EFFECTS OF CULTIVAR, EXPLANT TREATMENT, AND MEDIUM SUPPLEMENTS ON CALLUS INDUCTION AND PLANTLET REGENERATION IN PERENNIAL RYEGRASS

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1 International Turfgrass Society Research Journal Volume 9, EFFECTS OF CULTIVAR, EXPLANT TREATMENT, AND MEDIUM SUPPLEMENTS ON CALLUS INDUCTION AND PLANTLET REGENERATION IN PERENNIAL RYEGRASS D. E. Bradley, A. H. Bruneau, and R. Qu* ABSTRACT Perennial ryegrass (Lolium perenne L.) is a widely used cool-season grass species as turf and forage. In order to use biotechnological approaches to improve the species, factors affecting tissue culture responses of mature caryopses of perennial ryegrass were studied. Callus induction and regeneration from mature caryopses of thirteen recentlyreleased turf-type perennial ryegrass cultivars were evaluated, and significant differences were found among the cultivars with Roadrunner having the highest callus induction and regeneration rates. Callus induction rates increased more than five fold when the caryopses were longitudinally sliced before plating. Callus quality was also improved by this treatment resulting in substantially higher callus regeneration rates. Supplement of BAP (6- benzylaminopurine) ( mg L -1 ) in callus culture medium enhanced callus regeneration ability. Copper supplement up to 10 µm in the MS (Murashige and Skoog) medium did not have any effect and 50 µm was toxic. INTRODUCTION Perennial ryegrass (Lolium perenne L.) is a diploid (2n=14), cool-season grass species used in cool temperate climates all over the world as both turf and forage [Torello and Symington, 1984]. As a turf species, perennial ryegrass is most commonly used as a rapidly establishing component of mixtures with slower growing species such as Kentucky bluegrass (Poa pratensis L.), and for winter overseeding of warm-season turfgrasses. Perennial ryegrass is a wind-pollinated, out-crossing species. Conventional breeding efforts in recent years have significantly improved turf-type perennial ryegrass [Mohr et al., 1998]. Biotechnological approaches involving genetic transformation of perennial ryegrass [Spangenberg et al., 1995; Wang et al., 1997; Dalton et al., 1998; Altpeter et al., 2000] can be integrated into conventional breeding efforts to enlarge the germplasm pool and to enhance agronomic traits. Current transformation techniques for grasses depend heavily on development of reliable and repeatable methods for regeneration of fertile plants from tissue culture [Zaghmout and Torello, 1992]. Good quality callus and efficient regeneration of plants in grass species is a prerequisite to the grass transformation techniques. Thus, development of an optimized tissue culture protocol plays an essential role for successfully transforming the species. Department of Crop Science, North Carolina State University, Raleigh, NC *Corresponding author: rongda_qu@ncsu.edu Many factors could affect tissue culture responses of cereals and grasses, particularly formation of embryogenic callus and plant regeneration. These factors include genotype, explant tissue, culture medium and its supplements [Bhaskaran and Smith, 1990]. In perennial ryegrass tissue culture, 2,4-D concentrations in mature caryopsis culture [Torello and Symington, 1984], use of other explant tissues such as immature inflorescence [Dale and Dalton, 1983], meristems [Dalton, 1988] and anthers [Olesen et al., 1988] have been tested. Suspension culture performance of 21 commercial cultivars of perennial ryegrass was evaluated [Olesen et al., 1996]. Several groups reported successful protoplast isolation from suspension cell cultures [Dalton, 1988; Creemers-Molenaar et al., 1989; Zaghmout and Torello, 1992; Wang et al., 1993; Olesen et al., 1995]. Despite these efforts and achievements, some important factors have not been studied intensively in perennial ryegrass tissue culture. For example, tissue culture responses of turf-type cultivars released in the past decade have not been evaluated. These elite cultivars are most likely the ones to be genetically transformed for future improvement. Moreover, effects of medium supplements have not been well studied in perennial ryegrass tissue culture. The objectives of this study were to evaluate tissue culture responses of elite turf-type perennial ryegrass cultivars and to optimize turf-type perennial ryegrass tissue culture conditions for improved callus induction and regeneration. Plant Materials MATERIALS AND METHODS Breeder or foundation stock seeds of thirteen turftype cultivars were tested on their tissue culture responses

2 153 (Table 1). Mature caryopses were stirred in 500 ml L -1 sulfuric acid [Lowe and Conger, 1979] for 30 min for dehusking. The dehusked caryopses were rinsed five times with water, followed by a rinse with 700 ml L -1 ethanol. Caryopses were then surface sterilized in full-length Clorox (52.5 g L -1 sodium hypochlorite) and 1 ml L -1 Tween 20 (Fisher, Pittsburgh, PA, USA), with stirring, for 30 min. Caryopses were rinsed five times with sterile, distilled water before plating. For sliced caryopsis culture, sterilized mature caryopses were sliced longitudinally along the groove into halves aseptically with a scalpel, and cultured with the sliced side in contact with the medium. Culture Conditions The basal culture medium for callus induction and subculture contained MS medium salts and vitamins (M5519, Sigma, St. Louis, MO, USA) supplemented with 2 mg L -1 2,4-D, 30 g L -1 sucrose and 3 g L -1 phytagel. The medium was adjusted to ph 5.8 prior to autoclaving. When preparing medium with supplements, cupric sulfate (CuSO 4 5H2O) was added to the medium prior to ph adjustment, whereas sterile BAP stock solution (1 mg ml -1 ) was added to the autoclaved medium when it cooled down to 50 C. The regeneration medium was an MS basal medium supplemented with 30 g L -1 sucrose, 3 g L -1 phytagel, and 2.5 mg L -1 BAP [Li et al., 1993]. The rooting medium was a half-strength MS basal medium with 30 g L -1 sucrose, 3 g L -1 phytagel, and no plant growth regulator. All the chemicals used in the experiments were purchased from Sigma unless specified otherwise. Callus induction was conducted in a culture chamber (I-36NL, Percival, Boone, IA, USA) in the dark at 25 C for 4 wk. The induced calli were then excised from the explant and subcultured under the same conditions for an additional four wk before the calli were transferred Figure 1. Plantlets regenerating from callus in the regeneration medium (cv. Majesty ). to the regeneration medium. Calli on regeneration medium and plantlets in the rooting medium were maintained in a lighted incubator (CU-32L, Percival) at 25 C under 16 hr photoperiod with light intensity of 140 µmol m -2 s -1. Scoring and statistical analysis Callus induction was scored four wk after plating. An explant with unorganized cell clusters growing at least 1 mm in size was considered callusing. The callus induction rate was calculated as the number of caryopses with induced callus over the total number of explants plated Table 1. Callus induction and regeneration of mature caryopses of thirteen turf-type perennial ryegrass cultivars. Cultivars Seed Sources Callus Induction % Callus Regeneration % Achiever Scott s Company, Marysville, OH 11.3 bcd 6.5 bc Brightstar Turf-Seed Inc., Hubbard, OR 10.7 bcde 47.9 a Caravelle Scott s Company 14.0 abcd 10.2 bc Charger II Turf-Seed Inc abc 15.8 bc Cutter Pickseed West, Tangent, OR 10.3 cde 4.8 c Gator International Seeds, Halsey, OR 6.3 de 12.5 bc Gator II International Seeds 14.0 abcd 34.6 ab Greenland Pickseed West 13.0 abcd 13.9 bc Lowgrow Pickseed West 11.0 bcd 6.7 bc Majesty Scott s Company 19.0 ab 17.5 bc Regal II International Seeds 14.3 abc 11.1 bc Roadrunner Turf-Seed Inc a 58.0 a Sunshine Pickseed West 2.3 e 0.0 c Note: Each value in the table is the average of three replicates. Values sharing the same letter in each column are not significantly different from each other by protected LSD analysis (a=0.05 for callus induction, and a= 0.10 for callus regeneration).

3 154 Table 2. Effects of slicing on callus induction and regeneration of mature caryopses culture of perennial ryegrass. Treatment Callus Induction % Callus Regeneration % Sliced 74.6 a 46.0 a Intact 13.7 b 25.3 b Note: 1. Cultivar: Brightstar and Majesty, 2. Each value in the table is the average of three replicates. Significance of difference was analyzed by protected LSD (a= for callus induction, and a=0.05 for callus regeneration). x 100. Callus regeneration was scored six wk after the calli were transferred to the regeneration medium. The criterion used to determine regeneration was the formation of a distinguishable shoot(s) at least one centimeter in length. Callus regeneration rate was defined as the percentage of callus that had regenerated shoots. For sliced caryopses, callus induction and regeneration rates were scored on a per caryopsis basis. A completely randomized design was used for all experiments. Each experiment was replicated three times with 100 samples in each replicate. Fisher s protected least significant difference (LSD) analysis was used to separate means. RESULTS AND DISCUSSION Evaluation of tissue culture responses for thirteen cultivars Mature caryopses of 13 turf-type cultivars of perennial ryegrass were evaluated for their tissue culture responses in MS basal medium. Germination rates of these cultivars ranged from 78 to 98.5% (data not shown). Callus started appearing 7 to 10 days after plating. After eight wk in culture, callus size was from 4 mm to more than 10 mm. Callus could be divided into two major morphological categories: 1) watery, yellowish to translucent, and friable, and 2) compact, nodular, yellowish to opaque. The translucent watery callus usually grew faster than the compact callus. Some calli had hard, white compact scutellumlike structures, an indicator of somatic embryogenesis [Bradley et al., 2001]. Red splotches formed on the calli about five days after transferring to regeneration medium [Torello and Symington, 1984], and green shoots appeared after 10 to 14 days (Fig. 1). Most regenerated shoots were green while less than 1% were albino or a mixture of albino and green shoots. Calli were induced from all 13 cultivars but the induction rates were low (ranging from 2.3% to 21%), and callus regeneration rates were variable (from 0 to 58%, Table 1). Callus induction (α=0.05) and regeneration rates (α=0.10) were significantly different among the thirteen cultivars. Cultivars Roadrunner, Majesty, Charger II, Regal II, Caravelle, Gator II and Greenland were among the best in callus induction. Roadrunner had the highest callus induction rate (21%), which was significantly better than six other cultivars. Roadrunner also had the highest callus regeneration rate (58%), which was not significantly different from Brightstar (47.8%) and Gator II (34.6%). Effects of caryopsis slicing on culture response To explore ways to improve the callus induction rate from mature caryopses, two cultivars ( Brightstar and Majesty ) from two harvest years were cultured on MS basal medium either as intact caryopses or as two halves sliced longitudinally. With the sliced caryopses, callus were induced from most explants and appeared earlier. Clear watery calli induced from sliced caryopses were first observed four days after plating and off-white or yellowish compact callus became evident approximately three days later. Statistical analysis showed no significant differences between the two cultivars and between the two harvest years. Thus, the data were combined for comparison of intact and sliced caryopses (Table 2). The callus induction rates of the sliced caryopses were 74.6% on a percaryopsis basis, compared to 13.7% for intact caryopses. The difference was highly significant (α=0.0001). The callus quality was also improved by the slicing. The callus regeneration rate of the sliced caryopses (46.0%) was significantly higher (α=0.05) than the intact ones (25.3%). Effect of BAP in callus culture medium Inclusion of low levels of cytokinins, particularly BAP, in callus culture medium improved callus regeneration in several grass species [Zhong et al., 1991; Chaudhury and Qu, 2000]. To improve callus regeneration in perennial ryegrass, effects of BAP as a supplement to the callus culture medium were investigated. Intact mature caryopses of cultivar Majesty were plated on culture medium supplemented with 0, 0.02, 0.1, and 0.5 mg L -1 BAP. A noticeable difference was the increased number of somatic embryos [Bradley et al., 2001] that developed on calli cultured with higher concentrations of BAP in the medium. Data indicated that the callus regeneration rates increased when the BAP concentration in the medium was elevated (Table 3). Calli from medium containing 0.5 mg L -1 BAP Table 3. Effect of supplemented BAP in MS callus culture medium on mature caryopses culture of perennial ryegrass BAP Level (mg L -1 ) Callus Induction % Callus Regeneration % ab 74.1 a b 46.1 ab a 38.8 b b 20.0 b Note: 1. Cultivar: Majesty, 2. Each value in the table is the average of three replicates. Values sharing the same letter in each column are not significantly different from each other by protected LSD analysis (a=0.05).

4 155 Table 4. Effects of copper (CuSO 4 5H 2 O) supplement to MS callus induction medium on mature caryopses culture of perennial ryegrass Copper Supplement (µm) Callus Induction % Callus Regeneration % a 43.0 ab a 46.3 a a 32.6 ab a 0.0 c Note: 1. Cultivar: Majesty ; 2. Each value in the table is the average of three replicates. Values sharing the same letter in each column are not significantly different from each other by protected LSD analysis (a=0.05). had the highest regeneration rate (74.1%). The 0.5 mg L -1 rate was not significantly higher than the 0.1 mg L -1 BAP rate, but it was significantly higher (α=0.05) than media containing 0.02 mg L -1 BAP or with no BAP added. Effects of copper concentration in callus culture medium The original MS medium contained 0.1 mm cupric sulfate [CuSO 4 5H 2 O, Murashige and Skoog, 1962]. Reports in wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) indicated that the optimal copper concentration for these two cereal species was 5 µm [Purnhauser, 1991; Dahleen, 1995]. To determine the appropriate copper level for perennial ryegrass tissue culture, sliced mature caryopses of cv. Majesty were cultured on callus induction medium with no copper supplement or with cupric sulfate supplemented at 5, 10 and 50 µm, respectively. No obvious morphological difference was observed among the calli of the 0, 5 and 10 µm supplements, whereas induced calli on medium supplemented with 50 µm cupric sulfate were much smaller than other treatments, and none of these calli were regenerable (Table 4). The data suggested that the copper level in MS medium was optimal for perennial ryegrass tissue culture. Supplement of copper up to 10 µm did not have much impact on callus induction or regeneration and 50 mm was toxic to the callus. CONCLUSIONS Differences in tissue culture responses were observed among the thirteen turf-type perennial ryegrass cultivars. Roadrunner was the best in both callus induction and regeneration, and could serve as a model cultivar for transformation experiments. Longitudinally slicing caryopses prior to plating profoundly improved the callus induction rate as well as its regeneration ability. Similar results were observed in tall fescue (Festuca arundinacea Schreb.) [Bai and Qu, 2001], and may have general application in grass caryopsis culture. As in several other grass species [Zhong et al., 1991; Griffin and Dibble, 1995; van der Valk et al., 1995; Chaudhury and Qu, 2000; Bai and Qu, 2001], inclusion of a low concentration of BAP in callus culture medium also improved callus quality and regeneration ability in turftype perennial ryegrass culture, and 0.5 mg L -1 was the optimum rate among the tested concentrations. Although the optimal copper level in wheat and barley tissue culture was shown to be 5 µm [Purnhauser, 1991; Dahleen, 1995], the experiment performed with perennial ryegrass did not find significant differences on callus induction and regeneration using 0 to10 µm copper supplements to the MS medium. Copper level in the MS medium was sufficient for perennial ryegrass tissue culture. High concentration of copper (50 µm) was toxic. In conclusion, turf-type perennial ryegrass cultivars with the best callus induction and regeneration rates have been identified. Tissue culture conditions of turftype perennial ryegrass have been optimized. The results will definitely facilitate the transformation efficiency of the turf-type perennial ryegrass. ACKNOWLEDGMENTS The authors would like to thank all the companies and their representatives who provided seeds for this project. Sincere appreciation is extended to Dr. M. L. K. Fraser for her continual support. Special thanks are extended to Dr. C. 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