the growth of carrot callus cultures at various concentrations and composition of saline water

Department of Field and Vegetable Crops and Department of Soil Science, Faculty of Agriculture, The Hebrew University, Rehovot, Israel

The Growth of Carrot Callus Cultures at Various Concentrations and Composition of Saline Water

RINA GOLDNER, NAKDIMON UMIEL and YON A CHE!\~

With 8 figures

Received June 15, 1977· Accepted June 29,1977

Summary

Diploid callus cultures of carrots (Daucus carota ssp. sativa) were grown on media which contained various concentrations and compositions of salts. Salt stresses were induced by adding various concentrations of the following salt(s) to the media: sea water, synthetic sea water, NaCl, KCl, MgCI2, CaCI2, Na2S04, HaBOa. In addition, Mannitol was used in some experiments for the induction of osmotic stress. The salinity stress inhibited the growth of the calli, prevented the formation of green color in the tissue, and caused necrosis and death. We have concluded from these experiments that growth inhibition was caused mainly by the increased osmotic pressure while the inhibition of green color formation and the necrosis were caused mainly by the high concentration of salt(s). Potassium was more toxic to the calli than sodium. The results are discussed in terms of selection systems for the isolation of salinity resistant mutants.

Key words: Carrot callus, sea water, salinity, resistance.

Introduction

The most abundunt group of environmental chemicals which affect living organisms are simple inorganic salts, and mainly NaCl. These salts exist in the soil as well as in water, sometimes at such high concentrations that prevent their use for agriculture. Nevertheless, BOYKO (1967) has envisioned the possibility of practical sea-water agriculture. The removal of the polluting chemicals is costly, and not always possible. However, at times, it is possible to overcome the toxic effects by breeding resistant cultivars (e. g. ENGLE and GABELMAN, 1966). MELCHERS (1972) has suggested that tissue culture techniques could be gainfully employed for the selection and isolation of salt resistant crop plants. ZENK (1974) and NABORS et al. (1975) have used this system and have selected NaCI resistant cell lines from tobacco cell cultures. DIX and STREET (1975) used the tissue culture techniques to select NaCl resistant cell lines from both Nicotiana sylvestris and Capsicum annuum. These studies were not yet followed by tests on the derived plants and their progenies for salinity resistance.

Z. P/lanzenphysiol. Ed. 85. S. 307-317. 1977.

308 RINA GOLDNER, NAKDIMON UMIEL and YONA CHEN

Genetic variation in salt tolerance of some plants was reported repeatedly (e. g.: ABEL, 1969; DEWEY, 1960; RUSH and EpSTEIN, 1976; TAL, 1971). BERNSTEIN (1953) did not find such variation in carrots, but HARARI and UMIEL (1975) found variation in sea-water tolerance of germinating carrot seeds. The nature of the physiological processes in plants which are affected by salinity are only partly understood (ZENK, 1974). Moreover, at this stage we are unable to determine the relative importance of high osmotic pressure effects vs. the effects of specific ions in salinity stress (BERNSTEIN, 1975).

The purpose of the present work was to develop a model system that might be used in future studies on salinity resistance in plants. Also, to uncover some of the factors which affect the growth of plant tissue cultures under a salinity stress. We employed carrot callus cultures and sea water salinity as models. Diluted sea water solutions were chosen because of their similarity in ionic composition to many saline water wells (U.S. Salinity Laboratory Staff, 1954).

Material and Methods

The plant material consisted of two genetic types of carrot: the commercial cultivar Chantenay and the inbred line W93A. Seeds of line W93A were generously supplied by Prof. W. H. GABELMAN from the University of Wisconsin, Madison, Wisconsin, USA. Seeds were germinated on filter paper, the seedlings were surface sterilized, explants were cut from the hypocotyls and planted on MURASHIGE and SKOOG (1962) medium (MS medium) containing 2.5 mg/l kinetin and 4.0 mg/l NAA. Some cultures were also grown on B5 medium (GAMBORG et aI., 1968). When used in experiments, the B5 medium gave results which were similar to those obtained with the MS medium. Cultures were incubated in continuous light (450-500 ft. c.) at 26 ± 1°C, and the calli were transferred at 30 days intervals to fresh medium for maintenance. Salinity treatments consisted of either one of the following groups: sea water (SW), synthetic sea water (SWS), or specific salt(s) solutions, which were added to the MS (or B5) medium, replacing part of the distilled water in the medium. A control medium of MS only was employed as a reference level. The effects of each type of solution were tested in a range of concentrations equivalent to 0-60 010 sea water. Each solution was tested by 6 replications. In each replication, 4-6 pieces of callus were placed in a petri dish (58 mm in diameter) with total weight of inoculum averaging 40 mg per dish. Increase in fresh weight of the tissue in each dish was measured at the end of each 30 days growth cycle.

The composition of sea water was determined as followed: Na by an EEL-140 flame photometer; Mg and Ca by a Perkin Elmer model 303 atomic absorption spectrophotometer; CI was measured with Buchler Cotlove automatic chloridometer; and B concentration was determined by the azomethin-H method (WOLF, 1974). Total salts content was determined gravimetrically after drying sea water in an oven at 105°C. Electrical conductivities of the solutions were measured with a Radiometer Copenhagen type CDM 2b conductivity meter. Osmotic pressure was estimated from the electrical conductivity (EC) of the solutions by the following approximation (US Salinity Laboratory Stuff, 1954): 0.36XEC = OP, where EC = electrical conductivity in milimhoslcm at 25°C, and OP = osmotic pressure in atmospheres.

Concentrations of the major ions in the sea water, and the composition of the synthetic sea water are listed in Table 1. Electrical conductivities and osmotic pressure of diluted

z. Pjlanzenphysiol. Ed. 85. S. 307-317. 1977.

Growth of carrot callus cultures in sea water 309

Table 1: Major ions present in sea water, and composition of synthetic sea water. The sea water used contained 4.52 010 of total salts.

The major ions present in sea water

Ion meq/l Na+ 555 K+ 15.3 Mg++ 263 Ca++ 25 Cl- 815

Composition of the synthetic sea water

Salt Na2S04 KCl MgCI2 • 6H2O CaCl2 • 2H2O NaCl

_ 30 u &, N

"0

gil Ion 1.989 Na+ 1.147 K+

26.727 Mg++ 1.844 Ca++

29.926 Cl-

meq/l 540.

15 263

26 816

10.8

E "0

~ 20. III o

.r:.

72 OJ :; III III ~

!L

Fig. 1: Electrical conductivities and the calculated osmotic urn of diluted sea water solutions and the basal growth media.

E .5 ~ 10. ";; '-8

36 :g E

:::J "0 C o

U O+---~--~~--~---r--~r---~----~O o 10 20 30 40 50 60 75

Sea Water (Of.)

III o

Table 2: Salinity treatments and solutions composition.

Treatment designation

No.

1 2 3 4 5 6 7 8 9

10.

Salt solution type and concentration (the final concentration in the media was 0.-60. 0/0 of the indicated concentration).

Sea water (SW) from the Mediterranean Synthetic sea water (see Table 1) Mannitol (O.P. equals that of SW) NaCI- 555 meq/l (Na+ concentration as in SW) NaCI- 815 meq/l (Cl- concentration as in SW) KCl- 555 meq/l (K+ concentration is equal to that of Na+ in SW) Na2S04 - 815 meq/l (S04-- concentration equals to that of Cl- in SW) MgCl2 • 6H20 - 263 meqll + mannitol (Mg++ in concentration as in SW) CaCl2 • 2H20 - 25 meq/l + mannitol (Ca++ in concentration as in SW) HaBOa - 61.8 mg!l + mannitol (B concentration as in SW)

Z. P/lanzenphysiol. Bd. 85. S. 307-317.1977.


sea water are presented in Fig. 1. The sea water contained 3.72 mg!l boron. Specific ions were tested in the same dilution range of stock solutions in which the ion concentration equaled its concentration in sea water. The osmotic pressure of these solutions was adjusted to that of the equally diluted sea water solution by the addition of mannitol. The amount of mannitol which was necessary to adjust the osmotic pressure of specific ions solutions was calculated using data presented by WOLF and BROWN (1966). Details on the salinity treatments are presented in Table 2.

Results

Sea water

Calli from the two carrot types were grown on media contammg 0-60 % sea water. After 30 days in culture the increase in fresh weight was measured (Fig. 2) and a portion of each callus was transferred for a second 30 days growth cycle at the same sea water concentrations (Fig. 3). Salinity not only supressed growth (Figs. 2 & 3), but also affected the color of the calli. On 10-20 % sea water the calli developed a darker green color when compared to cultures grown on 0 % sea water. On concentrations higher than 30 % sea water the calli developed a necrotic brown color. After a similar third growth cycle the cultures were rated visually for colors and growth. The third growth cycle gave results which were qualitatively similar to those obtained in the first two cycles. The most drastic effects on growth and color was exerted during both cycles in the treatments range of 20-40 % sea water.

~ c o u

'0 ~

100

E CJ)

.~ 50

.c til

~ .!:

Cl> til o ~ u c

10 20 30 40 50 60 Sea Water ("!o)

z. Pjlanzenphysiol. Bd. 85. S. 307-317. 1977.

Fig. 2: Effects of increasing sea water concentration in the medium, on growth of carrots calli during the first 30 days in culture.


Fig. 3: Effects of increasing sea water concentration in the medium, on growth of carrots calli during the second 30 days growth cycle in culture.

e c o u

l' OJ

100

.~ 50

.J:: III

~ .s

(IJ III o ~ u 1:

0---0 Chantenay

~ W93

O+---.---.---.----.---.---r~ o 10 20 30 40 50 60

Sea Water ("!oj

Beyond 40 Ofo sea water there was almost no growth, and the color of the calli was necrotic brown.

After three growth cycles of 30 days each in various levels of salinity (see Figs. 2 & 3), the calli were tested for their ability to recover from the effects of sea water (Fig. 4). In general, good recovery was obtained for calli which were previously

Fig. 4: Recovery of carrots calli from the effects of sea water salinity. Calli were grown for 3 growth cycles (30 days each) on media containing various sea water concentrations. After 90 days on sea water the calli were transferred to MS medium for 30 days of recovery. A multiple range test indicate that values having different letters differ significantly at the 5 Ofo level.

e c

100

8 80 '0

3: 20

~ u 1:

o

0 0 a a a a • Chantenay o W 93

b b b r>

bbc

be b

b

0 o 10 20 2S 30 40 so 60

Recovery from Sea Water ( "!o)

z. PJlanzenphysiol. Bd. 85. S. 307-317. 1977.


grown on 0-25 % sea water. After treatments with 30-60 % sea water, however, the calli grew significantly less than those previously grown on 0 % sea water (Fig. 4). These results indicated that in the necrotic brown calli which were obtained at high salinity levels (e. g. 50-60 Ufo sea water - Figs. 2 & 3) at least some cells were still alive even after 90 days. These cells were able to grow upon transfer to MS medium which did not contain sea water.

Synthetic sea water and mannitol

Synthetic sea water which contained the major components present in sea water (Table 1) was used in tests with Chantenay calli. The results (Fig. 5) resembled those obtained with sea water. Mannitol solutions were used to test whether salinity exerted its effects only through changes in the osmoticum of the media. At low osmotic pressures (equivalent to 0-25 % sea water) the effects of salts and mannitol on growth were similar (Fig. 5). In contrast, at higher osmotic pressures (30-60 Ofo sea water) mannitol was less toxic to growth than the salts solutions (Fig. 5). Over

"2 c o u

100 0

10 20

0---0 5 W

~ SWS

.. - .... Mannitol

~ 1\

I \ \ \ \ \ \

30

\ \ \ \ \ \ \ \

A""

40

'~-

50 Sea Water ("!o)

--.. 60 0 10 20 30 40

E.C. (mmhos/cm at 25°C)

Fig. 5: Effects of mannitol, sea water (SW), and synthetic sea water (SWS), on the growth of calli from the Chantenay carrot type. The mannitol was added to the media in concentrations which gave the same osmoticum as that produced by the indicated sea water concentration. The results are expressed both in terms of sea water concentrations (A), and in terms of electrical conductivity (E.C.) of the media (B).

z. Pjlanzenphysiol. Ed. 85. S. 307-317. 1977.


the whole range of mannitol concentrations the calli developed green color which was darker than the green color of cultures grown on media free of mannitol or salts.

Specific ion effects

Effects of calcium, magnesium and sodium cations were tested separately by the addition of their chloride salts to the media (Fig. 6). Similarly, effects of chloride and sulfate anions were studied, using their sodium salts (Fig. 7). The osmoticum of the single salt solutions was kept similar to that of the appropriate sea water solution by the addition of mannitol. NaCI treatments inhibited growth (Fig. 6) to approximately the same extent as sea water (Fig. 2). Growth inhibition by MgCl2 and CaCl2 treatments were similar to that obtained with equivalent concentrations of mannitol (Fig. 6). The inhibition of the green color formation in NaCl and MgCl2

treatments at high concentrations resembled sea water, while in this respect CaCl2

resembled the mannitol treatments.

Fig. 6: Effects of NaCl, MgCI2, CaCl2

and mannitol on growth of Chantenay calli cultures. Concentrations of cations are ex pressed as percentage of their concentrations in sea water. The osmotic pressure was adjusted to that of sea water solutions by the addition of mannitol.

100

e C 0 u

'0 ~ E Cl

~ 50 L Vl

~ .!; OJ Vl 0 e u

.£;

0 0

----A L>------6 NoCI \

0----0 MgCI2+ Mannitol

.-.. CaCI2+ Mannitol

Mannitol

Chontenoy

10 20 30 40 50 60 Salt concentration. Percent of Sea-Water

Tests were also performed by using Na2S04 in comparison to NaCl (Fig. 7), and KCI vs. NaCI (Fig. 8). No differences were found between Na2S04 and NaCl treatments (Fig. 7). In contrast, KCi inhibited the growth of Chantenay calli stronger than NaCi did (Fig. 8).

Effects of boron (as HaBOa) were tested, using concentrations equivalent to those of B in sea water solutions. We did not observe any significant effect of boron on the growth and color of both types of carrots.

Z. P/lanzenphysiol. Bd. 85. S. 307-317. 1977.


e c 8 '0 ~ 1: OJ

100

.~ 50

..c til

~ .!:; Q) til

~ U C

Chantenay

o~ __ ~ __ ~ __ ~ __ ~ ____ -+ __ ~ __ ~ __ ~ __ ~ o 01 0.2 03

Anion concentration (eq/ll 0.4 o 10 20 30

E.c. (mmhos/cm at 25°C)

Fig. 7: Effects of NaCl and Na2S04 on the growth of Chantenay calli, expressed both In

terms of anion concentration (A) and electrical conductivity, E.C. (B).

e c o u

L OJ

100

~ 50 ..c VI

~ .!:; Q) VI o Q)

t; E

Chantenay

0.1 0.2 0.3 Cation concentration (eq/l)

®

o 10 20 30 E.C.

(mmhos/cm at 25°C)

Z. Pjlanzenphysiol. Bd. 85. S. 307-317. 1977.

Fig. 8: Effects of NaCl and KCl on growth of Chantenay calli, expressed both in terms of cation concentration (A) and electrical conductivity, E.C. (B).


Discussion

Chemical stress of the herbicide amitrole has been found to affect three separate phenotypic processes in tobacco callus cultures; growth, green color formation and differentiation (BARG and UMIEL, 1977). Similarly, in the present work we found that salinity stress affected growth and green color formation. Furthermore, BARG and UMIEL (1977) have isolated several types of amitrole resistant mutants which exhibited various phenotypic expressions of resistance. It was thus concluded that amitrole affects the cultures through several different modes of action. The results of the present study also indicate that salinity stress involves at least three different types of stress a follow:

1. Osmotic stress

The fact that the increase in osmoticum of the media is causing growth inhibition is clearly seen in the comparison between SW and SWS treatments vs. mannitol (Fig. 5). This osmotic stress has the strongest effect on growth in the range of salinities equivalent to 0-40 % sea water.

2. Stress caused by the high concentration of total salts

The increased osmoticum caused by salinity has inhibited growth. However, the increased osmoticum per se can not fully account for the growth inhibition observed, especially at high salinities. This is clearly evident from the comparison between the SW and SWS vs. the mannitol treatments (Fig. 5). Moreover, the increased osmoticum has caused the development of darker green color in all the mannitol treatments as well as in the low salts treatments (10-20 Ofo SW and SWS). But, at high osmotic pressures caused by salt treatments the calli lost their green color and developed necrotic brown tissues, indicating specific salt effects. With respect to the browning of calli at high osmotic pressures, we did not find any differences between treatments of SW, SWS, NaC!, Na2SO. and MgCl2 •

3. Stress caused by specific ions

From the comparison between SW and SWS (as well as NaC!), we conclude that the minor elements which are present in sea water contribute little if any, to the salinity stress in the present experimental systems. The only specific ion effect found was the strong growth inhibition caused by potassium (Fig. 8). This effect is probably caused by the unbalanced solutions with the high K+ to Na+ ratio. The relatively high K+ content might interfere with ion uptake by the cells, and/or other physiological processes. Alternatively, if more K+ penetrates the cells, and is followed by Cluptake, then it might result in chloride poisoning. It is noteworthy that natural salts solutions such as sea water, contain relatively low K+ concentrations. Therefore, such specific K+ effects are not expected in experiments with sea water.

z. P/lanzenphysiol. Bd. 85. S. 307-317.1977.


An agreement that the stress caused by salinity is composed of several different types of stresses, will necessarily lead to the suggestion that different types of salinity resistant mutants must exist. Assuming that the previously isolated cell lines (ZENK, 1974; NABORS et al., 1975; DIX and STREET, 1975) are salinity resistant mutants, then what type(s) of resistance(s) these cell lines might represent? Lets consider the selection techniques used for the isolation of these mutants, as well as the reported range of salinities (ZENK, 1974; NABORS et al., 1975; DIX and STREET, 1975). From the results of the present work it is most reasonable to suggest that these mutants are the first type of salts' resistant mutations, namely, resistance against moderate osmotic stres of about 1 0J0 NaCI (which is equivalent to 20-25 % SW). These first type mutants can be best selected by employing the cell suspension technique (ZENK, 1974; NABORS et al., 1975; DIX and STREET, 1975) with gradually increasing the salinity in the medium.

This discussion is limited to the phenotypic nature of the salinity resistance, and does not include the mechanisms by which a mutant becomes resistant (e.g. rate of salt penetration or accumulation, etc). Other types of salt resistant mutants, in addition to the osmotic mutants mentioned above, might also exist, but were not yet clearly demonstrated in plant tissue cultures. Such mutants will enable the cells to overcome the toxic effects of specific ions, to prevent the inhibitory effects of high salts content on growth, and to enable the cells to form their normal green color in the presence of high concentrations of salts. Another type of mutant will resist the lethal effects of prolonged presence in high salinities. DIX and STREET (1975) have obtained colonies from cells which were not killed by 21 days in the presence of 3 0J0 NaCl. However, the recovery of carrot cells after 90 days in 60 % SW in the present work, tends to suggest that the colonies obtained from the 3 0J0 NaCI treatment (DIX and STREET, 1975) were just survivors and not mutants. Nevertheless, such mutants might exist, but stronger salinity stresses might be required for their successful isolation.

For the isolation of each type of resistant mutation, the appropriate selection technique should be devised. Several mutational events and selection steps might be required for the production of a plant (BARG and UMIEL, 1977) which is resistant to salinity under field conditions. It is possible that many of the mutants which were, and will be, isolated in culture, will not express resistance under field conditions. Nevertheless, studies with such mutants will greatly contribute to our present knowledge on salinity stresses.

Acknowledgements

We extend our thanks to Dr. M. TAL for many helpful suggestions during the preparation of this manuscript.

This work was supported by a research grant from The Israel National Commision for Basic Research, and a scholarship (to R. G.) from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel.

z. P/lanzenphysiol. Bd. 85. S. 307-317. 1977.


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z. Pjlanzenphysiol. Bd. 85. S. 307-317. 1977.

the growth of carrot callus cultures at various concentrations and composition of saline water

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