Effects of different nitrogen sources in culture media on protoplast release from plant cell suspension cultures

Plant Science Letters, 11 (1978) 241--250

241

© Elsevier/North-Holland Scientific Publishers Ltd.

E F F E C T S O F D I F F E R E N T N I T R O G E N SOURCES IN C U L T U R E MEDIA ON P R O T O P L A S T RELEASE F R O M PLANT CELL SUSPENSION C U L T U R E S *

YUKIO F U K U N A G A and JOHN KING

Department of Biology, University of Saskatchewan, Saskatoon, Sask. S7N OWO (Canada) ( R e c e i v e d July 23rd, 1977)

(Revision received and accepted October 21st, 1977)

SUMMARY

The release of protoplasts from alfalfa, soybean, flax, tobacco, wheat and rice cells grown in liquid media containing either nitrate, ammonium, L-glutamine or L-arginine as the sole nitrogen source was investigated. Growth o f all cell types in nitrate medium was comparable or greater than that in other media b u t protoplast yield was poor. Alfalfa, soybean and flax cells grew better in nitrate medium supplemented with 2 mM NH4+ than in medium containing only nitrate, and protoplast yield was also increased. T o b a c c o cells did not respond to this regime. Growth in ammonium medium was usually p o o r but protoplast yield was consistently as high or higherthan for any other medium. Growth in L-glutamine medium was comparable to that in a m m o n i u m medium in all cases as was protoplast yield except for flax and wheat cultures where protoplast yield was as low as in nitrate medium. Arginine supported growth comparable to that in nitrate medium except in the case of t o b a c c o culture where growth was poor. Protoplast yield from arginine-grown cells was higher than from nitrate-grown cells except in the case of soybean and t o b a c c o cultures where yield was poor. The effect o f nitrogen starvation for 3 days on protoplast yield was examined in wheat and rice cultures. Starvation was found to enhance the yield substantially when compared to that in nitrate medium. Results demonstrated a relationship between nitrogen metabolism and wall organization in these plant cells.

INTRODUCTION

In recent years more and more attention has been paid to plant protoplasts * This work w a s s u p p o r t e d by a grant-'in-aid of research t o o n e o f us (J.K.) Abbreviation: MES, 2(N-morpholine)ethanesulfonic acid; TCA, trichloroacetic acid.

242

because of their usefulness in physiological, biochemical and, especially, genetical work. In fact, their use in interspecific and intergeneric fusion as well as in transformation studies may further our knowledge of plants and improve crop yields significantly [ 1--3 ]. Protoplasts have been isolated from a wide variety of plant cells most successfully by enzymatic digestion of cell walls. The degree of success in these isolation procedures was influenced by a number of factors including the physiological condition of the plant cells, their source, and the combination of enzymes used to digest cell walls [4--7]. Some general observations have been made regarding the influence of the physiological state of plant cells on the efficiency with which protoplasts can be released from them. These include the fact that cells in suspension culture yield protoplasts more readily upon enzymatic digestion if the cells are growing faster, while the presence of lignin~in cell walls renders them more resistant to enzymatic digestion [ 4--7 ]. However, there has not been a systematic study of the influence of cell physiology on protoplast isolation despite the fact that knowledge of how efficiency of protoplast isolation might be increased would be of great advantage to those interested in higher protoplast yields. The nitrogen metabolism of cells would be expected to have an effect on the composition of plant cell walls and, hence, on the ease with which protoplasts could be obtained. For example, nitrogen metabolism is linked to the production of cell wall polysaccharides via the production of specific enzymes of cell wall formation [8]. The biosynthesis of lignins is closely related to phenylalanine and tyrosine formation [9], precursors of lignins. Plant grbwth substances such as auxins and cytokinins are known to affect cell wall properties either directly or indirectly, e.g. by stimulating cell division, and the activity of these substances can be affected by the nitrogen status of cells [10]. In this study, cells of a variety of plant species were exposed to several nitrogen regimes in sterile, liquid culture and the effect of these regimes on protoplast isolation was observed. The results indicated that the response of cell walls to hydrolytic enzyme attack could be altered sharply and rapidly by these treatments, and that this led, in turn, to dramatic changes in the efficiency with which protoplasts could be isolated. MATERIALS AND METHODS

Suspension cultures of alfalfa (Medicago sativa L. cv. Canadian No. 1), soybean (Glycine max L. cv. Mandarin), flax (Linum usitatissirnurn L. cv. Red Wing), tobacco (Nicotiana tabacum L. cv. Humflis), wheat (Triticum monococcum L. cv. Einkorn), and rice (Oryza satire L.), cells originally derived from root explants, were obtained from the Prairie Regional Laboratory in Saskatoon. All cells were grown routinely in YK(NO3) medium which was modified from B~ [11] medium as follows (rag 1-1): KNO3(2500), MgSO4.7H20(370), KH2PO4(340), CaCI~.2H20(150); Fe(Sequestrene) (28), MnSO4.H20(10), H3BO3(3), ZnSO4.7H20(2), Na2MoO4.2H20(0.25), CUSO4(0.025), COC12.6H20-

243 (0.025); nicotinic acid (1.0), thiamine--HC1 (10), pyridoxine--HC1 (2), myoinositol (100); 2,4
244

source of nitrogen into YK(NO3) medium invariably resulted in a considerably reduced protoplast yield within 24 h regardless of how high the yield had been previously. When alfalfa, soybean and flax cells were cultured for 4 days in YK(NO3) medium supplemented with" 2 mM NH4+, growth and protoplast yield was greater than in YK(NO3) medium alone (Fig. 2A--C) whereas tobacco cells did not respond to NH4+ addition either in their growth or in protoplast yield (Fig. 2D). A.

N Source

50 l

i

2

NO 3-

Dry Weight (gl -])

Protoplast Yield (%)a

Day

100 I

1 I

2 !

3 I

4 I

5 I

Medium pH 5.9

i

6.1

4

6.4

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Protoplast Yield (%)a

Day

Dry Weight (g 1-I)

50

I00

I

I

1 I

2 I

3 I

2

4 I

5 I

Medium pH 6.1

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m

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mml

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245

Most plant cells cannot grow on ammonium as the sole nitrogen source unless the culture medium is supplemented with TCA cycle acids [12,13]. Except for alfalfa and tobacco (Fig. 1A, D) all cell types failed to grow well in YK(NH4) medium, which also contained 5 mM of a-ketoglutarate, and soybean cells (Fig. 1B) failed to grow at all in this medium. In all cases, however, the protoplast yield was substantially greater from these cells than from those grown in YK (NO3) medium. Protoplast yield maxima varied from 85% in flax C.

N Source

Protoplast Yield (%)a

Day

50 I

Dry Weight i00 I

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4 l

g 1-i 6 I

8 I

Medium i0 ;

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pH

5.3

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D.

N Source

50

4 6

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i

2

I

=zzzmmmz=m

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5.4 5.4

Ill

4 ii 6b

4 i

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=z~zz~zzz~

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3 l

Medium pH

5.7 JH.

4

2 Ars

2 I

I

~,

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1 I

i00 I

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Dry Weight (g1-11

Protoplast Yield (%)a

Day

!

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246 E.

N Source

D~y!

Protoplast Yield

(%)a

Dry Weight

50

100

I

I

(g i-])

1

2

3

4

I

I

I

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.

Medium pH

|

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4.6

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6

4.9

Fo

N Source

Protoplast Yield (%)a

Day

Dry Weight

1,o0

50

I •

2

(gl -]) ~

2 N03-

+ NH 4

6.2

n

6.2

6

m

6.2

2

=zzz~zm

4.7

4

Ff 111/i/JA

4.0

i i i

. . . . . . . . . . tl

2

i

4

11111~11111|

6 2 Arg

Medium pH

4

6

Gin

4 i

5 I

4 6

°

=e==

IIII I I i

3.8 4.8 4.4 4.4 4.7

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4.2 3.9

Fig. 1. Growth of and protoplast yield from plant cells grown in liquid media containing different nitrogen sources. Alfalfa (A), soybean (B), flax (C), tobacco (D), wheat (E) and rice (F) cultures, grown in liquid mediaunder different nitrogen regi.-nes, aMeasured after 8 h of e n z y m e treatment, bprotoplut yield less than 1%. The initial pH of the culture media was 5.5. The vertical dotted line in each histogram indicates either the initial p r o t o p l ~ t yield or the inoculum size as the case may be. The standard deviation of dry weight m e ~ u ~ e n ~ was leas than 2% in all c s ~ s , and t~e duplicate protoplast countings always agreed to within 10%.

247

A.

+ NH4

Protoplast Yield (%)a 50

Day

I

i00

I

!

2

+c

2 4

I

!

I

5

Medium pH

I

6.0

.........•"__.

I i 4 I:

b

Dry Weight (g i-I]" 1 2 3 4

6.2 5.2 5.0

!

B. + NH4.

5O I

b

Dry Weight (g i-I )

Protoplast Yield (%)a Day

i00 I

2

4

6

i

i

i

8

i0 I

I

-i

2 4

6.3 5.4 5.9

2

+c

Medium pH 6.1

4 i

C. + NH 4

Protoplast Yield (%)a

Day

Dry Weight (g i-I)

~lO

5O i

b

2

~

2 4

+c

+ NH 4

: l

4 ~

Day

Medium

pH 5.6 5.6 5.8 6.0

Protoplast Yield (%)a

Dry Weight (g iml)

Medium pH 5.5

b 4

~

5.6 5.3

+c 4

5.1

Fig. 2. G r o w ~ o f and protopla~ yield from plant cells grown in liquid medium containing nitrate and ammonium. Alfalfa (A), soybean (B), flax (C), tobacco (D). aMeasured after 3 h of e n z y m e treatment, bNOs* (2 raM) or (c) NH4 ÷ (2 raM) added to YK(NOs) medium• See legend to Fig. 1 for other experimental details.

248 (Fig. 1C) to ca. 25% in soybean {Fig. 1B) or tobacco (Fig. 1D). Maxima were achieved at different times after inoculation in different cultures. Thus, high protoplast yield from YK(NH4)-grown cells did not parallel successful growth on this medium at all. This was especially clear in the case of wheat and rice cells which did not grow well in YK(NH4) medium but protoplast yield maxima were 57% and 66%, respectively, whilst the maxima obtained from YK(NO3)grown cells were 2% and 27%, respectively (Fig. 1E, F). Growth and protoplast yields were in many cases similar in cultures grown in YK(Gln) medium and those grown in YK(NH4) medium. However, in the case of wheat {Fig. 1E) protoplast yield from YK(Gln)-grown cells was considerably lower than that from YK(NH4)-grown cells, although growth was comparable. Protoplast yield from YK(Gln)-grown flax cells was also much lower than that from YK(NH4)-grown cells despite the comparable growth (Fig. 1C). It was also observed that although soybean cells failed to grow in either YK(NH4) or YK(Gln) medium, protoplast yield after 2 days was high although it later declined (Fig. 1B). In five of the six cases, arginine was also a suitable source of nitrogen for growth, and cells grown in YK(Arg) medium, in general, yielded substantial amounts of protoplasts except for soybean and tobacco (Fig. 1B, D). In the case of tobacco (Fig. 1D) cells could not grow in YK(Arg) medium, and protoplast yield which was as high as 14% after 2 days rapidly declined to 2% by the fourth day. On the other hand, although soybean cells grew well in YK(Arg) medium, protoplast yield remained low (Fig. 1B). Especially notable was the good growth of and high protoplast yield from wheat cells grown in YK(Arg) medium {Fig. 1E). Wheat cells have been kept growing in YK(Arg) medium for over 6 months in this laboratory without any appreciable reduction in growth rate. In all experiments cells were starved of nitrogen for 48 h before being used for protoplast isolation tests (see METHODS) in order to reduce the size of nitrate pools of cells which might have been carried over from YK(NO3) medium on which the cells had previously been grown. Yields of protoplasts from cells starved of nitrogen for 48 h are indicated in Fig. 1 (A--F) by vertical dotted lines in each histogram. Quite high yields after starvation were noted in wheat (26%) and rice (57%) cultures (Fig. 1E, F). A similar tendency, although to a much lesser extent, was also found in alfalfa and soybean cultures in which maxima of protoplast yield from YK(NOs)-grown cells occurred in initial yields after starvation (Fig. 1A, B). In these cultures yields diminished sharply after a short exposure to YK(NO3) medium. The effects of N-starvation on growth and protoplast yields from wheat and rice cells are given and compared to those grown in YK(NOs) medium in Fig. 3 (A, B). In both cases, growth continued through 3 days of N-starvation, although not to the same degree as in YK(NO3) medium. Protoplast yield, however, was enhanced sharply by N-starvation over that found in YK(NO3) medium. After only 1 day in N-free growth medium wheat cells yielded 38% protoplasts compared to 9% at inoculation (Fig. 3A). In rice, protoplut yield

249 A° N Source

Protoplast Yield (%)a

Day

Dry Weight (g~l-i)

Medium

50

i00

i

2

3

4

5

i

I

I

I

I

;

I

i N0 3-

5.7

:.

2 3'

-

pH

-i

5.8

5.8

|

i

5.5

2

5.2

3

4.9

B.

N Source

1 N0 3-

2 3 1

-

Dry Weight (gl -I)

Protoplast Yield (%)a

Day I

50

i00

I

!

.

i

2

3

I

I

I

4 I

5

Medium pH

I

5.9 ~

:

6.1

~

:

6.2 5.5

2

5.3

3

5.0

Fig. 3. Effect of nitrogen starvation on protoplast release from plant cells grown in liquid media. Wheat (A) and rice (B) cells, aMeasured after 3 h of enzyme treatment. Cells from 3-day-old cultures were transferred to either YK(NOs) medium or N-free growth medium (see Fig. 1 for details) after washing thoroughly with N-free medium. The initial pH of the culture media, the standard deviation of dry weight measurements and the deviation of protoplast countings were the same as in Fig. 1. increased progressively to 68% after 3 days from 26% at inoculation (Fig. 3B). Both cell type remained viable even after several days of N-starvation and continued to grow as before upon transferring to YK(NO3) medium. DISCUSSION The yield of protoplasts from six types of plant cells grown in suspension culture was sharply and rapidly affected by the nitrogen source in the medium. In all cases except flax, cells grown in nitrate medium were resistant to attack by hydrolytic enzymes and produced low yields of protoplasts, although nitrate was generally the most suitable source of nitrogen for growth. The yield from 'nitrate-grown cells was not improved by either a prolonged digestion (up to 10 h) or pipetting given at intervals during digestion (unpublished observations). It has been shown in tobacco cultures that incubation periods longer than 2 or 3 h were without benefit [ 1 4 ] . In contrast to nitrate, ammonium gave rise to quite high yields of protoplasts in all cases despite the fact that it

250 did not support growth in m a n y cases. In fact, there was no consistent correlation between growth and protoplast yield regardless of the source of nitrogen in the medium, suggesting that faster growth is not necessarily a criterion for high protoplast yield as has been suggested by others [4--7]. This last point is well illustrated in soybean and rice cultures in which little or no growth on a m m o n i u m occurred and yet protoplast yield was much greater than that from nitrate-grown cultures where growth was substantial. This does not imply that enhanced growth never leads to higher protoplast yields. In fact, enhanced growth of alfalfa, soybean and flax cells on nitrate by a supplementary a m o u n t of a m m o n i u m was paralleled by increase in protoplast yield. It can also be seen that changes in the cell walls allowing enzyme attack can occur rapidly; within 24 h in the case of N-starvation and 48 h in other media. Although time course studies shorter than these have not been performed, i t is probable that changes begin to appear much earlier than 24 h. Whatever wall components are affected by the nitrogen source supplied to cells can change rapidly giving rise to cells more susceptible to hydrolytic enzyme attack. In general, it would seem useful to investigate the effects of nitrogen regime, including N-starvation, on protoplast production, since wide differences in yields could be demonstrated and could, perhaps, with further manipulation, be enhanced even further. Experiments with various nitrogen sources might also be of use to those interested in cell wall biogenesis, for the dramatically variable responses of cells to hydrolytic enzymes observed in these studies may reflect rapid and repeatable modifications to cell wall composition. ACKNOWLEDGEMENTS The authors wish to thank Dr. K. O h y a m a for his encouragement and helpful advice and Dr. F. Constable for useful discussions. We would like to thank Mr. J.P. Shyluk for providing suspension cultures. REFERENCES 1 2 3 4

P.S. Carlson, H.H. Smith and R.D. Dearing, Proc. Natl. Acad. SCi. USA, 69 (1972) 2292. K. Ohyama, O.L. Gamborg and R.A. Miller, Can. J. Bot., 50 (1972) 2077. K.N. Kao and M.R. Miehayluk, Planta (Bed.), 115 (1974) 355. E.C. Cocking and P.K. Evans, in H.E. Street (Ed.), Plant Ti~ue and Cell Culture, Blackwell, Oxford, 1973, pp. 100--120. 5 T. Eriksson, H. Bonnett, K. Glimelius and A. Wallin, in H.E. Street (Ed.), Ti~ue Culture and Plant Science, Academic Press, London, 1974, pp. 213--231.

6 F. Constabel, in O.L. Gamborg and L.R. Wetter (Eds.), Plant Tissue Culture Methods, National Research Council of Canada Publication No. 14383, 1975, pp. 11--21. 7 E. Thomas and M.R. Davey, in From Single Cells to Plants, Wykehsm Publications, London, 1975, Chapter 5.

8 D.T.A. Lamport, Annu. Rev. Plant Physiol., 12 (1.970) 235. 9 A.C. Neish, in J. Bonner and J.E. Varner (Eds.), Plant Biochemistry, Academic Press, New York, 1965, pp. 581--617. 10 L. Beevers, in Nitrogen Metabolism in Plant~, Arnold, London, 1976. 11

O.L. Gamborg, in O,L. Gamb0rg and L.R. Wetter (Eds.), Plant Tissue Culture Methods, National Research Council of Canada Publication No. 14383, 1975, pp. 1--10. 12 O.L. Gsmborg and J.P. Shyluk, Plant Physiol., 45 (1970) 598, 13 J. Behrend and R.I. Matelas, Plant Physiol., 56 (1975) 584. 14 H. Uchiyama and T. Murashige, Plant Physiol., 54 (1974) 936.