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Ginova A., Tsvetkov I., Kondakova V., 2012. Rosa damascena Mill.- An overview for evaluation of propagation methods. Bulg. J. Agric. Sci., 18 (4): 545-556

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Rosa damascena Mill. f.Trigintipetala, named kazanlak oil – bearing rose is an emblematic plant species for Bulgaria with important economical sense. its importance is determined by the extremely high quality of rose oil, which is obtained after dis-tillation of rose flowers. The methods of propagation play a general role in maintenance and improving the quality of oil rose. Traditionally, the oil-bearing rose is vegetative propagated. in this review are considered the advantages and disadvantages of classical methods of propagation (by grafting, layering, cutting) and in vitro methods for propagation. special attention is directed to the problems that accompany the use of these methods. The layering and grafting are not preferred for use, because their more time consuming and low multiplication rate. The cutting is the preferable way for oil rose propagation in Bulgaria. This method has high generative rate, but obtained plants are not always healthy. In vitro techniques are used for rapid multi-plication with high rate, production of healthy and disease-free plants, but they are costly.Key words: Rosa damscena Mill., classical methods of propagation, in vitro micro propagation, bioreactorsAbbreviations:Ms - Murashige & skoog Medium, QL- Quirin and Lepoivre Medium, BAP - 6-Benzylaminopu-rine, BA - Benzyl Adenine, nAA - 1-naphthaleneacetic acid, iAA - indole-3-acetic acid, GA3 - Gibberellic acid, iBA - indole-3-butyric acid, PGRs - plant growth regulators, kin - kinetin, TdZ - Thidiazuron, WPM – Woody Plant Medium, PvP –Polyvinylpyrrolidone, PvPP -Polyvinylpolypyrrolidone Ginova A., Tsvetkov I., Kondakova V., 2012. Rosa damascena Mill.- An overview for evaluation of propagation methods. Bulg. J. Agric. Sci., 18 (4): 545-556. Available from: https://www.researchgate.net/publication/256669695_Ginova_A_Tsvetkov_I_Kondakova_V_2012_Rosa_damascena_Mill-_An_overview_for_evaluation_of_propagation_methods_Bulg_J_Agric_Sci_18_4_545-556 [accessed Feb 6, 2017].
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545
ROSA DAMASCENA MILL. - AN OVERVIEW
FOR EVALUATION OF PROPAGATION METHODS
A. GINOVA*, I. TSVETKOV and V. KONDAKOVA
AgroBioInstitute, 8, Dragan Tsankov blvd., BG - 1164 Soa, Bulgaria
Abstract
GINOVA, A., I. TSVETKOV and V. KONDAKOVA, 2012. Rosa damascena Mill. - an overview for evaluation of
propagation methods. Bulg. J. Agric. Sci., 18: 545-556
Rosa damascena Mill. f.Trigintipetala, named Kazanlak oil – bearing rose is an emblematic plant species for Bulgaria with
important economical sense. Its importance is determined by the extremely high quality of rose oil, which is obtained after dis-
tillation of rose owers. The methods of propagation play a general role in maintenance and improving the quality of oil rose.
Traditionally, the oil-bearing rose is vegetative propagated. In this review are considered the advantages and disadvantages
of classical methods of propagation (by grafting, layering, cutting) and in vitro methods for propagation. Special attention is
directed to the problems that accompany the use of these methods. The layering and grafting are not preferred for use, because
their more time consuming and low multiplication rate. The cutting is the preferable way for oil rose propagation in Bulgaria.
This method has high generative rate, but obtained plants are not always healthy. In vitro techniques are used for rapid multi-
plication with high rate, production of healthy and disease-free plants, but they are costly.
Key words: Rosa damscena Mill., classical methods of propagation, in vitro micro propagation, bioreactors
Abbreviations: MS - Murashige & Skoog Medium, QL- Quirin and Lepoivre Medium, BAP - 6-Benzylaminopu-
rine, BA - Benzyl Adenine, NAA - 1-Naphthaleneacetic acid, IAA - Indole-3-acetic acid, GA3 - Gibberellic acid,
IBA - Indole-3-butyric acid, PGRs - plant growth regulators, Kin - Kinetin, TDZ - Thidiazuron, WPM – Woody
Plant Medium, PVP –Polyvinylpyrrolidone, PVPP -Polyvinylpolypyrrolidone
Bulgarian Journal of Agricultural Science, 18 (No 4) 2012, 545-556
Agricultural Academy
E- mail: a.ginova@gmail.com
Introduction
Rosa damascena Mill. is a beautiful aromatic ower
with immense horticultural importance, signication
from economical and research point of view. The Rosa
genus belongs to the family Rosaceae and has over 130
species (Cairns, 2001).
Roses are grouped into classications based on
the botanical characteristics: hybrid teas, grand oras,
polyanthas, oribundas, miniatures, climbing, shrub,
old roses, but only a few species are scented (Gudin,
2000). These include Rosa damascena Mill., R. gallica
Linn., R. centifolia Linn., R. bourboniana Desportes., R.
chinensis Jacq., R. moschata Herrm. and R. alba Linn.
The initially rose species used for oil production
are: Rosa damascena Mill, Rosa gallica Linn., Rosa
centifolia Linn. and Rosa moschata Herm. (Tucker and
Maciarello, 1988). Hurst (1941) classied the today
Damask roses into two types according to their ower-
ing habit: summer Damask that blooms only in early
summer, and autumn Damask that blooms during the
autumn. Traditionally, highly scented summer Damask
roses are cultivated in commercial rose gardens. There
are several main prospects for production of Damask
rose (R.. damascena) as rose water, attar of rose and
other essential oils in the perfume industry (Lavid et
al., 2002). It has application in pharmacology such as
anti-HIV, antibacterial, antioxidant, hypnotic, antidi-
546 A. Ginova, I. Tsvetkov and V. Kondakova
abetic activities, and relaxant effect on acheal chains.
(Boskabady et al., 2011). Rose petals are used for prep-
aration of jams, jellies, conserves, liqueurs. Distillation
waste can be used for livestock feed and composting.
In other countries, Damask rose is also used as an orna-
mental plant in parks and gardens.
The world main producers of rose oil are Bulgaria,
Turkey and Iran and to a less extent France, India, Chi-
na and Northern Africa. As Rosa damascena was origi-
nally introduced from the Middle East into Western Eu-
rope. In Bulgaria, the cultivation of oil – bearing rose
was initiated in the 16th century after the expansion of
the Ottoman Empire (Topalov, 1978). Kashan, Shiraz,
Fars, Meshed and Azerbaijan are the major cultivation
areas in Iran; Isparta, Burdur, Denizli, Afyonkarahisar
in Turkey and the Kazanlak valley in Bulgaria. The soil
and climatic properties of the Kazanlak region such as
temperature, humidity, cloudiness, and precipitation in
the owering season - May and June have important
contribution in obtaining rose oils with high quality in
accordance with the world standards, so, the planting
of rose was sustained there. During the 19th century
the industrial cultivation of R. damascena (called also
‘Kazanlashka roza’: the rose of town of Kazanlak, Bul-
garia) spreads throughout the completely Sub-Balkan
valley from the town of Sliven to the town of Klisura.
Oil rose plantations were also established on the south
slopes of the mountain Sredna Gora Strelcha, Zele-
nikovo and the north slopes of the Rhodopa Mountain
– Bratsigovo, Peshtera. The maximum planted area
has been reached in 1917 (8951 ha) and after that it
gradually decreased. (Kovatcheva et al., 2010). These
days the spreading of oil bearing rose is expanded
further to the west of Klisura to Mirkovo village (Soa
regions).
In Turkey, Isparta province (latitude 37o45’ N, lon-
gitude 30o33’E, altitude 997 m) is accepted as the val-
ley of the oil rose. The climatic conditions of Isparta
proved to be favorable for the cultivation of the rose.
Eighty percent of oil rose production of Turkey is from
Isparta and 20% both from Burdur, Afyonkarahisar and
Denizli districts. Cuttings, by budding and grafting
which is a difcult and long process with unsatisfactory
multiplication rate, usually do the commercial propa-
gation of roses. Some rose genotypes have problems
with rooting. Bhattacharjee, 2010 claimed that there is
a correlation between plant propagation, soil and cli-
mate conditions.
In this context, we dene Rosa damascena as recal-
citrant species and looking for alternative methods for
propagation with high multiplication rate and ensuring
the genetic stability.
The aim of this review is to do a comparative
evaluation of all methods for propagation and to
determine their positive and negative sides.
Classical methods for propagation
Traditionally, Rosa damascena have been propa-
gated vegetative; since the time of Theophrastus, one
has been advised against growing roses from seeds,
cuttings, grafting, layering or budding (Widrlechner,
1981). Several methods for propagation are used.
These methods are very slowly and time-consuming
and usually associated with various problems such as
limitation of stock plant and prolonged production time
(Skirvin et al., 1990). Some methods are suitable for
some roses and not for others.
In the past in Bulgaria have used layering and graft-
ing as a means of propagation but they were not effec-
tive enough. In nowadays only way used for the propa-
gation of oil rose is through green cuttings.
Initially for the vegetative propagation methods
head been used as “kesme” (Topalov et al., 1967; To-
palov, 1978) and including branches from old planta-
tion in trenches 40 cm deep and 35 cm wide, which
after a certain time to develop shoots (aboveground or-
gans), is launching a new rose bushes. In this method,
except branches from rose bushes are used and com-
pletely uprooted trees, which are laid horizontally at
the base of the trenches. Ordered rose bushes are cov-
ered with 10cm soil and 5-6 cm animal manure. Dur-
ing the springtime from the buds of the laid bushes are
growing shoots which develop an own root system. The
propagation rate is 1:8 to 1:10 (Figure 1).
The other method known as “Chinese layer” was
developed from V. Topalov (1978). In this case, the
roses from the row are bending and place in pre-digged
trench in row spacing, close to the row. They are cov-
ered with soil and animal manure. In the autumn roses
are removed, separated by native bush and planted in a
Rosa Damascena Mill. - An Overview for Evaluation of Propagation Methods 547
permanent place. The propagation rate reaches 1:10 to
1:15 (Figure 2).
In order to accelerate reproduction and increase the
breeding rate of mother crops in the 70s year of the last
century has been established suitable rootstock for the
production of grafted material from oil-bearing rose.
It was found that the most suitable rootstock is Rosa
canina Brogs, which has good afnity and compatibility
with Rosa damascena Mill. f. trigintipetala (Zlatev et
al., 2001).
Since 1986 in Bulgarian Institute of Roses, Essen-
tial and Medical Cultures, Kazanlak, was implemented
new technology for production of seedlings by rooting
of green cuttings in cultivation facilities (Zlatev et al.,
2001). By this method, parts of healthy leaved shoots
placed under favorable conditions to form roots and
above ground organs. In this method, each rooted cut-
ting is developing new plant. The best root cuttings are
obtained from strongly and moderately growing shoots.
This method has high generative rate. It is the only way
of propagation in Bulgaria, yet.
Layers and cuttings realize the propagation of rose
in Turkey. Rose twigs or cuttings from old gardens cut
at the soil level, they are placed into ditches horizon-
tally, and their ends are overlapping in the trenches,
which are then covered with soil containing manure.
The cuttings necessary for planting are procured from
old rose elds, not younger than six years. It is less la-
bour consuming than other methods in which seedlings
are used (Baydar H, 2006).
Primarily cuttings and suckers achieve damask
rose propagation, but micropropagation is a develop-
ing method in Iran (Nikbakht et al., 2004). The cut-
tings have not to be younger than six years. Cuttings
were taken from the base of rose shrubs and carefully
inspected. Dry and diseased parts were removed. An
older method for procuring cuttings was to uproot the
entire rose shrub and use the roots as well. Later, this
method was abandoned as inefcient, because the old
rose garden died, the roots were not reliable planting
material, and the procedure was too labour intensive.
The woody 30-100 cm long cuttings were placed into
ditches horizontally and their ends are overlapping
(Haghighi et al., 2008).
The successful application of classical vegetative
propagation methods requires knowledge of the
strengths and weaknesses of all above description
methods. Using of this methods will be well to know
that they require more time, they are labor intensive
and more of the case performance is not satisfactory.
Propagation by layers requires rather large area for
Fig. 1. Vegetative propagation by layers:
A. Pull branch down for simple layer;
B. Make wound or cut at bend;
C. Stake tip to hold upright
(http://extension.missouri.edu/p/G6970)
Fig. 2. Vegetative propagation by “chinese layers”
(http://extension.missouri.edu/p/G6970)
548 A. Ginova, I. Tsvetkov and V. Kondakova
a lay-bed, and weed control among the layers is a
problem. The advantages of using green cuttings for
propagation is greater efciency (1:50 to 1:60) and
material is genetically identical (Kovatcheva et al.,
2009). The weakness of this method is the root system,
which is not always well developed. Another negative
side is the possibilities for transmission of bacterial
and viral diseases that can cause a problem in future
plantings.
In vitro micro propagation
In vitro methods have also been employed for
achieving faster rates of multiplication, (Khosh-Khui
and Sink, 1982; Ishioka and Tanimoto, 1990; Kornova
and Michailova, 1994). The tissue culture method is a
widely applicable for study, selection and propagation
of plants. The potential of the cells to grow and develop
into multicellular organisms is known as cell totipoten-
cy (Wetzstein and He, 2000). It can be employed for
large-scale propagation of disease free clones and gene
pool conservation.
Clonal stability of the micropropagated plants is
essential for in vitro germplasm conservation. Skirvin
and Janick (1976) were among the rst who directed
attention to the fact that clonal variation in genotypes
is very important for improvement in horticultural spe-
cies. Subsequently, Thorpe and Harry (1997) evidence
that in vitro culture techniques have played on impor-
tant role in the breeding programme, production and
improvement of plant quality. Various types of changes
were reported in cell cultures at phenotypes, karyo-
typic, physiological, biochemical and molecular level.
Larkin and Scowcroft (1981) reviewed extensively and
reported the phenotypic variation among plants regen-
erated after a passage through tissue and cell culture.
Recently, the propagation of R. Damascena by in
vitro techniques is used mainly in Iran and India. Ac-
cording to Debergh and Read (1991) and Altman
(2000), the micropropagation process can be divided
in ve different stages: selection of the mother plant,
initiation of culture, multiplication, rooting and adapta-
tion. Often, clear relations of the physiological status of
the original tissue, and the reactions of explants taken
thereof can be observed. In addition, the reaction of
explants correlates with the growth conditions of the
mother plant used to obtain explants for cultivation.
Plants used for apply of tissue culture methods must
be healthy, genetic identity and actively growing.
The correct choice of explants material can have an
important effect on the success of tissue culture. For
micropropagation of rose, the most commonly used ex-
plants is a nodal stem segment with size - 9.0–10.0-mm
long; 3.0–4.0-mm diameter (Figure 3).
First report for efcient and rapid propagation
of Rosa damascena Mill., using nodal explants from
natural grown plants was reported by Bhoomsiri and
Masomboon (2003). After sterilization, nodal explants
were transferred on MS (Murashige and Skoog, 1962)
and QL (Quirin and Lepoivre, 1977) medium with dif-
ferent concentration of BA and NAA. The highest num-
ber of shoot per explant was obtained on QL medium
supplemented with 4.0 mg.l-1 BA and 0.1 mg.l-1 NAA
(8.27 shoots per explants).
In Bulgaria, rst report for using of apical and ad-
ventitious buds was described by Kornova et al. (2001).
It was tested 11 different media on the base of MS with
Fig. 3. In vitro propagation of R. Damascena
(a. induction of plant; b. multiplication; c. rooting; d. adaptation)
Rosa Damascena Mill. - An Overview for Evaluation of Propagation Methods 549
various concentration of BAP (0.5-1.5 mg.l-1) and par-
ticipation of auxins NAA and IAA (0.1 mg.l-1). The
most appropriate medium for micropropagation was
MS containing BAP 0.5 – 1 mg.l-1 with or not 0.1 mg.l-1
IAA with optimal rate of multiplication 2.2 -2.6.
A liquid culture system using nodal segments was
used for shoot proliferation and root induction in Rosa
damasena and Rosa burboniana (Pati et al., 2005). For
efcient and large-scale induction of roots in micro
shoots, a rooting vessel was designed and developed
to facilitate the micro propagation protocol. Their work
highlights the signicance of osmotic potential in re-
lation to enhanced growth and development in liquid
cultures, vis-à-vis agar-gelled cultivars, especially in
relation to root induction during micro propagation.
In research of Nikbakht et al. (2005) was investigat-
ed the regeneration of two Iranian cultivars of Damask
rose (Rosa damascena Mill.), “Azaran” and “Ghamsar”
under in vitro conditions. The shoot single node seg-
ments included lateral buds were taken from bushes.
Results of the study showed that among 12 different
media, a liquid modied MS medium (with eliminated
Cl- and reduced NH4 + ions) caused the best growth of
newly proliferated shoots and no aging occurred. The
choice of suitable medium with optimal multiplication
effect were made between tested 32 different combina-
tion including growth regulators in different concentra-
tion- BA (0, 1, 2 and 3 mg.l-1), GA3 (0, 0.1, 0.25 and 0.5
mg.l-1) and NAA (0 and 1 mg.l-1).
Best results as proliferation, multiplication rate, ap-
pearance and leaf color were registred on medium with
BA (1-2 mg.l-1), GA3 (0.1 mg.l-1) and NAA (0-0.1 mg.l-1)
for cv.”Azaran” and with the same concentrations of
BA and GA3, but without NAA for cv.”Ghamsar”. In
the same time, Jabbarzadeh and Khosh-Khui (2005)
reported that the combination of BA at concentrations
of 2.5–3 mg.l-1 with a low rate of IBA was the most
suitable treatment for in vitro multiplication of Dam-
ask rose. (3.75-4.00 shoot number per single-node ex-
plants). The explants tolerated a higher concentration
of plant growth regulators (PGRs) without showing
any abnormality in morphology. Shoot orientation did
not inuence on shoot multiplication rate. Mamaghani
et al. (2010) announced the effects of culture medium
and combination of various plant growth regulators on
shoot proliferation of three elite Iranian R. damascena
Mill accessions - M6 (Kashan), G1 (East Azerbyjan)
and G2 (West Azerbyjan). First, the effect of culture
media, MS and WPM (Lloyd and McCown, 1980)
containing 1.5, 2.5, 5 mg.l-1 BAP on shoot multiplica-
tion (shoot number per explants) and shoot length were
studied. Then, the effect of BAP (5 mg.l-1) combina-
tion with 0.1 and 0.1 mg.l-1 as well as IAA (0.1 and
0.5 mg.l-1) and IBA (0.1 and 0.5 mg.l-1) was studied.
Finally, three levels of BAP (2, 2.5 and 5 mg.l-1) and
Kin (2, 2.5 and 5 mg.l-1) alone and their combination by
adding TDZ (0.05 mg.l-1) and IBA (0.1 mg.l-1) on shoot
proliferation and shoot length in three accession were
examined. The explants had higher shoot multiplica-
tion, shoot length and better green leaves on MS than
WPM medium. The highest shoot proliferation (5.9)
was obtained at a combination of 5 mg.l-1 BAP and
0.1 mg.l-1 TDZ. Maximum shoot length was observed
in the medium with 0.5 mg.l-1 IAA and 5 mg.l-1 BAP
and 0.01 mg.l-1 TDZ. Shoot multiplication and shoot
length were varied with genotypes and BAP as well as
Kin concentration. M6 accession needed 2 mg.l-1 BAP
and 2mg.l-1 Kin for maximum shoot multiplication and
shoot length, G1 and G2 needed 2.5 mg.l-1 BAP and
2.5 mg.l-1 Kin. Anther culture of Rosa has been suc-
cessfully done (Tulaeezadch and Khosh-khui, 1981).
The MS media with 2.0 g.1-1 IAA and 0.4 mg.l-1 kinetin
was the best for anther culture of R .damascena.
Some explants placed on culture medium exude
dark colored compounds into the culture medium (phe-
nols, pigments) that are released from the cut ends of
the explants. This can cause browning of tissue and the
medium, which is often connected with poor culture
establishment and reduced regeneration ability. The
basal media enriched with, such as activated charcoal
(1-2%), various antioxidants - ascorbic acid or citric
acid (50 - 100 mg.l-1), or polyvinylpyrrolidone (PVP),
polyvinylpolypyrrolidone (PVPP), or the ethylene in-
hibitor, silver nitrate are often employed to nullify
this effect. Frequent subculture, incubation in shaking
liquid culture, reduced culture temperature or the use
of etiolated explants, are also methods that have been
used to deal with this problem (Iliev et al, 2010). The
addition of PVP helps in oxidizing polyphenols leached
in the medium, and promotes high rate of organogen-
550 A. Ginova, I. Tsvetkov and V. Kondakova
esis (Rout et al., 2006). In order to decrease leaves and
shoot tip necrosis additional vitamin complex, contain-
ing biotin (1 mg.l-1), calcium pantothenate (0.5 mg.l-1),
riboavine (0.5 mg.l-1), and folic acid (0.5 mg.l-1) were
used (Mamaghani et al., 2010).
Roberts and Schum (2003) indicated some type of
roses require more calcium salts as calcium decien-
cy may lead to shoot tip necrosis, hyperhydration of
stem and leaf tissues. They showed that adding calcium
in the multiplication medium signicantly increased
shoot multiplication rates and stem length. Application
of calcium pantothenate complex may enhance shoot
quality and root regeneration. Calcium regulated cellu-
lar process such as cell division and elongation (Ghor-
banli and Babalar, 2003). Arnold et al. (1995) have sug-
gested that calcium and potassium have a role in auxin
absorption.
Vitrication is physiological disorder, which can
be a serious problem in plant micropropagation. Vit-
ried microplants lose their ability to propagate and/
or present difculties of ex vitro acclimatization. As
cause of vitrication has been suggested high concen-
tration of cytokinins, high concentration of potassium
and ammonium, calcium deciency (Mohamed –Yas-
sen et al., 1992).
The main problem in Damask rose in vitro
propagation, however, is the rooting step. The auxin,
3-indole butyric acid (IBA), is usually used with great
success for rooting in plant tissue culture, but the other
common auxins such as 1-naphthaleneacetic acid (NAA)
and indole-3-acetic acid (IAA) have been used for root
induction with less percentage of success (Saffari et
al., 2004). Rooting is improved in many woody and
herbaceous species by lowering the concentration of
sucrose from 2 or 3 to 0.5 to 1% in rooting medium
(Jabbarzadeh and Khosh khui, 2005). The effect of
reduced sucrose and organic salt concentration may be
important for various plants root initiation (Mirza et
al., 2011). Another problem which previous researcher
were faced with was short lifelong of scions in rooting
medium because of the appearance of brownish ends
of cuttings and dying off after few days. However, we
overcame this by applying activated charcoal 0.1% in
MS medium to absorb phenolic compounds that evolve
from woody plant part in MS medium, and considered
as toxic elements for explant. Also, in parallel, it seems
that darkness through applying charcoal MS medium
was suitable for rooting of damask scions as well. Mirza
et al. (2011) also reported highest percentage of root
formation 89% on MS medium supplemented with 0.5
mg.l-1 IBA which is similar with present study results.
A lower rooting ability was also recorded in old world
spp. (Rosa canina and Rosa damascena) when com-
pared with Rosa hybrida (Pati et al., 2006). Kirichenko
et al. (1991) reported that rooting of micro shoots of the
essential oil bearing roses was difcult when compared
with the ornamental varieties. Current studies indicate
that, there are genes responsible for increased number
of bud initials and shoot proliferation. Moreover, the
possible involvement of the gene in modulating hor-
mone levels has also been reported (Tantikanjana et al.,
2001). In R. damascena and R. bourboniana rooting
was initiated as a two-step procedure where IBA (2
mg.l-1) was used in MS medium and in the second
step the shoots were transferred to PGR free medium
containing half strength of MS (Pati et al 2005).
Bhoomsiri and Masomboon (2003) reported that root-
ing was obtained only on MS medium supplemented
with 0.5 mg.l-1 NAA (87.5 % of the explants exhibited
root development with 2.71 roots per explant). The
experimental medium of Kornova et al. (2001) with a
base on modied MS and 0.1 mg.l-1 NAA was created
good conditions for rooting. Strongly inuenced by
the coverage of rooting medium with a liquid lm of
hormone free medium. Jabbarzadeh and Khosh-Khui
(2005) announced for problems in rooting too. Applica-
tion of different media (MS, 1/2 MS, 1/3 MS and 1/4
MS) with different concentrations of auxins did not
produce satisfactory results. Similarly, explants failed
to produce roots in quick-dip method using sterilized
0–2000 mg.l-1 auxin solutions. Other treatments such as
using various concentrations of ABA with various con-
centrations of IAA, IBA and NAA also applying dif-
ferent concentrations of sucrose and agar did not result
in rooting. At last, among the treatments the success-
ful treatment for rooting of shoots was using 2.5 mg.l-1
2,4-D for 2 weeks in MS medium and then transferring
the explants to MS medium without any PGRs. When
shoots were kept in the rooting medium for more than
2 weeks, root-tips became brown in color and plant-
Rosa Damascena Mill. - An Overview for Evaluation of Propagation Methods 551
lets died after a few days. The problem was solved by
transferring the plantlets from rooting medium to root
elongation medium (without any PGRs). It may be con-
cluded that auxins are necessary just for root initiation,
but not for subsequent root development of Damask
rose. The report by Nikbakht et al. (2005) for in vitro
rooting include quick deep treatment of micro shoots’
bottom in 2000 ppm IBA solution and then rooting in
liquid half strength of the same MS medium showed
the best result compared with 1000 ppm.
Most rooting media involve a modication of the
MS high mineral salt medium with or without growth
regulators (Khosh-khui and Sink, 1982). The most
common auxins used for rose root induction are NAA
(naphthaleneacetic acid 0.03-0.1 mg.l-1), IAA (Indole-3
-acetic acid 0-1 mg.l-1) and IBA (3.0 mg.l-1 IBA indole-
3-butyric acid). All are effective in rooting of rose in
vitro (Khosh-Khui and Sink, 1982). Another factor that
affects rooting of rose is the salt concentration of the
nutrient medium. Many roses rooted well in diluted
medium; half or quarter strength MS salt concentrations
often promote rooting (Hasegawa, 1980 Environmental
factors also affect the ability of roses to root. According
to Khosh-Khui and Sink (1982) rose shoots grown at
low light intensity (1.0 Klux) gave a higher rooting
percentage (84%) than those grown under higher light
intensities (3.0 Klux). Skirvin et al. (1990) reported
that red light can have positive effect on rooting of
miniature roses (R. chinensis) (Skirvin and Chu, 1984).
They also reported their miniature roses proliferated
better under cool white uorescent light than under
warm white uorescent.
In the report of Mamaghani et al. (2010) was uti-
lized modied MS medium contained 1/2, 1/3 and 1/4
concentrations of macronutrients, sucrose as well as
full concentration of micronutrient supplemented with
different concentrations of NAA (0.1, 0.2, 0.5 and 1
mg.l-1). The results indicated that root regeneration of
shoots under in vitro condition were difcult. 25% of
shoots in G1 (East Azerbyjan) accession were rooted
on modied 1/3 MS medium supplemented with 0.1
mg.l-1 NAA. On the other hand, root regeneration of G2
(West Azerbyjan) accession was found on modied 1/2
MS medium by adding 0.2 mg.l-1 NAA . Using TDZ
with high concentration of BAP decreased adventitious
root formation on micro shoots. BAP can be accumu-
lated in plant tissue at high concentration in the form of
conjugates, inactive glucosides, as well as physiologi-
cally active ribosides and ribotides (Podwyszynska,
2003). Horan et al. (1995) reported that the establish-
ment of rooted plantlets was dependent on the age of
the micro shoots. Huettman and Preece (1993) sug-
gested that rooting of micro shoots were difcult be-
cause of carry over effect from cytokinin. Podwyszyn-
ska (2003) formulate the hypothesis that the problem
with root formation of some rose genotypes may result
from inadequate endogenous level of auxin and other
growth regulators, phenolics or enzymes. Low rooting
attributed to mature explants might have some inhibi-
tory materials (Podwyszynska, 2003). The result indi-
cates there were different responses among accessions
for rooting. Hasegawa (1980) suggested that there is
a difference in rooting requirements and responses to
culture condition, as well as in rooting capacity.
The last and most important step in micropropagation
is the establishment of plantlets to ex vitro conditions
(Rahman et al., 1992; Rout et al., 1999). Once plantlets
are well rooted, they must be acclimatized in normal
greenhouse environment. The compost containing
a high amount of organic and inorganic nutrients
could increase the nutrient availability for the plants.
Therefore, plants grown in sand, soil, organic matters
and vermiculite mixture develop better than in other
medium tested. Roses can be successfully grown on
a wide range of soils, but they do best on well-drained
soils, with a soil pH of 6.0 to 6.5 (Jabbarzadeh and
Khosh khui, 2005).
The presence of organic matter and vermiculite tend
to keep more moisture in the potting medium.This is
important for plant survival during the rst week after
the transfer to outdoor conditions. The potted plants
are covered with perforated plastic bag that can pro-
vide a favorable environment for the damask growth. It
could be concluded that the composition of sand, soil,
organic materials, vermiculite (2:2:1:1) was the suitable
medium for acclimatization of damask rose plantlets.
Pati et al. (2005) indicate that the period of incuba-
tion of micro shoots in rooting vessel also inuenced
on the survival percentage of R. damascena plantlets
under greenhouse conditions. Signicantly higher sur-
552 A. Ginova, I. Tsvetkov and V. Kondakova
vival percentage (96.66%) was observed after 6 weeks
of incubation compared to the lowest (3.3%) after 1
week, suggesting a clear correlation of incubation pe-
riod during root induction upon survival of plantlets
during hardening or acclimatization.
During in vitro process, plantlets grow under very
special conditions in relatively air-tight cultivation ves-
sels, e.g., air humidity is higher and irradiance lower
than in conventional culture. The use of closed vessels
in order to prevent microbial contamination decreases
air turbulence, which increases leaf boundary layers
and limits the inow of CO2 and outow of gaseous
plant products from the vessels. The cultivation media
are often supplemented by saccharine as carbon and en-
ergy sources. This addition decreases considerably the
water potential of the medium and increases the risk of
bacterial and fungal contamination.
Other application of biotechnological tools is regen-
eration from protoplasts culture. This system allows
applying protoplast fusion technology for facilitating
gene transfer between incompatible rose species holds
great potential. Application of this technique allows
one to bypass sexual incompatibilities thus facilitating
widening of the gene pool available for rose improve-
ment. Pati et al. (2004) reported for successful proto-
col for protoplast isolation from microspores of Rosa
damascena Mill.
This technology is more complicate, requires knowl-
edge in several research disciplines and mainly apply-
ing for genetic study.
Plant regeneration
Indirect organogenesis
First report for indirect shoot regeneration from rose
callus using Rosa damascena was reported by Ishioka
and Tanimoto (1990) who tried to examine the plant
regeneration from rose callus. In this investigation was
showed that adventitious buds could be successfully in-
duced from Bulgarian rose callus tissues. Adventitious
bud formation could be successfully attained, depend-
ing on the kinds of mineral salts used in the medium,
auxin and cytokinin used. When callus tissues were
cultured on the medium without ammonium nitrate and
contained indoleacetic acid and benzyladenine, buds
were formed on the callus. The number of buds was
signicantly increased by the simultaneous addition of
calcium ionophore. When the cultures were transferred
to the medium without cytokinin, roots were formed
in the basal part of the buds. For induction of adventi-
tious bud was tested various concentrations of auxins
- NAA (0.02 – 0.2 mg.l-1), IAA (0.02 – 0.2 mg.l-1) and
cytokinins – BA (0.2 – 2 mg.l-1), K (0.2 – 2 mg.l-1). The
best results for bud formation were observed on the MS
medium containing 0.2 mg.l-1 IAA and 2 mg.l-1 BA (2.6
buds per culture). The cultures with buds were trans-
ferred to the rooting medium containing 1 mg.l-1 IAA.
Direct organogenesis
The rst report on direct shoot regeneration in scent-
ed rose was describe by Pati et al. (2004) using direct
induction of shoot buds from leaf explants of in vitro-
raised shoots of R. damascena var. Jwala.( Institute of
Himalayan Bioresource Technology, Palampur) with-
out the intervening callus phase. Petioles from fully
developed young leaves, obtained from 4 wk pruned
old shoots, were found to be ideal for regeneration of
shoots. Initially the explants were cultured in an in-
duction medium ½ MS including 1.5 mg.l-1 TDZ, 0.05
mg.l-1 α-NAA and 2.5 mg.l-1 AgNO3 and subsequently
transferred to the regeneration medium MS with 0.5
mg.l-1 BA and 0.01 mg.l-1 NAA for 21 days. In vitro
rooting of micro-shoots was accomplish within 2 wk
on ½ MS liquid medium containing 2 mg.l-1 IBA for 1
wk in the dark and later transferred to hormone – free
medium and kept in the light. Plantlets were transferred
to soil.
Somatic embryogenesis
In roses, somatic embryo genesis has been ob-
tained from a variety of explants such as calli de-
rived from leaf tissue, immature leaf and stem seg-
ments, immature seeds, petioles and roots and anther
laments (Bhattacharjee, 2010). It has been reported
for induction of somatic embryogenesis from Rosa
damascena in Bulgaria (Borisova, A. and Goturanov.
G, unpublished data).
The use of in vitro technology for propagation
of vegetative plants is an effective alternative to
traditional methods. The plants thus obtained were free
of pathogens, genetically identical but their price is
higher. In vitro method is expensive and therefore its
use should be very well grounded and motivated.
Rosa Damascena Mill. - An Overview for Evaluation of Propagation Methods 553
Bioreactors
Automation of tissue culture will depend on the
use of liquid cultures in bioreactors; allow fast prolif-
eration, mechanized cutting, separation, and automated
dispensing (Sakamoto et al., 1995). These techniques
were used for some plants, which involve minimal hand
manipulation and thus reduce in vitro plant production
costs. Eide et al. (2003) reported two liquid culture sys-
tems for plant propagation i.e. temporary immersion
systems and permanent submersion of the plant cells/
tissue that requires oxygen supply through rotary shak-
ers or bioreactors. Temporary immersion system, e.g.
RITA bioreactor, seems to be better than the permanent
submersion system for shoot proliferation (Figure 4).
Other approach of plant cells and tissue culture tech-
niques is production of secondary metabolites under
controlled and reproducible conditions, independent
of geographic and climatic conditions. Studies on the
production of plant metabolites by callus and cell sus-
pension cultures have been carried out on an increasing
scale since the end of the 1950’s. The prospect of using
such culturing techniques is for obtaining secondary
metabolites, such as active compounds for pharmaceu-
ticals and cosmetics, hormones, enzymes, proteins, an-
tigens, food additives and natural pesticides from the
harvest of the cultured cells or tissues.
As for the clonal propagation of a plant in large
scale, regeneration from cells belonging to explants
of plants is potentially very useful. (Yesil-Celiktas et
al, 2010) Liquid media have been used for plant cells,
somatic embryos, and organ cultures in both agitated
asks and various types of bioreactors. Scale up of
plant regeneration in a liquid medium is easier than on
a solid medium (Okamoto et al. 1996). Although the
use of bioreactors has been directed mainly for cell sus-
pension cultures and secondary metabolite production,
research directed at improving bioreactors for somatic
embryogenesis has been reported for several plant spe-
cies as well. Utilization of bioreactors for clonal propa-
gation has been attracting interest recently due to scale-
up and automation advantages (Gurel, 2009). Pavlov
et al (2005) investigate the low molecular metabolites
(volatiles and polar compounds) produced by Rosa
damscena Mill. 1803 cell suspension cultured in biore-
Fig. 4. Temporary immersion bioreactor system (RITA®): Components and assembly of the RITA
Bioreactors (Kintzios, 2010); b. RITA® c, d. multiplication of roses in bioreactor
554 A. Ginova, I. Tsvetkov and V. Kondakova
actor and in asks. It was found that the main groups of
volatiles were hydrocarbons and free acid and their es-
ters. The main components of polar fraction were free
acids, especially amino acids and oxidized acids.
Bioreactors have also been used for the cultivation
of hairy roots mainly as a system for secondary metab-
olite production (Ziv, 2000). Furthermore, innovative
processes have been proposed for producing secondary
metabolites selectively by enzymatic reactions.
Conclusion
Rosa damascena have great importance to the
Bulgaria economy. Based on our review of the methods
used for generation of this culture, we can summarize
their application until now having mind positive and
negative side of each multiplication method.
The expansion of the areas and increase the yield of
rose owers is a priority for manufacturers and scien-
tists dedicated knowledge and experience of it.
Choosing an appropriate method for breeding
should be subordinated to the goal that we set ourselves
and know that there are no perfect methods. Best result
would be obtained if combining the use of different
methods and the ability to use the best of them.
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It is concluded from a review of the literature that plant cell culture itself generates genetic variability (somaclonal variation). Extensive examples are discussed of such variation in culture subclones and in regenerated plants (somaclones). A number of possible mechanisms for the origin of this phenomenon are considered. It is argued that this variation already is proving to be of significance for plant improvement. In particular the phenomenon may be employed to enhance the exchange required in sexual hybrids for the introgression of desirable alien genes into a crop species. It may also be used to generate variants of a commercial cultivar in high frequency without hybridizing to other genotypes.
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The effect of several factors on in vitro rooting of the Kazanlak oil-bearing rose has been studied. Using the Murashige Skoog nutrient medium, the concentrations of naphthalene acetic acid (0.05 and 0.1 mg/L), the presence of phloroglucinol and variation in the quantity of mineral salts used were evaluated. Also, the conditions of microplant cultivation such as temperature, light and photoperiod, as well as the changes during growing in a two-phase-nutrient medium (solid, including the tested variants, plus liquid) were studied. Optimum results were obtained using a nutrient medium with 25% of the recommended mineral salts normally encountered in the medium plus 0.1 mg/L naphthalene acetic acid and 126 mg/L phloroglycinol in the presence of light. Analogous results were obtained for both the single and two-phase-nutrient media.
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Basal medium strength, auxins, sucrose, agar, pH, photoperiod and culture room temperature substantially affected the development of roots from proliferated rose (cultivar ‘Tajmahal’) shoots. Half strength Murashige and Skoog (MS) medium fortified with 0.1 mg l−1 naphthaleneacetic acid (NAA) and 0.5 mg l−1 indolyl-3-acetic acid (IAA), sucrose level of 40 g l−1, agar concentration at 6 g l−1, a 16 h photoperiod and 28°C culture room temperature were required for optimal in vitro rooting of the excised shoots. Gradual acclimatization was essential for subsequent establishment of plantlets in natural condition.
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Anthers of 2 tetraploid Rosa species (4n=28) were plated on selected basis media, supplemented with various amounts of auxins and kinetin, at different bud stages and using various light conditions. Among the media tested, MS medium with 2.0 mg/l IAA and 0.4 mg/l K was generally the best for anther culture of R. damascena Mill., while medium with 7.5 mg/l IAA and 0.8 mg/l K was found optimum for R. hybrida L. Culture of anthers of R. damascena when a few petals are visible on the flower bud, and of R. hybrida when the flower bud is completely closed, are recommended. Cytological examination showed that most cells of both species had 2n=14 chromosomes and thus were of pollen origin.