Romanian Biotechnological Letters Vol. 15, No.1, Supplement, 2010 Copyright © 2010 University of Bucharest Printed in Romania. All rights reserved ORIGINAL PAPER

In vitro culture initiation and phytohormonal influence on henteri – a Romanian endemic

Received for publication, November 10, 2009 Accepted, February 8, 2010

VICTORIA CRISTEA1, ALEXANDRA-TIMEA BRUMMER2, LILIANA JARDA1, MIHAI MICLĂUŞ3 1”Babeş-Bolyai” University, “Alexandru Borza” Botanical Garden, phone/fax: 0264.592152, E-mail: [email protected] 2 ”Babeş-Bolyai” University, Faculty of Biology and Geology, Republicii 3 Waksman Institute of Microbiology, Rutgers University

Abstract

The biotechnology of in vitro culture plays an important role in the conservation of biodiversity nowadays. In vitro collections represent a viable alternative for species that have phytogeographic and sozologic importance and that are difficult to conserve outdoors. Dianthus henteri Heuff. ex Griseb. & Schenk (fam. ) is a strictly endemic species for the Meridional branch of the Romanian Carpathians, being threatened nationwide. The in vitro culture was initiated from hydrogen peroxide-sterilized seeds. The disinfection rate was 93.75% and 75% of the seeds germinated 15 days after inoculation. Three media were tested for multiplication and three others for rhizogenesis, having different balances of NAA and BAP phytohormones. The best results for multiplication were on NAA 0.1 mg l-1 and BAP 1 mg l-1 (hormonal balance 1/10) medium, while the NAA 1 mg l-1 and BA 0.1 mg l-1 medium gave the best results for rhizogenesis (hormonal balance 10/1). 83 days of in vitro cultures generated 110 to 240 new-explants/inoculum that could further be acclimatized to ex vitro conditions.

Keywords: in vitro culture, Dianthus henteri, micropropagation, rhizogenesis, ex situ conservation

Introduction

Nowadays in vitro plant tissue cultures have a wide range of applications in biodiversity conservation, among others. Endemic plant species or the ones vulnerable at different levels – cormophytes in general, but not only – can be multiplied and conserved through aseptic cultures. Later on they can be used in repopulating natural sites when this is considered appropriate. These studies have been initiated in our country in the 90s and are still ongoing now, using the biotechnology of in vitro cultures. Dianthus henteri Heuff. ex Griseb. & Schenk (fam. Caryophyllaceae) is a strictly endemic species for the Romanian Carpathians – the Meridional branch – and is also considered a vulnerable species [16], being threatened nationwide [15]. Studying this species by means of modern technologies proves to be even more important because it is reported as missing from the collections of all botanical gardens in the country. Due to the fact that it is a mountainous species, its maintenance on the rocks of the botanical gardens over time is difficult, therefore its conservation and multiplication through in vitro cultures is of great interest. This biotechnology has already been used for in vitro micropropagation and conservation of other Romanian or European Dianthus species [1, 2, 4, 7-11, 14, 19] but studies regarding the in vitro conservation of D. henteri have not been undertaken yet. This paper presents the results we have regarding the multiplication and rhizogenesis for this species, after its in vitro induction. 25

In vitro culture initiation and phytohormonal influence on Dianthus henteri – a Romanian endemic species

Materials and methods

Vegetal material In vitro culture was initiated with seeds (code XX-0-M-2004/1268) from the München Botanical Garden Nymphenburg, harvested in 2006.

Seed disinfection A 13 h pre-sterilization with 4% H2O2 was done initially. H2O2 plays the double role of being a good sterilizing agent but also getting the seeds out of dormancy. The sterilization itself was done with 96% ethanol – 2 min – followed by 10% H2O2– 19 min.

Culture media A basal Murashige-Skoog media (macro- and microelements, and FeEDTA) was used for germinating the sterilized seeds, supplemented with thiamine, pyridoxine, and nicotinic acid, 1 mg l-1 each, myo-inositol 100 mg l-1, sucrose 30 g l-1 and agar 8 g l-1. Gibberellic acid -1 (GA3) 100 mg l was either added or not to the above medium (Table 2). GA3 is known as a phytohormone playing a role in getting the seeds out of dormancy. It was added using a Millipore filter after autoclaving the culture media. For the multiplication and rhizogenesis experiments same media as above were supplemented with auxin (α-naphthalene acetic acid – NAA) and cytokinin (6-benzylaminopurine - BAP) is the different phytohormonal variants presented in Table 1.

Table 1. Phytohormonal content of culture media for multiplication and rhizogenesis Medium Hormonal balance Phytohormones (mg l-1) variants (auxin/cytokinin) NAA BAP For multiplication V1 0/1 0 1 V2 1/1 1 1 V3 1/10 0.1 1 For rhizogenesis V4 1/0 1 0 V5 1/1 1 1 V6 10/1 1 0.1

Culture conditions and studied parameters Half of the inoculated seeds were kept in light and the other half in darkness, in the growth chamber, which had a 16 h light/8 h dark photoperiod, 90 µmol m-2 s light intensity, and 25±2°C. Infection and germination rates were measured 15 days after inoculation. From the generated neo-plantlets, apical and nodal explants were transferred on the 3 multiplication media variants, observations being conducted at 44 and 83 days after transfer. The influence of the hormonal balance on the length of the generated neo-plantlets and the number of neo- plantlets/inoculum was recorded. Later on, uni-nodal explants from these neo-plantlets were transferred on the 3 media variants for rhizogenesis. Observations were then recorded at 29 and 60 days after inoculation. The multiplication rate, the average length of the roots, and the average number of roots/explant were monitored.

Acclimatization In vitro generated , with a good radicular system, were planted for acclimatization in: (i) water-imbibed perlite, (ii) mixture of perlite and sterilized soil (50%- 26 Romanian Biotechnological Letters, Vol. 15, No. 1, Supplement (2010)

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50%) imbibed with Murashige-Skoog macroelements solution, (iii) mixture of perlite and sterilized soil (50%-50%) imbibed with water. After potting the plants were sprayed with 1% paclobutrazol. The pots were initially covered under a transparent hood, to maintain a high atmospheric humidity, and then gradually being uncovered.

Statistical analysis Statistical analysis was performed using t and ANOVA tests, with 95% confidence intervals. p-values < 0.05 were regarded as indicating statistical significance (*).

Results and discussion

In vitro culture induction Results at 15 days after disinfection and inoculation of the seeds on the 2 medium variants (with and without GA3) are presented in Table 2.

Table 2. The germination and infection rates of the seeds 15 days after inoculation Germination Basal medium with GA3 (100 Basal medium without GA3 Species conditions mg l-1) % infected % germinated % infected % germinated Dianthus Light 0 100 0 75 henteri Dark 0 50 25 75

The disinfection method has good results, as seen in Table 2, with a 6.25% infection rate only. The germination rate was also good, 75% of the seeds germinating 15 days after inoculation. These results are similar to those recorded for other species of the Dianthus genus, Centaurea reichenbachii DC, or Aquilegia nigricans Baumg. [2, 3, 5, 10]. In vitro culture initiation by means of sterilized seeds is obviously a much easier method than sterilizing vegetal material [5-7]. The germination rate in light overpassed the one in darkness although the latter one is considered to stimulate germination. No differences in the influence of GA3 were noteworthy, maybe because the seeds of this species were not in physiological dormancy. For other species though, that have a low germination rate, GA3 had positive in vitro effect [12, 18].

In vitro multiplication The results of the biometric measurements at 44 days after the inoculation of the apical and nodal explants on V1, V2, and V3 media variants are presented in Fig. 1. No. neoplants/inoculum Lengths of neoplants (cm)

8 3.5 7 3 6 2.5 5 Node Node 2 4 Apex 1.5 Apex 3 2 1 1 0.5 0 0 V1 V2 V3 V1 V2 V3 A B Fig. 1. Influence of hormonal balance at 44 days after inoculation, on three culture media for multiplication: A. number of neo-plantlets; B. length of neo-plantlets. Statistical analysis (t test) shows that there are significant differences both in the case of the number of neo-plantlets and their length, between the two explants used (node and apex): (p=0.0097(**) and p=0.0016 (**), respectively

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In vitro culture initiation and phytohormonal influence on Dianthus henteri – a Romanian endemic species

As shown in Fig.1 A, the greatest number of neo-plantlets – 7.7/apical inoculum and 6.9/nodal inoculum – was generated on V3 medium, which was the variant containing 10x more cytokinin compared to auxin. This was in agreement with the expectations when choosing a media that has a favorable hormonal balance for cytokinins, known to stimulate cell division and the generations of shoots [17]. On V2 medium, where cytokinins have the same concentration to auxins, worse results were observed, as expected. Apical inoculi have a better generation rate of neo-plantlets in general. For another taxon of the Dianthus genus - D. petraeus Waldst. & Kit. ssp. simonkaianus (Péterfi) – a better multiplication rate was achieved on a medium with 1/1 hormonal balance (NAA 0.5 mg l-1 and kinetin (K) 0.5 mg l-1) versus a medium with 1/10 hornomal balance (NAA 0.05 mg l-1 and BAP 0.5 mg l-1) but in this instance the cytokinins on the 2 variants are different and K is often preferred to BA [11]. In the latter case, although the hormonal balance favors cytokinins, auxin concentration is very low. V1 and V3 media were also better for the length of the neo-plantlets (Fig. 1) meaning that more cytokinin leads to better results for this parameter, too. The average length of nodal inoculi was 3.2 cm on V1 and 2.9 cm on V3, respectively. Regarding the generation rate of neo-plantlets/inoculum, the apical explants gave better results than the nodal ones (Fig. 1 A) while for the length of neoplantlets (Fig. 1B), nodal explants gave better results compared to the apical ones. For other endemic/vulnerable Dianthus species, from Romania or other regions of Europe (D. glacialis Haenke ssp. gelidus Schott, Nyman & Kotschy, Dianthus spiculifolius Schur, Dianthus giganteus D’Urv., Dianthus giganteus D’Urv. ssp. banaticus (Heuff.) Tutin, Dianthus alpinus L., Dianthus gratianopolitanus Vill., Dianthus ferrugineus Mill., Dianthus gallicus Pers.) the nodes have a better multiplication rate than the apexes while the latter ones have a better rhizogenesis compared to the nodes [5]. Interestingly, no differences induced by the phytohormonal balance on apical inoculi are noted in this case. The aforementioned results are also shown in Figure 2, with V2 medium giving the lowest multiplication rate and the shortest length for the neo-plantlets, the evolution of the cultures being the same at 44 and 71 days after inoculation.

V1 V3 V2

Fig. 2. In vitro multiplication of D. henteri on 3 different culture media, 71 days after inoculation.

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Because the aim of in vitro cultures is to generate as many explants/initial inoculum as possible, and because the cultures still had potential for further development at 44 days, they were left in the culture flasks up to 83 days, having enough space requirements for growth inside (Fig. 3).

16 14 12 10 V1 8 V2 6 V3 4 2 0 No. neoplants/inoc. Length of neoplants (cm) Fig. 3. Influence of hormonal balance on D. henteri multiplication, 83 days after inoculation, on three culture media variants. P-values for the ANOVA test show statistically-significant differences among the 3 culture media both for the number and for the length of the neo-plantlets. p = 0.0002 (***) and p = 0.037 (*), respectively.

As shown in Fig. 3, the evolution trend on the culture media was the same even after doubling the time from inoculation. The greatest number of neo-plantlets was achieved on V3 and V1 media, i.e., 16 and 11/inoculum, respectively while the lengthiest neo-plantlets were present on the same V1 and V3 media, i.e., 3.6 cm and 3.1 cm, respectively. Analyzing the results of these multiplication experiments we can say that after 83 days, each neo-plant, having about 10-15 nodes, could generate 110 to 240 new explants, which can be further replicated. This multiplication rate can be considered as being very satisfactory. We recommend the use of V3 medium, with NAA 0.1 mg l-1 and BAP 1 mg l-1 (1/10 hormonal balance). This medium proves its value, with great results, in the case of in vitro multiplication of D. spiculifolius, as well [4]. D. fruticosus L. has a similar response to the hormonal balance of the culture media, with the best multiplication rate for a 1/5 balance (NAA 0.1mg l-1 and isopentenyladenine (2iP) 0.5 mg l-1) [13]. It appears to be a species-specific response to in vitro cultures though, with species like D. petraeus W. et K. spp. simonkaianus (Péterfi) Tutin generating 110 neo- plantlets/apical inoculum [11] at a 1/1 balance (IAA 1mg l-1 and BAP 1mg l-1), following successive transfers, while for another species, D. pyrenaicus Pourr., even a 1/2 balance (NAA 0.5mg l-1 and BAP 1mg l-1) can only generate 4.1 neo-plantlets/inoculum [10]. So, a species-specific variability inside the Dianthus genus was present regarding its in vitro multiplication.

In vitro rhizogenesis Having the ex vitro acclimatization as a final goal for the in vitro generated neo- plantlets, these is required to have a well developed root system. Knowing that an auxin-rich

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In vitro culture initiation and phytohormonal influence on Dianthus henteri – a Romanian endemic species phytohormonal balance stimulates rhizogenesis, a second experiment was set up, on culture media V4, V5, and V6. Table 3 summarizes the results regarding multiplication process on these 3 media.

Table 3. The influence of hormonal balance on multiplication and rhizogenesis on D. henteri, 29 days after inoculation Phytohormones Hormonal Multiplication (%) (mg l-1) balance Good Average Necrotic (10-30 neopl./inoc.) (5-10 neopl./inoc.) ANA=1 BA=0 1/0 (V4) 29 18 53 ANA=1 BA=1 1/1 (V5) 43 43 14 ANA=1 BA=0,1 10/1 (V6) 77 18 5

The results for these 3 new phytohormonal combinations confirmed the influence of the hormonal balance, noted in the multiplication experiment as well. Thus, on V4 medium with NAA 1 mg l-1 lacking cytokinin, the number of inoculi with a good or average rate of multiplication was low, 29% and 18% respectively, but still less than a half. On the other hand, the necrotic rate was 53%, so this medium should not be used, at least not for this species. The best results for multiplication were seen on V6 medium, with a hormonal balance rich in auxin. 95% of the inoculi grown on this medium generated 5-30 neo- plantlets/inoculum, 29 days after inoculation and the cultures had a good further development in general. V5 medium, with its balanced composition of hormones, was worse than V6, having a higher necrotic rate (14%), but still had good results, with 86% of the inoculi generating neo-plantlets. Still, the number of those generating 10-30 neo-plantlets (i.e., having a high multiplication rate) was under half of total (43%). V6 medium (NAA 1 mg l-1 and BAP 0.1 mg l-1) presented also the best results for rhizogenesis (Fig. 4, 5). Both at 29 and at 60 days after inoculation the number of roots/inoculum as well as their length was higher for an auxin-rich hormonal balance. On V4 medium, results for rhizogenesis after 60 days were similar to V6, but because of its lack of cytokinins (Table 3), the multiplication was affected. On V5 medium, with a balanced hormonal composition, rhizogenesis was absent. Judging on the results of this second experiment, regarding the in vitro multiplication and rhizogenesis for D. henteri, we could conclude that the V6 medium, having 10x more auxin compared to cytokinin, led to a rhizogenesis that allowed the acclimatization of the neo-plantlets and also satisfactory multiplication. The results for rhizogenesis fit the ones reported for other species of Dianthus that are important phytogeographically and sozologically. For D. pyrenaicus 2 auxins were tested in parallel: NAA (1 mg l-1, same as in this work, and IAA (indolilacetic acid) 1 mg l-1) [10]. The authors report that on the media containing IAA the rhizogenesis is better than on the one with NAA (73.2% rooted inoculi versus 11%). In both cases a 1/1 hormonal balance is used. NAA was used for this present work, instead of IAA, the latter one being photodegradable, negatively influencing its action during the experiments conducted under light. For D. spiculifolius though, the influence of 2 cytokinins, in different hormonal balances (1/1; 0.4/1; 0.2/1 and 0.1/1) was recorded with regard to their effect on rhizogenesis [4]. The best variant proved to be the 1/1 hormonal balance (NAA mg l-1 and K mg l-1), i.e. the one with the highest concentration of auxin. For D. petraeus spp. simonkaianus, the same hormonal balance of NAA and K has the best results, among several others that were tested [11].

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9 8 7 6 5 4 V4 V5 3 V6 2 1 0 ABAB

Rhizogenesis after 29 days Rhizogenesis after 60 days

Legend: A-Length of roots (cm), B-No. of roots/inoculum

Fig. 4. Influence of hormonal balance on rhizogenesis of D. henteri at 29 and 60 days after inoculation, on three media variables. These results are confirmed by p-values that show statistically significant differences among the 3 media at 29 days and 60 days, both for the number of roots and for their length. (p = 0.0106 (*) for number of roots at 29 day; p < 0.0001 (***) for length of roots at 29 day; p = 0.0025 (**) for number of rots at 60 day; p = 0.0001 (***) for length of roots at 60 day.

V4 V5 V6 Fig. 5. In vitro rhizogenesis for D. henteri, on V4, V5, and V6 culture media, 68 days after inoculation.

In the case of D. fruticosus, where the goal was only to induce rhizogenesis, the best medium is the one containing only auxin (2 mg l-1 indole-3-butyric acid (IBA) [13]. Starting from the above results, we used a novel medium for our present work, with more elevated auxin levels in the hormonal balance, leading to an average number of 8.6 roots/inoculum. It’s noteworthy that for the other studied Dianthus species, rhizogenesis is present even at a 1/1 hormonal balance while in the case of D. henteri 10x more auxins were needed, compared to cytokinins, for inducing this phenomenon. Therefore a species-specific variability inside the Dianthus genus was also present for the rhizogenesis process.

Acclimatization Observations, 12 days after ex vitro transplantation of the generated neo-plantlets, show that the most vigorous plants are the ones acclimatized on water-imbibed perlite (Fig. 6). Water-imbibed perlite-soil mixture also leads to viable plants but that are less vigorous. The worse results were on perlite-soil mixture, imbibed in culture media.

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In vitro culture initiation and phytohormonal influence on Dianthus henteri – a Romanian endemic species

A B

C

Fig. 6. Ex vitro acclimatized D. henteri vitroplant. A. Plant acclimatizeted on perlite – soil mixture. B. Plants acclimatizeted on perlit. C. Acclimatized vitroplants, in soil, in the greenhouse.

Studies of somaclonal variation as a result of in vitro culture are underway, using RAPD and SSR markers. Results will show if the culture conditions induced any genetic modification in the vegetal material. Based on the kind of phytohormones used, their concentration, and other studies on various species, it seems most likely that this species will not display any somaclonal variations either, that are due to its micropropagation. After confirming that the vegetal material is not genetically modified, in situ repopulation could proceed, when required.

Conclusions

D. henteri, strictly endemic for the Meridional branch of the Romanian Carpathians and threatened nationwide, was successfully micropropagated starting from seeds and is now part of the in vitro collection of endemic/endangered plants. The best medium for multiplication had a 1/10 hormonal balance with NAA 0.1 mg l-1 and BA 1 mg l-1. 83 days after inoculation each neo-plant generated 110 to 240 explants. The best results for rhizogenesis – 8.6 roots/inoculum – came out of a medium with NAA 1 mg l-1 and BAP 0.1 mg l-1 (1/10 hormonal balance). The multiplication rate on this medium was also satisfactory. In vitro generated plants were successfully acclimatized. This species behavior in vitro was in close correlation to the hormonal balance of the different media variants that were used.

Aknowledgements

Funding came partially from 31-008/2007 - PNCDI2-Parteneriate, CNMP project. The authors are thankful to the Biological Research Institute, Cluj-Napoca, for technical support.

References

1. A. BUTIUC-KEUL, C. DELIU, Rolul unor extracte naturale in multiplicarea in vitro la Leontopodium alpinum Cass. si Dianthus spiculifolius Schur, In: D. CACHITA-COSMA, A. BRAVU, A. BREZEANU, eds., Actualităţi şi perspective în biotehnologia vegetală, Ed. Ovidius, Constanţa, 2000, pp. 126-134. 2. V. CRISTEA, C. DELIU, B. OLTEAN, A. BUTIUC-KEUL, A. BRUMMER, C. ALBU, G.L. RADU, oilless cultures for pharmaceutical use and biodiversity conservation. Acta Hort. (ISHS), 843, 157-164 (2009). 3. V. CRISTEA, M. MICLĂUŞ, C. DELIU, A. HALMAGYI, The micropropagation of some endemic and rare taxa from Gilău – Muntele Mare Massif, Apuseni Mountains, România. Contribuţii Botanice, 39, 201-209 (2004).

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4. V. CRISTEA, M. MICLĂUŞ, M. PUŞCAŞ, C. DELIU, Influence of hormone balance and in vitro photoautotrophy on Dianthus spiculifolius Schur micropropagation. Contribuţii Botanice, 37, 145-153 (2002). 5. V. CRISTEA, M. PUŞCAŞ, M. MICLĂUŞ, C. DELIU, Conservative micropropagation of some endemic or rare species from the Dianthus genus. Acta Hort., (ISHS), 725, 357-364 (2006). 6. I. HOLOBIUC, R. BLÂNDU, V. CRISTEA, Researches concerning in vitro conservation of the rare plant species Dianthus nardiformis Janka. Biotechnol. & Biotechnol. Eq., 23 (2), 221-224 (2009). 7. I. HOLOBIUC, A. PAUNESCU, R. BLÂNDU, Ex situ conservation using in vitro methods in some Caryophyllaceae plant species from the Red List of vascular plants in Romania. Rom. J. Biol. – Plant Biol., 49- 50, 3-16 (2004-2005). 8. D. KLAVINA, A. GAILITE, G. JAKOBSONE, J. NECAJEVA, , G. GAVRILOVA, Tissue culture in conservation of threatened plant species of Latvia. Acta Universitatis Latviensis, Biology, 676, 183-188 (2004). 9. J. KOVAC, Micropropagation of Dianthus arenarius subsp. bohemicus – an endangered endemic from the Czech Republic. Botanic Gardens Micropropagation News, 1(8) (1995). 10. D. MARCU, V. CRISTEA, A. BUTIUC KEUL, Micropropagation of Dianthus pyrenaicus Pourr. – endemic species from Pyrenean Mountains. Contr. Bot., 41(2), 153-159 (2006). 11. M. MICLĂUŞ, V. CRISTEA, C. DELIU, Micropropagation on Dianthus petraeus W. et K. ssp. simonkaianus (Péterfi) Tutin. Contribuţii Botanice, 38 (1), 77-84 (2003). 12. T. D. NIKAM, R. B. BARMUKH, GA3 enhances in vitro seed germination in Santalum album. Seed Science and Technology, 37 (2), 276-280 (2009). 13. M. PAPAFOTIOU, J. STRAGAS, Seed germination and in vitro propagation of Dianthus fruticosus L.. Acta Hort.( ISHS), 813, (2009). 14. A. PĂUNESCU, I. HOLOBIUC, Conservation of endemic species Dianthus callizonus Schott&Kotsky using in vitro techniques. Rev. Roum. Biol. – Biol. Veget., 48 (1-2), 3-7 (2003) 15. A. SÂRBU, ed., Arii speciale pentru protectia si conservarea plantelor in România, Victor B Victor, Bucuresti. 2007. 16. I. SÂRBU, G. NEGREAN, A. OPREA, I. CRISTUREAN, Listele nationale elaborate in conformitate cu criteriul A al proiectului, A. SÂRBU, coord., Ghid pentru identificarea importantelor arii de protecţie şi conservare a plantelor din România, Alo, Bucuresti, 2003, pp 39-79. 17. J. STADEN, E., ZAZIMALOVA, E., F., GEORGE, Plant growth regulators II: Cytokinins, their Analogues and Antagonists, E. F. GEORGE, M. A. HALL, G. J. KLERK, eds., Plant propagation by tissue culture, 1, 2008, pp. 205-227. 18. S. TODOROVIC, D. GRUBISIC, Z. GIBA, D. MISIC, R. KONJEVIC, Sucrose effects on in vitro fruiting and seed production of Centaurium pulchellum. Biologia Plantarum, 50 (4), 771-774 (2006). 19. M. ZĂPÂRTAN, Specii endemice, rare si ocrotite, conservate prin tehnici de cultura in vitro Dianthus spiculifolius Schur. Analele Univ. Oradea, Biologie, 2, 36-48 (1995).

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