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Article January 2013


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Mina Bayanati
Ferdowsi University Of Mashhad


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International Journal of Agriculture: Research and Review. Vol., 3 (2), 241-245, 2013
Available online at
ISSN 2228-7973 2013 ECISI Journals




1- Graduate Student, Faculty Agriculture, University of Zanjan.

2- Assistant Professor, Faculty Agriculture, University of Zanjan.

*Corresponding Author: MINA BAYANATI

ABSTRACT: Roses have been one of the worlds most popular ornamental plants for a long
time. Recently, in vitro flower induction in roses was demonstrated. To reduce the intensive
labour requirement along with the production cost during plant propagation by tissue culture
technique, there is an immense need of developing scale-up system and automation. Progress
in tissue culture automation will depend upon the use of liquid culture in bioreactors. This
system usually involves a wetting and drying cycle which occur periodically in a given period
of time. Present investigation was carried out to determine the efficiency of in vitro
propagation of Rosa hybrida cv. Black baccara with tow type periodical bioreactor. For mass
propagation in bioreactors, 7-10 nodal segments were transferred into bioreactors containing
VS liquid medium supplemented with 3% sucrose and 2 M 6-Benzylaminopurine (BAP).
The nutrition cycle was set at 10 min every 24h. The results showed that the semi- continuous
system is more benefit than routine system (250ml jars) for shoot number, shoot length and
fresh/dry weight. Comparative studies between two types periodical bioreactor indicated that
there was not significant difference between the performances of two types of bioreactors.
Plants produced in Bioreactor had better grown (3.39 and 0.57 g for fresh and dry weight,
respectively) than plants which grown in 250 ml jars (0.56 and 0.02 g for fresh and dry
weight, respectively). Also these kinds of bioreactor could be used for mass propagation with
reduced cost, energy and handle work.

Key words: In vitro, Liquid media, Periodical Bioreactor, Rose, Shoot multiplication.

INTRIDUCTION advanced as a possible way of reducing costs

(Paek et al., 2005).Bioreactors are usually
Roses are the most economically important described in a biochemical context as self-
flowers in the world. There are than 20000 contained, sterile environments which capitalize
commercial cultivars, which are collectively on liquid nutrient or liquid/air inflow and
based on only 8 of the 200 wild species in the outflow systems, designed for monitoring and
genus rosa (Roberts and Smith, 1990). control over conditions (agitation, aeration,
Traditionally, most ornamental roses are dissolved oxygen, pH, etc.) (Ziv, 2000).
heterozygous and do not breed true to type, Conventional plants are mainly in solid and
They are therefore propagated vegetative. semi-solid culture mediums that takes time and
Miniature roses are usually propagated by cost. In this regard, it is recommended to use
cutting but the roses are usually propagated by moving fluid culture. Movements cause to get
budding or bench-grafting onto rootstocks of oxygen and nutrition to all tissues and make
species such as Rosa caninaInermis and Rosa grow faster (Etinne and Berthouly, 2002).The
multifloraSimplex(Khosravi et al., same technology can be readily adapted for the
2007).Tissue culture on the other band in growth of regenerable cells and tissues in liquid
becoming increasingly popular as an alternative media in bioreactors, as demonstrated by the
means of plant vegetative propagation. successful mass propagation of shoots in a 500
Automation of via organogenesis or somatic litre bioreactor by Akita et al (1994). They
embryogenesis in a bioreactor has been harvested64.6 kg of shoots of Stevia rebaudiana
Intl. J. Agric: Res & Rev. Vol., 3 (2), 241-245, 2013

from an initial inoculum of 460 g, 140- min at 121C and 1.2 kPa pressure. Cultures
foldincrease. Similarly, a 10 litre jar fermenter were placed under high pressure metal halide
was used for the production of potato tubers, lamps on a 16/8 hour light/dark cycle in a
which could be used directly as seed tubers. The culture room main-tained at 21 1C. Axillary
use of bioreactor for micropropagation was first shoots were detached and transferred to VS
reported in 1981 for Begonia propagation medium (Van der Salm et al., 1994) in which
(Takayama and Akita, 1994) and after that time FeNaEDTA was replaced by FeEDDHA as iron
there are a lot of reports about propagation plats source after 14 days. These culture were used as
by Bioreactor (Paek and Chakrabarty, 2005; plant material for shoot multiplication
Sajcetal, 2000). There are some reports about experiment.
asexual embryo production and produce
secondary metabolites in Bioreactors Shoot multiplication in routine system and
(Takayama and Akita, 1994; Ziv et al., bioreactor cultures
1994).Bioreactors has been used to propagate Nodal segments (3 per culture vessel) of
potato, gladiolus, lilies, strawberry, Hyacinth, about 2 cm in length were cultured in 250 ml
Amaryllis and severa Araceae.etc. (Ziv, jars containing 30 ml VS medium supplemented
2000).Three main classes of culture system in with 30g/l sucrose, 8 g/l agar and 2 MBAP.
bioreactors can be distinguished: those The pH of the medium was adjusted to 5.8with
production biomass, those production NaOH before autoclaving at 121C1.2 kPa
metabolites and enzymes and those used for pressure for 20 min. This experiment conducted
biotransformation of exogenously added in completely randomized design with three
metabolites (Paek et al., 2005). Bioreactors are replications. Integrate and Separate mini-
presently being used for commercial Bioreactor system using 1 lwere used for further
micropropagation in the USA, Japan, Taiwan, investigate shoot multiplication of Rosa hybrid
Korea, Cuba, Costa Rica, Holland, Spain, cv. Blackbaccara. 7-10 nodal segmentsabout 2
Belgium, and France for ornamental and cm in length were transferred into each
bulbous plants, pineapple, potato, and forest bioreactor containing a working volume of 500
trees. The cost per propagate unitof foliage ml VS medium supplemented with 30g/l
plants was estimated to be reduced from 17 to sucrose and 2 M BAPthe pH of the medium
67 cents (US) (Ziv, 2000). Micropropagation was adjusted to 5.8 with NaOH before
in bioreactors for optimal plant production will autoclaving at 121C1.2 kPa pressure for 20
depend on a better understanding of plant min. The nutrition cycle was set at 10 min every
responses to signals from the microenvironment 24h. Bioreactors and 250 ml jars maintained
and on specific culture manipulation to control under diffuse white light of 55mol m-2/s, with
the morphogenesis of plants in liquid cultures. an 18 h photoperiod at 21C for the culture
This study was conducted to investigate the period of 8 weeks(Figure 1).
potential for mass propagation of Rosa hybrid
cv. Black baccara. Experimental design
Experiments were set up in a completely
MATERIAL AND METHODES randomized design. Each treatment had three
replication. Several growth parameters were
Plant material and general procedures assessed including shoot length, shoot number,
Nodal segments (1-1.5 cm) were taken from fresh weight and dry weight. Dry weight was
the stems of Black baccara plants in the rose recorded after drying the material for 48 h at
garden of the Agricultural Biotechnology 70C. Data were subjected to ANOVA and
Research Institute of Iran (ABRII). They were Duncan,s Multiple Rang Test using the
washed thoroughly with running tap water for MSTAT-C program.
half an hour and surface sterilized for 30
seconds in 70% (v/v) ethanol, followed by a 15 RESULT AND DISCUSSION
min soak in 2.5% (v/v) sodium hypochlorite
solution with a few drops of Tween-20 as a The bioreactor studies revealed that shoot
wetting agent, and then rinsed three times with multiplication was highest (4.94 shoots) in
sterile distilled water. MS (Murashige and Integratedand (5.61) Separate (Table 1 and
Skoog, 1962) basal medium (without hormone) Figure 2). Highest average number of branches
was used for the in vitro of induction of explants gets in Bioreactor (Table 2).The results showed
in culture; the pH of the medium was adjusted that the semi- continuous system is more benefit
to 5.8 with 1 N NaOH or 1 N HCl before adding than routine system(250ml jars) for shoot
8 gl-1 plant agar. Media were autoclaved for 15 number, shoot length and fresh/dry weight.

Intl. J. Agric: Res & Rev. Vol., 3 (2), 241-245, 2013

Comparative studies between two types length, number if shoot and dry weight per
periodical bioreactor indicated that there was explant significantly different (P < 0.001) and
not significant difference between the highest length seen in Bio-reactor. Plants
performances of two types of bioreactors. Plants produced in Bioreactor had better grown (2.39
produced in Bioreactor had better grown (3.39 and 0.15 g for fresh and dry weight,
and 0.57 g for fresh and dry weight, respectively) than plants which grown in 250 ml
respectively) than plants which grown in 250 ml jars (0.12 and 0.01 g for fresh and dry weight,
jars (0.56 and 0.02 g for fresh and dry weight, respectively).
respectively).Results showed that mean shoot

Table 1.Effect of different culture dishes (Bio-reactor or popular culture dishes)on number of shoots,
shoot height, fresh weight and dry weight.
Treatment df Dry weight per Fresh weight Shoot length per No. of shoot per explant
explant (g) per explant (g) explant (cm)

Culture dishes 2 0.012** 2.23* 1.38** 7.62**

Error 6 0 0.053 0.012 0.15

**,* means significant of 1 and 5 percent of probability, respectively

Table 2.Compare means of on number of shoots, shoot height, fresh weight and dry weight in different
culture dishes.
Culture dishes Fresh weight (g) Dry matter (g) Shoot height (cm) Proliferation

Routine system(250 0.12b 0.01 c 2.47 b 1.74 b

ml jars)

Integrated Bioreactor 2.36ab 0.12 a 3.8 a 4.94 a

Separate Bioreactor 2.39 a 0.15 a 3.6 a 5.61 a

**,* means significant of 1 and 5 percent of probability, respectively

Lorenzo et al. (2001) showed better aeration metabolic activity of the growing cell biomass
and reduce CO2 and C2H4 in medium culture can affecte culture yield (Ziv, 2000). Bulblets of
cause higher bud production and shoot length in Lilium Oriental Hybrid Casablanca grew at a
Banana. Studies showed Bio-reactor can make faster rate when the medium was exchanged
more and heavier Potato tubers than culture in with new medium frequent in a BTBB
semi-solid medium. Akita et al., (1994), while (immersion type) (Lian et al., 2003). Fulzele et
propagating Stevia shoots in a bioreactor, al.,(1995) also reported the biomass of
observed a higher increase in fresh mass of Artemisia annua shoots increased 4 -5-fold in 1-
shoots than was obtained in the present study l capacity bioreactors under submerged
which could probably be due to the use of conditions after30 days. For production of cells,
different source of shoot culture and a different somatic embryos organogenic propagates (e.g.
type of bioreactor. The environment inside bulb lets, corms, nodules, micro-tubers or shoot
bioreactors normally used in plant clusters), bioreactor culture is one of the most
micropropagation is characterized by high promising ways for scaling up the system and
relative humidity, poor gaseous exchenge there have been several reports on large scale
between the internal atmosphere of the propagation of horticultural and medicinal
bioreactor and its surrounding environment, and plants by using bioreactors (Ziv, 2000). The
the accumulation of ethylene, conditions that cultivation of shoots in bioreactor offers several
may induce physiological disorders (Dewir et advantages. Some of them being large number
al., 2006).The available oxygen for plant cells of plantlets can be easily produced, easy
in liquid cultures determined by oxygen transfer handling, stimulation of growth rate by forced
coefficient (kLa values) is the part that dissolves aeration and suppression of apical dominance
in water. Its depletion as afunction of the leading to stimulation of growth of numerous

Intl. J. Agric: Res & Rev. Vol., 3 (2), 241-245, 2013

shoot buds into plantlets (Takayama and Akita, determine the efficiency of in vitro propagation
1994).Shoot cultures of Spathiphyllum of Rosa hybrida cv. Black baccara with tow
cannifolium (Dewir et al., 2007) and Stevia type periodical bioreactor The results showed
rebaudiana (Sreedhar et al., 2008) were carried that Also these kinds of bioreactor could be
out in bubble column bioreactors in order to used for mass propagation with reduced cost,
produce flowers and biomass, respectively. energy and handle work.
Present investigation was carried out to

Figure 1.Lateral bud growth in periodical Bioreactor after 6 week culture.

Figure 2. Compare proliferation of lateral bud for popular culture dish (a) and Bioreactor (b)

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