Air exchange rate affects the in vitro developed
leaf cuticle of carnation
J.P. Majada
*, M.I. Sierra, R. SaÂnchez-TameÂs
Lab. FisiologõÂa Vegetal, Dpto. BOS, Facultad de BiologõÂa, Universidad de Oviedo,C/Catedratico rodrigo Uria s/n, 33071 Oviedo, Asturias, Spain
Received 29 June 1999; received in revised form 27 October 1999; accepted 13 April 2000
Abstract
The leaf surfaces ofDianthus caryophyllusplants cultured in vitro in either airtight or ventilated vessels were examined using scanning electron microscopy (SEM). The resultant hyperhydrated, non-hyperhydrated and acclimatized plants were compared for stomatal density, cuticular wax development and stomatal function. The leaf surfaces of in vitro cultured plants were basically the same as those of acclimatized plants but less wax deposition was observed on their leaves. Stomata were found both open or closed after transfer of plants ex vitro. However, stomata of in vitro leaves grown in ventilated culture vessels were more functional than plants grown in other conditions. Acclimatized plants had a normal leaf epidermal surface, and were wholly covered with waxes; their stomatal density being similar to that of highly ventilated plants but lower than that of less ventilated plants. Leaves of plants grown in airtight culture vessels or under a low number of air exchanges per hour had less waxes than plants grown at a higher number of air exchanges per hour or than acclimatized plants. In contrast, hyperhydrated plants had abnormal, malformed stomata and no wax deposition was detected. The adaxial surface of non-hyperhydrated leaves seemed more normal than the abaxial, especially in the most ventilated vessels, and this may be due to the former receiving more light and so developing in a more favourable microenvironment.#2001 Elsevier Science B.V. All rights reserved.
Keywords: Dianthus caryophyllus; Scanning electron microscopy; Ventilation rates; Vitri®cation; Waxes
*
Corresponding author. Tel.:34-985-104834; fax:34-985-104867. E-mail address: [email protected] (J.P. Majada).
1. Introduction
The anatomical and physiological characteristics of carnation plants produced in vitro are a re¯ection of the limited gaseous exchange of the culture vessels (Majada et al., 1998). The degree to which these plants differ from ®eld or glasshouse grown plants depends on many factors including plant species and the variables of the culture environment, such as the number of air exchanges per hour and water availability (Ziv, 1991; Majada et al., 1997). Based on this, a range of abnormality severeness can be established from plantlets that appear normal to plants that look clearly malformed, the so-called vitri®ed or hyperhydrated (Debergh et al., 1992). Because of their peculiar status, in vitro tissue-cultured plants when transferred ex vitro must be treated with great care; the most abnormal plantlets do not survive while others can revert and follow normal development patterns.
Hyperhydrated plantlets are translucent, thick and brittle (Debergh et al., 1981; Leshem et al., 1988; Dillen and Buysens, 1989). Hyperhydrated lea¯ets from a range of species have been found to have abnormal or non-functional stomata (Werker and Leshem, 1986; Miguens et al., 1993), a discontinuous cuticle (Miguens et al., 1993) or an abnormal epidermis (Leshem, 1983; Leshem et al., 1988). Hyperhydrated plantlets tend to have higher fresh weight and a low dry weight relative to non-hyperhydrated plantlets (BoÈtcher et al., 1988). Anatomi-cally, hyperhydrated and airtight cultured plantlets have bigger intercellular spaces in their leaves and water compartmentation studies have produced evidence to suggest that additional water is located in these spaces (Paques and Boxus, 1987). Moreover, the amount of cuticular waxes recovered from airtight cultured plants was usually small, and poor epicuticular wax quality formation was observed (Sutter, 1985, 1988; Diettrich et al., 1992).
Hyperhydrated or normal airtight grown plants when transferred to ex vitro conditions are more susceptible to desiccation. Additional misting may be used to reduce losses but it enhances the risk of fungal attacks. However, ventilated plants reach an intermediate phenotype between airtight and acclimatized ones (Majada et al., 1997, 1998). Moreover, proliferation and/or rooting of the plants obtained in ventilated systems allowed acclimatisation with negligible stress, indicating that after transplanting to the soil, the degree of survival was directly related to the hydric relationship established in the culture vessels during the proliferation phase.
The aim of this study is to use scanning electron microscopy (SEM) to compare the leaves of Dianthus caryophyllus of acclimatized plants and hyperhydrated and non-hyperhydrated plants obtained under different ventilation conditions.
2. Material and methods
2.1. Plant material
Dianthus caryophyllus L. cv. Nelken was cultured in vitro as described
elsewhere (Majada et al., 1997). Cylindric culture vessels of 200 ml with diameter 13.194 cm, and an opening of 13.85 cm2 were used. The ¯asks were capped with different SUNCAP (Sigma) ®lters: 3 mm aluminium mounted Ref. C6795 (F3), 6 mm transparent plastic mounted Ref. C6920 (F6) and 10 mm
aluminium mounted Ref. C6670 (F10). Control ¯asks were capped with
aluminium foil (F0). Gaseous microenvironments in the culture vessels were previously characterized (Majada et al., 1997), showing that the number of air exchanges per hour were 0.11, 0.21, 0.68 and 0.86 forF0(airtight vessel) andF3, F6,F10(ventilated vessels), respectively. Ventilation rates of 18.7, 35.7, 115.6 and 146.2 ml hÿ1 in the vessels (200 ml less 30 ml of medium) cited above were calculated according to Kozai et al. (1986). At the end of the multiplication phase, shoots had 4±6 pairs of leaves but only 3 or 4 were fully expanded. Thus, after 4 weeks in culture, the third and fourth pair of leaves from the apical meristem were sampled. Thirty plants obtained in airtight vessels were acclimatized in the glasshouse for 12 weeks and leaves from the same position were also sampled. Three replicates of 10 plants per treatment were analysed.
2.2. Stomatal density
2.3. Response of stomata after leaf excision
To study the morphology and response of the stomata, the same pair of leaves was excised, and left for 0 (Control) or 60 min at 238C, PPFD 18mmol mÿ2sÿ1 (TL D Standard 36W/54) and 60% RH. These leaves were deep-frozen in liquid nitrogen, lyophilized and kept in the desiccator with silica gel to avoid possible effects of rehydratation (Robinson et al., 1987) and cell wall collapse until they were gold coated prior to SEM observation. At the same time, leaves were detached and held for 60 min to allow stomatal closure. In order to decrease the possibility of extracting internal lipids, the leaves were immersed in chloroform for wax extraction (Majada et al., 1998). The adaxial or abaxial surfaces of wax-containing leaves and the wax-free abaxial surfaces were gold coated and observed under SEM as above.
Quantitative data were analysed by ANOVA at a level of a0.05 (SPSS1
WinTM, Chicago, IL).
3. Results and discussion
Many authors have compared hyperhydrated and non-hyperhydrated in vitro grown plantlets; however, as even non-hyperhydrated plants obtained in traditional airtight vessels are not entirely normal, it is better to consider glasshouse grown plants as the basis for comparison. Therefore, for the purpose of this study plants will be referred to as either acclimatized plants (glasshouse), in vitro cultured plants, obtained under different ventilation conditions (plants that will survive ex vitro transfer, facilitated by ventilation of the culture vessels) or hyperhydrated plantlets (plants that normally will not survive transfer to ex vitro conditions).
The number of air exchanges per hour affected stomatal density in carnation leaves (Table 1). As the number of air exchanges per hour increased, the stomatal density decreased, the values obtained at the higher air exchange being similar to those of acclimatized plants (Ac). The decrease of stomatal density was observed on both the adaxial and abaxial surfaces of the leaves, but in all cases the adaxial surface shows a lower density than the abaxial surface and this may be due to the adaxial surfaces receiving more light than the abaxial (Lee et al., 1985; Zacchini et al., 1997).
environments in our experiments, except when hyperhydrated leaves were taken into consideration. We did not observe any difference in stomatal size between in vitro cultured and acclimatized plants, in contrast with what has been described previously (Preece and Sutter, 1991; Santamaria et al., 1993). In hyperhydrated leaves, the stomata have ellipsoidal guard cells sited at the same level as the epidermal cells. However, some stomata are raised above the leaf surface, supported on epidermal cells (Fig. 1C). Different samples of hyperhydrated leaves showed great variability in stomatal density (Table 1) in agreement with Werker and Leshem (1986) and Ziv (1991).
After 12 weeks in the glasshouse, the leaves of acclimatized plants were completely covered with waxes (Fig. 1A and B), whereas those grown in airtight culture vessels or at low number of air exchanges per hour (0.21 changes per hour) had less waxes which were structured as ®lamentous strings (Fig. 1C and D). Hyperhydrated leaves showed poor wax deposition and no pattern was observed (Fig. 1C). As ventilation increased, the amount of waxes increased too, and bigger deposits were observed up to a point at which some guard cells looked like those of acclimatized plants (Fig. 1E). Moreover, there were always more waxes on the adaxial than the abaxial leaf surface, and therefore adaxial surfaces of carnation lea¯ets resemble more those of normal plants than the abaxial ones, in agreement with previous reports (Zacchini et al., 1997).
As shown in Fig. 2, detached leaves from plants grown in airtight culture vessels when submitted to desiccation (Fig. 2A and D), had a lower degree of stomatal closing in agreement with Marin et al. (1988) and Sutter (1988) or even a lag period, when compared with detached leaves from acclimatized plants (Sutter and Langhans, 1982; Shackel et al., 1990). However, in carnation leaves, stomatal closure was greater if they came from the most ventilated culture vessels (Fig. 2G and J). Gribble et al. (1996) found both opened or closed stomata
Table 1
In¯uence of the number of air exchanges (changes per hour) on stomatal densitya
Normal plantsb Hyperhydrated
Signi®cant differences in rows are indicated by different letters (Duncan test,a0.05). Each value is the mean for 30 plantsstandard error of the mean.
b
Normal leaves developed after 4 weeks, cultured in vitro in closed (0.11 changes per hour) or ventilated vessels at a rate of 0.21, 0.68 or 0.86 changes per hour.
c
Leaves after 4 weeks of acclimatization in the glasshouse.
depending on the relative humidity maintained in the specimen chamber of an environmental SEM. However, stomata of airtight in vitro grown plants have been observed to be non-functional, even when detached leaf epidermis was exposed to ABA, mannitol, polyethylene glycol, Ca2, CO2 or darkness (Wetzstein and Sommer, 1983; Conner and Conner, 1984; Ziv et al., 1987), whereas acclimatized
plants were sensitive to these agents. The ®ndings of the present SEM study con-®rm previous results (Majada et al., 1998) indicating that stomatal morphology and physiology were in¯uenced by the microenvironmental conditions.
Differences in cuticle development were also shown when the waxes of the leaves were washed with chloroform. Those grown at a low number of air exchanges (0.11 or 0.21 changes per hour) suffered deformations (Fig. 2B and E)
Fig. 2. SEM of the adaxial surface of carnation leaves grown under different levels of ventilation. (A±C) 0.11 changes per hour. (D±F) 0.21 changes per hour. (G±I) 0.68 changes per hour. (J±L) 0.86 changes per hour. Leaves developed at a low number of air exchanges per hour (A, D) show collapsing of the epidermal cells, produced during sample preparation. Leaves washed for 15 s in chloroform are shown in B, E, H and K. Detached leaves kept for 60 min after removal from the culture vessel at 238C, 18mmol mÿ2
sÿ1
while the rest kept their shapes (Fig. 2H and K). Non-washed leaves immediately ®xed after removal from the ¯ask had open stomata independent of the number of air exchanges per hour (Fig. 2C, F, I and L). However, differences were found in the degree of closure, it being higher at the lower number of air exchanges per hour (data not shown). Several authors have found that acclimatized plants often have a well-developed wax layer whereas in vitro cultured plantlets have no wax deposition or a poor wax layer (Sutter and Langhans, 1979; Ritchie et al., 1991; Majada et al., 1998). This phenomenon has been related to the high RH in airtight culture vessels or to low agar or sugar content in the culture medium (Ziv, 1991). Other authors have described that wax deposition can increase in in vitro acclimatisation in airtight cultures by reduction of RH using drying agents (Sutter and Langhans, 1982; Wardle et al., 1983), polyethylene glycol or changing the composition of the culture medium (Ziv, 1991). However, ventilation of the culture vessels during the proliferation phase lowers RH and increases evapotranspiration, diminishing in turn the water potential of the culture medium, and creating an environment favourable for epicuticular wax deposition. Moreover, if the proliferation stage proceeds in ventilated culture vessels, the anatomical and physiological characteristics of the plants produced are better than those obtained in airtight cultured vessels, as con®rmed by their higher survival after soil transplantation (Majada et al., 1997). This higher stomatal density and lower wax deposition support previous results (Majada et al., 1998) indicating that in vitro plants grown in airtight culture vessels had lower stomatal functionality and higher water loss under ex vitro conditions.
The development of plants grown in ventilated vessels approximates that of the acclimatized plants and there is no disadvantage or decrease in the micropropagation rate compared to that of plants grown in airtight or less-ventilated vessels (Majada et al., 1998). This suggests that in vitro development is an early function of environmental conditions in spite of the fact that the basic form is genetically controlled. Thus, control of morphogenesis in micropropa-gated plants can be achieved through proper management of the environmental factors in the culture vessels.
Acknowledgements
This research was partially supported by the DireccioÂn General de Investiga-cioÂn Cientõ®ca y TeÂcnica (PB 92-0999) of Spain.
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