Stomata of leaves from in vitro grown rose plantlets remain opened in the dark. The ultrastructure of their guard cells was studied after a 7 h light and a 7 h dark ...
Plant Cell, Tissue and Organ Culture 32: 227-233, 1993. © 1993Kluwer Academic Publishers. Printed in the Netherlands.
The ultrastructure of micropropagated and greenhouse rose plant stomata Huguette Sallanon, Maryse Tort & Alain Coudret Laboratoire de Physiologie et GEnEtique VOgEtale, UniversitE Blaise Pascal, 4 rue Ledru, 63038 Clermont-Ferrand, France
Received 15 July 1991; accepted in revised form 21 July 1992
Key words: in vitro, stomata, ultrastructure, rose
Abstract
Stomata of leaves from in vitro grown rose plantlets remain opened in the dark. The ultrastructure of their guard cells was studied after a 7 h light and a 7 h dark period, and compared to that of functional stomata from plants which have been acclimatized to greenhouse conditions. Qualitative and quantitative observations concerning the shape of the guard cells, mitochondria, plastids and starch grains, demonstrated the similarity in guard cell ultrastructure. The peculiarity of guard cell ultrastructure of in vitro cultured plants was the inability to close in the dark; vacuolar area was 40% of the whole guard cell area during both light and dark period whereas, in guard cells from greenhouse plants, the vacuolar area was 40% of the whole guard cell area during the light and only 25% during the dark period. These results indicate that stomata from in vitro plants are duly developed and possess an ultrastructure suitable for a typical functioning. The inability to close in the dark results from atypical water relation.
Introduction
The successful transfer of regenerated plants from in vitro culture to the greenhouse is an essential step for successful micropropagation systems. This transfer stage is still problematic for many crops: most of the difficulties could result from the poorly developed vascular system (Grout & Aston 1977; Leshem 1983), and of an excessive dessication immediately after removal from the culture (Brainerd & Fuchigami 1981; Conner & Conner 1984). The dessication is thought to be caused by a reduced epicuticule on the leaf surface (Grout & Aston 1977; Grout 1975; Suther & Langhans 1982) and an abnormal stomatal functioning (Blanke & Belcher 1989; Brainerd & Fuchigami 1981; Ziv et al. 1981).
Failure of stomata to close in response to darkness or externally applied A B A or high level of CO 2 were reported in apple and cauliflower plants propagated in vitro (Brainerd & Fuchigami 1982). Environmental changes in the culture container such as a decrease in the humidity and increase of the irradiance, reduced water losses (Maene & Debergh 1987) and induced normal leaf and stomata development (Cappelades et al. 1990). The cause of the failure of stomata to close could be due to the guard cell wall (Ziv et al. 1987) and/or result from the stomata deformation (Blanke & Belcher 1989). The purpose of the present work is to study the ultrastructure of the guard cells from plants propagated in vitro in view to determine if these
228 guard cells are completely developed or not and to observe if their internal structure is different from that of stomata on leaves developed in the greenhouse.
Materials and methods
Plant material The greenhouse rose cultivar Madame G. Delbard (R) deladel was cultivated both in vitro and under greenhouse conditions by the Nurseries G. Delbard, France. The axillary budding medium used for micropropagation consisted of Murashige and Skoog salts (Murashige & Skoog 1962), supplemented with benzylamino purine (6.6 I~M1-1) sucrose (87.7mM1-1) then solidified with Bacto agar (7 g 1-1). After adjustment to pH 5.5, 120 ml of medium were pourred in 850ml glass containers (Verreries Champenoises) closed with polycarbonate lids that allow gas exchange and water loss (3 to 6g during a micropropagation cycle of 21 days). Culture conditions were 16-h photoperiod, irradiance 50 Ixmol m -z s -1 (Mazda, day light), temperature 26--+ 1 °C during the day and 22 + 1 °C during the night. The relative humidity inside the growth vessels, measured with a Wescor HP 115 was 9 8 - 2%. Axillary shoots were rooted in the same medium listed above, except that the only growth regulator was indol acetic acid at 5.7 ixM1-1. Cultures were incubated in the same light and temperature conditions. The rooted shoots were acclimatized in a growth chamber at the same temperature, under a light intensity of 120 ~mol m - 2 s - I (Mazda, day light) and 80% RH. Two-month-old plants were transferred in greenhouse and incubated under a natural light period at the same temperature conditions and at relative humidity varying between 70 and 80%. Samples were taken at the end of the in vitro multiplication phase (21-day-old shoots) and on 4 months old plants cultivated in greenhouse. All samples were studied after a 7 h light or 7 h dark period. These samples were taken from the middle of the terminal leaflet grown on the third leaf under the apical bud of the in vitro and acclimatized plants.
Light microscopy, scanning (SEM) and transmission electron microscopy ( TEM ) Stomatal aperture of guard cells during the light and the dark period were determined microscopically on fresh samples under a light microscope. For scanning and electron microscopy, leaf samples were fixed in 5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.5) for 2h and washed 3 times for 15 rain in the same buffer. For SEM, samples were dehydrated through an ethanol and acetone series and critical-pointdried with CO 2. The samples were viewed under a Leica 360 S. For TEM, samples were postfixed in osmium tetroxide (1% in 0.1 M cacodylate buffer) for 1 h, then rinsed again 4 times in distilled water. After dehydration through a graded series of ethanol, the material was embedded in Epon resin. Ultrathin sections were cut on a Reichert OMU2 ultramicrotome with glass knives, collected on copper grids and stained for 30 min in a 2% acqueous solution of uranyl acetate, followed by 3 or 4 min in 0.08 M lead citrate. Sections were examined with a JEOL 1200 EX electron microscope. Micrographs of paradermal sections of guard cells, cutting the nucleus, were analysed quantitatively with an imaging analysis system LEITZ ASM 68K. For TEM, results are the mean from 12 guard cells of three different plant leaves. For SEM, 12 samples of in vitro and acclimatized plants were examined.
Results
Stomata from leaves grown after the acclimatization phase were open during the day and closed during darkness, whereas stomata of leaves grown during in vitro culture were never functional (Fig. 1). No significant differences were observed between guard cells ultrastructure of in vitro and of acclimatized plants, either after a 7 h light or dark period. The cytoplasm of guard cells from both treatments contains a full assortment of organelles and cellular components, such as nuclei, mitochondria, endoplasmic reticulum, plastids and vacuoles (Fig. 2A, B, C).
229
Fig. i. Scaningelectron micrographs of stomata: (A) in vitro plants in light, (B) in vitro plants in dark, (C) acclimatized plants in
light, (D) acclimatized plants in dark.
The nucleus is similar in size and is centrally positioned close to the ventral wall (Fig. 2A, B , C ) . Plasmodesmata are never observed between guard cells themselves or between guard cells and contiguous cells. Mitochondria are numerous, their number and their size are the same in guard cells of in vitro and acclimatized plants; their shape and appearance are also the same: the stroma is poorly coloured and cristae are frequently swollen (Fig. 3B). Each paraderma section of guard cell contains 6 to 7 plastids, with thylakoid structures and large amounts of starch (Fig. 2A, B, C and 3A); no difference could be found between the size of plastids and starch grains in light and dark from
in vitro and acclimatized leaves. The endoplas-
mic reticulum is associated with many ribosomes and is present in great quantity (Fig. 3A, B, E). Dictyosomes are not numerous but they are present in all the guard cells; small cytoplasmic inclusions, associated with the vacuole, sometimes occur (Fig. 3A, E). Microtubules located close to the plasmalemma are also observed (Fig. 3C, D). One major difference was observed between in vitro and acclimatized guard cells: in the light the vacuolar area is 40% of the whole guard cells whereas it decreases to 20% during the dark period in acclimatized leaves. In guard cells of in vitro plants, the vacuolar area remains the same
230
Fig. 2. Paradermal sections of guard cells of acclimatized plants in dark p~riod (A) ( × 7760), and light period (B) ( × 9600), and of in vitro plants in dark period (C) ( × 7200). Abbreviations: GC - guard cell; EPC - epidermal cell; W - wall; N - nucleus; V - v a c u o l e ; M-mitochondria; P - plastid; S - starch grain; T-thylakoid; R E R - r o u g h endoplasmic reticulum; D - dictyosomes; M t - microtubule; I - inclusion.
231
Fig. 3. (A, B, E): part of guard cells of in vitro plants; (A) ( x 18900): plastids with starch grain, rough endoplasmic reticulum and cytoplasmic inclusion associated with a small vacuole: (B) ( x 27000): mitochondria; (E) ( × 16200): dictyosome; (C) (x8100): cross section of a guard cell of in vitro plants, the box is enlarged in (D) (x65700) and shows microtubules. Abbreviations: GC - guard cell; EPC - epidermal cell; W - wall; N - nucleus; V - vacuole; M - mitochondria; P-plastid; S - starch grain; T-thylakoid; R E R - r o u g h endoplasmic reticulum; D - dictyosomes; M t - microtubule; I - inclusion.
232 in both the dark and light period (about 40% of the whole guard cell area).
Discussion The ultrastructure of guard cells of in vitro and acclimatized rose plants is similar to that of many other species (Louguet et al. 1990; Willmer 1983). In all the plants examined, guard cells contain dense cytoplasm and all the usual cell organelles (Willmer 1983). Mitochondria are always numerous (Pallas & Mullenhauer 1972) and do not show any configurational changes in light or dark situations. The most important variation between species concerns the chloroplast shape, their number, the starch content and the amount of the thylakoid (Faraday et al. 1982). During stomatal differentiation, the endoplasmic reticulum is developed but in mature guard cells rough endoplasmic reticulum and polysomes are prevalent (Louguet et al. 1990); mature guard cells are devoid of plasmodesmata (Louguet et al. 1990). Both the abundance of rough endoplasmic reticulum and of polysomes and the lack of plasmodesmata in guard cells from in vitro plants, indicate that guard cells are mature and consistent with a high rate of protein synthesis. The guard cells of in vitro plants exhibit the structural equipment to function. The distinguishing feature of stomata from in vitro plants is their inability to close in the dark: the vacuolar area is constant either in the dark or in the light period. Therefore light does not induce water movements between the guard cells and the contiguous cells. Stomatal function is caused by change in guard cells volume which occurs as a result of water movement due to the water potential gradient between the guard cells and the epidermal cells. The water potential in the cells is mainly controlled by osmotic potential and wall pressure. Much attention has been given to the mechanism of osmotic regulation in the guard cells (Zeiger 1983) but cell wall metabolism might play a role in the regulation of stomatal movements (Meidner 1982; Pantoja & Willmer 1986; Takenchi & Kondo 1988). It was demonstrated that
volume changes in the guard cells have been affected by the elasticity of the guard cell walls and suggested that a decrease in Ca 2+ was implied in wall relaxation and water uptake (Meidner 1982). The similarity between ultrastructure of guard cells of in vitro and acclimatized plants makes it not possible to distinguish between the most important role of osmotic and pressure potential in the inability of stomata to close. Numerous authors suggested that the cause of the failure of stomata to close lie mainly in the guard cell wall and not in the protoplast (Sutter 1985; Ziv et al. 1987). By reducing the relative humidity in the culture container environment or with elevated Ca 2+ in media it was possible to induce in vitro functioning guard cells development (Cappelades et al. 1990; Ziv et al. 1987). These results could indicate that in the guard cells from leaves raised in vitro plants, the protoplast could been functioning, but the physiological functions related to the wall structure and implied in water movements are modified. Stomata from leaves raised in vitro never become functional: this fact has been estabished by measurements with an automatic porometer Mk3. These results show that stomata raised in vitro get structural or physiological malfunctioning which cannot be repaird. To be functional, stomata ontogenesis must occur under a not too high relative humidity; in this study, these conditions only occur in a greenhouse.
Acknowledgement We are grateful to the Nurseries G. Delbard (France) for the production of rose plants propagated in vitro and acclimatized in greenhouse.
References Blanke MM & Belcher AR (1989) Stomata of apples leaves cultured in vitro. Plant Cel Tiss. Org. Cult. 19:85-89 Brainerd KE & Fuchigami LH (1981) Acclimatization of aseptically cultured apple plants to low relative humidity. J. Amer. Soc. Hort. Sci. 106:515-518 Brainerd KE & Fuchigami LH (1982) Stomatal functioning of in vitro and greenhouse apple leaves in darkness, mannitol, ABA and CO:. J. Exp. Bot. 33:388-392
233 Cappelades M, Fontarnau R, Carulla C & Debergh P (1990) Environment influences anatomy of stomata and epidermal cells in tissue-cultured Rosa multiflora. J. Amer. Soc. Hort. Sci. 115:141-145 Conner LN & Conner AJ (1984) Comparative water loss from leaves of Solanum laciniatum plants cultured in vitro and in vivo. Plant Sci. Let. 36:241-246 Faraday CD, Thomson WW & Platt-Aloia KA (1982) Comparative ultrastructure of guard cells of C 3, Ca and CAM plants. In: Ting IP & Gibbs M (Eds) Crassulacean Acid Metabolism (pp 18-30) Grout BWW & Aston MJ (1977) Transplanting of cauliflower plants regenerated from meristem culture: I. Water loss and water transfer related to changes in leaf wax to xylem regeneration. Hort. Res. 1 7 : 1 - 7 Grout BWW (1975) Was development on leaf surfaces of Brassica oleracea vat. Currawong regenerated from meristem culture. Plant Sci. Let. 5:401-405 Leshem B (1983) Growth of carnation meristems in vitro: anatomical structure of abnormal plantlets and the effect of agar concentration in the medium and their formation. Ann. Bot. 52:413-415 Louguet P, Coudret A, Couot-Gastelier J & Lasceve G (1990) Structure and ultrastructure of stomata. Biochem. Physiol. Pflanzen. 186:273-287 Maene lJ & Debergh P (1987) Optimization of the transfer of tissue cultured shoots to in vivo conditions. Acta Hort. 212:335-348 Meidner H (1982) Guard cell pressures and wall properties during stomatal opening. J. Exp. Bot. 33:355-359 Murashige T & Skoog F (1962) A revised medium for rapid
growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15:474-497 Pallas JE & Mollenhauer HH (1972) Physiological implications of Vicia faba and Nicotiana tabacum guard cell ultrastructure. Amer. J. Bot. 59:504-514 Pantoja O & Willmer CM (1986) Pressure effects on membrane potentials of mesophyll cell protoplasts and epidermal cell protoplasts of Commelina communis L. J. Exp. Bot. 37:315-320 Sutter E & Langhans RW (1982) Formation of epicuticular wax and its effect on water loss in cabbage plants regenerated from shoot-tip culture. Can. J. Bot. 60:2896-2902 Sutter EG (1985) Morphological, physical and chemical characteristics of epicuticular wax on on~amental plants regenerated in vitro. Ann. Bot. 55:321-329 Takeuchi Y & Kondo N (1988) Effect of abscissic acid on cell wall metabolism in guard cells of Vicia faba L. Plant Cell Physiol. 29:73-580 Willmer M (1983) Stomatal development and differenciation and the ultrastructure of guard ceils. In: Willmer M (Ed) Stomata (pp 28-50). Longman Zeiger E (1983) The biology of stomatal guard cells. Ann. Rev. Plant Physiol. 34:441-475 Ziv M, Meir G & Halevy A (1981) Hardening carnation plants regenerated from tips cultured in vitro. Environ Expt. Bot. 21:423-428 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. Let. 52:127-134
