A Protocol for Mass Production of Rosa hybrida cv. Iceberg Through in vitro Propagation

INTRODUCTION
Roses are the most economically important flowers in
the world. There are more than 20,000 commercial
cultivars, which are collectively based on only 8 of the
approximately 200 wild species in the genus rosa
(Roberts and Smith, 1990).
Traditionally, most ornamental roses are heterozygous and do not breed true to type, they are therefore
propagated vegetatively. Miniature roses are usually
propagated by cuttings but the roses are usually propagated by budding or bench-grafting onto rootstocks
of species such as Rosa. canina ‘Inermis’ and Rosa
multiflora ‘Simplex’ (Short and Roberts, 1991). The
conventional propagating methods are very slow, time
consuming, and tiring. Tissue culture on the other hand
is becoming increasingly popular as an alternative
means of plant vegetative propagation.
Although the presence of a cytokinin is almost
always advantageous, and is often all that is required,
optimum rates of shoot initiation generally occur with
combinations of auxin and cytokinin (George, 1993).
Natural and synthetic auxins have been used extensively in vitro in plant cell tissue and organ culture to
obtain specific morphogenic responses. One of the
most important applications of auxins is the induction
of adventitious root formation. Induction of adventitious roots in roses has been demonstrated by workers
such as Skirvin et al. (1990). But the presence of auxin
in defined combinations with cytokinins in the culture
medium is also necessary to obtain adventitious shoot
formation (Caboni and Tonelli, 1999).
‘Iceberg’, also known as ‘Fée des Neiges’, a repeat
reblooming floribunda, was bred by Kordes in
Germany and is the result of a cross between ‘Robin
Hood’, a Pemberton bred hybrid musk (1927) and
‘Virgo’ a large flowered hybrid tea rose (1947). It’s
ever present double white flowers, often with a flush
of pink in spring and fall, are lightly fragrant. This rose
has won many awards including the National Rose
Society Gold Medal in 1958, the Baden-Baden Gold
Medal in 1958, the ADR Anerkannte Deutsche Rose
(Germany) in 1960, the World’s Favorite Rose in 1983
and the Royal Horticulture Society Award of Garden
Merit in 1993 (Figure 1a).
Although tissue culture of roses has been reported by
many authors but procedures for mass production at
commercial level has not been reported in the scientific
publications. In the present study, attempts were made
to assess the interactive influence of BAP (6-
Benzylaminopurine) and NAA (1-Naphtalene acetic
acid) concentrations on the growth rate of Rosa hybrida cv. Iceberg for the first time, and thus increase
growth rate to a level suitable for commercial use. We
also describe a procedure for root initiation, root growth
and acclimatization of plantlets to in vivo conditions.
MATERIALS AND METHODS
Plant material and general procedures: Nodal segments (1-1.5 cm) were taken from the stems of
‘Iceberg’ plants in the rose garden of the Agricultural
Biotechnology Research Institute of Iran (ABRII).
They were washed thoroughly with running tap water
for half an hour and surface sterilized for 30 seconds in
70% (v/v) ethanol, followed by a 15 min soak in 2.5%
(v/v) sodium hypochlorite solution with a few drops of
Tween-20 as a wetting agent, and then rinsed three
times with sterile distilled water. MS (Murashige and
Skoog, 1962) basal medium (without hormone) was
used for the in vitro of induction of explants in culture;
the pH of the medium was adjusted to 5.8 before
adding 8 gl-1 plant agar. Media were autoclaved for 15
min at 121ºC and 1.2 kPa pressure. Cultures were
placed under high pressure metal halide lamps on a
16/8 hour light/dark cycle in a culture room maintained at 21 ± 1ºC. Axillary shoots were detached and
transferred to VS medium (van der Salm et al., 1994)
in which FeNaEDTA was replaced by FeEDDHA as
iron source after 14 days.
Shoot proliferation: The shoot proliferation media
contained full strength VS salts and vitamins with various levels of BAP (0, 2, 4 and 8 µM) in combination
with NAA (0, 0.05, 0.25 and 0.5 µM). Each treatment
involved 5 repeats with 5 explants (25 explants).
Number of axillary shoots and number of new leaves
were recorded after 21 days for three subsequent subcultures and the averages were calculated.
Root initiation: Shoots were cultured on shoot elongation medium (VS mineral salts and vitamins without
hormones) for 21 days prior to rooting treatments. For
rooting, three concentrations of VS mineral salts and
vitamins (full-, half-, and quarter-strength) containing
IBA (3-Indolebutyric acid) (0.25 µM) and NAA (0.25
µM) were tested in semi-solid and liquid media. For
liquid medium, sorbarods (Cellulose support plugs;
Sorbarod, Ilacon, UK) were used (Figure 1d). Each
treatment involved 5 repeats with 5 explants (25
explants). After 21 days, number of roots and their
lengths were recorded and data for different concentrations of VS media (full, 1/2 and 1/4) and state of media
(semi-sold and liquid) were recorded.
Transfer to soil: In vitro rooted plantlets either in
semi-solid medium or in sorbarods containing liquid
medium were taken out from the jars and gently
washed under running water and then transferred to
plastic cups containing a mixture of pit and perlite
(1:1) each covered with a transparent plastic cup. A
hole was made in the covering cup every day, until day10, when the cover was removed completely. Plantlets
were treated with NPK (nitrogen-phosphate-potassium) (7 g/l) fertilizer every 14 days and the percentage
of survivors was recorded after 60 days.
Experimental design and statistical analysis: Shoot
proliferation experiment was analyzed in a factorial
based completely random design and root initiation
experiment was as a completely random design. Each
experiment was repeated twice. Analysis of variance
was performed and comparisons of means were conducted using Duncan’s Multiple Range Test. All analyses were regarded as significant at P ≤ 0.05.
RESULTS
Shoot proliferation: Micropropagation of rose cultivars range from easy to difficult; however multiplication rate (axillary shoots and new leaf production) of
R. hybrida cv. Iceberg was occasionally high, up to 13
shoots and 34 leaves (Figure 1b), depending on the
cytokinin content of the medium. Results presented in
Table 1(a, b) indicate that when 4 µM BAP was used
in combination with 0.05 and 0.5 µM NAA, significant
differences were not observed in the average number
of axillary shoots, but there was a significant difference in the average number of new leaves produced. A
multiplication rate of 10 fold with an average number
of axillary shoots (10.1) and new leaves per explant
(25) were obtained in the medium containing 4 µM
BAP plus 0.5 µM NAA.
The results of the present study demonstrated that
as the concentration of BAP was increased the number
of axillary shoots and new leaves per explant were
increased. Although in some cases the significant differences in the increase of growth rates were not
apparent, but it was statistically established that the
maximum growth rate (number of axillary shoots and
new leaves per explant) was achieved when 4 µM of
BAP and 0.50 µM of NAA were used. As the concentration of BAP was raised to 8 µM, a reduced growth
rate was noted with all of the NAA concentrations,
(Table 1).
Root initiation and acclimatization of in vitro
plantlets: Figure 1c illustrates the morphogenetic
responses of the shoots treated with three (full, 1/2 and
1/4) strengths of VS salts and vitamins. The results
indicate that the average percentage of rooting was
higher in 1/4 strength VS (85%), although it was not
significantly different from the plants treated with
1/2 VS (Table 2). The average number of roots (4.35)
and root length (0.82 cm) were significantly higher in
1/4 strength VS.
Table 2 compares the effect of semi-solid and liquid media. Statistical analysis indicates that there was
not a significant difference between the average root
length in semi-solid and liquid media. However, the
highest percentage of rooting (93.33%) and number of
roots (4.45) were observed in the semi-solid medium.
The survival rates of the rooted plantlets transferred to
soil were 70% and 90% in plants treated with semisold and liquid media, respectively.DISCUSSION
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. The
most commonly used explant in shoot proliferation of
roses is the nodal stem segment, wherein the axillary
bud is made to proliferate and form multiple shoots.
The performance of nodal segments is much better
than the shoot tips (Horn, 1992).
In the present study, the highest multiplication rate
of 10 fold was obtained in the medium containing BAP
(1.0-10.0 mg l-1) which was essential for bud break and
shoot multiplication of R. hybrida. Pati et al. (2001)
optimized conditions by using a BAP concentration of
5 µM for shoot proliferation in Rosa damascena and
Rosa bourboniana. Bressan et al. (1982) reported maximum promotive effects using BAP as compared with
2-isopentyladenine (2-ip). Rout et al. (1990) reported
that the presence of cytokinin in the culture medium
helped in the year round multiplication of shoots in
hybrid roses. Roberts and Schum (2003) indicated that
for introduction into culture and multiplication, BAP at
a concentration of 2 µM was adequate for most species
and cultivars of rose. They reported that a maximum
multiplication of two to five folds every 4-6 weeks is
possible for most rose species and cultivars, although
using the mother plant method increased the multiplication rate by an average of 28 fold.
Variation in growth rate (number of axillary shoots
and new leaves per explant) was noted with all of the
NAA concentrations when the BAP concentration was
changed, suggesting that increasing BAP levels were
more effective in stimulating growth rate than changing the NAA levels. This is in accordance with Vijaya
et al. (1991), who reported that BAP was the most
effective growth regulator in stimulating shoot proliferation. Kim et al. (2003) obtained the best shoot proliferation in the present of 2 mg l-1 of BAP and 0.01 mg
l-1 of NAA in full-strength MS salts. They also showed
that in vitro shoot proliferation and multiplication are
largely based on media formulations containing
cytokinins as a major plant growth regenators, whereas, in some cases, low concentrations of auxins and
gibberellic acid 3 (GA3) were also used. Statistical
analysis of our data showed that a moderate BAP level
(4 µM) combined with 0.5 µM NAA resulted in the
highest growth rate, which is achieved only when the
concentrations of endogenous or exogenous auxins
and cytokinins are balanced (George, 1993).
Although rose shoots often proliferate readily in
vitro, rooting of those shoots is proved to be more difficult. Kim et al. (2003) suggested that rooting is
affected by genotype; MS medium salts concentration,
cold dark treatment, and auxin type. The average number of roots and root length were significantly higher
in 1/4 strength VS medium which is in accordance
with Skirvin et al. (1990) who reported that the
reduced salt concentration generally increased rooting
in MS medium. Faisal and Al-Amin (2000) also
reported that using 0.2 mg l-1 of IBA and 0.2 mg l-1 of
IAA with half strength MS were the best combination
for root formation in Chrysanthemum morifolium.
Enhanced root initiation and growth in 1/4 strength
medium could be attributed to a more favorable nitrogen concentration availability and thus a higher rate of
rhizogenesis than provided by full VS mineral salts
(Hyndman et al., 1982).
Our investigation showed that the highest percentage of rooting and number of roots were observed in
semi-solid medium. Ebrahim and Ibrahim (2000) also
reported that solid medium supported the fastest
growth and development of both roots and shoots.
Ghashghaie et al. (1991) showed that in liquid medium, vitrification was a response to osmotic shock, and
therefore reduced rate of rooting, although in the present investigation due to the use of sorbarods, vitrification was not observed but our results also showed that
lower percentage of rooting and number of roots were
observed in plants treated with liquid medium. Rout et
al. (1990) also reported that rooting of microshoots
was better in solid medium than that in liquid medium
too.
The survival rate of the rooted plantlets transferred
to soil was higher in plantlets treated in liquid medium
than the ones treated with semi-sold medium. Roberts
et al. (1990) reported that after transfer to plastic cups,
plantlets that were rooted in sorbarod plugs wilted less
but transpired more water than plantlets taken from
agar. The protective environment of the sorbarods may
have contributed to less damaged roots, higher uptake
of water and therefore higher survival rates.
Acknowledgments
This work was funded by the Agricultural Biotechnology
Research Institute of Iran (ABRII) (Project number: 2-013-
140000-01-0000-84004).