Scientia Horticulturae 160 (2013) 4953

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Scientia Horticulturae
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Short communication

Establishment of photomixotrophic cultures for raspberry

micropropagation in Temporary Immersion Bioreactors (TIBs)
Ariel D Arencibia , Carolina Vergara, Karla Quiroz, Basilio Carrasco,
Rolando Garca-Gonzales
Faculty of Agricultural and Forestry Sciences, Catholic University of Maule, Ave San Miguel 3605, Talca, Chile

a r t i c l e

i n f o

Article history:
Received 22 January 2013
Received in revised form 7 May 2013
Accepted 14 May 2013
Keywords:
Temporary immersion bioreactors
Photomixotrophic
Sucrose-reduced medium

a b s t r a c t
A novel protocol for raspberry (Rubus spp.) micropropagation in photomixotrophic conditions was optimized for the commercial genotypes Heritage, Meeker and Amity. Plant cultures were established in
Temporary Immersion Bioreactors (TIBs) under a controlled environment: 550 ppmv CO2 ; light intensity
80 M m2 s1 ; sucrose concentrations 15 gr/L and 30 gr/L. Results showed that both CO2 ux and chlorophyll uorescence were increased in plants cultured in TIBs with sucrose-reduced medium (15 gr/L) in
comparison with those plants micropropagated in TIBs + 30 gr/L sucrose, conrming a consistent photomixotrophic stage since the 5th day of culture. Raspberry plants multiplied in TIBs + 15 gr/L sucrose
demonstrated the highest values of plant size, total number of internodes, and percent of acclimatization
to greenhouse conditions. Studied variables could be related to an increase of the in vitro photosynthetic
activity which might prime plants for the ex vitro environment.
2013 Elsevier B.V. All rights reserved.

1. Introduction
The genus Rubus belongs to the family Rosaceae and contains cultivated raspberries, blackberries, hybrid berries, and others
species (Jennings, 1988). Raspberry fruits have been considered
nutraceuticals containing vitamins, bers, and phytochemical compounds that function as antioxidants and antimicrobial protective,
among other health-related properties (Poiana et al., 2010). The
wide diversity of Rubus species provides a potential source of novel
traits whereas breeding programs are actively working on releasing cultivars with excellent quality, high yields, greater adaptation
to adverse environmental conditions, and increased pest and disease resistance (Castillo et al., 2010). Rubus species and cultivars are
clonally propagated and maintained in greenhouses, screenhouses,
eld collections, and as tissue-cultured plants and cryopreserved
shoot tips (Wang et al., 2005; Gupta and Reed, 2006).
Clonal multiplication based on plant tissue culture is preferred
for the production of pathogen-free and genetic-delity commercial plants (Arencibia et al., 2011). Raspberries are susceptible to
numerous virus diseases, and sensitive cultivars may be killed or
severely weakened by virus infection; latent infections on tolerant
cultivars may shorten the planting life of a eld through reduced
yield 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).
0304-4238/$ see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.scienta.2013.05.010

bushy dwarf idaeovirus (RBDV), tobacco streak ilarvirus (TSV) and

tomato ringspot nepovirus (TomRSV) have been determined as
5.77%, 2.76% and 0.31%, respectively (Tsao et al., 2000).
The regeneration of adventitious shoots from different types of
explants has been reported for some Rubus species. Genotype, type
of explants, source, and balance of plant growth regulators (PGRs)
constitute the main factors inuencing regeneration (Fiola et al.,
1990; Cousineau and Donnelly, 1991; Turk et al., 1994; Mezzetti
et al., 1997). Moreover, conventional (agar-based) micropropagation 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 commercial raspberry cultivars.
For plant propagation biofactories, liquid cultures and automation has the potential to resolve the manual handling of the various
stages reducing the high production costs that render conventional
systems (agar-based) less suitable (Paek et al., 2005; Arencibia
et al., 2011). In this way, a two-step protocol for in vitro multiplication 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 efcient micropropagation procedure, this paper reports the establishment of photomixotrophic
raspberry cultures in a two-separate-vessel design of Temporary
Immersion Bioreactors (TIBs). There are several types of TIBs where
choice/design depends on the sensitivity of plant materials to
hyperhydricity and the costs for scaling up (Ziv, 1999, 2000; Kozai,
2006). In this study, two-vessel bioreactors have been standardized

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A.D. Arencibia et al. / Scientia Horticulturae 160 (2013) 4953

for photomixotrophic raspberry micropropagation, corroborating

that this approach could be suitable for large-scale productive process.
2. Materials and methods

(height), and in vitro shooting. The adaptability rate was determined after 30 days of planting (acclimatization). Data were
analyzed by ln(x). Variances were calculated for the micropropagation traits related to productivity and were compared between
treatments using Bartletts homogeneity of variances test STATISTICA V.6 software package (STATSOFT, Inc, 2003).

2.1. Plant stocks

3. Results and discussion
Shoot tips (5 cm) of raspberry plantlets of the genotypes
Heritage, Meeker and Amity were surface disinfected in a 10%
commercial solution NaOCl plus 0.1% Tween 20 (15 min). Explants
were immersed in 70% ethanol (5 min.) followed by three rinses
in sterile distilled water. Shoot tips were cultured for 8 weeks in
medium containing MS salts plus vitamins (Murashige and Skoog,
1962), 1 mg/L BAP, 0.3 mg/L IBA, 30 gr/L sucrose. Culture medium
was solidied with 8 gr/L agar and the pH was adjusted to 5.2
before autoclaving at 121 C. Plant cultures were maintained at
23 2 C, for a 16-h photoperiod under a combination of both
natural light/cool-white uorescent tubes at a light intensity of
50 M m2 s1 .
2.2. Temporary Immersion Bioreactors (TIBs)
Two-vessel bioreactors were set up following established
designs (Lorenzo et al., 2001; Yabor et al., 2007; Arencibia et al.,
2008, 2012). For each raspberry cultivar, a total of 10 internodes from vigorous plants of 20 days old (after subculture) were
transferred to a sterile transparent bottle (500 mL capacity). The
bioreactors contained 250 mL of liquid medium (as described above
but without sucrose). Experimental treatments for carbon (C)
source were: TIBs containing liquid medium plus 30 gr/L sucrose;
TIBs with liquid medium plus 15 gr/L sucrose. The immersion frequency was 3 min each 8 h. Air quality in the TIBs working station
was improved with 550 ppmv CO2 , and bioreactors were maintained during 30 days at 23 2 C under a combination of both
natural light and cool-white uorescent tubes at a light intensity
of 80 M m2 s1 . For each genotype, ve vessels (conventional
agar-cultures) subcultured with 10 internodes were the control
treatments. Each TIBs treatment included ve separate replicas. The
experiments were repeated at three different dates.
2.3. Photosynthesis research
Both CO2 ux and chlorophyll uorescence were measured
using the CIRAS-2 with a Chlorophyll Fluorescence Module (CFM).
Five leaves per treatment were randomLy selected at 0; 5; 10; 15;
20 days of TIBs cultures. The controls treatments for each cultivar
included: in vitro plants multiplying in agar-medium plus 30 gr/L
sucrose; ex vitro plants growing in greenhouses.
2.4. Acclimatization to greenhouse (ex vitro rooting)
Populations of raspberry plants multiplied by both TIBs and
agar-base (control) treatments were carefully separated, washed in
1 L of water, and planted in 128-cell plug trays (cell volume 25 cm3 )
containing a mixture of composted pine bark and zeolite (2:1).
Treatments/replicas were randomLy distributed in the greenhouse,
and the relative humidity was gradually reduced: 90% 80% 70%
in 10 day intervals. Luminosity was 100 M m2 s1 under the
natural photoperiod of JanuaryFebruary, latitude 35 30 altitude
71 30 , locality of Talca, Chile.
2.5. Data collection and statistical analysis
The following variables were recorded after the micropropagation cycle (30 days): total number of internodes, plants size

A procedure for raspberry micropropagation in programmed

bioreactors (TIBs based on two separate bottles) was developed
for the commercial genotypes Heritage, Meeker and Amity. Raspberry plants grew and multiplied in a sucrose-reduced culture
medium, with CO2 -rich and high luminosity environment. During
the experiment, variables of CO2 ux and chlorophyll uorescence were increased in plants cultured in TIBs + 15 gr/L sucrose
in comparison with those plants micropropagated in TIBs + 30 gr/L
sucrose (Fig. 1). Raspberries cultured in TIBs with sucrose reduce
medium (15 gr/L) demonstrated photosynthesis-related values
closely to plants growing in ex vitro conditions (control of autotrophy). Meanwhile, raspberry vitroplantlets propagated in TIBs with
sucrose-standard medium (30 gr/L) showed photosynthesis values
slightly higher than plants growing in conventional agar-base cultures + 30 gr/L sucrose (control of heterotrophy). Altogether, results
evidenced a consistent photomixotrophic stage in raspberry plants
from TIBs + 15 gr/L sucrose since the 5th day of culture. In parallel, the experiments corroborated the effects of TIBs (frequent air
exchange) enhancing the photosynthetic pathway.
For the rst time, photomixotrophic in vitro cultures of Rubus
spp. have been established by applying the TIBs technology. A
related paper from Debnath (2005) studied the effect of carbon
source (glucose, sorbitol, or sucrose) and concentrations using
shoots cultivated in vitro from nodal explants of lingonberry (Vaccinium 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-based
cultures without CO2 enrichment, concluding that carbohydrate
concentration had little effect on shoot vigor in a genotypedependent manner.
Previously, a system for in vitro multiplication of raspberry cultivars Festival, Heritage, and Latham has been reported using
the RITA bioreactors (Debnath, 2010). For that case, adventitious shoots multiplication was accomplished in thidiazuron (TDZ)
supplemented medium, while shoots elongation was reached in
a medium containing 6-benzyladenine (BA). Thereafter, shoots
rooted in the bioreactor vessel containing the same medium, but
without any plant growth regulators. At this time, our results support an advanced one-step methodology increasing the in vitro
photosynthetic activity and using a simple TIBs device; which could
be home-made and designed to reach the highest productive volume in the most efcient manner. Additionally, raspberry shoots
rooted in greenhouse in ex vitro conditions.
After 30 days of culture in TIBs, populations of raspberry plants
multiplied in both TIBs (15 gr/L sucrose; 30 gr/L sucrose) and
agar-base (control) treatments were transferred to greenhouse
for adaptability (ex vitro rooting). The following variables were
recorded per cultivar: plant size; total number of shoots; total
number of internodes, and percent of adaptability to greenhouse
conditions (Table 1).
Results show that Bartletts variance homogeneity test (P < 0.01)
was signicant for the studied variables. Raspberry plants multiplied in TIBs + 15 gr/L sucrose showed the highest values related to
plant size, total number of internodes and percent of adaptability
to greenhouse, overall variables that could be related to an increase
of the in vitro photosynthetic activity, which might prime plants
for the ex vitro environment. For both TIBs treatments, the total

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51

Fig. 1. Photosynthesis-related variables determined in raspberry plants (cv. Heritage) micropropagated in Temporary Immersion Bioreactors (TIBs). (ad) Signicant
differences (P < 0.01) determined by Duncan test at 5, 10, 15 and 20 days.

number of shoots was demonstrated to be higher in comparison with the agar-base procedure (control), evidencing the
enhancement effect of TIBs environment on plant multiplication.
In summary, the higher results for traits linked to plant micropropagation yields were demonstrated in raspberries multiplied
in CO2 -rich TIBs + 15 gr/L sucrose.
Fig. 2 illustrates the overall increase of micropropagation
efciency using the TIBs in comparison with the conventional

agar-based methodology (2A; B). In parallel, highest raspberry

biomass was produced in TIBs supplemented with sucrosereduced medium (2C); additionally the percent of adaptability was
improved in greenhouse conditions (2D).
Both the enhancement of the plantair contact in a CO2
enrichment atmosphere and placement under a light intensity of
110 mM m2 s1 were signicant factors to improve the phenolic metabolites secretion during sugarcane multiplication in TIBs

Table 1
Micropropagation traits evaluated during raspberry (Rubus spp.) micropropagation in CO2 -rich TIBs and agar-based procedures (control treatment).
Genotype

In vitro approacha

Plant size (cm)b

Total number of shootsb

Total number of internodesb

Adaptability (%)b

Heritage

Agar S 30 gr/l

5.78 2.41
(4.62)
5.97 3.14
(4.01)
9.21 3.67
(0.37)
4.98 2.59
(3.78)
6.73 3.21
(5.65)
10.67 5.35
(0.42)
5.65 3.12
(4.45)
6.41 2.71
(5.01)
12.07 5.54
(0.60)
6.52*

15.75 5.62
(12.77)
36.12 9.34
(21.44)
38.51 9.46
(28.52)
18.22 6.74
(9.22)
39.52 12.78
(30.45)
39.54 11.82
(27.83)
19.05 8.45
(10.75)
32.64 11.01
(28.32)
37.84 10.42
(32.74)
4.75*

74.65 21.10
(38.43)
186.40 32.97
(85.01)
345.52 40.35
(124.85)
84.32 28.43
(42.67)
198.43 41.55
(96.02)
360.21 61.64
(142.84)
74.65 21.10
(35.75)
186.40 32.97
(90.45)
345.52 40.35
(140.55)
5.62*

71.45 11.76
(35.76)
78.32 13.43
(10.54)
98.45 1.38
(7.31)
74.04 8.42
(42.56)
83.23 15.98
(23.78)
97.12 2.06
(16.51)
75.12 9.74
(40.65)
80.92 10.62
(19.43)
96.38 2.48
(11.83)
6.34*

TIBs S 30 gr/l
TIBs S 15 gr/l
Meeker

Agar S 30 gr/l
TIBs S 30 gr/l
TIBs S 15 gr/l

Amity

Agar S 30 gr/l
TIBs S 30 gr/l
TIBs S 15 gr/l

Bartletts test
a
b
*

S: Sucrose; TIBs: Temporary Immersion Bioreactors.

Population mean and standard error (in brackets: variance).
Signicant for P < 0.01 in the Bartlett variance homogeneity test.

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A.D. Arencibia et al. / Scientia Horticulturae 160 (2013) 4953

Fig. 2. Micropropagation of raspberry (Rubus spp.) in Temporary Immersion Bioreactors (TIBs) under 550 ppmv CO2 and light intensity 80 M m2 s1 . (A and B) Plantlets (cv.
Heritage) of 15 days old growing in agar-base medium (control) and TIBs. (C) Raspberries (cv. Amity) of 25 days old multiplying in TIBs + 15 gr/L sucrose (L) and TIBs + 30 gr/L
sucrose (R). (D) Raspberries (cv. Heritage) after 3 weeks of transplanted to greenhouse.

(Arencibia et al., 2008, 2012). The differential expression of Rubisco

transcripts in increased CO2 concentration in parallel with a reduction of sucrose in the culture medium indicated the change from
a heterotrophic to a photomixotrophic metabolic stage in vitroplantlets micropropagated in TIBs (Yang et al., 2010).
Cultures in two-vessels TIBs under a controlled environment
with CO2 -enrichment, higher luminosity, and sucrose-reduced
medium should be considered as a novel advance for plant
micropropagation priming the further acclimatization/rooting step
(Conrath et al., 2006; Arencibia et al., 2011). Data collected for three
commercial cultivars conrmed the TIBs plasticity as an integrative
tool toward raspberry high-scale production. Moreover, TIBs technology proves to be a useful tool to manage major environmental
factors toward an improvement of in vitro plant processes, in this
case the photosynthesis.
Considering photosynthesis as a complex process involving a
range of environmental factors determining carbon assimilation,
these results could be a starting point for further research in largescale raspberry and other bush berry micropropagation in specic
localities. CO2 concentration of 550 ppmv could be considered as
optimal; however, achievement of higher luminosity in the TIBs
working-stations in an efcient and economical way should be
based on the exploitation of the natural sunlight.

Acknowledgments
To Anne Bliss PhD (University of Colorado, USA) and Patrick Matzler for the language revision and copyediting of the manuscript.
To the Regional Government of Maule (Chile) for nancial support.

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