Planta

Planta 144, 509-510 (1979)

9 by Springer-Verlag 1979

Short Communication

Photoautotrophie Growth of Marchantia polymorpha L. Cells in Suspension Culture K e n j i K a t o h 1, Y o s h i m o t o O h t a 1, Y o s h i o H i r o s e

1,

and Tashuichi Iwamura 2

Institute of Food Chemistry, Shimamoto-cho, Mishima-gun, Osaka 618, and 2 Research Institute for Biological Regulation, Faculty of Agriculture, Nagoya University, Chikusa-ku, Nagoya 464, Japan

A b s t r a c t . A cell line o f M. polymorpha was g r o w n p h o t o a u t o t r o p h i c a l l y in liquid s u s p e n s i o n culture using 1% CO2 in air as sole c a r b o n source. T h e g r o w t h rate in t e r m s o f cell d r y - w e i g h t d u r i n g the e x p o n e n t i a l p h a s e was 0.171 a n d the d o u b l i n g time was 1.76 d. The rate o f increase in c h l o r o p h y l l was 1.6 times higher t h a n the g r o w t h rate. The highest c o n t e n t o f chlor o p h y l l was 24 m g g - 1 d r y weight, a n d the p h o t o s y n thetic activity o f the cells in the e x p o n e n t i a l phase, as c a l c u l a t e d f r o m the g r o w t h rate, was at least 60 g m o l m g - 1 c h l o r o p h y l l h - 1 K e y words: Cell s u s p e n s i o n cultures Photoautotrophic growth

Marchantia -

Cell s u s p e n s i o n cultures have been e s t a b l i s h e d for quite a n u m b e r o f p l a n t s b u t in m o s t cases the cells contain only incompletely developed chloroplasts and g r o w only in m e d i a c o n t a i n i n g a suitable source o f r e d u c e d c a r b o n , u s u a l l y a sugar. S o m e cell lines have been d e s c r i b e d t h a t possess, u n d e r certain c o n d i t i o n s , m o d e r a t e l y w e l l - d e v e l o p e d c h l o r o p l a s t s a n d are c a p a ble o f m a k i n g p h o t o a u t o t r o p h i c growth. H o w e v e r , the c h l o r o p h y l l c o n t e n t o f these cells is very low a n d p r e s u m a b l y b e c a u s e o f this, the a u t o t r o p h i c g r o w t h is g e n e r a l l y quite p o o r . M o r e o v e r , it seems to be difficult to s u b c u l t u r e m o s t o f these c h l o r o p h y l l o u s cell lines a u t o t r o p h i c a l l y for a long time. O n l y in Chenopodium rubrum a n d Nicotiana tabacum (Berlyn a n d Zelitch, 1975; H i i s e m a n n a n d Barz, 1977; Berlyn e t a l . , 1978; Y a m a d a a n d Sato, 1978) has it been possible to m a i n t a i n p h o t o a u t o t r o p h i c g r o w t h for s o m e m o n t h s , a l t h o u g h the g r o w t h rate was still low. I n a p r e v i o u s p a p e r , we r e p o r t e d the e s t a b l i s h m e n t o f a highly c h l o r o p h y l l o u s cell s u s p e n s i o n culture o f Marchantia polymorpha ( O h t a et al., 1977); when cult u r e d p h o t o h e t e r o t r o p h i c a l l y (using a c h e m i c a l l y de-

fined m e d i u m ) the cells grew at quite a high rate ( g e n e r a t i o n time in the e x p o n e n t i a l phase was 4.4 d, u n d e r the c o n d i t i o n s used). W e can n o w r e p o r t t h a t the s a m e c h l o r o p h y l l o u s cell line can be g r o w n u n d e r p h o t o a u t o t r o p h i c c o n d i t i o n s at a c o n s i d e r a b l y higher rate t h a n the c h l o r o p h y l l o u s cell lines so far described, a n d be r o u t i n e l y s u b c u l t u r e d every 2 weeks. For photoautotrophic culture of the chlorophyllous cell line the mineral salts of Murashige and Skoog's medium (1962) were used with a slight modification, namely, increasing phosphate to 11.28 mM (1.23 g KH2PO 4 and 0.39 g K2HPO4 per liter). The only organic constituent was 1 mg/1 2,4-dichlorophenoxyacetic acid (2,4-D). The pH was adjusted to 6.5 with KOH, and the medium autoclaved at 120~ C for 15 rain. The pH after autoclaving was 6.4. A flat culture flask (inner height, 28mm; 700ml capacity; see Tamiya et al., 1953) was filled with 500 ml of the medium, inoculated with 20ml of 7-d-old stock culture, and was placed in a 25~ C water bath illuminated by a bank of fluorescent lamps (FL40W; Matsushita Electric Works, Osaka); the fluence rate at the surface of the flask was 23.2 W m 2. CO2 at 1% in air was bubbled through the cell suspension at a rate of ca. 500 ml/min. Cell dry-weight and chlorophyll content were measured throughout the culture 1. Cell dry-weight was measured by filtering the culture with a membrane filter (Floropore-1000; Sumitomo Electric Industries, Osaka) under vacuum and drying the cells so collected for 3 h at 105~ C. Chlorophyll was determined in 90% methanol extracts by measuring the absorbance at 666 and 653 nm (Iwamura et al,, 1970). The rate of increase in cell dry weight or in the chlorophyll amount was calculated by the following formula: K= (dQ/dt)/Q = log1o(Q2/Q 1)/(t2 - tl). in which Q2 and Q1 are the quantities in question at day tz and q, respectively.

T h e g r o w t h o f the cells u n d e r p h o t o a u t o t r o p h i c c o n d i t i o n s is illustrated in Fig. 1. P r o l i f e r a t i o n was 1 Reduction of the culture volume because of evaporation of water was less than 0.5% per day. Therefore, we did not correct the measured values in calculating the growth rate

0032-0935/79/0144/0511/$01.00

K. Katoh et al. : Photoantotrophic Marchantia Cell Culture

510

O

100

O

v

~z tO

CD

"r" n O or

W

q n

0.1

10

20 DAYS

Fig. 1. Growth of M. archantia polymorpa cells in photoautotrophic conditions: 1% CO2, 23.2 W m -2, 25 ~ C. 9 Dry weight; o chlorophyll content

exponential from the start of the culture until day 5, and then almost linear until day 18 when a nearly stationary phase was reached. The growth rate in terms of cell dry weight during the exponential phase was 0.171; the doubling time was calculated to be 1.76 d. The net increase by the end of the culture was 3.7 mg/ml starting from a density of 0.09 mg/ml. The chlorophyll amount decreased briefly but rapidly at the start of the culture, reaching a minimum, almost half the initial value, after 1 d, and then increased roughly like cell dry-weight, but at a ca. 1.6 times higher rate. The maximum value in the stationary phase was 106 ~tg/ml. The ratio of chlorophyll a: b remained fairly constant at 2.3. The pH decreased gradually from 6.4 during the exponential phase, went down rapidly toward the end of this phase, to reach a value of 3.4 by day 6, and remained unchanged during the rest of the culture period. The chlorophyllous cell lines of Chenopodium rubruin and tobacco mentioned before contained about 100 ~tg chlorophyll g- ~ fresh weight and had a photosynthetic activity of 50-200 gmol CO2 mg- ~ chlorophyll h-1 under the exponential conditions used (2-40 W m- 2, 1-3% CO2, 25-30 ~ C). While the chloroplasts were moderately well-developed and had a reasonably high photosynthetic activity, the growth rate was only 0.015-0.03 (doubling time, 10-20 d), evidently because of the very low level of chlorophyll content in the cells. The growth rate of the chlorophyllous M. polymorpha cell line under photoautotrophic conditions was 0.171, i.e., ca. 6-11 times higher

than that of the photoautotrophically grown Chenopodium and tobacco cells; the photosynthetic rate was at least 60 gmol CO2 mg- 1 chlorophyll h- 1 and even in the linear growth phase it was not below 20 ~tmol CO2. The higher value is comparable to that found in the chlorophyllous cell lines of Chenopodium rubruin and tobacco, and also to that of Chlorella cells growing under similar conditions (e.g. Tamiya et al., 1953 ; Takabayashi et al., 1977). The chlorophyll content was 11-24 mg g- 1 dry weight, 10-20 times higher than that for the chlorophyllous cell lines, and 1/3--1/2 of that for Chlorella cells. Evidently, the photoautotrophic growth of green cells grown in culture is determined by the total photosynthetic activity of the culture, and this is proportional to or limited by the number of chloroplasts or the total chlorophyll amount in the entire cell population. Under the autotrophic conditions described above, we have been subculturing these chlorophyllous Marchantia cells for over 1 year, making transfers every other week. The cells can grow exponentially at least for the first 5 d or for nearly three generations. This feature may be salient for synchronizing the growth of these cells.

References Berlyn, M.B., Zelitch, I. : Photoautotrophic growth and photosynthesis in tobacco callus cells. Plant Physiol. 56, 753-756 (1975) Berlyn, M.B., Zelitch, I., Beaudette, P.B.: Photosynthetic characteristics of photoautotrophically grown tobacco callus cells. Plant Physiol. 61, 606-610 (1978) Hiisemann, W., Barz, W. : Photoautotrophic growth and photosynthesis in cell suspension culture of Chenopodium rubrum. Physiol. Plant. 40, 77 81 (1977) Iwamura, T., Nagai, H., Ichimura, S. : Improved methods for determining contents of chlorophyll, protein, ribonucleic acid, and deoxyribonucleic acid in planktonic populations. Int. Res. Ges. Hydrobiol. 55, 131-147 (1970) Murashige, T., Skoog, F.: A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473 497 (1962) Ohta, Y., Katoh, K., Miyake, K. : Establishment and growth characteristics of a cell suspension culture of Marchantia polymorpha L. with high chlorophyll content. Planta 136, 229~32 (1977) Takabayashi, A., Nishimura, T., Iwamura, T.: Incorporation of radioactive tracers of some labelled compounds into DNA, RNA, and protein in Chlorella cells at various stages of the cell cycle in synchronous culture. J. Gen. Appl. Mierobiol. 22, 183-196 (1976) Tamiya, H., Iwamura, T., Shibata, K., Hase, E., Nihei, T. : Correlation between photosynthesis and light-independent metabolism in the growth of Chlorella. Biochem. Biophys. Acta 12, 23~,0 (1953) Yamada, Y., Sato, F. : The photoantotrophic culture of chlorophyllous cells. Plant Cell Physiol. 19, 691~599 (1978) Received 13 November; accepted 1 December 1978