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Analele ştiinţifice ale Universităţii „Al. I. Cuza” IaşiTomul LVII, fasc. 1, s. II a. Biologie vegetală, 2011

IN VITRO MULTIPLICATION OF ROSA CANINA L.

SMARANDA VÂNTU

Abstract: The studies are based on vegetative multiplication of Rosa canina L., using meristematicexplants. It were tested the axillary buds reactivity on different variants of MS medium, supplemented withgrowth regulators: two types of auxins (2,4 diclorphenoxiacetic acid and naphtalenacetic acid) and twocytokinins (kinetine and benzylaminopurine). The caulogenesis induction was based on the use of variouscombination of auxins and cytokinins (variants I – 0,02 mg/l 2,4 D – 2 mg/l K and variants II – 2 mg/l BAP-0,02 mg/l NAA). The evaluation of explants reactivity was the number of developed shoots encountered oninitial explant. After two month from the initiation of culture, the shoots were isolated and cultivated separatelyon the MS medium, for roots induction. The whole plant reconstruction protocol lasted 10-12 weeks.

Keywords: Rosa canina, micropropagation, axillary bud.

Introduction

Rosa canina L. is a species native to Europe, northwest Africa and westernAsia. The plant prefers light (sandy), well-drained soil and can grow in heavy clay soil.

The plant prefers acid, neutral and basic (alkaline) soils. Is a scamblingdeciduous shrub with erect or arched stems, up to 5 m long, that are covered in hookedthorns.

Leaves are pinnate and divided into five to seven leaflets. Flowers are 30-50mm across and comprise of five pink or whitish petals. Flowers often solitary butsometimes produced in small groups. The plant is high in certain antioxidants. The seedis a good source of vitamin E. The fruit is a very rich source of vitamins and minerals,especially in vitamins A, C and E, flavanoids and other bio-active compounds[1],[7],[8],[9],[13],[14].

The cultivation of Rosa canina L. through unconventional techniques offermany advantages arising from opportunities to improve, conserve and perpetuate a plantwith food and pharmaceutical value [2],[3],[4],[10],[11],[12].

This paper aims to approach one of these techniques: vegetative multiplicationfrom meristematic explants, that allows a clonal micropropagation.

Materials and methods

Explants used in this experiment consisted axillary buds taken from joungbranches of Rosa canina L. (Photo 1 and 2).

Preparation of explants for inoculation was the chemical treatment using asequence of reagents: ethanol 70%, 5 seconds, sodium hypochlorite 3%, 10 minutes,sterile distilled water to remove chemicals used previously.

Alexandru Ioan Cuza University, Faculty of Biology, Bd. Carol I, no. 11, Iasi - 700506 Romania,s_vantu@yahoo.com

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The initiation of cultures aimed assesing the possibilities of direct regeneration,using meristematic explants. Consequently it has worked on five variations of thenutrient medium MS (Table 1). Culture conditions were a 16 h photoperiod and 23±2 0C.

Table I. Variations of MS medium to induce morphogenesis: 2,4 D – 2,4 diclorphenoxyacetic;NAA – naphtalenacetic acid; BAP – benzylaminopurine; K – kinetine

Variations ofMS medium

Growthregulators

Concentration mg/l

2,4 D 0,02IK 2BAP 2IINAA 0,02

III BAP 2IV K 2V - -

Results and discussions

The studies have focused on developing a protocol for rapid vegetativemultiplication using meristematic explants of Rosa canina L., collected fromindividuals belonging to the wild flora.

Reactivity test was performed on axillary buds variants of MS medium,supplemented with growth substances, in different concentrations and combinations(Table 1).

Were tested two types of auxins: 2,4 diclorphenoxyacetic acid andnaphtalenacetic acid and two types of cytokinins: kinetine and benzylaminopurine.

An important role in inducing this process is often associated with cytokininsand auxins. Can trigger caulogenesis by reducing the concentrations of auxins [5],[6].

Explants taken from a same plant show different organogenous potential. Themost common reaction, recorded 10 days after initiating the culture, was shootelongation. This process was achieved using relatively high concentrations of cytokinins(variants I, II).

Caulogenesis induction (Photo 3, 4) was recorded on a nutrient substrate withkinetine in excess (variants I- 0,02 mg/l 2,4 D- 2 mg/l K), but also with an excess ofbenzylaminopurine (variants II- 2 mg/l BAP- 0,02 mg/l NAA).

Main parameter in evaluating the effect caulogen was growth rate of shootsdeveloped from the bud explants. Explants reactivity to chemical stimuli was similar interms of the type used cytokinine.

After about two month after initiation of culture, shoots were isolated andcultured separately in order to rooting, on MS medium without growth regulators (Photo5).

Cultivating bud explants on inductive media for direct caulogenesis ended withthe formation of shoots and rooting them afterwards, the whole plant reconstructiongoing through a period of 10 to 12 weeks after initiating of culture.

Roots development depends on a series of limiting factors such as:carbohydrates, temperature (20-25 °C), pH, other nutrients, light.

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Conclusions

Micropropagation method applied to the species analyzed in this paper wasbased on the use of Rosa canina L. type bud explants.

Use excess of BAP in combination with NAA and 2,4 D in combination with Kproved beneficial for caulogenesis and thus triggering for direct micropropagation.

This pathway of regeneration “in vitro” is the most effective to increase themultiplication efficiency.

The presence of a large number of buds on the shoots cause economicefficiency, since from each bud can be regenerated a new one.

REFERENCES

1. BANTHORPE D. V., GREY T. J., 1986 – Monoterpene metabolism in cultures of Rosa species.Phytochemistry, 25, 10: 2321-2326.

2. GUSTAFSON A., 1944 – The constitution of the Rosa canina complex. Hereditas, 30: 405-428.3. KAMO K., JONES B., BOLOR J., SMITH F., 1996 – Regeneration from long term embryogenic callus of

the Rosa hybrida cultivar Kardinal J. In Vitro Cellular and Developmental Biology, 41: 32-36.4. KIM C. K.., CHUNG J. D., BURRELL A. M., BYRNE D. H., 2004 – Somatic embryogenesis and plant

regeneration from in-vitro-grown leaf explants of rose. Hort Science, 391: 1378-1380.5. KUCHARSKA D., GRUCHALA A., ORLIKOWSKA T., 2006 – In vitro propagation of four rose

rootstocks. Propagation of ornamental plants, 6, 1: 44-50.6. PATI P. K., RATH S. P., SHARMA M., SOOD A., AHUJA P. S., 2005 – In vitro propagation of rose - a

review. Biotechnology Advances, 24, 1: 94-114.7. RAZUNGLES A., OSMIANSKI J., SAPIS J. S., 2008 – Determination of Carotenoids in Fruits of Rosa sp.

(Rosa canina and Rosa rugosa) and of Chokeberry (Aronia melanocarpa). Journal of Food Science,54, 3: 774-77

8. ROSSNAGEL K., WILLICH S.N., 2001 –Value of complementary medicine exemplified by rose-hips.Gesundheitswesen 2001 Jun, 63, 6: 412-6.

9. SCHUM A., HOFMANN K., GHALIB N., TAWFIK A., 2001 – Factors affecting protoplast isolation andplant regeneration in Rosa sp. Gartenbauwissenschaft, 66, 3: 115-122.

10. VAN D. M. F., PIJNAKER H. J. P., 1990 – In vivo transformation of clonal Rosa canina rootstocks withAgrobacterium rhizogenes. Journal of Genetics and Breeding, 44, 4: 263-268.

11. VISESSUWAN R., KAWAI T., MII M., 1997 – Plant regeneration systems from leaf segment culturethrough embryogenic callus formation of Rosa hybrids and Rosa canina. Breed. Sci., 47: 217-222.

12. VANISREE M., CHEN YUE LEE, SHU FUNG LO, NALASWODE S. M., CHIEN ZIH LIN, HSINTSAY, 2004 – Studies on the production of some important secondary metabolites from medicinalplants by tissue cultures. Bot. Bull. Acad. Sin., 45: 1-22.

13. WAGNER H., BLADT S., ZGAINSKY E. M., 1984 – Plant Drug Analysis - A Thin LayerChromatography Atlas. Springer Verlag: 20-50.

14. ZIELINSKI J., PETROVA A., TAN K., 2004 – Taxonomic status of the roses (Rosa) described by S. G.Dimitrov from Bulgaria. Ann. Bot. Fennici , 41: 449-451.

Explanation of the PlatePhoto 1. Explant sourcePhoto 2. Axillary budPhoto 3. Shoot developmentPhoto 4. Shoot elongationPhoto 5. Roots development

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Smaranda Vântu PLATE I

Photo 1 Photo 2

Photo 3 Photo 4

Photo 5