ORGANIC AND INORGANIC CONSTITUENTS OF SALT TOLERANT TARO (COLOCASIA ESCULENTA VAR ANT/QUORUM) TISSUES CULTURED IN SALINE MEDIA

Abstract Salinity tolerant taro tissues were selected and cultured in vitro on saline media (50–350 mOsm). Salt tolerance in these tissues was associated with increased levels of calcium oxalate, chlorophyll, protein and some secondary alkaloids. The concentration of other secondary alkaloids as well as the levels of quaternary alkaloids decreased. Calcium, potassium, magnesium and sodium content of the tissues decreased with increased salinity. These findings are discussed in relation to salinity tolerance in taro.


INTRODUCTION
HALOPHYTES and salt tolerant cultivars of several crop plants have been studied extensively in recent yearsY· 17 • 20 J These studies suggest that adaptation to salinity may involve a number of mechanisms. One of these appears to be an accumulation or change in the constitution of components that affect the ionic and osmotic balance of cells. ( 16 ' 281 These components include organic acids, nitrogenous compounds, carbohydrates and mineral ions. ( 4 • 5 • 22 · 25 1 Determining which mechanisms are involved in salt tolerance in previous work is difficult because information has been obtained from diverse experimental systems. These include: ( l) several naturally-occurring halophytes, (2) a number of plants selected for salt tolerance from standard lines, (3) cultured tissues, seedlings and field-grown plants, and ( 4) inter-specific hybrids between halophytes and glycophytes. In addition, plants or cultures were often maintained under various environmental conditions, and with different sources of salinity, as for example, NaCl, natural seawater, artificial seawater or several salt mixtures. (4-7, 15, 19,22,25,32,33,35) Improved knowledge of the basis for salt tolerance in callus and in plantlets produced in vitro may aid in the development of more direct selection methods. Such knowledge can be obtained from studies involving tissues and/or plants of a single glycophyte selected for tolerance to a graded series of salinity levels. Salt tolerant taro tissues selected in our laboratory( 23 1 are such a system. Using this system we have attempted to limit variables for the purpose of gaining a better understanding of adaptation to salinity by glycophytes.

Tissue culture
Tissues, including protocorm-like bodies and undifferentiated cell masses, ( 24

Salinity
To select for salinity tolerance, tissue sections ca 0.1 cm 3 were transferred to successively higher concentrations (5% or 50 mOsm increments) of saline water (SW) approximately every 2.5 months. ( 29 ) Sections were taken from the most vigorously growing cultures. At least 20 new cultures were made every time. SW stock solution was prepared by dissolving 13.96 g NaCl, 0.39 g KC!, 2.25 g MgS0 4 7H 2 0 and 3.80 g MgCI 2 in 100 ml of double distilled water. ( 2 ) This solution was used at dilutions of 50--350 mOsm (equivalent to approximately 5-35% natural seawater).

Organic analyses
Organic compounds were assayed using methods we have used before. ( 231 This facilitated direct comparisons between the levels of organic constituents in mature plants and seedlings ( Table 2) and cultured tissues. Protein content was determined by multiplying nitrogen levels by 6.25. Nitrogen determinations were obtained with a Coleman Model 29 analyzerY 1 l All tissues from two replicate samples were analyzed for each SW concentration. The samples (i.e. culture vessels) were selected at random from among all cultures on a certain SW level.
Calcium oxalate (CaO) was assayed by permanganate titration. 111 • 31 l Alkaloid concentrations were measured with Wagner's and Mayer's reagents. 18 • 16 l This technique was modified only to the extent that a Klett colorimeter was used to measure turbidity of thoroughly agitated samples.13 11 Chlorophyll content was determined by methods adapted for use with orchid protocorms and employed previously for taro. l 31 l

Inorganic analyses
Tissues were dried at 50 ± 2°C until there were no further weight losses (usually 3 days), extracted with 10 N HCI and adjusted to an equal volume. Ionic concentrations in tissues were obtained by transferring samples to two dram polyvials (Van Waters and Rogers) containing I ml ofO. l N HCI and the following additives: none for sodium; 2 mg of cesium chloride for potassium; and 10 mg lanthanum chloride for calcium and magnesium. Concentrations were determined using a Varian AA-27 5 Series Atomic Absorption Spectrophotometer. ( 26 ) Three replicates, each consisting of all the tissue in a cul tu re vessel, were used for every SW concentration. Culture vessels for assays were selected at random.

Calcium oxalate
Levels ofCaO in all cultured tissues were much lower (regardless of the SW concentrations used) than those in leaves and corms of greenhouse grown plants (Table 2). CaO content in the tissue generally increased with SW concentration (

Hydration value
There were no differences between hydration values of tissue on 0, 50, I 00 and 150 mOsm, 50 and 100 mOsm and those on 250, 300 and 350 mOsm, and 250 and 300 mOsm (Fig. IA). The differences between tissues on 200 mOsm and 0, 50, 150, 250, 300 and 350 mOsm were more than one standard deviation.

Chlorophyll
In tissues cultured on medium which did not contain SW, chlorophyll concentration was within the range recorded for petioles of greenhouse-grown plants ( Table 2). The level of chlorophyll increased gradually in tissues from 0 t SD8339 is an experimental cytokinin which as of this writing is no longer available and our supply is exhausted. It is listed here because of its use in the original research. Our current work indicates that 6-DMAP is a satisfactory substitute and available. If SD8339 becomes available again in the future one or the other, not both, should be used. The stock solution should be refrigerated.  lB).

Protein
The protein content (nitrogen level x 6.25) in tissues cultured on 0-150 mOsm and 250-350 mOsm SW was lower than that in corms of mature plants (Fig. lB). Tissues grown on 200 mOsm had more protein than greenhouse-grown  ( Table 2, Fig. IC). SAMR content in corms ofa seedling population was extremely variable; some seedlings contained three times as much as others ( Table 2). Levels of SAMR in leaves of mature plants and seedlings were higher than those of cultured tissues ( Table 2, Fig. 1 C). SAMR concentrations of tissues grown on 0, 50, 100, 300 and 350 mOsm SW were not different from each other (Fig. 1 C). The most dramatic differences in SAMR levels occurred between 150 mOsm and 300 and 350 mOsm (Fig. IC). Generally, the levels of secondary alkaloids in corms of parents and seedlings as measured by Wagner's reagent (SAWR) were lower than in cultured tissues except on 250 mOsm SW ( Table  2, Fig. IC). SAWR content in leafblades of parent and seedling plants was variable. In cultured tissues, SA WR levels were within the range, but lower than the means observed in parents and seedlings ( Table 2, Fig. IC). The only notable difference in SA WR levels in cultured tissues was between 0 and 300 mOsm SW (Fig. IC).
Quarternary alkaloid content of cultured tissues as measured by Mayer's reagent (QAMR) was higher than in corms of mature plants ( Table  2, Fig. 1 C). In mature plants (leaves) and in seedlings (leaves and corms), QAMR levels were higher than in cultured tissues ( Table 2, Fig. IC). Differences, if any, between QAMR levels over the entire SW range were small (Fig. IC).
Levels of quarternary alkaloids in cultured tissues as measured by Wagner's reagent (QA WR) were lower than in leaves and higher than in corms of mature plants, and in the range of seedlings ( Table 2, Fig. IC). The differences between QAWR content of tissues grown on 0-150 mOsm and 250-350 mOsm SW were dramatic (Fig. 1 C).

Inorganic constituents
Levels of inorganic ions per unit of water in the tissue (mg ion/g water) generally dropped with increasing SW concentrations (Fig. ID). Sodium levels remained essentially unchanged from 0 to 100 mOsm SW; at I50 mOsm they were lower than the controls, but no different than in tissues grown on 50, 100, 200 and 250 mOsm. On 300 and 350 mOsm, the sodium concentration was less than that of tissues on standard medium. It was also lower than the levels in all other tissues cultured on SW. Magnesium levels were stable  ID). The Na+ /K + ratio was the same on 0 and 50 mOsm. It increased after that, reaching a peak on 250 mOsm. On 300 and 350 mOsm the ratio dropped to values which were similar to those on 0 and 50 mOsm (Fig. ID).

Growth
The initial transfer to SW brought about a substantial decrease in growth of the tissues (Fig.  2). This was followed by a further decrease. Growth on I 50 and 200 mOsm increased and afterward dropped to its lowest point before increasing again (Fig. 2).

Calcium oxalate
Higher oxalic acid levels were recorded in several halophytes including Halogenton glomeratus, Bassia, Kochia, Rhagodia and A triplex species. ( 7 ) Several reasons, some suggested in the literature and others by our data, could explain these levels. One suggestion is that CaO may play a role in the ionic balance of tissues. ( 7   •  9 • 10 l The fact that CaO content was higher in tissues grown in 100-350 mOsm SW than in those maintained at 0-50 mOsm supports this suggestion. CaO levels may reflect the concentration of oxalic acid in the cells.
If this is the case production of oxalic acid essentially doubled between 50 and 100 mOsm SW and increased less dramatically from 150 to 200 mOsm SW. Oxalic acid is toxic at excessively high concentrations. Therefore it is reasonable to assume that it would be restricted to vacuoles(?) where it will precipitate as CaO in the presence of Ca 2 + due to the low solubility product ofCa0:( 13 l Ca 2 + x C 2 0~-= 1.8 x 10-9 indicates that this must take place. The increased percentage of calcium which is incorporated in CaO when levels of the latter increase suggests that this may indeed be occurring. If so it is possible to speculate that the role played by oxalate and/or CaO in the ionic balance of salt tolerant plants may be of limited importance.
Only a very small proportion of the total calcium is bound as CaO. Such a small proportion probably has negligible effects on the uptake and utilization of calcium ions despite an almost twofold change in the level of this ion over the range of 0-300 mOsm. The decrease in total calcium from 0 to 200 mOsm SW and the subsequent plateau indicate that uptake and/or release of these ions by the tissues vary with adaptation to salinity.
The levels of CaO were lower in cultured taro tissues than in whole plants. This relationship may occur in other species as well since CaO idioblasts have often been observed to be ( 1) restricted in distribution, (2)smaller, or (3) nonexistent in callus derived from crystal-containing whole plants. (l0,l 8 ) Reduced levels of CaO in cultured tissues may result from a number of physiological differences between callus and whole plants, ( 9 • 18 l or from a reduced availability of cal-cium in vitro relative to the amounts available in soil. ( 12,27)

Hydration value
The lack of significance and the large standard deviations ofHV at 0, 50, 100, 150 and 200 mOsm suggest that the tissues, but not necessarily all individual cells, adapt well to these salinity levels. Adaptation to 250 mOsm is apparently more difficult and this may account for the drop in HV from that on 200 mOsm. The subsequent lack of significant differences and small standard deviation are indicative of a more uniform response by individual cells in the tissue. Except at 200 mOsm, the tissues essentially maintain the same HV over a wide range of SW salinity. This is an indication of effective water content regulation.

Chlorophyll content
In glycophytes exposed to saline conditions, the envelope and internal lamellae of some plastids may be affected adversely. ( 32 l These effects were most severe in cultured taro tissues at SW concentrations above 200 mOsm. The ratio of amyloplasts to chloroplasts in these tissues increased with SW. However, chlorophyll content in tissues cultured in 100-350 mOsm SW was higher than in the controls. The effects of SW on plastid structure and chlorophyll content appear contradictory and this may imply a complex relationship.

Protein content
Amino acids and amines accumulate as a result of exposure to salinity in higher plants. ( 7 ) In tomatoes, amino acid nitrogen can increase under salinized conditions from 19 to 82% over control plants. ( 28 ) The protein content of seawater tolerant barley can be as high as 12.9%, as opposed to up to 11.5% in fresh water varieties.( 6 ) NaCl salinization, however, causes a decrease in protein accumulation by pea plants. ( 321 In taro tissues, protein levels remained the same in the range of 0-150 mOsm SW and increased after that. Thus, our results conform only to studies which report increases in protein content with salinization. However, it is also necessary to keep in mind that our protein assay is based on the assumption that a certain proportion of tissue nitrogen is bound in proteins (and that the 6.25 multiplication factor is an accurate reflection of this). In tissues grown under saline conditions this proportion may vary due to the presence of more free amino acids. Also it is not clear at this point whether salinity can affect nitrogen availability and/or uptake, and if the taro tissues produce excessive amounts of a specific protein and/or amino acid (such as praline, for example).

Alkaloids
Horseradish, corn, peas and other plants accumulate alkaloids and amines under conditions of salinity and/or high NaCl levels. 1 32 1 Our finding with Mayer's but not Wagner's reagent, seems to confirm these reports. These reagents are non-specific and their sensitivities for different alkaloids may vary. 18

Inorganic constituents
A number of reports are concerned with the ability of glycophytic species to withstand saline conditions. In many instances, these conditions were generated by the addition of NaCL 1 4 . 5 ,i 4 ,i 5 ,i 9 ,zi, 33 -35 l Natural or artificial seawater was used only in a few experiments. 1 3 . 4 · 27 · 28 1 Attempts have also been made to select salt tolerant strains of a number of glycophytes. Of particular relevance to our work is the selection of NaCl tolerant alfalfa cellsYI The concentrations of NaCl employed in the selection of alfalfa cells are roughly the same as those in 200 and 350 mOsm SW. However comparisons between the alfalfa cells and taro tissues must be made with caution due to the presence of other salts in SW and differences between the two plants. Still it is interesting to note that on a dry weight (DW) basis: (I) Na+ content in taro is generally higher than in alfalfa except in 300 and 350 mOsm, (2) the Na+ content in taro remains relatively stable between 0 and 250 mOsm SW whereas in alfalfa there is a marked increase, (3) K + levels decrease in both plants, but concentrations of this ion in taro are five-six times higher than in alfalfa, ( 4) the Na+ /K + ratio in both plants increases up to 0. 75% Na+ and 250 mOsm SW (which are roughly equivalent) and then stabilizes in alfalfa and drops in taro. The comparison (above) between Na+ and K + content are on a DW basis to allow for comparisons with published data on alfalfa.' 5 1 However, ions are actually dissolved in water and the osmotically significant concentrations are the molarities (Fig. 2).
A common plant response to salinity is increased succulence, 1301 which may be accompanied by cell enlargement, and is characterized by increased water content which prevents excessive accumulation of salts in the cell sap. 151 Our results and those obtained with alfalfa 151 tend not to confirm the latter because water content does not increase dramatically. However, large increases in water content may not be necessary in taro tissues since total ion content is inversely related to salinity.
The Na+ /K + ratio increased with increasing salinity, reaching a peak at 250 mOsm SW. This is probably due to the fact that sodium content remained relatively stable on 0-250 mOsm SW whereas potassium levels dropped precipitously (Fig. 2). In alfalfa the Na+ /K + ratio shows a similar increase before reaching a plateau.151 These differences in Na+ and K + content may be due to salinity and sulfate ion effects on uptake. 141 In alfalfa, for example, salt selected cells accumulated more K + than standard lines. 151 The capacity to maintain high levels of potassium under saline conditions 15 1 and to utilize this ion in the regulation of sodium may be correlated with salt tolerance in halophytes.
Differences between the ion levels in standard and salt tolerant taro tissues may also be the result of transport systems which have been altered by the selection process. The affinities for and transport of ions may differ. 151 Therefore, comparisons between different plants, tissues, organs, and cell lines require caution.

Growth
The drastic reduction in growth following transfer to 50 mOsm, the subsequent increase and the decrease after that might be explained by two factors. One possibility is that the initial transfer resulted in the killing of all cells which were sentitive to salinity. A second is that the subsequent increase (100, 150, 200 mOsm) could be due to good growth of cells and tissues which have been selected for a certain degree of salinity tolerance. The same, but at a higher salinity threshold, could have happened in the 200, 250, 300 and 350 mOsm range. These data suggest that during the initial 4 weeks the tissues are adapting to the saline medium, but have not yet become fully tolerant to it.
It is interesting to note that the general shape of the growth curve is paralled by the curves for CaO,, hydration value, protein level, chlorophyll content, mineral concentration (especially K + + Mg 2 + + Ca 2 + +Na+ and K + alone) and some of the alkaloids. This is not surprising since the levels of ions and/or other constituents may reflect or be a function of growth.