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Journal of Applied Phycology ISSN 0921-8971Volume 26Number 2 J Appl Phycol (2014) 26:819-823DOI 10.1007/s10811-013-0130-4

Effects of plant growth regulators on growthand morphogenesis in tissue culture ofChondracanthus chamissoi (Gigartinales,Rhodophyta)

Nair S.Yokoya, Marcela vila, MariaI.Piel, Fabiola Villanueva & AngelicaAlcapan

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Effects of plant growth regulators on growth and morphogenesisin tissue culture of Chondracanthus chamissoi (Gigartinales,Rhodophyta)

Nair S. Yokoya & Marcela vila & Maria I. Piel &Fabiola Villanueva & Angelica Alcapan

Received: 26 May 2013 /Revised and accepted: 22 August 2013 /Published online: 27 September 2013# Springer Science+Business Media Dordrecht 2013

Abstract Chondracanthus chamissoi (C. Agardh) Ktzing(Gigartinales, Rhodophyta) is an edible species and commer-cialised for carrageenan production in Chile. Investigations ongrowth and development are needed to improve its cultiva-tion; therefore, this study aims to evaluate the effects of plantgrowth regulators (PGRs) on its growth and morphogenesis.PGRs tested were two auxins [indole-3-acetic acid (IAA) and2,4-dichlorophenoxyacetic acid (2,4-D)] and one cytokinin(benzylaminopurine (BA)) in concentrations of 0.5, 5.0 and50.0 M. These PGRs were added to seawater enriched withhalf strength of von Stosch solution and were gelled with0.6 % agar, and treatment control (without PGR addition)was conducted simultaneously. Apical and intercalary seg-ments were used as initial explants. Each treatment was testedwith six replicates of five axenic explants, and statisticalanalyses were performed. After culturing in a solid mediumfor 10 weeks to induce growth and callus formation, explantswere cultured in liquid medium with the same experimentalconditions for 10 weeks. Effects of auxins (IAA and 2,4-D)and the cytokinin BA on growth rates of apical segments ofC.chamissoi were not significant, while low concentration ofIAA stimulated the growth of intercalary segments. On theother hand, high concentrations of BA and IAA stimulated thecallus formation in apical and intercalary segments, respec-tively. In liquid medium, PGR did not have a significant effecton growth rates of apical segments, while 2,4-D in concentra-tions from 0.5 to 50.0 M stimulated growth of intercalary

segments, and formation of lateral branches was stimulated bylow 2,4-D in apical segments. These results suggest that PGRshave a regulatory role on callus formation and growth ofspecific explants of C. chamissoi . Furthermore, the formationof lateral branches stimulated by auxin could be used forseedling production under controlled conditions and couldimprove the micropropagation and cultivation ofC. chamissoiin the Chilean coast.

Keywords Auxins . Chilean seaweeds .Chondracanthus .

Cytokinins . Gigartinales . Seaweed tissue culture

Introduction

Seaweed tissue culture is an important tool for micropropagation,a technique which produces, within a short period, a largenumber of individuals that can be used as seedlings for seaweedcultivation rather than collecting them fromnatural beds (Yokoyaand Yoneshigue-Valentin 2011) and, also, for application ingenetic engineering (Stevens and Purton 1997). To improvetissue culture techniques, it is fundamental to understand thehormonal regulation of seaweed growth and development and,also, to characterise the hormone profiles in seaweeds (Yokoyaet al. 2010).

Endogenous plant hormones have been detected in green,brown and red seaweeds as well as in some extracts of variouskelp species that are used commercially as growth stimulantsin agricultural crops (Stirk and Van Staden 2006). Besides,endogenous cytokinins, including both isoprenoid and aro-matic groups, were identified in 5 Chlorophyta, 7 Phaeophytaand 19 Rhodophyta species from South Africa (Stirk et al.2003). Endogenous cytokinins, auxins and abscisic acid wereidentified and quantified in 11 red algae collected from theBrazilian coast (Yokoya et al. 2010). However, the physiolog-ical functions of these hormones need to be elucidated inorder to understand their regulation role on seaweed growth,

N. S. Yokoya (*)Ncleo de Pesquisa em Ficologia, Instituto de Botnica,Av. Miguel Estfano, 3687, 04301-012 So Paulo, Brazile-mail: nyokoya@hotmail.com

M. vila :M. I. Piel : F. Villanueva :A. AlcapanInstitute of Science and Technology, University Arturo Prat,Ejercito 443, Puerto Montt, Chile

M. vilae-mail: marcela.avila@unap.cl

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development and morphogenesis. This knowledge could beuseful in the commercial breeding and cultivation of importantseaweeds. Considering this aspect, Chondracanthuschamissoi (C. Agardh) Ktzing (Gigartinales, Rhodophyta)is an important species for Chilean and Peruvian industries forcarrageenan production, and it has been exploited to Asiancountries for human consumption (Avila et al. 2011; Bulboaand Macchiavello 2006). Several studies on C. chamissoihave reported different aspects of vegetative propagation(Macchiavello et al. 2003; Fonck et al. 2008; Sez et al.2008), sexual reproduction (Avila et al. 2011), reproductivephenology and populational dynamics (Gonzlez et al. 1997;Vsquez and Vega 2001), growth and development undercontrolled conditions (Gonzlez and Meneses 1996; Bulboaand Macchiavello 2001; Bulboa et al. 2010) and cultivationattempts (Bulboa and Macchiavello 2006). However, micro-propagation and tissue culture techniques have not been inves-tigated in this species. Therefore, this study aims to evaluate theeffects of plant growth regulators (PGRs) on the growth andmorphogenesis in tissue culture of C. chamissoi .

Material and methods

Algal material Female gametophytes with cystocarps of C.chamissoi (C. Agardh) Ktzing (Gigartinales, Rhodophyta)were collected in October 2007, in Coliumo, Chile (3631 S,7257W). Reproductive specimens were collected using hoo-kah divers and were transported in a cooler with gel pack tokeep the algal samples cold and humid until the laboratory ofthe Institute of Science and Technology of Arturo PratUniversity, in Puerto Montt, Chile. In the laboratory, maturecystocarpic specimens were washed four times with filteredseawater to eliminate epiphytes. The procedures to stimulatecarpospore liberation and culture methods were described byAvila et al. (2011). Isolated tetrasporophytes were cultured inhalf strength of Provasoli medium (Provasoli 1968), replaced atweekly intervals, under light/dark cycle of 16:8 h, temperatureof 13 C, photon flux density of 10 mol photons m2 s1 andsalinity of 33 practical salinity unit (psu).

Experiments with plant growth regulators PGRs tested weretwo auxins [indole-3-acetic acid (IAA) and 2,4-dichlo-rophenoxyacetic acid (2,4-D)] and one cytokinin (ben-zylaminopurine (BA)) in concentrations of 0.5, 5.0 and50.0 M. These PGRs (Sigma, USA) were added to seawaterenriched with half strength of von Stosch solution (VSES/2medium) as described by Oliveira et al. (1995) and modifiedwith the reduction of 50 % in the vitamin concentrations(Yokoya 2000) and were gelled with 0.6 % agar (Equilab,Chile). Treatment control (without PGR addition) wasconducted simultaneously.

To obtain axenic explants, thallus segments (1015 mmlong) were cut from tetrasporophytes and were cultured for48 h in solution composed by VSES/2 with penicillin(3.75 mg mL1) and nystatin (3.125 mg mL1). Inside alaminar flow chamber, apical and intercalary segments(5 mm long) were isolated and washed for 10 s in a solutionof sterile seawater with 0.5 % sodium hypochlorite and0.002% of organic detergent (Detlab, Chile) and were washedseveral times in sterile seawater to remove this solution.Before inoculation, these explants were washed with solutionof ciprofloxacin (5 mg L1) in sterile seawater. These explantswere inoculated into 30 mL autoclaved VSES/2 solid mediumsupplemented with PGR. Experiments were conducted underthe same laboratory conditions described for culturing isolatedtetrasporophytes. Each treatment was tested with six replicatesof five explants. After culturing in a solid medium for10 weeks, explants were transferred into a liquid mediumwiththe same experimental conditions and were cultured for10 weeks. This experimental period was chosen based onthe growth of C. chamissoi in laboratory conditions (Avilaet al. 2011). Mediumwas renewed weekly, and morphologicalvariations were recorded under a stereomicroscope (StemiDV4; Carl Zeiss, Germany) using the ImageJ software.

Specific growth rate was calculated as [ln (B fBo1)t1],

where Bo is the initial fresh biomass, B f is the fresh biomassafter t days, and t corresponds to the experimental period(Yokoya et al. 2007). Morphological data include the numberand length of lateral branches and the percentage of callusformation (number of calluses per explants).

Statistical analysis Data were analysed by one-way ANOVAand test of planned comparison, which allow performingparametric comparisons between each levels of an experimen-tal factor and a control, making the necessary adjustments tocorrect the type I error generated by the extensive use ofthe same set of data in multiple comparisons (Chambers andHastie 1992).

Results

Effects of auxins (IAA and 2,4-D) and the cytokinin BA ongrowth rates of apical segments of C. chamissoi were notsignificant, while low concentration of IAA stimulated thegrowth of intercalary segments cultured in a solid medium(Fig. 1(a, b)). On the other hand, high concentrations of BAand IAA stimulated the callus formation in apical and inter-calary segments, respectively (Fig. 1(c, d)).

Apical and intercalary segments ofC. chamissoi developedcalluses in different thallus regions: apical calluses developedin apical regions of branches (Fig. 2a), intermediate callusesdeveloped in intermediate parts of the explant (Fig. 2b, c) and

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basal calluses developed in the basal (proximal) pole of theexplant (Fig. 2d, e).

In liquid medium, auxins and cytokinin did not have asignificant effect on the growth rates of apical segments(Fig. 3(a)), but low 2,4-D concentration stimulated the highestnumber of lateral branches (Fig. 3(c)). However, differentresponses were observed in intercalary segments, where 2,4-D in concentrations from 0.5 to 50.0 M stimulated thegrowth rates (Fig. 3(b)), but PGR did not have significanteffects on the number of lateral branches (Fig. 3(d)).

Discussion

Explants of C. chamissoi developed apical, basal and intermedi-ate calluses without a tendency in relation to PGR type orconcentration. Gracilaria tenuistipitata Chang and Xia andGracilaria perplexa Byrne and Zuccarello (Gracilariales,Rhodophyta) also developed the same types of calluses(Yokoya et al. 2004). Formation of basal calluses is common indifferent species of Rhodophyta, since they are usually inducedby wounding process (Gusev et al. 1987; Polne-Fuller and Gibor

Fig. 1 Effects of indole aceticacid (IAA), 2,4-dichlorophenoxiacetic acid (2,4-D) and benzylaminopurine (BA)on growth rates (a , b) and callusformation (c , d) in apical (a , c)and intercalary segments (b , d) ofC. chamissoi cultured in halfstrength of von Stosch mediumwith 0.6 % agar for 10 weeks,under light/dark cycle of 16:8 h,temperature of 13 C, photon fluxdensity of 10 molphotons m2 s1 and salinity of33 psu. Each data point is themean of six replicates. Significantdifferences are indicated bydifferent letters

a b c d

e

Fig. 2 Different callus types inexplants of C. chamissoi culturedin half strength of von Stoschsolid medium (0.6 % agar) for10 weeks, under light/dark cycleof 16:8 h, temperature of 13 C,photon flux density of 10 molphotons m2 s1 and salinity of33 psu. Apical callus (a),intermediate calluses (b , c) andbasal calluses (d , e). Callus wasindicated by arrows . Scale =1 mm

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1987; Kaczyna andMegnet 1993; Huang and Fujita 1996, 1997;Yokoya and Handro 1996; Yokoya et al. 1999). On the otherhand, the development of apical calluses was described for somespecies as Solieria filiformis (Ktzing) Gabrielson (Robledo andGarcia-Reina 1993), Meristotheca papulosa J. Agardh (Huangand Fujita 1997), Gracilariopsis tenuifrons (Bird and Oliveira)Fredericq and Hommersand (Yokoya 2000), Kappaphycusalvarezii (Doty) Doty ex P.C. Silva (Reddy et al. 2003) andtwo colour strains of Hypnea musciformis (Wulfen in Jacqu.)J.V. Lamour. (Yokoya et al. 2003). Calluses of C. chamissoishowed a low potential for regeneration ofwhatwere observed inother species of Gigartinales (Gusev et al. 1987; Huang andFujita 1996). For micropropagation purpose, the formation oflateral branches is an alternative morphogenetic response andcould be used for seedling production instead of thallus regener-ation from calluses.

Our results showed that auxins have stimulatory effects onthe growth of intercalary segments of C. chamissoi ; specifi-cally, low concentration of IAA and 2,4-D in concentrationsfrom 0.5 to 50.0 M stimulated the growth in solid and liquidmedia, respectively. IAA also stimulated the callus formationin intercalary segments of C. chamissoi . These stimulatoryeffects of auxins could be related to their function on process-es of cell division and elongation (Krikorian 1995), withincreases in DNA, RNA and protein content, indicatingchanges in gene expression (Hagen 1995). Similar responseswere also observed in Gracilaria vermiculophylla (Ohmi)

Papenfuss (Yokoya et al. 1999) and in G. tenuifrons(Yokoya 2000). On the other hand, apical segments of C.chamissoi showed different responses: high concentrationsof BA stimulated the callus formation, and low concentra-tion of 2,4-D stimulated the formation of lateral branches.Distinct responses between apical and intercalary segmentsof C. chamissoi could be explained by the presence ofapical cells in apical segments, which result in a physio-logical and biochemical gradient along the thallus, andconsequently, this factor could influence the cell sensitivityto PGR (Davies 1995). The stimulatory effect of BA (anaromatic cytokinin) on callus formation is a result of itsrole on cell division.

In conclusion, our results suggest that PGRs have a regu-latory role on callus formation and growth of specific explantsof C. chamissoi . Furthermore, the formation of lateralbranches stimulated by auxin could be used for seedlingproduction under controlled conditions and could improvethe micropropagation and cultivation of C. chamissoi in theChilean coast.

Acknowledgments The authors thank supports from the FONDEF,Chile (projects D06I1058 and D08I1067 to MA), and the CONICYT(projectMEC 80100021 toNSY). The first author thanks the Universidadde Magallanes (Chile) for the stay as an invited researcher and grantsfrom the Fapesp and from the CNPq (Brazil). We also thank valuablesuggestions made by two anonymous reviewers who helped to improvethe manuscript.

Fig. 3 Effects of indole acetic acid(IAA), 2,4-dichlorophenoxiaceticacid (2,4-D) andbenzylaminopurine (BA) on growthrates (a, b) and formation of lateralbranches (c, d) in apical (a, c) andintercalary segments (b, d) of C.chamissoi cultured in half strengthof von Stosch liquid medium for10 weeks, under light/dark cycle of16:8 h, temperature of 13 C,photon flux density of 10 molphotons m2 s1 and salinity of33 psu. Each data point is the meanof six replicates. Significantdifferences are indicated bydifferent letters

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References

Avila M, Piel MI, Caceres JH, Alveal K (2011) Cultivation of the red algaChondracanthus chamissoi: sexual reproduction and seedling produc-tion in culture under controlled conditions. J Appl Phycol 23:529536

Bulboa C, Macchiavello J (2001) The effects of light and temperature ondifferent phases of the life cycle in the carrageenan producing algaChondracanthus chamissoi (Rhodophyta, Gigartinales). Bot Mar44:371374

Bulboa C, Macchiavello J (2006) Cultivation of cystocarpic, tetrasporicand vegetative fronds of Chondracanthus chamissoi (Rhodophyta,Gigartinales) on ropes at two localities in northern Chile. InvestigMar 34:151154

Bulboa C,Macchiavello J, Veliz K, Oliveira E (2010) Germination rate andsporeling development of Chondracanthus chamissoi (Rhodophyta,Gigartinales) varies along a latitudinal gradient on the coast of Chile.Aquat Bot 92:137141

Chambers JM, Hastie TJ (1992) Statistical models. In: Chambers JM,Hastie TJ (eds) Statistical models in S. Wadsworth & Brooks/Cole,Pacific Grove

Davies PJ (1995) The plant hormone concept: concentration, sensitivity andtransport. In: Davies PJ (ed) Plant hormonesphysiology, biochem-istry and molecular biology. Kluwer Academic, Dordrecht, pp 1338

Fonck E, Martinez R, Vsquez J, Bulboa C (2008) Factors that affect there-attachment of Chondracanthus chamissoi (Rhodophyta,Gigartinales) thalli. J Appl Phycol 20:311314

Gonzlez J, Meneses I (1996) Differences in the early stages of develop-ment of gametophytes and tetrasporophytes of Chondracanthuschamissoi (C. Ag.) Ktzing from Puerto Aldea, Northern Chile.Aquaculture 143:91107

Gonzlez J, Meneses I, Vsquez J (1997) Field studies in Chondracanthuschamissoi (C. Agardh) Ktzing: seasonal and spatial variations in life-cycle phases. Biol Pesq 26:312

Gusev MV, Tambiev AH, Kikova NN, Shelyastin NN, Aslanyan RR(1987) Callus formation in seven species of agarophyte marinealgae. Mar Biol 95:593597

Hagen G (1995) The control of gene expression by auxin. In: Davies PJ(ed) Plant hormonesphysiology, biochemistry and molecular bi-ology. Kluwer Academic, Dordrecht, pp 228245

Huang W, Fujita Y (1996) Callus induction and thallus regeneration insome species of red algae. Phycol Res 45:105111

Huang W, Fujita Y (1997) Callus induction and thallus regeneration ofthe red algaMeristotheca papulosa (Rhodophyta, Gigartinales). BotMar 40:5561

Kaczyna F, Megnet R (1993) The effects of glycerol and plant growthregulators on Gracilaria verrucosa (Gigartinales, Rhodophyceae).Hydrobiologia 268:5764

Krikorian AD (1995) Hormones in tissue culture and micropropagation.In: Davies PJ (ed) Plant hormonesphysiology, biochemistry andmolecular biology. Kluwer Academic, Dordrecht, pp 774796

Macchiavello J, Bulboa C, Edding M (2003) Vegetative propagation andspore recruitment in the carrageenophyte Chondracanthus chamissoi(Rhodophyta, Gigartinales) in northern Chile. Phycol Res 51:4550

Oliveira EC, Paula EJ, Plastino EM, Petti R (1995) Metodologias paracultivo no axenico de macroalgas marinhas in vitro. In: Alveal K,Ferrario ME, Oliveira EC, Sar E (eds) Manual de MtodosFicolgicos. Universidad de Concepcin, Concepcion, pp 429447

Polne-Fuller M, Gibor A (1987) Calluses and callus-like growth inseaweeds: induction and culture. Hydrobiologia 151/152:131138

Provasoli L (1968) Media and prospects for the cultivation of marine algae.In: Watanabe A, Hattori A (eds) Cultures and collection of algae.Proceedings of the US Japanese Society of Plant PhysiologistsConference, Hakone, pp 6375

Reddy CRK, Raja Krishna Kumar G, Siddhanta AK, Tewari A (2003) Invitro somatic embryogenesis and regeneration of somatic embryosfrom pigmented callus of Kappaphycus alvarezii (Doty) Doty(Rhodophyta, Gigartinales). J Phycol 39:610616

Robledo DR, Garcia-Reina G (1993) Apical callus formation in Solieriafiliformis (Gigartinales, Rhodophyta) cultured in tanks. Hydrobiologia260/261:401406

Sez F, Macchiavello J, Fonck E, Bulboa C (2008) The role of secondaryattachment disc in vegetative propagation of Chondracanthuschamissoi (Rhodophyta, Gigartinales). Aquat Bot 89:6365

Stevens DR, Purton S (1997) Genetic engineering of eucaryotic algae:progress and prospects. J Phycol 33:713722

Stirk WA, van Staden J (2006) Seaweed products as biostimulants inagriculture. In: Critchley AT, Ohno M, Largo DB (eds) Worldseaweed resources. ETI Information Services Ltd, Wokingham, pp132, DVD ROM

Stirk WA, Novak O, Strnad M, Van Staden J (2003) Cytokinins inmacroalgae. Plant Growth Regul 41:1324

Vsquez J, Vega JA (2001) Chondracanthus chamissoi (Rhodophyta,Gigartinales) in northern Chile: ecological aspects for managementof wild population. J Appl Phycol 13:267277

Yokoya NS (2000) Apical callus formation and plant regeneration con-trolled by plant growth regulators on axenic culture of the red algaGracilariopsis tenuifrons (Gracilariales, Rhodophyta). Phycol Res48:133142

Yokoya NS, Handro W (1996) Effects of auxins and cytokinins on agarculture of Grateloupia dichotoma (Gigartinales, Rhodophyta).Hydrobiologia 326/327:393400

Yokoya NS, Yoneshigue-Valentin Y (2011) Micropropagation as a toolfor sustainable utilization and conservation of populations ofRhodophyta. Braz J Pharmacogn 21:334339

Yokoya NS, Kakita H, Obika H, Kitamura T (1999) Effects of environ-mental factors and plant growth regulators on growth of the red algaGracilaria vermiculophylla from Shikoku Islands, Japan.Hydrobiologia 398/399:339347

Yokoya NS, Plastino EM, Artel R (2003) Physiological responses andpigment characterization of two colour strains of the carrageenophyteHypnea musciformis (Rhodophyta). In: Chapman ARO, AndersonRJ, Vreeland VJ, Davison R (eds) Proceedings of the 17th interna-tional seaweed symposium. Oxford University Press, New York, pp425433

Yokoya NS,West JA, Luchi AE (2004) Effects of plant growth regulatorson callus formation, growth and regeneration in axenic tissue cul-tures of Gracilaria tenuistipitata and G. perplexa (Gracilariales,Rhodophyta). Phycol Res 52:244254

Yokoya NS, Necchi Jnior O, Martins AP, Gonzalez SF, Plastino EM(2007) Growth responses and photosynthetic characteristics of wildand phycoerythrin-deficient strains of Hypnea musciformis(Rhodophyta). J Appl Phycol 19:197205

Yokoya NS, Stirk WA, van Staden J, Novak O, Tureckova V, Pencik A,StrnadM (2010) Endogenous cytokinins, auxins and abscisic acid inred algae from Brazil. J Phycol 46:11981205

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Effects...AbstractIntroductionMaterial and methodsResultsDiscussionReferences