Plant Cell Reports (1983) 2:126-128

Plant Cell Reports © Springer-Verlag 1983

Selection for NaCI Tolerance in Cell Cultures of Medicago sativa and Recovery of Plants from a NaCI-Tolerant Cell Line M. K. Smith s a n d J. A. M c C o m b School of Environmental and Life Sciences, Murdoch University, Murdoch, Western Australia 6150 Received March 5, 1983 / May 10, 1983 Communicated by A. H. Ellingboe

ABSTRACT

Medicago s a t i v a lines with a high incidence of r e g e n e r a tion were esta--B-fi~ed as suspension cultures and used to s e l e c t for NaCI t o l e r a n t lines. A t t e m p t s were then made to r e g e n e r a t e plants Irom t h e s e lines. R e g e n e r a t i o n . was severely depressed in NaCI t o l e r a n t calli and t h e only plants t h a t were successfully r e g e n e r a t e d were from one callus of M. s a t i v a cv. Regen S which grew in 62.5 mM NaCI. Plants from this callus, and new calli derived from the r e c o v e r e d plants, have shown a t o l e r a n c e to NaCI c o m p a r a b l e to calli and plants from the initial seed stock r a t h e r than an improved level of t o l e r a n c e . INTRODUCTION

Systems have been developed for r e g e n e r a t i n g plants in Medicago sativa from callus c u l t u r e s (Bingham e t al. 19751 Saunders and Bingham 1975, Walker et al. 1978, 19791 S t a v a r e k e t al. 1980), cell c u l t u r e s (McCoy and Bingham 1977) and protoplasts (Kao and Michayluk 19801 dos Santos e t al. 1980). These systems provide a means by which cultured cells may be s c r e e n e d for NaCI t o l e r a n t v a r i a n t s and plants r e g e n e r a t e d from the t o l e r a n t lines. A N a C l - t o l e r a n t cell line of M. sativa, s e l e c t e d by Croughan e t al. (1978), was capable of growth a t 171 mM NaCI and plants were subsequently r e g e n e r a t e d from longt e r m cultures of this line (Stavarek e t al. 1980). Rains (1982) l a t e r revealed t h a t t h e r e g e n e r a t e d plants were very weak and he was not able to d e m o n s t r a t e any i n c r e a s e in salt t o l e r a n c e . We report an independent a t t e m p t at r e g e n e r a t i n g plants from cell cultures of M. s a t i v a which t o l e r a t e NaCI. D a t a are p r e s e n t e d on t h e genotypie v a r i a t i o n in callus cultures of d i f f e r e n t M. sativa strains, the incidence of NaCIt o l e r a n t cell lines a f t e r plating on saline media and a comparison of NaC1 t o l e r a n c e of callus and plants b e f o r e and a f t e r r e g e n e r a t i o n from a N a C l - t o l e r a n t cell line. MATERIALS A N D METHODS

A. Plant Material Seeds of M. s a t i v a L. cv. Regen S (line W75RS), a line s e l e c t e d for its ability to r e g e n e r a t e plants from callus, were obtained from Professor E. T. Bingham, University of Wisconsin, who also supplied callus from a diploid line of Regen S (line HG2) which has e n h a n c e d r a t e s of r e g e n e r a tion from cell suspensions (McCoy and B i n g h a m 1977). Seeds of M. s a t i v a cv. C u l l 0 1 were obtained from t h e

Western Australian D e p a r t m e n t of Agriculture and from t h e s e a line (R3), with good r e g e n e r a t i o n was s e l e c t e d . B. Growth Conditions 1. Callus and suspension cultures The growth conditions and growth m e a s u r e m e n t s Ior callus cultures were as described by Smith and McComb (1981a). A minimum of 7 clones per line were t e s t e d with an a v e r a g e of t h r e e r e p l i c a t e s per t r e a t m e n t . To i n i t i a t e cell suspension% a p p r o x i m a t e l y 250 mg of callus was t r a n s f e r r e d to 35 ml of liquid medium (Kao e__tt al. 1974) which gave g r e a t e r cell densities and r e g e n e r a t i o n t-han liquid BII medium (Saunders and Bingham 1975). Liquid cultures were shaken a t 100 rpm under continuous illumination (5.2 Klux). A f t e r i0 days t h e suspension was f i l t e r e d through a :200 l~m stainless s t e e l mesh and the f i l t r a t e was spun down a t 200 G Ior 10 rain. To e s t i m a t e cell number, one ml of c o n c e n t r a t e d suspension was added to 5 ml of 10% c h r o m i c trioxide and held at 70°C for 15 min. The sample was t h e n a g i t a t e d on a flask shaker to c o m p l e t e the s e p a r a t i o n to single cells before counting. A second sample was used to e s t i m a t e cell viability using t h e fluorescein d i a c e t a t e t e s t (Widholm 1972). Cells were plated a t a final density of 50,000 viable ceils m1-1 in P e t r i plates (5 cm d i a m e t e r ) containing 5 ml of solidified BII media with 91aM 2,g-D and 9 tam Kinetin (Saunders and Bingham, 1975). Cultures were placed in t h e dark and growth of callus could be observed in 2-3 weeks.

Callus, w h e t h e r freshly i n i t i a t e d 9 subcultured or passed through a suspension culture cycle, was t r a n s f e r r e d to t h e r e g e n e r a t i o n medium, designated BOi2Y by Saunders and Bingham (1975). This was t h e same as BII medium e x c e p t t h a t it lacked hormones and had 2 g1-1 yeast e x t r a c t and 100 mg1-1 inositol. Cultures were housed at 25°C under continuous illumination (7.8-9.0 Klux). 2. Whole plants The growth conditions, h a r v e s t i n g and growth analyses for whole plants were as described by Smith and McComb (1981a). The plants r e c o v e r e d from a NaCIt o l e r a n t cell line were t e s t e d for t h e i r response to NaCI by placing two plants per 5 liters of c u l t u r e solution with a t o t a l of four plants per t r e a t m e n t . The plants were 4 weeks old a t t h e s t a r t of t h e e x p e r i m e n t and t h e duration ol growth in a e r a t e d culture was 5 weeks.

Present address: ARCO Plant Cell Research Institute, 6560 Trinity Court, Dublin, CA 94568, USA

127 C. Screening for NaCI-Tolerant Cell Variants A suspension of cells was plated directly onto saline media in 5 cm d i a m e t e r P e t r i plates, so t h a t the final NaCI c o n t e n t of the media was 0.1, 62.5, 125 or 250 mM. Plates were scored a f t e r 2 months and the callus t r a n s f e r r e d to r e g e n e r a t i o n media (BOi2Y). An average of 10 plates per t r e a t m e n t was used each t i m e a line was s c r e e n e d , and the screening was r e p e a t e d a minimum of five t i m e s for each line.

Table 1. ClonaI variation in Medicago sativa lines; t h e e f f e c t of NaC1 on the growth of callus. Growth is fresh weight increase p e r c e n t a g e of the fresh weight increase at Callus for W75RS is from 19 seedlings, r e g e n e r a t e d plants (calli-clones) and for calliclonal plants.

Line

Clone

RESULTS Callus from t h e t h r e e lines of Medicago sativa exhibited differing degrees of t o l e r a n c e to NaCI when placed d i r e c t l y onto saline media (Table 1). The e x i s t e n c e of higher levels of N a C l - t o l e r a n c e in W75RS callus cultures and whole plants have been r e p o r t e d previously (Smith and McComb 1981b), but an additional point of i n t e r e s t from Table i is the variation b e t w e e n clones. It is this variability t h a t one wishes to exploit when using cell cultures to isolate NaCIt o l e r a n t lines.

62.5 mM NaCI

W75RS

Calli developed from suspension cells of the t h r e e lines of M. sativa, when plated onto saline media, showed t h a t t h e number of m a c r o s c o p i c colonies was highest in W75RS (Table 2). The N a C l - t o l e r a n t line r e p o r t e d by Croughan e t al. (1978) also c a m e from a Regen S line (W74RS), fro-m which the line we used (W75RS) was developed. R e g e n e r a t i o n potential was lowered through a cell suspension-plating cycle. Using W75RS as an example, 73% of 4 week old calli were capable of r e g e n e r a t i n g plants but when cultures had been through a suspension culture and then r e p l a t e d they had been in culture an additional l0 weeks and only 16% could r e g e n e r a t e on BOi2Y medium. Even so, plants could be r e g e n e r a t e d reliably from calli produced from cells plated onto the control O NaCI media. Approximately 500 calli from the t h r e e M. sativa lines, including 300 ealli t h a t grew at NaCI levels of 62.5 - 250 mM, were placed on r e g e n e r a t i o n medium. Though many colonies showed signs of greening and d i f f e r e n t i a t i o n , plants could be r e g e n e r a t e d only from a single colony of W75RS callus which had grown on a plate with 62.5 mM NaCI. Plants from this particular callus, and new calli derived from the r e g e n e r a t e d plants, were grown at various NaCI levels (the plants in a e r a t e d culture solutions, the calli on solid media) and the response did not differ from the original population response seen for seedlings and callus of W75R5 (Fig. 1 a,b).

2 3 4 5 6 7 8 10 11 13 14 15 16 17 18 19

The screening indicated t h a t it was relatively easy to s e l e c t for N a C l - t o l e r a n t cell variants, without the use of mutagens, using a suspension culture and plating onto solid media (Table 2). Regeneration from the N a C l - s e l e c t e d cells was exceedingly difficult, however, and the few plants t h a t were r e g e n e r a t e d were not significantly more t o l e r a n t than t h e original population (Fig. ib). These results are similar to those r e p o r t e d by Rains (1982). More encouraging results are given by Nabors e t al. (1980) who successfully raised s a l t - t o l e r a n t plants from s a l t - t o l e r a n t callus of t o b a c c o and d e m o n s t r a t e d on improved level of t o l e r a n c e in the whole plants.

FRESH WEIGHT 125 mM 250 mM NaCI NaCI

84 133 66 54 103 97 93 95 96 146 181 98 136 114 135 109

52 73 56 24 65 60 89 56 71 55 91 54 63 97 37 106 66

33 37 21 17 I1 45 43 41 74 40 24 35 31 52 19 26 3#

HG2

1 2" 3 4 5 6 8

60 45 51 61 59 74 26 5#

20 24 28 20 14 27 32 24

18 18 19 17 15 22 22 19

Cur R3

1 2 3 4. 5 6 7 8

31 75 46 71 64 "101 62 68 6#

54 60 34 48 45 45 43 38 #5

8 10 13 5 7 8 10 8 9

DISCUSSION This study showed t h a t the N a C l - t o l e r a n t t r a i t of M. sativa cv. Regen S (W75RS) was r e t a i n e d through d-ed i f f e r e n t i a t i o n to calli and cell suspensions and d i f f e r e n t i a tion back to the whole plant. However, as plants w e r e r e c o v e r e d only from one callus growing at 62.5 mM NaCI, generalizations are difficult, e x c e p t to suggest t h a t t h e s e results provide e n c o u r a g e m e n t for further e x p e r i m e n t s to be conducted on the transmission of NaCI t o l e r a n c e from cultures to whole plants.

expressed as a O NaCI (control). for HG2 from 7 Cuf R3 from 8

Table 2. Callus growth a f t e r plating from suspension cultures onto solid media with d i f f e r e n t levels of NaCI. The p e r c e n t a g e is shown of plates with at least t h r e e 3 mm d i a m e t e r colonies a f t e r eight weeks on BII media.

NaC1 c o n c e n t r a t i o n (mM) Medicago sativa

line

W75RS HG2 Cur R3

0

62.5

i00% 98% 100%

79% 34% 38%

125 45% 28% 18%

250 3t~% 17% 3%

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Fig. 1. The effect of NaCI on the growth of whole plants and callus from Medica$o sativa cv. Regen S (W75RS). (a) O callus from seedling% • callus from plants regenerated from callus growing on 625 mM NaCI. (b) O plants raised from s e e d , • p l a n t s regenerated from callus growing at 62.5 mM NaCI. A comparison of the value for whole plants and callus showed no significant difference at each NaCI level (t-test for two means).

ACKNOWLEDGEMENTS M. K. Smith acknowledges support from a Commonwealth Postgraduate Research Award and 3. A. McComb acknowledges funding from the Australian Research Grants Scheme. REFERENCES Bingham ET, Hurley LV~ Kaatz DM, Saunders 3W (1975) Crop Sci 15;719-720 Croughan TP, Stavarek $3, Rains DW (1978) Crop S c i 18:959-963 Kao KN, Michayluk MR (1980) Z Pflanzenpbysiol 96:135-141 Kao KN~ Constabel F, Michalyuk MR, and Gamborg OL (197#) Planta 120"215-227 McCoy T3, Bingham ET (1977) Plant Sci Lett I0:59-66

Nabors MW, Gibbs SE, Bernstein CS, Meis ME (1980) Z Pflanzenphysiol 97:13-27 Rains DW (1982) California Agric 36:30-31 Saunders 3W, Bingham ET (1975) Am 3 Bot 62:850=855 Smith MK, McComb 3A (t981a) Aust 3 Plant Physiol 8:267275 Smith MK, McComb 3A (1981b) Aust 3 Plant Physiol 8:#37= ¢#2 dos Santos AVP, Outka DE, Cockin 8 EDi Davey MR (1980) Z Pflanzenphysiol 99:261-270 Stavarek $3, Croughan TP, Rains DW (1980) Plant Sci Lett 19:253-261 Walker KA, Yu PC, Sato $3, 3aworski EG (1978) Am 3 Bot 65:65#-659 Walker KA, Wendeln ML, 3aworski EG (1979) Plant Sci Lett 15:23-30 Widholm 3M (1972) Stain Technol #7"18%19#