Hort. Sci. (Prague) Vol.

45, 2018 (2): 83–91

https://doi: 10.17221/12/2017-HORTSCI

Essential factors for in vitro regeneration of rose

and a protocol for plant regeneration from leaves
Inas Mohamed Ali Mahmoud1, Anber Mahmoud Ahmed Hassanein1,2*
1
Horticulture Department (Ornamental Plants), Agriculture Faculty, Sohag University, Egypt
2
Plant Production and Protection Department, College of Agriculture and Veterinary
Medicine, Qassim University, Kingdom of Saudi Arabia
*Corresponding author: [email protected]

Abstract
Mahmoud I.M.A., Hassanein A.M.A (2018): Essential factors for in vitro regeneration of rose and a protocol for plant
regeneration from leaves. Hort. Sci. (Prague), 45: 83–91.

In vitro propagation of Rosa hybrida, L. cv. ‘Eiffel Tower’ was improved by the addition of thidiazuron (TDZ) and sil-
ver nitrate (AgNo3) to the culture medium. The combination of auxin and cytokinins was indispensable for inducing
response from leaf discs. Maintaining cultures under dark was better than light for callus formation and quality. The
source of explants was vital in the regeneration process wherein situ explants produced callus while, in vitro explants
regenerated somatic embryos and shoots. Gibberellic acid (GA3) had a favorable effect where in vitro explants showed
somatic embryogenesis with no shoots on media containing TDZ however, 37% of explants regenerated shoots directly
on medium containing GA3. The presence of benzyl adenine (BA) was essential for shoot elongation, and indole butyric
acid (IBA) was better than indole acetic acid (IAA) for rooting. The optimum conditions produced rooted plants from
leaf discs within ten weeks. The reported results clarify factors controlling in vitro regeneration of R. hybrida, and
provide a rapid protocol allowing further improvements of rose.

Keywords: Rosa hybrida; leaf discs; embryogenesis; organogenesis; direct regeneration

Rose is the most economically important flowering the generation of somaclonal variants and the in-
plant cultivated in the world. It includes more than sertion of interesting genes via transformation
200 species which are used for inside and outside dec- technology. The establishment of an efficient and
oration, cut flowers, essential oils as well as medici- reliable protocol for plant regeneration is an im-
nal and aromatic purposes. Rosa hybrida is the most portant prerequisite step needed for the successful
economically important species which includes most implementation of biotechnological techniques for
commercial cultivars. ‘Eiffel Tower’ is a scented rose plant improvement.
cultivar produced commercially in Egypt and other Many trials were conducted for in vitro regenera-
countries for its cut flowers and essential oil. tion of different rose species. R. damascena was re-
Conventional breeding of rose is limited by its generated indirectly from callus derived from stem
polyploid and highly heterozygous nature (Kim et tissue (Ishioka, Tanimoto 1990), or directly via
al. 2004), long and slow propagation (Pati et al. leaf explants of in vitro-raised shoots (Pati et al.
2006). It is also affected by climatic changes and do 2004). Direct regeneration was also reported on
not ensure healthy or uniform plants. Biotechnol- R. canina (Moallem et al. 2012). For R. hybrida,
ogy provides a tool capable of overcoming some of shoot regeneration has been reported from imma-
these restrictions. In vitro culture can offer a great ture embryos (Burger et al. 1990), callus derived
potential for rapid mass production of cultivars, from in vitro and in vivo leaves and shoots (Rout et

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al. 1992) and embryogenic cell-derived protoplasts 10 mg/l to culture medium was investigated (Aka-
(Kim et al. 2003). In plants regenerated from vari- saka-Kennedy et al. 2005). Five explants were cul-
ous vegetative embryogenic tissues of Meirutral tured per Petri dish (95 mm) containing 25 ml of
cultivar, somaclonal variants were found (Arene et medium. Each treatment consisted of nine dishes
al. 1993). In vitro leaf explants were used for plant in three replicates, and the experiment was repeat-
regeneration in a two-stage procedure by the in- ed twice. Cultures were maintained in the growth
cubation of leaf discs on induction medium before chamber under 16 hours daily light (70 µmol/s/m2),
transferring to regeneration medium (Ibrahim, 25 ± 1°C and 60% humidity.
Debergh 2001). Somatic embryogensis and plant Explant preparation and culture medium. Leaf
regeneration were also obtained using in vitro leaf explants of R. hybrid cv. ‘Eiffel Tower’ were obtained
explants after incubation on induction medium fol- from in vitro mother plants, one month after subcul-
lowed by several subcultures (Kim et al. 2004). ture, or two-year old plants grown in the greenhouse
Few reports deal with direct regeneration from (in situ). Young fully developed leaves of in situ grown
R. hybrida. In previous studies, we found in vitro plants were washed under tap water then surface
leaves efficient for direct and indirect regeneration sterilized using 70% ethanol before transferring to
of pelargoniums, but somaclonal variants were re- 15% sodium hypochlorite solution containing drops
sulted from callus formation and the long regenera- of Tween-80 for 20 min and finally rinsed three times
tion procedure (Hassanein, Dorion 2005, 2006). in sterile distilled water. Leaves from both sources
A rapid regeneration system is needed for its effi- were cut into leaf discs of 0.5 to 0.75 cm diameter,
cient use in a plant improvement program. Determi- then placed with abaxial side down, in Petri dishes of
nation of factors controlling regeneration procedure 95 mm diameter containing 25 ml medium, 7–8 leaf
can also help in further improvement and applica- discs per dish. The basal medium(BM) contained MS
tion on other species. To the best of our knowledge, macronutrients at half strength, MS micronutrients,
there is no report on plant regeneration of the com- vitamins of Morel, Wetmore (1951), Fe EDTA,
mercially important scented hybrid tea rose (R. hy- 20 g/l sucrose, 10 mg/l AgNo3and 9 g/l bacto agar. The
brida cv. ‘Eiffel Tower’). We previously reported an pH was adjusted to 5.8 and autoclaving was achieved
efficient protocol for the in vitro propagation of that at 113°C for 20 minutes.
cultivar (Mahmoud et al. 2011). Here, we report on Shoot regeneration from leaf discs and stud-
the factors controlling its direct or indirect regen- ied factors. For plant regeneration from leaf discs,
eration, and a protocol for rapid plant regeneration many factors were studied. The type and concen-
allowing mass production and further improvement tration of plant growth regulators (PGR) were in-
using the transformation approach. vestigated in a factorial experiment including three
PGRs and four concentrations. The PGRs included
naphthalene acetic acid (NAA), benzyl adenine
MATERIALS AND METHODS (BA) and thidiazuron (TDZ) at concentrations of 0,
0.5, 1 and 2 mg/l. The leaf discs were obtained from
Production of in vitro mother plants as source in situ grown plants and prepared as mentioned
of explants. Mother plants of hybrid tea rose, above then cultured in Petri dishes containing the
R. hybrid cv. ‘Eiffel Tower’, were introduced un- basal medium (BM) and one of the treatments.
der in vitro conditions following our previously Cultures were maintained in the growth chamber
reported propagation protocol (Mahmoud et al. under dark conditions for a week then exposed to
2011). Nodal segments of 1 cm length were taken a 16 h photoperiod. The daily temperature and hu-
from two-year old in situ grown plants. Explants midity were 25 ± 1°C and 60%, respectively.
were sterilized using 20% sodium hypochlorite so- The combination among the three PGR (NAA,
lution, then cultured on MS (Murashige, Skoog BA and TDZ) at three concentrations (0.5, 1 and
1962) modified medium containing half strength 2 mg/l) were studied under two different culture
macronutrients, micronutrients, vitamins, Fe conditions (dark and light). After preparation and
EDTA, 20 mg/l sucrose, 1 mg/l indole acetic acid sterilization as described above, leaf discs of in situ
(IAA), 9 g/l bacto agar and 2 g/l active charcoal. To grown plants were cultured in Petri dishes contain-
improve the protocol, the addition of thidiazuron ing the BM medium and one of twenty seven NAA
(TDZ) at 0.5 mg/l and/or silver nitrate (AgNo3) at + BA + TDZ combinations. Cultures were either

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covered to create dark conditions, or exposed to lus compared to the total cultivated leaf discs was
16 h of light (70 μmol/s/m2) in a growth chamber recorded. The response percentage was calculated
of 25 ± 1°C and 60% humidity. as the percent of leaf discs showing callus forma-
The source of explants was also studied under the tion or embryogenesis related to the initial cultivat-
best PGR combinations as determined in the previ- ed number of leaf discs. Shoot regeneration (%) was
ous experiment. Leaf discs were taken from in vitro similarly recorded as the percent of explants regen-
grown plants or in situ grown plants after disin- erating shoots directly. The mean of shoot length
fection, and prepared as previously described. Ex- and number of leaves per shoot were determined
plants were cultivated on BM medium containing per elongated shoot. Rooting percentage was re-
one of six PGR combinations including NAA + BA corded as the percent of rooting plantlets related to
in combination with thidiazuron (TDZ) or gibber- the cultivated ones. Data were subjected to analy-
ellic acid (GA3) at 0.5, 1 and 2 mg/l, to study the sis of variance (ANOVA) to determine significant
effect of GA3 on plant regeneration. Cultures were differences, and the least significant difference at
kept in the dark under 25 ± 1°C and 60% humidity. 0.05 were used for the comparison of means using
All the experiments performed in three replicates SAS 9.1.3 program. For certain results, means were
and repeated twice. shown with the confidence interval at significance
Shoots elongation and plantlets rooting. After level of 0.05 using Excel 2010 program.
four weeks of culture, growing buds or regenerat-
ing shoots were individually transferred to jars con-
taining 25 ml of fresh basal medium (BM) contain- RESULTS AND DISCUSSION
ing BA at 0, 0.5 and 1 mg/l in combination with IBA
or IAA at 0, 0.5 and 1 mg/l for shoot elongation. Production of in vitro mother plants
Twenty explants in four replicates were cultured
per treatment. Cultures were kept in the growth The production of mother plants under in vitro
chamber with 16 h photoperiod from white fluo- conditions using nodal segments showed signifi-
rescent light, 25 ± 1°C and 60% humidity. cantly higher grown buds on media containing th-
For rooting, elongated shoots with 3–4 nodes were idiazuron or silver nitrate compared to control (Ta-
individually transferred to jars containing 25 ml of ble 1). The highest rate of bud growth (100%) was
fresh basal medium (BM) supplemented with IAA obtained with medium including both compounds
or IBA at 0, 0.5, 1 or 2 mg/l. Sixteen plantlets in four where all buds developed shoots of good length. It
replicates were tested per treatment. Cultures were was also noticed that media containing TDZ have
maintained under similar elongation conditions. no phenolic compounds compared to other tested
Collection of data and statistical analysis. For media. These obtained results proved the possi-
all experiments, data were collected four weeks af- bility of improving our previously reported pro-
ter culture. The percentages of green or grown buds tocol (Mahmoud et al. 2011) by the addition of
and the percent of buds showing phenolic com- thidiazuron(TDZ) and silver nitrate (SV). The pro-
pounds (phenols %) were calculated related to the moting effect of TDZ and SV on shoot proliferation
total cultivated explants (Mohmoud et al. 2011). was reported on other plant species (Geetha et al.
The mean of shoot length was measured for grow- 2016). The beneficial effect of SV may be related to
ing buds. The percentage of leaf discs showing cal- its inhibition of ethylene action (Ozudogru et al.

Table 1. Propagation of in vitro mother plants from nodal segments of R. hybrid cv. ‘Eiffel Tower’ as affected by
thidiazuron (TDZ) and silver nitrate (AgNo3), four weeks after culture

Treatments Green buds (%) Grown buds (%) Phenols (%) Shoot length (cm)
BM (control) 100.0 88.9 ± 0.07 0.7 ± 0.15 2.3 ± 1.04
BM + 0.5 mg/l TDZ 100.0 93.3 ± 0.05 0.0 2.0 ± 0.61
BM + 10.0 mg/l AgNO3 100.0 93.3 ± 0.05 0.3 ± 0.10 2.7 ± 0.77
BM + 0.5 DZ + 10 mg/l AgNo3 100.0 100.0 0.0 3.2 ± 0.96

BM – basal medium containing (mg/l) indole acetic acid (IAA); means of two repetitions with confidence interval at 0.05

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Table 2. Effect of type and concentration of plant growth regulators on callus formation (%) from leaf discs of R. hybrid
cv. ‘‘Eiffel Tower’’

TDZ (mg/l)
NAA (mg/l) BA (mg/l) Mean (NAA × BA) Mean (NAA)
0 0.5 1 2
0.0 0.0 0.0 0.0 0.0 0.0
0.5 0.0 0.0 0.0 0.0 0.0
0.0 0.0
1.0 0.0 0.0 0.0 0.0 0.0
2.0 0.0 0.0 0.0 0.0 0.0
Mean (NAA × TDZ) 0.0 0.0 0.0 0.0
0.0 0.0 85.7 96.4 85.7 67.0
0.5 78.6 82.1 92.9 92.9 86.6
0.5 85.9
1.0 89.3 89.3 96.4 100.0 93.8
2.0 96.4 96.4 96.4 96.4 96.4
Mean (NAA × TDZ) 66.1 88.4 95.5 93.8
0.0 0.0 89.3 92.9 92.9 68.8
0.5 89.3 89.3 89.3 92.9 90.2
1.0 87.5
1.0 100.0 92.9 96.4 96.4 96.4
2.0 89.3 89.3 100.0 100.0 94.7
Mean (NAA × TDZ) 69.7 90.2 94.7 95.6
0.0 60.7 92.9 92.9 92.9 84.9
0.5 92.9 92.9 96.4 96.4 94.7
2.0 94.0
1.0 96.4 96.4 100.0 100.0 98.2
2.0 96.4 96.4 100.0 100.0 98.2
Mean (NAA × TDZ) 86.6 94.7 97.3 97.3
0.0 15.2 67.0 70.6 67.9
0.5 65.2 66.1 69.7 70.6
Mean (BA × TDZ)
1.0 71.4 69.7 73.2 74.1
2.0 70.5 70.5 74.1 74.1
Mean (TDZ) 55.6 68.3 71.9 71.7
Mean (BA) 55.1 67.9 72.1 72.3
The least significant difference (LSD) for means at significant level 0.05
Factors NAA BA TDZ NAA × BA NAA × TDZ BA × TDZ NAA × BA × TDZ
LSD 0.05 7.40 3.01 2.99 5.98 5.95 5.95 11.91

NAA – naphthalene acetic acid; BA – benzyl adenine; TDZ – thidiazuron

2005), and that of TDZ may be due to its inhibition cytokinins concentration (Fig. 1). In the presence of
of phenolic compounds (Table 1). cytokinins, the rate of callus formation percentage in-
creased with increasing concentration of NAA. Callus
production also increased with increasing cytokinins
Effect of plant growth regulators where more leaf discs formed callus with 1 and 2 mg/l
of either BA or TDZ. The presence of cytokinin was
The type and concentration of growth regulators also essential for callus formation where no callus was
are important factors for the in vitro regeneration observed on media free of both BA and TDZ. Howev-
from rose leaf discs (Table 2). No callus was formed er, auxin seems to be more important than cytokinin
on media free of growth regulators. Auxin was found as some leaf discs produced callus with 2 mg/l NAA
to be important for callus formation regardless of the alone. The importance of auxin comes from its role

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(a) (b)

(c) (d)

Fig. 1. Callus formation from in situ leaf discs of hybrid tea rose (‘‘Eiffel Tower’’)

(a) no response on media free of growth regulators, (b) callus formation on media containing PGR combinations, (c)
callus formed under light condition, (d) callus formed under dark condition

in cell division (Mather, Roberts 1998). The com- for the regeneration efficiency of other ornamental
bination between auxin and cytokinin at any concen- and medicinal plants (Mendi et al. 2009; Mun, Mun
tration gave good response, however, the best results 2016). In the present study, all leaf discs produced
were obtained when NAA was combined with both good callus but no shoot regeneration was observed
cytokinins (BA and TDZ). Most leaf discs formed (Fig. 1). So, the combination of the three growth regu-
callus on media containing the three growth regula- lators was studied under different culture conditions.
tors and the least percentage was recorded on media
containing the minimum levels; 0.5 mg/l of NAA in
combination with 0.5 mg/l of BA and/or TDZ. Our Effect of culture conditions (dark and light)
earlier studies showed the importance of the combi-
nation between auxin and cytokinin in plant regener- Higher callus formation percentage was obtained
ation from leaf discs and protoplasts of pelargoniums in dark compared to light conditions (Table 3).
(Hassanein, Dorion 2005, 2006). The presence of Callus that formed under light was compact and
auxin with cytokinins was also found to be essential green in color compared to less compact and white

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Table 3. Effect of culture conditions (dark or light) on callus formation (%) from leaf discs of R. hybrid cv. ‘‘Eiffel Tower’’,
cultivated on different combinations of plant growth regulators (PGR)

PGR combinations (mg/l ) Culture conditions Mean

(PGR combina-
NAA BA TDZ Light Dark tions)
0.5 0.5 0.5 57.1 81.0 69.1
0.5 0.5 1.0 76.2 76.2 76.2
0.5 0.5 2.0 85.7 85.7 85.7
0.5 1.0 0.5 81.0 90.5 85.8
0.5 1.0 1.0 85.7 90.5 88.1
0.5 1.0 2.0 90.5 90.5 90.5
0.5 2.0 0.5 95.2 95.2 95.2
0.5 2.0 1.0 90.5 100.0 95.3
0.5 2.0 2.0 100.0 95.2 97.6
1.0 0.5 0.5 76.2 85.7 81.0
1.0 0.5 1.0 85.7 76.2 81.0
1.0 0.5 2.0 85.7 90.5 88.1
1.0 1.0 0.5 90.5 90.5 90.5
1.0 1.0 1.0 95.2 100.0 97.6
1.0 1.0 2.0 95.2 100.0 97.6
1.0 2.0 0.5 90.5 90.5 90.5
1.0 2.0 1.0 100.0 95.2 97.6
1.0 2.0 2.0 100.0 100.0 100.0
2.0 0.5 0.5 81.0 90.5 85.8
2.0 0.5 1.0 90.5 90.5 90.5
2.0 0.5 2.0 90.5 100.0 95.3
2.0 1.0 0.5 90.5 95.2 92.9
2.0 1.0 1.0 95.2 95.2 95.2
2.0 1.0 2.0 100.0 100.0 100.0
2.0 2.0 0.5 100.0 100.0 100.0
2.0 2.0 1.0 100.0 95.2 97.6
2.0 2.0 2.0 100.0 100.0 100.0
Mean (culture conditions) 89.9 92.9
The least significant difference (LSD) for means at significant level 0.05
Factors Culture conditions PGR combinations Conditions × PGR
LSD 0.05 2.62 8.42 18.42

callus under dark conditions (Fig. 1). Less cal- 0.5 mg/l BA. The interaction between PGR and cul-
lus formation under light conditions may be re- ture conditions was also important where callus for-
lated to the degradation of light sensitive PGRs mation percentage varied from 57% with the mini-
(Mather, Roberts 1998). Similar results on the mum level (0.5 mg/l) of the three PGRs under light
enhancing effect of dark were previously reported to 100% with the combinations containing high lev-
in Pelargonium (Hassanein, Dorion 2005) and els of cytokinins under either dark or light. The re-
R. multiflora (Canli 2003). All PGRs combina- sults showed that 0.5 mg/l of NAA in PGR combina-
tions induced callus formation of leaf discs with the tions, containing higher concentration of cytokinins,
least callus formation on media containing 0.5 or is sufficient for maximum callus formation however
1 mg/l of both NAA and TDZ in combination with no shoot was regenerated. Therefore, PGR combina-

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Table 4. Effect of explants source (In vitro or In situ mother plants) and growth regulators combination on response
and shoot regeneration from leaf discs of R. hybrid cv. ‘‘Eiffel Tower’’

PGR combinations (mg/l) Response (callus or embryos) (%) Shoots regeneration (%)
NAA BA TDZ GA3 In situ In vitro Mean In situ In vitro
0.5 0.5 0.5 – 80.0 83.3 81.7 – –
0.5 1.0 1.0 – 93.3 93.3 93.3 – –
0.5 2.0 2.0 – 100.0 93.3 96.7 – –
0.5 0.5 – 0.5 96.7 93.3 95.0 – –
0.5 0.5 – 1.0 100.0 96.7 98.4 – 36.7 ± 0.6
0.5 0.5 – 2.0 96.7 76.7 86.7 – –
Mean (explant source) 94.5 89.4
The least significant difference (LSD) for means of response (%) at significant level 0.05

Factors Explants source PGR combinations Source × PGR Factors Explants source

LSD 0.05 4.15 5.83 8.25 LSD 0.05 4.15

tions including 0.5 mg/l NAA with varying concen- the hormonal balance in such plantlets as they are
trations of BA and TDZ were studied with several sterile and experience less stress due to treatment
concentrations of GA3 and two explant sources. needed for surface sterilization. Such explants al-
lowed shoot regeneration from some rose cultivars
after incubation or several subcultures (Ibrahim,
Effect of explant source and gibberellic acid Debergh 2001; Kim et al. 2004). The age and the
source of explants were also important variables in
Generally, explants taken from mother plants the micropropagation of rose and other ornamental
grown under in situ conditions showed similar or and aromatic plants (Mahmoud et al. 2011; Ozel
higher callus formation as compared to those taken et al. 2015). In our case, shoots regenerated directly
from in vitro mother plants. However, embryos and after four weeks of culture, which is more rapid
shoots were developed only on in vitro explants than the reported protocols. The somatic embryos
(Table 4). Most tested PGR combinations allowed can also develop shoots after two weeks of transfer
good callus production except for the treatment to medium containing GA3. The favorable role of
with 0.5 mg/l of both NAA and BA in combina- GA3 on direct regeneration from leaf explants was
tion with 0.5 mg/l TDZ or 2 mg/l GA3. Leaf discs reported on R. canina (Moallem et al. 2012).
of in situ mother plants showed callus formation
with no regeneration regardless of the type or con-
centration of growth regulators however, somatic Shoots elongation and plantlets rooting
embryos and shoots were regenerated from in vitro
leaf discs (Fig. 2). In vitro explants showed somatic The transfer of individual shoots to elongation me-
embryogenesis with no shoots on media contain- dium containing both indole acetic acid (IAA) and
ing TDZ however, 36.7% of explants regenerated benzyl adenine (BA) encouraged more shoot elonga-
shoots directly on medium containing 0.5 mg/l tion and leaf development than the medium free of
of NAA and BA in combination with 1 mg/l GA3 growth regulators or without BA (Table 5). Leaves in
(Table 4 and Fig. 2). All embryogenic callus ma- these treatments turned to yellow then died within
tured and began to regenerate shoots after trans- 15 days. These results indicate the importance of
ferring to fresh medium containing the same PGR growth regulators, especially cytokinin, for good
combination. These findings agree with those re- shoot elongationin rose. However, kinetin was bet-
ported on other cultivars of R. hybrida (Kim et al. ter than BA for the elongation of other cultivars of
2004). Results showed the vital role of using in vitro R. hybrida (Ibrahim, Debergh 2001), and zea-
mother plants for plant regeneration. This might tin was efficient for in vitro multiplication of other
be explained by the juvenility of in vitro plantlets, horticultural crops (Paprštein, Sedlák 2015). The

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(a) (b)

(c)

Fig. 2. Callus formation, somatic embryogenesis and shoot

regeneration from leaf discs of hybrid tea rose (‘‘Eiffel
Tower’’). (a) Non-embryogenic callus from in situ leaf discs,
(b) embryogenic callus from in vitro leaf discs, (c) shoot
regeneration from in vitro leaf discs using GA3

longest shoots with the most leaves per shoot were taining 1 or 2 mg/l IBA, three weeks after transfer
obtained with 1 mg/l of IAA and BA (Fig. 3). Simi- (Fig. 3). This result is in agreement with Shekha-
lar results were obtained on shoot elongation when wat et al. (2015) who found that IBA was the best
IAA was replaced by IBA (data non shown). auxin for rooting of Passiflora shoots. It should be
No roots were developed on media free of growth mentioned that root tips turned to brown when
regulators or containing IAA whatever its concen- shoots were kept on rooting medium more than two
tration. However, all plantlets rooted on media con- weeks. This problem was resolved by transferring
(a) (b)

Fig. 3. Shoot elongation (a) and plantlet rooting (b) of hybrid tea rose ‘‘Eiffel Tower’’

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plantlets to medium free of growth regulators. Simi- Mahmoud I.M.A., Hassanein A.M.A., El-keltawi N.E. (2011):
lar result was reported on R. damascene where aux- Factors affecting in vitro propagation efficiency of hybrid
ins were needed for root initiation only but not for tea rose. American-Eurasian Journal of Agricultural &
subsequent root development (Ginova et al. 2012). Environmental Sciences, 11: 847–853.
Mather J.P., Roberts P.E. (1998): Introduction to Cell and Tissue
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