Plant Cell Reports

Plant Cell Reports (1987) 6:414-416

© Springer-Verlag 1987

Plant regeneration from protoplasts of Dimorphotheca and Rudbeckia J. S. AI-Atabee and J. B. Power Plant Genetic Manipulation Group, Department of Botany, University of Nottingham, Nottingham NG7 2RD, UK Received July 28, 1987 / Revised version received October 26, 1987 - Communicated by I. Potrykus

ABSTRACT Protoplasts were isolated from leaves, shoots, cotyledons, ray florets and callus cultures of Dimorphotheca aurantiaca (syn. D. sinuata) (Cape Marigold, Star of the Veldt) and Rudbeckia hirta, R. laciniata and R. purpurea; species of ornamental value. For Dimorphotheca, plants were regenerated from protoplasts of all sources apart from the ray floret, whilst for the Rudbeckia species, although protoplast division was induced in most cases, only leaf mesophyll protoplasts of R. hirta c.v. Marmalade gave plants. The establishment of plant regeneration for these ornamental species, from protoplasts, now provides a basis for their somatic hybridisation. ABBREVIATIONS BAP, 6-benzylaminopurine; IAA, indole-3-acetic acid; NAA, naphthaleneacetic acid; K, kinetin; GA_, gibberellic acid; MS, Murashige and Skoog (~962); f.wt., fresh weight. INTRODUCTION Plant regeneration from protoplasts has been described for members of several genera within the family Compositae including Gaillardia, Cichorium, Senecio (Binding et al., 1981, Crepy et al., 1982), Helianthus (Bohorova et al., 1986), Chrysanthemum (Otsuka, 1986), and Lactuca species (Brown et al., 1987). With the exception of Chrysanthemum there are few ornamental species where regeneration has been established yet the Compositae is recognised as containing a vast number of commercially important ornamentals. The establishment of plant regeneration from such species will progressively allow for their realistic incorporation into somatic hybridisation programmes designed, ultimately, to foster their improvement in relation to their floral and marketable qualities. MATERIALS AND METHODS Plant Material Dimorphotheca Seeds of D. aurantiaca (cvs. Giant Orange, Olistening White and 'New Hybrids', Suttons Seeds Ltd., Torquay, U.K.) were surface sterilised in 10%

Offprint requests to: J. B. Power

Domestos solution CLever Bros., U.K.) (25 min) followed by six changes of sterile tap water and germinated, in the dark, on MS medium lacking growth regulators but with 0.8% agar (Sigma), 3% sucrose. Cotyledons, of 12-14 day old seedlings, were used for protoplast isolation. Plants were also grown in the greenhouse (6000 lux, 18h daylength, mercury vapour lights, 20-25~C) and leaves (3rd/4th leaves of 25-40 day old plants), stems and fully expanded ray florets taken for protoplast isolation. Materials were surfaced-sterilised as for seeds (15 min). Leaf callus, for protoplast isolation, was established on MS medium with 2.0 mg/1 IAA, 1.0 mg/l BAP, 3% sucrose, 0.8% agar (pH 5.8) (MSD~) medium (Power et al., 1984), and grown routinel~ at 23°C under continuous illumination (2000 lux, daylight fluorescent tubes) with subculturing every 3-4 weeks. Rudbeckia Seeds of R. hirta (cv. Marmalade), R. laciniata (cv. Irish Eyes) and R. purpurea (syn. Echinacea purpurea) (cv. Brilliant Star) (Suttons Seeds Ltd, Torquay, U.K.) were sterilised (50% Domestos, lh) with in vitro cultured seedlings and greenhouse plants maintained as for Dimorphotheca. Callus cultures were established on MS medium with 2.0 mg/l BAP, 1.0 mg/1 IAA, 3% sucrose and 0.8% agar pH 5.8). Protoplast isolation and culture: Rudbeckia The lower epidermis of surface-sterilised leaves, cotyledons and ray florets was removed by peeling and tissues were plasmolysed, for lh in CPW salts,( Frearson et al., 1973) with 13% mannitol for the ray florets or 8% mannitol for the others (pH 5.8) and transferred to an enzyme mixture which consisted of 0.8% Cellulase RIO (Kinki Yakult, Nishinomiya, Japan), 0.4% Driselase (Kyowa Hakkokogya, Tokyo, Japan), 0.8% Rhozyme HP 150 (Rohm and Hass, Philadelphia, U.S.A.), 0.2% Macerozyme RIO (Kinki Yakult), CPW salts 8% mannitol for the leaves and cotyledons, 13% mannitol for the ray florets (pH 5.8) with antibiotics, 400.0 mg/l ampicillin (Boots, Pure Drug Co. Ltd., Nottingham U.K.), 10.0 mg/1 gentamycin, 10.0 mg/l tetracycline (Sigma). Leaves, cotyledons and ray florets were incubated at 23°C (3-4h) on a rotary shaker (30 cycles/min) followed by a stationary incubation in the dark (8-I0h).

415 Protoplasts were released from callus using the same enzyme mixture but with Macerozyme RIO at 0.4% (12h on a rotary shaker). After incubation tissues were passed through a nylon sieve (64 bm pore size), washed twice in CPW salts, with 8% mannitol for the leaves and cotyledons, 13% mannitol for the others, by resuspension and centrifugation (80 x g; 5 min) and finally freed of debris by fioatation in CPW21S medium (CPW salts with 21% sucrose) coupled with centrifugation (100 x g; 5 min). Protoplasts were transferred to the appropriate culture medium (see Results) for 24h in the dark andLsubsequently diluted to a density of 2.5 x 10"/ml and plated as liquid cultures (4 ml per 5 cm Petri dish, A/S Nunc, Roskilde, Denmark), agarose bead cultures (Shillito et al., 1983) (0.2 ml beads surrounded hy 2.0 ml of liquid medium (5 cm Petri dishes). Dishes were sealed with Nescofilm and maintained in the dark or with a continuous illumination of 700 lux (daylight fluorescent tubes) (23°C). Protoplast culture media were replaced every 5-7 days with the same medium before reduction of the osmotic pressur~ was initiated. The following three media regimes were used with a progressive reduction in the osmotic pressure (every 5 days) being initiated at 16 days for callus protoplasts and 30 days for the other systems by the replacement of the protoplast culture medium with medium of lower osmotic pressure:MSP19M medium (MS medium with 2.0 mg/1 NAA, 0.5 mg/l BAP, 9% mannitol, pH 5.8) diluted with MSPIOM medium (as MSP19M but no mannitol) in the ratios (ml) 3:1, 2:2, 1:2. 0:4; KM8P medium (Kao and Michayluk, 1975) with KM8 and K8P medium with K8 (Kao, 1977), the latter two media systems being utilised as for MSPI9M medium in terms of handling for dilution. Plating efficiency was asssessed after 9 days. Protoplast-derived calli were finally transferred to MS medium with 1.0 mg/l IAA, 2.0 mg/1 BAP, 0.8% agar, 3% sucrose (pH 5.8) for 3 weeks for further growth (culture conditions as for protoplasts) and then to plant regeneration medium (MS medium with 0.1 mg/l IAA, 0.4 mg/1 K, 0.8% agar, Table I

3% sucrose (pH 5.8). Shoots were rooted in MS medium with 0.00875 mg/1 IAA, 0.03 mg/1 K, 0.001 mg/1 folic acid, 3% sucrose, 0.7% agar (pH 5.8) (BGS) medium (Power et. al., 1984) whereupon they could be transferred to the open greenhouse. Dimorphotheca Leaves, cotyledons, ray florets and stem segments were plasmolysed for lh in CPW salts (with 8% mannitol for leaves/cotyledons or 13% mannitol for stems/ray florets). Protoplasts were released from leaf/cotyledon tissues using an enzyme mixture which consisted of 2.0% Meicelase (Meiji Seika Kaisha Ltd, Japan), 2.0% Rhozyme HP 150, 0.03% Macerozyme RIO, CPW salts, 8% mannitol (pH 5.8). For callus/stem/ray floret tissues the enzyme mixture was as for Rudbeckia callus. Incubation (dark, 23°C) was static for leaves (5-6h), cotyledons (I0-12h) and ray florets (1Oh), whilst for stem/callus tissues incubation was on a rotary shaker (4-5h, 30 cycles/min) followed by static conditions (8-I0h). The purification and culture of protoplasts was as for Rudbeckia. Protoplast derived calli (1.5-2.00 mm diam.) were transferred to MSDz medium (3-5 weeks) for proliferation and then ~o MS medium (with 1.0 mg/l IAA, 0.5 mg/1 BAP, 0.3 mg/l GA_, 0.8% agar, pH 5.8) for 5-7 weeks to induce shootsJ Shoots, detached from the callus, were grown on BGS medium (3-5 weeks) and then rooted by transfer to MS medium (with 0.5 mg/l NAA above) (3-5 days) followed by MS medium lacking hormones. RESULTS AND DISCUSSION The results are summarised in Table I. Division of Dimorphotheca protoplasts was initiated after 2-3 days and had reached the 8 celled colony stage by day 12. Protoplasts from ray florets failed to divide and division was delayed by 2 days for the other systems when grown in the dark.

Yields, viabilities, plating efficiencies and culture media responses for protoplasts of Domorphotheca aurantiaca and Rudbeckia species. Data is given for the optimum tissue sources only.

Species (cultivar)

D. aurantiaca (Giant Orange) (Glistening White) ('New Hybrids') Rudbeckia hirta (Marmalade)

R. lacinata (Irish Eyes)

R. purpurea (Brilliant star)

Yield (x I06/g f.wt.)

Viability

Source

(%)

Tissue

Plating Efficiency (%) MSPI9M

K8P

L

D

L

4.1" 29.0*

3.7* nt

30.2 80.1

KM8P D

L

D

25.1 nt

25.5 73.7*

23.5 nt

4.1 4.3

88 87

Leaf Callus

6.6 4.9

81 86

Leaf Leaf

4.0* 3.8*

2.9* 2.5*

28.9 30.4

25.7 26.2

25.4 27.3

23.5 23.3

2.3 2.8 2.5

74 84 67

Leaf Callus Cotyledon

1.9" 0 2.7*

1.3" 0 1.9"

12.6 2.0 18.7

20.4 1.1 24.0

11.8 1.7 14.2

20.1 2.1 24.2

1.8 2.7 3.2

77 90 73

Leaf Callus Cotyledon

2.1" 0 1.9"

1.3" 0 2.6*

13.9 1.0 18.5

19.5 1.2 23.7

10.9 1.6 14.0

21.2 2.5 24.8

1.3 1.3

89 81

Callus Floret

nt nt

nt nt

nt 0

10.8" 0

nt 0

8.5* 0

L, culture in light; D, dark conditions; nt: not tested. Best results were generally obtained for agarose solidified media except as shown* (liquid media). All experiments were replicated three times.

416 Like sunflower (Bohorova et al., 1986), MSP19M medium did not support div-~s~on. There was no requirement to reduce colony density, during culture, unlike the lettuce {Brown et al., 1987) or sunflower (Bohorova et al., 1986) protoplast systems. Upon transfer to regeneration medium, shoot bud formation was very rapid. The efficacy of plant regeneration was variety dependent with 26%, 20% and 30% of leaf protoplast derived calli undergoing regeneration for the varieties Glistening White, Giant Orange and 'New Hybrids' respectively. Callus protoplasts of the variety Giant Orange also gave plants. Shoots were rooted within 5 days. The efficiency of regeneration for Dimorphotheca was superior to that described for the related species Chrysanthemum morifolium (Otsuka, 1986) where only 1.49% of leaf protoplast derived calli gave shoots. The pattern of development for protoplast division of the Rudbeckia species was similar to DimorphothecaJbut, although callus was a superior source tissue in terms of protoplast viability, it was only leaf protoplasts of R. hirta cv. Marmalade that gave callus capable of plant regeneration, where regeneration efficiency was found to be 100% after two subcultures on regeneration medium. This is the first report of successful plant regeneration of Rudbeckia protoplasts. The only previous report (Binding et al., 1981) showed that protoplast division in R. fulgida could not be sustained. Regenerated plants of Dimorphotheca and Rudb~ckia flowered normally and were fertile. This study forms a basis for somatic hybridisation of ornamental Compositae species. Although plant regeneration has been established for Dimorphotheca,

Rudbeckia, the fact that it is variety dependent can form the basis of a partial selection strategy. For example, in preliminary experiments leaf protoplasts of R. laciniata, electrofused with non-dividing ray floret protoplasts of D. aurantiaca, gave heterokaryons capable of sustained division in agarose solidified KM8P medium. Stem protoplasts of the related ornamental species, Ageratum hostonianum (cv. Blue Mink) have also, in preliminary experiments, entered sustained division using the cultural regimes developed for Dimorphotheca and Rudbeckia protoplasts. REFERENCES Binding H, Nehls R, Kock R, Finger, J, Mordhorst, G (1981) Z Pflanzenphysiol 101:119-130 Bohorova NE, Cocking EC, Power, JB (1986) Plant Cell Rep 5:256-258 Brown C, Lucas JA, Power,JB (1987) Plant Cell Rep 6: 180-182 Crepy L, Chupeau MC, Chupeau Y {1982) Z Pflanzenphysiol 107:123-132 Frearson EM, Power JB, Cocking EC (1973) Dev Biol 33:130-137 Kao KN (1977) Mol Gen Genet 150:225-230 Kao KN, Michayluk MR (1975) Planta 126:105-110 Murashige T, Skoog F (1962) Physiol Plant 15:473-497 Otsuka M (1986) Agric Hort 41:50-51 Power JB, Chapman JV, Wilson D (1984) Laboratory Manual: Plant Tissue Culture, University of Nottingham, UK Shillito RD, Paszkowski J, Potrykus I (1983) Plant Cell Rep 2:224-247

Plant regeneration from protoplasts of Dimorphotheca and Rudbeckia.

Protoplasts were isolated from leaves, shoots, cotyledons, ray florets and callus cultures of Dimorphotheca aurantiaca (syn. D. sinuata) (Cape Marigol...
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