Developmental and photosynthetic characteristics of a photoautotrophic Chrysanthemum culture

:J

PHOTOSYNTIIETICA 37 (r): 53-59, 1999

Developmental and photosynthetic characteristics of a photoautotrophic Chrysanthemum culture v. CRISTEA*,

F. DALLA VECCHIA**, ffidN. LA ROCCA**

Biological Research Institute, Republicit 48, 3400 Clqj-Napoca, Romania, Department of BiologSt, University of Padova, G. Colombo 3, 35 j21 padova, Italy,.

Abstract Chrysanthemlz plantlets were cultivated in vitro on media with 2.0, 0.3, or 0%o sucrose, or photoautotrophically without an organic carbon source but with supplementation of the culture vessel atnosphere with 2 o Coz. The photoautotrophically cultivated plantlets showed a better growth and multiplication, higher contents of chlorophyll (Chl) and carotenoids, higher Chl a/b ratio, net photosynthetic rate and ribulose-l,5-bisphosphate carboxylase/oxygenase and phosphoenolpymvate carboxylase activities than plantlets grown on the medium with sucrose. Additional key words: carotenoids; chlorophyll;

in vitro CO, supply; net

photosynthetic rate;

phosphoenolpyruvate carboxylase; photosynthetic pigments; ribulose- 1,S-bisphosphate carboxylase; suncaps.

Introduction From a photosynthetic point of view, in vitro active cultures represent a step forward cultures. The theoretical and practical applications of these culture types are multiple (Kozai et ol. 1992, widholm 1992, 1995, Hiisemann 1995, Pospi5ilov6 et al. 1997). To regard the specific environmental conditions, classical in vitro cl,ltures are heterofiophic or photomixofiophic. However, at present, the capacity of plantlets to photosynthesise in vitrohas been proved in several species: sweetgnm, tobacco, rose, potato, strawberry (e.g., Pospi5ilovh et al. 1987, Tich6 et al. 1988,Kozai et al. 1992, Desjardins et al. 1995). The achievement of photosynthetically active aseptic cultures is important for the obtaining of an increased efficiency in plantlets acclimatisation (Fujiwara et al. 1988, Kozai et al. 1995, van Huylenbroeck and De Riek 1995, Van Huylenbroeck and Deberg 1996).

in the field of plant

Received ll March 1999, accepted 25 August 1999. Abbreviations'. Car - carotenoids; Chl - chlorophyll; FM - fresh mass; p6 gross photosynthesis; p.g - net photosynthetic oxygen release rate; PEPC - phosphoenolpyruvate carboxylase; Rp - dark respiration rate; RuBPC - ribulose- 1,5 -bisphosphate carboxylase. Aclcttowledgements'. TIte authors are grateful to Prof. Nicoletta Rascio for her valuable advice and help. This work was supported by a grant from the University of padova.

53

V. CRISTEA et a/.

The photosynthetically micropropagated plantlets generally require the supplementation of culture vessel atnosphere with CO2 @.g., Kozai 1991, Debergh et al. 1992, Solarov6 and Pospi5ilov6 1997). The closure systems of the classical in vitro culture vessels are as tight as possible, in order to prevent infections favoured by the presence of sucrose in the culture medium. This is why this gas disequilibrium is created, which inhibits photosynthesis. The use of suncaps for the closure of vessels allows gas exchange with the exterior atrnosphere enriched in CO2, but prevents from infecting. The present work aimed to induce photoautrophy in Chrysanthemum plantlets cultivated in vitro and to study them from the point of view of some developmental and physiological parameters.

Materials and methods Plants and conditions of growth: The Chrysanthemum morifolium Ramat. apical cuttings were taken from plantlets developed in vitro and maintained for 22 d under 16 h photoperiod. The irradiance was 250 pmol m-2 s-1. The Murashige and Skoog (1962) culture medium contained I g m-3 cr-naphtaleneacetic acid, 0.5 g m-3 kinetin, and 7 kg m-3 agar. Three experimental variants were without CO2 supplementation of the culture vessel atrnosphere, with 2.0, 0.3, or 0 %6 sucrose in the culture medium. The first variant represented the control, i.e., classical in vitro culture conditions. The fourth variant consisted of a culture medium without sucrose but with supplying of the culture vessel atrnosphere with 2 yo CO2. High CO2 partial pressures were established using a system similar to Tich6 (1996). These culture vessels were closed with suncaps. The vessels of the three other variants were closed hermetically with polyethylene film. The development of plantlets, i.e., their growth, multiplication, organogenesis and rhizogenesis, was studied.

Total pigment analysis: Total Chl and Car from leaf tissues were analysed in a double array spectrophotometer (model 124, Perkin Elmer, Norwalk, CT, USA) after exfraction with N,N-dimethylformamide. The extinction coefficients proposed by Wellburn (1994) were used to determine the pigment concentrations. Leaflenvironment oxygen exchanges (orygen release or uptake) were measured with an oxygen electrode (I^91model 53, Yellow Spring Instruments, Ohio, USA) on small pieces ofleaftissues according to Ishii et al. (1977). RuBPC (ribulose-l-5-bisphosphate carboxylase) activity was determined according to Di Marco and Tricoli (1983). The specfophotometric method is based on the reduction of D-3-phosphoglycerate formed in the carboxylase reaction. Changes in absorbance were measured at 340 nm.

PEPC (phosphoenolpyruvate carboxylase) activity was determined spectrophotometrically according to Usuda et al. (1984). The method nrcasures a change in absorbance at 340 nm as a result ofreduction ofoxaloacetate to malate.

54

CHARACTERISTICS OF A CHRYSANTTIEMUM CULTURE

Results

The photoautotrophically cultivated Chrysanthemum plantlets grew and developed better than those cultivated on a 2.0,0.3, or 0 %" sucrose medium without CO2 (Table l). The reduction of sucrose concentration from 2.0 to 0.3 %io caused a 2.5-fold reduction in the stem growth and a 2-fold reduction in the number of the newly formed leaves (all values were compared to the control ones). The plantlets grown photoaubophically showed a 2.8-fold growth and l.7-times as marry leaves compared to the control. The highest percentage ofinocula generating new plantlets appeared in the photoautrophic culture as well. The rhizogenesis occurred significantly in 67 %o of the initial inoculate in photoautofiophic conditions. Table

l.

The effect of different sucrose and CO2 concentrations on the developmental

and

photosynthetic parameters of Chrysanthemum plantlets,22

Chl

-

chlorophyll;

P6 -

d after the inoculation. Car - carotenoids; gross photosynthetic rate; PN - net photosynthetic rate; PEPC -

-

phosphoenolpyruvate carboxylase; Rp dark respiration rate; RuBPC carboxylase. Means from 15 plants, with standard deviations. Parameter

Sucrose [%] 2.0 0.3 %

% -coz

Plant growth [trunJ Leaf number Root number per inoculum Regenerated plantlets per inoculum Chl a+b [mg kg-t(FM)] Chl a/b

Car [mg kg-t(FM)] PN [pmol(Oz) kg-t(FM) s-t] Pc [pmol(O2) kg-t(FM) s-t] Ro [pmol(Oz) kg-t(FM) s-t] Pp/Rp RUBPC [pmol(Coz) kg-t(FM;

PEPC [% of control]

5-t1

0%

0%

-Coz

+COz

0.91+1. t6 0.6+0.5 0.08 0 143 t.75 39+25

8.00* I .56

3.16*2.64 2.5+ I .0

0

0

I

069

2.05

ribulose-1,5-bisphosphate

-coz

4.8+l .2 0.3 3

-

0.t7 531 2.21

240+10

123+t2

2.5 5*0.5 5

6

4.91

22.5*3.5

8.2*t.2 0.67 0.67

l5l9 276 301+13

2.t7+0.75

3.06

8.9510 61

8.34

2,66+0.20

| .67+.0.27

0.8910.32

2.08*0. l6

.67*0.

8

3

I

I 03

0.91

4.21

2.43

0.48*0.05

0.42+0.04

0.08+0.04 l.6l+0.03

100.0

180.6

70.9

4.72 t45 .2

Similar influence of the diflerent experimental variants occurred in the contents of Chl o+b and Car (Table l). By reducing the sucrose concentration from 2.0 to 0.3 %o,

the pigment contents decreased two-times, and, in photoautotrophic cultures, they increased l.4-times. The Chl a/b ratro was 2.76 in the leaves of photoautotrophic plantlets and 2.05 in those developed on the 2.0 oZ sucrose medium. Reduction in sucrose concentration from 2.0 to 0.3 yo increased the net photosynthetic rate (PN) by 2.6-times, and the presence of Co2 and the absence of carbon organic source from the medium caused a 3.5-fold increase in this parameter (Table 1). Dark respiration rate (Rp) was lower in all variants compared to the control one. In plantlets developed on the medium with 0.3 7o sucrose and in those developed under

55

V. CRISTEA et aL

was l'7- aJ|ld2'2-ti'lrres gross photosynthetic rate (P6) ohotoautrophic condrtions, the was 3.3-times higher than o""autrophic conditions pnpt activity was l.8-times higher in the case of sucrose that of the control, *a,rrc .L tigh"t in photoautotrophic conditions

itff:t3;JffiT["|i1li reduction from 2.0

,i.Z

and l.4-iime,

(Table 1).

I)iscussion

Asfoundinotherstudies(Cristeae,lal.|999),thepholoaut3trophicatlycultivated ite than those cultivated on 2'0' pf*,ti,"r"J;;;diff.t""t

"fr"rtt""t

Chrysanthem,r,

COr0.3, or 0 7o sucrose medium without photoautotropiotosynthetic activity occur in and The best growth, *ithoot sucrose and with .urtitf phicalty cultivated Cristea'et al' (1996)' and

*"lilii";,il, *.Cot d;i:;;:;;"'i",frl ftels;' *ii Desjardin' n'i' Co2 supply, u, .o*tJ-Ly n ve el s normalptitt"J * n* n'v' c "llo::lil r ich6 ( e6). w., nol*"i medium do"' not allow gas exchange' on a int'i'h "u i

o

re

s s

closed witr, potyetrtyi;;;;' abiftlttf"t" C52 supply allows' in thenatuwithout sucrose dirl not grow and develop. under plants a devellpment similar to that of sence of a carbon -g*il**.", the point of view soperiot,.from o"u.rofrn.rriis ral conditions. This;fr;i|l,ffiht. cultures in vi*o' y*ur,L, including classical of all studied cb2 supplementation inhibited

lv

ilA; n.r-#;;;; concentration #;;;;

The decrease in sucrose

thegrowthanddevelopmentofthetlf:t"-tt*"u"t"a-areductionof5Oo/"ofCN as found by in vitro d;;ti;"phic conditions' (ree2) fot content. P11 is enhan""d *dt' at' et for rose fi;l;giv TitYT Langford and wainwright (1e87) et al' (1992) Fujiwara However, f,"-"nsfated by oJresults. potato cultures, *a than of 2'5 %" | ", at a sucrose concentation of '5 found a higher go*th oi ttre plantlets from medium in the culture of sucrose of %. We found that the decreaie amount "oi""tttutio" a gooa piJto growth-:Y:i^if the al' 2.0 to 0.3 oh drd;;;lt* et i- aT^ a ol' (1938) andTanakavessels photosynthetic 02 production was i*r""r"JSt in o.t ci'v'oitnemum pr'antrel: crolvn (leela,b) studied ;;;;;i;d;t

o CO2 on *"!i1 with 3 or 0 "/" sucrose' 0'04 with an afinosphere supplemented with 15, ot2lo/o'PN' oxygen_concenfation of 5,. 10, where the variable ru"to, *as the grown on the *.r" .i!rin;6 htgh"r in plantlets RuBpc activity -fi;;,* and soluble chl the on tt. 3-%;;;",i"dio-,6ut medium without ;;;; with a plantlets of not higher' y:,l"i;good dwelopmel: protein contents were

significantincrease.inCo2concentration.u",,o''themediumwithoutsucrose. dweloped plantlets that in is higher in photouutorophically -'a*o'[oated The RuBpc actrvity and Widholm ior 9f^^9"1d:tein control plantlets, n' sucrose to "th;;-;'*itt (1994a), adding by

ogstb) Hdider and Desjardins (1990) or Tanaka without sucrose' "i'rl ,;;bt;-pi*trot initiatlv gg*tt,oo 1.T:i1* the medium the ca11lvtrc.hffnover rate of the pN. Howey.I, "f induced a decrease in 9v_l"ltiring that the PN inhibition was state of RuB-PC, tft.V sriggested enzyme and the due to deactivation' According "rti*tiii, in RuBPi a consequenc. 56

"f

;;-;;;,i";

"rtii"*V

CHARACTERISTICS OF A CHRYSANTHEMUM CULTURE

to Desjardins et al. (1995), under uncontrolled environmental conditions the CO2 exhaustion in the culture vessels or the feedback inhibition of the Calvin cycle by exogenous sucrose lead to RuBPC deactivation.

Hdider and Desjardins (l99ab) have confirmed that the presence of sucrose stimulated the CO2 fixation mediated by PEPC. They correlated the presence of sucrose in the culture medium with the enhancement of the respiratory acnity rn in vitro plantlets. In our experiments the Rp was directly proportional to the sucrose

amount

in the

culture medium. Unlike

in the

control, where

the

photosynthetic/respiratory 02 ratio was almost l, in photoautotrophic plantlets this ratio significantly increased. In a closed space with equilibrium between the two 02 types, a development on photoslmthetic basis is not possible, therefore these cultures are photomixotrophic or photoheterotrophic. Moreover, because the photosynthetic 02 released by the plantlets developed without the CO2 supply on media containing 2 or 0 %o sucrose was similar, a weak contribution of photosynthesis at the development of classical ln

vilro cultures was eyident. Hence we conclude that Chrysanthemum plantlets cultivated in vitro on media without an organic carbon source but with a supply of Co2 have a photoautotrophic development with all the studied parameters superior to those of the plantlets developed in other experimental conditions.

References Cristea, V., Dalla Vecchia, F., Criciun, C.: Biochemical and ultrastructural aspects of carrot (Daucus carotaL.) tissue culture in different experimental grorth conditions. - G. bot. ital. l3O 924-926, 1996.

Cristea, V., Dalla Vecchia, F., Criciun, C., La Rocca, N.: Ultrastructural aspects of photoautotrophic Chrysanthemun culture. - In: Gariib, G. (ed.): Photosl,nthesis: Mechanisms and Effects. pp. 41754178. Kluwer Academic Publ.. Dordrecht - Boston - London 1999.

Debergh, P.C., De Meester, J., De Riek, J., Gillis, S., Var Huylenbroeck, J.: Ecological and physiological aspects oftissue-cultured plants. - Acta bot. neerl. 41: 417-423,1992. Desjardins, Y., Hdider, C., De Riek, J.: Carbon nutrition in vitro - regulation and manipulation of carbon assimilation in micropropagated systems. - In: Aitken-Christie, J., Kozai, T., Smith, M.A.L. (ed.): Automation and Environmental Control in Plant Tissue Culture. Pp.44l-471. Kluwer Academic Publ., Dordrecht - Boston - London 1995. Di Marco, G., Tricoli, D.: RuBP carboxylase determination by enzymic estimation of D-3-PGA formed. - Photosynth. Res. 4: 145-149, 1983. Fujiwara, K., Kira, S., Kozai, T.: Time course of co, exchange of potato cultures in vitro \rlith different sucrose concentrations in the culture medium. - l. agr. Meteorol. 48 49-56, 1992. Fujiwara, K., Kozai, T., Watanabe, I.: Development of a photoautotrophic tissue culture system for shoots and./or plantlets at rooting and acclimatization stages. - Acta Hort. 230: 153-158, 1988. Goldstein, C.S., Widholm, J.M.: Photosynthetic characterization of photoautotrophic cells cultured in a minimal medium. - Plant Physiol. 94 164l-1646, 1990. Hdider, C., Desjardins, Y.: Changes in ribulose-1,5-bisphosphate carboxylase/oxygenase and phosphoenolpyruvate carboxylase activities and laCO, fixation during the rooting of strawberry shoots in vitro. -Can. J. Plant. Sci. 74: 827-831.1994a.

57

V. CRISTEA eral.

carboxyEffects ofsucrose on photosynthesis and phosphoenolpynrvate 27-33' 36 cvlt' organ Tissue cell Plant plantlets. lase activity of in vitro cultured strawberry

Hdider, c., Desjardins, 1994b.

Htisemann,

y.:

W.: Establishment of photoautotrophic cell cultures.

-

In: Plant Tissue Culture Manual

Hl. Pp. l-30. Kluwer Academic Publ', Dordrecht 1995' y.: on a method for measuring photoslmthesis and respiration of Ishii, R., yamagishi, T., M;;;, 46: 53'57 1977 ' leaf slices with an oxygen electrode' - Jap' J' Crop Sci' -' In: Deberg, P.C., Zimmerman, conditions. photoautotrophic una., Kozai, T.: Micropropagat,oi 447-469' Kluwer Academic Publ'' R.H. (ed.): Micropropagation' Teciurology and Application 'Pp'

Dordrecht - Boston - London I99l' The in vitro environment and its control in Kozai, T., Fujiwara, r., H"v*r'i M., Aitken.Christig, J : Production Systems. Pp.247-282' (ed.): Transplant . T. micropropagation. In: K"'utu, K- Kozai, 1992' London Boston Dordrecht Publ., Kluwer.tcJOernl" control for large scale production of Kozai, T., Kitaya, Y., F"ji;;; K., Adelberg, J': Environmental

invitroplantlets.-In:Terzi,tr,t.,cetta,R.,Falavigna,A'(ed.):ClrrreltlssuesinPlantMolecular

Pubtishers, Dordrecht 1995. and Cellular Biology. Pp. 659.667 . Kluwer Academic on the photosynthetic ability of rose concentration r--gf"O, p.J., Wainftghi, H.: Effects of sucrose 633-640,1987 60: ' Bot. Autrt. shoots in

vito.'

Murashige,T.,Skoog,F.:Arevisedmediumforrapidgrowthandbioassayswithtobaccotissue cultures. - Physiol. Plant. lE: 473-497, 1962'

In: Pessarakli, M. * 8"rrr.v, i., s*a,, Z.: photosynthesis in plant cultivated ytr:.- Hong Kong Basel York New Dekker, 52S-S+0. Marcel t"a l, HandLook oipf,otoryntft.sis. lp.

p;#;;;, --

1997.

l-

ofplant regenerants' Specificity of

ro.pisiioua, J., datskj', J., Solarov6, J', Tich6, I: Photosynthesis plant. 29 415-421,1987. invitroconditions and plantlet response. - Biol. o, concentration on net photoslmthesis of C3 |ow of Effects T.: Kozai, F., Shimada, N., Tanaka, plantlets invifto. - ActaHort. 230: l7l-175' 1988' during in vitro cultivation and Sol6rov6, J', Pospi5ilov6, J.: Effect of carbon dioxide enrichment 1997 ' acclimation to ex vitro conditions - Biol Plant' 39 23-30' on photorespiration in Tanaka, F., Watanabe, y., Shimada, N.: [Effect of Or concentrations

Chrysanthemummorifoliumplantletsin.vitroculfuredphotoautotrophicallyand

Jap ] phoiomixotrophically.l - PlantTissue Cult Lett' 8: 87-93' l99la' [In growth of Chrysanthemum on concentration 02 of r*ut.u, F., Watanabe,-Y., Shimada, N.: [Effect - Environ' photomixotrophically'] and photoautotrophically vitro cultured in morifolium plantlets Control Biol. 29: I07-l 16, l99lb [In Jap ] effect of suncaps closures on Tich6, I.: Optimization of photoautotiophic tobacco in vitro culture: 1996' 475-479, Photosynthetica.,32 plantlet growth. pospiSilov5, J., Sol6rov6' J.: Photosyrthesis Tich6, I., ondiej, M., H;l;, M., bur.ty, I ,Zima,J., I Morphology, anatomy and Agrobacterizm of plasmids by in regenerants ortouacco tiansformed 8' 98 350-363' I gas Jtchange of transformants - Photosynth etica 22:

Usuda,H.'Ku,M.S.B.,Edwards'G.E.:ActivationofNADP-malatedehydrogenase,pyruvate,Pi - Plant

photospthetic rate in maize' dikinase, and fructose l,6-bisphosphatase in relation to Physiol. 7 6: 238-243, 1984. in,vitro on photosynthesis and van Huylenbroeck, J.M., beberg, c.p.: Impact of sugar concentration plantlets. - Physiol. Plant 96: spathiphyllum of acclimatization uiitro carbon metabolism during 298-304,1996. during er vitro rooting and van Huylenbroeck, J.lM., De fuek, J.: Sugar and starch metabolism Plant S-ci' lll: 19-25 ' 1995 ' acclimatization of micropropag ut"a Spotiipnyilum'Petite'plantlets as total carotenoids' well b' as a and Wellburn, A. R.: The .p."irui Olter-ination oi chlorophylls Plant Physiol' 144: J' resolution' different of spectrophotometers with using various solvents 307-313, 1994.

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CHARACTERISTICS OF A CHRYSANTHEMUM CVLTURE

/ 1

r32: Widholm, J.M.: properties and uses of photoautotrophic plant cell cultures. to9-175,1992. plant cell cultures: an overview. - In: Carre, F., Chagvardieff, P (ed ): widholnu JJvl.: Photosynthetic -ltrotosyntttetic in Vitro Cultures. Saint-Paul-lez-Durance, France. Pp. 15-24' Ecophysiology anO CEA, Cadarache 1995.

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