Int. J. Agricult. Stat. Sci. Vol. 16, No. 1, pp.

259-263, 2020 ISSN : 0973-1903

ORIGINAL ARTICLE

IN VITRO MULTIPLICATION OF SHOOTS’ TIP AND NODES IN DAHLIA

HYBRYIDA

Aysar M. S. Almemary1*, Waad S. Faizy2 and Bashar Z. Kassab Bashi1

1
Department of Horticulture & Landscape, College of Agriculture & Forestry, Mosul University, Iraq.
2
Department of Plant Production Techniques, College of Agriculture, Northern Technical University, Iraq.
E-mail: aysaralsalim@yahoo.com

Abstract : This investigation was carried out at plant tissue culture laboratories of Agriculture College, University of Duhok
during the period 1/4/2018 to 1/12/2018. The objective of this study was to investigate the influence of different concentrations
of BA or Kinetin on multiplication of shoot tips or nodes of Dahlia hybryida cv. Karma choc and the effect of IBA on rooting
shoots propagation in vitro. The results can be summarized as follows: the highest shoots number 2.5, 2.25 were achieved
from cultured shoot tips on MS medium supplemented with 2.0 mg/L BA, 4.0 mg/L Kinetin respectively after eight weeks,
otherwise cultured nodes on MS medium supplemented with 0.5 mg/L BA, 4.0 mg/L Kinetin gave the highest shoots number
2.87, 4.00 shoot/ explant respectively after eight weeks. The best rooting of shoot produced in vitro were achieved from
cultured shoots on half strength medium supplemented with 0.4 mg/L IBA, which gave the highest rooting percentage 60%
with highest root number 7.00 root/explant and root length 2.43 cm.
Key words : Dahlia hybryida, Shoot multiplication , IBA, Complete Randomized Design (C.R.D).

1. Introduction [Al-Taey (2017), Al-Taey and Majid (2018)].

Dahlia plant belongs to Asteraceae family and is
diversified in terms of colors, sizes, shapes, forms and
the profusion of flowering [De Hertogh (1989)]. The
native of this plant is Mexico which got spread through
cultivation to all global countries at the beginning of
17th century. Dahlia flowers are cut flowers and the
plant is a dicotyledonous one which has composite leaf
type with 3-7 leaflets [Dole and Wilkins (1999)]. Dahlia
plants reproduce both by sexually via seed and by
vegetatively via tuberous roots. This plant can be easily
infected by fungal, bacterial and viral diseases [Bose
and Yadav (1989)]. The in vitro culture technique is
the best method to successfully treat these infections
so that healthy dahlia plants can be cultivated [Sediva
et al. (2006), George et al. (2008)].
Phytohormones are considered as the most
important endogenous substances in the modulation of
physiological and molecular responses, a critical
requirement for the survival of plant as sessile
organisms. Phytohormones act at its site of synthesis
*Author for correspondence Received November 11, 2019
Cytokinins are known to significantly improve the
growth of crop plants. A number of researchers
conducted investigations on the ability of Cytokines to
improve the plant growth and cell division [Al-Taey and
Saadoon (2012)]. It plays a vital role in organogenesis
by stimulating cell division in vitro whereas the root
development depends on the addition of plant growth
regulators in culture medium especially auxin [Skoog
and Muller (1957)]. Majid and Israa (2015) found the
highest regeneration percentage for shoot 86.67% when
the shoot tips of Dahlia explants were cultured in MS
medium supplemented with 2.0 mg.L-1 for each of BA
and NAA, respectively. The study attained the best
effects in shoot tips rooting (96.67%) which gave 3.00
roots per explant and 1.63 cm root length when used
0.6 mg.L-1 IBA. Fatima et al. (2007) found that the
maximum response of shooting was obtained to be
13.00% shoot when the shoot tip of Dahlia explants
were cultured in MS medium supplemented with a
combination of BA and NAA 0.3 mg.L-1. Sharma et
Revised April 19, 2020 Accepted May 06, 2020
260 Aysar M. S. Almemary et al.
al. (2001) found the largest number of shoots when
the shoot tip of the Dahlia explants were cultured in
MS medium supplemented with 0.41 mg.L-1 for each
of BA and NAA, respectively.
2. Materials and Methods
The study was conducted in plant tissue culture
laboratory, College of Agriculture, University of Duhok,
Iraq, cv. The shoot tips and nodes of ‘Karma choc’
plant with dark red flowers were used in this study.
The samples were sterilized with 10% sodium
hypochlorite for a period of 10 minutes and washed
thrice in sterile distilled water. Then the samples were
transferred to the Laminar Air-flow Cabinet for
disinfestation. The explants were cut into 1.0 cm length
and then cultured in MS medium [Murashige and Skoog
(1962)] containing different concentrations of BA (such
as 0.0, 0.5, 1.0, 1.5 and 2.0) mg.L-1 or Kin (such as
0.0, 2.0, 4.0, 6.0 and 8.0) mg.L-1 for multiplication and
shoot regeneration. From the previous experiment, the
samples were cultured in MS full strength or half
strength of MS medium supplemented with IBA at (0.0,
0.2, 0.4, 0.6 and 0.8) mg.L-1 for rooting. All the media
were dispensed in 150 ml that had 20 ml media. The
cultures were incubated under 1,000 Lux light intensity
provided by white fluorescent lamps for 16/8 hrs
photoperiod and temperature 27 ± 1ºC. The plantlets,
produced in vitro, were acclimatized by culturing it in
10 cm diameter-plastic pots containing peat moss and
sand in the ratio of 1:2, placed in growth room under
controlled conditions. The recorded characteristics of
multiplication included the number of shoots, length of
the shoots and number of leaves after 4 or 8 weeks.
Further, for rooting record, the rooting percentage (%),
number and length of roots, and the length of shoots
were measured after 4 weeks. The investigation was
designed as Complete Randomized Design (C.R.D).
The comparison between the means was carried out
according to Duncan’smultiple range test (p < 0.05).
3. Results and Discussion
Table 1 infers that the treatment of 2.0 mg/L BA
yielded the highest number of shoots per explant i.e.,
1.76 and 2.5 shoots for establishment and multiplication
stages, respectively. The highest number of shoots and
leaves were achieved in control treatment after 8
weeks.
Table 2 shows that the treatment of 4.0 mg/L Kin
produced the highest number of shoots per explant i.e.,
2.25 and 2.25 shoots for establishment and multiplication
stages, respectively. The highest shoot length achieved
from the control treatments were (1.68, 3.30) cm for
establishment and multiplication stages, respectively
when 2.0 mg/L Kin was added, it yielded the higher
number of leaves i.e., 8.87 and 11.50 leaf/explant,
respectively. These results confirmed the findings of
AL-Qudaht et al. (2008) i.e., BA is the most important
growth regulator for shoot multiplication and a
relationship exists between BA levels and shoot number
[Messeguer et at. (1993)]. The reason behind the
positive role of cytokinins in multiplication stage might
be due to the vital role played by cytokinins in releasing
the lateral buds from the dominance of terminal buds
without removing the apical bud. It is clear from the
data on Tables 1 and 2 that BA was more effective
than Kin on multiplication traits. This can be due to
internal structure of BA and the number of double bonds
[Mohammed (1985)]. The side chain of BA contains
three double bonds whereas Kin has only two.
Furthermore, the activity of cytokinins got increased
with the increase in the number of double bonds in their
side chain [Krishnamoorthy (1981)]. Further, the
presence of benzene ring on BA structure increases its
efficiency and makes it the most effective cytokinine
[Wasfy (1995)].
Table 3 infers that the addition of BA was effective
on establishment and multiplication stages in terms of
number of shoots and the concentration of 0.5 mg/L
produced 2.62 and 2.87 shoots per explant respectively,
while the highest shoot length was achieved in the control
treatment which yielded 1.12 and 2.17 cm for
establishment and multiplication stages. This treatment
0.5mg/L BA also produced the highest number of leaves
for these stages.
Table 4 concludes that the highest number of shoots
i.e., 3.00 and 4.00 were achieved from cultured nodes
on MS supplement 4.0 mg/L Kin, respectively. This
seems to be significantly different when compared with
other treatments where the longest shoots reached were
1.28 and 2.81 cm with the addition of 2.0 mg/L Kin.
This treatment produced the highest number of leaves
during for these stages and are clarified in the discussions
regarding (Tables 1 and 2).
Table 5 shows that the concentration of 0.4 mg/L
IBA produced the highest rooting percentage (50%)
and this treatment yielded the highest number of roots
 In vitro Multiplication of Shoots’ Tip and Nodes in Dahlia hybryida 261

Table 1 : Effect of BA on initiation and multiplication of Dahlia hybryida shoot tips cultured on MS medium.
Establishment stage after 4 weeks Multiplication stage after 8 weeks
BA (mg.L-1)
Shoots Shoots Leaves Shoots Shoots Leaves
Number Length (cm) Number Number Length (cm) Number
0.0 1.25b 1.68 a 7.12 a 1.62 a 3.30 a 10.50 a
0.5 1.37a 0.56b 8.75 a 2.00a 1.43 a 9.25 a
1.0 1.62a 0.48b 5.00 a 2.00a 1.10 a 8.62 a
1.5 1.62a 0.61b 6.50 a 2.00a 1.17 a 8.12 a
2.0 1.76a 0.40b 4.87 a 2.50a 1.15 a 9.12 a
Different letters within the columns represent significant differences according to Duncan’s multiple range test at 5% level.
Table 2 : Effect of Kin on initiation and multiplication of Dahlia hybryida shoot tips cultured on MS medium.
Establishment stage after 4 weeks Multiplication stage after 8 weeks
Kin (mg.L-1)
Shoots Shoots Leaves Shoots Shoots Leaves
Number Length (cm) Number Number Length (cm) Number
0.0 1.25bc 1.68 a 7.12 ab 2.00a 3.30 a 10.50 a
2.0 1.25bv 1.17b 8.87 a 2.00a 2.01 a 11.50 a
4.0 2.25 a 0.65 bc 6.00b 2.25 a 1.48 a 6.50 ab
6.0 1.62ab 0.66bc 6.62 ab 2.12a 1.61 a 10.87 a
8.0 0.75c 0.46c 2.37 c 1.37a 1.01 a 9.25 a
Different letters within the columns represent significant differences according to Duncan’s multiple range test at 5% level
Table 3: Effect of BA on initiation and multiplication of Dahlia hybryida nodes cultured on MS medium
Establishment stage after 4 weeks Multiplication stage after 8 weeks
BA (mg.L-1)
Shoots Shoots Leaves Shoots Shoots Leaves
Number Length (cm) Number Number Length (cm) Number
0.0 1.75b 1.12a 5.50a 2.00b 2.17 a 8.50 ab
0.5 2.62a 0.63ab 6.25 a 2.87 a 1.61 ab 11.75a
1.0 2.12a 0.63ab 5.87 a 2.25ab 1.63 ab 11.62 a
1.5 2.12a 0.75ab 5.37 a 2.12ab 0.97 b 6.87 b
2.0 2.37a 0.43b 5.25a 2.75 ab 1.08 b 8.62 ab
Different letters within columns represent significant differences according to Duncan’s multiple range test at 5% level.
Table 4 : Effect of Kin on initiation and multiplication of Dahlia hybryida nodes cultured on MS medium.
Establishment stage after 4 weeks Multiplication stage after 8 weeks
BA (mg.L-1)
Shoots Shoots Leaves Shoots Shoots Leaves
Number Length (cm) Number Number Length (cm) Number
0.0 1.88bc 1.21a 6.55 a 1.88 bc 2.35 ab 9.11 ab
2.0 2.14b 1.28a 8.14 a 2.28 b 2.81 a 12.71 a
4.0 3.00a 0.90a 7.37a 4.00 a 1.73 bc 8.87 ab
6.0 1.37 cd 0.30b 2.75b 2.37 b 1.26 c 7.75 b
8.0 1.00d 0.11b 2.25b 1.25c 1.01 c 7.25 b
Different letters within columns represent significant differences according to Duncan’s multiple range test at 5% level.

(2.15 roots) with highest root length (5.60 cm) from highest root percentage (60%) was achieved from
culture shoots on full strength MS media. culture shoots on half strength MS media that was
As per the Table 6, it can be concluded that the supplemented with 0.4 mg/LIBA. This treatment
262 Aysar M. S. Almemary et al.

Fig. 1 : Propagation of Dahlia. A- multiplication of shoot tips cultured on MS medium supplement with (2.0 mg/L) BA after
eight weeks. B- multiplication of nodes cultured on MS medium supplement with (4.0 mg/L) BA after eight weeks.

Fig. 2 : A- Plant let to pots, B- Acclimatization.

Table 5 :Effect of different of IBA on rooting culture in full Table 6 :Effect of different of IBA on rooting shoots of
MS medium. Dahlia cultured in half strength MS medium.
IBA mg.L-1 Root% Nunber root Length root IBA mg/L Root% Number of roots Length root
0.0 30 1.70 a 5.57 a 0.0 40 4.55 a 2.00 a
0.2 40 1.75 a 5.16 a 0.2 50 5.42 a 2.02 a
0.4 50 2.15 a 5.60 a 0.4 60 7.00 a 2.43 a
0.6 40 2.12 a 3.43 b 0.6 50 6.80 a 2.30 a
0.8 20 2.00 a 2.55 b 0.8 40 6.16 a 2.00 a
Different letters within columns represent significant Different letters within columns represent significant
differences according to Duncan’s multiple range test at 5% differences according to Duncan’s multiple range test at 5%
level. level.
 In vitro Multiplication of Shoots’ Tip and Nodes in Dahlia hybryida
yielded the highest number of roots (7.0 roots) and the
longest root length (2.43 cm).
The results proved that the IBA has a role in rooting
process since it promotes the budding of adventitious
roots in the basses of cultured shoots [Abdul (1987)
and Saleh (1991)].
The results further inferred that the shoots that were
cultured in half strength-MS medium were more
effective than the roots that were cultured in full strength
MS medium (Fig. 1). This is because of the C:N ratio
that increases the sucrose and reduces the nitrogen in
the source medium [Hartmann (2002)]. The plantlets
produced in vitro were acclimatized at the laboratory,
in plastic pots covered with glass flask for two weeks
and then transferred to green-house where the cover
was gradually removed in one month (Fig. 2). All the
acclimatized plants were transferred to lath house
successfully.
Acknowledgement
Authors are thankful to the anonymous referee for
his critical comments for the much improvement on the
earlier draft of this manuscript.
References
Abdul, K.S. (1987). Plant Growth Regulators. Salahaddin
University. Ministry of Higher education and Scientific
Research, IRAQ (In Arabic).
AL-Qudaht, A., K. Al-Maarri and R. Shibli (2008).
Micropropagation of Ajami and Baladi by tissue culture.
Damascus University Journal, 25, 275-194.
Al-Taey, D.K.A. and Z.Z. Majid (2018) Study Effect of Kinetin,
Bio-fertilizers and Organic Matter Application in Lettuce
under Salt Stress. Journal of Global Pharma Technology,
10(1), 148-164.
Al-Taey, D.K.A. (2017). Mitigation of Salt Stress by Organic
Matter and GA3 on Growth and Peroxidase Activity in
Pepper (Capsicum annum L.). Advances in Natural and
Applied Sciences, 11(10), 1-11.
Al-Taey, D.K.A. and A.H. Saadoon (2012). Effect of treatment
of kinetin to reduce the salinity damage by drainage
water irrigation on the growth and nitrate accumulation
in the leaves of spinach, Spenacia oleracea L. Euphrates
Journal of Agriculture Science, 4(4), 11-24.
Bose, T.K. and L.P. Yadav (1989). Commercial Flowers, Naya
Prakash, Calcutta, India, 267.
De Hertogh, A.A. (1989). Holland Bulb Forcer’s Guide. 4th
263
ed., Int. Flower Bulb Center, Hillegom, The Netherlands.
369.
Dole, J.M. and H.F. Wilkins (1999). Floriculture principles
and species. Prentice Hall, New Jersey, 287-291.
Fatima, B.M., T. Usman, R. Ashraf Waseem and M.A. Ali
(2007). In vitro regeneration from cotyledon and
hypocotyl explants of dahlia cultivars. Pak J Agri Sci.,
44(2), 312-316.
George, E.F.M.A. Hall and G.J. De Klerk (2008). Plant
propagation by tissue culture. Springer, 1, 206-217.
Hartmann, H.T., D.E. Kester, F.T.J. Davies and R.L. Geneve
(2002). Plant Propagation, Principles and Practices.
7th edition. Prentice upper saddle river- Hall, Iac., New
Jersey.
Krishnamoorthy, H.N. (1981). Plant Growth Substances. Tata
Mc Grow Hill Publishing Company Limited, New Delhi.
Majid, A. I. and A. D. Israa (2015). Micropropagation of
dahlia plants Dahlia variabilis wild (Desf.) Effect of
explant and plant growth regulators on shoot
regeneration and growth. University of Basra In Iraq.
AAB Bioflux, 7(1), 1-6.
Messeguer, J., M.C. Arconada and E. Mele (1993).
Adventitious Scientia Hort., 54, 153-163.
Mohammed, A.K. (1985). Plant Physiology. 2nd Part.
Ministry of Higher Education and SCI. Research, Uni.
Of Mosul, Iraq. (In Arabic).
Murashige, T. and F. Skoog (1962). A revised medium for
rapid growth and bioassays with tobacco tissue culture.
Physiol Plant, 15, 437-497.
Saleh, M.M.S. (1991). Physiology of Plant Growth
Regulation. Mosul University Publishers. Ministry of
Higher Education, Iraq (In Arabic).
Sediva, J., P. Novak, J. KanKa and J. Laxa (2006).
Micropropagation, detection and elimination of DMV
in the Czech collection of dahlia. Act Hort., 725, 495-
498.
Sharma, H.P.A.K. and K.K. Sinka Nag (2001). Callus induction
and regeneration from shoot apex of Dahlia pinnata
var. white alvas. Perspectives in Biotechnology,
Proceedings of a National Symposium, Warangal, India,
26-27 February 1999, 121-124
Skoog, F. and C.O. Miller (1957). Chemical regulation of
growth and organ formation in plant tissue cultured in
vitro. Sym. Soc. For Exp. Biol., 11, 118-131.
Wasfy, E. (1995). Plant Growth Regulators and Flowering
and their Use in Agriculture. Academic Library, Cairo-
Egypt.