In vitro separation of a rose chimera

Abstract

“Fairmount 1 thorny” (“FM1 thorny”) (a Rosa multiflora Thunb ex. J. Murr.) and a thornless sport of “FM1 thorny” (“Fairmount 1” (“FM1”)) were established invitro to investigate
chimeral segregation under various levels of BA and to obtain a pure thornless rose. While the chimeral thornless sport was
expected to segregate invitro and yield both thorny and thornless plantlets, “FM1 thorny” was to yield only thorny plants.
“FM1” segregated invitro into its constituent genotypes and yielded thorny and thornless plantlets, suggesting that “FM1”
is chimeral. “FM1 thorny” produced only thorny plants invitro. These results indicate that the “FM1 thorny” clone was not chimeral (pure thorny) and that the thornless regenerates of “FM1”
did not develop via somaclonal variation. There was a significant linear relationship between increasing BA concentration
and the percentage of thorny plants. Among a population of 690 tissue culture derived plants from all the BA experiments,
6 plants were classified as pure thornless plants 1year later.

Full text

ORIGINAL PAPER
In vitro separation of a rose chimera
Fatih A. Canli ÆRobert M. Skirvin
Received: 21 January 2008 / Accepted: 27 August 2008 / Published online: 7 September 2008
ÓSpringer Science+Business Media B.V. 2008
Abstract ‘‘Fairmount 1 thorny’’ (‘‘FM1 thorny’’) (a
Rosa multiflora Thunb ex. J. Murr.) and a thornless
sport of ‘‘FM1 thorny’’ (‘‘Fairmount 1’’ (‘‘FM1’’))
were established in vitro to investigate chimeral
segregation under various levels of BA and to obtain
a pure thornless rose. While the chimeral thornless
sport was expected to segregate in vitro and yield
both thorny and thornless plantlets, ‘‘FM1 thorny’’
was to yield only thorny plants. ‘‘FM1’’ segregated
in vitro into its constituent genotypes and yielded
thorny and thornless plantlets, suggesting that ‘‘FM1’’
is chimeral. ‘‘FM1 thorny’’ produced only thorny
plants in vitro.These results indicate that the ‘‘FM1
thorny’’ clone was not chimeral (pure thorny) and that
the thornless regenerates of ‘‘FM1’’ did not develop
via somaclonal variation. There was a significant
linear relationship between increasing BA concentra-
tion and the percentage of thorny plants. Among a
population of 690 tissue culture derived plants from
all the BA experiments, 6 plants were classified as
pure thornless plants 1 year later.
Keywords Thornless rose Chimera 
In vitro Tissue culture Breeding
Abbreviations
BA 6-benzylaminopurine
MS Murashige and Skoog
NAA Naphthaleneacetic acid
Introduction
Rose (Rosa spp.) is a member of Rosaceae family and
one of the most important commercial flower crops in
the world (Short and Roberts 1991). Most roses have
thorns and the rose thorn is used to refer to the hard
multicelled epidermal appendages. Since rose thorns
develop from epidermis and have no vascular tissue
they are technically known as prickles (Nobbs 1984;
Rosu et al. 1995). Thornless roses would be preferred
by growers, merchandisers, and by the public,
because thorns make roses difficult to grow, handle
and harvest; retailers remove rose thorns prior to sale
(Nobbs 1984; Canli 2003).
Spontaneous thornless sports of rose have been
reported (Morey 1969; Nobbs 1984; Oliver 1986;
Druitt and Shoup 1991; Canli 2003). However, the
best thornless roses revert to the thorny condition
following high temperature shock or freezing (Nobbs
1984). These reversions suggest that thornless roses
are chimeral in nature (Nobbs 1984; Rosu et al. 1995;
Canli and Skirvin 2003). The reversion to the thorny
F. A. Canli (&)
Department of Horticulture, Faculty of Agriculture,
Suleyman Demirel University, Cunur, Isparta, Turkey
e-mail: canlifat@ziraat.sdu.edu.tr
R. M. Skirvin
University of Illinois at Urbana-Champaign,
Urbana, IL, USA
123
Plant Cell Tiss Organ Cult (2008) 95:353–361
DOI 10.1007/s11240-008-9449-y

state and unstable nature of the chimeral roses could
be caused by possible unstable mutations involving
transposable elements. The genetics of thornlessness
in roses remain unknown because most thornless
roses are infertile thus making genetic studies and
thornless rose breeding studies difficult (Nobbs 1984;
Morey 1969). A tissue culture method to obtain a
pure thornless rose from chimeras would enable us to
transfer gene(s) controlling thornlessness into desir-
able cultivars.
Segregation of chimeras into constituent genotypes
can occur in several ways: (a) the result of changes in
the histogenic layer composition (b) selective growth
of cells from one histogenic layer (c) differentiation
of a single cell to form an entire plantlet of a pure
type, or (d) mutation (McPheeters and Skirvin 1989).
Among these, induction of rapid multiplication in
shoot tips and adventitious shoot formation in vitro
are the most common methods used for separation of
chimeras into pure types.
There are several reports on adventitious shoot
regeneration and somatic embryo formation from
different explants of roses (Tweddle et al. 1984; Lloyd
et al. 1988; Burger et al. 1990; Noriega and Sondahl
1991; Kunitake et al.1993; Arene et al. 1993;
Firoozabady et al. 1994; Rosu et al. 1995; Hsia and
Korban 1996; Kim et al. 2003; Pati et al. 2004), but
most protocols are useful only for specific genotypes,
or occur at such low frequencies thus limiting value of
these protocols for most roses. Fortunately, researchers
also succeeded in obtaining pure forms from chimeras
by rapid proliferation in vitro (Hall et al. 1986;
McPheeters and Skirvin 1989; Rosu et al. 1995).
A pure ‘‘Thornless Loganberry’’ (Rubus loganob-
accus L.H. Bail.) from a periclinal thornless parent
was obtained in vitro using meristem tips (Hall et al.
1986); McPheeters and Skirvin (1983,1989) suc-
ceeded in obtaining pure thornless blackberries from
chimeral thornless plants using tissue culture. Rosu
et al. (1995) and Canli and Skirvin (2003) modified
the procedures used earlier by McPheeters and
Skirvin (1983,1989) for chimeral blackberries to
obtain putative pure thornless R. multiflora Thunb ex.
J. Murr. roses. They both obtained putative thornless
roses, but no verifications of pure thornlessness were
performed since these plants do not produce root
suckers. If thornlessness in roses is analogous to
thornlessness in blackberries, roses could also be
forced to yield pure thornless forms in vitro. The
thorny parts of the roses may overcome the newly
mutated thornless shoot(s) due to their unstable
nature and the thornless mutant shoot(s) might be
lost. Therefore the isolation of pure thornless roses
from chimeral ones is also important to maintain
thornless roses.
In this study, we demonstrate that chimeral
thornless roses can be separated into pure types using
relatively high levels of BA and that an in vitro
verification protocol can distinguish between pure
thornless and chimeral plants. A pure thornless rose
can be used in traditional breeding cycles of crosses
to pass the thornless character or eventually, gene(s)
for thornlessness could be introduced into outstand-
ing thorny cultivars by genetic transformation.
Materials and methods
Plant material and establishment of cultures
‘‘Fairmount 1 thorny’’ (‘‘FM1 thorny’’), a thorny
R. multiflora rose, was used as a control to study the
stability of the thorny condition (Fig. 1a, b). ‘‘Fair-
mount 1’’ (‘‘FM1’’), a chimeral thornless sport of
‘‘FM1 thorny’’ was used to isolate a pure thornless
rose in vitro (Fig. 1b).
‘‘FM1’’ and ‘‘FM1 thorny’’ shoot tips were
harvested and were cut into about 4 cm sections.
These explants were washed with tap water and
surface sterilized twice with 15% bleach (0.787%
sodium hypochlorite) for 15 min, each followed by a
10–15 min rinse using sterile distilled water (SDW).
Then shoots of ‘‘FM1 thorny’’ and ‘‘FM1’’ were
cut into 1.5–2 cm sections containing one or two
buds and explanted on Skirvin and Chu’s modifica-
tion (1979) of Murashige and Skoog (1962)
proliferation medium. The modified medium is called
standard MS (SMS) containing MS high mineral salts
supplemented with Staba (1969) vitamins, Na
2
EDTA
(37.25 mg/l), FeSO
4
7H
2
O (27.85 mg/l), myo-inosi-
tol (100 mg/l), BA (8.8 lM), NAA (0.54 lM), 30
sucrose and 7.5 g/l agar. The pH was adjusted to 5.5
with HCl and KOH before autoclaving at 121°Cat
18 atm for 20 min. Then the medium was dispensed
into 25 9150 mm culture tubes.
The cultures were placed in a culture room main-
tained at a 16 h photoperiod supplied by cool-white
fluorescent lambs (131 lMm
-2
s
-1
as measured with
354 Plant Cell Tiss Organ Cult (2008) 95:353–361
123

a Li-cor, Inc. integrating quantum/radiometer/pho-
tometer, model LI-188B) and 24 ±1°C.
After 4 weeks, the cultures were transferred into
jars (the shoots were harvested and cut into 1 cm
stem segments) containing 30 ml of the same
medium described above. Then, cultures were sub-
cultured onto the same medium every 6 weeks.
Preliminary studies and selections
In preliminary studies, we observed that some of the
cultures of ‘‘FM1’’ developed a deep red color
especially in rapidly proliferating cultures on SMS
(8.8 lM BA). Most of the color was observed on
young leaves and as well as on leaves of the shoots
that originated from the submerged calluses at the
base of main shoots. Leaves developing higher on the
stems were less intensively pigmented. Once the
shoots began to elongate and grow rapidly, most of
the red color disappeared. When the population of
roses was large enough, whole jars were classified as
the red selected group and the green group.
Experimental media, explanting and experiments
All media described in these experiments were SMS,
except different treatments had different levels of BA
(0, 1.25, 2.5, 5, 10, or 20 lM).
Shoots of the green group, the red selected group
and ‘‘FM 1 thorny’’ were harvested from 6-week-old
tissue cultures growing on SMS proliferation medium.
Shoots of each group were cut into 1 cm pieces (each
piece had one node) one node and explanted onto the
experimental medium containing different levels of
BA (0, 1.25, 2.5, 5, 10, or 20 lM) and placed in the
culture room in a completely randomized experimen-
tal design. Each jar contained five explants. Each
concentration was replicated five times (each replica-
tion contained 5 jars =25 plants) for each group.
Data collection and evaluation of shoots
for thornlessness
Collection of in vitro data and evaluation of individ-
ual plants for the presence or absence of thorns were
performed 7 weeks after the initiation of experiment
(in vitro evaluation).
Then, all tissue culture plants (all plants that are
derived from all BA experiments) were moved to a
greenhouse and they were acclimatized using a trans-
parent lid (Fig. 2a). After 6 weeks of acclimatization,
the plants were evaluated again for the presence or
absence of thorns (acclimatization evaluation) (Fig. 2b).
After acclimatization, some of the thornless plants
were potted and placed in a greenhouse. After 3–
4 months of greenhouse growth, they were evaluated
again for the presence and absence of thorns (first
greenhouse evaluation) and a fourth evaluation was
performed again after 7–12 months of growth in the
greenhouse (second greenhouse evaluation).
In vitro verification studies for pure thornlessness
Among the thornless greenhouse plants (derived from
in vitro BA experiments) of ‘‘FM1’’ rose, we selected
Fig. 1 A thorny Rosa multiflora, its chimeral thornless sport
and effect of different levels of BA in vitro. (a) A rose thorn is
actually a prickle that develops from epidermis and has no
vascular tissue. (b) ‘‘Fairmount 1 thorny’’ (‘‘FM1 thorny,’’ on
the left) and ‘‘Fairmount 1’’ (‘‘FM1’’), the thornless sport of
‘‘FM1 thorny’’ (on the right). (c) Effect of different levels of
BA on ‘‘FM1’’ in vitro
Plant Cell Tiss Organ Cult (2008) 95:353–361 355
123

12 putative thornless clones for further in vitro
verification. The objective of in vitro verification
was to put these 12 putative thornless plants back
in vitro and to force them to segregate on a BA
containing medium again in order to distinguish pure
ones from the chimeral ones. Actively growing
shoots of the 12 selected clones were surface
sterilized with 15% bleach (0.787% sodium hypo-
chlorite) for 15 min and rinsed with sterile distilled
water (SDW) for 10 min. Explants were again
sterilized with 15% commercial bleach for 20 min
and rinsed with SDW for 15 min.
Then shoots were cut into 1.5–2 cm pieces con-
taining one or two buds and explanted on SMS media
supplemented with BA (8.8 lM), NAA (0.54 lM) in
25 9150 mm culture tubes (10 ml/tube). Ten
explants were used for each thornless clone. After
2 weeks, shoots were transferred into jars containing
the same medium. In vitro derived shoots of the 12
thornless greenhouse clones were evaluated 6 weeks
later for the presence of thorns.
Statistical analysis
Analysis of variance (ANOVA) was performed on
the data and means were subjected to LSD tests at the
5% level using the SAS.
Results
In vitro evaluations and effects of BA on chimeral
segregation of ‘‘FM 1’’ rose
In the green group, there were no significant differ-
ences among the BA levels with respect to the
percentage of thorny plants (Table 1). BA signifi-
cantly affected shoot length (Fig. 1c), shoot number,
rooting percentage, root number, root length, callus
formation, and callus diameter (Table 1). The highest
numbers of shoots per explants were obtained at the
highest BA concentrations (10 and 20 lM) (Table 1).
For all BA levels, shoot lengths were higher than the
control ones (0 lM BA) (Table 1). The shoots of the
control treatment (0 lM BA) did not elongate and
were too small to be evaluated for the presence of
thorns (Fig. 1c) in in vitro evaluation. The longest
roots were observed in the control, lower and
intermediate levels of BA (0, 1.25, 2.5, and 5 lM)
(Table 1). Root number per explant was highest for
control and decreased as the BA concentration
increased (Table 1). All BA levels induced callus
formation, except the control (Table 1). The largest
callus diameters were obtained at intermediate and
high levels of BA (2.5, 5, 10, and 20 lM) (Table 1).
In the red selected group, BA significantly affected
the percentage of the thorny shoots (Table 2). The
percentages of thorny plants were highest at the four
highest levels of BA. BA had no significant effect on
shoot length. However, BA significantly affected the
shoot number, rooting percentage, root number, root
length, callus formation and the callus diameter. The
highest numbers of shoots per explants were obtained
Fig. 2 Acclimatization and evaluation of tissue culture
derived plants of ‘‘Fairmount 1’’ (‘‘FM1’’). (a) Acclimatization
of tissue culture derived plants of ‘‘FM1.’’ (b) Evaluation of
tissue culture derived plants of ‘‘FM1’’ for the presence or the
absence of thorns after acclimatization. (c) Evaluation of tissue
culture derived plants of ‘‘FM1’’ for the presence or absence of
thorns in the greenhouse. This figure shows a reversion of a
thornless plant to its thorny state. This plant had been classified
as thornless in the first greenhouse evaluation
356 Plant Cell Tiss Organ Cult (2008) 95:353–361
123

from intermediate and the high BA concentrations
(2.5, 10, and 20 lM) (Table 2). The percentage of
rooted explants and mean root lengths were highest
for control and 5 lM BA (Table 2). The mean root
number per explant was highest for control and
decreased as the BA concentration increased
(Table 2). All BA levels induced callus formation,
but no callus formed in the control treatment
(Table 2). The largest callus diameters were obtained
at intermediate and high levels of BA (5, 10, and
20 lM) (Table 2).
BA significantly affected shoot length and shoot
number of ‘‘FM 1 thorny’’ in vitro. The highest mean
shoot number was obtained at 10 lM BA (Table 3).
Mean plant height was higher in the presence of BA
than that of the control (Table 3). ‘‘FM 1 thorny’’
clone did not segregate in vitro and no thornless
shoot was obtained from it (Table 3).
Acclimatization evaluations and effects of BA on
chimeral segregation of ‘‘FM 1’’ rose
In the green group, there was a slight increase in the
percentage of thorny plants as BA concentration
increased (Table 4). Higher BA concentrations (10
and 20 lM BA) yielded more thorny shoots than
either control or the lowest concentrations of BA
(1.25 and 2.5 and 5), however, there was no
Table 1 Effects of different BA concentrations on stability and chimeral segregation of the green group of ‘‘FM1’’ rose in vitro
Treatment
BA (lM)
Shoots/
explant
Shoot
length (cm)
Thorny
shoots (%)
Rooted
shoots (%)
Length of the
longest root (cm)
Root # Callus
a
(cm)
Callus forming
shoots (%)
0.0 1.08c
b
1.70b –
c
100.0a 3.50a 13.52a 0.00c 0c
1.25 1.78b 4.10a 40.0 91.0a 2.9ab 6.68b 0.41b 91b
2.5 1.74b 3.82ab 48.2 68.0b 1.80b 4.12c 0.78a 100a
5.0 1.96ab 3.66ab 58.0 84.0ab 3.59a 2.4cd 0.71a 100a
10.0 2.47a 2.28ab 75.0 34.0c 0.61c 0.79d 0.83a 100a
20.0 2.37a 2.00ab 69.8 19.6c 0.29c 0.30d 0.84a 100a
n=25 (5 95)
a
Callus diameter was measured at the stem base
b
Numbers within columns followed by different letters are significantly different by ANOVA at 5%; mean separation by LSD (least
significant difference)
c
Since the shoots were too small to evaluate and thorns were not easily observable, data was not collected
Table 2 Effects of different BA concentrations on stability and chimeral segregation of the red selected group of ‘‘FM1’’ rose
in vitro
Treatment
BA (lM)
Shoots/
explant
Shoot
length (cm)
Thorny
shoots (%)
Rooted
shoots (%)
Length of the
longest roots (cm)
Root # Callus
a
(cm)
Callus forming
shoots (%)
0.0 1.09d
b
2.58 –
c
100a 4.4ab 12.85a 0.00c 0c
1.25 1.63bc 2.66 16c 75c 3.1bc 4.74b 0.32b 87b
2.5 2.8abc 3.02 67ab 80bc 2.04c 3.44b 0.44b 96ab
5.0 1.60cd 3.58 58b 96ab 4.0ab 4.84b 0.70a 100a
10.0 2.18a 2.62 95a 25d 0.46d 0.60c 0.77a 100a
20.0 2.14ab 2.58 72ab 5e 0.07d 0.05c 0.81a 100a
n=25 (5 95)
a
Callus diameter was measured at the stem base
b
Numbers within columns followed by different letters are significantly different by ANOVA at 5%; mean separation by LSD (least
significant difference)
c
Since the shoots were too small to evaluate and thorns were not easily observable, data was not collected
Plant Cell Tiss Organ Cult (2008) 95:353–361 357
123

significant linear relationship between BA levels and
the percentage of thorny plants of the green group
(Table 4) that survived acclimatization.
Although there was a slight increase in the
percentage of thorny plants as the BA concentration
increased in the red group, there was no significant
linear relationship between BA levels and the
percentage of thorny plants that survived (Table 4).
If the green group and the red selected group were
combined and evaluated for the presence of a
relationship between BA treatments and the percent-
age of thorny plants, there was a significant linear
relationship (r
2
=0.73) between increasing BA con-
centrations and the percentage of thorny plants
overall (Table 4).
The ‘‘FM1 thorny’’ yielded only thorny plants and
gave no thornless plants in any of the treatments
(Table 3).
Greenhouse evaluations and effects of BA
on chimeral segregation of ‘‘FM 1’’ rose
Among the 66 thornless greenhouse plants selected
from the green group of ‘‘FM1’’ rose (Table 5), only
four plants reverted to the thorny condition (Fig. 2c).
These all came from the control or lower levels of BA
(0, 1.25, and 5 lM).
Out of 67 greenhouse plants of the red selected
group, only four (which were obtained from control
or lower levels of BA (0, 1.25, and 1.25 lM)) had
recurved thorns at the tips of their shoots in the
greenhouse (Table 5).
In vitro verification studies for pure thornlessness
Among 690 tissue culture derived plants (from all BA
experiments), 203 plants were classified as thornless
at the acclimatization stage. One year later 38 of them
were still classified as thornless ex vitro.
About 12 of these greenhouse clones, which had
been derived from in vitro BA experiments earlier,
were re-introduced in vitro and tested for in vitro
segregation again. Of the 12 clones tested, 6
remained both thornless over a year in the green-
house, and did not segregate in vitro (yielded only
thornless plants on the medium containing BA)
(Table 6). These results suggest that these six clones
are pure thornless.
Table 3 Effects of different BA concentrations on stability
and segregation of ‘‘FM1 Thorny’’ rose
BA treatment
(lM)
Number of
shoots/explant
Shoot length
(cm)
Thorny
shoots (%)
b
0.0 1.00d
a
0.95b 100
1.25 1.3cd 4.09a 100
2.5 1.6bc 3.56a 100
5.0 1.89b 3.74a 100
10.0 2.20a 3.43a 100
20.0 1.92b 4.30a 100
n=30 (6 95)
a
Numbers within columns followed by different letters are
significantly different by ANOVA at 5%; mean separation by
LSD (least significant difference)
b
Acclimatization results
Table 4 Effects of different BA concentrations on stability and chimeral segregation of ‘‘FM1’’ rose. The plants were evaluated for
the presence of thorns after acclimatization
Treatment
BA (lM)
Green group Red selected group Two groups combined
Total shoots Thorny shoots Total shoots Thorny shoots Total shoots Thorny shoots
Total % Total % Total %
0.0 22 1 4.5 18 5 27.0 40 6 15
1.25 22 9 40.9 22 2 9.0 44 11 25
2.5 17 8 47.0 16 10 62.5 33 18 54.5
5.0 19 11 57.8 21 10 50.0 40 21 52.5
10.0 18 13 72.2 19 18 94.7 37 31 81
20.0 9 7 77.7 16 14 82.3 25 21 84
Significance NS NS *, r
2
=0.73
NS,* Nonsignificant or significant at P\0.05, respectively. Linear regresssion (y=b
0
?b
1
x?e)
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123

Discussion
Pure thornless roses from a chimeral thornless rose
were isolated in vitro and the isolation of pure
thornless roses was confirmed by an in vitro verifi-
cation protocol.
All tissue culture experiments with ‘‘FM1,’’ the
thornless sport of the ‘‘FM1 thorny,’’ yielded both
thorny and thornless regenerants suggesting that it is
a chimera consisting of thorny and thornless tissues
growing together. ‘‘FM1 thorny’’ did not segregate
and yielded only thorny plants in vitro. No thornless
plants of ‘‘FM1 thorny’’ were observed in any of the
treatments. These indicate that ‘‘FM 1 thorny’’ clone
was not chimeral, but a pure thorny plant.
The results from the current study agree with the
reports on the separation of chimeras in close
relatives of rose (Hall et al. 1986; McPheeters and
Skirvin 1989); Hall et al. (1986) reported separating
chimeral ‘‘Thornless Loganberry,’’ a periclinal chi-
meral blackberry, into its component parts by using
meristem tips in vitro.McPheeters and Skirvin
Table 5 Reversion rates at first greenhouse evaluation of thornless plants, which had been classified as thornless at acclimatization
stage
Treatment
BA (lM)
Green group Red selected group
Total plants Plants with
thorny shoots
(reverted)
Location of the
first thorn
Total plants Plants with
thorny shoots
(reverted)
Location of the
first thorn
0.0 23 2 Tip section 13 1 Tip section
1.25 15 1 Middle section 20 2 Tip section
2.5 8 0 15 1 Tip section
5.0 8 1 Middle section 9 0
10.0 5 0 6 0
20.0 4 0 4 0
Table 6 In vitro verification of the clones of ‘‘FM1’’ rose, which were classified as thornless in the first greenhouse evaluation
Thornless
selection code
Treatment from
which plants are
derived (BA lM)
Second
greenhouse
evaluation
Segregation
in vitro
a
Pure
thornless
b
64 0.0 Thorny ?No
9 0.0 Thorny ?No
17 1.25 Thornless -Yes
37 2.5 Thornless -Yes
83 2.5 Thornless -Yes
38 2.5 Thorny ?No
106 5.0 Thornless -Yes
91 10.0 Thornless -Yes
115 10.0 Thornless -Yes
41 20.0 Thorny -No
93 20.0 Thorny -No
39 20.0 Thorny ?No
The in vitro derived greenhouse plants were re-introduced in vitro and were forced to segregate one more time on a medium
containing 8.8 lMBA
a
?segregated in vitro and yielded both thorny and thornless plants, -remained thornless
b
To be considered pure thornless, the clone had to remain thornless ex vitro for 1 year (column 3) and produce only thornless
regenerants in vitro (column 4)
Plant Cell Tiss Organ Cult (2008) 95:353–361 359
123

(1989) evaluated 900 plants from in vitro shoot tip
cultures of the ‘‘Thornless Evergreen’’ (‘‘TE’’),
another periclinal chimeral blackberry, and obtained
thornless plants. Rosu et al. (1995) and Canli and
Skirvin (2003) modified the procedures of McPheet-
ers and Skirvin (1989) to obtain putative pure
thornless roses (R. multiflora thunb ex. J. Murr.).
The term putative pure thornless rose was used by the
researchers, because it was not possible to confirm
the genetic make up of the internal layers with respect
to thornlessness. Unlike roses, the genetic make up of
internal tissues can be confirmed in blackberries since
they produce root suckers. A chimeral thornless plant
possessing a thornless epidermis (LI) that overlayed a
core of thorny cells (LII and LIII) should produce
thorny root suckers. McPheeters and Skirvin (1989)
reported that among the meristem tip cultures of a
periclinal chimeral blackberry plants, the pure thorn-
less ones produced thornless root suckers, whereas
the chimeral plants gave thorny suckers. The authors
assumed that the pure thornless blackberries devel-
oped from the epidermis (LI) adventitiously, while
the chimeral ones arosed from axillary buds. Since
roses do not sucker, an in vitro protocol developed in
this study was used to verify the chimeral nature of
the in vitro derived rose plants. Some of the thornless
regenerants were chimeral, another portion were pure
thornless as confirmed by the in vitro verification
protocol. By using this protocol, putative pure
thornless plants (grown in the greenhouse for over a
year) were forced to segregate in vitro again. Some of
the in vitro derived putative pure thornless clones did
not segregate in vitro when they were re-put in vitro
and re-forced to segregate. This indicates that they
are pure thornless.
BA at concentrations of 5–20 lM yielded the
highest number of shoots and was most suitable for
the in vitro rapid multiplication of ‘‘FM1’’. This is
in agreement with the results of the reports in other
rose species (Khosh-Khui and Sink 1982; Skirvin
et al. 1990; Compas and Pais 1990; Kumar et al.
2001; Jabbarzadeh and Khosh-Khui 2005).BA
induced chimeral segregation in vitro. Combined
BA data shows a significant linear relationship
(Table 5) between increasing BA concentrations and
the percentage of thorny plants, suggesting that to
separate chimeras into their component genotypes,
relatively high concentrations of BA should be used
in vitro. When the effect of BA was considered
individually for certain groups (the green group and
the red selected group), its effect was found non
significant (Table 5). The lack of significance for
BA in these particular experiments is probably due
to the fact that the explants had been growing on
SMS proliferation medium containing 8.8 lM BA,
which we now know is capable of stimulating
chimeral segregation. Therefore, even our controls
probably had pre-segregated thorny and thornless
segregants which arose on the SMS proliferation
medium.
Callus formation at the base of the explants and the
percentages of callus forming explants were higher at
higher concentrations of BA. The pure thornless
plants obtained in vitro were originated most prob-
ably by putative adventitious shoots formed on these
nodular calluses. Similar findings were also reported
by Rosu et al. (1995) for another chimeral thornless
type of R. multiflora. They reported that shoots
harvested from MS proliferation medium, supple-
mented with gibberellic acid (GA
3
, 0.5–1.0 mg/l
-1
)
and silver nitrate (3.4 mg/l), formed nodular callus
and occasional putative adventitious shoots when
subcultured on the same media supplemented with
different levels of thidiazuron (TDZ).
Since no thorns were observed on the stems of
some regenerants over a year, and six of the in vitro
derived plants did not yield any thorny plants when
they were re-forced to segregate in vitro, we assume
that we succeeded in obtaining pure thornless roses
by separating the chimeral tissues. A pure thornless
rose may enable us to better understand the genetics
of thornlessness and the thornless character may also
be introduced into commercially important rose
cultivars by hybridizations.
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