Effects of artificial seawater on morphology and anatomy of taro (Colocasia esculenta var Antiquorum, araceae) tissues in vitro

NYMAN L. P. and ARDITTij. A. 1'-jfects ef artificial seawater on morphology and anatomy of taro ( Colocasia escuknta var antiquorum, Araceae) tissues in vitro. ENVIRONMENTAL AND EXPERIMENTAL BoTANY 28, 215-224, 1988. Taro tissues were cultured in 50-500 milliosmolcs (mOs) artificial seawatn ( ASW). Only those in 50 350 were stable and survived prolonged culture. Organogenesis of shoots, leaves and leaf-like structures was similar in all ASW concentrations, although more abnormal growth was observed at 300-350 mOs. All cultures had highly organized tissues including procambium or vascular tissue, tannin-containing idioblasts and vacuolate paren chyma cells. Differentiation of a recognizable epidermis with stomata was most pronounced in liquid culture.


INTRODUCTION
TARO tissues cultured on standard media undergo reversible structural changes from a slow-growing callus to a teratoma-like mass we have termed calloid. 191 Most of the calloid growth for the first 6-9 months of culture was tumor-like. ( 91 When the calloids were cultured on a graded series of artificial seawater (ASW) concentrations it was possible to select for salt tolerant tissues which produced plantlets. However, the number and quality of plantlets produced per calloid culture varied with ASW concentration, hormone balance and age of the tissue. 18 • 91 Cultures in the middle concentrations of ASW ( 150-250) produced higher numbers of plantlets which appeared normal. 01 Plantlets produced in vitro had stunted shoots, under-developed root systems and survived in soil for limited periods. This study was undertaken to obtain more information about the effects ofincreasing salinity on (I) shoot morphogenesis and leaf development, (2) tissue differentiation within the calloid, and (3) production of tumor-like structures.
Subcultures were made every 2-4 months. Liquid cultures were initiated on a rotary shaker at 1 rpm and transferred to a reciprocal one at 29 oscillations min -1 • The media containing ASW were prepared as described previously. 12 • 8 • 91 To select for salt tolerance, tissue was transferred from standard LS medium to successively higher levels of A.SW at 215 50 milliosmole (mOs) increments. Transfers to higher concentrations of ASW were made only after the tissues became established and grew well. The highest concentration of ASW in which tissues remained stable was 350 mOs.

Microscopy
Plant material for light microscopy was cut into segments no larger than 0.25 cm 2 , fixed overnight in 4% glutaraldehyde buffered with 0.1 M sodium cacodylate (pH 7.2), washed and dehydrated in acetone at 0°C. Tissue was gradually brought to room temperature, infiltrated with Spurr resin and embedded at 70°C for 18 hr.
For SEM observations tissue was sectioned into 1 mm 3 segments and fixed for 14--18 hr in 4% glutaraldehyde buffered with sodium cacodylate at pH 7.2. After a slow ethanol dehydration ( 10% increments of ethanol for 20-30 min at each concentration) and a similarly slow infiltration with freon, the tissue was dried in a Bomar 900 EX critical point dryer. The sections were then mounted and coated with gold for 3 min in a Hummer 2 Sputter coater. Observations were made in a Hitachi S-500 scanning electron microscope.
To stabilize tissues taken from samples grown in seawater-containing media, sucrose was added to the fixation solution. A concentration of0.63 M sucrose was empirically found suitable for tissues grown on 350 mOs ASW. Sucrose concentrations necessary to stabilize tissues from other levels of ASW were calculated mathematically (i.e. 0.09 M sucrose for 50 mOs ASW, 0.18 M sucrose for 100 mOs ASW, 0.27 M sucrose for 150 mOs ASW, etc.)

Effects of ASW on calloid and plantlets
Healthy green calloids were established in ASW concentrations of 50-350 mOs. No differences in gross morphology were noted in young ( 4--8 weeks) calloid cultures placed on 0 or 50-350 mOs ASW. Tissues survived for a brief period on 400-500 mOs ASW, but necrosis usually developed after about 1 month of culture.
Plantlets generated on all ASW concentrations varied in ( 1) overall size, (2) leaf dimension and shape, (3) extent of internodal elongation, (4) degree of basal shoot formation, and (5) root development. These differences were evident in plantlets of similar ages produced synchronously in the same culture vessel. After transfer to indi- vidual ft.asks, differences between plantlets often remained constant.

Morphological features of ASW calloids
Observation of 32 week-old calloids under the SEM showed that protocorm-like bodies, multiple meristem regions, abnormal shoot and leaflike structures as well as normally-appearing plantlets were produced at all levels of ASW The meristematic regions and the primordia produced from them were structurally variable. Variability seemed to be independent of ASW concentration. Leaf'.-like primordia which did not differentiate beyond small projections [ Fig. 1  Fm. 4. Light microscope images of sections of the calloid surface from tissues grown in artificial seawater-containing media. (a) Section cut perpendicular to the calloid surface from tissue grown on semi-solid Linsmaier-Skoog medium containing 100 mOs artificial seawater. A small pocket of cells (arrows) with oblique and anticlinal divisions disrupts the uniseriate outer layer surrounding it. This could represent a region close to an active meristemoid or an area which had a short period of meristematic activity which has been lost. Tannin idioblasts (d) can be seen in the ground tissue surrounded by vacuolate parenchyma cells containing amyloplasts.
x 128. (b) Section cut perpendicular to the calloid surface in a reg'ion similar to the one shown in Fig. 3(b). Tissue was grown on semi-solid Linsmaier-Skoog medium containing 150 mOs artificial seawater. A disruption in the uniseriate appearance of the outer calloid layer is present (arrow). x 128. (c) Section cut perpendicular to the calloid surface. This tissue was grown on semi-solid Linsmaier-Skoog medium containing 300 mOs artificial seawater. Many tannin-containing idioblasts are visible (d) and numerous small or degenerating plastids (arrows) are present in the ground parenchyma cells. The outer layer ( e) appears to be primarily uniseriate.
x 128. (d) Section cut perpendicular to the calloid surface. This tissue was grown on semi-solid Linsmaier-Skoog medium containing 350 mOs artificial seawater. A tannin-containing idioblast ( d) is present, a uniseriate outer layer ( e), and a number of light stained plastids (arrows) are visible in ground parenchyma cells. x 128.
ity of plantlets produced from callus subcultured for more than a few months may diminish in some species, whereas other plants like taro 17 • 8 ) and alfalfa 1121 actually require long culture periods for plantlet development. The physiological constraints on rapid plantlet production and the mechanisms which control development (i.e. organogenesis or embryogenesis) are often unknown. Since prolonged culture of tissues may lead to spontaneous genetic changes, 13 ) the time required for plantlet production becomes an important consideration in determining the strategy and outcome of in vitro selection. Even when in vitro selection pressures can be used successfully to produce cells or tissues tolerant to an introduced factor, there are differences in the degree to which traits expressed in culture can be transferred to plantlets generated from them. Success of in vitro selection appears to depend both on innate qualities of the tissues used and on the character(s) studied. 14 • 5 • 1 u 4 .t7.t 9 i Most studies of salinity effects have been limited to the biochemical, physiological or cytological responses of plant tissues exposed to increasing concentrations of NaCl. 1131 Exogenously supplied mixtures of salts are more likely to approximate field conditions than single salts. Hence responses in cultured tissues exposed to increasing concentrations of seawater may be more reliable indicators of potential adaptation to natural saline conditions. Cell and tissue responses are useful in assessing the components of the physiological adaptation to saline conditions, but cannot always be used as a measure of successful plant establishment in soil at comparable levels of salts. Therefore it is important when using in vitro systems to understand the mechanism(s) of plantlet regeneration in standard conditions, and the manner in which salinity affects normal plantlet development.
Initial studies of small numbers of taro calloid cultures on standard media 171 did not reveal the regions responsible for plantlet development.
Observations of ASW cultures of taro made in this study helped to clarify the nature of in vitro plantlet initiation and growth in taro cultures. Time-dependent changes in production oflateral organs on calloid meristemoids (i.e. from linear through spatulate, and finally hastate or peltate forms resembling leaves of seedling-grown plants), became clear only after large numbers of ASW cultures were examined.
Observations of tissues from all media indicate that plantlets, abnormal leaves and shoots, as well as teratoma-like structures, arise from analogous meristematic regions. Further, the development of meristemoids is dependent upon tissue age, exogenous growth regulator supply and most probably local endogenous supplies of growth active substances. This explanation might account for the heterogeneous appearance of calloid tissues, the diversity of differentiated organs observed, and the increase in normal plantlet production with aging and tissue crowding. The multilayered arrays of cells noted in the calloid surface may represent meristemoids which have become inhibited early in their development.
The differentiation ofa recognizable epidermis with well developed stomata appeared to be correlated with and/or a reflection of a more rapid transition to normal plantlet development (e.g. in 150-250 mOs ASW). No other major qualitative differences in general structure were detected in calloid over the range of ASW concentrations used. For example dark stained idioblasts occurred in the ground tissue of all calloids. These were similar to cells observed in taro leaves.' 151 Previous reports 116 ' and more recent studies in our laboratory' 91 indicate that salinity may affect quantitative characteristics and ultrastructural features of cells. Such effects may include variations in cell size, wall thickness, chloroplast size and structure, numbers and size of starch grains and the appearance of the nucleus.
The similarities in morphological and anatomical features of calloids grown on 0 and 50-350 mOs ASW suggest that other than selection for increased tolerance to salinity, growth in AS\V is not associated with gross phenotypic transformation of the tissues. This conclusion is supported by studies of growth kinetics 17 ' and the organic constituents of the tissue. 161 It seems clear therefore that the general problems related to plantlet development are a function of calloid growth rather than ASW concentration. However, both abscisic acid and endogenous cytokinin levels within plants can be affected by exposure to saline conditions. (lo Therefore greater production of tumor-like formations in taro and tissue death at very high ASW concentrations may be related at least in part to changes in endogenous hormone levels. Studies to determine the nature of the observed anomalous and slow plantlet growth from calloid cultures are continuing in the hope of clarifying the mechanism(s) involved. In vitro selection of ASW tolerance does not appear to significantly alter patterns of organogenesis in taro tissues. Therefore this system holds considerable promise as a means for the production of salt tolerant strains of taro.