Directory UMM :Data Elmu:jurnal:S:Scientia Horticulturae:Vol87.Issue1-2.Jan2001:
Scientia Horticulturae 87 (2001) 121±130
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 of Dianthus caryophyllus plants 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).
0304-4238/01/$ ± see front matter # 2001 Elsevier Science B.V. All rights reserved.
PII: S 0 3 0 4 - 4 2 3 8 ( 0 0 ) 0 0 1 6 2 - X
122
J.P. Majada et al. / Scientia Horticulturae 87 (2001) 121±130
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). Anatomically, 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.
New systems for environmental control inside the culture vessels will improve
plant quality, autotrophy, automation (Kozai and Smith, 1995) and will also
make acclimatisation safer (Majada et al., 1997). However, few data are
available on the control of culture vessel environments during micropropagation,
and even less information is available about the physiological characteristics
of the in vitro produced plants when the internal environment is modi®ed.
J.P. Majada et al. / Scientia Horticulturae 87 (2001) 121±130
123
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 for F0 (airtight vessel) and F3,
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
The number of stomata was studied by SEM. The sampled leaves were
prepared as described by Robinson et al. (1987), with slight modi®cations. The
leaves were ®xed for 16 h in 3% glutaraldehyde in 0.1 M phosphate buffer pH 7.2
and washed three times with the same buffer. Afterwards, the samples were
dehydrated in an ethanol series and ®nally passed to amilo iso-acetate. Critical
point drying was made (Blazers CDP030), handling the samples very quickly to
avoid rehydration, and they were ®xed with Dotite CX-12 (Jeol/S-C). Finally,
they were coated with gold in an argon atmosphere for 90 s and 22 mA (Blazers
SCD 004 Sputter Coater) and were observed in a Jeol SEM (JSM 6100) at 15 kV.
The adaxial and abaxial surfaces of samples were examined to determine
stomatal density (number of stomata/cm2).
124
J.P. Majada et al. / Scientia Horticulturae 87 (2001) 121±130
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 18 mmol mÿ2 sÿ1
(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 waxcontaining 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).
Stomatal density in carnation leaves was higher in less ventilated plants than in
the more ventilated or acclimatized ones (Table 1), as has been described for
other species (Wetzstein and Sommer, 1983; Blanke and Belcher, 1989).
Although some authors found differences in stomatal morphology between in
vitro grown plants and those grown in the glasshouse or in the ®eld (Donnelly and
Vidaver, 1984; Blanke and Belcher, 1989; Capellades et al., 1990), no differences
were observed in stomatal morphology among the plants obtained in different
J.P. Majada et al. / Scientia Horticulturae 87 (2001) 121±130
125
Table 1
In¯uence of the number of air exchanges (changes per hour) on stomatal densitya
Normal plantsb
0.11
0.21
0.68
0.86
Hyperhydrated Acclimatized
leaves
leavesc
Density of
Adaxial 103 21a 116 15a 69 7.5b 45 10c 34 7.5c
stomata
(number/cm2)
Abaxial 161 20a 149 14b 138 12b 103 11c ±d
41 3.5c
92 4.3c
a
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.
d
Data not collected.
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
126
J.P. Majada et al. / Scientia Horticulturae 87 (2001) 121±130
Fig. 1. SEM of the third and fourth pair of abaxial leaves (Dianthus caryophyllus cv. Nelken).
Leaves after 4 weeks of acclimatization in the glasshouse (A, B). In vitro leaves developed after
four weeks in culture (C±E). (A) Abaxial surface of a wholly expanded leaf showing elliptical
stomata (acclimatized plant). (B) Epicuticular waxes on leaves of an acclimatized plant previously
grown in airtight conditions. (C) Stomata of a hyperhydrated leaf. (D) Stomata in a leaf grown in an
airtight culture vessel. (E) Stomata on the abaxial surface of a leaf grown under ventilation (0.86
changes per hour). Bar represents 10 mm.
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
J.P. Majada et al. / Scientia Horticulturae 87 (2001) 121±130
127
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, 18 mmol mÿ2 sÿ1 and 60% RH (A, D, G, and J) with different opening of
stomata. Bar represents 10 mm.
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)
128
J.P. Majada et al. / Scientia Horticulturae 87 (2001) 121±130
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 lessventilated 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 micropropagated 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 InvestigacioÂn Cientõ®ca y TeÂcnica (PB 92-0999) of Spain.
References
Blanke, M.B., Belcher, A.R., 1989. Stomata of apple leaves cultured in vitro. Plant Cell Tiss. Organ.
Cult. 19, 85±89.
J.P. Majada et al. / Scientia Horticulturae 87 (2001) 121±130
129
BoÈtcher, I., Zoglauer, K., Goring, H., 1988. Induction and reversion of vitri®cation of plants
cultured in vitro. Physiol. Plant. 72, 560±564.
Capellades, M., Fontarnau, R., Carulla, C., Debergh, P.C., 1990. Environment in¯uences anatomy
of stomata and epidermal cells in tissue cultured Rosa multi¯ora. J. Am. Hort. Sci. 115, 141±
145.
Conner, L.N., Conner, A.J., 1984. Comparative water loss from leaves of Solanum laciniatum plants
cultured in vitro and in vivo. Plant Sci. Lett. 36, 241±246.
Debergh, P.C., Harbaoui, Y., Lemeur, R., 1981. Mass propagation of globe artichoke Cynara
scolymus: evaluation of different hypotheses to overcome vitri®cation with special reference to
water potential. Physiol. Plant. 53, 181±187.
Debergh, P.C., Aitken-Christie, J., Cohen, J., Grout, D.B., von Arnold, S., Zimmerman, R., Ziv, M.,
1992. Reconsideration of the term `vitri®cation' as used in micropropagation. Plant Cell Tiss.
Organ. Cult. 30, 135±140.
Diettrich, B., Mertinat, H., Luckner, M., 1992. Reduction of water loss during ex vitro
acclimatisation of micropropagated Digitalis lanata clone plants. Biochem. Physiol. P¯anz.
188, 23±31.
Dillen, W., Buysens, S., 1989. A simple technique to overcome vitri®cation in Gypsophila
paniculata L. Plant Cell Tiss. Organ. Cult. 29, 181±188.
Donnelly, D.J., Vidaver, W.E., 1984. Leaf anatomy of red raspberry transferred from culture to soil.
J. Am. Hort. Sci. 109, 172±176.
Gribble, K., Sara®s, V., Nailon, J., Holford, P., Uwins, P., 1996. Environmental scanning electron
microscopy of the surface of normal and vitri®ed leaves of Gypsophila paniculata (Babies
Breath) cultured in vitro. Plant Cell Rep. 15, 771±776.
Kozai, T., Smith, M.A.L., 1995. Environmental control in plant tissue culture. In: Aitken-Christie,
J., Kozai, T., Smith, M.A.L. (Eds.), Automation and Environmental Control in Plant Tissue
Culture. Kluwer Academic Publishers, Dordrecht, pp. 301±318.
Kozai, T., Fujiwara, K., Watanabe, I., 1986. Fundamental studies on environments in plant tissue
culture vessels. Effects of stoppers and vessels on gas exchange rates between inside and outside
of vessels closed with stoppers. J. Agric. Met. 42, 119±127.
Lee, N., Wetzsein, Y., Sommer, H.E., 1985. Effects of quantum ¯ux density of photosynthesis and
chloroplast ultrastructure in tissue-cultured plantlets and seedlings on Liquidambar styraci¯ua
L. towards improved acclimatization and ®eld survival. Plant Cell Environ. 78, 637±641.
Leshem, B., 1983. Growth of carnation meristems in vitro: anatomical structure of abnormal
plantlets and the effect of agar concentration in the medium on their formation. Ann. Bot. 52,
413±415.
Leshem, B., Werker, E., Shalev, D.P., 1988. The effect of cytokinins on vitri®cation in melon and
carnation. Ann. Bot. 62, 271±276.
Majada, J.P., Fal, M.A., SaÂnchez-TameÂs, R., 1997. The effect of the number of air exchanges per
hour on proliferation and hyperhydricity of Dianthus caryophyllus L. in vitro. Cell Dev. Biol. 33,
66±69.
Majada, J.P., Centeno, M.L., Feito, I., FernaÂndez, B., SaÂnchez-TameÂs, R., 1998. Stomatal and
cuticular traits on carnation tissue culture under different ventilation conditions. Plant Growth
Regul. 25, 113±121.
Marin, J.A., Gella, R., Herrero, M., 1988. Stomatal structure and functioning as a response to
environmental changes in acclimatizated micropropagated Prunus cerasus L. Ann. Bot. 62,
663±670.
Miguens, F.C., Pereira, R., Dodsworth, R., 1993. A scanning electron microscope study of normal
and vitri®ed leaves from Datura insignis plantlets cultured in vitro. Plant Cell Tiss. Organ. Cult.
32, 109±113.
130
J.P. Majada et al. / Scientia Horticulturae 87 (2001) 121±130
Paques, M., Boxus, P., 1987. Vitri®cation: review of literature. Acta Hort. 212, 155±166.
Preece, J.E., Sutter, E.G., 1991. Acclimatization of micropropagated plants to the greenhouse and
®eld. In: Debergh, P.C., Zimmerman, R.H. (Eds.), Micropropagation: Technology and
Application. Kluwer Academic Publishers, Dordrecht, pp. 71±93.
Ritchie, G.A., Short, K.C., Davey, M.R., 1991. In vitro acclimatization of Chrysanthemum and
sugar beet plantlets by treatment with paclobutrazol and exposure to reduced humidity. J.
Exp. Bot. 42, 1557±1563.
Robinson, D.G., Ehlers, U., Herken, R., Herrmam, B.H., Mayer, F., SchuÈrmam, F.W., 1987.
Methods for SEM. In: MuÈhlethaler, K. (Ed.), Methods of Preparation for Electron Microscopy.
Springer, Berlin, pp. 145±172.
Santamaria, J.M., Davies, W.J., Atkinson, C.J., 1993. Stomata of micropropagated Delphinium
plants respond to ABA, CO2, light and water potential but fail to close fully. J. Exp. Bot. 44, 99±
107.
Shackel, K.A., Novello, V., Sutter, E.G., 1990. Stomatal function and cuticular conductance on
whole tissue-cultured apple plants. J. Am. Soc. Hort. Sci. 115, 468±472.
Sutter, E., 1985. Morphological physical and chemical characteristics of epicuticular wax on
ornamental plants regenerated in vitro. Ann. Bot. 55, 321±329.
Sutter, E., 1988. Stomatal and cuticular water loss from apple cherry sweetgum plants after removal
from in vitro culture. J. Am. Soc. Hort. Sci. 113, 234±238.
Sutter, E., Langhans, R.W., 1979. Epicuticular wax formation on carnation plantlets regenerated
from shoot tip culture. J. Am. Soc. Hort. Sci. 104, 493±496.
Sutter, E., Langhans, R.W., 1982. Formation of epicuticular wax and its effect of water loss in
cabbage plants regenerated from shoot-tip culture. Can. J. Bot. 60, 2896±2902.
Wardle, K., Dobbs, E.B., Short, K.C., 1983. In vitro acclimatization of aseptically cultured plantlets
to humidity. J. Am. Soc. Hort. Sci. 108, 383±389.
Werker, E., Leshem, B., 1986. Structural changes during vitri®cation of carnation plantlets. Ann.
Bot. 59, 377±385.
Wetzstein, H.Y., Sommer, H.E., 1983. Scanning electron microscopy of in vitro cultured
Liquidambar styraci¯ua plantlets during acclimatization. J. Am. Soc. Hort. Sci. 108, 475±480.
Zacchini, M., Morini, S., Vitagliano, C., 1997. Effect of photoperiod on some stomatal
characteristics of in vitro cultured fruit tree shoots. Plant Cell Tiss. Organ. Cult. 49, 195±200.
Ziv, M., 1991. Vitri®cation: morphological and physiological disorders of in vitro plants. In:
Debergh, P.C., Zimmerman, R.H. (Eds.), Micropropagation: Technology and Application.
Kluwer Academic Publishers, Dordrecht, pp. 45±69.
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,
127±134.
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 of Dianthus caryophyllus plants 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).
0304-4238/01/$ ± see front matter # 2001 Elsevier Science B.V. All rights reserved.
PII: S 0 3 0 4 - 4 2 3 8 ( 0 0 ) 0 0 1 6 2 - X
122
J.P. Majada et al. / Scientia Horticulturae 87 (2001) 121±130
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). Anatomically, 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.
New systems for environmental control inside the culture vessels will improve
plant quality, autotrophy, automation (Kozai and Smith, 1995) and will also
make acclimatisation safer (Majada et al., 1997). However, few data are
available on the control of culture vessel environments during micropropagation,
and even less information is available about the physiological characteristics
of the in vitro produced plants when the internal environment is modi®ed.
J.P. Majada et al. / Scientia Horticulturae 87 (2001) 121±130
123
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 for F0 (airtight vessel) and F3,
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
The number of stomata was studied by SEM. The sampled leaves were
prepared as described by Robinson et al. (1987), with slight modi®cations. The
leaves were ®xed for 16 h in 3% glutaraldehyde in 0.1 M phosphate buffer pH 7.2
and washed three times with the same buffer. Afterwards, the samples were
dehydrated in an ethanol series and ®nally passed to amilo iso-acetate. Critical
point drying was made (Blazers CDP030), handling the samples very quickly to
avoid rehydration, and they were ®xed with Dotite CX-12 (Jeol/S-C). Finally,
they were coated with gold in an argon atmosphere for 90 s and 22 mA (Blazers
SCD 004 Sputter Coater) and were observed in a Jeol SEM (JSM 6100) at 15 kV.
The adaxial and abaxial surfaces of samples were examined to determine
stomatal density (number of stomata/cm2).
124
J.P. Majada et al. / Scientia Horticulturae 87 (2001) 121±130
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 18 mmol mÿ2 sÿ1
(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 waxcontaining 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).
Stomatal density in carnation leaves was higher in less ventilated plants than in
the more ventilated or acclimatized ones (Table 1), as has been described for
other species (Wetzstein and Sommer, 1983; Blanke and Belcher, 1989).
Although some authors found differences in stomatal morphology between in
vitro grown plants and those grown in the glasshouse or in the ®eld (Donnelly and
Vidaver, 1984; Blanke and Belcher, 1989; Capellades et al., 1990), no differences
were observed in stomatal morphology among the plants obtained in different
J.P. Majada et al. / Scientia Horticulturae 87 (2001) 121±130
125
Table 1
In¯uence of the number of air exchanges (changes per hour) on stomatal densitya
Normal plantsb
0.11
0.21
0.68
0.86
Hyperhydrated Acclimatized
leaves
leavesc
Density of
Adaxial 103 21a 116 15a 69 7.5b 45 10c 34 7.5c
stomata
(number/cm2)
Abaxial 161 20a 149 14b 138 12b 103 11c ±d
41 3.5c
92 4.3c
a
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.
d
Data not collected.
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
126
J.P. Majada et al. / Scientia Horticulturae 87 (2001) 121±130
Fig. 1. SEM of the third and fourth pair of abaxial leaves (Dianthus caryophyllus cv. Nelken).
Leaves after 4 weeks of acclimatization in the glasshouse (A, B). In vitro leaves developed after
four weeks in culture (C±E). (A) Abaxial surface of a wholly expanded leaf showing elliptical
stomata (acclimatized plant). (B) Epicuticular waxes on leaves of an acclimatized plant previously
grown in airtight conditions. (C) Stomata of a hyperhydrated leaf. (D) Stomata in a leaf grown in an
airtight culture vessel. (E) Stomata on the abaxial surface of a leaf grown under ventilation (0.86
changes per hour). Bar represents 10 mm.
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
J.P. Majada et al. / Scientia Horticulturae 87 (2001) 121±130
127
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, 18 mmol mÿ2 sÿ1 and 60% RH (A, D, G, and J) with different opening of
stomata. Bar represents 10 mm.
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)
128
J.P. Majada et al. / Scientia Horticulturae 87 (2001) 121±130
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 lessventilated 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 micropropagated 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 InvestigacioÂn Cientõ®ca y TeÂcnica (PB 92-0999) of Spain.
References
Blanke, M.B., Belcher, A.R., 1989. Stomata of apple leaves cultured in vitro. Plant Cell Tiss. Organ.
Cult. 19, 85±89.
J.P. Majada et al. / Scientia Horticulturae 87 (2001) 121±130
129
BoÈtcher, I., Zoglauer, K., Goring, H., 1988. Induction and reversion of vitri®cation of plants
cultured in vitro. Physiol. Plant. 72, 560±564.
Capellades, M., Fontarnau, R., Carulla, C., Debergh, P.C., 1990. Environment in¯uences anatomy
of stomata and epidermal cells in tissue cultured Rosa multi¯ora. J. Am. Hort. Sci. 115, 141±
145.
Conner, L.N., Conner, A.J., 1984. Comparative water loss from leaves of Solanum laciniatum plants
cultured in vitro and in vivo. Plant Sci. Lett. 36, 241±246.
Debergh, P.C., Harbaoui, Y., Lemeur, R., 1981. Mass propagation of globe artichoke Cynara
scolymus: evaluation of different hypotheses to overcome vitri®cation with special reference to
water potential. Physiol. Plant. 53, 181±187.
Debergh, P.C., Aitken-Christie, J., Cohen, J., Grout, D.B., von Arnold, S., Zimmerman, R., Ziv, M.,
1992. Reconsideration of the term `vitri®cation' as used in micropropagation. Plant Cell Tiss.
Organ. Cult. 30, 135±140.
Diettrich, B., Mertinat, H., Luckner, M., 1992. Reduction of water loss during ex vitro
acclimatisation of micropropagated Digitalis lanata clone plants. Biochem. Physiol. P¯anz.
188, 23±31.
Dillen, W., Buysens, S., 1989. A simple technique to overcome vitri®cation in Gypsophila
paniculata L. Plant Cell Tiss. Organ. Cult. 29, 181±188.
Donnelly, D.J., Vidaver, W.E., 1984. Leaf anatomy of red raspberry transferred from culture to soil.
J. Am. Hort. Sci. 109, 172±176.
Gribble, K., Sara®s, V., Nailon, J., Holford, P., Uwins, P., 1996. Environmental scanning electron
microscopy of the surface of normal and vitri®ed leaves of Gypsophila paniculata (Babies
Breath) cultured in vitro. Plant Cell Rep. 15, 771±776.
Kozai, T., Smith, M.A.L., 1995. Environmental control in plant tissue culture. In: Aitken-Christie,
J., Kozai, T., Smith, M.A.L. (Eds.), Automation and Environmental Control in Plant Tissue
Culture. Kluwer Academic Publishers, Dordrecht, pp. 301±318.
Kozai, T., Fujiwara, K., Watanabe, I., 1986. Fundamental studies on environments in plant tissue
culture vessels. Effects of stoppers and vessels on gas exchange rates between inside and outside
of vessels closed with stoppers. J. Agric. Met. 42, 119±127.
Lee, N., Wetzsein, Y., Sommer, H.E., 1985. Effects of quantum ¯ux density of photosynthesis and
chloroplast ultrastructure in tissue-cultured plantlets and seedlings on Liquidambar styraci¯ua
L. towards improved acclimatization and ®eld survival. Plant Cell Environ. 78, 637±641.
Leshem, B., 1983. Growth of carnation meristems in vitro: anatomical structure of abnormal
plantlets and the effect of agar concentration in the medium on their formation. Ann. Bot. 52,
413±415.
Leshem, B., Werker, E., Shalev, D.P., 1988. The effect of cytokinins on vitri®cation in melon and
carnation. Ann. Bot. 62, 271±276.
Majada, J.P., Fal, M.A., SaÂnchez-TameÂs, R., 1997. The effect of the number of air exchanges per
hour on proliferation and hyperhydricity of Dianthus caryophyllus L. in vitro. Cell Dev. Biol. 33,
66±69.
Majada, J.P., Centeno, M.L., Feito, I., FernaÂndez, B., SaÂnchez-TameÂs, R., 1998. Stomatal and
cuticular traits on carnation tissue culture under different ventilation conditions. Plant Growth
Regul. 25, 113±121.
Marin, J.A., Gella, R., Herrero, M., 1988. Stomatal structure and functioning as a response to
environmental changes in acclimatizated micropropagated Prunus cerasus L. Ann. Bot. 62,
663±670.
Miguens, F.C., Pereira, R., Dodsworth, R., 1993. A scanning electron microscope study of normal
and vitri®ed leaves from Datura insignis plantlets cultured in vitro. Plant Cell Tiss. Organ. Cult.
32, 109±113.
130
J.P. Majada et al. / Scientia Horticulturae 87 (2001) 121±130
Paques, M., Boxus, P., 1987. Vitri®cation: review of literature. Acta Hort. 212, 155±166.
Preece, J.E., Sutter, E.G., 1991. Acclimatization of micropropagated plants to the greenhouse and
®eld. In: Debergh, P.C., Zimmerman, R.H. (Eds.), Micropropagation: Technology and
Application. Kluwer Academic Publishers, Dordrecht, pp. 71±93.
Ritchie, G.A., Short, K.C., Davey, M.R., 1991. In vitro acclimatization of Chrysanthemum and
sugar beet plantlets by treatment with paclobutrazol and exposure to reduced humidity. J.
Exp. Bot. 42, 1557±1563.
Robinson, D.G., Ehlers, U., Herken, R., Herrmam, B.H., Mayer, F., SchuÈrmam, F.W., 1987.
Methods for SEM. In: MuÈhlethaler, K. (Ed.), Methods of Preparation for Electron Microscopy.
Springer, Berlin, pp. 145±172.
Santamaria, J.M., Davies, W.J., Atkinson, C.J., 1993. Stomata of micropropagated Delphinium
plants respond to ABA, CO2, light and water potential but fail to close fully. J. Exp. Bot. 44, 99±
107.
Shackel, K.A., Novello, V., Sutter, E.G., 1990. Stomatal function and cuticular conductance on
whole tissue-cultured apple plants. J. Am. Soc. Hort. Sci. 115, 468±472.
Sutter, E., 1985. Morphological physical and chemical characteristics of epicuticular wax on
ornamental plants regenerated in vitro. Ann. Bot. 55, 321±329.
Sutter, E., 1988. Stomatal and cuticular water loss from apple cherry sweetgum plants after removal
from in vitro culture. J. Am. Soc. Hort. Sci. 113, 234±238.
Sutter, E., Langhans, R.W., 1979. Epicuticular wax formation on carnation plantlets regenerated
from shoot tip culture. J. Am. Soc. Hort. Sci. 104, 493±496.
Sutter, E., Langhans, R.W., 1982. Formation of epicuticular wax and its effect of water loss in
cabbage plants regenerated from shoot-tip culture. Can. J. Bot. 60, 2896±2902.
Wardle, K., Dobbs, E.B., Short, K.C., 1983. In vitro acclimatization of aseptically cultured plantlets
to humidity. J. Am. Soc. Hort. Sci. 108, 383±389.
Werker, E., Leshem, B., 1986. Structural changes during vitri®cation of carnation plantlets. Ann.
Bot. 59, 377±385.
Wetzstein, H.Y., Sommer, H.E., 1983. Scanning electron microscopy of in vitro cultured
Liquidambar styraci¯ua plantlets during acclimatization. J. Am. Soc. Hort. Sci. 108, 475±480.
Zacchini, M., Morini, S., Vitagliano, C., 1997. Effect of photoperiod on some stomatal
characteristics of in vitro cultured fruit tree shoots. Plant Cell Tiss. Organ. Cult. 49, 195±200.
Ziv, M., 1991. Vitri®cation: morphological and physiological disorders of in vitro plants. In:
Debergh, P.C., Zimmerman, R.H. (Eds.), Micropropagation: Technology and Application.
Kluwer Academic Publishers, Dordrecht, pp. 45±69.
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,
127±134.