Sodium Sulfate Tolerance and Sensitivity in Tissue Cultures and Regenerated Plants of Nicotiana tabacum

J.PlantPhysiol. Vol. 132.pp. 155-159{1988}

Sodium Sulfate Tolerance and Sensitivity in Tissue Cultures and Regenerated Plants of Nicotiana tabacum STEPHEN

F.

CHANDLERI), ELENA RAGOLSKY,

and TREVOR A.

THORPE

2

)

Plant Physiology Research Group, Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada T2N 1N4 Received February 25,1987' Accepted June 1, 1987

Summary Control (C), NazS04-normal (N), NazS04-tolerant (T) and NazS04-sensitive (S) shoot cultures of Nico· tiana tabacum L. cv. Wisconsin 38 were transferred to medium containing NaZS04 after 15 or 19 monthly subcultures in the absence of the salt. There were no significant differences among the four shoot cultures in respect to the inhibitory effect of NaZS04 on shoot growth. Rooting was inhibited by NaZS04; only explants from N cultures rooted on 105 or 140 mM NaZS04. Growth of callus cultures initiated from each of the four shoot cultures was inhibited by NaZS04, and all were killed on 140 mM NaZS04. The T callus culture appearad to be more necrotic than C, N or S cllus, but produced occasional adventitious shoots. After exposure to NaZS04, the concentration of proline increased and reducing sugars decreased in C, Nand S callus and all shoot cultures. The opposite response was observed in T callus. Seed was obtained from selfed plants regenerated from C, N, T and S shoot cultures. Although viable, seed from S shoot cultures was much more sensitive to 70 mM NaZS04 than seed from any other source. Similar observations were made on KZS04, NaCI and KCI in comparison to control seed. Shoot cultures initiated from this sensitive seed were also more sensitive to salt during rooting than those initiated from control seed.

Key words: Nicotiana tabacum cv. Wisconsin 38, seed, sensitive, shoot culture, sodium sulfate, stability, tobacco, tolerant. Abbreviations: C = Control; CP = Callus proliferation; DW = Dry weight; FW == Fresh weight; FW: DW == Fresh weight: dry weight ratio; GVI == Germination velocity index; N = Normal; S = Sensitive; SP = Shoot proliferation; T = Tolerant.

Introduction In an earlier report (Pua and Thorpe, 1986 a) it was shown that plants regenerated from NazS04-grown tobacco callus showed phenotypic variation and produced seed that was either normal (N, similar in behaviour to control), sensitive (S) or tolerant (T) to NaZS04 (70 and 140 mM). These classifications were obtained in germination tests by comparison to control (C) seed, produced from plants regenerated from shoot-tip derived, unstressed shoot cultures (Pua and Thorpe, 1986 a). The tests included percent and rate of germination, and seedling survival to Na ZS04' Shoot cultures I) Present Address: Calgene Pacific Pty Ltd., P.O. Box 53, Ivanhoe Victoria 3079, Australia. 2) To whom correspondence should be addressed. '9 1988 by Gustav FIScher Verlag, Stuttgart

from seedlings of each of the four categories (C, N, T, S) retained the characteristics of their respective grouping. However, after eight subcultures in the absence of salt some parameters of tolerance or sensitivity were no longer expressed (Pua and Thorpe, 1986 b). In the present study, we assessed the stability of tolerance in the shoot cultures, and callus derived from them, after an even longer (15 to 19 subcultures) period of maintenance in the absence of salt. Plants have also been regenerated and the selfed seed obtained used to assess the heritability of the original selected phenotypes in an additional tissue culture generation. Despite extensive studies on the effect of salinity on cell cultures (Chandler and Thorpe, 1986) relatively few report extend observations to regenerated plants. There are only a handful of reports where seed from plants regenerated from selected cultures has been obtained (Bhaskaran et al., 1986;

156

5TEPHEN F. CHANDLER, ELENA RAGOLSKY, and TREVOR A. THORPE

McHughen and Swartz, 1984; Nabors, 1983; Nabors et al., 1980; Pua and Thorpe, 1986a; Woo et al., 1985; Ye et al., 1986). Seed must be obtained, and tested over several generations, if the often-stated potential of tissue culture for improving salinity tolerance is to be realistically assessed.

Materials and Methods Cultures The isolation of the C, N, T and 5 shoot cultures from aseptically germinated seedlings of tobacco (Nicotiana tabacum L. cv. Wisconsin 38) has been described previously (Pua and Thorpe, 1986a). The control shoot cultures used in the present study are control I cultures, initiated from shoot tips of greenhouse-grown plants (Pua and Thorpe, 1986 a). All shoot cultures were maintained by monthly subculture on 5P (shoot proliferation; Pua and Thorpe, 1986a) medium without additional salt. Callus was initiated from 8 month-old shoot cultures (after 8 subcultures) on excess salt-free CP (callus proliferation; Pua et al., 1985 b) medium, and maintained by subculture onto CP medium by monthly subculture. Callus initiated from C, N, T and 5 shoot cultures were designated C, N, T and 5 callus cultures respectively. After maintenance through ten subcultures 2 or 3 plants were regenerated from the C, N, T and 5 shoot cultures using methods previously described (Pua and Thorpe, 1986a). These plants were designated C1 and C2 (from C shoot culture), N1 and N2 (from N shoot culture), T1 and T2 (from tolerant shoot culture) and 51, S2 and S3 (from sensitive shoot culture). Seed lots collected after selfing each of these plants were identified in the same way. Shoot tips from C1, 51 and 52 seedlings were used to initiate three new shoot cultures (C1, 51 and 52), which were maintained by monthly subculture on 5P medium (Pua and Thorpe, 1986 a).

Tolerance tests The effect of salt on shoot culture proliferation (shoot number, shoot survival, explant fresh weight), rooting of shoot explants (percentage rooting) and seed germination (percentage germination, percentage seedling survival, germination velocity index) was quantified as previously described (Pua and Thorpe, 1986 a). The additional parameters, maximum explant height during shoot culture proliferation and shoot height during rooting tests, were also measured. In both cases these data were taken after four weeks. For tolerance/sensitivity tests of callus cultures, explants (ca. 30-50mg, fresh weight) were grown on CP medium containing 0, 70 or 140 mM Na2S04 for one month. Fresh weight per explant was measured, and the 14 replicates used for each treatment were pooled for determination of fresh weight : dry weight (FW: DW). Dry weights were obtained after drying at 80°C for 24 hours.

Culture conditions All cultures were maintained in the light (16 h photoperiod; photon fluence rate ca. 80JLmolm- 2s- 1; 380-800nm) at 27±1 °C during culture maintenance and in tolerance tests, including germination.

Determination

0/proline and reducing sugars

Fresh leaf (from shoot cultures) or callus tissue (in both cases ca. 100 mg fresh weight) was frozen in liquid nitrogen and homogenized in methanol: chloroform: water (12: 5: 1; v/v/v). After partition against water and chloroform aliquots of the clear upper layer were assayed for proline using a method previously described (Chandler and Thorpe, 1987) and for reducing sugars by the method of Somogyi (1952). Proline and glucose standards were included with each batch of extracts and taken through all procedures, including partitioning.

Results

Effect o/Na2S04 on shoot cultures Table 1 shows the effect of Na2S04 on growth of shoot explants from 15 subculture-old C, N, T and S shoot cultures. Sodium sulfate was inhibitory to all cultures, however, fresh weights were higher on 140mM Na2S04 in C explants than in explants from N, T and S. Although healthy, the sensitive shoot culture was different from the other three cultures, having shorter stems and smaller leaves. This was reflected in a lower fresh weight in the absence of salt (Table 1). Shoots from every category survived on 140mM Na2S04 and only those leaves in direct contact with the medium became necrotic. Fresh weight: dry weight ratios of N, T and S shoot cultures were higher than control in the absence of Na2S04, but with one exception (tolerant explants on 70 mM N a2S04) were lowerthan control in its presence (Table 1).

Table 1: Effect of Na2S04 on fresh weight (g) and fresh weight: dry weight (FW: DW) of explants from 15-subculture-old C, N, T and S shoot cultures of tobacco. Data was taken after 4 weeks. All shoots from one flask (there were four explants per flask) were pooled for determination of FW : DW. Parameter

Na2S04 (mM) C

Fresh weight a)

0 70 140

FW:DWb)

0 70 140

N

3.58±0.29 1.91±0.12 0.77±0.12

Culture T

3.15±0.10 1.08±0.09 OAO±0.06

4.02±0.30 1.45±0.05 OA7±0.02

S 1.81±0.17 1.09±0.17 0.22±0.01

17.30 ± 3.70 19.90±2.20 22.10±0.80 22.10±2.30 18AO±0.20 16.80±0.60 19.20± 1.90 16AO±0.90 14.90± 1.10 10.70±0.70 11.90±OAO 12.10±0.70

a) Mean ± S.E. of 12 replicates. b) Mean ± S.E. of 3 replicates.

A

E !SO ~

01

'i 4:

.. 40 0 0

4:

I/J

0 SO 01 .~

(; 0

a:

40

0~

0

0

35

70

105

140

Na2S04 (mM)

Fig. 1: The effect of 0-140 mM Na2S04 on shoot height (A) and % rooting (B) of explants from 19 subculture-old C (O), N (~), T (181) and S (_) shoot cultures of tobacco. There were 15 replicates per treatment. Percentage rooting was assessed after 3 weeks and shoot height after four. Bars represent standard error.

Sodium sulfate tolerance in tobacco Table 2: Effect of Na2S04 on fresh weight (mg) and fresh weight: dry weight (FW: DW) of explants from callus cultures derived from C, N, T and S shoot cultures of tobacco. Callus had been established for eight subcultures on salt-free medium prior to this experiment. Data shown was obtained after one month. FW : DW was obtained from pooled callus for each treatment. Culture

Na2S04 (mM)

C

N

T

S

Fresh weight a)

0 70 140

395±40 117± 17 41± 3

536±42 199±27 35± 3

840±78 108±24 48± 4

604±52 285±33 37± 4

FW:DW

0 70 140

9.85 6.78 7.44

9.59 7.20 7.58

11.45 7.97 7.04

11.05 8.68 7.20

Parameter

a) Mean ± S.E. of 14 replicates.

Effect ofNa 2 S04 on callus cultures Callus cultures initiated from the C, Nand S shoot cultures were green and compact. In contrast, the T callus culture was friable, had a higher water content, appeared light brown and tended to become necrotic. Furthermore, this culture occasionally produced single adventitious shoots (in 10-20% of callus pieces), even after 15 maintenance subcultures. No adventitious shoots were observed in the other 3 lines after six subcultures. Sodium sulfate inhibited growth of all four callus cultures (Table 2). There was approximately 50 % survival on 70 mM NaZS04 and complete necrosis on 140mM NaZS04.

Table 3: Concentrations of proline and reducing sugars in C, N, T and S shoot cultures oftobacco grown on 0 and 140 mM NaZS04 and III C, N, T and 5 callus cultures grown on 0 and 70 mM Naz50 4. Proline a ) (!'mol· gFW- 1) no salt Shoot

C N

T S Callus

C N

T S

Reducing Sugarsa) (!'mol glucose equivs' gFW- 1)

plus Na2S04 14.8±1.5 24.8 ±4.8 20.4±4.4 35.9±2.2

203.4±21.1 202.4±24.6 150.3± 12.9 229.1±38.2

25.8± 1.3 62.0±2.7 32.6±2.1 47.7±3.9 10.2±3.8 9.0±4.1 15.0±1.4 34.5±6.3

W.2± 4.1

1.0±0.1 0.8±0.1 0.7±0.1 1.3±0.6

a) Mean ± 5.E. of 3 determinations.

Proline and reducing sugars in tissue cultures Table 3 shows the concentrations of proline and reducing sugars in the C, N, T and S callus and shoot cultures in the absence and presence of Na2S04. In all four shoot cultures exposure to NaZS04 resulted in an increase in proline concentration and a decrease in reducing sugars concentration. The same observation was made in C, Nand S callus. However, in tolerant callus grown on Na2S04 there was a slight decrease in the amount of proline, accompanied by a large increase in reducing sugars concentration, to levels over 10 times greater than C, N or S callus (Table3). In both the absence and presence of NaZS04, control callus contained 5 -6 x less reducing sugars than N, T or S callus (Table3).

Effect of salt on seed obtained from shoot cultures

Sodium sulfate inhibited rooting of explants from all four shoot cultures (Fig. 1). Only explants from the N shoot culture were able to root on 105 or 140 mM Na2 S04. All explants survived on 140 mM N a2 S04, but shoot height was reduced (Fig. 1). There was no significant difference between the different categories of shoot cultures in the pattern of reduction, although because of the different morphology of the S culture, shoots from this source were smaller, even in the absence of salt (Fig. 1).

Culture Category

157

78.9± 17.1 52.5±30.5 21.9±21.5 38.6± 10.7

4.3± 0.9 60.3± 9.4 20.1± 4.8 73.8± 18.7 236.9±53.6 81.3± 6.5 25.5 ± 3.6

Table 4 shows the effect of NaZS04 during germination of seed from plants regenerated from C, N, T and S shoot cultures. Germination was virtually completely inhibited on 140mM Na2 S04, while on 70mM NaZS04 the percentage germination and germination velocity index were much lower for seed lots from the sensitive culture than those from other sources. Seed lots T1 and T2 were slightly more sensitive to salinity than C1, C2, N1 or N2. Germination velocity index was lower in the absence of NaZS04 for seed lots S 1- S3 than for any other seed lot (Table 4), though viability was 95-100%. The response of Sl, S2 and S3 seed lots to NaZS04 was quite variable (Table 4) and this was also seen when germination of S 1 and S2 was compared to C on Na2S04, K ZS0 4 , NaCI and KCI (Table 5). On all salts, the percentage germination and germination velocity index were lower for S 1 and S2 than for the control. At equimolar conTable 4: Effect of NaZS04 on germination (%), seedling survival (SURV) and germination velocity index (GVI) in seeds lots from plants regenerated from C, N, T and S shoot cultures of tobacco. Means within each source are indicated in parenthesis. Naz504 (mM)

5eed Lot

0

70

%a) 5URVb ) GVI

C

)

%

5URV GVI

140 % SURV GVI

Cl C2

99 100 99 100 (99) (100)

41.5 42.1 (41.8)

100 100 (97) (100)

30.4 100 29.4 1 0 (29.9) (1) (50)

0.10 0.05 (0.08)

Nl N2

99 100 99 100 (99) (100)

40.3 98 100 42.3 100 100 (41.3) (99) (100)

24.1 0 30.0 1 50 (27.1) (1) (25)

0.15 0.05 (0.10)

T1

95 100 98 100 (97) (100)

39.8 40.4 (40.1)

85 100 82 100 (84) (100)

25.0 0 20.3 1 (22.7) (1)

0.10

96 100 95 (97)

31.7 28.0 29.4 (29.7)

73 54 9 (45)

T2 51 52 S3

100 100 100 (100)

98

96

100 100 100 (100)

8.5 6.1 1.45 (5.4)

0 0 0 (0)

50 -

-

a) Percentage germination. 50 seeds from each seed lot were tested at each level of Na2S04 and germination was measured at 20 days.

b) Percentage of germinated seedlings surviving after 35 days. C) Germination Velocity Index = EGIt, where G is % germination at 2 day intervals and t is the total time of germination (after Pua and Thorpe, 1986 a).

158 158

STEPHEN F. F. CHANDLER, CHANDLER, ELENA ELENA RAGOLSKY, RAGOLSKY, and and TREVOR TREVOR A. A. THORPE THORPE STEPHEN

NaCI or Table 5: 5: Effect Effect of of 70 70 mM mM Na Na2250 S04, K2250 S044,, NaCI or KCI KCl on on germinagerminaTable 4, K tion (%), (%), seedling seedling survival survival (SURV) (SURV) and and germination germination velocity velocity index index tion (GVI) in in tobacco tobacco seed seed lots lots Cl, C1, 51 Sl ands ands 52. S2. Means Means of of 51 Sl and and S2 S2 are are (GVI) of %, %, 5UR SURV and GVI GVI are are given given indicated in in parenthesis. parenthesis. Definitions Definitions of indicated V and in Table Table 4. 4. in

80 ..§..§.80

Parameter Parameter

40 .c~ 40

Seed Seed Lot Lot

% %

SURV 5URV

GVI

.c.c• 011

~

none none

KCl

NaCI NaCl

C1 Cl

99 99

100 100

69 69

97 97

99 99

Sl 51 S2 52

88 88

13 13

11 11

82 82

78 78

(94) (94)

11

(34) (34)

(6) (6)

10 10 (46) (46)

(72) (72)

C1 Cl

100 100

100 100

38 38

100 100

100 100

Sl 51 S2 52

100 100 (100)

80 100 (90)

50

63

100 100 (100)

100 100

54 54

o

(25)

98 (81)

C1 Cl

37.6 37.6

26.9

12.0

27.6 27.6

26.9 26.9

16.1 28.0 (22.1)

2.2

1.7 0.8

11.5 1.2 (6.4)

12.0 12.3 (12.2)

6.1 6.1 (4.2)

(1.3)

13r-------------------------------, A

12

...

~

~

.~

•'"

011

oo oo ~

~

II)

(/)

oo

66 66

Sl

S2 52

..

Salt 5alt

E E

A

8

EE :I

880 0

'"= C>

"0 0= 00 40 a:a: 40 ~

0 0~

00 Na C I

KCI

Fig.3: The effect of no salt and 70 mM supplements of Na2 50 S04, 4, K250 S044,, NaCI or KCI KCl on shoot height (A) and % rooting (B) of explants from 8 subculture-old Cl C1 (D), (0), 51 Sl (~) or 52 S2 (_) shoot cultures of tobacco. tobacco. There were 15 replicates per treatment. treatment. Percentage rooting was assessed after 3 weeks and shoot height after four. four. Bars Barsrepresent standard error error..

c:c~

"0oo

Effict ofsalt on shoot cultures established from seedlings of C 1, 51 SI and 52 S2 seed lots

o

~ ~

II) til

01

...c:~ OJ .!!

~011~

•

•

7 5

33

~

GiQ. Co

3:~

u. II..

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.§.

...

11

80

~

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~ 40

.-E ... )( c: :I

OJ OJ

:Eo. )(

011

0 No No sai l salt

Na S04 Na 2S04 2

K 2 S04 K 2S04

NaCI NaCI

KCI KCI

Fig. 2: Effect of no salt and 140 mM supplements of Na22S04, 504 , K22S0 5044,, NaCI or KCl KCI on shoot number (A), fresh weight per explant (B) and maximum explant height (C) for explants from 8 subculture-old subculture-old Cl Cl (0), (D), S51 (~) (~) and and S2 52 (_) shoot cultures of tobacco. For each treatment there were 55 flasks, each each with 33 explants. Data was taken taken after after 44 weeks weeks and and bars bars represent represent standard standard error. error.

centrations, centrations, sulfates sulfates were were more more inhibitory inhibitory than than chlorides chlorides for all all seed seed lots lots (Table (Table 5). 5). 5eed 5eed from from 51 51 and and 52 52 which which failed failed to to germinate germinate on on Na2S04 Na2S04 or or K K22S0 S044 were to salt-free salt-free were transferred transferred to germination germination solution. solution. Within Within one one day day radicles radicles emerged emerged and and after after 14 14 days days the the percentage percentage germination germination was was 56 56 % % and and 82 82 % % for for seeds seeds from from SS11 and and 52 52 respectively. respectively.

5hoot cultures derived from C1, 51 and 52 seedlings were Shoot used in a test of salt effects on both shoot proliferation (Fig.2) and rooting (Fig. 3). In shoot proliferation tests, shoot number did not vary significantly between the three cultures, although in the absence of salt S2 appeared to multiply slower than the other two cultures (Fig. 2 A). On both sulfates, fresh weight of C 1 explants was greater than C1 explants was 51 or 52 (Fig. 22B). On all salts the height of Cl greater than 51 or 52 (Fig. 2 C). More consistent results were obtained in the rooting test, where on all salts C 1 explants were taller than S 1 or 52 and showed greatest % rooting (Fig. 3). In both tests sulfates were more inhibitory than equimolar concentrations of their respective chlorides. In the rooting test, 100 % survival was obtained on all salts. In the shoot proliferation test survival was 100% on NaCI, KCI Sl culture which died and Na2504 (except one shoot from 51 S04 survival was 60 %, 67 % and on the latter salt). On K250 C1, 51 and 52 respectively. 60% for Cl,

Discussion The C, C, N, N, T T and and 55 shoot shoot cultures used in this study were The defined on on the the basis basis of of response response to to salt salt during during gergeroriginally defined mination of of seed seed from from aa number number of of plants plants regenerated regenerated from from mination salt-grown callus callus (Pua (Pua and and Thorpe, Thorpe, 1986 1986 a). a). To To test test whether whether salt-grown this original original variability variability was was genetic genetic in in origin, origin, selfed selfed seed seed was was this used in in similar similar germination germination tests. tests. Plants Plants regenerated regenerated from from used gave rise rise to to non-tolerant non-tolerant seed, seed, while while tolerant shoot shoot cultures cultures gave tolerant that from from sensitive sensitive shoot shoot cultures cultures was was clearly clearly more more sensitive sensitive that

Sodium sulfate tolerance in tobacco to salt during germination, particularly as measured by the germination velocity index. It appears therefore that sensitivity, but not tolerance, was inherited in our system. Overall, all seed raised was more sensitive to salt than that used in the study determining the four categories (Pua and Thorpe, 1986a). The reason for this is unclear, but it may be in part due to differences in the growing conditions. Stability of tolerance has been briefly reported in several species (Nabors, 1983; McHughen and Swartz, 1984; Ye et al., 1986) and in more detail in Samsum tobacco (Nabors et al., 1980). The mechanism of inheritance is probably quite complex as there was wide variability in the three sensitive seed lots. Inheritance does not seem to be a simple Mendelian phenomenon in NaCl-tolerant Samsum tobacco (Nabors et al., 1980; Nabors, 1983). Loss of tolerance in the present study may be due to reversion during either maintenance of the shoot culture or during meiosis. It is also possible genetic changes during either process could affect genes controlling expression of genes conferring Na zS04 tolerance. To test the first possibility we used rooting to assess stability of tolerance in long-term shoot cultures, as previous work established that this parameter was a stable characteristic of tolerance, whereas others were lost (Pua and Thorpe, 1986 b). This was confirmed in the present study, as shoot cultures from seedlings of seed obtained from S shoot cultures were more sensitive to salt during rooting than a control. However, this was not obvious in shoot proliferation tests, a finding which points out that selection of a correct parameter for assessing tolerance is very important. The rooting test indicated that the T shoot culture was no longer tolerant after 19 subcultures maintenance in the absence of stress. This raises the possibility that genetic changes occurred during this period, although proliferation via axillary bud break is considered to be stable genetically (Hu and Wang, 1983). That physiological factors causing reversion may be more important, is indicated by the fact that the S shoot culture did not appear to be sensitive during rooting, even though sensitivity was inherited from these cultures. The possibility that tolerance was lost during recombination at meiosis is currently being assessed, using both selfing and controlled crosses. Callus cultures were initiated from C, T, Nand S shoot cultures to determine whether the selected phenotypes were also expressed at the cellular level, like tolerance in Samsum tobacco (Nabors, 1983). There was no evidence for this, but T callus was quite different from the other three lines, both morphologically and in physiological response to salt. Neither distinction could be made in comparison of T to N, S and C shoot cultures. However, it is still not possible to determine whether the variability at the callus culture level is incidental, or because of some factor inherent to T shoot cultures, but only expressed in callus. It is interesting that the T callus produced adventitious shoots, as this is a characteristic of Wisconsin 38 callus grown on CP medium plus NazS04 (Pua et al., 1985 b). Possibly the original selection pressure was sufficient to induce a stable change leading to this phenotype, but only carried through the T shoot culture. Our results have shown that salt tolerance selected in vitro in tobacco is not necessarily stable through selfed generations. This is probably dependent on species, and may be

159

compounded in tobacco which is amphidiploid, and also liable to give rise to aneuploid or tetraploid plants (Pua et al., 1985 a). In addition, this study points out the need to use a variety of measures to examine the stability of specific traits in in vitro derived regenerants. Stability of selected traits is an absolute requirement if tissue culture technology is to be useful for production of agronomically important somaclonal variants. Acknowledgements This work was supported by Natural Sciences and Engineering Research Council of Canada Grant No. G1642 to T. A. Thorpe, D. M. Reid, and E. C. Yeung. We wish to thank S. Miller for enlightening discussion.

References BHASKARAN, S., R. H . SMITH, and K. F. SCHERTZ: Progeny screening of Sorghum plants regenerated from sodium chloride-selected callus for salt tolerance. J. Plant Physio!. 122, 205-210 (1986). CHANDLER, S. F. and T. A. THORPE: Variation from plant tissue cultures: biotechnological application to improving salinity tolerance. Biotech. Advs. 4, 117 -135 (1986). - - Characterization of growth, water relations and proline accumulation in sodium sulfate tolerant callus of Brassica napus L. cv. Westar (Canola). Plant Physio!. 84,106-111 (1987). Hu, C. Y. and P. J. WANG: Meristem, shoot tip and bud cultures. In: EVANS, D. A., W. R. SHARP, P. V. AMMIRATO, and Y. YAMADA (Eds.): Handbook of Plant Cell Culture, Vo!' 1, pp. 177-227, Macmillan, New York (1983). McHuGHEN, A. and M. SWARTZ: A tissue-culture derived salt-tolerant line of flax (Lmum usztattssimum). J. Plant Physio!. 117, 109-117 (1984). NABORS, M. W.: Increasing the salt and drought tolerance of crop plants. In: RANDALL, D. D. (Ed.): Current topics in plant biochemistry and physiology Vo!' 2, pp. 165-184. University of Missouri Press (1983). NABORS, M. W., S. E. GIBBS, C. S. BERNSTEIN, and M. E. MEIs: NaCltolerant tobacco plants from cultured cells. J. Plant Physio!. 97, 13-17 (1980). PUA, E.-C., E. RAGOLSKY, and T. A. THORPE: Retention of shoot regeneration capacity of tobacco by NaZS04. Plant Cell Rept. 4, 225 - 228 (1985 a). PUA, E.-C., E. RAGOLSKY, S. F. CHANDLER, and T. A. THORPE: Effect of sodium sulfate on m vttro organogenesis of tobacco callus. Plant Cell Tissue Organ Culture 5, 55-62 (1985 b). PUA, E.-C. and T. A. THORPE: Differential Na2S04 tolerance in tobacco plants regenerated from Na2S04-grown callus. Plant Cell Environ. 9, 9 -16 (1986 a). - - The effect of various salts on multiplication of Ntcottana taba· cum L. shoot cultures of different Na2 S04 tolerance. J. Plant Physio!. 124, 465 - 472 (1986 b). SOMOGYI, M.: Notes on sugar determinations. J. Bio!. Chern. 195, 19-23 (1952). Woo, S.-c., S.-W. Ko, and C.-K. WONG: In vttro improvement of salt tolerance in a rice cultivar. Bot. Bull. Acad. Sin. 26, 97 -104 (1985). YE, J. M., B. L. HARVEY, and K. N. KAo: Screening salt-tolerant barley genotypes via anther culture in high salt media. In: SOMERS, D. A., B. G. GENGENBACH, D. D. BIESBOER, W. P. HACKETT, and C. E. GREEN (Eds.): Abstracts of VI International Congress of Plant Tissue and Cell Culture, p. 414, IAPTC, Minneapolis (1986).