PlantCell Reports

Plant Cell Reports (1996) 16:18-21

© Springer-Verlag1996

Plant regeneration from mesophyli protoplasts of Dianthus superbus Joon Chul Kim and Eun Ae Lee Department of Biology, Kangwon National University, Chuncheon 200-701, Republic of Korea Received 17 October 1995/Revised version received 16 February 1996 - Communicated by I. K. Vasil

Abstract Leaf mesophyll protoplasts of Dianthus superbus were cultured at a density of 5 x 104 protoplasts/ml and divided at about 18% plating efficiency in MS liquid medium supplemented with 0.5 mg/L BAP, 2.0 mg/L NAA and 9% mannitol after 2 weeks. Protocolonies formed after 3 to 4 weeks of culture in the dark at 27°C. These colonies were transferred to continuous illumination (21.5 ~tE m2 secI) for 2 weeks where most of the colonies divided to form microcalli, about 2 mm in diameter. Subsequently, green microcalli were transferred to MS solidified medium with 2.0 mg/L 2,4-D that induced shoot-forming calli after 4 weeks. These calli were transferred onto N6-2 medium containing 0.1 mg/L 2,4-D, 0.1 mg/L NAA, 2.0 mg/L kinetin and 2.0 g/L casein hydrolysate and were cultured under light. After 5 weeks the calli gave rise to multiple shoots (10 to 15 per callus). Upon transfer to MS medium containing 2.0 mg/L NAA, individual shoots were rooted in 4 weeks. The regenerants were successfully transplanted into potting soil.

Somatic hybridization and transformation of protoplasts offers a novel means for the genetic improvement of some ornamental species in relation tc their floral and market qualities (Sakamoto and Taguchi, 1991; Nakano and Mii, 1993). Dianthus superbus, a member of the Dianthus genus which contains carnation, Indian carpet, Chinese pink and D. chinensis, is an important omamental crop. It is alsG used widely for analgesic, sedative and urinary effects. Meristem culture and somatic embryogenesis in Dianthus species have been successfully used for micropropagation and virus elimination (Raojevie et al., 1990; Miller et al., 1991; Lee et al., 1994). Plant regeneration from protoplasts of some omamental Dianthus species, but not D. superbus, has been reported (Nakano and Mii, 1992). We now describe a protocol for protoplast culture and plant regeneration of D. superbus. In this respect, D. superbus is now a potential germplasm source for somatic hybridizations. Materials and Methods

Abbreviations. MS, Murashige and Skoog; BAP, 6benzylaminopurine; 2,4-D, 2,4-dichlorophenoxyacetic acid; NAA, 1-naphthaleneacetic acid; N6, Chu basal salt mixture; MES, 2-N-morpholinoethanesulfonic acid Introduction

To date, successful plant regeneration from protoplasts has been reported in several ornamental species (Griesbach, 1988; Kunitake and Mii, 1990; Matthews et al., 1991; Nakano and Mii, 1992). Correspondence to." J. C. Kim

Protoplast isolation. Seeds of Dianthus superbus were obtained from the Alpine Experiment Station, RDA, Korea. They were germinated in soil and grown in the greenhouse at 15-17°C under natural daylight (18 h photoperiod). When the fourth-sixth leaves unfolded, they were surface sterilized in 20% bleach(Clorox) containing 2 drops of Tween 20 for 20 min and subsequently rinsed five times in sterile distilled water. After the lower epidermis was peeled off, 1 g FW of leaf fragments were transferred into Petri dishes (100 ram) containing CPW 13M solution

19 (Frearson et al., 1973). The CPW 13M solution was removed and replaced by 10 ml of filter sterilized enzyme solution with various combinations of 1 or 2% Cellulase R-10, 1 or 2% Macerozyme R-10 and 0.1 or 0.2% Pectolyase Y-23 in CPW 13M solution containing 5 mM MES at pH 5.8. The tissues were incubated in static condition or on a rotary shaker (60 rpm) at 27°C in the dark. After 12 h incubation, the mixture was passed through a 45 grn nylon sieve to separate undigested debris, and the filtrate was centrifuged at 80 g for 3 min. The protoplast pellet was washed twice with CPW 13M solution. The protoplast suspension was loaded gently on the top of CPW 21% sucrose solution and centrifuged at 100 g for 5 min. After centrifugation, the purified protoplasts floating on the sucrose solution were collected and washed by centrifuging twice with protoplast culture medium (MS medium containing 0.5 mg/L NAA and 9% mannitol). Viability of isolated protoplasts was determined using fluorescein diacetate.

Protoplast Culture. Protoplast yield was calculated with a haemocytometer and the culture density of protoplasts was adjusted to 5 x 104/ml. Five ntis of protoplasts were cultured in 60 x 15 mm NUNCLON Petri dishes placed in the dark. In order to stimulate sustained division, and colony formation, 0.4 ml of fresh liquid medium with a lower osmolarity (3% mannitol) was added at one-week intervals. Plating efficiency was determined as percent of dividing cells per total number of protoplasts after 2 weeks of culture. The protoplast-derived colonies were transferred to continuous illumination (21.5 ~tE m2 sec-1) for 2 weeks. The influence of light vs. dark conditions on protoplast division was investigated at different culture steps.

Induction of Shoot-Forming Callus from Protoplast-Derived Colonies. When colonies were about 2 mm in diameter, the green microealli were placed on MS solid medium (containing 0.8% agar) supplemented with 1 or 2 mg/L of 2,4-D or NAA, alone or in combination with 0.5 mg/L BAP or kinetin. Twelve microealli were used per Petri dish (100 ram). Cultures were maintained at 27°C under continuous illumination for 4 weeks. Shoot Formation and Plant Regeneration. After 4 weeks of culture, the developing shoot-forming calli were transferred onto regeneration media containing

MS or N6 basal salts and then kept at 27°C under continuous illumination. All media contained 0.1 mg/L NAA, 0.1 mg/L 2,4-D and 2 g/L casein hydrolysate. The effects of 2 mg/L BAP, 2 mg/L kinetin and 1, 2 or 3 mg/L zeatin were investigated. Regeneration frequency (regenerated shoots, 5 mm) was determined for each treatment after 2 months of culture. Regenerated shoots were cut and placed in MS with 0, 0.5, 1.0 or 2.0 mg/L NAA for further elongation of shoots and root formation. Regenerated plants were acclimatized and potted in autoclaved soil. Results and Discussion

Protoplast Isolation and Culture. The enzyme solution which contained 2% Cellulase R-10, 1% Macerozyme R-10 and 0.1% Pectolyase Y-23 was most effective for degrading cell walls and releasing mesophyll protoplasts. Fully expanded fourth-sixth leaves yielded 8.2 x 10 6 protoplasts/g tissue with 91% viability (Fig. 1A). Optimum incubation condition for yielding intact protoplasts was 12 h at 27°C in dark using stationary condition. Whereas poor yields (about 1.4 x 106 protoplasts/ g tissue) were obtained from lower old leaves and upper young leaves, the isolated protoplasts often were easily broken in the culture medium. In Dianthus superbus, tissue maturity has been reported to have a significant effect on the yield of isolated intact protoplasts (Xu, 1991). Protoplasts were initially cultured at a density of 5 x 104 protoplasts/ml. After 5 to 6 days of culture, sustained mitotic cell divisions were observed (Fig. 1B). After 14 days of culture they divided at about 18% plating efficiency in MS liquid medium supplemented with 0.5 mg/L BAP, 2.0 mg/L NAA and 9% mannitol. The protoplastderived colonies became visible to the naked eye within 30 days of culture (Fig. 1C) in the dark at 27°C, but they did not continue growth in the dark. For further proliferation, the 30 day-dark cultured colonies were transferred to continuous illumination (21.5 ~E m"2sec-1) for 2 weeks (Table 1). The division frequency of protoplast-derived colonies could be dramatically increased in the illuminated condition and protoplast-derived microcalli reached about 2 mm diameter in size (Fig. 1D). However, as shown in Table 1, 15 day-dark cultured colonies that were cultured continuously in the light turned dark-brown and eventually died.

Induction of Shoot-Forming Callus. Microcalli derived from the 30 day-dark cultured colonies were

2O forming calli showed a high frequency of organogenesis(Fig. 2B) when subcultured under illumination on N6 medium with 2.0 mg/L kinetin (Table 3). Shoot-forming calli cultured in the dark turned brown and lost organogenic potential. These results showed that induction and developement of these shoot-forming calli were dependent upon low auxin concentration under illumination (Table 2).

Figure 1. Mesophyll protoplasts and protoplast-derived calli in D. superbus. A, Intact purified protoplasts; B, Protoplast division after 5 to 6 days of culture; C, Colony formed after 30 days; D, Protoplast-derived calli. Bars indicate 30 ~tm in A, B and C, and 0.4 mm in D. Table 1. Influence of different culture methods on protoplast divisiona . Culture Method

au

0

Time of Culture (day) 15 30

Division

2.

Shoot

and

plant

regeneration from

Death

shoot-forming calli were formed on MS medium containing 2 mg/L 2,4-D under continuous light condition at 27°C; B and C, Green spots(GS) on shoot-forming calti and initial shoots; D, Regenerated 10 to 15 shoots per Division Colony Callus ===============================================================================callus; ===========E, =====Regenerants ======= potted in soil. Bars indicate 1 mm in A and B, and 10 mm in C and D, and 50 mm in E. ~]Rn ~:7

C

Figure

calli in D. superbus. A, Pale-yellow iiiiiiiiiiiiiiiiiiiiiiiiiiiJiiiiiiiiiiiiiiisiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiprotoplast-derived iiiiiiiiiiiiiiiiiii ~ . , . . . . . . . . .

B

45

~

~Division

:::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::

, ~ . . . . . . .

:::5:5 : : : : : : : : ::::::::::::::::::::::::::::::::::::: :::5 ::: : : : : ............. :+" .........................

Colony

J, Dark; ~, Light (21.5 txE m -2 secq), bA, 15 day-dark and then light; B, 30 day-dark and then light; C, Continuous dark. transferred omo solidified medium with 2,4-D under illumination for the induction of shoot-forming calli as in Table 2 (Lee et al., 1994). They formed nodular, mucilaginous, pale-yellow organogenic calli (Fig. 2A). However, on MS medium with 1 or 2 mg/L NAA alone and in combination with 0.5 mg/L BAP or kinetin, shoot-forming calli were not induced; the tissues became dark-green and compact. Morphogenic capacity could be observed only upon subsequent culture on MS medium with 2,4-D (Table 2). Shoot-

Shoot and Plant Regeneration. After 4 weeks of culture on MS medium with 2 mg/L 2,4-D, shootforming calli were transferred to MS supplemented with different combinations of cytokinins under illumination (Table 3). After 7 to 8 weeks of subculture, the shoot-forming calli showed green spots and shoot initiation (Fig. 2B and 2C). They eventually formed 10 to 1-5 shoots per callus (Fig. 2D). N6 medium (Table 3)was more suitable than MS medium for initiating shoots from such protoplast-derived shoot-forming calli. The difference in the total nitrogen ratio of these two media suggests that it plays an important role in obtaining plant regeneration from Dianthus superbus protoplasts. The type and concentration of cytokinins have a critical effect on plant regeneration from embryogenic callus of soybean

21 (Chen et al., 1985; Wei and Xu, 1988; Kawahara and Komamine, 1991). Table 2. Effect of plant growth regulators on shootforming callus induction under illumination in D.

superbus Plant Growth Regulators (mg/L) 2,4-D

NAA

BAP

Kinetin

1.0 2.0 1.0 2.0 1.0 2.0 0 0 0 0 0 0

0 0 0 0 0 0 1.0 2.0 1.0 2.0 1.0 2.0

0 0 0.5 0.5 0 0 0 0 0.5 0.5 0 0

0 0 0 0 0.5 0.5 0 0 0 0 0.5 0.5

Frequency of Shoot-forming Calli" ++ +++ + + + +

As shown in Table 3, kinetin was more effective than zeatin and BAP for shoot formation from the protoplast-derived calli in D. superbus. When the shoots having 2 - 3 leaves were transferred to MS medium with 2.0 mg/L NAA, roots were initiated at the base of shoots after 4 weeks of transfer. So far, more than 60 plants were acclimatized and then transferred to potting soil (Fig. 2E) and finally 45 plants were cultivated in a green house. In these plants, the leaf shape and growth habit were normal at least during the early stages of growth, but some morphological differences at flowering stage were observed, including reduced plant height, thinner leaf shape and early flowering. The regeneration of plants from D. superbus protoplasts has encouraged us to attempt somatic hybridization between D. superbus and D. caryophyllus. Acknowledgement This work was supported by KNURF '94 to J. C. Kim.

References

Chen TH, Lain L, Chen SC(1985). Plant Cell Tiss Org Cult 4:51-54

-, none; +, less than 30%; ++, 30-70%; +++, more than 70%. Protoplast-derived colonies were tested with 10 replications for each experiment. Table 3. Effect of plant growth regulators on shoot regeneration from protoplast-derived callus under illumination in D. superbus Culture Medium~

Growth Regulator, mg/L

MSB 2 MSK2 MSZ 1 MSZ 2 MSZ 3 N6- 1

2.0 2.0 1.0 2.0 3.0 2.0 1.0 2.0

N6 - 2

BAP Kinetin Zeatin Zeatin Zeatin Kinetin + ABA Kinetin

Frequency of Shoot Regeneration 0 18.0 +_3.1 0 8.3 + 2.4 16.7 + 3.4 41.7 + 6.8 75.0 + 8.5

All the media contained 0.1 mg/L NAA, 0.1 mg/L 2,4-D and 2.0 g/L casein hydrolysate. b The values represent the mean + SE of at least 3 independent experimets.

Frearson EM, Power JB, Cocking EC(1973) Dev Biol 33:130-137 Griesbach RJ(1988) Sci Hort 37:246-256 Kawahara R, Komamine A(1991) Kor J Plant Tiss Cult 18:334-339 Kunitake H, Mii M(1990) Plant Sci 70:115-119 Lee EA, Kim JC, Kim WB, Kim BH(1994) J Plant Biol 37:381-385 Lu C, Nugent G, Chandler S, Young R, Dalling M(1991) Biotech 9:864-868 Matthews D, Mottley J, Horan I, Roberts AV(1991) Plant Cell Tiss Org Cult 24:173-180. Miller RM, Kaul V, Hutchinson JF, Richards D(t991) Ann Bot 67:35-42

Murashige T, Skoog F(1962) Physiol Plant 15:473-497 Nakano M, Mii M(1992)Plant Cell Reports 11:225-228 Nakano M, Mii M(1993) TheorAppl Genet 86:1-5 Raojevic L, Nevena D, Petrovic J(1990) Acta Hort 280:163-167

Sakamoto K, Taguchi T(1991) Theor Appl Genet 89:509513 Tsay HS(1993) In: So WY, KomamineA (eds) Advances in Developmental Biology and Biotechnology of Higher Plants. Press ofKor J Plant Tiss Cult, pp. 128-149 Wei ZM, Xu ZH(1988) Plant Cell Reports 7:348-351 Xu ZH(1991) In: Sun YR, An XP (eds) Plant Protoplast Culture. SciencePress, Beijing,pp. 72-85