al., 2006), and Curcuma amada (Prakash et al., 2004). However, addition of auxin may cause excessive formation of callus tissues, causing somaclonal variation of in vitro shoots.
Effects of carbenicillin and cefotaxime on shoot regeneration.
No significant interaction was found between cultivar and antibiotics. Shoot regeneration was increased when Cb was added into regeneration medium alone or combined with Cf (Table 3). Addition of Cf by itself did not affect the regeneration rate. Higher concentration (500 mg·L−1) of Cb gave rise to more shoots regenerated from each explant (estimated data, not shown).
Effects of carbenicillin and cefotaxime on shoot regeneration of two Buddleia cultivars.z
Carbenicillin and Cf, two most common antibiotics used to eliminate Agrobacterium in plant transformation, have been reported to be effective on callus formation and plant regeneration in several species with a controversial result. Cefotaxime had a stimulatory effect on plant regeneration of Maize (Danilova and Dolgikh, 2004), apple (Yepes and Aldwinckle, 1994), and durum wheat (Borrelli et al., 1992) and inhibitory or no effect on plant regeneration of common snapdragon (Holford and Newbury, 1992), tobacco (Nauerby et al., 1997), and poplar (Ding et al., 2006). Carbenicillin, on the other hand, has shown opposite effects. For example, carbenicillin inhibited regeneration of apple but enhanced callus growth of tobacco and apple (Yepes and Aldwinckle, 1994; Nauerby et al., 1997), indicating that the effect of Cb and Cf on in vitro culture is species-dependent. The synergistic effect of Cb on shoot regeneration of buddleia in this study suggests that Cb could be broken down to release phenylacetic acid or phenylmalonic acid that have auxin activities in the tissue culture (Holford and Newbury, 1992).
Rooting, acclimatization, and greenhouse performance.
In vitro shoots of both cultivars were easy to root in auxin-free medium (Table 4; Fig. 1B). However, application of a low concentration of NAA (0.1 to 1.0 μm) increased the root number (data not shown). In contrast, high NAA (10 μm) significantly lowered the rooting percentage of in vitro cuttings. More than 95% of rooted plants survived after they were potted and grown in the greenhouse (Fig. 1C).
Effects of auxin on in vitro rooting of two Buddleia cultivars.z
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