Stomatal Response of in vitro Cultured Plantlets. I. Responses in Epidermal Strips of Chrysanthemum to Environmental Factors and Growth Regulators

Biochem. Physiol. Pflanzen 178, 619- 624 (1983)

Stomatal Response of in vitro Cultured Plantlets. 1. Responses in Epidermal Strips of Ohrysanthemum to Environmental Factors and Growth Regulators K. W AROLE and K. C. SHORT Department of Biology, North East London Polytechnic, England Key Term Index: environment, growth regulators, stomata, tissue culture; Chrysanthemum

morifolium

Summary Abaxial epidermal strips removed from ti!)sue cultured Chrysanthemum plantlets during the light or dark revealed open stomata and the gnarcl cells contained starch. Closure of the stomata was not induced by incubation of epidermal strips in darkness or by the presence of ABA. Further stomatal opening was induced by CO2 -free air and by kinetin. Following further opening using C02~ free air, the stomata could be induced to exhibit partial closure to their intiallevel of opening using ABA and darkness.

Introduction III vitro cultured plantlets often exhibit poor water relations when transferred to so if conditions which may result in high mortality rates. Acclimation of the plantlet to low humidity is possible in many cases and the use of film antitranspirants has also been used (CLARE and COL!.IN 1973; WALKEY and MATTHEWS 1979; WARDLE et al. 1979). The cause of the water imbalance may be due to a combination of poor water uptake and excessive water loss (GROUT and ASTON 1977 ; WARDLE et al. 1979). The effect of tissne culture conditions on epicuticular wax formation has been studied in several species with the general conclusion that· high levels of high humidity severly restrict wax formation leading to greatly enhanced cuticular transpiration (GROUT and ASTON 1977; SUTTER and LANGHANS 1979; WARDLE et al. 1979; BRAINERD and FUCHIGAMI 1981). Recently BRAINERD and FUCHlGAMI (1982) have suggested that the stomata of in vitro arc unable to exhibit rapid closure when the cultured plants are transferred to soil conditions and in addition arc unresponsive to agents which normally induce stomatal closure (ABA, mannitol). This study was an attempt to investigate more fully the stomatal responses of tissue cultured plantlets by using detached epidermal strips of Chrysanthemum.

Materials and Methods Tissue cultures of Chrysanthemum rnorifoliurn Ram. cv. "Snowdon" were established as previously described (WARDLE et al. 1983). The plantlets used in this investigation were maintained by the subculture of axillary buds which ga.ve rise to single plantlets on hormone-free MURASHIGE and Abbreviations: ABA, abscisic acid

620

K.

WARDLE

and K. C. SHORT

SKOOG (1962) medium. The plantlets were maintained in a. controlled environment room at 25 ± 2 °0 with a 161ight period (fluorescent lighting) giving a light intensity at plant level of 20- 25,uE m- 2 8- 1 , The method used for incubation of abaxial epidermal strips has previously been described (WARDLE and SHORT 1981). The epidermal strips were floated on an incubation solution consisting of 10 mM KCl and 10 mM morphalino ethanesulphonic acid (pH 6.1, KOH) contained in 5 em Petri dishes.

The dishes were aerated using ambient (411 ppm CO2 ) or CO2 -free a.ir (soda. lime) at 30 ems min-to Light intensity was lOO,uE m- 2 8 - 1 and temperature 25 ± 1 °0. Growth rculators were added to give a final eoncentartion of 2 X 10-4 AI. Stomatal apertures were determined microscopically using a graticule eyepiece on 40 stoma.ta.. All experiments were repeated on at least two separate occasions and similar results were obtained. All results presented are from single experiments and not pooled data.

Results

Freshly removed epidermal strips revealed that the stomata of stissue cultured

Chrysanthemum plantlets were open during both the light and dark phases of the photoperiod. From a single leaf stomatal aperture was relatively uniform although a wide variation occurred between leaves. Although the stomata were open the presence of starch was indicated by staining with iodine. The variation in stomatal aperture between leaves was further examined by determining the mean stomatal aperture for every leaf from single plantlets. Some differences were noted between plantlets although a distinct trend wa~ apparent in all plantlets Siomaial aper ture

Ljim] 20

r-

1

0_ /0'0_0/1..'0

\~o

15

o\

o :\.

'0 . . . . 0...... ,,0

10

5

0

0

\-

b-

o

o

\0

I

2

,

12 8 10 I. Leaf number (from base) 6'

10

18

Fig.!. Rela'ionship between leaf position (age) and stomatal aperture in an 8-week-old Chrysanthemum plantlet.

621

Stomatal Responses of Tissue Cultures

Table 1. Effect of growth regulators, CO2 , and light on changes i n stomatal aperture in epidermal strips from in vitro Chrysanthemum planUets

(Initial aperture 15.8

± 0.50 I'm;; ± s.e. mean)

Light')

CO,')

Growth regulator

Aperture (,urn) after an incuba.tion period of

3h

+ +

+ + + + + ')

+ present, -

+ + +

kinetin

ABA famesol

16.7 16.2 22.8 22.2 16.6 4.6

6h

± 0.67 ± 0.45 ± 0.40 ± 0.36 ± 0.64 ± 0.26

16.2

± 0.53

17.4

± 0.55

absent

Table 2. ABA-induced closure of stomata follo wing opening in CO 2-free air and light Treatment

Aperture (I'm)

. Initial 3 h CO2-free air, light 3 h ambient air, darkness, 2xlO- f, M ABA

12.6 ± 0.41 20.6 ± 0.57 13.2 ± 0.39

examined in that stomatal aperture decreased from the lower (mature) leaves to the upper (immature) leaves (Fig. 1). Using detached epidermal strips from leaves with a similar initial aperture it was possible to examine the ability of the stomata to respond to environmental factors and to growth regulators. The results (Table 1) demonstrated that further opening was possible (e.g. CO 2-free air, kinetin) whcreas closure was not (e.g. darkness, ABA) even after extended incnbation periods. Closure was observed using farnesol but this was presumably due to its known ability to disrupt membranes. The apparent inability of stomata to close in epidermal strips from Chrysanthemum leaves wa£ examined further by inducing further opening with CO 2-free air and light, after which the strips were transferred to a fresh incubation solution containing 2 X 10- ' M ABA and incubated in darkness using ambient air for aeration (Table 2). This latter treatment did result in a reduction in stomatal aperture from that obtained using CO 2free air. However, the stomata only returned to their original aperture and did not close further. Discussion

The physical environment within the tissue culture vcssel may differ from the normal atmosphere in two major respects. Firstly, many tissue cultures do not cxhibit net CO 2 uptake (PAMPLIN and CHAPMAN 1975), and secondly the atmosphere is generally

622

K.

WARDLE

a.nd K. C.

SHORT

saturated with water vapour. Assuming normal stomatal responses, then any lack of photosynthetic capacity and the accumulation of CO2 in the tissue culture container would be expected to induce stomatal closure although in the case of Chrysanthemum plantlets a net uptake of CO 2 does occur (unpublished). With respect to the humidity level, it is known that a decrease in humidity can induce stomatal closure by a hydropassive mechanism which is subsequently followed by changes in the guard cell contents of osmotically active solutes (LOSCH and SCHENK 1978). Thus the high humidity that inevitably occurs in the tissue culture atmosphere would be predicted to enhance stomatal opening. The finding that the stomata of in vitro grown Chrysanthemum plantlets are open, in the reasoning of the above, is not therefore surprising. The presence of starch in the guard cells of open stomata has also previously been observed (PE,rADAsA 1981). However, this was caused by using levels of KCI in excess of that normally required to achieve stomatal opening, and it is also possible that CI was used as a counterion to K+ thus eliminating the requirement to produce malate from starch. With tissue culture the total level of inorganic ions in the culture medium is also high (K+ > 20 mM, NH.+ > 20 mM, electroneutrality maintained with N03 -, CI- , SO;- ) in MURASHlGE and SKOOG (1962) medium and it has been demonstrated that some plant cultures absorb significant quantities of any ions present (NASH and DAVIES 1972; WARDLE et al. 1981). The finding that stomata are open in tissue cultured plantlets may be partially explained by the high level of atmospheric humidity. However, the high levels of potentially available ions for guard cell moyements in the culture medium can only be considered as having a lesser effect. Several workers have demonstrated that using epidermal strips from conventionally propagated plants stomatal opening can be induced by high levels of KCI even in the presence of factors which normally cause stomatal closure (WILLMER et al. 1978; WILSON et al. 1978; TRAVIS and MANSFIELD 1979; WARDLE and SHORT 1981). However, stomata induced to open by high levels of KCI in the incubation ,olution do not respond further to stimuli known to cause opening (WARDLE and SHORT 1981) since a "maximal" aperture has been attained. Epidermal strips removed from the in vitro cultured plantlets of Chrysanthemum did exhibit further opening under appropriate conditioned (e.g. CO,-free air or kinetin) which therefore suggests that the ion content of the guard cells is not maximal at the time the epidermal strips were removed. Failure of the guard cells to deflate and result in stomatal closure when epidermal strips were incubated in the presence of ABA or in darkness is a result typified by the presence of excess ions although this possibility has been largely excluded. A high degree of closure was observed in the presence of farnesol which is known to cause a loss of membrane integrity (FENTON et al. 1976; WILUlER et al. 1978). Farnesolinduced stomatal closure can be reversed although high concentrations of farnesol cause irreversible stomatal closure (FENTON et al. 1976; FENTON et al. 1977). Significantly, farnesol did not induce total stomatal closure of in vitro Chrysanthemum plantlets as was observed in epidermal strips from seedling plants of Commelina communis by FENTON et al. (1976).

Stomatal Responses of Tissue Cultures

623

Following further stomatal opening, closure of in vitro Chrysanthemum stomata was observed to their original, or slightly higher, aperture. This result clearly demonstrates that the guard cells are capable of both inflation and deflation , and suggests that the biochemical events of guard cell movements are essentially unimpaired. The exact cause(s) of open stomata from in vitro plantlets cannot be fully elucidated from the results presented here. However, it is possible from these results to postulate a mechanism which may account for the findings. During leaf maturation of in vitro Chrysanthemum cultures the stomata exhibit opening presumably due to the high level of humidity (o n immature leaves the narrower stomatal aperture is caused partially by the level of guard cell maturity in that it is physically not possible for the stomata to open furth er). During stomatal development the deposition of cellulose in the cell walls and possibly of the radial microfibrils is arranged for the position of stomatal closure. However, the stomata 'during this time arc open and thus the normal "closed" state of the guard cells has in effect a significant a nd mea~ureable aperturc. From this "closed" state opening may occur and full closure would only be to the original "closcd" state. This situation may almost fully ex plains the observed results and in the case of farn esol-induced closure, the disruption of membrane integrity and associated loss 01 ions and water would allow an "enhanced" closure of the stomatal pore causcd by pressure from the surrounding epidermal cells. It is essential, however, that ultrastructural studies be performed to either confirm or disprove this proposed mechanism. References K. E., and FUCIIIGAMI, L. H.: Acclimatization of aseptically cu ltured apple pla.nts to low rel.tive humidity. J. Am. Soc. Hort. Sci. 106, 515- 518 (1981). BRAINERD, K. E., and FUCIIIGAMI, L. H.: Sto matal functioning of in vitro and greenhouse apple leaves in darkness, mlmnitol, ABA, and CO2 , J. Exp. Bot. 33, 388- 392 (1982). CLARE, ~I. V., and COLLIN, H. A.: l\[eristem culture of Brussels sprouts. Hort. Res. 13, 111- 118 (1973). FENTON, R., MANSFIELD, T. A., and WELLB UKN, A. R.: Effects of isoprenoid alcohols on oxygen exchange of isolated chloropla.sts in relation to their possible physiological effects on stomata.. J. Exp. Bot. 27, 1206- 1214 (1976). FE NTON, R., DAVIES, W. J., and MANSFIELD, T. A.: The rol e of farnesol as a regulator of stomatal openiug in Sorghum. J. Exp. Bot. 28, 1043- 1053 (1977). GROUT, B. W. W. , and A STON, 1\1. J.: Transplanting of cauliflower plants regenerated from meristem culture. 1. Water loss and water transfer re lated to changes in leaf wax and to xylem regene ration. Ho rt. Res. 17, 1-7 (1977). LOSCH, R., and SCHENK, B.: Humidity respo nses of stomata and the potassium content of gua.rd cells. J. Exp. Bot. 29, 781- 787 (1978). MURASIIlG E, '1'., and SKOOG, F.: A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physio!. Plant. 15, 473- 497 (1962). NASH, D. T., and DAVIES, W. E.: Some aspects of growth and metabolism of Paul's Scarlet Rose cell suspcusions. J. Exp. Bot. 23, 75-91 (1972). PAMPLI N, E. J., and CHAPMAN, J. M.: Sucrose suppressio n of chlorophyll sy nthesis in tissue cu ltures: Changes in the activity of the enzymes of the chlorophyll biosynthetic pathway. J. Exp. Bot. 26, 212-220 (1975). PEMA DASA, M. A.: Photocontrol of stomatal movements. BioI. Rev. 56, 551-588 (1981). BRAI~ERD,

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K. WARDLE and K. C. SnoRT, Stomatal Responses of Tissue Cultures

E., and LANGHANS, R. W.: Epicuticular wax formation on carnation plantlcts regenerated from shoot tip culture. J. Am. Soc. Hort. Sci. 1M, 493- 496 (1979). TRAVIS, A. J., and MANSFIELD, T. A.: Stomatal responses to light and CO 2 are dependent on KCl concentration. Plant Cell Environ. 2, 319-323 (1979). WALKEY, D. G. A., and MATTHEWS, K. A.: Rapid clonal propagation of rhubarb (Rheum rhaponticum L.) from meristem-tips in tissue culture. Plant Sci. Lett. 14, 287-290 (1979). WARDLE, R., QUINLAN, A" and SUIPKINS, I.: Abscisic acid and the regulation of water loss in plant. lets of Brassica oleracea L. var. botrytis regenerated through apical meristem culture. Ann. Bot. 45,745- 752 (1979). WARDLE, K., and SnoRT, K. C.: Responses of stomata in epidermal strips of Vicia {aha to carbon dioxide and growth hormones when incubated on potassium chloride and potassium iminodiacetate. J . Exp. Bot. 32, 303-309 (1981). WARDLE, K., DIXON, P. A., and SI:MPKINS, 1.: Sodium accumulation by leaves of cauliflower plantlets and the effect of the mode of plant formation. Ann. Bot. 47, 453- 459 (1981). WARDLE, K., DALSOU, V., SIMPKINS, 1., and SnoRT, K. C.: Redistribution of rubidium in plants of Chrysanthemum mori{olium RAM. cv. Snowdon derived from tissue cultures and transferred to soil. Ann. Bot. iiI, 261 - 264 (1983). WILLMER, C. M., DON, R., and PARKER, W.: Levels of short-chain fatty acids and of abscisic acid in water-stressed and non-stressed leaves and their effects on stomata in epidermal strips and excised leaves. Planta. 139, 281- 287 (1978). WILSON, A. J., OGUNK.\NMI, A. B., and MANSFIELD, T. A.: Effects of external potassium supply on stomaBal closure induced by abscisic acid. Plant Cell Environ. 1, 199- 201 (1978).

SUTTER,

Received December 28, 1982; accepted March 26, 1983 Authors' address: K. WARDLE and K. C. SHORT, Department of Biology, North East London Polytechnic, Romford Road, London E15 4LZ, England.