effect of light wavelength on in vitro organogenesis

7/28/2019 Effect of Light Wavelength on in Vitro Organogenesis

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INTRODUCTION

European greenhouses are filled with many epi-phytic orchid species and hybrids produced overmany years of growing and hybridizing. Orchids

are propagated both from seeds and vegetatively,but the process is slow. Multiplication of micro-propagated material is faster than by any conven-tional method of propagation, and in vitro culturetechniques currently are standard practice fororchid culture (Chen and Chang, 2004; Chen et al.,2004; Tuong and Tanaka, 2004). Plant morpho-

genesis is influenced by many environmental fac-tors such as nutrients and the quality, durationand intensity of light, temperature regime, type ofcarbon source, gas composition in the culture ves-sel (especially CO2 concentration), and the numberof air exchanges during culture (Kirdmanee et al.,1992; Hahn and Paek, 2001; Li et al., 2002). There

are many reports on the effects of nutrition andgrowth regulators used for in vitro cultivation of avariety of orchids, but much fewer focusing on theeffect of irradiation on bud initiation and subse-quent growth (Ichihashi, 1990; Adelberg et all,1997; Tanaka et al., 1998; Islam et al, 2000).Modifying the duration and intensity of particular

wavelengths has been shown to affect morphogene-sis in different ways (Vince-Pure, 1994).

Here we report research on the effects of lightquality on micropropagation of a valuable interspe-cific Cattleya hybrid. We also estimated chlorophyll

and carotenoid content in this material propagatedunder different light regimes. The long-term pur-pose is to obtain high qualityCattleya intermedia C. aurantiaca plantlets for further multiplicationand transfer to soil.

MATERIALS AND METHODS

The plant material used was a Cattleya Lindl.hybrid obtained by crossing Cattleya intermediaand C. aurantiaca, originating from the OrchidCollection of the Botanical Garden of JagiellonianUniversity. Explants were obtained from mericloned

cultures, cloned pathogen-free and subcultured at 8-week intervals. They were maintained in tissue cul-ture for about a year. Shoots ~5 mm high with threeor four leaves, regenerated from a mass of proto-corm-like bodies, were excised and placed on 50 ml

*e-mail: [email protected]

Abbreviations: BA 6-benzylaminopurine; NAA 1- naph-thaleneacetic acid; MS Murashige and Skoog medium; PLBs protocorm-like bodies.

EFFECT OF LIGHTWAVELENGTH ON INVITRO ORGANOGENESIS

OF ACATTLEYA HYBRID

TERESACYBULARZ-URBAN1*, EWAHANUS-FAJERSKA1, ANDADAM WIDERSKI2

1Department of Botany, 2Department of Biochemistry,

Agricultural University, Al. 29 Listopada 54, 31425 Cracow

Received November 20, 2006; revision accepted July 4, 2007

The effect of light wavelength on multiplication, tissue growth and pigment content was studied in Cattleya inter-media C. aurantiaca microcutting cultures. The initial explants were shoots regenerated from protocorm-likebodies. Modified MS medium containing 5.0 mgl-1 BA, 0.2 mgl-1 zeatin and 1.0 mgl-1 NAA, solidified with Difcoagar, was used for adventitious regeneration of shoots and aerial roots. The rate of organ initiation depended onthe wavelength of the monochromatic light applied. Red and blue treatments were effective in triggering photo-morphogenesis in the evaluated material. The propagation coefficient reached 11.7 under red light, 10.6 under

blue, 8.3 under white and 6.2 in darkness. Total chlorophyll and carotenoid content were highest in culturesilluminated with white light, gradually decreasing from the blue to the red and the far red treatments. Blue lighttreatment improved the efficiency of micropropagation and benefitted initiation of rhizogenesis and aerial rootelongation, and the resulting plants were true to type.

Key words: Cattleya, monochromatic light, adventitious buds, rhizogenesis, chlorophylls,carotenoids.

ACTA BIOLOGICA CRACOVIENSIA Series Botanica 49/1: 113118, 2007

PL ISSN 0001-5296 Polish Academy of Sciences, Cracow 2007


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solidified modified MS medium in a 250 mlErlenmeyer flask. The proliferation medium wascomposed of MS salts and vitamins, supplied with2 mgl-1 adenine sulphate, 5.0 mgl-1benzylaminop-urine, 0.2 mgl-1 zeatin, 1.0 mgl-1 naphthaleneaceticacid and 3% (w/v) sucrose, solidified with 0.8% (w/v)agar (Bacto Agar, Difco). The pH was adjusted to5.5. Cultured material was transferred every six

weeks onto fresh medium of the same compositionfor three months. The cultures were maintained at232C with a 16 h photoperiod under light of dif-ferent spectra: the control, white (390760 nm,Tungsram F33 40 W lamp); far red (770800 nm,100 W incandescent light and filters: standard filterno. 405 orange + standard filter no. 420 deep blue,Compact light B. V. Amsterdam), red (647770 nm,Philips TLD 36 W) and blue (450492 nm, PhilipsTLD 36 W). Radiometric measurements were madein the horizontal plane 35 cm above the cultures.Solar spectral irradiance in the range 3001100 nm,expressed in mol m-2s-1, was measured (with wave-

length drive intervals of 2 nm) with an LI1800 spec-troradiometer (LI-COR U.S.A.). The measurementsand data integration in quantum units (mol m-2s-1)in the 3001000 nm range and photosyntheticallyactive radiation (PAR 400700 nm) were graphicallyinterpreted using PC188 ver. 3.01 (LI-COR) byBach and Malik (1999). The photosyntheticallyactive radiation was 60 mol m-2s-1 except for redand far red light (20 mol m-2s-1). Additional controlmaterial was maintained under continuous dark-ness throughout the whole course of the experiment,

with the rest of the incubation conditions as givenearlier. Each experimental combination was donetwice in 25 replicates. A single replicate was a flask

with 25 explants. The biometric data recorded afterthree months of culture for the whole experimentaldesign were as follows: number of regeneratedadventitious shoots and aerial roots, shoot lengthand root length. From each treatment, ten samplesconsisting of several shoots were collected at ran-dom from different flasks at the end of the three-month culture period, and fresh and dry matter

were measured. For dry weight determination the

material was dried at 105C for 30 min and thenkept at 60C to constant weight. Additionally,chlorophyll a and b as well as carotenoid content

were determined by light absorption spectroscopy inacetone extracts of 200 mg plant samples, accordingthe method described by Bach and widerski(2000). Absorbance values at 662, 645 and 470 nm

were recorded with a Jasco V530 spectrophotome-ter. For light microscopy, material fixed in 3% glu-taraldehyde followed by post-fixation in 2% osmiumtetroxide was sectioned transversely. Sections 1 mthick were cut from randomly selected Epon 812

blocks with a Tesla 490 A ultramicrotome. Theresults were subjected to ANOVA using STATISTICA6.1. Duncan's multiple range t-test was used; signif-icance was assumed at p = 0.05.

RESULTS

The presented experiments yielded information on

the differential morphogenetic response ofCattleyamicropropagated under different monochromaticlight regimes. Adventitious bud formation wasenhanced by red and blue light, and after threemonths of culture the propagation coefficients ofmaterial exposed to those light wavelengths were sig-nificantly higher: 11.7 under red and 10.6 under

blue light, only ~8 under white light, and ~6 in con-tinuous darkness (Tab. 1). Elongation of initiatedshoots was most pronounced under red light, butelongation of aerial roots in regenerated plantlets

was greatest under blue light. Blue light was alsomost effective in initiation of rhizogenesis (Fig. 1).The weight of cultures was depended upon the num-

ber of regenerated organs. The highest fresh weightwas for cultures under red light, and dry weight washighest for cultures under blue light. The propaga-tion coefficient and biomass weight under far redtreatment were comparable to the results under con-stant darkness (Tab. 1). Light wavelength stronglyaffected the anatomy of regenerated vegetativeorgans. Cross sections of leaves initiated in vitroshowed that blue light favored proper anatomical

Cybularz-Urban et al.114

TABLE 1. Effect of light wavelength on the number and length of adventitious shoots and aerial roots, and on the weightof regenerated Cattleya plantlets after 90 days of exposure for 16 hours a day

*Means followed by the same letter in each column do not significantly differ at p = 0.05.


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Influence of light wavelength on Cattleyaculture in vitro 115

Fig. 1. Effects of white (W), blue (B), red (R), far red (FR) light and continuous darkness (D) on organogenesis inCattleya intermedia C. aurantiaca shoot explants cultured in vitro.


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development, whereas red light caused collapse ofpart of the mesophyll cells, and reduction of leaf

blades (Fig. 2). The concentration of pigments inCattleya leaves also depended upon the wavelengthof the applied light. Data on pigment content perfresh weight of plants are shown in Figure 3. Bothtotal chlorophylls and total carotenoids were highest

in control plants illuminated with white light, ~50mg/100 g and ~10 mg/100 g, respectively, anddecreasing gradually from the blue to the red andthe far red treatments. The lowest values were inplants grown in darkness. The same correlations

were found for chlorophyll a and b treated sepa-rately (Fig. 4). Photosynthetic pigment levels werelower under illumination with monochromatic lightthan under white light, but in terms of the analyzed

plant material's morphological and anatomical fea-tures, irradiation with blue monochromatic light

was most favorable for Cattleya intermedia C.aurantiaca cultures. Total chlorophyll a and b forthe blue treatment was ~42 mg/100 g fresh weight,not much lower than under white light; it was appar-ently sufficient for the photosynthetic activity of

mixotrophic culture, and at the same time beneficialto photomorphogenesis.

DISCUSSION

Orchids grown in vitro are generally not photosyn-thetically autonomous and frequently depend on asource of organic carbon such as sucrose (Chen


Fig. 2. Transversal section of middle part of leaf blades regenerated under white (a), blue (b), and red (c) light, and con-tinuous darkness (d). abep abaxial epidermis; adep adaxial epidermis; vb vascular bundle; me mesophyll.Bar = 74 m.


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and Chang, 2004; Chen et al., 2004; Tuong andTanaka, 2004), though in recent years it has beenshown that CO2 enrichment and the use of highphotosynthetic photon flux can speed the growthrates of both C3 and CAM orchids in tissue culture(Hahn and Paek, 2001; Li et al., 2002). Light, as amain environmental trigger, plays a central role inregulating plant development. Here we studiedlight wavelength as a factor in morphogenesis and

growth in mixotrophic cultures of Cattleya inter-media C. aurantiaca as an experimental model.

We demonstrated that it can alter organogenesis inthis slow-growing material, and can alter the mor-phology and anatomy of orchid plantlets obtainedadventitiously in vitro. Applying superbright red

and blue light-emitting diodes (LED) toCymbidium cultures, Tanaka et al. (1998) foundthat the fresh and dry weight of plantlets was sig-nificantly higher when grown under red and redplus blue LEDs, and that leaves were longest underred. This is consistent with our results with incan-descent light. However, attention should be paid tolight intensity, another important variable. Culturing

Phaius and Vanda in vitro, Soontornchainaksaenget al. (2001) observed that light intensity significant-ly affected dry weight and plantlet height, leaf num-

ber, shape and area. In our study the differences infresh and dry weight of biomass obtained under

blue and red light may have been due in part to dif-ferences in light intensity.

InBetula, as in our Cattleya hybrid, light qual-ity was shown to affect not only proliferation rateand shoot elongation but also the anatomy of cul-tured plantlets (Sb et al. 1995). Red light signifi-cantly enhanced adventitious bud formation andstem elongation inPetunia and Gerbera, while blueincreased the number of adventitious buds and thefresh weight of obtained shoots of Freesia,

Hyacinthus and Cyclamen (Gabryszewska andRudnicki, 1995; Bach and Malik, 1999; Bach et al.,2000; Bach and widerski, 2000; Witomska andKoszewska, 2002).

Blue light treatment improved the efficiency ofthe in vitro propagation system used. The morphol-ogy and anatomy of the obtained plantlets was alsopositively affected, and the resulting plants were trueto type. The plantlets obtained under red were notacceptable, because the total chlorophyll andcarotenoid content of leaves was much reduced anddegenerative changes occurred in the mesophyll. Weconclude that light spectra can be used in Cattleya

organ culture to control morphogenesis and thegrowth of tissues in vitro. This is important, becausetechniques successfully adopted for mass propaga-tion of orchids are now widely used not only forclonal propagation but also during genetic manipu-lation aimed at plant improvement (Tanaka et al.,2005).

ACKNOWLEDGEMENTS

We thank Prof. Anna Bach, head of the OrnamentalPlant Department of the Agricultural University,Cracow, for valuable advice and for providing use ofthe climate chamber in the Department.

REFERENCES

ADELBERG JW, DESAMERO NW, HALE A, and YOUNG RE. 1997.Long-term nutrient and water utilization during micro-propagation of Cattleya on liquid/membrane system.

Plant, Cell, Tissue and Organ Culture 48: 17

Influence of light wavelength on Cattleyaculture in vitro 117

Fig. 3. Chlorophyll and carotenoid content in shoots ofhybrid Cattleya cultured under monochromatic lightregimes.

Fig. 4. Chlorophyll a and b content in Cattleya interme-dia C. aurantiaca shoots regenerated adventitiously in

vitro under monochromatic light regimes. Values forplants grown in darkness were treated as blanks and weresubtracted.


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BACHA, and MALIKM. 1999. Effect of light quality on somaticembryogenesis ofCyclamen persicum Mill. 'Medium'. In:Schwenkel HG [ed.], Reproduction of Cyclamen per-

sicum Mill. through somatic embryogenesis using sus-

pension culture systems, 2736. Directorate General forResearch of European Commission, COST Action 822.

BACH A, MALIK M, PTAKA, and KDRA M. 2000. Effect of lightquality on morphogenesis of bulbous ornamentalmicroplant.Acta Horticulturae 53: 173180.

BACH A, and WIDERSKI A. 2000. The effect of light quality onorganogenesis ofHyacinthus orientalis L. in vitro.Acta

Biologica Cracoviensia Series Botanica 42/1: 115120.CHEN JT, and CHANGWC. 2004. TIBA affects the induction of

direct somatic embryogenesis from leaf explants ofOncidium. Plant, Cell, Tissue and Organ Culture 79:315320.

CHEN TY, CHEN JT, and CHANGWC. 2004. Plant regenerationthrough direct shoot bud formation from leaf cultures of

Paphiopedilum orchids.Plant, Cell, Tissue and OrganCulture 76: 1115.

GABRYSZEWSKAE, and RUDNICKI R. 1995. The influence of lightquality on the shoot proliferation and rooting ofGerbera

jamesonii in vitro.Acta Agrobotanica 48/2: 105111.

HAHN EJ, and PAEKKY. 2001. High photosynthetic photon fluxand high CO2 concentration under increased number ofair exchanges promote growth and photosynthesis offour kinds of orchid plantlets in vitro.In Vitro Cellular &

Developmental Biology Plant 37: 678682.ICHIHASHI S. 1990. Effects of light on root formation ofBletilla

striata seedlings.Lindleyana 5/2: 140143.ISLAM OM, MATSUI S, and ICHIHASHI S. 2000. Effects of light

quality on carotenoid contents of in vitro growingseedlings of Cattleya. Journal of India Orchid Society14: 717.

KIRDMANEE C, KUBOTA C, JEONG B, and KOZAI T. 1992.Photoautotrophic multiplication of Cymbidium proto-corm-like bodies.Acta Horticulturae 319: 243248.

LI CR, GAN L.J, XIAK, ZHOUX, and HEWCS. 2002. Responsesof carboxylating enzymes, sucrose metabolising enzymesand plant hormones in a tropical epiphytic CAM orchidto CO2 enrichment. Plant, Cell and Environment 25:369377.

SB A, KREKLING T, and APPELGREN M. 1995. Light qualityaffects photosynthesis and leaf anatomy of birch plantletin vitro. Plant Cell, Tissue and Organ Culture 41:177185.

SOONTORNCHAINAKSAENG P, CHAICHAROEN S, SIRIJUNTARUT M, andKRUATRACHUE M, 2001. In vitro studies of the effect oflight intensity on plant growth ofPhaius tankervilliae(Banks ex L'Herit) Bl. and Vanda coerulea Griff. Science

Asia 27: 233237.TANAKAY, KATSUMOTO Y, BRUGLIERA F, and MASON J. 2005.

Genetic engineering in floriculture. Plant Cell, Tissueand Organ Culture 80: 124.

TANAKA M, TAKAMURA T, WATANABE H, ENDO M, YANAGI T, andOKAMOTO K. 1998. in vitro growth of Cymbidiumplantlets cultured under superbright red and blue light-emitting diodes (LEDs).Journal of Horticultural Scienceand Biotechnology 73: 3944.

TUONG HL, and TANAKAM. 2004. Callus induction from proto-

corm-like body segments and plant regeneration inCymbidium (Orchidaceae). Journal of HorticulturalScience and Biotechnology 79/3: 406410.

VINCE-PURE D. 1994. Photomorphogenesis and plant develop-ment. In: Lumsden PJ, Nicholas JR and Davies WJ[eds.],Physiology, growth and development of plants inculture, 1930. Kluwer Academic Publishers, Dordrecht.

WITOMSKA M, and KOSZEWSKAA 2002. Wpyw jakoci wiata icytokinin na organogenez in vitro petunii ogrodowej(Petunia hybridaVlim.)Zeszyty Problemowe Postpw

Nauk Rolniczych 483: 271280.


effect of light wavelength on in vitro organogenesis

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