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.

REFERENCES Abdullah, R., C. Cocking, and J. A. Thompson. (1986) Bio/Technology. 4:1087-1090. Al-Khayri, J. M., F. H. Huang, L. F. Thompson, and J. W. King. (1989) Crop Science. 29: 1324-1325. Asano, Y. (1989) Plant Cell Reports. 8: 141-143. Asano, Y. and K. Sugiura. (1990) Plant Science. 72: 267-273. Chu, C. C., C. C. Wang, C. S. Sun, C. Hsu, K. C. Yin, C.Y. Chu and F. Y. Bi. (1975) Sci. Sinica. 18: 659-668. Dalton, S. J. (1988) Plant Cell, Tiss. Org. Cult. 12 : 137-140. Ha, S. B., F. S. Wu, and T. K. Thorne. (1992) Plant Cell Reports. 11 : 601-604. Harms, Ch. T. and I. Potrykus. (1978) Theor. Appl. Genet. 53: 57-63.

Plant regeneration from protoplasts of Japanese lawngrass.

Embryogenic callus of Japanese lawngrass (Zoysia japonica Steud.) was induced from sterile mature seeds on LS medium with 5 mg / l of 2,4-D. Embryogen...
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