TISSUE CULTURE AND EX-VITRO ACCLIMATION OF RHODODENDRON sp.

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1 Buletin USAMV-CN, 64/2007 (-) ISSN TISSUE CULTURE AND EX-VITRO ACCLIMATION OF RHODODENDRON sp. Clapa Doina, Al. Fira Fruit Research Station Cluj, 5 Horticultorilor Str. Horticultorilor nr.5, Cluj-Napoca, Romania, doinaclapa@yahoo.com, Key words: Rhododendron, tissue culture, initiation, multiplication, acclimation. Abstract:The majority of Rhododendron species reproduce by seeds, but the cultured varieties the production of planting material is done vegetatively by greenwood cuttings or by grafting. Another, much faster and more economical propagation method is propagation by tissue culture. In this paper aspects regarding the micropropagation of this species are presented, respectively, initiation, multiplication and acclimation. For initiation two variants of media were used: V1-Murashige & Skoog and V2-Woody Plant Medium (McCown). The biologic material used for initiation is represented by 2-5 cm long young shoots taken from plants isolated in the greenhouse, juvenilized by repeated prunings. For multiplication, two variants of medium were used, also, V1 - Woody Plant Medium (McCown)+ 2 mg/l Zeatin and V2 - Woody Plant Medium (McCown)+ 5 mg/l 2-Ip. INTRODUCTION The great number of flowers that it yields, their pleasant colour and the long duration of the flowering period make the Azalea to be used more and more for decorating interiors, balconies, terraces. The Azalea is usually reproduced by seed, by cuttings or by grafting, but this species also fully benefits from the advantages of micropropagation. It has a high multiplication rate, the in vitro rooting phase is eliminated from the micropropagation technology, which considerably reduces the costs and it can be easily acclimated. The nutritive media used for the Azalea are Woody Plant Medium, Anderson s Rhodoenndron Medium, Economou & Read medium in different variants (1,2,3,4,5). MATERIAL AND METHOD I.Initiation: The plant material used: young shoot fragments 2-5 cm in length taken from healthy plants isolated in the greenhouse, juvenilized by repeated prunings. From these shoots one-node microcuttings were taken, which were disinfected with 20 % bleach solution and rinsed with sterile distilled water in several steps. The nutritive medium (table 1) : two variants of nutritive media were used, V 1 Murashige & Skoog all the components from stock solutions) ph adjusted to 5,7 and V 2 - Woody Plant Medium (after Lloyd and McCown) + 5 mg/l 2-Ip, from prepackaged powder, stock-solutions of vitamins B1, B6 and nicotinic acid, the ph of the medium was adjusted to 5. In both variants the carbon source was castor sugar and powder agar was used as gelling agent (Caisson Laboratories Inc, catalog A 03, lot 630). All the components were added after media autoclavation. The media were dispensed into test-tubes and autoclaved at 121 C for 25 minutes. One single-node microcutting was inoculated into each test-tube.

2 The cultures were incubated in the growth room, in artificial light provided by fluorescent tubes. Light intensity was of cca lux, 16-hour photoperiod and the temperature was of C.100 test tubes were inoculated for each variant. Table 1 Variants of media used for initiation Components Variant 1 Variant 2 Microelements MS* WPM** Macroelements MS WPM Vitamins MS WPM 2-Ip 5 mg/l Sugar 25 g/l 25 g/l Agar 5 g/l 5 g/l ph 5,7 5 Sequestrene mg/l FeNa EDTA 36,7 mg/l - * Murashige & Skoog **Woody Plant Medium (McCown) II.Multiplication. The second stage, multiplication was done after 40 days, using 2 variants, V 1 -WPM + 3 mg/l Zeatin and V 2 - WPM + 5 mg/l 2-Ip (Table 2). For multiplication, the nutritive media were dispensed into Magenta vessels (approx. 50 ml/vessel), following the standard procedure of media preparation. 9 inoculi were planted into each vessel. Variants of media used for multiplication Table 2 Components V 1` Concentration WPM* salts 2,3 g/l 2,3 g/l Sequestrene mg/l 100 mg/l Myo inozitol 100 mg/l 100 mg/l Vitamin B1 2 mg/l 2 mg/l Vitamin B6 1 mg/l 1 mg/l Nicotinic acid 1 mg/l 1 mg/l 2-Isopentenyladenin 5 mg/l Zeatin 3 mg/l Sugar 30 g/l 30 g/l Agar 5 g/l 5 g/l ph 5 5 * Woody Plant Medium RESULTS AND DISCUSSION In the initiation phase the evolution of the inoculi was very weak on variant V 1 -MS, whereas on variant V 2 -WPM the regeneration percentage was of 60 %. In the second phase (multiplication) the shoots obtained in the initiation phase were fragmented into single-node microcuttings and planted into Magenta vessels, inoculi/vessel.for both variants (fig. 3). V 2`

3 Fig. 3. Multiplication phase 6 weeks after inoculation the evolution of the microcuttings was the following: -in variant V1 -WPM + 3 mg/l Zeatin from the microcuttings from a vessel 2 plantlets were obtained, which could be either transferred ex-vitro for acclimation or propagated further in vitro (Fig. 4). Fig. 4. V 1 Plants resulted onv 1` WPM + 3 mg/l zeatin -in variant V2 -WPM + 5 mg/l 2-Ip, from the microcuttings in a vessel a number of 14 plantlets were obtained, which could either be transferred ex-vitro for acclimation or propagated further in vitro (Fig. 5). Fig. 5. Plants resulted on V 2` WPM + 5 mg/l 2-IP

4 Some of the plantlets were transferred for acclimation into plastic trays (Fig. 6), using as substrate either perlite or peat-perlite mixture in a 1:1 volume to volume ratio in order to watch the acclimation of the plantlets (Table 3). Rootless plantlets were used for acclimation, the in vitro rooting stage being eliminated from the propagation technology of this species. Table 3 Acclimation pe of mixture peat+perlite 1:1 Total of plantlets transferred for acclimation 100 Plants without evolution 11 Plants with evolution 9 Acclimation rate (%) 9 Figure 6. Peat+perlite substrate 1:1 In optimal conditions of temperature, light and humidity, even a 100 % acclimation rate can be obtained. After acclimation, the plants can be planted into pots in order to produce plant material for sale. Some vessels were kept for another 2 weeks in order to obtain material for micropropagation. The evolution of the inoculi was the following: - in variant V1 4 shoots resulted on the average from each inoculum, which were again transferred to fresh medium - in variant V2 2 shoots resulted on the average from each inoculum, 1-4 cm in length., 1 microcuttings resulted from these shoots on the average, which were again transferred to fresh medium. The multiplication rate in the two variants is presented in table 4. Table 4 Multiplication rate Variant V1` WPM + 3 mg/l Zeatin V2` WPM + 5 mg/l 2-IP Initial no. of inoculi/vessel No. of inoculi obtained after weeks Multiplication rate 15

5 CONCLUSIONS For tissue culture initiation in Rhododendron sp. 2-5 cm long shoots were used, harvested immediately after sprouting, in April, from mother plants isolated in the greenhouse, from which single-node cuttings were made. On variant V2-WPM +5 mg/l 2-Ip regeneration was of 60 %. In vitro multiplication was done by using microcuttings taken from plants grown in vitro, using 2 variants of medium: V1 - WPM + 3 mg/l Zeatin and V2 - WPM + 5 mg/l 2-Ip. Six weeks after inoculation, in variant V1 from the microcuttings from a vessel 2 plantlets were obtained and in variant V2 from the microcuttings/vessel 14 plantlets were obtained, which could be either acclimated ex-vitro or propagated further. Acclimation was done in caooed plastic trays, using either perlite or peat+ perlite mixture. Rootless shoots were used for acclimation, the in vitro rooting phase being eliminated from the micropropagation technology of this species. The acclimation rate was of 9 %. weeks after inoculation, the multiplication rate was of 15 for variant V1 and for variant V2 For the micropropagation of Rhododendron sp we recommend the use of Woody Plant Medium after Lloyd and McCown and, as plant hormones, either Zeatin or 2-Ip, Zeatin being far more effective. BIBLIOGRAPHY 1. Fordham, I.; Stimart, D. P.; Zimmerman, R. H Axillary and adventitious shoot proliferation of Exbury azaleas in vitro, HortScience 17(5): ; 2. George, E.F., , Plant Propagation by Tissue Culture, Part 2, Exegetics Limited, Edington, Wilts; 3. Mercure E. W, Jones C. S., Brand M. H. and Auer C. A Anatomy of shoots and tumors of in vitro habituated Rhododendron 'Montego' (Ericaceae cultures with tissue proliferation, American Journal of Botany 5(5): ; 4. Singh K. K., Kumar S., Rai L. K. and Krishna A. P Rhododendrons conservation in the Sikkim Himalaya, Current Science, vol. 5, no.5, 10 September; 5. Tomsone S., Gertnere D., Novikova D The influence of thidiazuron on shoot regeneration and proliferation of rhododendrons in vitro, Acta Universitatis Latviensis, Biology,, Vol. 676, pp