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					International Turfgrass Society                                                                                          152
Research Journal Volume 9, 2001.


                                   D. E. Bradley, A. H. Bruneau, and R. Qu*


       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 recently-
released 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 in-
creased 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) (0.1- 0.5 mg L-1) in callus culture medium enhanced callus regeneration ability. Copper supple-
ment up to 10 µM in the MS (Murashige and Skoog) medium did not have any effect and 50 µM was toxic.

                                                                 genic callus and plant regeneration. These factors in-
                                                                 clude genotype, explant tissue, culture medium and its
                                                                 supplements [Bhaskaran and Smith, 1990]. In perennial
          Perennial ryegrass (Lolium perenne L.) is a dip-
                                                                 ryegrass tissue culture, 2,4-D concentrations in mature
loid (2n=14), cool-season grass species used in cool tem-
                                                                 caryopsis culture [Torello and Symington, 1984], use of
perate climates all over the world as both turf and forage
                                                                 other explant tissues such as immature inflorescence [Dale
[Torello and Symington, 1984]. As a turf species, peren-
                                                                 and Dalton, 1983], meristems [Dalton, 1988] and anthers
nial ryegrass is most commonly used as a rapidly estab-
                                                                 [Olesen et al., 1988] have been tested. Suspension culture
lishing component of mixtures with slower growing spe-
                                                                 performance of 21 commercial cultivars of perennial
cies such as Kentucky bluegrass (Poa pratensis L.), and for
                                                                 ryegrass was evaluated [Olesen et al., 1996]. Several groups
winter overseeding of warm-season turfgrasses. Peren-
                                                                 reported successful protoplast isolation from suspension
nial ryegrass is a wind-pollinated, out-crossing species.
                                                                 cell cultures [Dalton, 1988; Creemers-Molenaar et al., 1989;
Conventional breeding efforts in recent years have sig-
                                                                 Zaghmout and Torello, 1992; Wang et al., 1993; Olesen et
nificantly improved turf-type perennial ryegrass [Mohr et
                                                                 al., 1995].
al., 1998].
                                                                          Despite these efforts and achievements, some
         Biotechnological approaches involving genetic
                                                                 important factors have not been studied intensively in
transformation of perennial ryegrass [Spangenberg et al.,
                                                                 perennial ryegrass tissue culture. For example, tissue cul-
1995; Wang et al., 1997; Dalton et al., 1998; Altpeter et al.,
                                                                 ture responses of turf-type cultivars released in the past
2000] can be integrated into conventional breeding efforts
                                                                 decade have not been evaluated. These elite cultivars are
to enlarge the germplasm pool and to enhance agronomic
                                                                 most likely the ones to be genetically transformed for fu-
traits. Current transformation techniques for grasses de-
                                                                 ture improvement. Moreover, effects of medium supple-
pend heavily on development of reliable and repeatable
                                                                 ments have not been well studied in perennial ryegrass
methods for regeneration of fertile plants from tissue cul-
                                                                 tissue culture. The objectives of this study were to evalu-
ture [Zaghmout and Torello, 1992]. Good quality callus
                                                                 ate tissue culture responses of elite turf-type perennial
and efficient regeneration of plants in grass species is a
                                                                 ryegrass cultivars and to optimize turf-type perennial
prerequisite to the grass transformation techniques. Thus,
                                                                 ryegrass tissue culture conditions for improved callus in-
development of an optimized tissue culture protocol plays
                                                                 duction and regeneration.
an essential role for successfully transforming the species.
                                                                            MATERIALS AND METHODS
         Many factors could affect tissue culture responses
of cereals and grasses, particularly formation of embryo-
                                                                 Plant Materials
Department of Crop Science, North Carolina State University,
Raleigh, NC 27695-7620. *Corresponding author:                            Breeder or foundation stock seeds of thirteen turf-
rongda_qu@ncsu.edu                                               type cultivars were tested on their tissue culture responses

(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      Figure 1. Plantlets regenerating from callus in the regeneration
preparing medium with supplements, cupric sulfate             medium (cv. ‘Majesty’).
(CuSO4·5H2O) was added to the medium prior to pH ad-
justment, 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 me-          to the regeneration medium. Calli on regeneration me-
dium supplemented with 30 g L-1 sucrose, 3 g L -1 phytagel,   dium and plantlets in the rooting medium were main-
and 2.5 mg L-1 BAP [Li et al., 1993]. The rooting medium      tained in a lighted incubator (CU-32L, Percival) at 25°C
was a half-strength MS basal medium with 30 g L-1 su-         under 16 hr photoperiod with light intensity of 140 µmol
crose, 3 g L-1 phytagel, and no plant growth regulator. All   m-2 s-1.
the chemicals used in the experiments were purchased
from Sigma unless specified otherwise.                        Scoring and statistical analysis

         Callus induction was conducted in a culture                   Callus induction was scored four wk after plat-
chamber (I-36NL, Percival, Boone, IA, USA) in the dark        ing. An explant with unorganized cell clusters growing at
at 25°C for 4 wk. The induced calli were then excised from    least 1 mm in size was considered ‘callusing’. The callus
the explant and subcultured under the same conditions         induction rate was calculated as the number of caryopses
for an additional four wk before the calli were transferred   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.                                       15.0 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.                                       21.0 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 (α=0.05 for callus induction, and α= 0.10 for
callus regeneration).

 Table 2. Effects of slicing on callus induction and regeneration      highest callus induction rate (21%), which was significantly
 of mature caryopses culture of perennial ryegrass.                    better than six other cultivars. ‘Roadrunner’ also had the
 Treatment      Callus Induction %       Callus Regeneration %         highest callus regeneration rate (58%), which was not sig-
 Sliced                74.6 a                    46.0 a                nificantly different from ‘Brightstar’ (47.8%) and ‘Gator
 Intact                13.7 b                    25.3 b
                                                                       II’ (34.6%).
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 (α=0.0001 for callus induction,          Effects of caryopsis slicing on culture response
and α=0.05 for callus regeneration).
                                                                                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
x 100. Callus regeneration was scored six wk after the calli           basal medium either as intact caryopses or as two halves
were transferred to the regeneration medium. The crite-                sliced longitudinally.
rion used to determine regeneration was the formation of
a distinguishable shoot(s) at least one centimeter in length.                   With the sliced caryopses, callus were induced
Callus regeneration rate was defined as the percentage of              from most explants and appeared earlier. Clear watery
callus that had regenerated shoots. For sliced caryopses,              calli induced from sliced caryopses were first observed
callus induction and regeneration rates were scored on a               four days after plating and off-white or yellowish compact
per caryopsis basis.                                                   callus became evident approximately three days later.
          A completely randomized design was used for all                        Statistical analysis showed no significant differ-
experiments. Each experiment was replicated three times                ences between the two cultivars and between the two har-
with 100 samples in each replicate. Fisher’s protected                 vest years. Thus, the data were combined for comparison
least significant difference (LSD) analysis was used to sepa-          of intact and sliced caryopses (Table 2). The callus induc-
rate means.                                                            tion rates of the sliced caryopses were 74.6% on a per-
                                                                       caryopsis basis, compared to 13.7% for intact caryopses.
              RESULTS AND DISCUSSION                                   The difference was highly significant (a=0.0001). The
                                                                       callus quality was also improved by the slicing. The callus
Evaluation of tissue culture responses for thirteen                    regeneration rate of the sliced caryopses (46.0%) was sig-
cultivars                                                              nificantly higher (a=0.05) than the intact ones (25.3%).
          Mature caryopses of 13 turf-type cultivars of pe-            Effect of BAP in callus culture medium
rennial ryegrass were evaluated for their tissue culture
responses in MS basal medium. Germination rates of these                         Inclusion of low levels of cytokinins, particularly
cultivars ranged from 78 to 98.5% (data not shown). Callus                  ,
                                                                       BAP in callus culture medium improved callus regenera-
started appearing 7 to 10 days after plating. After eight wk           tion in several grass species [Zhong et al., 1991; Chaudhury
in culture, callus size was from 4 mm to more than 10 mm.              and Qu, 2000]. To improve callus regeneration in peren-
Callus could be divided into two major morphological                   nial ryegrass, effects of BAP as a supplement to the callus
categories: 1) watery, yellowish to translucent, and friable,          culture medium were investigated. Intact mature cary-
and 2) compact, nodular, yellowish to opaque. The trans-               opses of cultivar ‘Majesty’ were plated on culture medium
lucent watery callus usually grew faster than the compact              supplemented with 0, 0.02, 0.1, and 0.5 mg L-1 BAP A     .
callus. Some calli had hard, white compact scutellum-                  noticeable difference was the increased number of somatic
like structures, an indicator of somatic embryogenesis [Bra-           embryos [Bradley et al., 2001] that developed on calli cul-
dley et al., 2001].                                                    tured with higher concentrations of BAP in the medium.
                                                                       Data indicated that the callus regeneration rates increased
         Red splotches formed on the calli about five days             when the BAP concentration in the medium was elevated
after transferring to regeneration medium [Torello and                 (Table 3). Calli from medium containing 0.5 mg L-1 BAP
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.                                                                Table 3. Effect of supplemented BAP in MS callus culture
                                                                       medium on mature caryopses culture of perennial ryegrass

         Calli were induced from all 13 cultivars but the              BAP Level (mg L-1) Callus Induction % Callus Regeneration %
induction rates were low (ranging from 2.3% to 21%), and               0.5                       12.8 ab                74.1 a
callus regeneration rates were variable (from 0 to 58%,                0.1                       10.5 b                 46.1 ab
                                                                       0.02                      15.1 a                 38.8 b
Table 1). Callus induction (a=0.05) and regeneration rates
                                                                       0                         11.8 b                 20.0 b
(a=0.10) were significantly different among the thirteen
                                                                       Note: 1. Cultivar: ‘Majesty’, 2. Each value in the table is the
cultivars. Cultivars ‘Roadrunner’, ‘Majesty’, ‘Charger II’,            average of three replicates. Values sharing the same letter in
‘Regal II’, ‘Caravelle’, ‘Gator II’ and ‘Greenland’ were               each column are not significantly different from each other by
among the best in callus induction. ‘Roadrunner’ had the                                         α
                                                                       protected LSD analysis (α=0.05).

Table 4. Effects of copper (CuSO4·5H2O) supplement to MS          improved callus quality and regeneration ability in turf-
callus induction medium on mature caryopses culture of            type perennial ryegrass culture, and 0.5 mg L-1 was the
perennial ryegrass                                                optimum rate among the tested concentrations.
Copper                     Callus                Callus
Supplement (µM)         Induction %         Regeneration %
                                                                           Although the optimal copper level in wheat and
0                           39.0 a              43.0 ab
5                           36.0 a              46.3 a
                                                                  barley tissue culture was shown to be 5 µM [Purnhauser,
10                          37.6 a              32.6 ab           1991; Dahleen, 1995], the experiment performed with pe-
50                          28.1 a               0.0 c            rennial ryegrass did not find significant differences on
Note: 1. Cultivar: ‘Majesty’; 2. Each value in the table is the   callus induction and regeneration using 0 to10 µM copper
average of three replicates. Values sharing the same letter in    supplements to the MS medium. Copper level in the MS
each column are not significantly different from each other by    medium was sufficient for perennial ryegrass tissue cul-
protected LSD analysis (α=0.05).                                  ture. High concentration of copper (50 µM) was toxic.

                                                                           In conclusion, turf-type perennial ryegrass culti-
had the highest regeneration rate (74.1%). The 0.5 mg L      -1   vars with the best callus induction and regeneration rates
rate was not significantly higher than the 0.1 mg L-1 BAP         have been identified. Tissue culture conditions of turf-
rate, but it was significantly higher (α=0.05) than media         type perennial ryegrass have been optimized. The results
containing 0.02 mg L -1 BAP or with no BAP added.                 will definitely facilitate the transformation efficiency of
                                                                  the turf-type perennial ryegrass.
Effects of copper concentration in callus culture
medium                                                                           ACKNOWLEDGMENTS

          The original MS medium contained 0.1 mM cu-                      The authors would like to thank all the compa-
pric sulfate [CuSO4·5H2O, Murashige and Skoog, 1962].             nies and their representatives who provided seeds for this
Reports in wheat (Triticum aestivum L.) and barley (Hor-          project. Sincere appreciation is extended to Dr. M. L. K.
deum vulgare L.) indicated that the optimal copper con-           Fraser for her continual support. Special thanks are ex-
centration for these two cereal species was 5 µM                  tended to Dr. C. Brownie for her assistance in statistical
[Purnhauser, 1991; Dahleen, 1995]. To determine the ap-           analysis. We are grateful to Dr. L. Li for critical reading
propriate copper level for perennial ryegrass tissue cul-         of the manuscript. This work was supported by grants
ture, sliced mature caryopses of cv. ‘Majesty’ were cul-          from the Turfgrass Council of North Carolina to R. Q.
tured on callus induction medium with no copper supple-           and A. B.
ment or with cupric sulfate supplemented at 5, 10 and 50
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