Plant Cell Reports
Plant Cell Reports (1996) 15: 737- 741
9 Springer-Verlag 1996
Plant regeneration from protoplasts of Japanese lawngrass Chie Inokuma, Kiyoyuki Sugiura, Chol Cho, Ryoji Okawara, and Seiji Kaneko Japan Turfgrass Inc., 3-6-2 Akanehama, Na'rashino-shi, Chiba, 275 Japan Received 5 August 1995/Revised version received I0 September 1995 - Communicated by K. Shimamoto
the plant regeneration was successful from isolated protoplasts
ABSTRACT
(wheat ; Harris et al. 1988, Hayashi and Shimamoto 1988, maize Embryogenic callus of Japanese lawngrass (Zoysia japonica Steud.) was induced from sterile mature seeds on LS medium with 5 mg / 1 of 2,4-D. Embryogenic callus selected visually
; Rhodes et al. 1988, rice ; Abdullah et al. 1986, Yamada et al. 1986, Kyozuka et al. 1987 etc.). As for turfgrass species,
under microscope was proliferated in liquid N6 medium with
recently the plant regeneration from isolated protoplasts has been successful in several species of cool-season turfgrasses
amino acids (N6-AA medium). Protoplasts were isolated from
(tall fescue, perennial ryegrass and Italian ryegrass ; Dalton
suspension cells by the treatment of enzyme mixture containing
1988, tall fescue ; Takamizo et al. 1990, kentucky bluegrass ;
pectolyase Y-23 and cultured in K8p medium with 2 mg / 1 of
Valk et al. 1988, redtop ; Asano and Sugiura 1990, creeping
2,4-D at the density of 106 / ml. Plants were regenerated by
bentgrass ; Terakawa et al. 1992, meadow fescue ; Wang et al.
transferring the protoplasts derived callus to MS medium and
1993 etc.). Also, transgenic plants of turfgrasses have been
incubating at 28 ~ under light for two months. Plantlets were
obtained by a microprojectile bombardment of embryogenic
successfully transplanted in the soil.
callus of creeping bentgrass (Zhong et al. 1993) and a electroporation (Sam-Bong Ha et al. 1992) or polyethylene glycol treatment (Zeng-yu Wang et al. 1992) of protoplasts
Key Words:
Zoysia japonica, embryogenic callus, protoplasts, plant
isolated from embryogenic callus of tall fescue.
regeneration About Japanese lawngrass, although plant regeneration from embryo derived callus has been reported (Asano, 1989 ; AI-
Abbreviations. 2,4-D ; 2,4-dichrolophenoxyacetic acid
Khayri et al., 1989), the plant regeneration from isolated
MES ; 2-(N-Morpholino) ethanesulfonic acid
protoplasts has not been successful (Asano, 1989). It is necessary for the establishment of plant regeneration system from protoplasts to obtain an embryogenic suspension cell line
INTRODUCTION
and to isolate a high yield of protoplasts from suspension cells.
Japanese lawngrass (Zoysia japonica Steud.), which is the most popular warm-season turfgrass in Japan, has several attractive
Amino Acids (AA) medium, which was efficient to obtain fine suspension cultures of rice (Toriyama and Hinata, 1985 ;
characteristics for a turf, such as a traffic tolerance and low
Abdullah et al, 1986) and redtop (Asano and Sugiura, 1990),
water and fertilizer requirements.
was applied to obtain suspension cells of Japanese lawngrass in this report. We report here induction of embryogenic callus,
However, further genetic
improvement on disease and insect tolerances is still desired. Plant regeneration system from isolated protoplasts is necessary for applying genetic manipulation like cell fusion and protoplast transformation techniques to Japanese lawngrass improvements. In gramineae species, there have been several reports in which
Correspondence to: C. Inokuma
isolation of protoplasts and regeneration of plants by using regenerable embryo derived Zoysia japonica cell suspension.
MATERIALS
AND METHODS
738 Commercially available seeds of Japanese lawngrass were soaked in 50% H2SO4 for 30 min. and rinsed in tap water.
densities of the proloplasts were changed from lxl05 / ml to 2x106 / ml.
Seeds were surface sterilized with 70% ethanol and 2% sodium hypochrolite for 3 and 20 rain., respectively. After rinsing with
The colonies derived from the protoplasts was proliferated in
sterile water, seeds were placed on plastic petridishes (90 mm
MS liquid medium (pH 5.8) supplemented with 1 m g / 1 2,4-D
diameter) containing 20 ml of medium solidified with 0.7%
and 60 g / 1 sucrose (Kyozuka et al., 1987) for 2 weeks and then
agarose (Type I, Sigma Chemical Co., ST. Luis, MO, USA).
transferred onto the MS regeneration medium without 2,4-D
For the initiation of callus, MS (Murashige and Skoog, 1962),
(0.7% agarose, type I, Sigma Chemical Co., ST. Luis, MO,
N6 (Chu et al., 1975), or LS (Linsmaier and Skoog, 1965) media
USA, pH 5.8). Regenerated plants were transferred to soil after
were used. Modified LS medium about thiamine (0, 0.1, 0.4,
two months incubation at 28 ~ under fluorescent light (40
and 1.0 mg / 1) were also used to clarify the effect of thiamine
l.tEm-2s-1, 12 h photoperiod) and grown in glasshouse.
on the developement of embryogenic callus. All media were supplemented with 2,4-dichrolophenoxyacetic acid (2,4-D) at 5 nag / 1 and sucrose at 30 g / 1 and adjusted to pH 5.8 prior to
RESULTS AND DISCUSSION
autoclaving at 121~ for 15 rain. Seeds were incubated at 28 ~ in the dark for 1 month. Calli were maintained by subculturing
Embryogenic callus was induced most efficiently when LS
on the same medium monthly.
medium was used as an induction medium and subcultured on the same medium (Table 1). When subeultured on LS medium
Embryogenic callus developed on LS medium with 2,4-D at 5
for 2 months, 13% of the calli had embryogenic features
rug / 1 was collected under microscope and cultured in N6-AA
(i.g.white to yellow in color, compact, smooth surface (Asano,
medium which consists of N6 medium supplemented with
1989)) [Fig. I-A]. Lower induction rates for the embryogenic
amino acids (876 mg / 1 L-glutamic acid, 266 nag / 1 L-aspartic
callus were observed in N6 (2.3%) and MS (2,0%) media (Table
acid, 174 mg ] 1 L-arginin, and 7.5 m g / I glycine), 3 mg ] 12,4-
1).
D, and 30 g / 1 sucrose and adjusted to pH 5.8 before autoclaving at 121 ~ for 15 rain. Suspension cultures were
Subtraction of thiamine from LS medium resulted in no
initiated in a small volume of N6-AA medium (10 to 20 ml) and
formation of embryogenic callus, suggesting the necessity of
finally subcultured by transferring 10 ml of suspension to 30 ml
thiamine for embryogenic callus induction (Table 2). Twenty
of fresh N6-AA medium in 200 ml conical beaker sealed by two
suspension cultures were established by incubating embryogenic
layers of aluminum foil. The culture was maintained on the
callus originated from individual seeds in N6-AA medium. One
rotary shaker (120 rpm) at 28 ~ in the dark. Suspension
suspension culture, which had the ability to regenerate plants
cultures were tested for the plant regeneration ability and the
(data not shown) and fine texture suitable for the isolation of
cell line Z2a was selected for the further studies.
protoplasts, was selected and named Z2a (Fig. I-B, l-C). This suspension culture was maintained as described in materials and
One gram of cells were collected from the suspension line Z2a
methods for the isolation of protoplasts 40 to 50 weeks after the
and incubated with the enzyme mixture consisting of 3 mM CaCI2, 0.7 mM NaH2PO4, 3 mM MES (2-(N-morpholino)
induction.
ethansulfonic acid, monohydrate), 0.5 M mannitol, 2% cellulase "Onozuka"RS (Yakulto Co. Ltd.) and varied combinations of
Table 1. Effect of media on the induction of embryogenic callus
macerozyme R-10 (Yakulto Co. Ltd.),
from mature seeds, Percentage of callus and embryogenic callus
or pectolyase Y-23
(Seishin Co. Ltd.) and adjusted to pH 5.6.
In Table 3, the
concentration of three enzymes is fixed. After 4 h's incubation on a rotary shaker (40 rpm) at 28~ in the dark, protoplasts were filtered through 20 gm nylon mesh and collected by
calculated on seed and callus basis respectively, and shown in parentheses. . . . .
centrifugation at 800 rpm for 10 rain. Protoplasts were washed
media MS
N6
LS
No. of seeds
80
80
80
No. of callus
51 (63.8)
44 (55.0)
54 (67.5)
I (2.0)
1 (2.3)
7 (13.0)
twice with 20 ml of KMC solution (Harms and Potrykus, 1978) and resuspended in the K8P medium (Kao, 1977) with or without 2 m g / I 2,4-D. The effect of the addition of t.25% agarose (Sea Plaque agarose, FMC Co., Rockland, ME, USA) on the plating efficiency was also examined. P~aling efficiency was calculated as the number of formed microcalli (>lmm) from plated protoplasts afler two weeks incubation. The
No. ofembryogenic callus
739 Table 2. Effect of LS medium with varied concentration of thiamin-HCl on the induction of embryogenic callus from mature seeds. Percentage of callus and embryogenic callus calculated on seed and callus basis respectively, and shown in parentheses. thiamin-HCl (mg/1)
No. of seeds No. of callus No. of embryogenic callus
0
0.1
0.4
1.0
160
160
160
160
80 (50)
90 (56.3)
115 (71.9)
94 (58.8)
0 (0)
14 (15.6)
22 ( 19.1 )
15 (16.0)
Fig. 1. Establishment of embryogenic suspension culture of Japanese lawngrass. A Close-up of an embryogenic callus initiated from seeds in LS medium. Bar = 5 mm.
B Suspension culture established from embryogenic callus (Z2a). Bar = 10 mm.
C Plant
regenerated from suspension cells (Z2a). Bar = 10 mm
Table 3. Effect of enzyme solution on the yield of protoplasts
yield of 2-6 x 107 / g fresh weight. The isolated protoplasts
from Z2a suspnsion culture
were shown in Fig. 2-B. The protoplasts were collected after washing and transferred to the culture medium with varied cell
composition of enzyme solution (%) macerozyme R-10 2
2
pectolyase Y-23
protoplasts yield *
densities as described in Table 4 .
(x 107 / g. fresh weight)
0.1
4.2•
Table 4.
0.1
2.7•
formation of Z2a protoplasts
Effect of plating density and media on colony
0.6•
* Protoplast yield shows mean of three replications with standard deviation.
colony formation * protoplast density (xl05 / ml )
liquid
solidified
Three kinds of enzyme mixtures were applied for the isolation
l
- -
- -
of protoplasts from Z2a suspension culture (Fig.2-A). One gram
5
++
++
of cells were collected 3-5 days after subculture and incubated
10
+++
+++ (0.15%)
with three enzyme mixtures. The addition of 0.1% pectolyase
20
+++
+++
Y-23 increased the yield of protoplasts (Table 3). The best yield was obtained with enzyme mixture supplemented with 2% cellulase 'Onozuka' RS, 2% macerozyme R-10, and 0.1% pectolyase Y-23. Thus, this enzyme mixture was applied for the protoplast isolation in the following experiments giving stable
* - - to +++ denotes the degree of colony formation from none to maximum (Colony formation observed at the density of 106 / ml and cultured in K8P medium with 2 mg 2,4-D). The observation was made two weeks after incubation at 28~ in the dark.
740
Fig.2. Protoplast culture and plant regeneration from Japanese lawngrass. A Z2a suspension cell. Bar = 200 I.tm. B Freshly isolated protoplasts. Bar = 20 ~tm. C Colonies formed on K8P medium. Bar = 20 I.tm. D Formation of green spots on MS medum. Bar = 10 mm. E Shoot formation on MS medium. Bar = 10 mm. F Establishment of plants regenerated from protoplasts in soil. Bar = 10 mm.
741 The division of Z2a protoplasts required the addition of 2 mg / 1 2,4-D to KSP medium (We could not find cell division without 2,4-D). Minimum cell density supporting cell division was 5 xl05 / ml when cultured in KSP medium ( 2 mg / 1 2,4-D) with
Harris, R., M. Wright, M. Byrne, J. Varnum, B. Brightwell, and K. Schubert. (1988). Plant Cell Reports. 7: 337-340. Hayashi, Y. and K. Shimamoto. (1988) Plant Cell Reports. 7: 414-417.
or without 1.25% sea plaque agarose (Table 4). When cultured
Kao, K. N. (1977) Mol. Gen. Genet. 150: 225-230.
at the density of 106 / ml in K8P medium ( 2 mg / I 2,4-D ),
Kyozuka, J., H. Hayashi, and K. Shimamoto. (1987) Mol. Gen.
solidified with 1.25% sea plaque agarose, the plating efficiency was 0.15% after two weeks incubation. The colonies formed
Genet. 206: 408-413. Linsmaier, E. M. and F. Skoog. (1965) Physiol. Plant. 18: 100127.
were shown in Fig. 2-C.
Murashige, T. and F. Skoog. (1962) Physiol. Plant. 15: 473-497. Colonies formed in K8P medium were proliferated well when cultured in liquid MS medium supplemented with 1 mg/ 12,4-D and 60 g / 1 sucrose for two weeks.
The calli formed were
transferred to hormone free MS agarose medium for plant regeneration and the development of green spots and embryogenic calli were observed in 2 weeks incubation at 28 ~
Rhodes, C. A., K. S. Lowe, and K. L. Ruby. (1988) Bio/Technology. 6: 56-60. Takamizo, T., K. Suginobu, and R. Ohsugi. (1990) Plant Science. 72 : 125-131. Terakawa, T., T. Sato, and M. Koike. (1992) Plant Cell Reports. 11 : 457-461.
under fluorescent light (40 laEm-2s-1, 12 h photoperiod, Fig. 2-
Toriyama, K. and K. Hinata. (1985) Plant Science. 41: 179-183.
D). Finally, plantlets formed were successfully developed after
Valk, P. vander, M. A. C. M. Zaal, and J. Creemers-Molenaar.
transplanting in soil (Fig. 2-E, 2-F).
(1988) Euphytica, Suppl. 169-176. Wang, Z. Y., T. Takamizo, V. A. Iglesias, M. Osusky, J. Nagel,
This is the first report showing the plant regeneration from
I. Potrykus, and G. Spangenberg. (1992)
protoplasts isolated from the embryogenic suspension cell line,
Bio/Technology. 10 : 691-696.
Z2a, of Japanese lawngrass belonging to warm-season turfgrasses. However, the rate of visible colony formation from protoplasts of Z2a was low (0.15%). Therefore, it is necessary to improve the method of protoplast culture such as nurse culture for high plating efficiency.
Wang, Z. Y., M. P. Montavon, I. Potrykus, and G. Spangenberg. (1993) Plant Cell Reports. 12 : 95-100. Yamada, Y., Z. Q. Yang, and D. T. Tang. (1986) Plant Cell Reports. 5 : 85-88. Zhong, H., M. G. Bolyard, C. Srinivasan, and M. B. Sticklen. (1993) Plant Cell Reports. 13 : 1-6.
The protocol for the establishment of plant regeneration system from isolated protoplasts described in this paper should faciliate "the application of protoplast transformation techniques to Japanese lawngrass improvements.
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