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Callus Cultures and Indirect Regeneration of Ruscus Hypoglossum in Vitro

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Callus Cultures and Indirect Regeneration of Ruscus Hypoglossum in Vitro 49 Bulgarian Journal of Agricultural Science, 19 (2) 2013, 49–51 Agricultural Academy CALLUS CULTURES AND INDIRECT REGENERATION OF RUSCUS HYPOGLOSSUM IN VITRO T. IVANOVA*, D. DIMITROVA, G. ANGELOV, Ch. GUSSEV, Y. BOSSEVA and T. STOEVA Bulgarian Academy of Sciences, Department of Plant and Fungal Diversity and Resources, Institute of Biodiversity and Ecosystem, BG – 1113 Sofi a, Bulgaria Abstract IVANOVA, T., D. DIMITROVA, G. ANGELOV, Ch. GUSSEV, Y. BOSSEVA and T. STOEVA, 2013. Callus cultures and indirect regeneration of Ruscus hypoglossum in vitro. Bulg. J. Agric. Sci., Supplement 2, 19: 49–51 Ruscus hypoglossum L. is a highly valued ornamental plant collected mainly from the wild and threatened in several Euro- pean countries. Slow growth and low germination rates hamper its cultivation. Micropropagation has been considered advanta- geous for rapid production of planting material and ex situ conservation of Ruscus species. Callus cultures of R. hypoglossum were induced on TDZ containing medium and indirect shoot regeneration rate was evaluated on different medium composi- tion. TDZ ensured higher regeneration rates, caused shoot, and cladode alterations in regenerates persisting throughout the cultivation. Callus induction was optimal at 30 g.l–1 sucrose and dropped down with the increase of sucrose concentration. In- crease of the sucrose (15–60 g.l–1) infl uenced positively shoot proliferation. DNA content of the morphologically altered plants was not signifi cantly different. Isozyme profi le patterns demonstrated loss of isoform bands and/or induction of new ones. Key words: genome size, isoenzymes, micropropagation, rhizome, Ruscaceae Abbreviations: 6-benzylaminopurine (BAP), α-naphtaleneacetic acid (NAA), acid phosphatase (AP), esterase (EST), growth index (GI), kinetine (KIN), polyacrilamide gel electrophoresis (PAGE), peroxidase (POD), polyacrilamide gel electrophoresis (PAGE), thidiazuron (TDZ) Introduction use of plant growth regulators (Elmaghrabi and Ochatt, 2007). Such variations can be detected using morphogical and mo- Ruscus hypoglossum L. (Ruscaceae) is a slow-growing lecular markers, which provide useful information about the rhizomatous shrub native to the Euro-Mediterranean area. genetic stability of the micropropagated plants (Larkin and The species is used as ornamental plant for its evergreen long- Scowcroft, 1981; Rani and Raina, 2000). Data about R. hypo- lasting branches, widely collected from the wild, thus causing glossum callus culture and the effectiveness of micropropaga- severe threat to the natural populations (Karlović, 2009). R. tion through indirect shoot regeneration are missing in litera- hypoglossum is included in the Habitat Directive 92/43/EEC ture. The applicability of R. hypoglossum micropropagation as an economically important plant species of conservation through organogenic callus cultures is discussed. Assessment value. In Bulgaria it is under the regulation of the Biodiversity of the morpho-physiological variation, DNA content and iso- Act (2002), however uncontrolled supply to local fl ower mar- zyme profi les of the cultures is presented. kets is frequent. Micropropagation is considered advantageous to counterpoise overexploitation of natural populations. How- Material and Methods ever, limited data on in vitro cultivation of R. hypoglossum are available (Abou Dahab, 2005а, b). Wide range of variations Callus cultures were induced on agar MS media (Mu- is observed in tissue culture and is mainly associated with the rashige and Skoog, 1962) supplied with 0.5 mg. l–1 TDZ *E-mail: [email protected] 50 T. Ivanova, D. Dimitrova, G. Angelov, Ch. Gussev, Y. Bosseva and T. Stoeva from rhizomatous tissue of in vitro germinated seeds col- Results lected from Bulgarian wild population of R. hypoglos- sum. Callus and shoot induction, morphological variation Initiation of callus cultures from seedling rhizome ex- and viability were evaluated on media with TDZ, KIN, plants of R. hypoglossum was prolonged for more than a BAP, NAA (Table 1). The subsequent growth and devel- year. Callus growth and shoot development were limited opment of the obtained organogenic callus was estimated on hormone-free media and shoot number was reduced on hormone-free media with 15-90 g. l–1 sucrose. Culture on media without TDZ (Table 1). Only TDZ promoted growth index (GI) was calculated: GI = (FW2-FW1)/FW1 culture growth while KIN, BAP and NAA suppressed the (FW1 – starting fresh weight, FW2 – fi nal fresh weight). development and caused necrosis. Highest proliferation Cultivation was performed in containers, with agar medi- rate was obtained at 1 mg.l–1 NAA and 0.5 mg.l–1 TDZ um (0.8%). Four repetitions with 10 explants were set for (average 5 shoots/explants). Despite of the good prolif- each treatment with growth regulators and 5 repetitions eration TDZ caused signifi cant morphological alterations. with 6 calli for the effect of sucrose concentration. All me- Most frequent variation was associated with overgrowth dia were autoclaved at 121ºC and 101.325 kPa for 20 min. of shoots and cladodes, bifurcation of cladode tips and pH was adjusted to 5.75 prior to sterilization. The cultures unusual fl ower position. Shoot proliferation in callus cul- were kept at 24±1ºC and 16/8 h photoperiod (40.5 μmol tures was best stimulated by transferring on hormone-free m–2.s–1) with passages at every 2 months. Four-month old media with 60 g.l–1 sucrose (Table 2). The highest sucrose rooted plants were transferred in pots with mineral mix- concentration 90 g.l–1 caused severe decline reaching 50% ture (Bentonite®, BG). DNA content of the cultures from necrotic explants. Molecular assessments of the cultures 2 clones was measured using fl ow cytometry with propid- 2C, pg ium iodide staining, on Partec CyFlow® SL and following 17.4 the protocol provided by Partec. Solanum pseudocapsi- cum (2C = 2.59 pg) was used as internal standard. Iso- 17.2 zyme profi les of anodal isoforms of EST, POD and ACP were resolved using discontinuous PAGE system accord- 17 ing to Angelov (2003). Data were processed statistically by single factor ANOVA followed by Duncan’s multiple 16.8 range test (SPSS Inc., 2000). 16.6 Table 1 Effect of growth regulators on growth and development 16.4 of callus cultures of R. hypoglossum: – no callus induc- tion/variation/ necrosis; + less than ¼ of explants with 16.2 callus induction/variation/ necrosis; ++ ½ of explants with callus induction/variation/ necrosis; +++ more than 16 2/3 of explants with callus induction/variation/ necrosis Control CloneA CloneB Growth regulator, mg.l–1 Shoot Callus Morph. Necrosis Fig. 1. DNA content of R. hypoglossum regenerants number induction variation Table 2 NAA BAP TDZ KIN % 0.25d – – +++ Effect of sucrose on callus growth and shoot proliferation 0.5 1 0.33d – – +++ in R. hypoglossum in vitro cultures 0.5 0.00c – – +++ Sucrose, Calluso GI Average Morph. Necrotic 1 1.28c + ++ + g.l–1 genesis, shoot variation, explants, 0.2 0.95c +++ +++ – % number % % 0.5 3.25b +++ +++ – 15 36.67a 2.07b 3.07c 40a 0.00b 0.5 0.89c – + ++ 30 13.33b 3.54a 5.79b 40a 0.00b 1 0.5 0.00d – – +++ 60 6.67b 2.74ab 8.38a 0b 6.67b 1 0.5 5.00a +++ +++ – 90 0.00b 0.88c 1.86c 0b 50.00a Values followed by same letters are insignifi cantly different Values followed by same letters are insignifi cantly different p ≤ 0.05 (Duncan’s multiple range test) p ≤ 0.05 (Duncan’s multiple range test) Callus Cultures and Indirect Regeneration of Ruscus hypoglossum In vitro 51 tion phase, also known source of variation in tissue culture (George at al., 2008). Conclusion The observed variation caused by indirect regeneration in R. hypoglossum could be considered positive for breeding of new forms for horticulture. However for conservation pur- poses, the proposed protocol could be a source for undesired genetic instability. References Abou Dahab, A. M., Afaf M. A. Habib, Y. A. Hosni and A. M. M. Gabr, 2005a. Effect of MS-salt strength, sucrose and IBA con- centration andacclimatization media on Ruscus hypoglossum L. micropropagation. Arab J. Biotech., 8 (1): 141–154. Abou Dahab, A. M., Afaf M. A. Habib, Y. A. Hosni and A.M.M. Gabr, 2005b. Effect of some sterilization treatments and growth regulators on Ruscus hypoglossum L. Arab J. Biotech., l. 8 (1): 127–140. Angelov, G., 2003. Isoenzyme variation and genetic relationships among Eliytrigia junceiformis, E. × litorea and E. repens (Tri- ticeae: Poaceae). Ann. Bot. Fenn., 40: 83–70. Banciu, C., M. Mitoi and A. Brezeanu, 2009. Biochemical peculiarity of in vitro morphogenesis under conservation strategy of Ruscus aculeatus L. Ann. For. Res. 52: 109–116. Elmaghrabi, A. and S. Ochatt, 2006. Isoenzymes and fl ow cytom- Fig. 2. Isozyme profi les (POD, EST, ACP) etry for the assessment of true-to-typeness of calluses and cell of R. hypoglossum regenerants suspensions of barrel medic prior to regeneration. Plant Cell Tissue And Organ Culture, 85: 31-43. showed that marked variability in shoot and cladode mor- George E., M. Hall and J. De Klerk, 2008. Plant Propagation by phology is not associated with signifi cant variation in Tissue Culture, 3rd Edition, Springer, 502 pp. DNA content (Figure 1). However profi les of the three Huetteman, C. and J. Preece, 1993. Thidiazuron: a potent cytoki- isozyme systems present evidence that in vitro clones did nin for woody plant tissue culture. Plant Cell Tiss. Org. Cult., not share common bands with the control and/or express 33: 105–119. Karlović, K., 2009. Introduction of Ornamental Native Plants into additional ones, especially in the ACP system (Figure 2). Commercial Production in Croatia. Acta Hort. (ISHS), 813: Adaptation of the cultures ex vitro conditions was 50% in 107–112. greenhouse conditions. Larkin, P. and W. Scowcroft, 1981. Somaclonal variation – A novel source of variability from cell cultures for plant improve- Discussion ment. Theor. Appl. Genet., 60: 197–214. Moyano, E., M.
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