71 ORGANOGENESIS IN CHRYSANTHEMUM MORIFOLIUM RAMAT

Analele ştiinţifice ale Universităţii “Al. I. Cuza” Iaşi
Tomul LII, s. II a. Biologie vegetală, 2006
ORGANOGENESIS IN CHRYSANTHEMUM MORIFOLIUM RAMAT (CULTIVAR
“ROMICA”) CALLUS CULTURES
SMARANDA VÂNTU
Abstract: The plants of Chrysanthemum morifolium Ramat. (cultivar Romica) have been regenerated
from callus cultures, established from stem and leaves explants. Callus cultures induced from stems had
a greater shoot differentiation than those obtained from leaves. Despite the great opportunity of genetic
variation in callus cultures, the regenerated plants differ not in their externel appearance from the
normal plants.
Key words: Chrysanthemum morifolium, callus, organogenesis
Introduction
In a previous paper it was reported a protocol for direct micropropagation at
Chrysanthemum morifolium Ramat. (cultivar Romica) (5).
Indirect regeneration through callus cultures offers the possibility to select and to
obtain new genotypes (4) .
One of the cultivars of Chrysanthemum morifolium Ramat., belonging to the
collection of Botanical Garden from Iasi was cultivated and regenerated “in vitro”. They
offer opportunities for rapid clonal propagation of some unique, superior genotypes. The
unconventional techniques permit the multiplication and maintenance of these genotypes
(2), (4),(6).
The micropropagation of Chrysanthemum morifolium Ramat. was achieved
through tissue culture technique and involved callus induction followed by shoot
multiplication, rooting and establishment of plantlets in soil.
The greatest disadvantage of some in vitro methods of vegetative propagation
(callus cultures) is the occurrence of genetic variation, because the more the organizational
structure of a plant is broken down, the greater is the chance of mutations.
Material and methods
The stem and leaves explants were sterilized for 10-15 minutes in sodium
hypochlorite solution 3 % and washed with sterilized distilled water
After desinfection, plant parts are excised in rectangular pieces approximately 5
mm in diameter. Stem explants were implanted apical side down to stimulate the natural
flow of auxins and carbohydrates.

“Al. I. Cuza” University, Faculty of Biology, B-dul Carol I no.11, 700506 - Iassy, Romania
71
The callus cultures were established from stem and leaves explants on MS
medium, supplemented with 2.4 D and K (Table 1).
The different variants of MS medium (I, III, IV) were used for callus induction.
The transfer of calluses to the same medium, containing lower levels of growth substances
resulted in some instances, in the neoformation of shoots and roots (3). The basal medium
for initiation of cultures was similar with the medium for regeneration , but the
concentration of auxin was reduced and the ratio auxin/ cytokinin was modified (1).
The regenerated plants tested for organogenesis contained in each case the same
growth substances as the initiation were transferred “ex vitro” to soil and the first few days
were protected with a transparent cover and watered with water at room temperature.
The culture substrate that has been used consisted of a mixture of soil. This
mixture has been sterilized by autoclaving.
Table 1-The variants of MS medium
The variants of basal MS
medium
I
II
III
IV
V
Auxins- 2,4 D mg/l
Cytokinins- K mg/l
0,2
0,2
2
2
-
0,2
2
0,2
2
-
Results and discussions
In practice is recommended the direct propagation for conservation the genetically
defined stoks.
The use of stem tips gives the best guarantee for the preservation of the original
genetic constitution.
This paper aims to study the possibility of vegetative propagation through callus
cultures at a cultivar of Chrysanthemum morifolium Ramat.
Visible proliferation can be recognized on the surface of explants after 5-10 days.
A compact, opaque, dark brown callus is formed within 2 weeks.
The rate of growth of the callus varies considerably with the amount of 2,4D and
the type of explants.
The dedifferentiation capacities were greater on stem explants, cultivated on
variants III and IV of MS medium.
The morphogenetical potential has been established to be greater in callus cultures
derived from stem explants. The organogenic callus grows rapidly and shows early an
extensive organization (Photo 1).
72
The callus differentiated into shoots when transferred to MS medium containing
2mg/l K and 0,2 mg/l 2,4 D (Photo 2, 3, 4, 5).. Callus cultures derived from stems had a
greater shoot differentiation than those derived from leaves.
After 30 days, green shoots appeared and roots were developed in MS medium
with no growth regulators (Table 1). Root induction on shoot cultures was achieved by
subculturing the shoots (Photo 6, 7).
The callus cultures for several weeks show signs of aging, noted as deceleration of
growth, necrosis and browning. It was observed the loss of regenerative potential in vitro
by repeated subculturing.
Aging is the result of exhaustion of nutrients, inhibition of nutrient diffusion,
evaporation accompanied by an increase in the concentration of some constituents of the
medium, accumulation of metabolites, some of which may be toxic.
The vigorous callus pieces, about 5 mm transferred to fresh medium at intervals of
4 weeks maintain the organogenic callus lines.
The acclimatization of regenerated plants is concerned in transfer to nonsterile
conditions with humidity control and temperature control.
The regenerated plants were transferred in soil and grown in a controlled
environment chamber with 16 hours photoperiod at 24°C. The regenerated plants obtained
through callus cultures were similar in appearance to the plants obtained by direct
micropropagation (Photo 8).
Conclusions
■ The capacity for callus formation depends on type of explants.
■ The morpho-genetical potential has been established to be greater in primary callus
cultures derived from stem explants.
■ The callus differentiated into shoots when transferred to MS medium containing 2mg/l K
and 0,2 mg/l 2,4 D.
■ The use a combination of a synthetic cytokines such as kinetin in excess and a auxine led
to a increase in the frequency of shoots differentiation in callus cultures.
■ Root induction on shoot cultures was achieved in MS medium without growth regulators.
■ The regenerated plants obtained from callus cultures were similar in morphology to the
direct micropropagated plants
■ The acclimatization of regenerated plants was achieved without difficulties.
■ The capacity for shoot differentiation depended on concentration of cytokinin in the
shoot-induction medium and age of the callus cultures.
■For root induction, shoots were excised and transferred to MS medium lacking growth
regulators.
■ The cultivar of Chrysanthemum morifolium Ramat. regenerated “in vitro”displayed an
vigorous growth capacity to the natural environment.
73
BIBLIOGRAPHY
1.
2.
3.
4.
5.
6.
ROUT, G.R., PALAI, S.K., PANDEY, P., DOS, P. 1997. Direct plant regeneration of Chrysanthemum
morifolium Ramat., influence of explant source, age of explant, culture environment, carbohydrates
nutritional factors and hormone regime, Proc. Nat. Acad. Sci. India, 67(8): 57-66
ROUT, G.R., DOS, P. 1997. Recent trends in the biotechnology of Chrysanthemum, a critical review,
Scientia Horticulturae 69: 239-257
SARKER, R.H., SHAHEEN, I. 2001. In vitro propagation of Chrysanthemum morifolium through callus
culture, Plant Tissue Cult. 11(1): 85-91
TRIGIANO, R.N., MAY, R.A., GRAY, D.J. 2001. Advances in tissue culture of Chrysanthemum
morifolium In: Dallos M.P. (Ed.)- Agricultural Biotechnology, a focus on the improvement of plants
ACEVIV
VÂNTU, S. 2005- In vitro multiplication of Chrysanthemum morifolium Ramat, An. Şt. Ale Univ. „Al. I.
Cuza”, Iaşi, t. LI, s. II a, Biologie vegetală, 75-81
ZĂPÎRŢAN, M., CACHIŢĂ-COSMA, D 1989. Date on the „in vitro” behaviour of several Chrysanthemum
cultivars, In vitro explant cultures- present and perspective, 55-57
Photo 1 - Stages of caulogenesis in callus
cultures of Chrysanthemum morifolium
Ramat. (Romica)
Photo 2 - Caulogenesis in callus
cultures
74
Photo 3 - Caulogenesis in callus
cultures
Photo 4 - Stages of caulogenesis in callus cultures of
Chrysanthemum morifolium Ramat. (Romica)
Photo 5 - Shoots differentiation from callus
Photo 6 - Regenerated plants of Chrysanthemum
morifolium Ramat. (Romica)
75
Photo 7 - Roots development at
Chrysanthemum morifolium Ramat.
(Romica)
Photo 8 - Acclimatization of plants
regenerated “via callus”
76