Volume 2 Part 2 July 1996
Acram M. Taji (l), Richard R. Williams (2) and Warren H. Sheather (3)
(1) Department of Agronomy and Soil Science, University of New
England, Armidale, NSW, 2351 Australia.
(2) Department of Horticulture, The University of Queensland, Gatton Campus, Lawes, Qld., 4343, Australia.
(3) Department of Botany, University of New England, Armidale, NSW, 2351. Australia.
Hyperhydricity (vitrification) is a physiological disorder of in vitrogrown plants resulting in tissue hypertrophies. We examined the anatomical basis of this response by comparing normal and affected stems and leaves of four rare Australian native plants, Olearia microdisca, Prostanthera calycina, Prostanthera eurybioides and Swainsona viridis All species developed symptoms of hyperhydricity on Gelrite medium with deFossard's minerals plus l0M BAP. Control plants were grown on growth regulatorfree agar medium. In hyperhydrated plants, stems were shorter and thicker with enlarged cells and large intercellular spaces in the cortical and pith parenchyma and hypolignification of the vascular system. Leaves were also broader, thicker and in some species had a succulent appearance with reduced palisade parenchyma and an increase in intercellular spaces in the spongy mesophyll.
Vitrification, translucency, hyperhydration, succulency and glassiness refer to a physiological and morphological disorder in tissue culturegrown plants. The affected plants appear glassy, have reduced or retarded growth, a bushy habit and thickened and malformed stems and leaves with hypertrophy of cortical and pith parenchyma cells (Kevers et al., 1984; Phan & Hegedus, 1986; Vieitez et al., 1985; Ziv et al., 1987). Hyperhydration is a major problem in the tissue culture industry since it can affect shoot multiplication and culture vigour (Hammerschlag, 1986) and can impede the successful transfer of micropropagated plants to in vivo conditions. Up to 60% of affected plants fail to acclimatise (Paques & Boxus, 1987), thereby limiting the application of in vitro techniques for mass propagation.
Hyperhydration can be caused by high concentrations of cytokinins e.g. in Picea abies (Bornman & Vogelmann, 1984), Dianthus caryophyllus (Lesham et al., 1988) and Olearia microdisca (Williams & Taji, 1991). The gelling agent used in the medium can be another factor inducing hyperhydration e.g. Gelrite induces hyperhydration in apple (Pasqualetto et al., 1988) and in Clianthus formosus (Taji & Williams, 1989).
In the work reported here, the anatomical responses to hyperhydration are compared in four rare and endangered Australian native plants (Leigh et al., 1981).
Shoot tips from existing in vitro cultures of four rare species of Australian native plants, Olearia microdisca J.M. Black (Compositae), Prostanthera calycina Benth. and P. eurybioides F. Muell. (Labiatae) and Swainsona viridis J.M. Black (Leguminosae) were grown on deFossard's MZZM medium (1976) either growth regulatorfree solidified with 0.8% Bacto BiTeck agar (control) or with 0.25% Gelrite® supplemented with l0M BAP (benzylamino purine) to induce hyperhydration.
Cultures were maintained in a growth room at 23 1°C under cool white fluorescent light (50mol m-2s-1) for 16 hours daily. Cultures were assessed after four weeks. Leaf and stem samples from normal and hyperhydrated cultures were harvested and fixed in freshly prepared FAA (formaldehyde: glacial acetic acid: 70% ethanol 5:5:90 by volume), dehydrated by passage through a tertiary butyl alcohol series, then embedded in paraffin. Sections 15 m thick were cut on a rotary microtome and stained with safranin followed by fast green (Jensen, 1962). Hyperhydrated shoots were identified on the basis of their succulent and translucent leaves and stem. Leaf and stem samples were of similar physiological age for normal and hyperhydrated explants within each species.
(1) Leaves (See micrographs of transverse sections of (a) normal leaf , (b) hyperhydrated leaf of Prostanthera.)
The anatomical structure of hyperhydrated leaves differed markedly from normal in vitro grown leaves in all species under study. In general there was a reduction in the number of palisade cell layers; the palisade cells resembling more those of spongy parenchyma with air spaces. The chloroplast density was also reduced in hyperhydrated leaves, possibly because of the large cell size. In all cases the intercellular space in the spongy parenchyma was large. The hyperhydrated leaves were broader and thicker than normal in vitro grown leaves with larger and smoother epidermal cells and more raised stomata. The vascular system of the hyperhydrated leaves was less developed than in normal leaves and the xylem bundles and phloem fibres less lignified. In the case of O. microdisca and P. eurybioides, which are considered to be more sensitive to hyperhydration, the cell walls of both palisade and spongy mesophyll were defective in affected plants.
(2) Stem (See micrographs of transverse sections of (c) normal stem and (d) hyperhydrated stem of Prostanthera.)
In general hyperhydrated plants stems were shorter and thicker. Parenchyma cells in cortical tissue were larger, with thinner cell walls and larger intercellular spaces. The vascular tissues, xylem and phloem fibres were hypolignified in hyperhydrated stems of all species under study. Epidermal cells were more compact and numerous in normal plants.
The process of hyperhydration results in plants taking up excess water and hence the abnormal structures of leaf and stem as observed here.
The lack of clear differentiation between the palisade and spongy mesophyll layers, and the presence of large intercellular spaces are the same characteristics described for affected shoots of Cynara scolymus (Debergh et al., 1981), Dianthus caryophyllus (Ziv et al., 1983) and Castanea sativa (Vieitez et al., 1985). It is well established that internal leaf structure is modified by environmental conditions. For example, many hydrophytes have undifferentiated mesophyll, and in aquatic plants mesophyll acquires characteristic aerenchyma. These changes correlate with high relative humidity (Vieitez et al., 1985).
In the species studied here both leaves and the stem were thicker in hyperhydric cultures. In leaves this is due to longer and wider cells in the palisade and spongy mesophyll and the occurrence of larger intercellular spaces. In stems it is due to an increase in cell size in both cortical and pith parenchyma and an increase in intercellular spaces. Bornman & Vogelmann (1984) proposed that the pronounced differences in cell size between affected and normal tissues could be due to enhanced water diffusion into the cells with a concomitant higher hygroscopic pressure. Other researchers (Daguin & Letouze, 1983; Vieitez et al., 1985) have shown decreased cell wall lignification in hyperhydric tissues, resulting in continuing cell enlargement. In the four species studied here there was a reduction in both phloem and xylem fibres in the stem with no corresponding increase in the number of cells in cortical and pith parenchyma. The increase in cell sizes and intercellular spaces were the only factors causing increased stem thickness, presumably due to the ability of cells to stretch more when filled with water under conditions which induce hyperhydration.
The work described above is the result of the first study on the anatomical status of normal and hyperhydric Australian native plants grown under in vitro conditions.
Bornman C.H. & Vogelmann T.C. (1984) Effect of rigidity of gel medium on benzyl adenine induced adventitious bud formation and vitrification in vitro in Picea abies. Physiol. Plant. 61: 505-512.
Daguin F. & Letouze R. (1985) Relations entre hypolignification et état vitreux chez Salix babylonica en culture in vitro. Rôle de nutrition ammoniacale. Can. J. Bot. 63: 324-326.
Debergh P., Harbaoui Y. & Lemeur R. (1981) Mass propagation of globe artichoke (Cynara scolymus): Evaluation of different hypotheses to overcome vitrification with special reference to water potential. Physiol. Plant. 53: 181-187.
deFossard R.A. (1976) Tissue culture for plant propagators. University of New England, Armidale, New South Wales.
Hammerschlag F.A. (1986) Temperate fruits and nuts. Pp. 221-236. In: Zimmerman R.H., Griesbach R.J., Hammerschlag F.A. & Lawson R.H. (eds.) Tissue culture as a plant production system for horticultural crops. Martinus Nijhoff, Dorbrecht.
Jensen W.A. (1962) Botanical histochemistry. Principles and practice. W.H. Freeman & Co. San Francisco & London.
Kevers C., Conmans M., ConmansGilles M.F. & Gasper T. (1984) Physiological and biochemical events leading to vitrification of plants cultured in vitro. Physiol. Plant. 61: 69-74
Leshem B., Werker E. & Shalev D.P. (1988) The effects of cytokinins on vitrification in melon and carnation. Ann. Bot. 62: 271276.
Leigh J., Briggs J. & Hartley W. (1981) Rare or threatened Australian Plants. Commonwealth of Australia Publication No 7.
Paques M. & Boxus P. (1987) Vitrification: review of literature. Acta Hort. 212:155165.
Pasqualetto P.L., Zimmerman R.H. & Fordham I. (1988) The influence of cation and gelling agent concentrations on vitrification of apple cultivars in vitro. Plant Cell Tiss. Org. Cult. 14: 3140.
Phan C.T. & Hegedus P. (1986) Possible metabolic basis for the developmental anomaly observed in in vitro culture called "vitreous plants". Plant Cell Tiss. Org. Cult. 6: 83-94.
Taji A.M. & Williams R.R. (1989) In vitro propagation of Clianthus formosus (Sturt's Desert Pea) an Australian native legume. Plant Cell Tiss. Org. Cult. 16: 61-66.
Vieitez A., Ballester A., SanJose M.C. & Vieitez E. (1985) Anatomical and chemical studies of vitrified shoots of chestnut regenerated in vitro. Physiol. Plant. 65: 177-184.
Williams R.R. & Taji A.M. (1991) Effect of temperature, gel concentration and cytokinins on vitrification of Olearia microdisca (J.M. Black) in vitro shoot culture. Plant Cell Tiss. Org. Cult. 26: 16.
Ziv M., Meir G. & Halevy A.H. (1983) Factors influencing the production of hardened glaucous carnation plantlets in vitro. Plant Cell Tiss. Org. Cult. 2: 5565.
Ziv M., Schwartz A. & Fleminger D. (1987) Malfunctioning stomata in vitreous leaves of carnation (Dianthus caryophyllus) plants propagated in vitro. Implications for hardening. Plant Sci. 52: 127134.
The help of Bronwen Clark in the histology laboratory is gratefully acknowledged. Financial support for this project was provided by an Australian Research Council Small Grant.