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Planta (Berl.) 99, 12-20 (1971) 9 by Springer-Verlag 1971

Plating of Isolated Tobacco Mesophyll Protoplasts on Agar Medium TOSHIYUKI NAGATA a n d ITARU TAKEBE Institute for Plant Virus l~esearch, Chiba, Japan Received April 5, 1971 Summary. A technique was developed to derive cell and plant clones from isolated mesophyll protoplasts of tobacco. The protoplasts, plated on a fully defined agar medium, divided and grew actively forming visible colonies after one month of culture. Efficiency of colony formation depended on cell density and light condition during incubation. Under standard conditions, 60 % of plated protoplasts formed colonies. Upon transfer onto suitable media, these colonies differentiated shoots and roots, and eventually regenerated whole plants. Advantages of mesophyll protoplasts as the source of clones as well as implication of the plating technique for genetical studies are discussed. Introduction P r e v i o u s w o r k from this l a b o r a t o r y showed t h a t single m e s o p h y l l cells i s o l a t e d e n z y m a t i e a l l y f r o m t o b a c c o leaves d i v i d e a n d grow in vitro (Usui a n d T a k e b e , 1969). N a k e d p r o t o p l a s t s of these ceils also u n d e r w e n t cell division after t h e y h a d r e g e n e r a t e d cell walls ( N a g a t a a n d T a k e b e , 1970). Since these cells are r e a d i l y o b t a i n e d as a p u r e p o p u l a t i o n of single cells a n d in v e r y large n u m b e r s , t h e y should p r o v i d e an excellent s u b s t i t u t e for c u l t u r e d ceils which h a v e h i t h e r t o been t h e o n l y a v a i l a b l e source of p l a n t cell clones. I n d i v i d u a l m e s o p h y l l p r o t o p l a s t s from t o b a c c o were r e c e n t l y shown to be c a p a b l e of growing on a n a g a r m e d i u m t o f o r m colonies (Takebe et al., 1971). H o w e v e r , t h e culture m e d i u m used was r a t h e r complex, a n d o n l y a small f r a c t i o n of t h e p l a t e d p r o t o p l a s t s y i e l d e d colonies. I n t h e p r e s e n t p a p e r we r e p o r t a n i m p r o v e d p l a t i n g m e t h o d a n d a fully defined m e d i u m which s u p p o r t vigorous colony formation by tobacco mesophyll protoplasts.

Materials and Methods Isolation o] Protoplasts. Protoplasts of palisade cells were aseptically isolated from leaves of Nicotiana tabacum L. according to the method previously described (Nagata and Takebe, 1970). Cultivar Xanthi nc was used throughout this study, although some experiments were also performed with cultivars Xanthi and Samsun. Unless otherwise stated, the results refer to the protoplasts from Xanthi nc. Inoculation and Incubation o/Plates. Freshly isolated protoplasts were suspended in the medium of Table 1 at a cell denstiy of 1-1.5 • 104 m1-1, as measured with a

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Table 1. Composition o] the culture medium a A. Mineral salts Major elements (rag/l) b

Minor elements (rag/l) c

NH,NO 3 825 KNO~ 950 CaC12 92H20 220 MgSO 4-7H20 1233 KH~P04 680 Na~-EDTA 37.3 FeSO 4 97H30 27.8

H3BO a 6.2 MnSO C 4H20 22.3 ZnSO 4 - 4H20 8.6 KI 0.83 Na~Mo0a'2H20 0.25 CuSO~" 5H20 0.025 CoSO 4. 7H~O 0.030

B. Organic constituents Sucrose

meso-Inositol Thiamine. HC1 1-NaphthMeneacetic acid 6-Benzylaminopurine D-Mannitol

10 g/1 100 rag/1 1 rag/1 3 rag/1 1 rag/1 0.7 M

a pH was adjusted with K O H to 5.8 before autoclaving. b Modified from Murashige and Skoog (1962). c According to Murashige and Skoog (1962) except that CoSO 4 was substituted for CoC12.

Coulter Electronic Counter (Aoki and Takebe, 1969). The suspension was mixed gently but quickly with an equal volume of melted agar medium (1.2% agar in the medium of Table 1) which had been kept at 45 ~ and 5-ml aliquots were poured into glass Petri dishes of 6 cm diameter. The cell density in the plates was thus 5-7.5 • 10 a m1-1. The dishes were sealed with Parafilm M and were placed upside down in an incubator of 28 ~ C. Continuous illumination was provided at 2300 lux with light from white fluorescent tubes. At least three replicate plates were prepared for each experiment. Estimatio~ o/Plating E/]iciency. Plating efficiency was defined as the percentage of protoplasts which formed visible colonies after one month. Since colonies were evenly distributed and were usuulIy very large in number (see Figs. 7-9), at least 500 cells or colonies in randomly selected areas of plates were counted to calculate plating efficiency.

Results Development o/ Colonies. Cell d i v i s i o n of t h e p r o t o p ] a s t s e m b e d d e d in t h e a g a r m e d i u m c o u l d be s e e n b e g i n n i n g on t h e 3rd d a y of c u l t u r e (Fig. 1). I n c r e a s e in cell v o l u m e as well as c h a n g e in t h e a r r a n g e m e n t of c h l o r o p l a s t s p r e c e e d e d cell d i v i s i o n , as was p r e v i o u s l y n o t i c e d in p r o t o p l a s t s c u l t u r e d in s u s p e n s i o n ( N a g a t a a n d T a k e b e , 1970). P r e s u m a b l y , t h e p r o t o p l a s t s h a d also r e g e n e r a t e d cell walls. T h e s e c o n d r o u n d of

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T. Nagata and I. Takebe: Plating of Isolated Protoplasts

division took place on and after the 5th day, and was followed by successive divisions resulting in the formation of masses of cells (Figs. 2, 3). The cells in this stage appeared to be rich in cytoplasm and with thin walls, so t h a t the boundaries between neighboring cells were not very distinct (Fig. 3). This situation, together with the 3-dimensional arrangement of the cells, made it difficult to count the number of cells in a mass. Chloroplasts also became less and less distinct, and were difficult to discern after several cell divisions. The cells contained, however, a number of gTanules which showed red fluorescence under near-ultraviolet light. A similar behavior of chloroplasts was reported in the actively dividing mesophy]l cells of Macleaya cultured in vitro (Kohlenbach, 1966). More than 70 % of the isolated, cultured protoplasts divided at least once during the first week of culture. Within 2 weeks practically all of the surviving protoplasts underwent division and some developed into small but visible colonies (Fig. 4). However, some of the initially living protoplasts had ejected their cytoplasm (Fig. 6) and died by this time without undergoing cell division. After 3 weeks of culture, the majority of the masses of progeny cells which originated from the individual protoplasts grew into readily visible colonies of light-green color (Fig. 5). The cells in these colonies were now more vacuolated and showed clearer boundary than in the earlier stages of colony formation. Macroscopic growth of these colonies during the succeeding weeks is illustrated in Figs. 7-9. Over 80 % of the colonies were 0.3-0.8 m m large after 4 weeks of culture (Fig. 10). Most of them reached a size of 0.5-1 m m after 6 weeks, and were now ready for transfer onto a differentiation medium (see below). The colonies could also be repeatedly subeultured as callus masses on, for example, Murashige and Skoog's medium (1962) with 1-naphthaleneacetic acid (3 rag/l) and 6-benzylaminopurine (1 rag/l). Plating E//iciency. Since no colony became newly countable after 1 month, plating efficiency was determined at this time. Under the standard condition of plating (see Materials and Methods) the efficiency averaged 60%. The percentage of colony-forming protoplasts varied, however, according to the conditions of inoculation and incubation. Among other factors, the cell density of the inoeulum had a marked influence on the plating efficiency, as had also been reported for the

Figs. 1-5. Successive stages of colony formation by tobacco mesophyll protoplasts embedded in agar medium. Figs. 1, 2 • 416, Figs. 3, 4 • 238, Fig. 5 • 108 Fig. 6. Protoplast ejecting cytoplasm. • 238 Figs. 7-9. Growth of colonies from tobacco mesophyll protoplasts. The same plate was photographed after 3 (Fig. 7), 4 (Fig. 8) ~nd 6 (Fig. 9) weeks of incubation. • 1.0

Figs. I-9

T. Nagata and I. Takebe:

16

~o

~3o "520

0

0.2 Q~ Oiometer

0.6

0.8

tO

12.

of colony (ram}

Fig. 10. Size distribution of colonies in 4-week-old plate Table 2. Influence o] cell density on plating e]]iciency Cell density in plate (cells/ml)

Plating efficiency (%)

Experiment 1

1.0 • 10a 3.0 • 102 1.0 • 102

49 0a 0

Experiment 2

1.2 X 104 7.3 X 10a 2.5 X 108 1.5 X 108

54 41 0a 0

Experiment 3

1.9 • 5.6 • 3.8 • 1.9 x 5.6 •

68 67 43 38 0a

104 l0 s 108 l0 s l0 S

a 10-30% of the protoplasts divided once or a few times.

p l a t i n g of c u l t u r e d cells (Blakely a n d Steward, 1964a; Gibbs a n d Dougall, 1965) ; t h e p l a t i n g efficiency of p r o t o p l a s t s d r o p p e d w i t h decreasing cell d e n s i t y (Table 2). N o colonies a t all were f o r m e d when p r o t o p l a s t s were p l a t e d below a certain d e n s i t y . This critical d e n s i t y v a r i e d somew h a t from one e x p e r i m e n t to a n o t h e r b u t was a r o u n d 1 • 103 m1-1 (Table 2). A t densities slightly below this level, some p r o t o p l a s t s underw e n t several cell divisions b u t failed to grow further. The p l a t i n g efficiency of t h e i s o l a t e d p r o t o p l a s t s was also influenced b y t h e m e t h o d of plating. F o r e x a m p l e , t h e d o u b l e - l a y e r m e t h o d emp l o y e d p r e v i o u s l y (Takebe et al., 1971) c o n s i s t e n t l y gave m u c h lower

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yield of colonies than the single layer method of the present study. This observation, as well as the marked dependency of plating efficiency on cell density, suggested that some factor(s) produced by the protoplasts is (are) necessary for their division and growth. When an agar layer without cells underlies the one containing protoplasts, or when the cell density in the plate is not high enough, leakage and diffusion into medium apparently make the cells deficient for this factor, thus resulting in cessation of cell growth. Based on this reasoning, some efforts were made to enhance colony formation by supplementing the medium with amino acids or growth regulators. No improvement of plating efficiency was obtained, however, with the following substances added individually to the medium (in rag/l): easamino acids (500), arginine, tiC1 (210), gibberrellic acid (1.0), 2-chloroethylphosphonie acid (4.1). Light intensity during incubation also affected colony formation by the plated protoplasts. Thus, when the plates were illuminated at 700 lux, their plating efficiency was less than half as high as that of the plates incubated under the standard light condition (2300 lux). The colony size was also much smaller in the former plates. On the other hand, colony formation was not promoted by raising the light intensity to 5000 lux. Colonies formed under the strong light were much less colored in green.

Regeneration o] Whole Plants. When the colonies formed by protoplasts grew to the size of 0.5-1 mm, individual colonies were picked out with a dentist's scalpel and were placed on agar plates containing the B 3 medium of Sacrists and Melehers (1969). The plates were incubated at 28 ~ C under continuous light of 700 lux. The colonies grew actively on this medium first as callus masses (Fig. 11) and then in about 3 weeks differentiated many shoots, mostly without roots (Fig. 12). These shoots subsequently produced roots on White's basal medium (White, 1963) containing agar, and developed into plantlets (Fig. 13) which could be transplanted into soil and grown in a greenhouse. The tobacco plants thus regenerated from individual mesophyll protoplats appeared to be normal in their gross morphology. Other Observations. Our procedure for preparing protoplasts involves the isolation of palisade cells (Nagata and Takebe, 1970). These isolated palisade cells formed also colonies, but in lower frequencies than their protoplasts. In one experiment in which the plating efficiency of protoplasts was over 50%, only 5% or less of palisade cells yielded colonies under identical conditions. Colony formation by palisade cells could be enhanced to some extent by lowering the mannitol concentration in the medium to 0.5 M. Nevertheless, the highest plating efficiency so far obtained with palisade cells did not exceed 40 %. 2

P l a n t a (Berl.), Bd. 99

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T, Nagata and I. Takebe:

Fig. 11. Colonies transferred onto a plate of B~ medium and growing as callus masses. An indication of shoot differentiation is seen in some individuals. • 0.6 Fig. i2. Shoots differentiated from callus masses on B3 medium. • 0.6 Fig. I3. Plantlet of tobacco regenerated from single mesophyll protoplast. • 0.8

Plating experiments were also extended to protoplasts from other cultivars of tobacco. Growth of the protoplasts from Xanthi was very similar to that of Xanthi nc protoplasts. I n contrast, the protoplasts from Samsun grew less vigorously under the present conditions, yielding a lower plating efficiency (22 % ) than the protoplasts from the other two cultivars. Whole plants were regenerated also from the protoplasts of Xanthi and Samsun.

Discussion Since the work of Bergmann (1960), single plant cells from various cultured tissues were plated to form colonies on agar media and thus yielding cell clones (Blakcly and Steward, 1964a; Earle and Torrey, 1965; Gibbs and Dougall, 1965; Eriksson, 1965). The usefulness oi this technique for establishing cell clones is, however, limited because of certain disadvantages of cultured cells which will be discussed below. We now showed that single, somatic cells directly isolated from the plant body are capable of forming colonies. The mesophyll protoplasts used in this study seem to have definite advantages over the cultured cells as the source of clones. Thus, the protoplasts are readily obtainable in very

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large numbers and, at the same time, as a population consisting entirely of single cells, which is not the ease for cultured cells. The present study showed that the protoplasts ean be plated in high effieieneies, while the frequency of colony formation by cultured single cells tends to be very low (Bergmann, 1960; Blakely and Steward, 1964a; Earle and Torrey, 1965). Finally, the mesophyll cells appear to be quite uniform with respect to chromosome number, virtually all the cells being diploid (Motoyoshi, unpubl.). This is in marked contrast to the wide range of caryological aberrations frequently encountered in cultured cells (Torrey, 1959; Mitra et al., 1960; Mitra and Steward, 1961; Fox, 1963; Blakely and Steward, 1964b; Shimada and Tabata 1967), and is obviously a matter of great importance when a material is considered as the source of elones. The plating technique developed in this study may be employed to supply elonal cells for many studies using plant-cell cultures. In particular, the genetieal scope of somatic plant cells should be greatly enlarged by this technique, since the latter permits the establishment of clones in practically unlimited numbers. Thus, the production of mutant clones by physical or chemical mutagens, for example, should be greatly facilitated. Most promising in this respect should be the protoplasts from haploid plants, since mutants with recessive eharaeters are easily deteetable using haploid cells (Carlson, 1970 ; Melehers and Labib, 1970). I t may also be possible to modify the genetic constitution of plant cells by introducing into them foreign genetic materials (Takebe et al., 1971). This approach makes use of the ability of protoplasts to take up nucleic-acid molecules (Aoki and Takebe, 1969) and nucleoprotein particles (Takebe and Otsuki, 1969) or to undergo fusion with protoplasts from other plants (Power et al., 1970). Using the technique of the present study, it should now be possible to follow the fate of genetic materials thus introduced into protoplasts. If they are integrated into the genetic materials of the host cell, this approach should find application for plant breeding, since whole plants can be readily regenerated starting from individual protoplasts. We are grateful to Prof. T. Yamaki, University of Tokyo, for his interest and encouragement. Thanks are also due to Dr. F. Motoyoshi of this Institute who kindly made his unpublished results accessible to us. Note added in proo[: The first tobacco plant regenerated in this work from Xanthi ne protoplast flowered and yielded seeds. References Aoki, S., Takebe, I. : Infection of tobacco mesophyll protoplasts by tobacco mosaic virus ribonucteic acid. Virology 39, 439-448 (1969). Bergmann, L. : Growth and division of single cells of higher plants in vitro. J. gen. Physiol. 43, 841-851 (1960). 2*

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T. Nagata and I. Takebe: Plating of Isolated Protoplasts

Blakely, L.M., Steward, F.C.: Growth and organized development of cultured cells. V. The growth of colonies from free cells on nutrient agar. Amer. J. Bot. 51, 780-791 (1964a). - - - - Growth and organized development of cultured cells. VII. Cellular variation. Amer. J. Bot. 51, 809--820 (1964b). Carlson, P.S.: Induction and isolation of auxotrophic mutants in somatic cell cultures of Nicotiana tabacum. Science 168, 487-489 (1970). Earle, D.E., Torrey, J. G. : Colony formation by isolated Convolvulus cells plated on defined media. Plant Physiol. 40, 520-528 (1965). Eriksson, T. : Studies on the growth requirements and growth measurements of cell cultures of Haplopappus gracilis. Physiol. Plantarum (Cph.) 18, 976-993 (1965). Fox, J.E. : Growth factor requirements and chromosome number in tobacco tissue cultures. Physiol. Plantarum (Cph.) 16, 793-803 (1963). Gibbs, J.L., Dougall, D.K.: The growth of single cells from Nicotiana tabacum callus tissue in nutrient medium containing agar. Exp. Cell Res. 40, 85-95 (1965). Kohlenbaeh, H.W. : Die Entwicklungspotenzen explantierter und isolierter Dauerzellen. I. Das Streckungs- und Teilungswachstum isolierter Mesophyllzellen yon Macleaya cordata. Z. Pflanzenphysiol. 55, 142-157 (1966). Melchers, G., Labib, G. : Die Bedeutung haploider h6herer Pflanzen fiir Pflanzenphysiologic und Pflanzenzfichtung. Ber. dtsch, bot. Ges. 83, 129-150 (1970). Mitra, J., Mapes, M.O., Steward, F.C.: Growth and organized development of cultured cells. IV. The behavior of the nucleus. Amer. J. Bot. 47, 357-368 (1960). - - Steward, F. C. : Growth induction in cultures of Haplopappus gracilis. II. The behavior of the nucleus. Amer. J. Bot. 48, 358-368 (1961). Murashige, T., Skoog, F. : A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plantarum (Cph.) 15, 473-497 (1962). Nagata, T., Takebe, I. : Cell wall regeneration and cell division in isolated tobacco mesophyll protoplasts. Planta (BEE.) 92, 301-308 (1970). Power, J.B., Cummins, S.E., Cocking, E. C. : Fusion of isolated plant protoplasts. Nature (Lond.) 225, 1016-1018 (1970). Sacristan, M.D., Melchers, G. : The caryological analysis of plants regenerated from tumorous and other callus cultures of tobacco. Molec. Gen. Genetics 105, 317-333 (1969). Shimada, T., Tabata, M. : Chromosome numbers in cultured pith tissue of tobacco. Japan. J. Genetics 42, 195-201 (1967). Takebe, I., Labib, G., Melchers, G.: Regeneration of whole plants from isolated mesophyll protoplasts of tobacco. Naturwissensehaften (in press). - - Otsuki, Y.: Infection of tobacco mesophyll protoplasts by tobacco mosaic virus. Proe. nat. Acad. Sci. (Wash.) 64, 843-848 (1969). Torrey, J.G.: Experimental modification of development in the root. In: Cell, organism and milieu, p. 189-222, D. Rudnick, ed. New York: Ronald Press 1959. Usui, H., Takebe, I. : Division and growth of single mesophyll cells isolated enzymatically from tobacco leaves. Development, Growth and Differentiation 11, 143-151 (1969). White, P.R.: The cultivation of animal and plant cells, 2nd cdn. New York: l~on~ld Press 1963. Toshiyuki Nag~t~ Institute of Biology College of General Education University of Tokyo Meguro-ku, Tokyo 113, Japan

Itaru Takebe Institute for Plant Virus l~esearch 959 Aobacho, Chiba 280, Japan