Plant Cell Reports

Plant Cell Reports (1991) 10:417-420

9 Springer-Verlag1991

Plant regeneration from callus-derived protoplasts of Pelargonium • domesticum K. B. Dunbar 1,z and C. T. Stephens 1 1 Department of Botany and Plant Pathology, Michigan State University, East Lansing, MI 48824, USA z Present address: USDA, ARS, SAA, REG. PL. INTRO. STN. 1109 Experiment Street Griffin, GA 30223-1797, USA

Abstract

A protocol for regenerating plants from callus-derived protoplasts of Pelargonium X domest icum (rega 1 g e r a n i u m cv. 'Melissa') has b e e n developed. Protoplasts were isolated from leaf-derived callus tissue on MS m e d i u m s u p p l e m e n t e d with 3.0 mg/l naphthalene acetic acid, 2.0 mg/l 6benzylaminopurine, and 3.0% sucroseff This callus yielded 2.7 X 10 ~ protoplasts/gram of tissue after a 6 hr i n c u b a t i o n in an e n z y m e s o l u t i o n consisting of 2.0% cellulysin, 0.5% macerase, and 0.5 M s u c r o s e Protoplasts were plated at 1 X 10 ~ protoplasts/ml in a mixture (i:i v/v) of KMP8/KP liquid medium layered on the same medium solidified with 0.6% agarose. Protoplast division was initiated within 2 days, and colonies of 15 to 50 cells developed 8 wk a f ter plating. P-calli 1-2 m m 3 developed 15 wk after plating, and plants regenerated from the p-calli have been transferred to the greenhouse. ~rev

iat ions:

NAA, napthaleneacetic acid; 6-BAP, 6benzylaminopurine; CW, Calcofluor White; FDA, fluorescein diacetate. Introduction

The three important

Pelargonium species most to the U.S. floriculture

Offprint requests to. C. T. Stephens

industry are P. X hortorum L. H. Bailey (garden geranium), P. X d o m e s t i c um L. H. Bailey (regal geranium), and P. peltat u m (iv y g e r a n i u m[-~. Pelargonium X domesticum is considered the most beautiful cultivated Pelargonium species; however, problems with cultivation and flowering have prevented it from becoming as popular as P. X hortorum in most parts of the U.S.A. (Hanniford and Holcomb 1982). Pelargonium X d o m e s t i c u m requires a long treatment of cool night temperatures to f l o w e r , but is r e s i s t a n t to b a c t e r i a l b l i g h t of geranium (Ewart 1980, Hanniford and Holcomb 1982). Pelargonium X hortorum does not require a cold treatment to flower, but is susceptible to bacterial b l i g h t (Ewart 1980). A t t e m p t s to sexually cross P. X hortorum with P. X d o m e s t i c u m h a v e b e e n unsuccessf--ul "iEwart 1980). Regeneration of plants from protoplasts provides new approaches to t r a d i t i o n a l p l a n t breeding including the generation of somatic hybrids, a s y m m e t r i c hybrids, and cybrids (Griesbach 1 9 88). Isolated protoplasts also can be used for direct introduction of DNA and g e n e t i c t r a n s f o r m a t i o n s (Griesbach 1988). Before such techniques can be utilized for whole plant improvement, protoplast isolation and regeneration procedures for a plant species are required. Over 45 flowering ornamental plant species have been regenerated from protoplasts; however, the majority of

418 these have been Nicotiana and Petunia species ( G r i e s b a c h 1988). Plant regeneration from protoplasts has been reported for P. X hortorum (Kameya 1975, Y a r r o w et al. 1987), and P. peltatum (Yarrow et al. 1987). This report describes procedures for the regeneration of P. X d o m e s t i c u m from callus-derivedpr-o-s M a t e r i a l s and methods Callus culture: Leaf explants from regal geranium cv. 'Melissa' were used to initiate green organogenic callus tissue as described by Dunbar and Stephens (1989). This callus was transferred (eight to ten 0.5 cm 3 pieces per Petri dish) to a Murashige and Skoog MS (1962) m e d i u m supplemented with 3.0 mg/l NAA, 2.0 mg/l 6-BAP, 3.0% sucrose, solidified with 0.8% agar (Sigma Chemical Co., St. Louis, MO), adjusted to pH 5.8, and poured 20 ml per 100 X 15mm polystyrene Petri dish (C-Medium). After 21 days incubation at 25 C in the dark, white friable callus was selected for subculture. White friable callus was subcultured every 3 wk, and was used for protoplast isolation after the fourth subculture. Protoplast isolation and culture: White friable callus tissue (7 to ii grams) was harvested 10 to 12 days after subculture and placed into a 100 X 15 m m polystyrene Petri dish. Th tissue was broken into pieces 1 m m with forceps and 15 ml of an enzyme solution consisting of 2.0% cellulysin (Behring Diagnostics, La Jolla, CA), 0.5% macerase (Behring Diagnostics, La Jolla, CA), 0.5 M sucrose, in CPW salts (Frearson et al. 1973) adjusted to pH 6.0 was poured over the tissue. Dishes were incubated for 6 hr at 23 C in the dark on a rotary shaker at 46 rpm. After incubation, protoplasts were released by slowly passing the callus suspended in enzyme solution in and out of a P a s t e u r pipet two times. Protoplasts were separated from undigested callus tissue by filtration t h r o u g h a 62 um n y l o n sieve. The

protoplasts were centrifuged at 100g for 10 min in 15 X 125 m m test tubes. Protoplasts were collected in 6 ml of W5 solution (Menczel et al. 1981) by layering the W5 solution on top of the e n z y m e s o l u t i o n and r e m o v i n g the protoplasts from the interface between the two solutions in the W5 solution. An equal volume of W5 solution was added to the protoplast suspension and the protoplasts were centrifuged at 36 g for 10 min. The W5 solution was discarded and the pellet was suspended into 3 ml of filter sterilized KMP8/KP liquid m e d i u m (Yarrow et al. 1987). The protoplasts w e r e c o u n t e d on a hemacytometer and diluted to 1 X 105 protoplasts/ml. Protoplasts in 4 ml of liquid medium were poured into 60 X 15 m m p o l y s t y r e n e Petri d i s h e s that c o n t a i n e d 3 ml of K M P S / K P m e d i u m solidified with 0.6% agarose (SeaPlaque, FMC, Rockland, MD). To visualize cell wall material, isolated protoplasts were plated into liquid KMP8/KM medium with 10 mg/l Calcofluor White (CW) (Polysciences, Warrington, PA) (Hahne et al. 1983) and observed immediately after isolation and after 48 hr. The viability of protoplasts was determined immediately after isolation and after 48 hr in culture w i t h f l u o r e s c e i n d i a c e t a t e (Sigma C h e m i c a l Co., St. Louis, MO) (FDA) (Wilholm 1972). Cell w a l l and viability staining were viewed with a Nikon fluorescence microscope equipped with epifluorescence optics (330 to 380 nm exciter, a 420 nm barrier and a 400 nm mirror). Protoplast cultures were fed every 7 to 10 days by the addition of 0.5 ml of fresh KMP8/KP liquid medium. After 8 wk, the glucose in the feeding m e d i u m was reduced to 30 g/l, and after the 12th wk, the glucose was reduced to 15 g/l and the sucrose was ~aised to 15 g/l. After 14 wk, 1-2 m m J protoplastderived calli were transferred to 100 X 15 m m Petri dishes containing an MS medium supplemented with 3.0 mg/l NAA, 2.0 mg/l 6-BAP, 1.0 g/l casamino acids, 3.0% sucrose, solidified with 0.8% agar (Sigma Chemical Co., St. Louis, MO), adjusted to pH 5.8 (pC-medium). After one month, callus tissue was transferred to a MS medium supplemented

419

with 0.2 mg/l 6-BAP, 2.0% sucrose, solidified with 0.8% agar and adjusted to pH 5.8 (S-medium). Callus with shoot primordia was subcultured onto the same medium for shoots to elongate. Shoots were transferred to culture tubes (25 X 150 ram) containing 15 ml of half strength MS m e d i u m supplemented with 0.1 mg/l NAA, 1.0% sucrose, and adjusted to pH 5.8, and solidified with 0.9% agar (R-medium). After 60 days, rooted plantlets were transferred to Bacto Professional Planting Mix (Michigan Peat Co., Houston, Texas) in 8 cm plastic pots and covered with a p l a s t i c bag for 1 wk. Isolated p r o t o p l a s t s a l s o w e r e cultured as described above with the cytokinin in the KMPS/KP liquid and solid media substituted with either 0.5 mg/l 6-BAP (KMP8/KP-B) or 1.0 m g / l zeatin

Standard error of means was used to evaluate the protoplast isolation and regeneration procedures. Two samples (300-600 p r o t o p l a s t s / s a m p l e ) per isolation were counted with a hemacytometer to determine the averages for protoplasts. The average number of protoplasts isolated per gram of callus was determined from data of seven isolations. The data for viability and cell wall stainings represents observations from four isolations. The average plating efficiency represents observations from five isolations. The average number of p-calli surviving the transfer to solid m e d i u m represents data from 30 Petri dishes with four pcalli per dish. The average number of p-calli developing shoot primordia represents data from 30 Petri dishes with 10 p-calli per dish.

(KMP8/KP-Z)

Figure i. The development of P. X d o m e s t i c u m cv. 'Melissa' from callus-derived protoplasts to plants (a-f). (a) Callus-derived protoplasts 1 hr after isolation. Bar = 10 um. (b) First division 3 days after isolation. Bar = 5.0 um. (c) Protoplast-derived colony 8 wk after isolation. Bar = 10 um. (d) Protoplastderived callus 3-4 wk after transfer to pC-medium. (e) Shoot development on callus tissue 30 days after transfer to S-medium. (f) Plant regenerated from a protoplast and acclimated to greenhouse conditions.

420 Results and D i s c u s s i o n Callus subcultured on MS medium with 2 mg/l NAA, 2 mg/l 6-BAP and 2% sucrose (Dunbar and S t e p h e n s 1989), and incubated in light developed numerous shoot primordia on the upper callus surface. W h e n this c a l l u s was transferred to medium with 3 mg/l NAA, 2 m g / l 6 - B A P and 3% sucrose, and incubated in the dark, a white friable callus was produced. This white nonorganogenic callus, s e l e c t e d f r o m cultures 10 to 12 days after subculture to C-medium, yielded 2.7 X 105 + 6.2 X 104 protoplasts/gram of -callus (+ s t a n d a r d e r r o r = SE) (Fig. la). These protoplasts were buoyant. The sucrose used to provide the osmotic pressure in the enzyme solution caused these protoplasts to float easily when centrifuged. The protoplasts also floated in liquid K M P 8 / K P m e d i u m , therefore W5 solution was used to wash the e n z y m e s f r o m the p r o t o p l a s t s . Immediately after isolation, protoplasts plated in KMP8/KP liquid m e d i u m with 10 mg/l CW did not show fluorescence. However, after 48 hr fluorescence was observed on protoplasts. This i n d i c a t e s the isolation procedure was successful at removing the cell walls, and new cell walls were being formed within 48 hr. Staining with FDA established that 75.5% + 3.6 (SE) of recently isolated protoplasts were viable, and 70.0% + 3.9 (SE) were viable after 48 hr in culture. First division of protoplasts w a s o b s e r v e d 2 days after plating (Fig. ib). A plating efficiency (% of dividing cells per total protoplasts) of 3.5% + 0.5 (SE) was observed 5 wk after plating protoplasts in KMP8/KP medium. Small colonies of 15-50 cells were observed 8 wk after plating ~Fig. ic). These colonies formed 12 m m ~ p calli 7 wk later. These p-calli were transferred onto pC-medium, and within 30 days 93% + 2.6 (SE) formed callus tissue (Fig. id). Callus was transferred to S-medium and within 30 days 63.4% + 5.3 (SE) had developed shoot primordia (2 m m long shoot tips with leaf primordia). Calli with shoot primordia were subcultured onto the same m e d i u m and the shoots elongated

(Fig. le). Shoots were transferred to R-medium and roots developed within 3-4 wk. Twelve plants were transferred to qreenhouse conditions (Fig. if) and abnormalities were not observed in these plants. Isolated protoplasts cultured in KMP8/KP-Z or KMP8/KP-B medium were not significantly different from protoplasts cultured in KMP8/KP medium with respect to plating efficiency (data not shown). However, p-calli did not develop from protoplasts plated in KMP8/KP-Z or KMP8/KP-B medium. A hybrid between P. X domesticum and P. X hortorum could be used to transfer the bacterial blight resistance of P. X d o m e s t i c u m to P. X hortorum, or the a b i l i t y to flo--wer w i t h o u t a cold treatment from P. X hortorum to P. X domesticum. Pelargonium X domesticum also has many flower colors not found in P. X h o r t o r u m ( E w a r t 1980). Protoplast fusion technology could be used to make this potentially useful hybrid. P r o t o p l a s t i s o l a t i o n and r e g e n e r a t i o n p r o t o c o l s h a v e been reported for P. X hortorum (Kameya 1975, Yarrow et al. 1987). With the protoplast isolation protocols for P. X domesticum reported here, regeneration of such a somatic hybrid is feasible. References

Dunbar KB, Stephens CT (1989) Plant Cell, Tissue and Organ Cult 19:13-21 Ewart LC (1980) HortScience 16:135-137 Frearson EM, Power JB, Cocking EC (1973) Dev Biol 33:130-137 Griesbach RJ (1988) Scientia Hortic 37: 247-256 Hahne G, Herth W, Hoffman F (1983) Protoplasma 115:217-221 Hanniford GC, Holcomb EJ (1982) In: M a s t a l e r z JW, H o l c o m b EJ (eds) Geraniums III. Pennsylvania Flower Growers. University Park, PA. Kameya T (1975) Jap J Genet 50:417-420 Menczel L, Nagy F, Kiss ZsR, Maliga P (1981) Theor Appl Genet 59:191-195 Murashige T, Skoog F (1962) Physiol Plant 15:473-597 W i d h o l m JM (1972) Stain Techn 47:189194 Yarrow SA, Cocking EC, Power JB (1987) Plant Cell Rep 6:102-104