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Scientia Horticulturae 160 (2013) 49–53

Contents lists available at SciVerse ScienceDirect

Scientia Horticulturae

journa l h om epage: www.elsev ier .com/ locate /sc ihor t i

hort communication

stablishment of photomixotrophic cultures for raspberryicropropagation in Temporary Immersion Bioreactors (TIBs)

riel D Arencibia ∗, Carolina Vergara, Karla Quiroz, Basilio Carrasco,olando García-Gonzales

aculty of Agricultural and Forestry Sciences, Catholic University of Maule, Ave San Miguel 3605, Talca, Chile

r t i c l e i n f o

rticle history:eceived 22 January 2013eceived in revised form 7 May 2013ccepted 14 May 2013

a b s t r a c t

A novel protocol for raspberry (Rubus spp.) micropropagation in photomixotrophic conditions was opti-mized for the commercial genotypes Heritage, Meeker and Amity. Plant cultures were established inTemporary Immersion Bioreactors (TIBs) under a controlled environment: 550 ppmv CO2; light intensity80 �M m−2 s−1; sucrose concentrations 15 gr/L and 30 gr/L. Results showed that both CO2 flux and chloro-

eywords:emporary immersion bioreactorshotomixotrophicucrose-reduced medium

phyll fluorescence were increased in plants cultured in TIBs with sucrose-reduced medium (15 gr/L) incomparison with those plants micropropagated in TIBs + 30 gr/L sucrose, confirming a consistent pho-tomixotrophic stage since the 5th day of culture. Raspberry plants multiplied in TIBs + 15 gr/L sucrosedemonstrated the highest values of plant size, total number of internodes, and percent of acclimatizationto greenhouse conditions. Studied variables could be related to an increase of the in vitro photosyntheticactivity which might prime plants for the ex vitro environment.

. Introduction

The genus Rubus belongs to the family Rosaceae and con-ains cultivated raspberries, blackberries, hybrid berries, and otherspecies (Jennings, 1988). Raspberry fruits have been consideredutraceuticals containing vitamins, fibers, and phytochemical com-ounds that function as antioxidants and antimicrobial protective,mong other health-related properties (Poiana et al., 2010). Theide diversity of Rubus species provides a potential source of novel

raits whereas breeding programs are actively working on releas-ng cultivars with excellent quality, high yields, greater adaptationo adverse environmental conditions, and increased pest and dis-ase resistance (Castillo et al., 2010). Rubus species and cultivars arelonally propagated and maintained in greenhouses, screenhouses,eld collections, and as tissue-cultured plants and cryopreservedhoot tips (Wang et al., 2005; Gupta and Reed, 2006).

Clonal multiplication based on plant tissue culture is preferredor the production of pathogen-free and genetic-fidelity commer-ial plants (Arencibia et al., 2011). Raspberries are susceptible toumerous virus diseases, and sensitive cultivars may be killed or

everely weakened by virus infection; latent infections on tolerantultivars may shorten the planting life of a field through reducedield and fruit quality. Incidence in Robus accessions of raspberry

∗ Corresponding author. Tel.: +56 71 210500; fax: +56 71 210500.E-mail addresses: arieldarencibia@gmail.com, arielarencibia@yahoo.com

A.D. Arencibia).

304-4238/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.scienta.2013.05.010

© 2013 Elsevier B.V. All rights reserved.

bushy dwarf idaeovirus (RBDV), tobacco streak ilarvirus (TSV) andtomato ringspot nepovirus (TomRSV) have been determined as5.77%, 2.76% and 0.31%, respectively (Tsao et al., 2000).

The regeneration of adventitious shoots from different types ofexplants has been reported for some Rubus species. Genotype, typeof explants, source, and balance of plant growth regulators (PGRs)constitute the main factors influencing regeneration (Fiola et al.,1990; Cousineau and Donnelly, 1991; Turk et al., 1994; Mezzettiet al., 1997). Moreover, conventional (agar-based) micropropaga-tion protocols including the optimization of both multiplication(Pedroso de Olivera and Pacheco, 2009) and rooting (Nolasco et al.,2009) steps have been reported for large-scale production of com-mercial raspberry cultivars.

For plant propagation biofactories, liquid cultures and automa-tion has the potential to resolve the manual handling of the variousstages reducing the high production costs that render conventionalsystems (agar-based) less suitable (Paek et al., 2005; Arencibiaet al., 2011). In this way, a two-step protocol for in vitro multiplica-tion in RITA® bioreactors studying plant growth regulators (PGRs)has been described for both Rubus chamaemorus and Rubus idaeus(Debnath, 2007, 2010).

As a step toward a more efficient micropropagation proce-dure, this paper reports the establishment of photomixotrophicraspberry cultures in a two-separate-vessel design of Temporary

Immersion Bioreactors (TIBs). There are several types of TIBs wherechoice/design depends on the sensitivity of plant materials tohyperhydricity and the costs for scaling up (Ziv, 1999, 2000; Kozai,2006). In this study, two-vessel bioreactors have been standardized

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or photomixotrophic raspberry micropropagation, corroboratinghat this approach could be suitable for large-scale productive pro-ess.

. Materials and methods

.1. Plant stocks

Shoot tips (∼5 cm) of raspberry plantlets of the genotypeseritage, Meeker and Amity were surface disinfected in a 10%ommercial solution NaOCl plus 0.1% Tween 20 (15 min). Explantsere immersed in 70% ethanol (5 min.) followed by three rinses

n sterile distilled water. Shoot tips were cultured for 8 weeks inedium containing MS salts plus vitamins (Murashige and Skoog,

962), 1 mg/L BAP, 0.3 mg/L IBA, 30 gr/L sucrose. Culture mediumas solidified with 8 gr/L agar and the pH was adjusted to 5.2

efore autoclaving at 121 ◦C. Plant cultures were maintained at3 ± 2 ◦C, for a 16-h photoperiod under a combination of bothatural light/cool-white fluorescent tubes at a light intensity of0 �M m−2 s−1.

.2. Temporary Immersion Bioreactors (TIBs)

Two-vessel bioreactors were set up following establishedesigns (Lorenzo et al., 2001; Yabor et al., 2007; Arencibia et al.,008, 2012). For each raspberry cultivar, a total of 10 intern-des from vigorous plants of 20 days old (after subculture) wereransferred to a sterile transparent bottle (500 mL capacity). Theioreactors contained 250 mL of liquid medium (as described aboveut without sucrose). Experimental treatments for carbon (C)ource were: TIBs containing liquid medium plus 30 gr/L sucrose;IBs with liquid medium plus 15 gr/L sucrose. The immersion fre-uency was 3 min each 8 h. Air quality in the TIBs working stationas improved with 550 ppmv CO2, and bioreactors were main-

ained during 30 days at 23 ± 2 ◦C under a combination of bothatural light and cool-white fluorescent tubes at a light intensityf 80 �M m−2s−1. For each genotype, five vessels (conventionalgar-cultures) subcultured with 10 internodes were the controlreatments. Each TIBs treatment included five separate replicas. Thexperiments were repeated at three different dates.

.3. Photosynthesis research

Both CO2 flux and chlorophyll fluorescence were measuredsing the CIRAS-2 with a Chlorophyll Fluorescence Module (CFM).ive leaves per treatment were randomLy selected at 0; 5; 10; 15;0 days of TIBs cultures. The controls treatments for each cultivar

ncluded: in vitro plants multiplying in agar-medium plus 30 gr/Lucrose; ex vitro plants growing in greenhouses.

.4. Acclimatization to greenhouse (ex vitro rooting)

Populations of raspberry plants multiplied by both TIBs andgar-base (control) treatments were carefully separated, washed in

L of water, and planted in 128-cell plug trays (cell volume 25 cm3)ontaining a mixture of composted pine bark and zeolite (2:1).reatments/replicas were randomLy distributed in the greenhouse,nd the relative humidity was gradually reduced: 90% → 80% → 70%n 10 day intervals. Luminosity was 100 �M m2 s−1 under theatural photoperiod of January–February, latitude 35◦30′–altitude1◦30′, locality of Talca, Chile.

.5. Data collection and statistical analysis

The following variables were recorded after the micropropa-ation cycle (30 days): total number of internodes, plants size

iculturae 160 (2013) 49–53

(height), and in vitro shooting. The adaptability rate was deter-mined after 30 days of planting (acclimatization). Data wereanalyzed by ln(x). Variances were calculated for the micropropa-gation traits related to productivity and were compared betweentreatments using Bartlett’s homogeneity of variances test STATIS-TICA V.6 software package (STATSOFT, Inc, 2003).

3. Results and discussion

A procedure for raspberry micropropagation in programmedbioreactors (TIBs based on two separate bottles) was developedfor the commercial genotypes Heritage, Meeker and Amity. Rasp-berry plants grew and multiplied in a sucrose-reduced culturemedium, with CO2-rich and high luminosity environment. Duringthe experiment, variables of CO2 flux and chlorophyll fluores-cence were increased in plants cultured in TIBs + 15 gr/L sucrosein comparison with those plants micropropagated in TIBs + 30 gr/Lsucrose (Fig. 1). Raspberries cultured in TIBs with sucrose reducemedium (15 gr/L) demonstrated photosynthesis-related valuesclosely to plants growing in ex vitro conditions (control of autotro-phy). Meanwhile, raspberry vitroplantlets propagated in TIBs withsucrose-standard medium (30 gr/L) showed photosynthesis valuesslightly higher than plants growing in conventional agar-base cul-tures + 30 gr/L sucrose (control of heterotrophy). Altogether, resultsevidenced a consistent photomixotrophic stage in raspberry plantsfrom TIBs + 15 gr/L sucrose since the 5th day of culture. In paral-lel, the experiments corroborated the effects of TIBs (frequent airexchange) enhancing the photosynthetic pathway.

For the first time, photomixotrophic in vitro cultures of Rubusspp. have been established by applying the TIBs technology. Arelated paper from Debnath (2005) studied the effect of carbonsource (glucose, sorbitol, or sucrose) and concentrations usingshoots cultivated in vitro from nodal explants of lingonberry (Vac-cinium vitis-idaea ssp. vitis-idaea L. and V. vitis-idaea ssp. minus.Lodd). In that case, photosynthesis variables were not studied,and the experiments were conducted in conventional agar-basedcultures without CO2 enrichment, concluding that carbohydrateconcentration had little effect on shoot vigor in a genotype-dependent manner.

Previously, a system for in vitro multiplication of raspberry cul-tivars ‘Festival’, ‘Heritage’, and ‘Latham’ has been reported usingthe RITA® bioreactors (Debnath, 2010). For that case, adventi-tious shoots multiplication was accomplished in thidiazuron (TDZ)supplemented medium, while shoots elongation was reached ina medium containing 6-benzyladenine (BA). Thereafter, shootsrooted in the bioreactor vessel containing the same medium, butwithout any plant growth regulators. At this time, our results sup-port an advanced one-step methodology increasing the in vitrophotosynthetic activity and using a simple TIBs device; which couldbe home-made and designed to reach the highest productive vol-ume in the most efficient manner. Additionally, raspberry shootsrooted in greenhouse in ex vitro conditions.

After 30 days of culture in TIBs, populations of raspberry plantsmultiplied in both TIBs (15 gr/L sucrose; 30 gr/L sucrose) andagar-base (control) treatments were transferred to greenhousefor adaptability (ex vitro rooting). The following variables wererecorded per cultivar: plant size; total number of shoots; totalnumber of internodes, and percent of adaptability to greenhouseconditions (Table 1).

Results show that Bartlett’s variance homogeneity test (P < 0.01)was significant for the studied variables. Raspberry plants multi-plied in TIBs + 15 gr/L sucrose showed the highest values related to

plant size, total number of internodes and percent of adaptabilityto greenhouse, overall variables that could be related to an increaseof the in vitro photosynthetic activity, which might prime plantsfor the ex vitro environment. For both TIBs treatments, the total

A.D. Arencibia et al. / Scientia Horticulturae 160 (2013) 49–53 51

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ig. 1. Photosynthesis-related variables determined in raspberry plants (cv. Herifferences (P < 0.01) determined by Duncan test at 5, 10, 15 and 20 days.

umber of shoots was demonstrated to be higher in compar-son with the agar-base procedure (control), evidencing thenhancement effect of TIBs environment on plant multiplication.n summary, the higher results for traits linked to plant micro-

ropagation yields were demonstrated in raspberries multiplied

n CO2-rich TIBs + 15 gr/L sucrose.Fig. 2 illustrates the overall increase of micropropagation

fficiency using the TIBs in comparison with the conventional

able 1icropropagation traits evaluated during raspberry (Rubus spp.) micropropagation in CO

Genotype In vitro approacha Plant size (cm)b Total num

Heritage Agar – S 30 gr/l 5.78 ± 2.41 15.75 ± 5.6(4.62) (12.77)

TIBs – S 30 gr/l 5.97± 3.14 36.12 ± 9.3(4.01) (21.44)

TIBs – S 15 gr/l 9.21 ± 3.67 38.51 ± 9.4(0.37) (28.52)

Meeker Agar – S 30 gr/l 4.98 ± 2.59 18.22 ± 6.7(3.78) (9.22)

TIBs – S 30 gr/l 6.73 ± 3.21 39.52 ± 12(5.65) (30.45)

TIBs – S 15 gr/l 10.67 ± 5.35 39.54 ± 11(0.42) (27.83)

Amity Agar – S 30 gr/l 5.65 ± 3.12 19.05 ± 8.4(4.45) (10.75)

TIBs – S 30 gr/l 6.41 ± 2.71 32.64 ± 11(5.01) (28.32)

TIBs – S 15 gr/l 12.07 ± 5.54 37.84 ± 10(0.60) (32.74)

Bartlett’s test 6.52* 4.75*

a S: Sucrose; TIBs: Temporary Immersion Bioreactors.b Population mean and standard error (in brackets: variance).* Significant for P < 0.01 in the Bartlett variance homogeneity test.

micropropagated in Temporary Immersion Bioreactors (TIBs). (a–d) Significant

agar-based methodology (2A; B). In parallel, highest raspberrybiomass was produced in TIBs supplemented with sucrose-reduced medium (2C); additionally the percent of adaptability wasimproved in greenhouse conditions (2D).

Both the enhancement of the plant–air contact in a CO2enrichment atmosphere and placement under a light intensity of110 mM m−2 s−1 were significant factors to improve the pheno-lic metabolites secretion during sugarcane multiplication in TIBs

2-rich TIBs and agar-based procedures (control treatment).

ber of shootsb Total number of internodesb Adaptability (%)b

2 74.65 ± 21.10 71.45 ± 11.76(38.43) (35.76)

4 186.40 ± 32.97 78.32 ± 13.43(85.01) (10.54)

6 345.52 ± 40.35 98.45 ± 1.38(124.85) (7.31)

4 84.32 ± 28.43 74.04 ± 8.42(42.67) (42.56)

.78 198.43 ± 41.55 83.23 ± 15.98(96.02) (23.78)

.82 360.21 ± 61.64 97.12 ± 2.06(142.84) (16.51)

5 74.65 ± 21.10 75.12 ± 9.74(35.75) (40.65)

.01 186.40 ± 32.97 80.92 ± 10.62(90.45) (19.43)

.42 345.52 ± 40.35 96.38 ± 2.48(140.55) (11.83)5.62* 6.34*

52 A.D. Arencibia et al. / Scientia Horticulturae 160 (2013) 49–53

F tors (TH erriess use.

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ig. 2. Micropropagation of raspberry (Rubus spp.) in Temporary Immersion Bioreaceritage) of 15 days old growing in agar-base medium (control) and TIBs. (C) Raspb

ucrose (R). (D) Raspberries (cv. Heritage) after 3 weeks of transplanted to greenho

Arencibia et al., 2008, 2012). The differential expression of Rubiscoranscripts in increased CO2 concentration in parallel with a reduc-ion of sucrose in the culture medium indicated the change from

heterotrophic to a photomixotrophic metabolic stage in vitro-lantlets micropropagated in TIBs (Yang et al., 2010).

Cultures in two-vessels TIBs under a controlled environmentith CO2-enrichment, higher luminosity, and sucrose-reducededium should be considered as a novel advance for planticropropagation priming the further acclimatization/rooting step

Conrath et al., 2006; Arencibia et al., 2011). Data collected for threeommercial cultivars confirmed the TIBs plasticity as an integrativeool toward raspberry high-scale production. Moreover, TIBs tech-ology proves to be a useful tool to manage major environmental

actors toward an improvement of in vitro plant processes, in thisase the photosynthesis.

Considering photosynthesis as a complex process involving aange of environmental factors determining carbon assimilation,hese results could be a starting point for further research in large-cale raspberry and other bush berry micropropagation in specificocalities. CO2 concentration of 550 ppmv could be considered asptimal; however, achievement of higher luminosity in the TIBsorking-stations in an efficient and economical way should be

ased on the exploitation of the natural sunlight.

cknowledgments

To Anne Bliss PhD (University of Colorado, USA) and Patrick Mat-ler for the language revision and copyediting of the manuscript.o the Regional Government of Maule (Chile) for financial support.

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