Analele ştiinţifice ale Universităţii Al. I. Cuza Iaşi Tomul LII, s. II a. Biologie vegetală, 006 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 (), (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.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-,4 D mg/l Cytokinins- K mg/l - - 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 weeks. The rate of growth of the callus varies considerably with the amount of,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). 7
The callus differentiated into shoots when transferred to MS medium containing mg/l K and mg/l,4 D (Photo, 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 4 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 mg/l K and mg/l,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.. 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: 39-57 SARKER, R.H., SHAHEEN, I. 001. 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. 001. Advances in tissue culture of Chrysanthemum morifolium In: Dallos M.P. (Ed.)- Agricultural Biotechnology, a focus on the improvement of plants ACEVIV VÂNTU, S. 005- 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 - 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