SOMATIC EMBRYOGENESIS IN RUBUS CAESIUS L. SUSPENSION CULTURES SMARANDA VÂNTU * Introduction

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1 Analele ştiinţifice ale Universităţii Al. I. Cuza Iaşi Tomul LIV, fasc. 2, s.ii a. Biologie vegetală, 2008 SOMATIC EMBRYOGENESIS IN RUBUS CAESIUS L. SUSPENSION CULTURES SMARANDA VÂNTU * Abstract: In vitro cultures technologies include many applications: plants improvement, micropropagation,, unconventional alternatives to exploit secondary metabolites. The regenerative potential of Rubus caesis L. was evaluated for the establishment of a propagation protocol. Not only the capacity of differentiation was tested, but also the regenerative potential. The initiation of in vitro cultures at Rubus sanctus Schreb was achieved from axillary buds, cultivated on different variants of Murashige- Skoog medium. The callus cultures obtained by axillary buds dedifferentiation were multiplicated on agar medium and liquid medium. The MS medium supplemented with 1mg/l benzylaminopurine and 0,1 mg/l IAA stimulated somatic embryogenesis at Rubus caesius L. suspension cultures,whereas the MS medium supplemented with 1mg/ kinetine generated an intensive proliferative reaction and callus development. The regeneration of whole plants was obtained in three steps: callus initiation, suspension cultures initiation, somatic embryogenesis induction. Key words: somatic embryogenesis, Rubus caesius, suspension cultures Introduction In this paper the procedure for the regeneration of Rubus caesius L. plants based on indirect somatic embryogenesis is presented.the studies are focused on the application of in vitro methods on this species, well known as a medicinal plant. The fruits are a good source of natural antioxidants [1], [5], [6]. Material and methods The initiation of in vitro cultures of Rubus caesius L. was achieved from axillary buds. The axillary buds were sterilized with ethanol 70 % and then sodium hypochlorite 0,5 % minutes. After rinsing with sterile distilled water, the explants were transferred to MS medium. 4 variants of MS medium were tested. (Table 1 and 2). The studies of the in vitro behaviour of this species is based on the use of auxins and cytokinins, in different combinations and concentrations [2], [3], [4]. The variants 1 and 3 (Table 2) stimulated callus induction and callus multiplication, whereas the variants 2 and 4 stimulated somatic embryogenesis and variants 5, without growth regulators induced embryos development and maturation. The axillary buds explants were cultivated on the callus induction media at 24 C in complete darkness. After 30 days the callus produced was used to establish cell suspensions on the same basal MS medium. Suspension cultures were established by transferring the section of calli 1 cm 3 each, from the exponential growth phase into 250 ml Erlenmeyer flasks containing 100 ml liquid medium. The flasks were rotated on rotatory shaker at 100 rpm. The cell suspensions were periodically subcultivated by filtration with a metallic sieve. The next step was the induction of somatic * Al. I. Cuza University, Faculty of Biology, 20 A Carol I Bd, Iasi, , Romania 104

2 embryogenesis. The suspension cultures were transferred on regeneration media and maintained at 24 C under 16 hours light cycle. Table 1. Chemical composition of MS medium COMPOSITION NH 4 NO 3 Concentration (mg/l) 1650 MACROELEMENTS KNO 3 CaCl 2 2H 2 O MgSO 4 7 H 2 O 370 KH 2 PO H 3 BO 4 6,2 MICROELEMENTS MnSO 4 4 H 2 O ZnSO 4 7 H 2 O Na 2 MoO 4 2H 2 O 22,3 8,6 0,25 CuSO 4 5 H 2 O 0,025 CoCl 2 6H 2 O 0,025 KI 0,83 VITAMINES SUCROSE Nicotinic acid 0,5 Pyridoxine HCl 0,5 Thiamine 0,1 Mezoinozitol g/l ph 5,8 105

3 Table 2. Variants of MS medium VARIANTS GROWTH REGULATORS BAP IAA NAA K 1 1 mg/l 1 mg/l mg/l 0,1 mg/l mg/l 1 mg/l ,1 mg/l 1 mg/l Results and discussions The main aim of the investigation was to select different cell lines from suspension cultures for embryogenetic capacity. The axillary buds of Rubus caesius L. were the explants for callus initiation. Five different media are used for plant propagation via somatic embryogenesis. These media differ by the type and level of plant growth regulators. The begin of callus proliferation was achieved on two variants of MS medium: variants 1 with a same concentration of benzylaminopurine and indolilacetic acid and variants 3 with the same concentration of kinetine and naphtalenacetic acid. The first cell proliferation were observed on the cantact surface of the explant with nutritive medium (Photo 1, 2). The primary callus cultures were obtained after 4 weeks from the in vitro cultivation, when the entire explant dedifferentiated (Photo 3). The callus cultures obtained on agar medium were multiplicated on liquid medium (Photo 4). The submerse cultures were periodically transferred on fresh medium. The suspension cultures were cultivated on rotatory shaker at 100 rpm and maintained in the dark. After 2 weeks, they were transferred on somatic embryogenesis induction medium (Photo 4, 5). The main factors for somatic embryogenesis induction were: the selection of cells, the use of growth regulators in some concentrations and combinations, the photoperiode. The embryogenic clusters were selected after each stage of subcultivation on induction media. Early stages of somatic embryogenesis were observed after a week from the initiation of the submerse cultures (Photo 6). The specific steps of the embryo development that were encountered were: globular, cardo, torpedo and cotyledonary stages. Embryogenesis was induced under various ratios of auxin to cytokinin. Despite the selection, the suspension cultures display not only a morphological heterogenity, but also differences in cell reaction: some cells multiplied and formed clusters, without regenerative potential, whereas some cells redifferentiated and developed somatic embryos. 106

4 The conditions that favor initial somatic embryogenesis may inhibit further development of the embryos. That is why the variants 2 and 4 are used for initiation of somatic embryogenesis and variant 5 is utilized to allow somatic embryos development. The use of MS medium supplemented with excess of benzylaminopurine in combination with indolilacetic acid and kinetine in combination with naphtalenacetic acid stimulated the somatic embryo development. The differentiated embryos could be converted into plants by transferring to the MS medium for maturation, without growth regulators. Conclusions Callus induction was stimulated on MS medium with an equal concentration of a cytokinine and auxine. Cell suspensions of Rubus caesius L. were obtained from callus cultures derived from axillary buds. The development and maturation of the somatic embryos occurred after 4 weeks in suspension cultures. The use a combination of a cytokinine in excess and an auxine led to a increase in the frequency of embryos differentiation in suspension cultures. Changing the growth regulators balance in the MS medium has major effects on in vitro dedifferentiation and redifferentiation at Rubus caesius L. REFERENCES 1. BENVENUTI, S., PELLATI, F., MELEGARI, M., BERTELLI, D., 2004-Polyphenols, anthocyans, ascorbic acid and radical scavenging activity of Rubus, Ribes and Aronia, Journal of Food Science 69(3): BUSBY, A., HIMELRICK, D.G., Propagation of blackberries (Rubus ssp.) by stem cuttings using various IBA formulations, Acta Hort, 505: FIOLA, J.A., SCHWARTZ, H., 1986-Somatic embryos organogenesis and proliferation in vitro from Rubus embryos, Acta Hort., 183: FIOLA, J.A., HASSAN, M.A., SWARTZ, H.J., BORS, R.H., MCNICHOLS, R., Effect of thidiazuron light fluence rates and kanamycin on in vitro shoot organogenesis from excised Rubus cotyledons and leaves, Plant Cell, Tissue and Organ Culture, 20: MOYER, R.A., HUMMER, K.E., FINN, C.E., FREI, B., WROLSTAD, R. E., Anthocyanins phenolics and antioxidant capacity in diverse small fruit: Vaccinium, Rubus and Ribes, Journal of Agricultural and Food Chemistry, 50: NIKITINA, U.S., KUZMINA, L., MELENTEV, A.I., SHENDEL, G.V., Antibacterial activity of polyphenolic compounds isolated from plants of Geraniaceae and Rosaceae families, Applied biochemistry and microbiology, 43(6):

5 SMARANDA VÂNTU PLATE Photo 1. Callus development Photo 2. Cell proliferation Photo 3. Primary callus cultures Photo 4. Suspension cultures Photo 5. Cell cluster Photo 6. Embryogenic cells cultures 108