See

discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/262150628

MASS PROPAGATION OF Rosa hybrid CV.

BLACK BACCARA BY PERIODICAL BIOREACTOR

Article January 2013

CITATION READS

1 100

2 authors, including:

Mina Bayanati
Ferdowsi University Of Mashhad
5 PUBLICATIONS 4 CITATIONS

SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Study on the expression and function of two longevity-associated genes in Rosa spp View project

All content following this page was uploaded by Mina Bayanati on 09 May 2014.

The user has requested enhancement of the downloaded file. All in-text references underlined in blue are added to the original document
and are linked to publications on ResearchGate, letting you access and read them immediately.
International Journal of Agriculture: Research and Review. Vol., 3 (2), 241-245, 2013
Available online at http://www.ecisi.com
ISSN 2228-7973 2013 ECISI Journals

MASS PROPAGATION OF Rosa hybrid CV. BLACK BACCARA BY

PERIODICAL BIOREACTOR

MINA BAYANATI1, SEYED NAJMEDIN MORTAZAVI2

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

Roses are the most economically important
flowers in the world. There are than 20000
commercial cultivars, which are collectively
based on only 8 of the 200 wild species in the
genus rosa (Roberts and Smith, 1990).
Traditionally, most ornamental roses are
heterozygous and do not breed true to type,
They are therefore propagated vegetative.
Miniature roses are usually propagated by
cutting but the roses are usually propagated by
budding or bench-grafting onto rootstocks of
species such as Rosa caninaInermis and Rosa
multifloraSimplex(Khosravi et al.,
2007).Tissue culture on the other band in
becoming increasingly popular as an alternative
means of plant vegetative propagation.
Automation of via organogenesis or somatic
embryogenesis in a bioreactor has been
(Paek et al., 2005).Bioreactors are usually
described in a biochemical context as self-
contained, sterile environments which capitalize
on liquid nutrient or liquid/air inflow and
outflow systems, designed for monitoring and
control over conditions (agitation, aeration,
dissolved oxygen, pH, etc.) (Ziv, 2000).
Conventional plants are mainly in solid and
semi-solid culture mediums that takes time and
cost. In this regard, it is recommended to use
moving fluid culture. Movements cause to get
oxygen and nutrition to all tissues and make
grow faster (Etinne and Berthouly, 2002).The
same technology can be readily adapted for the
growth of regenerable cells and tissues in liquid
media in bioreactors, as demonstrated by the
successful mass propagation of shoots in a 500
litre bioreactor by Akita et al (1994). They
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-
foldincrease. Similarly, a 10 litre jar fermenter
was used for the production of potato tubers,
which could be used directly as seed tubers. The
use of bioreactor for micropropagation was first
reported in 1981 for Begonia propagation
(Takayama and Akita, 1994) and after that time
there are a lot of reports about propagation plats
by Bioreactor (Paek and Chakrabarty, 2005;
Sajcetal, 2000). There are some reports about
asexual embryo production and produce
secondary metabolites in Bioreactors
(Takayama and Akita, 1994; Ziv et al.,
1994).Bioreactors has been used to propagate
potato, gladiolus, lilies, strawberry, Hyacinth,
Amaryllis and severa Araceae.etc. (Ziv,
2000).Three main classes of culture system in
bioreactors can be distinguished: those
production biomass, those production
metabolites and enzymes and those used for
biotransformation of exogenously added
metabolites (Paek et al., 2005). Bioreactors are
presently being used for commercial
micropropagation in the USA, Japan, Taiwan,
Korea, Cuba, Costa Rica, Holland, Spain,
Belgium, and France for ornamental and
bulbous plants, pineapple, potato, and forest
trees. The cost per propagate unitof foliage
plants was estimated to be reduced from 17 to
67 cents (US) (Ziv, 2000). Micropropagation
in bioreactors for optimal plant production will
depend on a better understanding of plant
responses to signals from the microenvironment
and on specific culture manipulation to control
the morphogenesis of plants in liquid cultures.
This study was conducted to investigate the
potential for mass propagation of Rosa hybrid
cv. Black baccara.
MATERIAL AND METHODES
Plant material and general procedures
Nodal segments (1-1.5 cm) were taken from
the stems of Black baccara plants in the rose
garden of the Agricultural Biotechnology
Research Institute of Iran (ABRII). They were
washed thoroughly with running tap water for
half an hour and surface sterilized for 30
seconds in 70% (v/v) ethanol, followed by a 15
min soak in 2.5% (v/v) sodium hypochlorite
solution with a few drops of Tween-20 as a
wetting agent, and then rinsed three times with
sterile distilled water. MS (Murashige and
Skoog, 1962) basal medium (without hormone)
was used for the in vitro of induction of explants
in culture; the pH of the medium was adjusted
to 5.8 with 1 N NaOH or 1 N HCl before adding
8 gl-1 plant agar. Media were autoclaved for 15
min at 121C and 1.2 kPa pressure. Cultures
were placed under high pressure metal halide
lamps on a 16/8 hour light/dark cycle in a
culture room main-tained at 21 1C. Axillary
shoots were detached and transferred to VS
medium (Van der Salm et al., 1994) in which
FeNaEDTA was replaced by FeEDDHA as iron
source after 14 days. These culture were used as
plant material for shoot multiplication
experiment.
Shoot multiplication in routine system and
bioreactor cultures
Nodal segments (3 per culture vessel) of
about 2 cm in length were cultured in 250 ml
jars containing 30 ml VS medium supplemented
with 30g/l sucrose, 8 g/l agar and 2 MBAP.
The pH of the medium was adjusted to 5.8with
NaOH before autoclaving at 121C1.2 kPa
pressure for 20 min. This experiment conducted
in completely randomized design with three
replications. Integrate and Separate mini-
Bioreactor system using 1 lwere used for further
investigate shoot multiplication of Rosa hybrid
cv. Blackbaccara. 7-10 nodal segmentsabout 2
cm in length were transferred into each
bioreactor containing a working volume of 500
ml VS medium supplemented with 30g/l
sucrose and 2 M BAPthe pH of the medium
was adjusted to 5.8 with NaOH before
autoclaving at 121C1.2 kPa pressure for 20
min. The nutrition cycle was set at 10 min every
24h. Bioreactors and 250 ml jars maintained
under diffuse white light of 55mol m-2/s, with
an 18 h photoperiod at 21C for the culture
period of 8 weeks(Figure 1).
Experimental design
Experiments were set up in a completely
randomized design. Each treatment had three
replication. Several growth parameters were
assessed including shoot length, shoot number,
fresh weight and dry weight. Dry weight was
recorded after drying the material for 48 h at
70C. Data were subjected to ANOVA and
Duncan,s Multiple Rang Test using the
MSTAT-C program.
RESULT AND DISCUSSION
The bioreactor studies revealed that shoot
multiplication was highest (4.94 shoots) in
Integratedand (5.61) Separate (Table 1 and
Figure 2). Highest average number of branches
gets in Bioreactor (Table 2).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.
242
 Intl. J. Agric: Res & Rev. Vol., 3 (2), 241-245, 2013

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).Results showed that mean shoot
length, number if shoot and dry weight per
explant significantly different (P < 0.001) and
highest length seen in Bio-reactor. Plants
produced in Bioreactor had better grown (2.39
and 0.15 g for fresh and dry weight,
respectively) than plants which grown in 250 ml
jars (0.12 and 0.01 g for fresh and dry weight,
respectively).

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

243
Intl. J. Agric: Res & Rev. Vol., 3 (2), 241-245, 2013

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

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)

REFERENCES GA3, sucrose and soil medium/bioreactor

culture on in vitro flowering of
Akita M, Shigeoka T, Koizumi Y, Kawamura M
Spathiphyllum and association of
(1994) Mass propagation of shoots of
glutathione metabolism. Plant Cell,
Stevia rebaudiana using a large scale
Tissue and Organ Culture. 90: 225-235.
bioreactor. Plant Cell Rep. 13: 180-183,
Etienne H, BerthoulyM (2002) Temporary
1994.
immersion systems in plant
Akita M, YO hata (1998) a simple method for
micropropagation. Plant Cell, Tissue and
mass propagation of potato (Solanum
Organ Culture 69: 215231.
tuberosum L.) using a bioreactor without
Fulzele DP, Heble MR, Rao PS (1995)
forced aeration. Plant Cell Rep. 18: 284-
Production of terpenoid from Artemisia
287.
annua L. plantlet cultures in bioreactor.
Dewir YH, Chakrabarty D, Ali MB, Hahn EJ,
Journal Biotechnology.40:43-139.
Paek KY (2006) Liquid peroxidant and
Khosravi P, Jafarkhani Kermani M,
antioxidant enzyme activities of
Nematzadeh G, Bihamta M (2007)
Euphorbia milliihy perhydric shoots.
Aporotocol for mass production of Rosa
Enviromental and Experimental
hybrid cv. Iceberg through in vitro
Botany.58:93-99.
propagation. Iranian Journal of
Dewir YH, Chakrabarty D, Ali MB, Singh N,
Biotechnology, Vol. 5.2: 100- 105.
Hahn E, Paek K (2007) Influence of

244
 Intl. J. Agric: Res & Rev. Vol., 3 (2), 241-245, 2013
Lian ML, Chakrabarty D and Peak KL (2003)
Bulblet Formation from bulbscale
segments of Lilium oriental Hybrid
Casablanca using bioreactor system.
Journal Biotchnology.Plant. 46:199-203.
Lorenzo JC, Ojeda A, Espinosa, Carlosborrot O
(2001) Field performance of temporary
immersion bioreactor-derived sugarcane
plants. In Vitro Cell. Dev.
Biotechnology. Plant 37:803-806.
Murashige T, Skoog F. A (1962) revised
medium for rapid growth and bioassays
with tobacco tissue cultures. Journal
Physiology Plant.15:97-473.
Paek KY, Chakrabarty D, EJ Hahn (2005)
Application of bioreactor systems for
large scale production of horticultural
and medicinal plant. Plant Cell, Tissue
and Organ Culture 81: 287-300.
Sajc L,Grubisic, D, Novakovic GV (2000)
Bioreactors for plant engineering: An out
for further research. Biochem. Eng. J. 4:
8999.
SreedharR,Venkatachalam V, Thimmaraju L,
Bhagyalakshmi R, Narayan N,
Ravishankar MS(2008) Direct
organogenesis from leaf explants of
Steviarebaudiana and cultivation in
bioreactor, Biologialogy Plantarum, 52.
(2): 355-360.
245
View publication stats
Takayama S, Akita M (1994) The type of
bioreactor used for shoots and
embryos Plant Cell, Tissue and
Organ Culture. 39: 147-157.
Van der Salm TPM. Van der Toorn CJG,
Hanischten Catech (1994) Improtannce
of the iron chelate formula for
micropropagation of Rosa hybrid L.
Moneyway, Plant Cell Tissue Organ
Culture; 37:73-77.
Wilken D, Gonzales E J, Hohe A, Jordan M,
Kosky RG, Hirschmann G, Gerth A(
2005) Comparison of secondary plant
metabolite production in cell suspension,
callus culture and temporary immersion
system. Liquid Culture Systems for in
vitro Plant Propagation, A.K. Hvoslef-
Eide and W. Preil (eds.). Chapter 39,
Springer, 525-537.
Ziv M (2000) Bioreactor Technology for Plant
Micropropagation. Horticultural
Reviews,Vol 24, Edited by Jules Janick;
Lohn Wiley & Sons, Inc.
Ziv M, Kahavy S, and Lilien-Kipnis H (1994)
Scale-up proliferation and regeneration of
Nerin in liquid cultures. Part I, The
induction and maintenance of
proliferating meristematic cultures by
paclobutrazul in bioreactors. Plant Cell,
Tissue and Organ Culture. 39: 109-117.