Research review paper
In vitro propagation of rose—a review
Pratap Kumar Pati a,*, Siba Prasad Rath b
, Madhu Sharma c
, Anil Sood c
,
Paramvir Singh Ahuja c
a
Department of Botanical and Environmental Sciences, Guru Nanak Dev University, Amritsar-143 005, India
b
P.G. Department of Botany, Utkal University, Vanivihar, Bhubaneswar-751004, India
c
Division of Biotechnology, Institute of Himalayan Bioresource Technology, Palampur-176 061 (H.P.), India
Received 15 November 2004; received in revised form 9 May 2005; accepted 5 July 2005
Available online 5 October 2005
Abstract
In vitro propagation of rose has played a very important role in rapid multiplication of cultivars with desirable traits and
production of healthy and disease-free plants. During the last several years, different approaches have been made for in vitro
propagation of rose. Micropropagation using apical buds or nodal segments and understanding the specific requirements at
different stages has been comprehensively covered in literature. New challenges for refinements of protocols for high rate of shoot
multiplication and development of cost effective methods has gained importance in the recent past. Importance of liquid static
culture for shoot proliferation and root induction for rose is also discussed in the present review. Further, the development of
protocol for in vitro plant regeneration which is considered as most important step for successful implementation of various
biotechnological techniques used for plant improvement programmes has been adequately addressed in literature. In rose, there are
several reports which indicate rapid regeneration and multiplication through organogenesis or somatic embryogenesis. On the
whole, the present review gives a consolidated account of in vitro propagation in rose.
D 2005 Elsevier Inc. All rights reserved.
Keywords: Rose; In vitro; Liquid medium; Shoot multiplication; Root induction; Rooting vessel; Micropropagation; Regeneration; Somatic
embryogenesis
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
2. Micropropagation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
2.1. Stages involved in micropropagation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
2.1.1. Initiation of aseptic cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
2.1.2. Shoot multiplication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
2.1.3. Rooting of microshoots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
2.1.4. Acclimatization and field establishment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
0734-9750/$ - see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.biotechadv.2005.07.001
Abbreviations: BAP, 6-benzyl aminopurine; IBA, indole-3-butyric-acid; NAA, a-naphthalene acetic acid; TDZ, Thidiazuron; IAA, indole-3acetic
acid.
* Corresponding author.
E-mail address: pkpati@yahoo.com (P.K. Pati).
Biotechnology Advances 24 (2006) 94–114
www.elsevier.com/locate/biotechadv
3. Plant regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
3.1. Organogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
3.1.1. Indirect organogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
3.1.2. Direct organogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
3.2. Somatic embryogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
1. Introduction
Rose is one of the most important commercial crops.
It is generally propagated by vegetative methods like
cutting, layering, budding and grafting. Seeds are used
for propagation of species, new cultivars and for production
of rootstocks (Horn, 1992). Although propagation
by vegetative means is a predominant technique in
roses, yet it does not ensure healthy and disease-free
plants. Moreover, dependence on season and slow multiplication
rates are some of the other major limiting
factors in conventional propagation.
In the last few years, in vitro propagation has revolutionized
commercial nursery business (Pierik, 1991).
Significant features of in vitro propagation procedure
are its enormous multiplicative capacity in a relatively
short span of time; production of healthy and diseasefree
plants; and its ability to generate propagules around
the year (Dhawan and Bhojwani, 1986). Martin (1985)
demonstrated that, using this technology, up to 400,000
plants could be cloned, from a single rose on annual
basis. Such a method has considerable implications for
the rose breeder as it allows rapid multiplication of new
varieties. Micropropagated plants are well suited for cut
flower production as they are more compact (Onesto et
al., 1985), branch better and sometimes yield more
flowers (Reist, 1985). In addition, tissue culture derived
dwarf roses used for pot plant production have a faster
rate of growth, early flowering, and exhibit shorter
shoots and more laterals than conventionally produced
plants (Dubois et al., 1988).
The history of rose tissue culture dates back to 1945,
when Nobecourt and Kofler succeeded in obtaining
callus and roots on the explanted buds. In the year
1946, Lamments for the first time reported the use of
embryo culture in rose breeding. Studies were initiated
by Nickell and Tulecke (1959) and Weinstein et al.
(1962) to culture cells, cell suspension and calli with
a view to understand differentiation and regeneration.
The first shoot organogenesis from callus tissue was
reported by Hill (1967) in a climbing Hybrid Tea rose
dThe Doctor.T The earliest references of rose micropropagation
were those of Jacob et al. (1969, 1970a,b) and
Elliott (1970) in R. hybrida cv. Superstar and R. multiflora,
respectively.
Since these pioneering efforts, a lot of data were
generated and a number of papers have been published
on different aspects of in vitro studies of rose with a
greater emphasis on micropropagation. A consolidated
account of tissue culture studies on rose is dealt with in
the present review.
2. Micropropagation
The most important technique in micropropagation
is meristem proliferation wherein apical buds or nodal
segments harbouring an axillary bud are cultured to
regenerate multiple shoots without any intervening callus
phase. Work on micropropagation of rose is summarized
in Table 1.
2.1. Stages involved in micropropagation
A successful micropropagation protocol proceeds
through a series of stages, each with a specific set of
requirements. These are (i) initiation of aseptic cultures,
(ii) shoot multiplication, (iii) rooting of microshoots,
and (iv) hardening and field transfer of tissue culture
raised plants.
2.1.1. Initiation of aseptic cultures
2.1.1.1. Choice of explant. The choice of explant for
initiation of culture is largely dictated by the method to
be adopted for in vitro propagation. Explants with
vegetative meristems are often suitable for enhanced
axillary branching (Table 1). The most commonly used
explant is a nodal stem segment, wherein the axillary
bud is made to proliferate to form multiple shoots (Fig.
1a). The performance of nodal segments is much better
than the shoot tips (Horn, 1992).
Different parameters influence the initial stage of
micropropagation. Mederos and Enriquez (1987) found
that buds taken from softwood stem were more responsive
than those from hardwood. Rout et al. (1989a) and
Bressan et al. (1982) observed significant differences
P.K. Pati et al. / Biotechnology Advances 24 (2006) 94–114 95
in the rate of shoot multiplication depending upon
position of node on the stem in different cultivars of
R. hybrida. The buds nearest to the apex and closest to
the base of the stem exhibited the slowest rate of
development, but those from the mid-stem region
grew very rapidly. The potential of axillary bud outgrowth,
which is related to position on the main axis,
appears to be determined by a balance among several
hormones (Sato and Mori, 2001). Further, the axillary
buds in the mid-region of plants have the potential to
grow; however, they cannot grow on intact plants due
to apical dominance. Removing apical dominance
could change this balance. The finding of Rout et al.
(1989a) and Bressan et al. (1982) on the differences in
the rate of shoot multiplication depending upon position
of node on the stem could be understood in this
context.
Mederos and Enriquez (1987) have reported that the
presence of petiole fragments in the nodal explants had
inhibitory effect on shoot growth. This report was
further endorsed by the work of Marcelis van Acker
and Scholten (1995) in R. hybrida. Salehi and KhoshKhui
(1997) found that the explant length and diameter
played significant roles in proliferation and shoot
growth of miniature roses (R. chinensis cv. Minima),
dLittle BuckarooT, dBaby MasqueradoT and dSouratiT.
They also reported that the best rates of shoot growth
and proliferation were obtained in explants with a
length of about 9.0–10.0 mm and diameter of about
3.0–3.5 mm. No such correlation was observed in Rosa
damascena and R. bourboniana (Pati, 2002). Alekhno
and Vysotskii (1986), while working with Hybrid Tea,
Floribunda and miniature rose cultivars, reported that
growing shoots in horizontal position during proliferation
almost doubled the axillary branching as compared
with growing shoots in vertical position. The
increased axillary shoot proliferation by placing the
explant in horizontal position could be attributed to
greater uptake of the medium constituents due to increased
contact with the medium (Mackay and Kitto,
Table 1
In vitro propagation of rose using different explants
Species/cultivars Explant Response Reference
Rosa multiflora apm Shoot multiplication Elliott, 1970
Rosa multiflora, Rosa indica st Shoot multiplication Graifenberg et al., 1975
Rosa hybrida cv. Forever Yours st Shoot multiplication, rooting Skirvin and Chu, 1979
Rosa hybrida cvs. King’s Ransom, Plentiful Parade,
Fragrant Cloud, Lili Marlene, Garnet Yellow,
Paul’s Lemon Pillar
am Shoot multiplication, rooting Davies, 1980
Rosa hybrida cv. Improved Blaze and Gold Glow am Shoot multiplication, rooting Hasegawa, 1980
Rosa hybrida cv. Improved Blaze st Shoot multiplication Bressan et al., 1982
st Rooting Hyndman et al., 1982a
Rosa hybrida cvs. Tropicana, Bridal Pink,
Rosa damascena, Rosa canina
st, am Shoot multiplication, rooting Khosh-Khui and Sink, 1982a
Rosa hybrida cvs. Tropicana, Bridal Pink st Shoot multiplication, rooting Khosh-Khui and Sink, 1982b
Rosa hybrida cvs. Crimson Glory, Glenfiditch am Shoot multiplication, rooting Barve et al., 1984
Rosa hybrida lb Shoot multiplication, rooting Sauer et al., 1985
Rosa hybrida cv. Queen Elizabeth apm Shoot multiplication, rooting Douglas et al., 1989
Rosa hybrida cvs. Landora, Virgo, Sea Pearl, Super
Star, Happiness, Queen Elizabeth
am Shoot multiplication, rooting Rout et al., 1990
Miniature rose dRosaminiT apm, am Shoot multiplication, rooting Campos and Pais, 1990
Rosa chinensis cv. Minima st Shoot multiplication, rooting Chu et al., 1993
Hybrid Tea cv. Dr. Verhage apm, am Shoot multiplication, rooting Voviatzi et al., 1995
Rosa hybrida cvs. Sweet Promise, Motrea am Shoot multiplication, rooting Marcelis van Acker and
Scholten, 1995
Rosa banksiae, Rosa laevigata, Rosa odorata,
Rosa rugosa, Rosa roxburghii, Rosa setigera,
Rosa wichuraina
st, lb Shoot multiplication, rooting Yan et al., 1996
Hybrid Tea rose cv. Peace st, am Shoot multiplication, rooting Ara et al., 1997
Miniature rose ’The Fairy’ am Shoot multiplication, rooting Sahoo and Debata, 1997
Rosa centifolia am Shoot multiplication Ganga et al., 1998
Rosa Âhybrida cv. Sonia am Shoot multiplication, rooting Singh and Syamal, 1999
Rosa damascena var. Jwala am Shoot multiplication, rooting Kumar et al., 2001
Rosa hybrida cv. Baronesse am Shoot multiplication Carelli and Echeverrigaray, 2002
Rosa damascena and Rosa bourboniana am Shoot multiplication, rooting Pati et al., in press
am—axillary meristems; apm—apical meristems; lb—lateral bud; st—shoot tip.
P.K. Pati et al. / Biotechnology Advances 24 (2006) 94–11496
1988). Similar results were also reported in pears
(Lane, 1979) and liliac (Hildebrandt and Harney,
1983).
2.1.1.2. Sterilization. For initiation of aseptic cultures,
a thorough knowledge of the physiological status
and the susceptibility of the plant species to
different pathological contaminants is required. In
most of the explants, the commonly adopted procedure
involves surface sterilization of initial explants
with 70% (v/v) ethanol for 20–30 s followed by 0.1%
HgCl2 for 5–7 min and rinsing in sterile distilled water
(Rout et al., 1989a,b, 1990; Skirvin et al., 1990).
However, Khosh-Khui and Sink (1982a,b), Skirvin
and Chu (1979) and Hasegawa (1979) sterilized the
shoot tips using sodium hypochlorite (5.25%) and
bTween 20Q or Triton X (0.1%) for 5–10 min followed
by washing in sterile distilled water. Salehi and
Khosh-Khui (1997) used sterile solution of different
antibiotics (gentamycin, ampicillin, tetracycline or
amoxicillin) at different concentrations and duration
for disinfection from internal contaminants. They noticed
that use of an antibiotic solution before surface
sterilization was unsuccessful. However, dipping in
100 mg/l solution of gentamycin or ampicillin after
surface sterilization resulted in the highest percentage
of disinfected explants. Such difference could be understood
as during surface sterilization, the exposed
ends of the explants are removed leaving the fresh
conducting tissue, which allows the antibiotic solutions
to penetrate deep inside the tissue resulting in
higher rates of disinfection.
2.1.1.3. Browning of the medium. Browning of the
medium is the result of oxidation of polyphenols exuded
from the cut surface of the explants which could be
overcome by adding substances such as PVP (polyvinyl
pyrrolidone), citric acid or ascorbic acid or resorting to
frequent subculturing (Rout et al., 1999) or incubating
cultures for a day or two in total darkness after inoculation
as polyphenol oxidase activity was found to be
induced by light (Pittet and Moncousin, 1981). Curir et
al. (1986) stated that 3 days of culture on the medium
containing charcoal followed by transfer to a fresh
medium was highly effective in enhancing the growth
of primary explants.
Fig. 1. (a–d) Micropropagation of Rosa damascena. (a) Axillary bud proliferation. (b) Shoot proliferation in liquid and agar-gelled medium,
necrosis of shoots in gelled medium. (c) Rooting in liquid medium. (d) Hardened plants in pots.
P.K. Pati et al. / Biotechnology Advances 24 (2006) 94–114 97
2.1.2. Shoot multiplication
This is the most crucial stage of micropropagation.
The success of a micropropagation protocol, to a large
extent, depends on the rate and mode of shoot multiplication.
Various factors that influence in vitro shoot
multiplication in rose are listed below.
2.1.2.1. Species/genotypes/cultivars. Horn (1992)
marked a clear effect of genotypes on in vitro propagation
in different cultivars of Floribunda and Hybrid
Tea rose. He observed that it was easy to propagate
cultivars Kardinal and Lilli Marleen, whereas it was
very difficult to propagate Anthena, Mercedes, Pasadena
and Golden times. Khosh-Khui and Sink
(1982a,b) observed that the rate of shoot multiplication
of R. hybrida cvs. Tropicana and Bridal Pink, R.
damascena and R. canina varied significantly in different
subculture periods. While, cultivar Bridal Pink
showed the highest number of shoots (5.5F0.2), R.
canina had only 3.6F0.15 shoots. In R. damascena
5.1F0.18, and in Tropicana 4.4F0.27 shoots were
produced per culture in a 4-week subculture period.
Bressan et al. (1982) reported varied responses of two
different cultivars of R. hybrida to BAP. At low
concentrations of BAP (0.03–0.3 mg/l), the development
of axillary buds was stimulated in cv. Gold Glow
but not in cv. Improved Blaze. The influence of
genotype on shoot proliferation could easily be interpreted
by linking it with the recent progress in functional
genomics of plants. 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 hormone
levels has also been reported (Tantikanjana et
al., 2001).
2.1.2.2. Media. Murashige and Skoog’s (1962) medium
(MS) was found to be the most commonly used for
rose propagation. Davies (1980) reported that the standard
MS medium induced the best rates of shoot proliferation
in different rose cultivars. However, the use of
other media has also been reported. Pittet and Moncousin
(1982) used Linsmair and Skoog’s medium supplemented
with BAP (0.5 mg/l) and IBA (0.1 mg/l) for
shoot initiation. Norton and Boe (1982) also used Linsmair
and Skoog’s medium and obtained faster rates of
proliferation with BAP (0.1–2.5 mg/l). Other media like
Gamborg’s and Lee and de Fossard’s were used by
Alekhno and Vysotskii (1986). Quorine Lepoivre
(QL) and woody plant medium (WPM) were used for
micropropagation of R. hybrida cv. Moneyway (van der
Salm et al., 1994).
2.1.2.3. Inorganic salts and organic compounds. Avramis
et al. (1982), Curir et al. (1986) and Valles and
Boxus (1987) found higher multiplication rates in different
species when the ammonium content of the
medium was reduced. Davies (1980) investigated the
effect of different nutrient salt concentrations on the
multiplication of seven cultivars in R. hybrida. He also
reduced (NH4)2 SO4 but found that unchanged MS salts
proved to be the best. Bressan et al. (1982), however,
mentioned a negative influence of low salt concentrations
on vigor and viability of shoots in R. hybrida cvs.
Improved Blaze and Gold Glow. The leaf yellowing
and shoot tip necrosis could be controlled by doubling
Ca concentration in the medium (Podwyszynska and
Olszewski, 1995). Moreover, increasing the concentration
of Ca, Mg, Fe and Mn in the medium markedly
improved the quality of microshoots. The better performance
of FeEDDHA, as compared to FeEDTA, was
observed in R. hybrida (van der Salm et al., 1994).
Martin et al. (1981) and Skirvin and Chu (1979) recommended
the use of organic compounds of MS medium
or with modifications, especially with respect to the
vitamin concentration.
2.1.2.4. Carbohydrates. Sucrose (3%) was commonly
used as a source of carbohydrate. In general for tissue
culture, Murashige and Skoog (1962) also stated that
the use of 3% sucrose is better than 2% or 4%. However,
there are many accounts on the use of higher
concentrations of sucrose both for shoot initiation and
proliferation. Davies (1980) had used a range of sucrose
concentration from 2 to 8% and indicated that 4 to 5%
was the best for culture shoot tips of seven rose varieties.
Choudhary (1993), on the other hand, indicated
that there was not much difference in the proliferation
of shoots in the variety Priyadarshini while using 3% or
4% sucrose. The use of glucose in the culture medium
has also been reported in one case (Marcelis van Acker
and Scholten, 1995).
2.1.2.5. Growth regulators. In vitro shoot proliferation
and multiplication are largely based on media
formulations containing cytokinins as a major PGR,
whereas, in some cases, low concentrations of auxins
and GA3 were also used (Table 2). Inclusion of BAP
(1.0–10.0 mg/l) in the culture medium was essential for
bud break and shoot multiplication of R. hybrida (Hasegawa,
1980; Wulster and Sacalis, 1980). Bressan et al.
(1982) reported maximum promotive effects with BAP
as compared to 2-isopentyladenine (2-ip). Addition of
IAA neither enhanced nor repressed shoot multiplication
regardless of the BAP concentration. Barna and
P.K. Pati et al. / Biotechnology Advances 24 (2006) 94–11498
Wakhlu (1995) and Kumar et al. (2001) have reported
the use of thidiazuron (TDZ) in the micropropagation of
R. hybrida and R. damascena var. Jwala, respectively.
Another report indicated that the presence of cytokinin
in the culture medium helped in the year round multiplication
of shoots in hybrid roses (Rout et al., 1990).
They observed a high percentage of bud break in a
hormone-free medium within 10–12 days but the rate of
growth was found to be very poor. Media supplemented
with BAP or BAP+GA3, however, induced early bud
break within 6–8 days of culture with enhanced rates of
shoot multiplication. Addition of BAP (2.0–3.0 mg/l) as
the only growth regulator in the culture medium
resulted in feeble callusing at the cut ends of the
explants and the shoot elongation was considerably
slow with 4.26F0.23 shoots per explant in a 60-day
culture period and the explant response varied from 63
to 80%. Incorporation of GA3 at low concentrations
(0.1–0.25 mg/l) in the BAP supplemented medium
improved explant response up to 95% with more than
seven shoots per explant. Vijaya et al. (1991) reported
that BAP was the most effective growth regulator in
stimulating shoot proliferation. Further they investigated
the use of 3 auxins in combination with BAP and
found that NAA was more effective than IAA or IBA in
the production of multiple shoots. Pati et al. (2001)
optimized the BAP concentration at 5 AM for shoot
proliferation in R. damascena and R. bourboniana.
Micropropagation of rose has also been reported to
be influenced by ethylene concentration. Kevers et al.
(1992) reported that pulse treatments of ethylene enhanced
shoot proliferation with 5 ppm concentration
being the most favourable. Ethylene also inhibited
shoot elongation. Similarly, Horn (1992) reported that
an increased ethylene level markedly increased senescence
while reducing shoot growth but enhanced shoot
number and fresh weight. Use of inhibitors of ethylene
biosynthesis, such as AVG (Aminoethoxy vinyl glycine)
and CoCl2, increased multiplication rate by inducing
more number of axillary shoots (Kevers et al.,
1992). Gaspar et al. (1989), however, did not find any
effect of AVG or CoCl2 on shoot proliferation.
2.1.2.6. Status of the medium. For plant tissue culture
media, both agar and phytagel, which are natural polysaccharides
with higher capabilities of gelation are
commonly used as gelling agents. Their gels combine
with water and absorb other compounds. Agar is most
frequently used because of its desirable characteristics
such as clarity, stability and its inertness (Ibrahim,
1994). However, phytagel, the alternative gelling
agent, is increasingly being used because it forms a
relatively clear gel and contains no contaminants
(Pierik, 1987). Marginally higher multiplication rates
in phytagel medium in R. damascena and R. bourboniana
(Pati et al., in press) have been attributed to the
Table 2
Commonly used growth regulators for in vitro shoot proliferation in rose
Species/Cultivars Growth regulators (mg/l) Reference
BAP TDZ NAA IAA IBA GA3
Rosa hybrida cv. Improved Blaze 3.0 0.3 Hasegawa, 1979
Rosa hybrida cv. Forever Yours 2.0 0.1 Skirvin and Chu, 1979
Rosa hybrida 2.0 0.004 0.1 Davies, 1980
Rosa hybrida cvs. Improved Blaze 1.0–10.0 0.3 Hasegawa, 1980
Rosa canina 0.1–2.0 0.1–1.0 Martin et al., 1981
Rosa hybrida cvs. Improved Blaze,
Gold Glow
1.0 0.3 Bressan et al., 1982
Rosa indica cvs. Major, Lusambo 2.0 Avramis et al., 1982
Rosa hybrida cvs. Tropicana, Bridal Pink,
Rosa damascena, Rosa canina
1.0–2.0 0.1 Khosh-Khui and Sink, 1982a
Rosa hybrida cvs. Tropicana, Bridal Pink 1.0–2.0 0.05–0.15 Khosh-Khui and Sink, 1982b
Rosa persica Âxanthina 1.0–2.0 0.005 Tweddle et al., 1984
Rosa hybrida cvs. Duftwolke, Iseta 2.0 0.1 Sauer et al., 1985
Rosa hybrida 0.22–2.20 Barna and Wakhlu, 1995
Cut rose cv. Samantha 2.0 0.1 He et al., 1996
Rosa hybrida cv. Crimson Glory 2.0 0.02 0.01 Syamal and Singh, 1996
Miniature rose dThe FairyT 0.25 0.1–0.25 0.25 Sahoo and Debata, 1997
Rosa hybrida cv. Sonia 2.0 0.1 0.01 Singh and Syamal, 1999
Rosa damascena var. Jwala 0.22 or 0.55 Kumar et al., 2001
Rosa hybrida cv. Baronesse 3.0 0.5 Carelli and Echeverrigaray, 2002
Rosa damascena and Rosa bourboniana 1.12 Pati et al., in press
P.K. Pati et al. / Biotechnology Advances 24 (2006) 94–114 99
above difference between the two gelling agents. A
similar study in R. hybrida was carried out by Podwyszynska
and Olszewski (1995), where increased shoot
length and fresh weight were observed in phytagel
medium but phytagel was not recommended for use
because of the poor quality of shoots. No such adverse
effect of phytagel was observed by Pati et al. (in press),
while working on micropropagation of R. damascena
and R. bourboniana.
While studying water status and growth of rose
plants cultured in vitro, Ghashghaie et al. (1991)
reported that vitrification impaired shoot proliferation
in liquid medium and that the shoot multiplication was
correlated with agar concentration. Chu et al. (1993)
reported that the growth of miniature roses in liquid
medium was greater as compared to those cultured in
two-phase (solid–liquid) medium or solid medium
alone. However, in our experiments with R. damascena
and R. bourboniana (Pati et al., in press), the
quantity of liquid medium was found to play an
important role in shoot growth and multiplication
which completely eliminated the possibility of vitrification
and facilitated better quality shoots in terms of
shoot length and thickness as compared to gelled
medium (Fig. 1b). The lowest multiplication rate
was recorded in 40 ml medium, which could be due
to hyperhydricity as a result of lower availability of
oxygen and submergence of shoots. Alternatively, 20
ml medium was found to be most suitable with respect
to optimal shoot proliferation as well as sustenance for
up to 6-week subculture period (Pati et al., in press).
A similar response was also observed in case of a
miniature rose (Chu et al., 1993), bamboo (Godbole et
al., 2002) and in tea (Sandal et al., 2001). Further,
elimination of agar in the liquid medium reduces the
cost (Sandal et al., 2001). Therefore, our effort in the
complete elimination of agar in the multiplication
medium, the use of lower volume of liquid medium,
prolong the culture period and increased rate of multiplication
of shoots in liquid medium all accounted
for substantial cost reduction (Pati et al., in press).
Moreover, better response in static liquid cultures
could be ascribed to (1) a better contact between
explants and the liquid medium which increases the
availability of cytokinins and other nutrients in liquid
state (Debergh, 1983), (2) dilution of any exudates
from the explants in liquid medium (Ziv and Halevy,
1983), and (3) adequate aeration in liquid media
which ultimately enhances growth and multiplication
(Ibrahim, 1994). Further, the successful establishment
of cultures in liquid medium has several other advantages
(Smith and Spomer, 1995; Chu et al., 1993) and
is an important step towards automation (AitkenChristie
et al., 1995).
2.1.2.7. Physical factors.
2.1.2.7.1. Light. Perusal of literature indicates that
light intensity plays an important role for satisfactory
shoot growth and 1 to 3 klux (ca. 16–46 Amol mÀ2
sÀ1
)
is reported to be sufficient for shoot proliferation in rose
(Horn, 1992). Bressan et al. (1982) described that 17
Amol mÀ 2
sÀ 1
for 12–14 h daily was optimal for shoot
multiplication. Capellades et al. (1990), however,
opined that cultured plantlets could resemble greenhouse
grown plants if these were cultured under high
light intensity (80 Amol mÀ2
sÀ 1
) as compared to the
light intensity that is normally available inside the
culture vessels (25 Amol mÀ 2
sÀ1
).
Usually 16-h light duration from Gro-lux type white
fluorescent lamps or a cool source of white light is
provided in culture conditions. Davies (1980) observed
that 16-h illumination showed better growth and multiplication
than 8 h and this fact is further corroborated
by the results of Bressan et al. (1982) which showed
improvements following an increase in light period
between 4 and 16 h.
2.1.2.7.2. Temperature. Leyhe and Horn (1994)
recorded optimal shoot formation at 21 8C for different
cultivars of R. hybrida. A similar observation
was also made by Rout et al. (1999) where they
noticed reduced shoot multiplication above 21 8C.
However, many researchers have used a temperature
of around 25 8C for shoot multiplication (Horn,
1992; van der Salm et al., 1996; Carelli and Echeverrigaray,
2002).
2.1.2.7.3. Growth room and vessel humidity. Begin-Sallanon
and Maziere (1992) reported that an increase
of the vapour pressure deficit (VPD) of the
growth room from 600 to 2000 Pa increased the air
VPD inside the culture vessels. Under high evaporating
conditions, the multiplication rate was significantly
higher, leaves were small, light green and slightly epinastic.
Shoots were long (1.5F0.2 cm) and pink–green,
due to the presence of anthocyanin pigments, thereby
indicating a juvenile characteristic of the plants in vitro.
2.1.2.7.4. CO2. Woltering (1990) reported that
CO2 had a significant effect on the growth of in vitro
rose shoots. Higher concentration of CO2 (5%), coupled
with low light, had a positive effect on plant growth.
2.1.3. Rooting of microshoots
For any micropropagation protocol, successful rooting
of microshoots is a pre-requisite to facilitate their
establishment in soil. Considerable work has been done
P.K. Pati et al. / Biotechnology Advances 24 (2006) 94–114100
to enhance rooting efficiency in different rose varieties.
Rooting of microshoots can be accomplished both
under in vitro and ex vitro conditions.
2.1.3.1. In vitro rooting of microshoots. The in vitro
rooting capacity depends on the interaction of internal
and external factors (Hyndman et al., 1982a). These are
described as follows.
2.1.3.1.1. Species/cultivars. According to Horn
(1992), rooting response in rose was cultivar dependent
and in certain species up to 100% success could be
achieved. Kirichenko et al. (1991), while optimizing
factors for in vitro multiplication of ornamental and
essential oil bearing roses, reported that rooting of
microshoots of the essential oil bearing roses was difficult
as compared to the ornamental varieties. A similar
kind of observation was also made by Khosh-Khui and
Sink (1982a) where they reported a lower rooting ability
in old world spp. (R. canina and R. damascena) as
compared to R. hybrida.
2.1.3.1.2. Age and size of microshoots. Rooting
also depended on the age and size of the microshoots.
Rout (1991) compared the performance of microshoots
0.5–1.0 cm (4 weeks old); 1.5–2.0 cm (6 weeks old)
and 2.0–2.5 cm (8 weeks old) of six cultivars of hybrid
roses for their rooting response. Better rooting (92–
98%) was achieved when 6-week-old shoots (1.5–2.0
cm) were dipped in IBA (1.0 mg/l) solution for 15–20
min before being placed in a sterile medium containing
sand–FYM–soil mixture in the ratio of 2:1:1.
2.1.3.1.3. Media. Badzian et al. (1991) reported
the use of MS medium with major elements reduced to
one quarter to one third strength for root induction.
Khosh-Khui and Sink (1982c) reported that half-strength
MS medium supplemented with NAA (0.54 AM) was
adequate for inducing rooting in cv. Bridal Veil of hybrid
rose. Sauer et al. (1985) reported that 10 different rose
cultivars rooted within 10–14 days on one third strength
of MS medium supplemented with IAA at 5.7 AM.
Subsequently, Douglas et al. (1989) achieved very high
percentages of rooting in cv. Queen Elizabeth using long
shoots (N2 mm) and by dilution of MS medium to one
fourth strength without a growth regulator.
2.1.3.1.4. Inorganic salts. Relatively low salt concentrations
in the medium are known to enhance rooting
of microshoots (Murashige, 1979). Hyndman et al.
(1982a) succeeded in enhancing root number and
length of in vitro grown shoots of R. hybrida cv.
Improved Blaze by lowering total nitrogen concentration
of MS salts (6.0 to 7.5 mM) in the culture medium
keeping other salt concentrations constant. Further,
when the NO3/NH4 ratio increased from 0.1 to 3.0,
there was an increase in the number of roots/explant.
Hyndman et al. (1982a) demonstrated that a decrease in
KNO3 and NH4NO3 concentration was the decisive
factor for improving the rooting percentage.
2.1.3.1.5. Carbohydrates. Sucrose concentration
acts as an enhancer of osmotic potential and also plays
a vital role in root induction. Lakes and Zimmerman
(1990) showed highest rooting percentage in apple on a
medium with high osmolarity. Rout et al. (1990) reported
that rooting of microshoots was better on a solid medium
as compared to the liquid medium. Damiano et al. (1989)
observed enhancement in in vitro rooting by using a
double layered medium, i.e., agar-gelled medium with
an upper layer of liquid medium. Lloyd et al. (1988)
reported that excised microshoots rooted well on sorbarod
plugs (Cellulose support plugs; Sorbarod, Baumgartner
Papiers SA, Switzerland) soaked in liquid
medium. On an average, about 45% of shoots were
rooted within 2 weeks of culture. It was further observed
that root length was short in solid medium as compared to
liquid medium (Ebrahim and Ibrahim, 2000). The retardation
in root length in the solidified medium may be
attributed to the relatively lower aeration in agar-gelled
medium for good root development as indicated earlier
by Davies (1980). However, Davies (1980) achieved
100% rooting in several cultivars of rose by using MS
medium devoid of growth regulators but supplemented
with 40 g/l sucrose. Hyndman et al. (1982b) also observed
more and larger roots with an increase in sucrose
concentration from 87.64 to 262.93 mM.
Suharsono (1995) observed that in different rose
cultivars viz., Eterna G, Lancome T10 and La Jocande
G2, root colour was influenced by the sugar used in
the medium. It was white when the medium contained
glucose and black when sucrose was used. In R.
damascena and R. bourboniana, it was observed that
besides the influence of PGRs, an increase in sucrose
concentration resulted in a corresponding increase in
the root length rather than root number (Pati et al., in
press). Moreover, beyond 3% sucrose, browning of
root tips occurred. Although, at this point of time, it
is difficult to establish the reason, yet one of the
reports in maize plant suggested that certain pigment
synthesizing genes were switched on in the presence of
sucrose (Jeannette et al., 2000) However, the same
report indicated that no pigmentation was observed
in the presence of sorbitol which was used as an
osmoticum. This indicates that the darkening of the
roots is due the presence of sucrose as carbohydrate
not as osmoticum. Perhaps this explanation could also
be extrapolated for browning of root tips in the presence
of higher concentration of sucrose in R. damasP.K.
Pati et al. / Biotechnology Advances 24 (2006) 94–114 101
cena and R. bourboniana. Further, experimental evidence
is required to establish this. When a comparison
was made on rooting response in both agar-gelled and
liquid media, there was only 5% rooting in the former
and 85% response in liquid culture medium. Addition
of mannitol (0.5 M) to an agar-gelled medium led to a
marked increase in percent rooting up to 81%. A
gradual decline of root induction beyond this concentration
of mannitol indicated a clear correlation of the
osmotic potential of the medium to root induction.
Critical analysis of the liquid and agar-gelled medium
with 0.5 M mannitol revealed that they had a similar
osmotic potential. This distinct role of the osmotic
potential in root induction clearly throws light on
subtle differences in induction of roots between varieties
that can now be easily managed by shifts in
osmotic potential of culture media. This also reflects
that agar-gelled media do not have an adverse effect on
the cultures or inhibit rooting but they are at a lower
osmotic potential than the liquid cultures.
2.1.3.1.6. Activated charcoal. Activated charcoal
when added to the culture medium was found to have
a remarkable positive influence on the rooting efficiency
of cultured rose microshoots in cv. Folklore, wherein
better rooting in terms of number of roots/shoot and
root length was obtained (Wilson and Nayar, 1995).
Moreover, addition of activated charcoal was found to
reduce the days required for root initiation from 8.5
days to 7.5 days.
2.1.3.1.7. Growth regulators. In most of the earlier
reports, varying concentrations of different auxins
were used for root induction (Table 3). However,
Skirvin and Chu (1979) induced rooting of microshoots
on growth regulators free solidified medium.
The roots were easily induced from excised mature
microshoots on MS medium supplemented with low
concentrations of auxins (IAA or IBA or NAA) in the
range of 0.1–0.5 mg/l (Hasegawa, 1979, 1980). Rooting
of microshoots was also achieved by dipping the
cut ends of shoots for a few hours in an aqueous
solution of 1 mM of IAA instead of being continuously
cultured on auxin containing medium (Collet, 1985).
Khosh-Khui and Sink (1982c) reported the effect of
different concentrations and combinations of auxins on
root formation in R. hybrida cv. Bridal Pink. They also
indicated that a combination of IAA (0.0–0.1 mg/l) and
NAA (0.0–0.1 mg/l) was quite effective in induction of
roots. Arnold et al. (1995) reported that microshoots of
R. kordesii cvs. John Franklin and Champlain rooted
well in MS medium with low or no auxin. Optimum
rooting in cv. Champlain was achieved at a high
concentration of IAA with low concentrations of salts
or intermediate concentrations of IBA and NAA with
low to medium concentrations of salts. Excised shoots
of R. hybrida cv. Peace rooted well on O MS supplemented
with 0.5 mg/l IBA. A close scrutiny of literature
indicates that rooting response with different
auxins is also cultivar dependent and it is rather diffiTable
3
Commonly used growth regulators for in vitro rooting in rose
Species/cultivars Growth regulators (mg/l) Reference
NAA IAA IBA 2,4-D GA3
Rosa hybrida cv. Improved Blaze 0.3 Hasegawa, 1979
Either 0.03–0.1 or 1.0 Hasegawa, 1980
Rosa hybrida 0.05–0.1 Davies, 1980
Rosa canina 0.1–1.0 0.1–1.0 Martin et al., 1981
Rosa indica 0.1 Avramis et al., 1982
Rosa hybrida cvs. Improved Blaze,
Gold Glow
1.0 Bressan et al., 1982
Rosa hybrida cvs. Tropicana, Bridal Pink,
Rosa damascena, Rosa canina
0.1 0.05 Khosh-Khui and Sink,
1982a
Rosa hybrida cvs. Tropicana, Bridal Pink 0.1 Khosh-Khui and Sink,
1982b
Rosa persica Âxanthina 0–0.3 Tweddle et al., 1984
Rosa hybrida cvs. Duftwolke, Iseta 2.0 Sauer et al., 1985
Rose cv. Folklore 1.0 1.0 Wilson and Nayar, 1995
Cut rose cv. Samantha 0.5 1.0 0.5 He et al., 1996
Rosa hybrida cv. Crimson Glory 0.2 0.5 Syamal and Singh, 1996
Miniature rose dThe FairyT 0.25 0.1 Sahoo and Debata, 1997
Rosa hybrida cv. Sonia 0.1 0.2 Singh and Syamal, 1999
Rosa damascena var. Jwala Kumar et al., 2001
Rosa damascena and Rosa bourboniana 2.0 Pati et al., in press
P.K. Pati et al. / Biotechnology Advances 24 (2006) 94–114102
cult to induce rooting in oil bearing rose cultivars
(Kirichenko et al., 1991). In R. damascena and R.
bourboniana rooting was initiated as a two-step procedure
where IBA (10 AM) 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., in press). Chu et al. (1993) reported that microshoots
of R. chinensis cultured in liquid medium with
BAP were difficult to root and only shoots that were
maintained on a medium without BAP developed
roots. However, we observed that there was no difficulty
in rooting of microshoots that are cultured in
liquid medium supplemented with BAP (Fig. 1c).
2.1.3.1.8. Culture vessel. The importance of culture
vessel in plant tissue culture has been well established.
McClelland and Smith (1990) showed that
woody plant explants routinely produced denser shoot
cultures when grown in larger vessels. The quality of
individual shoots was significantly better, shoot length
in many species was enhanced, and size of individual
leaves also increased with increase in the size of vessel.
The rooting potential for microshoots produced in large
sized vessels was also substantially improved, probably
in part due to enhanced leaf area and rooting co-factors
present in these leaves as per their hypothesis. However,
Mackay and Kitto (1988) found that culture vessels that
were excessively large also inhibited shoot length compared
to medium sized vessels. Campos and Pais (1990)
emphasized the choice of a container to be fundamental
in achieving a good rate of success on rooting of dwarf
rose cultivars dRosamani.T They observed that the
microshoots rooted in tubes were more vigorous and
with better roots with higher survival percentage than
those rooted in flasks. Kozai et al. (2000) suggested that
the use of large culture vessels for commercial scale
propagation could reduce production costs significantly
Fig. 2. (a–c) Culture vessel for rooting of microshoots of R. damascena. (a) Polypropylene box with bunches of shoots inserted in perforated
platform with their cut ends touching the medium. (b) Perforated platform turned upside down to show profuse and healthy rooting of microshoots.
(c) Rooted microshoots after 42 days of incubation in rooting vessel.
P.K. Pati et al. / Biotechnology Advances 24 (2006) 94–114 103
by reducing the labour cost nearly half that of conventional
system.
Considering the importance of culture vessel for
efficient rooting of microshoots, a rooting vessel was
designed and developed in our laboratory (Pati et al.,
in press; Patent File No. 0427 DEL2001, 23/03/01).
Such unique microchambers with platforms had several
advantages: (1) a large number of shoots could be
rooted concurrently at any given time (Fig. 2a–c), (2)
elimination of agar resulted in substantial savings in
cost (6-fold cost reduction), (3) the use of liquid
culture medium in the rooting vessel provided a suitable
microenvironment for rooting of bunches of
microshoots, (4) ease of operation because of having
a large sized container with a perforated platform, (5)
damage to roots was minimized as rooted plantlets
could be easily pulled out, and (6) the shape of the
vessel in the form of a box facilitates stacking of these
boxes in a culture shelf and hence, saves on space,
thereby reducing the cost of production.
It has also been demonstrated that the period of
incubation of microshoots in rooting vessel influenced
the survival percentage of R. damascena plantlets under
greenhouse conditions. Significantly higher survival
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 period
during root induction upon survival of plantlets during
hardening or acclimatization (Pati et al., in press).
2.1.3.1.9. Physical factors.
2.1.3.1.9.1. Light. There are only a few reports on
the role of culture environment such as light on rooting.
Khosh-Khui and Sink (1982b,c) achieved about
84% rooting in cv. Bridal Pink rose with an illuminance
of about 1.0 klux. They observed that higher
illuminances (3.0 klux) inhibited rooting. Bressan et al.
Table 4
Summary of work on indirect organogenesis in rose
Species/cultivars Explant Response PGRs (mg/l) Media Reference
Hybrid Tea rose
dThe DoctorT
ss c 2,4-D (0.05–5.0) and
coconut milk
Unspecified
synthetic medium
Hill, 1967
sp NAA (0.5), KN (0.2),
GA3 (20.0)
Rosa hybrida cv.
Super Star
ps c IBA KB Jacob et al.,
1969
ads IBA (0.1), Kn (5.0)
Rosa persica Â
xanthiana
ss c BAP (1.0–2.0), NAA
(0.1–0.3)
MS Lloyd et al.,
1988
c ads BAP (3.0), NAA (0.1–0.3)
Rosa hybrida cv.
Bridal Pink
em c BAP (0.22), NAA (0.05) Modified O MS Burger et al.,
1990
ads BAP (1.0), NAA (0.01)
Rosa damascena ss c NAA (1.86) MS gelrite 2.5 g/l Ishioka and
Tanimoto, 1990
c ads BAP (2.25), IAA (0.17) MS–NH4NO3
Rosa hybrida cv.
Landora
ls c BAP (0.5), NAA (0.1) Modified O MS Rout et al., 1992
ss BAP (2.0), NAA (0.5)
c ads BAP (0.2), NAA (0.01),
GA3 (10–20)
Modified O MS,
Addl. 600 mg/l
proline/glutamine
Rosa hybrida cv.
Melrutral
l, r c BAP (3.0), NAA (0.2) MS Arene et al.,
1993
ads
Rosa multiflora
dThornlessT
Is c TDZ (1.0) MS Rosu et al.,
1995
c ads
Rosa hybrida cv. ls, ss c 2,4-D (2.2–22.1) or MS Hsia and Korban, 1996
Carefree Beauty NAA (2.0–20.11)
and Rosa chinensis c ads TDZ (5.0), GA3 (1.0) O MS
cv. Red dSunblazeT
and dBaby KatieT
ads—adventitious shoot; c—callus; em—excised embryo; l—leaf; ls—leaf segment; ps—pith segment; r—root; ss—shoot primordial; ss—stem
segment.
P.K. Pati et al. / Biotechnology Advances 24 (2006) 94–114104
(1982), however, found that 66 Amol mÀ2
sÀ1
for 12–
14 h proved to be the best for rooting. Further, they
observed better rooting in the microshoots by shading
the lower portion of the culture vessels. Rooting enhancement
was also noticed in cultures kept in total
darkness (Norton and Boe, 1982). In R. damascena
and R. bourboniana, root initiation decreased by 20%
under light as compared to those in dark. Skirvin and
Chu (1984) found that red light had a beneficial effect
on the rooting of miniature roses.
2.1.3.1.9.2. Temperature. The effect of temperature
on root emergence and final rooting percentage
was assessed by Alderson et al. (1988). In cv. dDainty
DinahT on the IBA medium, roots emerged first at 25
8C and approximately 2 and 4 days later, at 20 8C and
15 8C, respectively. Horn (1992) obtained best rooting
response at 21 8C and plants cultured at this temperature
were also the most successful in the weaning stage.
However, Rahman et al. (1992) stated that a temperature
of 28 8C was optimum for in vitro rooting.
2.1.3.2. Ex vitro rooting
Considerable attempts have also been made for root
induction under ex vitro conditions. Pittet and Moncousin
(1982) obtained 100% rooting of cultured shoots
dipped in NAA (1.0 mg/l) for 1 h before planting these
in perlite. Avramis et al. (1982) obtained 100% rooting in
R. indica in peat vermiculite mixture after treating shoots
for 5 days in a shaken mineral solution supplemented
with NAA (5.5 mg/l) and sucrose (60 g/l). While treatment
of shoots with IBA (50–100 mg/l) resulted in good
rooting (Bini et al., 1983). Collet and Le (1987) also
studied root induction in microshoots and found that a
brief treatment of 16-h dip in 1 mM IAA gave good
results as compared to long treatment of 7 days.
2.1.3.3. Comparison of in vitro and ex vitro rooting
A comparison of in vitro and ex vitro rooting has
been made by Rogers and Smith (1992) in miniature
roses (R. chinensis cv. Minima). They indicated that
after 18 days of treatment, either in vitro (MS medium
+0.5 AM NAA) or ex vitro (dipping in 0.1% IBA in a
1:1Dpeat:sand mixture) resulted in 95–100% rooting
in three cultivars (cvs. Ginny, Red Ace and Tipper). In
contrast, in cultivar Debut, 73% of shoots rooted ex
vitro as compared to 93% microshoots. However, ex
vitro rooting produced longer and more number of
roots with increased root area. Ex vitro roots were
flexible, branched and had root hairs, whereas in vitro
roots were brittle, unbranched and without root hairs.
Table 5
Summary of work on direct organogenesis in rose
Species/cultivars Explant
source
Induction Medium (PGRs mg/l)
Regeneration
Additional information Reference
Rosa persica Âxanthiana r – MS+BAP (2.0) Shoot bud arose from
the region of pericycle
of the root segment
Lloyd et al., 1988
Rosa persica Âxanthiana l MS+BAP (0.5l) Shoots formed directly
on the petiole and mid
rib of leaflets
Rosa laevigata
Rosa hybrida l Following
unpublished
protocol
– Leffering and Kok,
1990
Rosa persica Âxanthiana r – MS+BAP (2.0) Shoot bud arose from
the region of pericycle
of the root segment
Lloyd et al., 1988
Rosa hybrida cv. Melrutral l – MS+BAP (2.2 AM) – Arene et al., 1993
r MS+BAP (8.9 AM)
Rosa hybrida cvs.
Madelon, Only Love,
Presto, Sonia, Tineke
l, pt Modified
MS+TDZ
(1.5)+NAA
(0.05)+casein
hydrlysate (100)
MS+BAP (0.5)+NAA
(0.01)+FeEDDAH (100)
Early and higher shoot
bud emergence in the
induction medium
with AgNO3
Dubois and de Vries,
1995
Rosa hybrida lt MS+TDZ
(1.5)+IBA (0.1)
MS+BAP
(0.5)+IBA (0.01)
Addition of AgNO3
enhanced regeneration
Ibrahim and Debergh,
1999
Rosa damascena pt O MS+TDZ
(1.5)+NAA (0.05)
MS+BAP
(0.5)+NAA (0.01).
Addition of AgNO3
enhanced regeneration
Pati et al., 2004
l—leaf; lt—leaflet; pt—petiole; r—root.
P.K. Pati et al. / Biotechnology Advances 24 (2006) 94–114 105
Table 6
In vitro somatic embryogenesis in rose
Species/cultivars Explant Response PGRs (mg/l) Media Reference
Rosa hybrida cvs. Domingo,
Vicket Brown, Tanja, Azteca
l, pt Embryogenic callus KN (0.1), NAA (0.05) or, KN
(0.1), +NOA (0.1)
O MS de Wit et al., 1990
Embryo development
Rosa hybrida f Callus induction 2,4-D (2.0), Zeatin (1.5) Modified B5 Noriega and Sondahl,
1991Embryogenic callus NAA (0.25), Zeatin (1.5),
GA3 (1.5)
Modified MS
Embryo development GA3 (1.0), ABA (2.0)
Somatic embryo
maturation
GA3 (1.0), ABA (0.2)
Somatic embryo
germination
– Modified O N6
Rosa hybrida cv. Landora ss, l Embryogenic callus BAP (0.5), NAA
(1.0), 2,4-D (0.5–2.0)
O MS Rout et al., 1991
Embryo development BAP (0.5), NAA
(0.01), GA3 (0.1)
O MS+l-proline (200–800 mg/l)
Somatic embryo
germination
BAP (0.5), GA3 (0.1) O MS+Adenine sulphate
(24.7 AM)
Rosa hybrida cv. Melrutral l, r Embryogenic callus BAP (3.0), NAA (0.2) MS Arene et al., 1993
Embryo development
Rosa rugosa Ims Embryogenic callus No PGRs MS Kunitake et al., 1993
Embryo development No PGRs MS, Fructose (0.1M)
Embryo germination No PGRs MS, sorbitol (0.1 M)
Rosa hybrida cvs. Glad
Tidings, Trumpeter
pt, r Embryogenic callus 2,4-D (1–4) MS or SH, l-proline
(300–1200 mg/l)
Marchant et al., 1996
Embryo development
Embryo maturation 2,4-D (1), ABA (1), GA3 (0.3) MS or SH, l-proline
(300–600 mg/l)
Embryo germination BAP (0.5–1.0), IBA (0.1) MS or SH, Maltose
(30 g/l), Agarose (4 g/l)
P.K.Patietal./BiotechnologyAdvances24(2006)94–114106
Rosa hybrida cv. Carefree Beauty
and Rosa chinensis cv. Red
dSunblazeT and dBaby KatieT
l, ss Embryogenic callus 2,4-D (2.2–22.1) or NAA
(2.0–20.11)
MS Hsia and Korban, 1996
Embryo development TDZ (5.0), GA3 (1.0) O MS
Rosa hybrida cv. Arizona pt Embryogenic callus 2,4-D (0.022), 2-iP (2.45) Modified MS Murali et al., 1996
Embryo maturation ABA (0.25) Modified O MS
Embryo germination NAA (0.5), Phloroglucinol (100) O MS
Rosa hybrida cv. Moneyway adr Embryogenic callus 2,4-D (11.0) SH van der Salm et al.,
1996Embryo development No PGR
Embryo germination BAP (4.44–6.75), IBA (0–0.1)
Rosa hybrida cv. Soraya, Baccara,
Mercedes and Ronto
l Embryogenic callus p-CPA (10.0), KN (1.0) MS Kintzios et al., 1999
Embryo develoment
Embryo maturation BAP (1.17), IAA (1.0)
Embryo germination
Rosa hybrida cv. Soraya l Embryogenic callus p-CPA(10.0), KN(1.0) MS+Nicotinic acid (0.1)/Cystein (10) Kintzios et al., 2000
Embryo development
Embryo maturation BAP (1.17), IAA (1.0)
Embryo germination
50 different rose cultivars of
Hybrid Teas, Floribundas,
Miniature roses, shrubs,
ramblers, rootstocks and
wild species
l Embyogenic callus 2,4-D or NAA MS Dohm et al., 2001
Embryo development Zeatin or TDZ
Embryo germination IBA (0.1), BAP (2.0), GA3 (0.1)
Rosa hybrida L. 4th of July l Embryogenic callus 2,4-D (1.0) or NAA(1.0) MS Kim et al., 2003
Embryo development Zeatin (4.0)
Embryo germination BAP (1.0)
adr—adventitious roots; f—filament; ims—immature seed; l—leaf; ls—leaf segment; pt—petiole; r—root; ss—stem segment.
P.K.Patietal./BiotechnologyAdvances24(2006)94–114107
Moreover, ex vitro rooting is a one-step procedure
comprising of both rooting and hardening (Taslim
and Patel, 1995), facilitating early transplantation to
soil.
2.1.4. Acclimatization and field establishment
The successful acclimatization of micropropagated
plants and their subsequent transfer to the field is a
crucial step for commercial exploitation of in vitro
technology. However, the acclimatization of micropropagated
roses was reported to be a difficult procedure
because of rapid desiccation of plantlets or their susceptibility
to diseases due to high humidity (Messeguer
and Mele, 1986). Preece and Sutter (1991) and Sutter et
al. (1992) have reviewed acclimatization of micropropagated
plants in the greenhouse and in the open field.
Smith et al. (1992) developed a new method of in vitro
hardening of micropropagated rose plants. They developed
a commercial kit, which consisted of cellulose
plugs (sorbarods), for support and protecting the roots
during transfer to soil, and ventilated culture vessel to
improve the resistance of the plantlets to desiccation.
The plantlets thus grown showed better survival when
transferred to ex vitro conditions.
Douglas et al. (1989) reported that the rooting of
micropropagated plants improved significantly in the
compost before they were precultured for 2 weeks in
vitro on sorbarods soaked in liquid medium containing
IAA. Sorbarods provided the necessary protection and
facility for proper handling of the microshoots and
helped in the production of larger roots, which facilitated
100% successful establishment. Roberts et al.
(1992) reported that the use of sorbarod transplantation
plugs and growth retardants in the culture medium
reduced endogenous gibberellins and prevented wilting
of in vitro grown rose plantlets when transferred to soil,
resulting in their better survival.
Davies (1980) achieved 15–85% rooting in a mixture
of coarse perlite, peat and loam (2:2:1) depending upon
the cultivar. Campos and Pais (1990) reported that about
83–100% of plantlets of dwarf rose cultivar Rosamini
established well in soil within 45 days of transfer. The
survival rate was higher than that reported in many other
rose species (Khosh-Khui and Sink, 1982a,b; Dubois et
al., 1988).
Relative humidity (RH) plays a crucial role in
acclimatization and survival of tissue culture raised
plants. The percentage survival of the micropropagated
hybrid roses cvs. Landora, Queen Elizabeth,
Virgo, Sea pearl and Happiness varied from 92 to
98% when transferred to the greenhouse at 80–85%
RH and planted in earthen pots containing sand:
soil:FYMD2:1:1 (Rout et al., 1989a). On the other
hand, Bhat (1992) described a procedure where
plantlets were acclimatized by incubating initially at
70–80% RH for 3 days before planting in vermiculite,
in which 95% survival was recorded. Plants
were transferred to earthen pots containing a
1:1Dsoil:leaf mould mixture and kept outdoors with
75.6% survival.
CO2 enriched environment and high photosynthetic
photon flux density (PPFD) can increase photosynthetic
capacity and improve the acclimatization of plantlets
under ex vitro conditions, even if they have no roots
(Gourichon et al., 1996). In our study (Pati, 2002),
these limitations were overcome by formulating a strategy
for the transfer of plantlets to the hardening chamber.
Large numbers of plantlets are normally transferred
during the month of October to March when the ambient
RH recorded to be 52% (Palampur conditions;
1300 m above msl, 3286VN; 76833VE), and hence,
chances of disease infestations are less. Further, keeping
the transplanted microshoots in a specially designed
hardening chambers enriched with CO2 (20/11Â10À5
mol lÀ 1
to 80/13Â10À7
mol lÀ 1
) favoured survival up
to 96.66%.
Age of the microshoots was also found to be important
for the establishment of rooted plantlets (Horan et
al., 1995). Our study (Pati et al., in press) indicates that
the period of incubation of microshoots in rooting
vessel also influenced the survival percentage of R.
damascena plantlets under greenhouse conditions
(Fig. 1d). Significantly higher survival 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 period during root
induction upon survival of plantlets during hardening
or acclimatization.
3. Plant regeneration
In vitro plant regeneration is often the most important
step for successful implementation of various biotechnological
techniques used for plant improvement programmes.
Induction of adventitious shoots and regeneration
from callus cultures are of importance for
somaclonal variation and, therefore, for breeding. Further,
explants can also be treated with mutagens, after or
prior to explanting, in order to obtain new genotypes by
mutation (Horn, 1992). In rose, there are many reports
which indicate rapid regeneration and multiplication
through organogenesis or somatic embryogenesis
(Tables 4–6).
P.K. Pati et al. / Biotechnology Advances 24 (2006) 94–114108
3.1. Organogenesis
3.1.1. Indirect organogenesis
Hill (1967) reported the formation of shoot primordia
on calli derived from stem explants of Hybrid Tea
rose cv. The Doctor Since then, many workers have
successfully induced shoot bud regeneration in rose by
employing appropriate combinations of auxins and
cytokinins. Summary of research work pertaining to
indirect organogenesis with a variety of species/cultivars
and different explants is presented in Table 2.
3.1.2. Direct organogenesis
There are few reports on direct regeneration of adventitious
buds in Rosa (Table 3). These reports indicate
that shoot regeneration response in the genus Rosa has
been obtained from roots, leaves, leaflets and petioles
(Lloyd et al., 1988; Leffering and Kok, 1990; Arene et
Fig. 3. Direct regeneration protocol from leaf explants of R. damascena.
P.K. Pati et al. / Biotechnology Advances 24 (2006) 94–114 109
al., 1993; Dubois and de Vries, 1995; Ibrahim and
Debergh, 1999). Further, studies on direct organogenesis
in the genus Rosa show the preponderance of use of
TDZ in inducing shoot morphogenesis on leaf explants.
On perusal of literature, it is also deduced that AgNO3
has enhanced the regenerative potential of the leaf
explants (Dubois and de Vries, 1995). However, all
these observations have been on ornamental roses. We
report that similar regeneration response can be evinced
on leaf petioles for R. damascena wherein TDZ was
effective in inducing shoot morphogenesis and AgNO3
hastened the regeneration response by at least a week. It
also highlights the importance of in vitro pruning for
obtaining uniform explants after 4 weeks of pruning.
Another feature of this study is the determination of
hypodermal origin of the regeneration response from
histological sections (Pati et al., 2004).
In our study (Pati et al., 2004), the regeneration
response was evinced from leaf petioles of R. damascena
(Fig. 3). Base of the petiole was recognized as the
regeneration site in R. hybrida (Dubois and de Vries,
1995) and many other members of the family Rosaceae
viz. Prunus canescens (Antonelli and Druart, 1990),
Rubus idaues (Cousineau and Donelly, 1991), Prunus
armeniaca and Prunus domestica (Escalettes and
Dosba, 1993). The regenerative capacity at the base of
petiole, however, could be ascribed to (a) basipetal
transport of endogenous auxins and/or carbohydrates
(Dubois and de Vries, 1995) and (b) position of the
regenerative target cells (Margara, 1982). It was also
found that addition of AgNO3 to the induction medium
advanced the emergence of shoot buds by 1 week. The
above response could be attributed to the inhibition of
ethylene synthesis by AgNO3. Moreover, ethylene plays
an important role in in vitro regeneration response of
many crops (Cambecedes et al., 1991; Escalettes and
Dosba, 1993; Table 5).
3.2. Somatic embryogenesis
Different explants such as leaf, petiole, stem and
even filaments have been used for somatic embryo
induction in scented roses and the medium was supplemented
with varied concentrations of PGRs (Table
6). However, in R. rugosa, where immature seeds
were used as explants, no PGRs were used in the
medium for embryo induction and embryo germination
(Kunitake et al., 1993). However, in R. bourboniana,
somatic embryogenesis was induced from
zygotic embryos in MS medium supplemented with
2,4-D (5–15 AM) (Fig. 4; Pati, 2002). Somatic embryos
thus formed germinated to form plantlets when
transferred to MS medium supplemented with BAP
(5 AM) (Table 6).
4. Conclusions
Rapid multiplication of elite clones, production of
healthy and disease-free plants and faster introduction
of novel cultivars with desirable traits are of urgent
need in rose improvement programme. In this regard,
in vitro propagation techniques are likely to play a
vital role. At present, there are many reproducible
protocols for in vitro propagation of rose. However,
the new challenges that are faced today by the tissue
culture industry include cost efficiency, automation,
control and optimization of the microenvironment,
etc. It is, therefore, important to bring about further
improvements in the existing tissue culture protocols.
The recent trend in shift of the status of the medium
from agar-gelled to liquid medium is a strategic step
in this direction. The effective use of liquid medium
during shoot multiplication and as rooting as well is a
cost effective proposition and a step towards automation
and commercialization. In vitro propagation of
Fig. 4. Somatic embryogenesis from zygotic embryos in R. bourboniana.
P.K. Pati et al. / Biotechnology Advances 24 (2006) 94–114110
rose via somatic embryogenesis offers a great potential
for rapid propagation and improvement, and direct
regeneration protocols using leaf explants from in
vitro raised shoots could be effectively used in maintaining
the clonal fidelity of elites and in genetic
transformation programmes.
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