Apical callus formation in Solieria filiformis (Gigartinales, Rhodophyta) cultured in tanks. D. R. Robledo & G. Garcia-Reina. Institute of Applied Algology, ...
Hydrobiologia 260/261: 401-406, 1993. A. R. O. Chapman, M. T. Brown & M. Lahaye (eds), Fourteenth InternationalSeaweed Symposium. © 1993 Kluwer Academic Publishers. Printed in Belgium.
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Apical callus formation in Solieria filiformis (Gigartinales, Rhodophyta) cultured in tanks D. R. Robledo & G. Garcia-Reina Institute of Applied Algology, University of Las Palmas of Gran Canaria,Box 550 Las Palmas, Canary Islands, Spain
Key words: callus, disorganization, Solieriafiliformis, Rhodophyta, seaweed, tank culture, turbulence
Abstract Loose-lying wild plants of the carragenophyte Solieria filiformis (Kltzing) Gabrielson were cultivated under greenhouse conditions in 6001 tanks in stationary and turbulent cultures, produced either by air bubbling or water jets at the bottom of the tanks. One week after inoculation 90.3 % of the apices of the plants grown in air turbulent cultures initiated the formation of callus. The apices were not broken and apparently non-wounded. No callus formation were observed from a few accidentaly broken apices in any culture. Only 4% of the apices in water turbulent cultures induced callus. Reorganization of branches from the calli took place after three weeks. Organogenetic calli detached from the mother plant after four weeks and formed spherical masses of 3 cm in diameter growing as unattached balls. Cellular disorganization (i.e. callus formation) in S. filiformis seems to be a consequence of intermittent abrasion or contact stimuli against tank walls produced by turbulence.
Introduction Studies on callus induction, culture and reorganization have played an important role in the development of higher plant tissue culture techniques and applications (Yeoman & Forche, 1980). In vitro attempts to induce and culture seaweed calli have been hitherto performed with little success, basically due to the lack of knowledge on the factors promoting callus formation and reorganization. Although the addition of plant growth regulators to the culture media has been described as an important factor for callus induction in seaweeds (Fries, 1973; Gusev et al., 1987; Bradley & Cheney, 1990; Liu & Kloareg, 1991), there are some reports emphasizing the efficacy of physical
parameters (i.e., solid vs liquid medium, osmolality) or species specific capabilities (Polne Fuller & Gibor, 1987; Garcia Reina etal., 1991). Even the concept of what is a callus in seaweeds is controversial due to the utilization of the term callus-like to define a wide range of abnormal neo-growths obtained in vitro and the absence of histological studies (Garcia-Reina et al., 1991). On the other hand, various types of warty outgrowths (galls, tumours) have been reported for seaweeds found in natural habitats or in laboratory cultures (reviewed in Apt, 1988). The high capacity of Solieria species to form secondary attachment (Shintani, 1988; Floc'h et al., 1987; Perrone & Cecere, 1991), and previous observations in our laboratory, lead us to
402 speculate on the potential ability of Solieriafiliformis apices to form calli in a response to an inductive physical factor (i.e. turbulence).
Table I. Frequency of apical callus in Solieriafiliformis after one week in culture under different turbulent conditions. Number of plants examined per treatment = 30 Treatment
Materials and methods Round-shaped loose-lying plants of Solieria filiformis growing in a sheltered sandy bottom at the east-coast of Gran Canaria (Canary Islands) were collected, hand cleaned of epiphytes and precultured for one week with running filtered seawater. The effect of turbulence on the cellular disorganization of the plants (i.e. callus formation) were tested by growing the unattached plants (1014 cm in diameter) for three months in three fiberglass tanks (600 1)at a density of 2-2.5 g wet wt 1- . The first tank was a 'stationary' culture without any agitation, but with a gentle water flow provided by an aquarium pump to prevent water stratification. The turbulence of 'air-turbulent' and 'water-turbulent' tanks were generated either by injecting air or recirculating water through a PVC pipe (1/2 inch diameter), with 1 mm diameter holes drilled at 10 cm intervals centered at the bottom of the tank. This created a circulation in the turbulent cultures that maintained the round-shaped plants in constant motion, rolling from the bottom to the top of the tanks in both turbulent cultures at approximately 3 rpm. However, the plants grown in the 'air-turbulent' tanks had a higher self-rotatory speed. All cultures were performed in a greenhouse exposed to daylight conditions. Highest irradiance (measured with a spherical sensor below the water surface) was in the range of 160-200 umol m-2 - , approximately the same irradiance the plants receive in their natural environment. Water temperature at noon was slightly lower in 'airturbulent' (17.5 C) than in 'water-turbulent' and 'stationary' cultures (19.9 and 19.2 C respectively). Seawater (37%0) enriched with NaNO3
Stationary Air turbulent Water turbulent
% Apices disorganized into callus
Number of apices examined
0
7113
90.3 4.0
5528 6305
(8 mM) and NaH 2 PO4 (0.8 mM) was renewed weekly. Daily fluctuations in pH were similar in the three tanks (8.2 + 0.11). Thirty plants were collected randomly from each tank at weekly intervals and the status (normal or callus) of the apices of each plant (100250 per plant) were examined under a stereomicroscope. Results were represented as the percentage of disorganized apices (of thirty plants) per culture system. The regeneration of accidentally broken apices were also examined. Tissues for light microscopic examination were fixed in 2.5% (w/v) glutaraldehyde in 0.1 M sodium cacodylate buffer (Peders6n etal., 1980), embedded in Araldite resin, sectioned (1 m) and stained with Toluidine blue (1%).
Results Apices of plants grown in both turbulent systems disorganized into callus after the first week. The frequencies of callus development were significantly higher in 'air-turbulent' cultures than in the other treatments (Table 1). Only the apical parts of the plants transformed into calli. No callus formation was detected in the 'stationary' culture after the complete experimental period. The development of callus structures from the apex of Solieriafiliformisis shown in Fig. 1. Nor-
Fig. 1. Sequential disorganization of the apex of Solieriafiliformis and its further reorganization (a) Initial callus development after one week in culture (b) Cross section of callus. (c) Mass of disorganized cells. (d) Transverse section of calli after two weeks. (e) Proliferation of branches from the callus after three weeks in culture. (f) Transverse section of branch developed from the callus. Scale bar = 0.5 mm for figures a, c, e. Scale bar = 8.5 tm for figures b, d, f.
403
404
Fig. 2. Solieriafiformis after two weeks in air-turbulent culture showing the callus formation at the apices.
mal acute to long acuminate apex exhibited a progressive deepening in pigmentation and initiated the proliferation of a mass of highly differentiated and disorganized cells (callus) (Fig. la, b). After two weeks in culture the callus becomes more evident (Fig. c, d). After the third week in cul-
ture, the wider calli, between 1-2 mm in diameter, started to proliferate radially arranged branches and to form spherical balls (Fig. le, f). Only the apices of the plants in turbulent cultures formed these calli (Fig. 2). Probably due to the turbulence or to fragmentation of the main axis, the organogenetic calli started to detach from the mother plant after four weeks. Free-floating calli continued the development of the branches forming spherical balls of up to 3 cm in diameter at the end of the experimental period. Regeneration of branches from accidentally broken apices took place from a thin layer of cells (Fig. 3a, b), but no callus masses were obtained from these areas.
Discussion Disorganization seems to be a consequence of the turbulence generated in the cultures, as other potentially inductive factors (i.e. irradiance, temperature, pH and nutrients) where identical in the
Fig. 3. Regeneration from accidentally broken apices. (a) Young bud regenerated from a broken area, scale bar = 0.5 mm (b) Longitudinal section of the regeneration surface, scale bar = 17.5 #m.
405 three tanks. Callus formation might be a response to abrasion of the apices against the tank walls whilst rolling around. The higher frequencies of callus formation in 'air-turbulent' cultures might be explained by the higher intensity of shear forces or the frequency of contact stimuli against the tank walls, as the plants in this culture were more frequently overturned, despite the same rpm of circular motion as 'water-turbulent' cultures. Norton & Mathieson (1983) suggested that loose-lying plants are modified versions of attached forms of the same species, and that these morphological changes could be a result of repeated tip damage. This response might be similar to those causing the adventituous formation of secondary attachment from cut surfaces and intact cortex of the apical region of Solieria filiformis described by Shintani (1988) and Perrone & Cecere (1991). As the contact stimuli in our turbulent cultures are not followed by continuous adherence to the substrate (but the opposite), the neogrowths develop and remain growing as callus. The lack of directional guidance and polarity may influence the morphogenetic flexibility of S.filiformis apices. Although the polarity gradients in the thallus of seaweeds are well marked they are easily disrupted if the plant is unattached and frequently overturned (Buggeln, 1981). Callus formation in Solieriafiliformis seems to be a consequence of its high cellular plasticity and induced by the intensity and frequency of contact stimuli in turbulent cultures. The histology and behaviour of the calli (structure, induction, pigmentation, starch content) are quite similar to in vitro cultured calli of Laurencia sp and Grateloupia doryphora (Montagne) Howe (GarciaReina et al., 1988; Robaina et al., 1990).
Acknowledgements The Consejo Nacional de Ciencia y Tecnologia (CONACYT # 57420, Mexico) is acknowledged for financial support to Daniel Robledo. This
work was partially (MAR91-1237).
supported
by CICYT
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