Different exotic dahlia cultivars namely Double Decorative and Double Giant were explored for shoot regeneration using cotyledon leaf and hypocotyl explants on MS medium supplemented with combinations of plant growth regulators (PGRs) viz. NAA and BA (A medium) and BA and IAA (B medium). Callus induction and shoot regeneration was found directly proportional while root formation was inversely proportionate to the addition of PGRs in the media. Maximum response for callus induction (60%) and shoot regeneration (13%) was found in cotyledon leaf and hypocotyl explants, respectively, on medium containing 3 mgL -1 each of NAA and BA. Significant genotypic variation was observed and cultivar Double Giant was found better for callus induction and organogenesis at both media types. Indirect shoot regeneration could be helpful for mass propagation of elite plant material and enhancing diversity in the ornamental plants

B Fatima

M A Ali

Muhammad Usman

R Waseem

T Ashraf

CiteSeerX

Pak. J. Agri. Sci., Vol. 44(2), 2007 
IN VITRO SHOOT REGENERATION FROM COTYLEDON AND HYPOCOTYL 
EXPLANTS OF DAHLIA CULTIVARS 
 
B. Fatima, Muhammad Usman*, T. Ashraf, R. Waseem and M.A. Ali 
Plant Tissue Culture Cell, Institute of Horticultural Sciences, University of Agriculture, Faisalabad. 
*Corresponding author’s E-mail: muhammadusman30@yahoo.com  
 
Different exotic dahlia cultivars namely Double Decorative and Double Giant were explored for shoot regeneration 
using cotyledon leaf and hypocotyl explants on MS medium supplemented with combinations of plant growth 
regulators (PGRs) viz. NAA and BA (A medium) and BA and IAA (B medium). Callus induction and shoot 
regeneration was found directly proportional while root formation was inversely proportionate to the addition of 
PGRs in the media. Maximum response for callus induction (60%) and shoot regeneration (13%) was found in 
cotyledon leaf and hypocotyl explants, respectively, on medium containing 3 mgL-1 each of NAA and BA. 
Significant genotypic variation was observed and cultivar Double Giant was found better for callus induction and 
organogenesis at both media types. Indirect shoot regeneration could be helpful for mass propagation of elite 
plant material and enhancing diversity in the ornamental plants.   
Keywords: Dahlia, shoot regeneration, NAA, BA, cotyledon leaf. 
 
INTRODUCTION 
 
Dahlia, member of Compositeae family, is a beautiful 
gift of nature and is an important garden plant due to 
its diversity in colors, size, shapes, forms and profusion 
of flowering (De Hertegh, 1989). It is grown as both 
annual and perennial plant. All the dahlia cultivars are 
derived from one immensely variable specie D. 
variables or D. pinnata (Chittenden, 1981). Dahlias are 
also medicinally important as its tubers contain high 
amount of insulin and fructose and small quantities of 
medicinally active compounds such as phytin and 
benzoic acid. Many important diseases of dahlia are 
caused by fungal, bacterial and viral sources leading to 
various types of impairment (Bose and Yadav, 1989) 
and can be successfully eliminated using micro-
propagated plant material (Sediva et al., 2006).  
Further, micropropagation of plants is a well known 
strategy for efficient production and propagation of the 
elite plant material. It helps in the development and 
rapid propagation of selected plants with desirable 
characters in shortest possible time and new cultivars 
can also be developed by protoplast fusion and genetic 
modifications (Pierik, 1987). A good micropropagation 
protocol for dahlia, therefore, could be useful for 
producing the low cost plant material in bulk. The 
regeneration of dahlia plants have been reported either 
directly from explants without callus formation or 
indirectly through callus induction and regeneration. 
There are a few reports available on in vitro 
propagation of Dahlia spp. However, in other flowering 
crops like carnation and gerbera, indirect regeneration 
is reported from certain explant sources like shoot 
meristems (Can and Koc, 1994; Poupet et al., 2006), 
floral buds (Radice and Marconi, 1998; Posada et al., 
1999) and hypocotyl (Sharma et al., 1999) on different 
media and the shoot and root regeneration occurred in 
the calli (Kongthong et al., 1999). The present study 
was therefore, aimed to develop an efficient protocol 
for mass propagation of exotic Dahlia cultivars to 
enhance their production for growers and the local 
markets. 
 
MATERIAL AND METHODS 
Plant material and sterilization procedures  
Seeds of exotic Dahlia cultivars viz. Double Decorative 
and Double Giant were sterilized using 70% ethanol 
(v/v) and 5% sodium hypochlorite (v/v) for 5 minutes 
followed by 3-5 times rinsing with sterilized water. 
Seeds were placed on MS medium (Murashige and 
Skoog, 1962) containing 30gL-1 sucrose and 8gL-1 for 
germination and plant development. These plants were 
multiplied using shoot tip and nodal explants on MS 
medium and were used as explant source for further 
callus induction experiments. Cotyledon leaf (CL) and 
hypocotyl (Hyp) explants were explored for callus 
induction and organogenesis on MS medium 
containing plant growth regulators (PGRs) NAA, BA 
and IAA alone and in combinations as given in table 1. 
Media preparation, sterilization and culture 
conditions 
Medium pH was adjusted at 5.8 and the media were 
sterilized in autoclave at temperature 121°C and 15 psi 
pressure for 15-20 minutes. After inoculation, cultures 
were placed in the growth room facilitated with 2500 
lux light intensity and 25 ± 2°C temperature for proper 
plant growth and development. 
312 
Fatima, Usman, Ashraf, Waseem and Ali 
Table 1. Media formulations for callus induction 
and organogenesis 
Plant Growth Regulators (PGRs) 
in mgL-1Treatments 
NAA BA IAA 
A medium 
To - - - 
T1 0.1 0.1 - 
T2 0.1 1.0 - 
T3 0.2 0.3 - 
T4 0.2 1.5 - 
T5 0.3 2.0 - 
T6 0.5 0.5 - 
T7 1.0 1.0 - 
T8 2.0 2.0 - 
T9 3.0 3.0 - 
B medium 
To - - - 
T1 - 0.05 0.05 
T2 - 0.1 0.1 
T3 - 0.2 0.2 
Plant acclimatization 
The regenerated plants were acclimatized following 
standard procedures (Khan et al., 2006) and were 
transferred to greenhouse for plant development. 
Data analysis 
The experiments were laid out following completely 
randomized design (CRD) using three replications per 
treatment. Means were compared using LSD test 
(Steel and Torrie, 1980)  
 
RESULTS AND DISCUSSIONS 
Plant development and multiplication 
Sterile plants of exotic dahlia cultivars Double 
Decorative and Double Giant were raised from 
sterilized seeds in vitro on MS medium (Murashige and 
Skoog, 1962) and germination was observed as 82% 
and 91% after 3 weeks of culture, respectively. Plants 
were multiplied using shoot tip and nodal explant to get 
explant (cotyledon and hypocotyl) material in bulk for 
further callus induction and shoot regeneration.  
Callus induction and organogenesis 
Different PGRs were used in combinations (NAA+BA; 
A medium) and (BA+IAA; B medium) to induce shoot 
regeneration in different explants of dahlia cultivars. 
However, it is worth mentioning that both the explants 
cotyledon leaf (CL) and hypocotyl (hyp) induced calli 
that subsequently regenerated into shoots when 
subcultured on the same medium. Significant 
genotypic differences among dahlia cultivars were 
observed for callus induction and organogenesis in CL 
explant whereas no variation was found for Hyp 
explant on both A and B media treatments (Fig. 1). 
However, different combinations of PGRs used 
significantly affected callus induction and plant 
regeneration.  
Callus induction was increased with the increase in the 
levels of PGRs in A (NAA+BA) and B (BA+IAA) media 
for both cotyledon leaf and hypocotyl explants. 
However, cotyledon leaf was found significantly better 
for callus induction (60.50%) compared to hypocotyl 
(50.50%) and the callogenesis was two fold high 
compared to control in both the explants in A (30.33% 
and 24.16%, respectively) and B medium (48.00% and 
38.33%, respectively). Similar trend was found for 
shoot induction which was also enhanced with the 
increasing PGR levels in both A (13.0% and 10.5%) 
and B (9.8 and 8.0%) media for calli derived from both 
hypocotyl and cotyledon leaf explants, respectively. 
Root formation started in the same medium and was 
inversely proportionate to the addition of PGRs in both 
A and B media. The efficiency of rooting was better in 
cotyledon leaf (23.50% and 21.83%) compared to 
hypocotyl explant (22.50% and 20.33%) in both B and 
A media, respectively (Table 2 and 3). 
Our results showed indirect shoot regeneration from 
calli induced on MS medium containing BA+NAA (A 
medium) and BA+IAA (B medium) instead of inducing 
direct shoot induction from cotyledon leaf and 
hypocotyl explants, respectively. Several researchers 
have also reported similar response of the explant 
tissues on medium containing either of these plant 
growth regulators (PGRs) alone or in combinations 
(NAA, BA and IAA) and obtained shoot regeneration 
through callus formation in Gerbra (Barbosa et al., 
1993; 1994) Asparagus (Pontaroli and Camadro, 
2005), Aster (Cammareri et al., 2002), Guar (Prem et 
al., 2005), Saussurea (Dhar and Joshi, 2005), Salvia 
(Ewa and Halin, 2004) and Piper (Bhat et al., 1992). 
These findings are in agreement to our results 
depicting indirect organogenesis. In contrast, there are 
reports on response of explants on media containing 
these PGRs and showing direct organogenesis instead 
of callus formation in Snapdragon (Cui et al., 2004), 
Dioscorea (Chen et al., 2007), Perilla (Hou and Jia, 
2005), Thapsia (Makunga et al., 2005) and chestnut 
rose (Wen and Deng, 2005) and the rooting was 
obtained on either same media devoid PGRs (MSO) or 
half strength media salts and either of NAA or IAA in 
reduced concentration.  
 313
Shoot regeneration in dahlia cultivars 
Table 2. Cal
me
A medium 
(MS mediu
Control 
0.1 + 0.1mg
0.1 + 1.0 m
0.2 + 0.3 m
0.2 + 1.5 m
0.3 + 2.0mg
0.5 + 0.5mg
1.0 +1.0 mg
2.0 + 2.0 m
3.0 + 3.0 m
LSD values
 
 
 
logenic and morphogenic response of explants to application of PGRs (NAA+BA) in the A 
dium  
Callus 
induction % 
Shoot 
induction % 
Root  
induction % m+NAA+BA) 
CL Hyp CL Hyp CL Hyp 
30.33h 24.16h 1.83g 1.00gh 5.33g 6.66e 
L-1 32.16gh 27.33g 3.16ef 1.83g 21.83a 20.33a 
gL-1 33.16fg 30.00f 4.16e 3.50fg 14.66c 14.50b 
gL-1 35.00f 34.66e 2.66fg 4.50f 17.66b 10.50c 
gL-1 40.16e 35.00e 3.50ef 6.83de 13.00c 9.00d 
L-1 41.16e 38.33d 6.33d 9.00bc 10.00de 8.00de 
L-1 44.66b 40.83c 7.50cd 9.50bc 7.50f 7.16e 
L-1 48.33c 41.83c 8.66bc 8.00cd 8.83ef 7.33e 
gL1 54.00b 43.83b 8.83b 10.66b 8.50ef 8.00de 
gL1 60.50a 50.50a 10.50a 13.00a 11.00d 11.00c 
 1.935 1.421 1.224 1.167 1.744 1.389 314
Fatima, Usman, Ashraf, Waseem and Ali Table 3. Callogenic and morphogenic response of explants to application of PGRs (BA+IAA) in the B 
medium  
Callus  
induction % 
Shoot 
induction % 
Root 
induction % B medium (MS medium+BA+IAA) 
CL Hyp CL Hyp CL Hyp 
Control 33.83d 31.00c 1.16d 3.50c 9.00c 5.66d 
0.05 + 0.05 mgL-1  41.83c 33.33d 3.50c 4.16c 23.50a 22.50a 
0.1 + 0.1 mgL-1 44.66b 36.66a 5.50b 7.50b 23.50a 18.00b 
0.2 + 0.2 mgL-1 48.00a 38.33a 8.00a 9.83a 19.33b 15.33c 
LSD values  1.450 1.731 1.172 1.030 1.999 2.357 Indirect organogenesis seems promisingly important in 
raising somaclonal variants and inducing diversity in 
the ornamental flowering plants like dahlia that usually 
lacks in the direct regeneration protocols. Further the 
system is efficient to be used for mass propagation of 
the elite cultivars in short time. However, further 
optimization of PGR levels and use of new explants is 
suggested for developing direct shoot regeneration in 
dahlia that could have been used for biotechnology 
applications.  
 
ACKNOWLEDGEMENT 
 
The authors are thankful to Dr. Muhammad Aslam 
Khan, Prof. Institute of Horticultural Sciences, UAF for 
provision of seeds of exotic dahlia cultivars.  
 
REFERENCES 
 
Barbosa, M.H.P., M. Pasqual, P. Jepb, E.F. Arello, B. 
Ide and D. Baros. 1993. Effects of 
Benzylaminopurine and Indol acetic acid on in vitro 
propagation of Gerbera jamesonii Bolus ex Hook 
cv. Applebloesem. Pequisa Agropecuaria 
Brasileira, 28: 15-19. 
Barbosa, M.H.P., P. Jebp, P. Capb and R. Innecco. 
1994. In vitro propagation of Gerbera jamesonii 
Bolus ex Hook cv. Applebloesem using young 
capitulums. Revista Ceres. 41: 386-395. 
Bhat, S.R., A.K. Kackar and K.P.S. Chandel. 1992. 
Plant regeneration from callus cultures of Piper 
longum L. by organogenesis. Plant Cell Rep., 11: 
52-5-528. 
Bose, T.K. and L.P. Yadav. 1989. Floriculture in India. 
Allied Publishers Ltd., New Delhi. 14: 41-48. 
Cammareri, M., A. Errico, E. Filippone, S. Esposito and 
C. Conicella. 2002. Induction of variability in 
chimeric Aster cordifolius ‘White Elegans’ through 
somaclonal variation. Euphytica, 128: 19-25. 
Can, C. and N.K. Koc. 1994. Investigation on in vitro 
micropropagation of carnation (Dianthus sp.). 
Dramancthk Dergisi, 16: 641-648. [Hort Absts., 64: 
2971]. 
Chen, F.Q., Y. Fu, D.L. Wang, X. Gao and L. Wang. 
2007. The effect of plant growth regulators and 
sucrose on the micropropagation and 
microtuberization of Dioscorea nipponica Makino. 
J. Plant Growth Regulators, 26: 38-45.   
Chittenden, F.J. 1981. Commercial Flowers. The Royal 
Horticulture Society of Gardening-II. 628-633. 
Cui, M.L., K. Takayanagi and T. Handa. 2004. High 
frequency from hypocotyl and stem segments of 
Antirrhinum majus (Snapdragon). Plant Cell, 
Tissue and Organ Culture, 78: 51-53.  
De Hertegh, A.A. 1989. Holland Bulb Forcer` s Guide. 
4th ed. Int. Flower bulb center, Hillegom. The 
Netherlands. 
Dhar, U. and M. Joshi. 2005. Efficient plant 
regeneration protocol through callus for Saussurea 
obvallata (DC.) Edgew. (Asteraceae): effect of 
explant type, age and plant growth regulators. 
Plant Cell Rep, 24: 195-200.  
Ewa, S. and W. Halina. 2004. In vitro regeneration of 
Salvia nemorosa L. from shoot tips and leaf 
explants. In Vitro Cell and Dev Biol, 40: 596-602. 
Hou, S.W. and J.F. Jia. 2005. In vitro regeneration of 
Perilla frutescens from hypocotyl and cotyledon 
explants. Biol Plant., 49: 129-132. 
Khan, M.S., M. Usman and M.I. Lilla. 2006. Facile plant 
regeneration from tomato leaves induced with 
spectinomycin. Pak. J. Botany, 38(4): 947-952. 
Kongthong, K., S. Komkris, P. Prutpongse and K. 
Kularb. 1999. In vitro culture of dahlia hybrids. Thai 
J Agri Science 32:9-18. 
Makunga, N.P., A.K. Jager and J. van Staden. 2005. 
An improved system for the in vitro regeneration of 
Thapsia garganica via direct organogenesis-
influence of auxins and cytokinins. Plant Cell 
Tissue and Organ Culture, 82: 271-280. 
 315
Shoot regeneration in dahlia cultivars 
Murashige, T. and F. Skoog. 1962. A revised medium 
for rapid growth and bioassays with tobacco tissue 
cultures. Physiol. Plant., 15: 473-497. 
Pierik, R.L.M. 1987. In vitro culture of higher plants. 
Martinus Nijhoff Pub., Dordrecht. The Netherlands. 
169-171. 
Pontaroli, A.C. and E.L. Camadro. 2005. Plant 
regeneration after long term callus culture in clones 
of Asparagus officinalis L. Biocell, 29: 313-317. 
Posada, M., N. Ballesteros, N. Obando, A. Angarita 
and G. Fischer. 1999. Micropropagation of 
Gerberas from floral buds. Acta Hort., 482: 329-
332. 
Poupet, R., L. Cardin, A. Henriand and J.P. Onesto. 
2006. Healthy in vitro propagation by meristem tip 
culture of Helleborus nigeris selected clone for cut 
flower. Acta Hort., 725: 301-310. 
Prem, D., S. Singh, P.P. Gupta, J. Singh and S.P.S. 
Kadyan. 2005. Callus induction and de novo 
regeneration from callus in Guar (Cyamopsis 
tetragonoloba). Plant Cell Tissue and Organ 
Culture, 80: 209-214. 
Radice, S. and P.L. Marconi. 1998. Micropropagation 
of several Gerbera jamesonii cultivars from 
capitula. Revista-de-la Facultaed-de-Agronomia-
La-Planta.103: 111-118. 
Sediva, J., P. Novak J. Kanka and J. Laxa. 2006. 
Micropropagation, detection and elimination of 
DMV in the Czech collection of dahlia. Acta Hort., 
725: 495-498 
Sharma, H.P., A.K. Sinka, K.K. Nag and S.M. Redy. 
1999. Callus induction and regeneration from 
shoot apex of Dahlia pinnata. Perspectives 
Biotech., pp.121-124. 
Steel, R.G.D. and J.H. Torrie. 1980. Principles and 
Procedures of Statistics. McGraw Hill Book Co. 
Inc., New York, USA.  
Wen, X.P. and X.X. Deng. 2005. Plant regeneration 
from immature zygotic embryos of chestnut rose 
(Rosa roxburghii Tratt) through organogenesis. 
Hort Science and Biotech., 80: 638-642. 
 316


IN VITRO SHOOT REGENERATION FROM COTYLEDON AND HYPOCOTYL EXPLANTS OF DAHLIA CULTIVARS

http://citeseerx.ist.psu.edu/viewdoc/download;jsessionid=E0F668316FD873398564C5547D8A8DBB?doi=10.1.1.1055.9869&rep=rep1&type=pdf

IN VITRO SHOOT REGENERATION FROM COTYLEDON AND HYPOCOTYL EXPLANTS OF DAHLIA CULTIVARS

Abstract

Similar works

Full text

Available Versions

CiteSeerX