Plant Cell Reports
Plant Cell Reports (1992) 11: 2 - 6
9 Springer-Verlag1992
In vitro shoot regeneration from leaf mesophyH protoplasts of hybrid poplar (Populus nigra x P. maximowiczil) Young Goo Park 1 and Sung Ho Son 2 1 Department of Forestry, College of Agriculture, Kyungpook National University, Daege 702-701, Korea 2 Department of Forestry, Iowa State University, Ames, IA 50011-1021 USA Received May 17, 1991/Revised version received December 5, 1991
Summary.
Protoplasts were isolated from leaf mesophyll of hybrid poplar (Populus nigra X P. maximowiczii) with a mean yield of 10.4 X 106 protoplasts per g fresh weight using 2.0% Cellulase 'Onozuka' R-10, 0.8% Macerozyme R10, 1.2% Hemice]lulase, 2.0% Driselase, and 0.05% Pectolyase Y-23 with CPW salts solution containing 0.6 M mannitol, 0.002 M DTT, 3 mM MES at pH 5.6. A liquid plating method produced the highest frequency of dividing protoplasts (48.6%) using an MS medium without NH4NO 3. The highest percent of colony formation was 22.8%, produced with fabric supported semi-solid (0.5% w/v) agar plating method using the same culture medium. Growing cell colonies and/or micro-calli were transferred to a fresh semisolid agar medium containing 0.44 pNi BAP and 9.0 p_M 2,4D. Multiple shoats were produced from protoplast-derived callus after culture on MS medium containing 6.8 }~M zeatin. After root induction on half-strength MS medium that lacked growth regulators, shoots were transferred to pots containing artificial soil mix.
Abbreviations:
cPw, Cell and Potoplast Wash solution; LPM, Liquid Plating Method; LDM, Liquid Drop Method; HDM, Hanging Drop Method; FSPM, Fabric supported Semi-solid agar P l a t i n g Method; DTT, Dithiothreitol; MES, 2-(N-morpholino) ethane sulfonic acid; BAP, 6-benzylaminopurine; 2,4-D, 2,4-dichlorophenoxy acetic acid; NAA, a - n a p h t h a l e n e acetic acid; MS, Murashige and Skoog (1962). Introduction For the purposes of genetic modification and physiological study of plant cells, protoplasts can serve as valuable experimental material (Park et al. 1990). Since the isolation of protoplasts from tree species was first reported in Acer pseudoplatanus by Rona and Grignon (1972), there have been many reports of protoplast isolation from forest tree species (Ahuja 1982; Chun 1985; Gupta 1986; Park and Son 1986). In recent years, progress has been made with the subsequent reformation of whole, dividing cells as well as successful establishment of regeneration systems from protoplasts of woody plants (Park and Son 1988a). Various methods such as liquid plating culture (Potrykus 1980), liquid drop culture (Vasil and Vasil 1979), hanging drop culture (Potrykus et al. 1979), micro droplet culture (Gelba 1978), as well as different feeder layer techniques (Raveh et al. 1973) have been adapted to plant
Offprint requests to. Y. G. Park
Communicated by G. C. Phillips
protoplast culture. In addition, a floating polyester screen disc method has been successfully demonstrated with poplar leaf protoplasts isolated from non-seedling tissue (Russell and McCown 1986). However, most of the previously reported protoplast culturing systems largely depend on the unique techniques developed by individual laboratories. No comparisons have been made among different culturing methods using similar donor cells and tissue derived protoplasts. In this paper, we report some critical factors for protoplast isolation and other important aspects of various culturing methods for each developmental stage of protoplasts from this hybrid poplar.
Materials and Methods Plant materials. Dormant branches (1-2 years old) bearing apical and axillary buds were collected during March, 1985 from same ortet of the upper crown of 20-yearold Populus nigra X P. maximowiczii at the Institute of Forest Genetics, Suwon, Korea. The shoot parts (3-4 cm) were sterilized using the method of Park and Son (1986). For sprouting shoots, the bud parts were plated on MS medium containing 0.05 ~VI NAA. After shoots were initiated, they were transferred to shoot multiplication medium containing MS basal salts, 0.3 p_M thiamine ttCI, 4.1 p~V[ nicotinic acid, 2.4 pM pyridoxine HC1, 26.6 ~Vl glycine, 108.2 ~M myo-inositol, 3% (w/v) sucrose, 0.75% (w/v) Difco bacto-agar, and 0.89 ~M BAP. After 5 subcultures, at approximately 4-week intervals, multiplied individual shoots were subcultured on half-strength MS medium lacking growth regulators for shoot elongation. Cultures for leaf expanding were maintained in 500 ml of transparent glass bottle (I.V. bottle) containing 50 ml of medium. Fully expanded leaves (aging from 6 to 8 weeks of culture) were used as the source material for protoplast isolation. The pH of the medium was adjusted to 5.8 prior to autoclaving at 121 ~ C for 15 rain. Cultures were maintained in a thermostatically controlled culture room with a photoperiod of 16 h, a photosynthetically active photon flux rate of 20-40 p.E m -2 s "1 and a constant temperature of 26+ 1~ C. P r o t o p l a s t isolation. One g fresh weight of expanded, healthy leaves from in vitro cultures were sliced (ca 1 X 1 mm size) and digested in 20 ml of enzyme solution (ES). Four different ESs were formulated by changing the level of Cellulase (Cel) and Macerozyme (Mac; both from Kinki
Yakult Ltd., Japan). ES-1, ES-2, ES-3, a n d ES-4 contained 2.0% Cel a n d 0.4% Mac, 1.0% Cel a n d 0.4% Mac, 2.0% Cel a n d 0.8% Mac, a n d 1.0% Cel a n d 0.8% Mac, respectively. All ESs c o n t a i n e d 1.2% Hemicellulase (Sigma Chem. Co., USA), 2.0% D r i s e l a s e (Kyowa H a k k o , J a p a n ) , 0.05% Pectolyase Y-23 (Seishin Ltd., J a p a n ) , 0.6 M mannitol, a n d CPW s a l t s ( c o n t a i n i n g 5.0 m M C a C 1 2 . 2 H 2 0 , 0.2 ram KH2PO4, 1.0 mlVl KNO3, 0.1 mM MgSO4.7H20, a n d 0.001 mM KI). After 20-min incubation at 30 ~ C on a reciprocal s h a k e r (80 strokes/ min), t h e first ES was replaced w i t h fresh E S of t h e s a m e composition for a n additional 30-min incubation period. This l a t t e r step was repeated two more t i m e s for a t o t a l of four i n c u b a t i o n s . After each i n c u b a t i o n , u n d i g e s t e d debris was r e m o v e d by filtering with a 54 ~'n nylon sieve. The filtrate, containing i n t a c t p r o t o p l a s t s , was c e n t r i f u g e d at 100 xg for 3 min, t h e s u p e r n a t a n t was discarded a n d t h e pellet was r i n s e d with CPW salt solution c o n t a i n i n g 0.6 M mannitol. Several osmotica (0.4 to 0.8 M mannitol) and pH levels (5.4 to 6.0) as well as t h e DTT a n d MES buffer in ES were tested for t h e i r influence on protoplast yield a n d viability (as detailed in Table 1). To collect intact, h e a l t h y protoplasts, t h e filtrate c o n t a i n i n g p r o t o p l a s t s was layered on t h e top of a 0.6 M sucrose solution a n d centrifuged at 80 xg for 5 rain. The p r o t o p l a s t b a n d on t h e top of t h e floating solution was collected u s i n g a p a s t e u r p i p e t t e ( P a r k a n d Son 1988; Russell a n d McCown 1988).
Protoplast culture.
Purified protoplasts were cultured at a density of 2.4 X 105 per ml in culture media (as detailed in Table 2). F o u r culture m e t h o d s were used for each d e v e l o p m e n t a l s t a g e ( s u c h as cell division a n d colony f o r m a t i o n ) of p r o t o p l a s t s : h a n g i n g drop (ca 1 m m in d i a m e t e r ) , liquid drop (ca 6 m m in d i a m e t e r ) , liquid plating, and fabric supported semi-solid agar plating. For the p r e p a r a t i o n of fabric supported semi-solid agar medium, double fold (each in 45 ~ angle) gauze (Sterilized bandage, Dong-A Paramercy, Korea; 2.5 X 2.5 cm with pore size of 0.1 cm) were placed on the top of the 0.5% (w/v) a g a r medium at 45~ u n d e r aseptic conditions. In addition, o t h e r factors such as m e d i u m a n d s u g a r types, a n d different levels of o s m o t i c u m were also t e s t e d to i n v e s t i g a t e t h e i r effects on p r o t o p l a s t division a n d colony formation. E v e r y 7d in culture, a k n o w n volume of fresh liquid culture m e d i u m (0.2 ml/ 2ml), lacking glucose or mannitol, was added to each 6 cm plastic petri dish to dilute t h e total osmotic potential. C u l t u r e s were m a i n t a i n e d in a dark, culture room at 26 _+ l~
regeneration. The growing micro-calli were t r a n s f e r r e d to a c a l l u s p r o l i f e r a t i o n m e d i u m of M S s u p p l e m e n t e d with 0.44 paVl BA and 9.0 pJVl 2,4-D. To obtain a sufficient a m o u n t of callus, t h i s proliferation step was repeated twice. The u p p e r parts of calli were sliced with a b l a d e into small pieces (ca 1 m m in d i a m e t e r ) a n d t h e n t r a n s f e r r e d to r e g e n e r a t i o n m e d i u m of MS s u p p l e m e n t e d with various concentrations of BA, 2iP, a n d zeatin (detailed in Table 3). E a c h of 5 pieces of calli was inoculated onto t h e t e s t media u s i n g glass petri dishes (12 cm in diameter) s e a l e d w i t h p a r a f i l m a n d exposed to l i g h t c o n d i t i o n s previously described for shoot cultures.
respectively. To obtain reliable d a t a a n d facilitate comparison, t h e liquid drop culture m e t h o d was used in all aspects of this study except for t h e culture type experiment. The d a t a for p l a n t r e g e n e r a t i o n from p r o t o p l a s t - d e r i v e d c a l l u s w e r e o b t a i n e d a f t e r 8 w e e k s of c u l t u r e o n r e g e n e r a t i o n media from each of 3 experiments. A n F test was used to detect differences b e t w e e n t r e a t m e n t s , a n d t h e D u n c a n ' s multiple r a n g e test was used to detect differences between levels of each t r e a t m e n t .
Results and Discussion The ANOVA F t e s t on each protoplast isolation t r e a t m e n t revealed highly significant differences at a=O.01. Further analysis to detect differences a m o n g t r e a t m e n t levels was conducted u s i n g D u n c a n ' s multiple r a n g e test. The best protoplast yield (10.4 X 106) was obtained with ES-3 followed by ES-1, ES-4, a n d ES-2 (Table 1). As t h e e n z y m e concentration was increased, t h e yield of protoplast also increased. The same t r e n d was seen with protoplast viability. To obtain intact viable protoplasts (Fig. 1-1), we did not t r e a t longer t h a n 110 min, b e c a u s e of t h e h i g h enzyme levels. Saito (1976) was successful in protoplast isolation from mesophyll cells of Paulownia fortunei a n d Populus euramericana u s i n g a 240 rain incubation period; however, a prolonged incubation period can be d e t r i m e n t a l to protoplast yield a n d viability (Russell a n d McCown 1986). The results of osmotica t r e a t m e n t shows t h a t protoplast yield was g r e a t e s t (10.9 X 106) with 0.6 M m a n n i t o l a n d decreased w h e n lower (0.4 a n d 0.5 M) or h i g h e r (0.7 a n d 0.8 M) m a n n i t o l c o n c e n t r a t i o n s were u s e d (Table 1). These results essentially comply with those of Saito (1976) a n d T a b l e 1. Effect of various factors for the isolation of leaf mesophyll protoplasts of hybrid poplar (Populus nigra X
P. rnaximowiczii ). Treatment
The effects of different types of ESs, osmoticum, pH, as well as DTT a n d IVIES on p r o t o p l a s t yield were e v a l u a t e d with a h e m o c y t o m e t e r a n d protoplast v i a b i l i t y was c o n f i r m e d w i t h 0.2% E v a n s ' blue s t a i n i n g (Kanai a n d E d w a r d s 1973) at t h e t i m e of isolation. The n u m e r i c a l v a l u e s of p r o t o p l a s t yield a n d viability were o b t a i n e d b y a v e r a g i n g 3 r e p l i c a t i o n s of e a c h i s o l a t e d protoplast m i x t u r e in each of 3 experiments. The d a t a for p r o t o p l a s t division a n d colony f o r m a t i o n are expressed as t h e percentage of dividing protoplast a n d cell colonies after 3 days a n d 3 weeks of culture,
Viability 2
E n z y m e solution
ES-1 ES-2 ES~ ES4
7.3b 6.4 c 10.4 a 6.4 c
91.7b3 90.0 c 94.8 a 88.5 c
Osmoticum
0.4M 0.5M 0.6M 0.7M O.8M
3.4 c 3.9bc 10.9 a 4.1b 2.2d
90.5 c 87.7d 95.1 a 92.9b 88.5d
pH
5.4
8.65
90.5ab
5.6 5~ 6.0
11.0a 6.2c 3.4d
92.2 a 9O.5ab 87.5b
Control 4 DTT MES DTT+MES
3.7b 3.9b 4.5 a 5.0a
89.7b 88.2b 94.2a 96.9a
Plant
Data collection.
Yield 1
DTT a n d M E S
1 The yield r e p r e s e n t s t h e total n u m b e r of protoplasts obtain -ed at t h e t im e of isolation (Xl0 6 per g fresh weight). 2 The viability represents t h e percentage of total protoplasts t h a t h a d a spherical intact a p p e a r a n c e without s t a i n i n g (Evans' blue) at t h e time of isolation. 3 M e a n s followed by' different letters are significantly different at a=0.01, following D u n c a n ' s multiple r a n g e test. 4. Control m e a n s ES-3 containing 0.6 M m a n n i t o l at pH 5.6.
Park and Son (1986) using Populus mesophyll cells, and Burger and Hackett (1982) using citrus cotyledonary cells. Although viability was best when 0.6 M mannitol was used, the other mannitol concentrations did not dramatically affect viability. Pretoplast yield and viability were greatest at pH 5.6 and decreased at lower and higher levels (Table 1). Park and Son (1988b) reported similar results with an other hybrid poplar (Populus alba X P. glandulosa). Protoplast yield and viability were increased by supplementing DTT as a reducing agent and IVIES buffer as a pH stabilizer (Table 1). This result is consistent with a previous report (Park and Son 1987). Stabilization of pH by MES was sufficient to achieve this improvement (Table 1). Osmotic potential in the culture medium during early stages of culture significantly affected protoplast stability and division. Osmotic conditions lower or higher than 0.6 M in early culture medium also adversely affected cell stability. Evans and Bravo (1983) also reported the important role of osmoticum in the early culture period. T a b l e 2. Effect of various factors for the culture of leaf mesophyll protoplasts of hybrid poplar (Populus nigra
X P. rnaximowiczii ). Treatment Medium type
Cell div. 1
Colony for. 2
MS (*NH4NO 3) ACM(-NH4NO 3) WPM(-NH4NO 3) B5 8p-KM
46.5 a 26.4c 32.3b 18.4d 44.5a
20.6a 14.5b 18.2ab 6.2c 8.3c
Osmoticum
0.4M 0.5M 0.6M 0.7M
15.0c 23.5b 36.1a 4.9 d
8.2c 12.0b 19.6a 2.1d
Sugar type
0.6M 0.6M 0.6M 0.2M 0.2M 0.4M 0.4M
42.4a 282 d 28.6d 31.0cd 32.4 c 34.2b 35.35
0.7f 2.1e 5.4d 6.2 cd 18.3 a 8.2c 12.2b
3.9d 10.2c 48.6a 16.4b
0,2d 8.2b 6.5c 22.8 a
Culture type
(M)3 (S) (G) (M)+O.4M (S) (M)+O.4M (G) (M)+O.2M (S) (M) 0.2M (G)
Hanging Drop Liquid Drop Liquid Plating FSSA plating
1 The cell div. represents the percentage of dividing protoplasts compared to total protoplasts cultured 3d. 2 The colony div. represents the percentage of colonies compared to total protoplasts cultured. 3 Values followed by different letters are significantly different at a=0.01, following Duncan's multiple range test. 4 Abbreviations for M, S, and G are Mannitol, Sucrose, and Glucose, respectively. When sucrose, glucose, and mannitol were used separa.tely, several cell divisions occurred but cells eventually ceased to divide in our study (Table 2). In contrast, 0.2 M mannitol plus 0.4 M glucose sustained cell division and a high rate of colonies were formed (Fig. 1-2 to 1-6). These findings suggest that there may be a synergistic effect when
glucose and mannitol are used in combination for protoplast culture (Gamborg et al. 1975). From previous results (Park and Son 1986; 1987; 1988a), 0.44 pM BA and 9.0 ~tM 2,4-D were found to be optimum for cell division and colony formation. Some colonies were also obtained in the treatment containing 2.22 IzM BA and 10.74 t ~ I NAA. Necrosis was apparent in cultures originating from media containing ammonium. The deleterious effects of ammonium have been reported in the protoplast culture of Lycopersicon species (Zapata et al. 1981), Broussonetia kazinoki (Hakman and Von Arnold 1983), and hybrid poplar and an aspen clone (Russell and McCown 1986; 1988). The problem was overcome by employing a modified medium in which NH4NO 3 was eliminated completely. This modified medium was suitable for sustained cell division leading to colony formation. Among the tested culture media, ammonium-free MS and WPM media were shown to work the best for colony formation (Table 2). So far, successful plant regeneration from protopIast culture of Populus species has been obtained using modified MS and WPM media (Park and Son 1988a; Lee et al. 1987; Russell and McCown 1986; 1988). In our study, MS was better than WPM for supporting cell division (Table 2). In the experiment using different culture methods for protoplast division and colony formation, we observed a high frequency of cell division in the LPM, but FSPM was T a b l e 3. Effect of cytokinins on shoot regeneration from leaf mesophyll protoplasts derived callus of hybrid poplar
(Populus nigra X P. maxirnowiczii ). Growth regulators (pM)
Number of shoots per calli 2
BA
22 4.4 6.7 89 11.1 13.3 15z 17~
0.0bl 0.3b 0.9a 0.0b 0.0b 0.0b o.0b 0.0b
2iP
2~5 49 7.4 9~ 12.3 14~ 172 19~
0.3 f 3.3a 2.7b 0.7e 0.6e 1.5c 1.3d 0.4 f
Zeatin
2.3 4~ 6~ 9.1 11.4 13.7 16.0 18.2
1.8g 7.1b 8.1a 5.8c 3.2d 2.4 ef 2.1f 2.6f
1 Values followed by different letters are significantly different at a=0.01, following Duncan's multiple range test. 2 Five pieces (ca 1 m m in diameter) were assigned to glass petri dishes (12 c m in diameter) with 3 replications. Data were collected after 8 weeks of culture on regenera -tion media in each of 3 experiments.
Fig.1. Regeneration of plants from protoplast derived callus of Populus nigra X P. maxirnowiczii cultured in vitro. (1) Freshly isolated protoplasts (X180), (2) Enlargement of protoplasts just before cell division (X150), (3-4) Cell division of enlarged protoplasts (X150), (5-6) Colony formation by vigorous cell division (X30), (7) Micro-callus formation after ca 10 weeks of culture, (8) Proliferated callus, (9) Shoot induduction, (10) Regenerated plant. superior for colony formation (Table 2). Cell colonies about 0.1 mm in diameter were obtained after 8 weeks in fabric supported semi-solid agar medium (Fig. 1-7). They were subsequently subcultured on fresh medium of same type for further growth. The most desirable callus growth response also occurred when the medium was supplemented with 0.44 p.M BA and 9.0 ~M 2,4-D. After two subcultures on callus proliferation media (Fig. 1-8), calli of about 1.0 mm in diameter were transferred to a solid regeneration medium. Shoot regeneration occurred with most 2iP and zeatin levels tested (Fig. 1-9), but not on many levels of BA. The highest frequency of shoot formation was achieved with 6.84 ~M zeatin in the shoot induction medium (Table 3). The excised shoots from the surface of callus were rooted on half-strength MS medium lacking growth regulators and then transferred to pots containing artificial soil mix (Fig. 1-10). There was no evident morphological variation among the protoclones but root sprouting ability and growing pattern differences are observed in low frequency. Further experiments to improve the protoplast culture system, analyze isozyme patterns of varied plantlets, and develop a protoplast-mediated transformation system are in progress.
Acknowledgement This research has been supported by a special grant from Korea Science & Engineering Foundation (KOSEP) and a part of the content of this manuscript was reported In: Breeding Research (1990): The Key to the Survival of the Earth, S. Lyama and G. Takeda (eds). Proc. of the 6th Internatl. Congr. of SABRAO. Tsukuba, Japan, pp 849-852. We thank Drs. T. Kameya and A. Saito for their helpful advice on pmtoplast culture, and Dr. Ned B. Klopfenstein for critical reading of this manuscript.
References Ahuja MR (1982) Silvae Genet 31(2-3):66-67 Burger DW, Hackett WP (1982) Physiol Plant 56:324-328 Evans DA, Bravo JE (1983) In: Evans DA, Sharp WA Ammirato DV, Yamada (eds) Handbook of Plant Cell Culture Vol.1 New York, Macmillan Publishing Co., pp 125-176
6 Chun YW (1985) J Kor For Soc 71:45-49 Gamborg OL, Shyluk JP, Kartha KK (1975) Plant Sci Lett 4"~285-292
Park YG, Son SH (1988b) In: Hanover JW, Keathley DE (eds) Genetic Manipulation of Woody Plants.Vol 44. Plenum Press, New York and London pp 483 Park YG, Son, SH, Hall RB (1990) Kor J Genet 12:167-189
Gelba YY (1978) Naturwissenschaften 65:158-159 Gupta PP (1986) Plant Sci 43:151-154 Hakman IC, Von Arnold S (1983) Plant Cell Rep 2:92-94
Potrykus I (1980) In: Advances in Protoplast Research. Akad Kiado Budapest pp 243-254. Potrykus I, Marms CT, Lorz H {1979) Plant Sci Lett 14:231239
Kanai R, Edwards GB (1973) Plant Physiol 52:484-490 Raveh D, Huberman E, Galun E (1973) In Vitro 9:216 Lee JK, Lee SK, Jang SS, Lee JJ (1987) Kor Inst For Genet Res Pep 23:143-148
Rona JP, Grignon C (1972) C R Acad Sci D 247:2976-2979
Park YG, Son SH (1986) J Kor For Soc 74:29-36
Russell JA, McCown BH (1986) Plant Sci 46:133-142
Park YG, Son SH (1987) Kor J Genet 9:133-140
Russell JA, McCown BH (1988) Plant Cell Rep 7:59-62
Park YG, SOn SH (1988a) J Kor For SOc 77 : 208-215
Saito A (1976) J Jap For Sci 58:301-305 Vasil V, Vasil K (1979) Z. Pflanzenphysiol 92:379-383 Zapata FJ, Sink KC, Cooking EC (1981) Plant Sci Lett 23:4-46
Plant Cell Reports (1992) 11: 2 - 6
9 Springer-Verlag1992
In vitro shoot regeneration from leaf mesophyH protoplasts of hybrid poplar (Populus nigra x P. maximowiczil) Young Goo Park 1 and Sung Ho Son 2 1 Department of Forestry, College of Agriculture, Kyungpook National University, Daege 702-701, Korea 2 Department of Forestry, Iowa State University, Ames, IA 50011-1021 USA Received May 17, 1991/Revised version received December 5, 1991
Summary.
Protoplasts were isolated from leaf mesophyll of hybrid poplar (Populus nigra X P. maximowiczii) with a mean yield of 10.4 X 106 protoplasts per g fresh weight using 2.0% Cellulase 'Onozuka' R-10, 0.8% Macerozyme R10, 1.2% Hemice]lulase, 2.0% Driselase, and 0.05% Pectolyase Y-23 with CPW salts solution containing 0.6 M mannitol, 0.002 M DTT, 3 mM MES at pH 5.6. A liquid plating method produced the highest frequency of dividing protoplasts (48.6%) using an MS medium without NH4NO 3. The highest percent of colony formation was 22.8%, produced with fabric supported semi-solid (0.5% w/v) agar plating method using the same culture medium. Growing cell colonies and/or micro-calli were transferred to a fresh semisolid agar medium containing 0.44 pNi BAP and 9.0 p_M 2,4D. Multiple shoats were produced from protoplast-derived callus after culture on MS medium containing 6.8 }~M zeatin. After root induction on half-strength MS medium that lacked growth regulators, shoots were transferred to pots containing artificial soil mix.
Abbreviations:
cPw, Cell and Potoplast Wash solution; LPM, Liquid Plating Method; LDM, Liquid Drop Method; HDM, Hanging Drop Method; FSPM, Fabric supported Semi-solid agar P l a t i n g Method; DTT, Dithiothreitol; MES, 2-(N-morpholino) ethane sulfonic acid; BAP, 6-benzylaminopurine; 2,4-D, 2,4-dichlorophenoxy acetic acid; NAA, a - n a p h t h a l e n e acetic acid; MS, Murashige and Skoog (1962). Introduction For the purposes of genetic modification and physiological study of plant cells, protoplasts can serve as valuable experimental material (Park et al. 1990). Since the isolation of protoplasts from tree species was first reported in Acer pseudoplatanus by Rona and Grignon (1972), there have been many reports of protoplast isolation from forest tree species (Ahuja 1982; Chun 1985; Gupta 1986; Park and Son 1986). In recent years, progress has been made with the subsequent reformation of whole, dividing cells as well as successful establishment of regeneration systems from protoplasts of woody plants (Park and Son 1988a). Various methods such as liquid plating culture (Potrykus 1980), liquid drop culture (Vasil and Vasil 1979), hanging drop culture (Potrykus et al. 1979), micro droplet culture (Gelba 1978), as well as different feeder layer techniques (Raveh et al. 1973) have been adapted to plant
Offprint requests to. Y. G. Park
Communicated by G. C. Phillips
protoplast culture. In addition, a floating polyester screen disc method has been successfully demonstrated with poplar leaf protoplasts isolated from non-seedling tissue (Russell and McCown 1986). However, most of the previously reported protoplast culturing systems largely depend on the unique techniques developed by individual laboratories. No comparisons have been made among different culturing methods using similar donor cells and tissue derived protoplasts. In this paper, we report some critical factors for protoplast isolation and other important aspects of various culturing methods for each developmental stage of protoplasts from this hybrid poplar.
Materials and Methods Plant materials. Dormant branches (1-2 years old) bearing apical and axillary buds were collected during March, 1985 from same ortet of the upper crown of 20-yearold Populus nigra X P. maximowiczii at the Institute of Forest Genetics, Suwon, Korea. The shoot parts (3-4 cm) were sterilized using the method of Park and Son (1986). For sprouting shoots, the bud parts were plated on MS medium containing 0.05 ~VI NAA. After shoots were initiated, they were transferred to shoot multiplication medium containing MS basal salts, 0.3 p_M thiamine ttCI, 4.1 p~V[ nicotinic acid, 2.4 pM pyridoxine HC1, 26.6 ~Vl glycine, 108.2 ~M myo-inositol, 3% (w/v) sucrose, 0.75% (w/v) Difco bacto-agar, and 0.89 ~M BAP. After 5 subcultures, at approximately 4-week intervals, multiplied individual shoots were subcultured on half-strength MS medium lacking growth regulators for shoot elongation. Cultures for leaf expanding were maintained in 500 ml of transparent glass bottle (I.V. bottle) containing 50 ml of medium. Fully expanded leaves (aging from 6 to 8 weeks of culture) were used as the source material for protoplast isolation. The pH of the medium was adjusted to 5.8 prior to autoclaving at 121 ~ C for 15 rain. Cultures were maintained in a thermostatically controlled culture room with a photoperiod of 16 h, a photosynthetically active photon flux rate of 20-40 p.E m -2 s "1 and a constant temperature of 26+ 1~ C. P r o t o p l a s t isolation. One g fresh weight of expanded, healthy leaves from in vitro cultures were sliced (ca 1 X 1 mm size) and digested in 20 ml of enzyme solution (ES). Four different ESs were formulated by changing the level of Cellulase (Cel) and Macerozyme (Mac; both from Kinki
Yakult Ltd., Japan). ES-1, ES-2, ES-3, a n d ES-4 contained 2.0% Cel a n d 0.4% Mac, 1.0% Cel a n d 0.4% Mac, 2.0% Cel a n d 0.8% Mac, a n d 1.0% Cel a n d 0.8% Mac, respectively. All ESs c o n t a i n e d 1.2% Hemicellulase (Sigma Chem. Co., USA), 2.0% D r i s e l a s e (Kyowa H a k k o , J a p a n ) , 0.05% Pectolyase Y-23 (Seishin Ltd., J a p a n ) , 0.6 M mannitol, a n d CPW s a l t s ( c o n t a i n i n g 5.0 m M C a C 1 2 . 2 H 2 0 , 0.2 ram KH2PO4, 1.0 mlVl KNO3, 0.1 mM MgSO4.7H20, a n d 0.001 mM KI). After 20-min incubation at 30 ~ C on a reciprocal s h a k e r (80 strokes/ min), t h e first ES was replaced w i t h fresh E S of t h e s a m e composition for a n additional 30-min incubation period. This l a t t e r step was repeated two more t i m e s for a t o t a l of four i n c u b a t i o n s . After each i n c u b a t i o n , u n d i g e s t e d debris was r e m o v e d by filtering with a 54 ~'n nylon sieve. The filtrate, containing i n t a c t p r o t o p l a s t s , was c e n t r i f u g e d at 100 xg for 3 min, t h e s u p e r n a t a n t was discarded a n d t h e pellet was r i n s e d with CPW salt solution c o n t a i n i n g 0.6 M mannitol. Several osmotica (0.4 to 0.8 M mannitol) and pH levels (5.4 to 6.0) as well as t h e DTT a n d MES buffer in ES were tested for t h e i r influence on protoplast yield a n d viability (as detailed in Table 1). To collect intact, h e a l t h y protoplasts, t h e filtrate c o n t a i n i n g p r o t o p l a s t s was layered on t h e top of a 0.6 M sucrose solution a n d centrifuged at 80 xg for 5 rain. The p r o t o p l a s t b a n d on t h e top of t h e floating solution was collected u s i n g a p a s t e u r p i p e t t e ( P a r k a n d Son 1988; Russell a n d McCown 1988).
Protoplast culture.
Purified protoplasts were cultured at a density of 2.4 X 105 per ml in culture media (as detailed in Table 2). F o u r culture m e t h o d s were used for each d e v e l o p m e n t a l s t a g e ( s u c h as cell division a n d colony f o r m a t i o n ) of p r o t o p l a s t s : h a n g i n g drop (ca 1 m m in d i a m e t e r ) , liquid drop (ca 6 m m in d i a m e t e r ) , liquid plating, and fabric supported semi-solid agar plating. For the p r e p a r a t i o n of fabric supported semi-solid agar medium, double fold (each in 45 ~ angle) gauze (Sterilized bandage, Dong-A Paramercy, Korea; 2.5 X 2.5 cm with pore size of 0.1 cm) were placed on the top of the 0.5% (w/v) a g a r medium at 45~ u n d e r aseptic conditions. In addition, o t h e r factors such as m e d i u m a n d s u g a r types, a n d different levels of o s m o t i c u m were also t e s t e d to i n v e s t i g a t e t h e i r effects on p r o t o p l a s t division a n d colony formation. E v e r y 7d in culture, a k n o w n volume of fresh liquid culture m e d i u m (0.2 ml/ 2ml), lacking glucose or mannitol, was added to each 6 cm plastic petri dish to dilute t h e total osmotic potential. C u l t u r e s were m a i n t a i n e d in a dark, culture room at 26 _+ l~
regeneration. The growing micro-calli were t r a n s f e r r e d to a c a l l u s p r o l i f e r a t i o n m e d i u m of M S s u p p l e m e n t e d with 0.44 paVl BA and 9.0 pJVl 2,4-D. To obtain a sufficient a m o u n t of callus, t h i s proliferation step was repeated twice. The u p p e r parts of calli were sliced with a b l a d e into small pieces (ca 1 m m in d i a m e t e r ) a n d t h e n t r a n s f e r r e d to r e g e n e r a t i o n m e d i u m of MS s u p p l e m e n t e d with various concentrations of BA, 2iP, a n d zeatin (detailed in Table 3). E a c h of 5 pieces of calli was inoculated onto t h e t e s t media u s i n g glass petri dishes (12 cm in diameter) s e a l e d w i t h p a r a f i l m a n d exposed to l i g h t c o n d i t i o n s previously described for shoot cultures.
respectively. To obtain reliable d a t a a n d facilitate comparison, t h e liquid drop culture m e t h o d was used in all aspects of this study except for t h e culture type experiment. The d a t a for p l a n t r e g e n e r a t i o n from p r o t o p l a s t - d e r i v e d c a l l u s w e r e o b t a i n e d a f t e r 8 w e e k s of c u l t u r e o n r e g e n e r a t i o n media from each of 3 experiments. A n F test was used to detect differences b e t w e e n t r e a t m e n t s , a n d t h e D u n c a n ' s multiple r a n g e test was used to detect differences between levels of each t r e a t m e n t .
Results and Discussion The ANOVA F t e s t on each protoplast isolation t r e a t m e n t revealed highly significant differences at a=O.01. Further analysis to detect differences a m o n g t r e a t m e n t levels was conducted u s i n g D u n c a n ' s multiple r a n g e test. The best protoplast yield (10.4 X 106) was obtained with ES-3 followed by ES-1, ES-4, a n d ES-2 (Table 1). As t h e e n z y m e concentration was increased, t h e yield of protoplast also increased. The same t r e n d was seen with protoplast viability. To obtain intact viable protoplasts (Fig. 1-1), we did not t r e a t longer t h a n 110 min, b e c a u s e of t h e h i g h enzyme levels. Saito (1976) was successful in protoplast isolation from mesophyll cells of Paulownia fortunei a n d Populus euramericana u s i n g a 240 rain incubation period; however, a prolonged incubation period can be d e t r i m e n t a l to protoplast yield a n d viability (Russell a n d McCown 1986). The results of osmotica t r e a t m e n t shows t h a t protoplast yield was g r e a t e s t (10.9 X 106) with 0.6 M m a n n i t o l a n d decreased w h e n lower (0.4 a n d 0.5 M) or h i g h e r (0.7 a n d 0.8 M) m a n n i t o l c o n c e n t r a t i o n s were u s e d (Table 1). These results essentially comply with those of Saito (1976) a n d T a b l e 1. Effect of various factors for the isolation of leaf mesophyll protoplasts of hybrid poplar (Populus nigra X
P. rnaximowiczii ). Treatment
The effects of different types of ESs, osmoticum, pH, as well as DTT a n d IVIES on p r o t o p l a s t yield were e v a l u a t e d with a h e m o c y t o m e t e r a n d protoplast v i a b i l i t y was c o n f i r m e d w i t h 0.2% E v a n s ' blue s t a i n i n g (Kanai a n d E d w a r d s 1973) at t h e t i m e of isolation. The n u m e r i c a l v a l u e s of p r o t o p l a s t yield a n d viability were o b t a i n e d b y a v e r a g i n g 3 r e p l i c a t i o n s of e a c h i s o l a t e d protoplast m i x t u r e in each of 3 experiments. The d a t a for p r o t o p l a s t division a n d colony f o r m a t i o n are expressed as t h e percentage of dividing protoplast a n d cell colonies after 3 days a n d 3 weeks of culture,
Viability 2
E n z y m e solution
ES-1 ES-2 ES~ ES4
7.3b 6.4 c 10.4 a 6.4 c
91.7b3 90.0 c 94.8 a 88.5 c
Osmoticum
0.4M 0.5M 0.6M 0.7M O.8M
3.4 c 3.9bc 10.9 a 4.1b 2.2d
90.5 c 87.7d 95.1 a 92.9b 88.5d
pH
5.4
8.65
90.5ab
5.6 5~ 6.0
11.0a 6.2c 3.4d
92.2 a 9O.5ab 87.5b
Control 4 DTT MES DTT+MES
3.7b 3.9b 4.5 a 5.0a
89.7b 88.2b 94.2a 96.9a
Plant
Data collection.
Yield 1
DTT a n d M E S
1 The yield r e p r e s e n t s t h e total n u m b e r of protoplasts obtain -ed at t h e t im e of isolation (Xl0 6 per g fresh weight). 2 The viability represents t h e percentage of total protoplasts t h a t h a d a spherical intact a p p e a r a n c e without s t a i n i n g (Evans' blue) at t h e time of isolation. 3 M e a n s followed by' different letters are significantly different at a=0.01, following D u n c a n ' s multiple r a n g e test. 4. Control m e a n s ES-3 containing 0.6 M m a n n i t o l at pH 5.6.
Park and Son (1986) using Populus mesophyll cells, and Burger and Hackett (1982) using citrus cotyledonary cells. Although viability was best when 0.6 M mannitol was used, the other mannitol concentrations did not dramatically affect viability. Pretoplast yield and viability were greatest at pH 5.6 and decreased at lower and higher levels (Table 1). Park and Son (1988b) reported similar results with an other hybrid poplar (Populus alba X P. glandulosa). Protoplast yield and viability were increased by supplementing DTT as a reducing agent and IVIES buffer as a pH stabilizer (Table 1). This result is consistent with a previous report (Park and Son 1987). Stabilization of pH by MES was sufficient to achieve this improvement (Table 1). Osmotic potential in the culture medium during early stages of culture significantly affected protoplast stability and division. Osmotic conditions lower or higher than 0.6 M in early culture medium also adversely affected cell stability. Evans and Bravo (1983) also reported the important role of osmoticum in the early culture period. T a b l e 2. Effect of various factors for the culture of leaf mesophyll protoplasts of hybrid poplar (Populus nigra
X P. rnaximowiczii ). Treatment Medium type
Cell div. 1
Colony for. 2
MS (*NH4NO 3) ACM(-NH4NO 3) WPM(-NH4NO 3) B5 8p-KM
46.5 a 26.4c 32.3b 18.4d 44.5a
20.6a 14.5b 18.2ab 6.2c 8.3c
Osmoticum
0.4M 0.5M 0.6M 0.7M
15.0c 23.5b 36.1a 4.9 d
8.2c 12.0b 19.6a 2.1d
Sugar type
0.6M 0.6M 0.6M 0.2M 0.2M 0.4M 0.4M
42.4a 282 d 28.6d 31.0cd 32.4 c 34.2b 35.35
0.7f 2.1e 5.4d 6.2 cd 18.3 a 8.2c 12.2b
3.9d 10.2c 48.6a 16.4b
0,2d 8.2b 6.5c 22.8 a
Culture type
(M)3 (S) (G) (M)+O.4M (S) (M)+O.4M (G) (M)+O.2M (S) (M) 0.2M (G)
Hanging Drop Liquid Drop Liquid Plating FSSA plating
1 The cell div. represents the percentage of dividing protoplasts compared to total protoplasts cultured 3d. 2 The colony div. represents the percentage of colonies compared to total protoplasts cultured. 3 Values followed by different letters are significantly different at a=0.01, following Duncan's multiple range test. 4 Abbreviations for M, S, and G are Mannitol, Sucrose, and Glucose, respectively. When sucrose, glucose, and mannitol were used separa.tely, several cell divisions occurred but cells eventually ceased to divide in our study (Table 2). In contrast, 0.2 M mannitol plus 0.4 M glucose sustained cell division and a high rate of colonies were formed (Fig. 1-2 to 1-6). These findings suggest that there may be a synergistic effect when
glucose and mannitol are used in combination for protoplast culture (Gamborg et al. 1975). From previous results (Park and Son 1986; 1987; 1988a), 0.44 pM BA and 9.0 ~tM 2,4-D were found to be optimum for cell division and colony formation. Some colonies were also obtained in the treatment containing 2.22 IzM BA and 10.74 t ~ I NAA. Necrosis was apparent in cultures originating from media containing ammonium. The deleterious effects of ammonium have been reported in the protoplast culture of Lycopersicon species (Zapata et al. 1981), Broussonetia kazinoki (Hakman and Von Arnold 1983), and hybrid poplar and an aspen clone (Russell and McCown 1986; 1988). The problem was overcome by employing a modified medium in which NH4NO 3 was eliminated completely. This modified medium was suitable for sustained cell division leading to colony formation. Among the tested culture media, ammonium-free MS and WPM media were shown to work the best for colony formation (Table 2). So far, successful plant regeneration from protopIast culture of Populus species has been obtained using modified MS and WPM media (Park and Son 1988a; Lee et al. 1987; Russell and McCown 1986; 1988). In our study, MS was better than WPM for supporting cell division (Table 2). In the experiment using different culture methods for protoplast division and colony formation, we observed a high frequency of cell division in the LPM, but FSPM was T a b l e 3. Effect of cytokinins on shoot regeneration from leaf mesophyll protoplasts derived callus of hybrid poplar
(Populus nigra X P. maxirnowiczii ). Growth regulators (pM)
Number of shoots per calli 2
BA
22 4.4 6.7 89 11.1 13.3 15z 17~
0.0bl 0.3b 0.9a 0.0b 0.0b 0.0b o.0b 0.0b
2iP
2~5 49 7.4 9~ 12.3 14~ 172 19~
0.3 f 3.3a 2.7b 0.7e 0.6e 1.5c 1.3d 0.4 f
Zeatin
2.3 4~ 6~ 9.1 11.4 13.7 16.0 18.2
1.8g 7.1b 8.1a 5.8c 3.2d 2.4 ef 2.1f 2.6f
1 Values followed by different letters are significantly different at a=0.01, following Duncan's multiple range test. 2 Five pieces (ca 1 m m in diameter) were assigned to glass petri dishes (12 c m in diameter) with 3 replications. Data were collected after 8 weeks of culture on regenera -tion media in each of 3 experiments.
Fig.1. Regeneration of plants from protoplast derived callus of Populus nigra X P. maxirnowiczii cultured in vitro. (1) Freshly isolated protoplasts (X180), (2) Enlargement of protoplasts just before cell division (X150), (3-4) Cell division of enlarged protoplasts (X150), (5-6) Colony formation by vigorous cell division (X30), (7) Micro-callus formation after ca 10 weeks of culture, (8) Proliferated callus, (9) Shoot induduction, (10) Regenerated plant. superior for colony formation (Table 2). Cell colonies about 0.1 mm in diameter were obtained after 8 weeks in fabric supported semi-solid agar medium (Fig. 1-7). They were subsequently subcultured on fresh medium of same type for further growth. The most desirable callus growth response also occurred when the medium was supplemented with 0.44 p.M BA and 9.0 ~M 2,4-D. After two subcultures on callus proliferation media (Fig. 1-8), calli of about 1.0 mm in diameter were transferred to a solid regeneration medium. Shoot regeneration occurred with most 2iP and zeatin levels tested (Fig. 1-9), but not on many levels of BA. The highest frequency of shoot formation was achieved with 6.84 ~M zeatin in the shoot induction medium (Table 3). The excised shoots from the surface of callus were rooted on half-strength MS medium lacking growth regulators and then transferred to pots containing artificial soil mix (Fig. 1-10). There was no evident morphological variation among the protoclones but root sprouting ability and growing pattern differences are observed in low frequency. Further experiments to improve the protoplast culture system, analyze isozyme patterns of varied plantlets, and develop a protoplast-mediated transformation system are in progress.
Acknowledgement This research has been supported by a special grant from Korea Science & Engineering Foundation (KOSEP) and a part of the content of this manuscript was reported In: Breeding Research (1990): The Key to the Survival of the Earth, S. Lyama and G. Takeda (eds). Proc. of the 6th Internatl. Congr. of SABRAO. Tsukuba, Japan, pp 849-852. We thank Drs. T. Kameya and A. Saito for their helpful advice on pmtoplast culture, and Dr. Ned B. Klopfenstein for critical reading of this manuscript.
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