Utilization of irradiated carrot cell suspensions as feeder layer for cultured Nicotiana cells and protoplasts

Plant Science Letters, 19 (1980) ~8----352 o lD.m~er/North-ltolland Scientific Pubndsms Ltd.

I Y r I U Z A T I O N OF I R R A D I A T E D C A R R O T C E I L 8USPIgNSION8 AS FEEDER LAYER FOR CULTIYRI~ NICOTIANA C E L I ~ A N D PROTOPLAffr8

RINO CBLLA* mad BSRA GALUN** Department of Plant Gcnetict The Weizmann Institute of Science, Rehovot (Israel)

(Received January 30th, 1980) (Revision received and aceepted May 80th, 1980)

SUMMARY

Non~lividing X-ray irradiatedcarrot suspension cellswere used as feeder cells to support the division of NicotJana protoplasts and cells plated at low densities. Pigmented carrot cellshave been employed because they are easily recognizable when occasional division and colony formation occurred in the feeder layer. Several parameters which influenced the plating efficiency have been analyzed. Optimal conditions, which allowed a high final plating efficiency of up to 70%, in the case o f Nicotiana tabacum cv Xanthi protoplasts, have been established.

INTRODUCTION

Plant protoplasts have usually to be plated at m i n i m u m densitiesin order to divide and produce colonies. Especially fortifiedmedia and micro-culture techniques have been devised to overcome this limitation [1,2]. A n alternativesolution, based on the use of X-irradiated protoplasts as feeder layer to support the division of an upper layer of cultured protoplasts,has been utilized in several studies [3--10]. The cross-feedingeffect is not species specificbut detectable even between cellsof different plant *Permanent addreu: Laboratorio di Genetica Bioch|mlea ed Evoluzionistica del C.N.R., Via S. Epifsnio, 14, 27100 Pavia, Italy. **The Irene and David Sehwsrz Profeesor of Plant Genetics. Address all correspondence to: Rino Celts, Iaboratorio di Genetica Biochimica ed Evoluzionistica del C.N.R., Via. 8. Epifanio, 14, 27100 Pavia, Italy

244 families [ 7]. Hence a feeding effect seems to exist between cells of different nutritional requirements and different sensitivities to toxic compounds. These features could be of advantage for the selection of metabolic mutants and of somatic hybrids following protoplast fusion. Suspension culture cells have a potential advantage over newly isolated leaf protoplasts as feeders since the former are readily available aseptically and in practically unlimited quantities. Their use will avoid hazard of contamination which is quite frequent with leaf protoplasts. Highly pigmented carrot ceil suspension seemed an especially favourable source of feeder layer for cultured Nicotiana protoplasts because such cells differ structurally from Nicotiana cells and can easily be identified when occasional division and colony formation occurs in the feeder layer. Moreover, carrot cell suspensions are easy to maintain, they are composed mostly of single and small groups of cells and show a much higher resistance to certain antimetabolites than Nicotiana cells [ 11 ]. In the present study we tested the ability of X-ray-irradiated carrot cell suspensions to serve as feeders to Nicotiana cells and protoplasts as well as to optimize the system by investigating several parameters related to preparation of the feeder layer cells. MATERIALS AND METHODS

Suspension cultures A high anthocyanin¢ontaining carrot (Daucus carota) cv 'Rote Ruben' cell line was obtained from Professor Dr. J. Reinert. It was maintained by weekly transferring 10 ml of suspension to 50 ml of Murashige and Skoog (1962) medium (MS) containing 1.8/~M 2,4-D as sole phytohormone. Suspension cultures of tobacco (Nicotiana tabacum) cv Xanthi and N. sylvestris were obtained from calli which were derived respectively from tobacco and haploid N. sylvestris mesophyll protoplast cultures [9]. These lines were maintained by weekly transferring 10 ml of suspension to 60 ml of MS medium containing 2.7 pM NAA and 0.9/~M BAP. Feeder layer Carrot cultures were started with a packed volume of 5 percent and reached 60 percent by day 10 with the exponential growth lasting until day 8. Unless otherwise specified, cells were collected from exponential suspension cultures, i.e. 5--7 days after transfer. Cells were exposed to 21 kR of X-rays as described previously [3,4]. This radiation dose prevented cell division without killing the cells as evaluated by viability tests and observation of cell enlargement after plating. After irradiation cells were washed twice with liquid medium, suspended at a density of about 106 cell/ml and incubated overnight at 22- -25°C on a rotary shaker (100 rev./min). Cells were then washed again with medium, counted and mixed with agarized medium to give a final 0.6% concentration.

245

Three milliliters o f this suspension were plated in each 5~m. petri dish and served as feeder layer. The cell densities used in different experiments are specified below. The medium used for washing and plating was the same required b y the cells or protoplasts cultured in the upper layer.

Isolationand culture of Nicotiana protoplastsand cells Xanthi protoplasts were isolated as previously described [9], suspended at given densities in either Nagata and Takebe [12] m e d i u m (NT) or Nagy and Maliga [13] medium (NM) containing 0.6% agar and plated in 2-ml quantities over the feeder layer. In some cases the protoplasts were first maintained for 5--6 days in liquid medium, then mixed with agar medium and plated over the feeder layer. In order to facilitatefurther growth, the plates were flooded with M S medium, for I h after 15--20 days of culture (approx. 2000 Lux, 26°C). Haploid N. sylvestrismesophyll protoplasts were isolated as described [9] and plated at optimal density (approx. 4 • 104/ml) in N M m e d i u m with 0.4% agar. After different time intervals, this soft~gar culture was diluted and mixed with N M agar medium, to give a final 0.6% of agar, plated over the feeder layer and further treated as described above. Nicotiana cell suspension cultures were harvested and filtered through a l-ram nylon mesh. The propagule density was determined by a l-ram deep counting chamber and cell number was counted after chromium trioxide treatment [14]. The filtered cells and aggregates were suspended in M S m e d i u m containing 13.5 ~ M N A A , 4.5 ~ M B A P and 0.6% agar and plated as 2-ml layers over the feeder layer. The plates were then incubated as described above. RESULTS AND DISCUSSION

Since previous studies indicated that Xanthi feeder layers induce division in Xanthi protoplasts plated at densities which are below the threshold of cell division, the capacity of carrot suspension culture cells to serve as feeders was tested (Table I). The feeding effect of carrot cells was irrespective of the culture media, i.e.feeding occurred in both N T [12], suitable for Xanthi protopiasts and in N M [13] suitable for the culture of both Xanthi and N. sylvestrisprotoplasts. Since exposure to X-rays is known to cause a release of toxic substances, we have tested the effect of washing the irradiated carrot cells on their feeding capacity. Table H indicate that irradiated carrot cells could serve as feeders for Xanthi protoplasts even without washing but that washing improved feeding capacity. W h e n Xanthi protoplasts were plated under identical conditions but at densities of 200 and 20 cells/ml, they resulted in 110 and 8.5 visible colonies/ml, respectively, provided that the feeder cells were washed twice before incubation and once before platin~ Similar results b u t with lower values were obtained when NT rather than NM medium was utilized.

246

TABLE I CELL DIVISION OF N. TABACUM ~ XANTHI, WITH AND WITHOUT AN X-IRRADIATED CARR(Yr FgBDBR LAYgR Cell divis/on was evaluated 15 days after plating. The final density of the feeder celk was 8 . 104/mL

Culture media

Plating density: p~otopl~ts/ml

Feed~ layer

Divided calls No./ml

Percent

of

initial number of protopluts NM

-

8500

85

+

6800

68

0

0

800

80

0

0

+

390

78

-

8100

81

10000 -

1000 + -

500

NT

10000 +

4000

40

--

0

0

+

680

63

--

0

0

320

64

1000 500

When selecting for metabolic mutants one m a y first culture m u t a t e d protoplasts in non,selective m e d i u m and after several days transfer t h e m to another medium in which only a small fraction o f t h e m will survive. We therefore tested the feeding effect o f carrot cells on Xanthi protoplasts which were precultured for several days and t h e n diluted and plated over feeder cells. The results o f this test (Table III) indicate t h a t freshly isolated as well as protoplast pre-cultured at high density and diluted after either 5 or 8 days are affected by the feeder cells. The lower plating efficiency after 8 days o f pre-culture could result from aging of feeder layers since in this experiment t h e y were prepared on t h e same day. We therefore tested this possible effect of aging in a further experiment presented in Table IV. The results obtained show t h a t the storage of feeder layers for 1 week at 25°C strongly affected their nursing capacity causing a sharp reduction in colony formation at those concentrations o f protoplasts such as 5 • 102 protoplasts/ml which required a feeder layer in order to grow. This loss

247

TABLE II CHANGES IN FEEDING CAPAC1TY OF CARROT SUSPENSION CULTURE CELLS, RESULTING FROM DIFFERENT WASHING PROCEDURES The final demdty of the feeder cells was 1.2 • 10S/ml. Xanthi protoplmfl~ were plated at a density of 2 • 10~/ml in the upper layer; culture medium was NM; cell divmion was evaluated after 15 daya Treatment of feeder cells

Division of Xanthi cells No./ml

No feeder layer

Irradiated celia restmpended in fresh medium Irradiated cells washed twice Irradiated celia washed twice incubated overnight and washed once again

Percent of initial protoplagr~

0 860

0 38

940 1100

47 55

of nursing capability is unlikely to be due to depletion of medium since cells, although alive, are not dividing. O n the contrary, storage of cells for the same period at 2°C allowed a satisfactory retention of feeding capability. Another parameter which was taken into account was the physiological age of the carrot cells.To this purpose we compared the feeding capability of cells harvested in exponential and early stationary phase of growth, but we were unable to detect any difference (not shown). Previous results [3] indicated that the protoplast density of a Xanthi feeder layer affects the division of cultured cells in the upper layer. Optimal feeding effect was observed when the density of the feeding cells was similar to the optimal density of protoplasts cultured without feeding cells, i.e. about 2--5 • 104 cells/ml. W e therefore tested the effect of the density of carrot feeder cells on the development of microcolonies derived from Xanthi protoplasts (Table V). The results indicate that the feeder cell density affected colony development in the upper layer especially when microcolonies were plated at low concentrations. Moreover it was found that the density of carrot cells in the feeder layer required to obtain a high plating efficiency was higher than the optimal density reported for feeder layers of Xanthi protoplasts. For instance when microcolonies were plated at a low density (5 • 102/ml) there was a higher percentage of cell division in the presence of 4 • 10 s than at 8 • 104 feeder cells/ml. One possible e x p l a n a t i o n o f this f a c t is t h a t c a r r o t cells have a ' n u r s i n g p o t e n t i a l ' l o w e r than that of Xanthi protoplasts and therefore a higher density of the former is r e q u i r e d t o o b t a i n similar effect. H o w e v e r at c o n c e n t r a t i o n s h i g h e r t h a n 4 • 1 0 s cells/ml in t h e f e e d e r l a y e r t h e scoring o f X a n t h i c o l o n i e s b e c a m e

248 TABLE HI PLATING EFFICIENCY OF XANTHI PROTOPLASTS AND PROTOPLAST DERIVED CELLS AS AFFECTED BY CARROT FEEDER CELLS Xanthi protoplasts were precuitured at 2 • 10'/ml in liquid NT as described in Materials and Methods and approached second and third divkion after 5 days and 8 days, respectively. Cell division was evaluated 18 days after plating in agar medium (with or without feeder cells in the underlayer), as number of colonies/ml; carrot cell density was 7 . lO'/ml. Pre-culture of Xanthi protoplasts s

Plating density of Xanthi protoplasts or microcolonies (number/ml)

Feeder cells

No./ml

-

No pr~cuiture

Division of Xanthi cells

0a

Percent of initially plated 0

5000 4 -

1600

32

0 a

0

+ -

160 0a

32 0

+

360

72

50O Pre-cultured in liquid medium for 5 days

5000 -

0 a

0

500 Pre-eultured in liquid medium for 8 days

+ -

400 0a

80 0

5000 + -

3000 O*

60 0

500 +

160

32

aThe microeolonies degenerated during further culture in the absence of feeder cells.

v e r y u n r e l i a b l e a n d t h e r e f o r e it was n o t possible t o f o l l o w t h e e f f e c t o f higher cell densities in t h e f e e d e r s u p o n t h e d e v e l o p m e n t o f colonies. E x p e r i m e n t s p e r f o r m e d using f r e s h l y isolated p r o t o p l a s t s i n s t e a d o f m i c r o c o l o n i e 8 gave similar r e m i t s . W h e n w e a t t e m p t e d t o p l a t e f r e s h l y i s o l a t e d N. sylvestris p r o t o p l a s t s o v e r c a r r o t f e e d e r l a y e r s we did n o t r e c o r d a n y f e e d i n g e f f e c t . M o r e o v e r t h e c a r r o t f e e d e r l a y e r r e d u c e d cell division o f N. sylvestris p r o t o p l a s t s p l a t e d a t high cell d e n s i t y ( 2 - - 3 • 104 p r o t o p l a s t s / m l ) . T h e adverse e f f e c t o f c a r r o t f e e d e r cells was u n e x p e c t e d d u e t o p r e v i o u s l y o b s e r v e d p o s i t i v e e f f e c t o f X a n t h i f e e d e r s o n N. sylvestris p r o t o p l a s t s [ 9 ] . We o b s e r v e d t h a t t h e a d d i t i o n o f casein h y d r o l y s a t e ( 3 0 0 ~ g / m l ) a n d t r y p t o p h a n e ( 2 5 ~ g / m l ) s u p p r e s s e d division o f freshly isolated N. sylvestris

249 T A B L E IV CHANGES IN P LAT I N G E F F I C I E N C Y OF PROTOPLAST D E R I V E D MICROCOLONIES R E S U L T I N G FROM S T O R A G E O F C A R R O T F E E D E R L A Y E R S Xanthi protoplasts were cultured for 1 week at 1 0 ' cells/ml in liquid NT medium and then diluted and plated on top of carrot feeder layers with a density of 8 • 10" cells/ml. Cell division was evaluated after 35 days by the determination o f the number of large colonies/ml. Feeder layer

No feeder layers Freshly prepazed feeder layer Feeder layer stored 1 week at 2°C Feeder layer stored 1 week at 25°C

Cell density: Number of microcolonies/ml

Cell division No./ml

Percent o f initial microcolonies

5000 500 5000 500 5000 500

0 0 1485 360 1850 277

0 0 29.7 72.0 37.0 55.4

5000 500

1859 44

37.2 8.8

TA B LE V CELL DIVISION IN XANTHI PROTOPLAST-DERIVED MICROCOLONIES CULT U R E D OVER C A R R O T F E E D E R CELLS PLATED AT D I F F E R E N T DENSITIES Protoplast-derived Xanthi cells were pre-cultured, as described in the text for 6 days. Cell division was evaluated under the microscope and colony formation macroscopically using a bacterial colony counter. Protoplastderived Xanthi cell density (cells/ml)

Cell Division observed after 14 days

Colony formation counted after 30 days

No./ml

Percent of initial microcolonies

No./ml

Percent of initial microcolonies

10' 10' 10 s 10" 10"

1000 1000 1000 100 100

0a 930 850 + --

0a 93 85 ¼ --

0a 450 470 0a 42

0a 45 47 0a 21

10 s

100

-

-

102

51

Feeder layer cell density (cells/ml)

2.5 8.0 2.5 2.5 8.0 2.5

aThe plated microcolonies (2--4 cells) degenerated during culture.

250

protoplasts but promoted division when added I week after plating. Based on this observation we assumed that some of the metabolites released from carrot cells could be inhibitory to fresh protoplasts. Therefore we first cultured N. sylvestrisprotoplasts at high concentration (2--3 • 104 protoplasts/ml) for different lengths of time, then diluted and plated the resulting microcolonies onto carrot feeder layers. U p to about 8 days the exposure of N. sylvestrismicrocolonies to carrot cells was deleterious while starting from day 10 the carrot feeders became beneficial. In addition we tested the supplement of animal bone activated charcoal with and without the addition of carrot cells to the underlayer. The results summarized in Table VI show that the dependence on feeder ceils was almost complete when charcoal was not present. Interestingly this dependence could be partially reversed by the addition of charcoal. However the presence of charcoal together with feeder cells did not improve the plating efficiency. The starting material for the selection of dominant metabolic mutants m a y be suspension culture cells rather than freshly isolated protoplasts or protoplast-derived microcolonies. W e therefore examined the ability of carrot feeder layers to support division of sparsely plated Xanthi and N. sylvestris suspension culture cells.The suspensions were routinely filtered through a nylon mesh which removed large cell aggregates leaving propagules of only a few ceils. Preliminary results showed that suspension cells of both Xanthi and N. sylvestris required a feeder layer when plated at low density. The plating TABLE VI GROWTH OF PROTOPLAST DERIVED MICROCOLONIES OF N. S Y L V E S T R I S ON CARROT FEEDER LAYERS WITH AND WITHOUT CHARCOAL Protoplasts obtained from haploid leaves were allowed to grow for 10 days in NM medium containing 0.4% agar. After this period colonies were diluted, plated on top of either carrot feeder layers or plain medium as a 2-ml agarized layer and flooded with 5 ml of MS medium for 1 h. Charcoal was included only in the b o t t o m layer. Results are expreued as number of large, visible colonies which developed after 30 days. Plating density No./plate

Total number of colonies/plate after 30 days Charcoal concentration None

0.25%

0.5%

1.0%

Feeder cells

Feeder cells

Feeder cells

Feeder cells

4

--

+

--

+

--

+

--

6 0

63 60

19 2

93 54

59 12

101 47

79 22

400

150

80

44

251

TABLE VII PLATING EFFICIENCY OF XANTHI 8USPKNBION CELLS CULTIYI~D OVER C A R R O T F E E D E R C E L L S P L A T E D A T V A R I O U S C E L L DENSYYIE8

Xanthi cells were obtained from late exponential suspension cultures, filt~ed, diluted and plated u 2-ml layer of 0.6% agar MS medium over 3 ml of carrot feeder celk. R~ults are expressed as number of Xanthi colonies/ml observed 20 days after plating. Dilution of cultured Xanthi suspemdon cells

Demfity of carrot feeder cells (No./ml) 9 ' 10'

1.8 • 10 s

3.6 • l0 s

1 1 1 1

125 40 10 7

130 64 13 5

129 54 10 7

: : : :

20 60 180 540

e f f i c i e n c y , e x p r e s s e d as p e r c e n t o f p r o p a g u l e s w h i c h gave rise t o visible c o l o n i e s a f t e r 20 d a y s o f c u l t u r e , was a b o u t 25% a n d 10% f o r X a n t h i a n d N. sylvestris r e s p e c t i v e l y . In p l a t e s w i t h o u t c a r r o t cells in t h e u n d e r l a y e r t h e p l a t i n g e f f i c i e n c y was usually n o u g h t . T h e f e e d i n g e f f e c t was a l r e a d y e v i d e n t a t f e e d e r cell c o n c e n t r a t i o n s which were 10 times lower than those required when plating protoplasts. A f u r t h e r t e s t w i t h X a n t h i s u s p e n s i o n cells clearly i n d i c a t e d t h a t increasing f e e d e r cell d e n s i t y f r o m 9 • 104 t o 3.6 • 1 0 s cells/ml n e i t h e r im-

TABLE VIII C O L O N Y F O R M A T I O N B Y X A N T H I SUSPENSION CELLS F R O M ErrHER EXPONENTIAL OR STATIONARY GROWTH PHASE PLATED OVER CARROT FEEDER

CELLS The mean number of cells/propagule was 20 and 10 for exponential and stationary phase suspensions, respectively. Cell density of the carrot feeder layer was 104/ml. Colonies were counted 20 days after plating. Growth phase of Xanthi cells

Dilution of stock suspemfion

Density of propagules (No./ml)

Colony formation No./ml

Percent of initial propagules

Exponential

1 : 270 1 : 810

100 33

46 16

46 48

Stationary

1 : 90 1 : 270

400 133

52 13

13 10

252

proved nor hampered division in the cultured upper layer irrespective of the density in the latterlayer (Table VII). The efficiency of irradiated carrot cellsto support division of very diluted Xanthi suspensions is indicated in another test (Table VIII) where the. plating efficiency of exponential and stationary phase Xanthi cells was compared. In either case, when the cells were diluted 3-fold no reduction in colony formation was observed. It should be noted that at dilutions employed in this test the Xanthi suspension cells would degenerate rather than result in colonies. This test also indicates that even stationary-phase Xanthi cells can be induced to form colonies when cultured over carrot feeder layers but their plating efficiency is rather lower than that of exponential-phase cells.In both cases more than 9 5 % of cells gave a positive response to the fluorescein diacetate test at the time of plating hence the decrease of plating efficiency in stationary cells could not be attributed to cell death. In conclusion, the results obtained in this work show that X-ray irradiated suspension cells of carrot can be used successfully as a feeder layer for the growth of Nicotiana leaf protoplasts and suspension cells plated at low densities. ACKNOWLEDGEMENTS

Grateful acknowledgement is made to the European Molecular Biology Organization for a Long Term Fellowship which enabled one of us (R.C.) to take part in this work. The authors are indebted to Dr. Dvora Aviv for her helpful advice during the course of this study and to Ms. Pazia Arzee for her help in part of the experimental work. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14

K.N. Kao and M.E. Michayluk, Planta (Berl.), 126 (1975) 105. Y.Y. Gleba, Naturwissenschaften, 65 (1978) 158. D. Raveh, E. Hubermann and E. Galun, In vitro, 9 (1973) 216. E. Galun and D. Raveh, Radiat. Bot., 15 (1975) 79. D. Raveh and E. Galun, Z. PflanzenphysioL, 76 (1975) 76. A. Vardi, P. Spiegel-Roy and E. Galun, Plant Sci. Lett., 4 (1975) 321. A. Vardi and D. Raveh, Z. PflanzenphysioL, 78 (1976) 350. D. Aviv and E. Galun, Plant Sci. Lett., 8 (1977) 299. A. Zelcer, D. Aviv and E. Galun, Z. Pflanzenphysiol., 90 (1978) 397. A. Wallin, K. Glimelitm and T. Eriksson, Z. Pflanzenphysiol., 91 (1978) 89. E. Nielsen, F. RolIo, B. Parisi, R. Celia and F. Sala, Plant Sci. Lett., 15 (1979) 113. T. Nagata and I. Takebe, Planta (Bed.), 99 (1971) 12. J.I. Nagy and P. Maliga, Z. PflanzenphysioL, 78 (1976) 453. R. Celia, F. Sala and H.E. Street, J. Exp. Bot., 27 (1976) 263.