Dianthus

7212019 Dianthus

httpslidepdfcomreaderfulldianthus 19

34 (2) 153-161 (2010)

UDK 582661510852

Original Scienti1047297c Paper

Received 12 November 2010 Revision accepted 24 December 2010

copy 2010 Institute o Botany and Botanical Garden Jevremovac Belgradecorrespondence ljradojevicsbbrs

In vitro propagation of Dianthus ciliatus ssp dalmaticus

and D giganteus ssp croaticus (Caryophyllaceae) fromstem segment cultures

Ljiljana R9831379831409831519831469831419831589831459833031 Dušica Ćalić-D9831549831379831439831519831559831379831589831379831391 Jovanka Š9831529831459831549831459833031Branka S9831569831419831589831379831509831519831589831459833032 and Vladimir S9831569831419831589831379831509831519831589831459833032

1 Institute or Biological Research lsquorsquoSiniša Stankovićrsquorsquo Bulevar Despota Steana 142 11000 Belgrade Serbia

2 Institute o Botany and Botanical Garden Faculty o Biology University o Belgrade akovska 4311000 Belgrade Serbia

ABSRAC Plant regeneration o carnation species Dianthus ciliatus ssp dalmaticus and Dianthus giganteus ssp croaticus was achieved through micropropagation rom apical and nodal segments culture on MS

2

Shoots multiplication was successul on the same medium via axillary buds Tere were differencesbetween multiplication index (MI) o shoots originating rom apical and nodal basal stem segmentsNodal segment shoots had a better MI (D ciliatus ssp dalmaticus = 786 D giganteus ssp croaticus = 068) than apical ones (D ciliatus ssp dalmaticus = 694 D giganteus ssp croaticus = 050) Shootso both species were rooted on MS

0without hormones MS

3 and MS

4 Adventitious buds (AB) and

somatic embryo like structures (ES) were ormed afer the transer o green-yellow callus rom MS5

to MS6 Further development and multiplication o AB and ES were achieved on medium MS

7

Plants ormation was brought about by micropropagation o shoots organogenesis andor somaticembryogenesis

In vitro plantlets o both carnation species were planted in rocky garden o the Belgrade Botanical

Garden lsquorsquoJevremovacrsquorsquo where they bloomed Subsequently these in vitro plantlets will be reintroducedin natural environment

Key words Dianthus ciliatus ssp dalmaticus Dianthus giganteus ssp croaticus adventitious buds somaticembriogenesis somatic embryo like structure micropropagation organogenesis stem segmentculture

Abbreviations BAP- 6-benzylamino purine 24-D-dichlorphenoxyacetic acid Kin- 6-ururylamino purine NAA-ala-napthaleneacetic acid IBA- 2-indiole-butyric acid GA

3- gibberellic acid DZ-thidiazuron

MS- Murashige and Skoogrsquos (1962) mineral solution AB- adventitous buds AS-apical segment NS-nodal segment EC-embryogenic callus ES-embryon like structure OC-organogenic callus

INRODUCION

Te genus Dianthus L (am Caryophyllaceae) includesmany perennial and a ew annual or biennial herbaceousspecies and very seldom low subshrubs with woody basalstems Nodes on the stem are swollen leaves are simple

linear opposite grey to blue green in colour Te 1047298owersare hermaphroditic terminal solitary or in dichasialcymes pale to dark pink Otherwise the genus was namedDianthus by C Linnaeus coined by Greek words dios ndashGod and anthos ndash 1047298ower ie bdquodivine 1047298owerldquo Te genusDianthus has about 300 species native to Europe and Asia

7212019 Dianthus


154 vol 34 (2)

In the Balkan Peninsula there were recorded about 170species o which almost 100 are endemics Tey inhabit

various types o geological substrata (sand loess limedolomite silicate serpentinite) in open habitats such assands steppes hilly and mountainous meadows and stony

grounds rom lowland regions to mountain tops over 2500m Te carnation plants preer neutral to slightly alkalinewell-drained soil Tey grow mostly in ull sun but alsotolerate light shade

Many species and more than 100 hybrids o the genusDianthus are used as ornamental plants o rocky gardensowing to its remarkably beautiul ragrant blossoms thatappear during the spring and summer and ofen deepinto the autumn Besides carnation plants are ofenused as cut-1047298owers and given that 1047298ower colour o dryplants is retained they are useul or pressed 1047298ower crafsand arrangements For 1047298ower industryhorticulture oparticular signi1047297cance are species having beautiul large

1047298owers o different shades o pink to red which aredistinguished by long blossoming period thriving onpoor acid soils and drought resistance (S983152983145983150983155983147983145 et al 1974C983144983151983157983139983144 amp 983158983137983150 S983156983137983140983141983150 1993 R983137983140983151983146983141983158983145983303 et al 2010)Such species and hybrids are common in parks especiallyin rocky gardens as 1047298oral edges o seedbeds or as coverso barren sites in settlements (R983137983140983151983146983141983158983145983303 2007)

Te endemic species are o particular interest bearingin mind the need o protection and preservation o speciesand genetic diversity o these more or less threatenedplants as well as their ornamental characteristics In theregion o the Balkan Peninsula to endemic carnationsbelong Dianthus ciliatus Guss and D giganteus Dum-Urville

D ciliatus ssp dalmaticus (Celak) Hayek is an endemicspecies spread in the Mediterranean and Submediterraneanzone o the Adriatic coast in Dalmatia (Croatia) andMontenegro (Fig1a) It is low semi-woody shrub

D giganteus ssp croaticus (Borbaacutes) utin is asubendemic species distributed in the western part o theBalkan Peninsula rom Slovenia eastward to Serbia (Fig1b) It is a perennial herb orming loose cushions

Hitherto only several papers concerned withconservative micropropagation o endemic species ocarnation were published (R983137983140983151983146983141983158983145983303 et al 1997 B983141983154983137983154983140983145

et al 2004 C983154983145983155983156983141983137 et al 2004 J983137983145983150 et al 2006 S983137983154983137983155983137983150et al 2006)Te aim o the present study was to orm a great

number o in vitro plants by applying stem segmentculture both or the purpose o preserving genopool othese species and or setting up an important resourceor the horticultural application o endemic carnations asornamental plants According to available data species Dciliatus ssp dalmaticus and D giganteus ssp croaticus tillnow have not been introduced to in vitro culture

MAERIAL AND MEHODS

Seed germination and seedling formationIn vitro culturewas initiated with seeds o D ciliatus ssp dalmaticus romMt Lovcen (Montenegro) and D gigantheus ssp croaticus rom Vlasina Lake (Serbia) Te seeds were washed withplenty o running water (2 h) and then immersed in 20 commercial bleach by modi1047297ed procedure o R983137983140983151983146983141983158983145983303et al (1990) or 20 min rinsed with sterile distilled waterand cultivated in Petri dishes (10 seeds per dish) containing

sterile 1047297lter paper with distilled water or germinationunder dark (10 days) Petri dishes were sealed withPara1047297lm (Pechiney Plastic Packing Chicago IL USA)Non-contaminated germinated seeds (Plate I Fig 1)reed rom seed coat were transerred in new Petri dishescontaining the MS

1=MS

0+ GA

3 (10 mg L-1) medium or

seedlings ormation and elongation (Plate I Fig 2)

Culture media Initial apical and nodal segments (about3-4 mm long) were cultivated on MS

2=MS

0+ NAA 10 +

Fig 1 Distribution o D ciliatus ssp dalmaticus (a) distri-bution o D giganteus ssp croaticus (b) (according to J983137983148983137983155 983078

S983157983151983149983145983150983141983150 1986)

7212019 Dianthus


155L Radojević et al In vitro propagation o Dianthus ciliatus ssp dalmaticus and D giganteus ssp croaticus (Caryophyllaceae) rom stem segment cultures

IBA 05 + BAP 10 (mg L-1 o each) or shoot inductionand ormation (Plate I Figs 3 and 9) Tese explants wereplaced in tube (100 x 10 cm) containing 8 ml o the MS

2

mediumIn the second subculture only some explants ormed

shoots about 1-2 cm (Plate I Figs 4 and 10) Tese shootswere subcultured on the same medium (40 mljar o 250ml) Cultures were continuously grown on MS

2medium

(100 ml o mediumjar o 500 ml with aeration) or shootmultiplication

Shoots o both species were rooted on MS0 without

hormones MS3 and MS

4= MS

0+ IBA (05-10 mg L-1

respectively) media

Culture media for organogenesis andor somatic

embryogenesis induction Sporadically some o basalpart o shoots in contact with media was ormed beingeither compact green andor loose white calli (Plate I

Fig 3) Separated calli were subcultured on MS5 =MS0+24-D 50 + BAP 10+ NAA 10 (mg L-1 each) and thentranserred on MS

6=MS

0+ 24-D 10 + BAP 10 + NAA 01

(mg L-1 each) media or urther differentiationOnly calli with adventitious buds (AB) andor embryo

like structure (ES) were transerred on MS7=MS

0+ 24-D

10 + BAP 10+ NAA 50+ DZ 02 (mg L-1 each) mediumDuration o this subculture was 30 days

All media (MS1-MS

7) contained MS mineral solution

(M983157983154983137983155983144983145983143983141 amp S983147983151983151983143 1962) supplemented with 2sucrose 07 (wv) agar and (in mg L-1) nicotinic acid10 panthothenic acid 01 olic acid 001 ascorbic acid10 vitamin B

120 ribo1047298avin 02 and myo-inositol 100

(medium MS0) Te media (MS1-MS7) were gelled with07 agar (wv) and pH was adjusted to 58 with 1 NNaOH beore sterilization All media were sterilized byautoclaving at 09 x 105 kPa and 1140C (25min) Cultureswere grown at 25plusmn10C under 16 h photoperiod and 8 hdark

Anatomical examination of the calli All types o calliie embryogenic callus (EC) and organogenic callus(OC) were 1047297xed in FAA (ormalin acetic acid ethanol5590) during 24 h at 4degC Te tissues were dehydrated inan ethanol series and the permanent preparations wereobtained by standard paraffi n technique and hematoxiline

andor toluidine blue staining procedures (M983137983154983156983151983146983137 ampM983137983154983156983151983146983137 1967)

Statistics and repetition Te results were assessed usingthe variation analysis in Statistica 100 Te means werecompared by the least signi1047297cant difference (LSD) test(signi1047297cance level plt005) Tree repetitions had beenperormed per each medium

Species Number of seeds in culture Medium for seedling formation(hormone in mg L-1)

Number of seedlings of seeds germination

D ciliatus ssp dalmaticus

149 MS + 3 sucrose + GA310 105 7046a

D giganteus ssp croaticus

158 MS + 3 sucrose + GA310 66 4177b

able 1 Formation o seedlings o D ciliatus ssp dalmaticus and D giganteus ssp croaticus in in vitro culture Each data is mean o threereplicatesvalues in each column marked by different letters are signi1047297cantly different at 005 using the LSD test

Species Date Mediumotal number of shoots formultiplication

Te average length ofshoots (in cm)

Te average No rootsplantlet


08022007MS

027 867 plusmn 035 733 plusmn 158a

MS3

15 943 plusmn 067 506 plusmn 077ab

29032007MS

038 885 plusmn 076 752 plusmn 100a

MS3



26062006 MS0

15 566 plusmn 089 300 plusmn 043b

MS3

30 822 plusmn 096 785 plusmn 091a

16082006MS

015 260 plusmn 035 0

MS3


able 2 Shoot multiplication index o apical (AS) and nodal (NS) segments in in vitro culture o D ciliathus ssp dalmaticus and D giganteus ssp croaticus seedlingsmeans plusmn SE (standard error) values in each column marked by different letters are signi1047297cantly different at 005 using the LSD test

7212019 Dianthus


156 vol 34 (2)

PLAE I Figs 1-15 Micropropagation in stem segment culture o D ciliatus ssp dalmaticus and Dianthus giganteus ssp croaticusGerminated seed (1) and seedling (2) o D ciliatus ssp dalmaticus the 1047297rst subculture and shoots multiplication o D ciliatus ssp dalmaticus (3) and o D giganteus ssp croaticus (4) on the medium MS

2 rooted shoot o D ciliatus ssp dalmaticus (5) on medium MS

3 in vitro plants

o D ciliatus ssp dalmaticus in early (6) and later acclimatation in greenhaus (7) 1047298owering in vitro plants o D ciliatus grown in BotanicalGarden lsquorsquoJevremovacrsquorsquo (8) the 1047297rst subculture (9) and shoots multiplication o D giganteus ssp croaticus on MS

2 medium (10) plants o the

same species in early and later acclimatization stage (11 12 13) 1047298owers (14) and mature seed capsules (15) o D giganteus ssp croaticus reintroduced in Belgrade Botanical Garden lsquorsquoJevremovacrsquorsquo

7212019 Dianthus



RESULS AND DISCUSSION

Protocol was designed or establishing initial culture oshoots rom apical segments (AS) and nodal segments(NS) o seedlings o the species D ciliatus ssp dalmaticus

and D giganteus ssp croaticus Seeds o these speciesgerminating on MS1 medium with 2 saccharose and GA

3

10 mg L-1 had different germination rate (able 1 Plate IFigs 1 and 2)

In the initial phase o seed cultures o D ciliatus sspdalmaticus there were 4642 o inected seeds whereasor D giganteus ssp croaticus seed inection was somewhatlower (3928) Inection considerably affected seedgermination given that it decreased a number o viableseeds Percentage o seed germination o D ciliatus sspdalmaticus was markedly higher (7046) in respect toseed germination o D giganteus ssp croaticus (4177)Species speci1047297city also affected multiplication o shoots

Apical segments (AS) and nodal segments (NS) o Dciliatus (Plate I Fig3) and D giganteus (Plate I Fig 9)explants have ormed clusters o small shoots already aferthe 1047297rst subculture on the medium MS

2

Multiplication o segments occurred via axillary budson the same medium afer the 1047297rst subculture Shootmultiplication was monitored rom the 1047297rst to the ourthsubculture (able 2 Plate I Figs 4 and 10)

Te positive gradient o multiplication index (MI) wasrecorded in the culture o AS and NS segments or both oD ciliatus ssp dalmaticus and o D giganteus ssp croaticus with the subsequent subcuturing (namely II III and IVsubculture) MI had the highest value in the subcultureIV being always signi1047297cantly higher in D ciliatus than inD giganteus ssp croaticus (able 2) In both species nodalsegments had a better MI than apical segments which is inagreement with the results o C983154983145983155983156983141983137 et al (2004 2006)or some other endemic species o the genus Dianthus rom Romania or other regions o Europe (D alpinus LD ferrugineus Miller D gallicus Pers D giganteus subsp banaticus (Heuffel) utin D gratianopolitanus Vill andD henteri Heuffel ex Griseb amp Schenk) Specially or Dhenteri the nodes have a better multiplication rate thanapexes segments (C983154983145983155983156983141983137 et al 2010)

For micropropagation o Dianthus species the most

ofen used o auxins are IAA and NAA and o cytokininsKin or BAP (S983156983151983150983141 1963 D983137983158983145983155 et al 1977) Combinationo two auxins (IBA and NAA) was used in the mediumor the multiplication o shoots in twelve varieties ocarnations important or horticulture (R983137983140983151983146983141983158983145983303 et al 1990 1994 R983137983140983151983146983141983158983145983303 2007) In addition both auxins(NAA and IBA) were used or the micropropagation oshoots o endemic species o carnations Dianthus petraeus Waldst amp Kit (R983137983140983151983146983141983158983145983303 et al 1997 J983137983145983150 et al 2006) D ciliatus ssp dalmaticus and D giganteus ssp croaticus

(R983137983140983151983146983141983158983145983303 et al 2006) Otherwise or the multiplicationo shoots o D ciliatus ssp dalmaticus and D giganteus sspcroaticus only cytokinin BAP was used which was in linewith the results or D caryophyllus L (W983151983151 amp P983137983154983147 1993G983144983137983154983138983145983137 et al 2008) D petraeus (R983137983140983151983146983141983158983145983303 et al 1997)

D superbus L (M983145983147983157983148983145983147 1999) D balbisii Ser (B983141983154983137983154983140983145et al 2004) and D elegance Dum-Urville (B983137983147983156983145983154 ampH983137983162983137983154 2004) In general in certain carnation speciescultures during multiplication phase vitri1047297ed shoots wereobserved which resulted in malormation o some shootsthat were excluded rom urther propagation Occurrenceo vitri1047297cation in shoots o carnations in the presenceo BAP was 1047297rst described by R983151983141983155983156 amp B983151983147983141983148983149983137983150983150(1981) Otherwise vitri1047297cation o carnation shoots is

very common phenomenon during micropropagationAccording to the results o M983141983155983155983141983143983157983141983154 et al (1993) andR983137983140983151983146983141983158983145983303 et al (1994) BAP concentration higher than 10mg L-1 increased a number o vitri1047297ed shoots Higher ratio

o NO3-NH4+ in the medium prevented the vitri1047297cationo carnation plantlets (983155983137983161 1998) However a suitableprotocol to overcome hyperhydracity in carnation duringmicropropagation was developed by Y983137983140983137983158 et al (2003)In our experiments the use o optimal BAP concentration(10 mg L-1) and growing o shoots in the aerated jars didnot lead to vitri1047297cation during propagation in D ciliatus ssp dalmaticus and D giganteus ssp croaticus

Carnation shoots were mostly rooted on media withouthormones (L983141983155983144983141983149 1986) or on those with auxins (P983141983156983154983157amp L983137983150983140983137 1974 R983137983140983151983146983141983158983145983303 et al 1997) Te shoots o Dciliatus ssp dalmaticus and D giganteus ssp croaticus wererooted on MS

0 MS

3 and MS

4 media Shoot rooting o both

carnation species depended on medium shoot length andthe season in which it occurred According to the resultsthe most suitable or shoot rooting were the shoots 885cm and 822 cm long o D ciliatus ssp dalmaticus andD giganteus ssp croaticus respectively (able 3) Temost avourable conditions or the rooting o D ciliatus ssp dalmaticus shoots was in March (752 rootsplantlet)on medium MS

0 without hormones whereas the most

avourable period o rooting or D giganteus ssp croaticus was June (785 rootsplantlet) on MS

3 medium (able 3

Plate I Fig5)Afer the 1047297rst passage a certain number o shoots in

the culture (about 10 ) has ormed (OC) organogenicgreenish-yellow compact (Plate II Figs 1 and 2) and (EC)loose white calli (Plate II Fig3) Tese calli were grownor the purpose o urther differentiation on the mediumMS

5 Afer one month these calli were transerred onto the

medium MS6 to induce organogenesis Afer the second

subculture adventitious buds (AB) and a smaller numbero proembryo like structures (ES) were ormed (Plate IIFig 4)

Only calli that ormed AB (Plate II Figs 1 2 and 5 )

7212019 Dianthus


158 vol 34 (2)

PLAE II 1-9 Different morphogenic response in callus cultures o D ciliatus ssp dalmaticus Organogenic callus with adventitious buds(AB) on the medium MS

5(12) embryogenic callus (EC) (3) EC with embryos like structure (ES) (4) AB EC ES and trichomes () (5)

on the medium MS6 emblings o D ciliatus ssp dalmaticus with two cotyledons on the medium MS

7(6) early stages o adventitious bud

ormation with tracheides (r) (7) latter stages o adventitious buds (AB) (8) Proembryo like structure (PrS) (9)

Remarks 7-9 Fixation FAA 7 and 8 Staining hematoxyline 9 Staining toluidine blue 7-9 Bar=50microm

7212019 Dianthus



andor ES structures (Plate II Fig 6) were transerred on

medium MS7 or urther development and multiplicationAccording to data rom available literature DZcontaining media are very effi cient or the inductiono somatic embryogenesis and or the multiplication osomatic embryos (V983145983155983155983141983154 et al 1992 M983145983156983144983145983148983137 et al 2003P983137983150983137983145983137 et al 2004)

Anatomical examinations o OC con1047297rmed that duringthe 1047297rst subculture on MS

5 and afer on MS

6 medium

meristem zones appeared and were quickly ormedinto initial nodular bumps like shoots During urtherdevelopment vascular elements were differentiated withinthese structures (Plate II Figs7 and 8) Serial sectionso regenerated structures did not reveal bipolarity thuscon1047297rming that morphogenesis proceeded in the directiono organogenesis In the present study in the culture ostem segments o both Dianthus species embryogeniccalli were developed on the same transer media (romMS

5 to MS

6) Reduction o 24-D in these media led to

teratological callus differentiation in OC EC and toormation o proembryo like structures at EC callus (PlateII Figs 6 and 9)

Micropropagation was possible via shoot culturederiving rom different segments o seedlings In vitro plantlets o D giganteus ssp croaticus had large ornamental1047298owers with long 1047298ower stalks o average length o

8614 cm while 1047298owers o D ciliatus were smaller withshorter 1047298ower stalks (4810cm) Due to its ornamentalcharacteristics and the duration o 1047298owering period thisspecies might be used in 1047298ower industryhorticultureeg ldquocut 1047298owersrdquo whereas in vitro plants may be used orurther research o hybridization (W983141983150 G983157983137983138983137983151 et al 1995) In the other hand the micropropagated plantletso D ciliatus might be used in park horticulture as1047298oral edges o seedbeds or as covers o barren sites insettlements (R983137983140983151983146983141983158983145983303 2007)

Preliminary anatomical studies concerned with callus

o D ciliatus indicate that simultaneous induction oorganogenesis and somatic embryogenesis is possible aspreviously has been reported or the embryos culture o Iris pumila (R983137983140983151983146983141983158983145983303 et al 1987) and Iris setosa (R983137983140983151983146983141983158983145983303amp S983157983138983151983156983145983303 1992)

Te regeneration protocol presented here orconservative micropropagation (R983137983140983151983146983141983158983145983303 et al 2010)and plant regeneration o two endemic Dianthus speciescan be useul or the ldquolarge scalerdquo multiplication o in vitroplantlets Tese plants were used or the reintroduction innatural habitats In addition in vitro plantlets may serve asa complementary resource or transgenic plant productionthat might to be a subject o uture studies

CONCLUSIONS

Micropropagation o two Dianthus species was achievedby stem segments culture Formation o adventitious budswas possible via organogenesis or endemic species Dciliatus ssp croaticus and D giganteus ssp croaticus Inwhite callus o D ciliatus ssp croaticus the ormation oembryogenic like structure is probably possible via somaticembryogenesis Also earlier preliminary cytologicalstudies showed that these two processes (orgnogenesisand somatic embryogenesis) may occur simultaneously in

the calli o D ciliatus (R983137983140983151983146983141983158983145983303 et al 2006 R983137983140983151983146983141983158983145983303et al 2010) but orgnogenesis was more pronounced thansomatic embryogenesis In vitro plantlets o both endemiccarnation species were planted in rocky garden o theBelgrade Botanical Garden lsquorsquoJevremovacrsquorsquo where they evenbloomed

Acknowledgement ndash Tis study was 1047297nancially supportedby grants o the Ministery or Sciences and echnologicalDevelopment o Serbia (No 1573 and 143015)

able 3 Shoots rooting o Dianthus giganteus subsp croaticus and Dianthus ciliatus ssp dalmaticus

Species

Segments

Medium

Shoots multiplication index (MI)

ype NumberSubculture

II III IV


AS 15

MS2

058 535 694

NS 126 137 741 786


AS 18 016 035 050

NS 34 032 050 068

7212019 Dianthus


160 vol 34 (2)

REFERENCES

B983137983147983156983145983154 I amp H983137983162983137983154 D 2004 In vitro propagation o Dianthuselegans 5th Symposium lsquorsquoIn vitro Culture and HorticultureBreedingrsquorsquo (IVCHB) 12-14 September 2004 DebrecenHungary p 162

B983141983154983137983154983140983145 G R983151983150983139983137983155983137983143983148983145983137 R S983139983137983154983137983158983141983148983148983145 D amp D983154983137983140983145 G2004 In vitro propagation o Dianthus balbisii Ser ssp liburnicus (Bartl) Pign by shoot tip culture 5th IVCHBSymposium I 12-14 September 2004 Debrecen Hungaryp 164

C983144983151983157983139983144 NR amp 983158983137983150 S983156983137983140983141983150 J 1993 In vitro culture oDianthus zeyheri subsp natalensis a South Aricancarnation Plant Cell Tissue and Organ Culture 35 81-85

C983154983145983155983156983141983137 V B983154983157983149983149983141983154 A J983137983154983140983137 L amp M983145983139983148ă983157ş M 2010In vitro initiation and phytohormonal in1047298uence onDiathus henteri - Romanian endemic species RomanianBiotechnological Letters 15(1) 25-33

C983154983145983155983156983141983137 V M983145983139983148atilde983157ş M P983157ş983139983137983155 M amp D983141983148983145983157 C 2004Conservative micropropagation o some endemic or rarespecies rom Dianthus Genus 5th IVCHB Symposium I12-14 September 2004 Hungary p 143

C983154983145983155983156983141983137 V P983157983155983139983137983155 M M983145983139983148ă983157ş M amp D983141983148983145983157 C 2006Conservative micropropagation o some endemic or rarespecies rom the Dianthus genus Acta Horticulturae 725357-364

D983137983158983145983155 MJ B983137983147983141983154 R amp H983137983150983137983150 JJ 1977 Clonal multiplicationo carnation by micropropagation J Am Soc Hort Sci 102 48-53

G983144983137983154983138983145983137 HD A983156983144983141983141983148 NY amp M983151983138983137983155983144983141983154 O 2008 Clonalpropagation o Dianthus caryophyllus L through tissues

culture J Duhok Univ 12(1) 91-95J983137983145983150 SM J983141983150983147983155 MA R983151983157983156 GR amp R983137983140983151983146983141983158983145983303 Lj 2006Micropropagation o ornamental potted plantsPropagation of Ornamental Plants 6(2) 67-82

J983137983148983137983155 J S983157983151983149983145983150983141983150 J (eds) 1986 Atlas Florae Europaeae7 Caryophyllaceae (Silenoideae) Te Committee orMapping the Flora o Europe and Societas BiologicaFennica Vanamo Helsinki

L983141983155983144983141983149 B 1986 Carnation plantlets rom vitri1047297ed plants asa source o somaclonal variation Hort Sci 21 320-321

M983137983154983156983151983146983137 R amp M983137983154983156983151983146983137 M 1967 Initiation aux techniquesde lrsquohistologie animal Masson et Ccedilie pp 163-203

M983141983155983155983141983143983157983141983154 J A983154983139983151983150983137983140983137 MC amp M983141983148983141 E 1993 Adventitious

shoot regeneration in carnation (Dyanthus caryophyllus) Ann Bot 68 563-568

M983145983147983157983148983145983147 J 1999 Propagation o endangered plant species bytissue cultures Acta Universitatis Palackianae OlmoucensisFacultas Rerum Naturalium Biologica 37 27-33

M983145983156983144983145983148983137 J H983137983148983148 JC V983145983139983156983151983154 JM amp S983137983160983141983150983137 PK 2003Tidiazuron induces shoot organogenesis at lowconcentrations and somatic embryogenesis at highconcentrations o lea and petiole explants o Arican violet (Saintpaulia ionatha Wendl) Plant Cell Report

21(5) 408-414M983157983154983137983155983144983145983143983141 amp S983147983151983151983143 F 1962 A revised medim or rapid

growth and bioassays with tabacco tissue cultures PhysiolPlant 15473-497

P983137983150983137983145983137 M S983141983150983137983154983137983156983150983137 D983145983160983151983150 KW amp S983145983158983137983155983145983156983144983137983149983152983137983154983137983149A 2004 Te role o cytokinins and thidiazuron in thestimulation o somatic embriogenesis in key memebers othe Restionaceae Aus J Bot 55 257-265

P983141983156983154983157 E amp L983137983150983140983137 Z 1974 Organogenesis in isolatedcaranation plant callus tissue cultivated in vitro BiolPlant 16 450-453

R983137983140983151983146983141983158983145983303 Lj 2007 Primena in vitro kulture u proizvodnjicveća Stanje i mogućnosti razvoja cvećarstva u SrbijiSeminar pejzažne hortikulture 8-9 ebruar 2007Šumarski Fakultet Beograd Zbornik predavanja pp 46-48

R983137983140983151983146983141983158983145983303 Lj amp S983157983138983151983156983145983303 A 1992 Plant regeneration oIris setosa Pall through somatic embryogenesis and

organogensis J Plant 139 690-696R983137983140983151983146983141983158983145983303 Lj D983146983151983154đ983141983158983145983303 N amp P983141983156983154983151983158983145983303 J 1990 Invitro culture techniques or carnation breeding ActaHorticulturae 180 163-168

R983137983140983151983146983141983158983145983303 Lj M983137983154983145983150983147983151983158983145983303 N amp J983141983158983154983141983149983151983158983145983303 S 1997Vegetativno raznožavanje u kulturi meristema i segmenatastabla Dianthus petreus Waldst et Kit subsp noeanusGlasnik Botaničke bašte Univerziteta u Beogradu 31 1-7

R983137983140983151983146983141983158983145983303 Lj S983151983147983145983303 O 983157983139983145983303 B 1987 Somaticembryogenesis in tissue culture o Iris (Iris pumila L) Acta Horticulturae 212 719-723

R983137983140983151983146983141983158983145983303 Lj M983137983154983145983150983147983151983158983145983303 N Z983140983154983137983158983147983151983158983145983303-K983151983154983137983303 S ampJ983141983158983154983141983149983151983158983145983303 S 1994 Primena različitih postupaka in vitro

kulture u mikropropagaciji aričke ljubičice hrizantema ikaran1047297la Savremena poljoprivreda 42(6)117-128R983137983140983151983146983141983158983145983303 Lj Š983152983145983154983145983303 J S983156983141983158983137983150983151983158983145983303 B amp S983156983141983158983137983150983151983158983145983303

V 2006 In vitro culture o several endemic speciesDhianthus in Balkan Peninsula XXII Inter Sym SectionOrnamentals 11-15 Sep Sanremo Italy p 61

R983137983140983151983146983141983158983145983303 Lj Ć983137983148983145983303-D983154983137983143983151983155983137983158983137983139 D S983156983141983158983137983150983151983158983145983303 B ampS983156983141983158983137983150983151983158983145983303 V 2010 In vitro culture o several endemicspecies o genus Dianthus rom Serbia and neighboringregions 10th Symposium on the Flora o SoutheasternSerbia and Neighbouring regions Vlasina 17-20 June2010 pp 75-76

R983151983141983155983156 S amp B983151983147983141983148983149983137983150983150 1981 Vegetative propagation o

carnation in vitro through multiple shoot developmentSci Hort 14357-366

S983137983154983137983155983137983150 VA C983154983154983145983152983155 R R983137983149983155983137983161 R A983156983144983141983154983156983151983150 MMM983139M983145983139983144983141983150 C P983154983141983150983140983141983154983143983137983155983156 M amp R983151983159983150983156983154983141983141 JK 2006Conservation in vitro o threatened plants-progress in thepast decade In vitro Cell Development Biol Plant 4220-25

S983152983145983150983155983147983145 P B983141983139983147 GE amp M983139 G983154983151983159983150 BH 1974 Callus cultureso Dianthus species Hort Science 914

S983156983151983150983141 OM 1963 Factors affecting the growth o carnation

7212019 Dianthus



plants rom shoots apices Ann Appl Biol 52199-209983155983137983161 HS 1998 Effecat o medium composition at different

recultures on vitri1047297cation o carnation (Dianthuscaryophyllus ) in vitro shoot prolieration Acta Hort 461243- 249

V983145983155983155983141983154 C Q983157983154983141983155983144983145 JA R983137983158983145983150983140983141983154 G amp S983137983160983141983150983137 PK 1992Morphology role o thidiazuron substitution o auxinand cytokinin requirements or the induction o somaticembryogenesis in geranium hypocotyl cultures PlantPhysiol 991704-1707

W983141983150 G Y983157 J Z983144983137983151 X F983137983150 G L983145983150 D amp Y983137983150983143 Z 1995Studies on carnation (Dianthus caryophillus) specieshybridization Acta Hort 404 82-90

W983151983151 S H amp P983137983154983147 J M 1993 Multiple shoot culture Dianthus caryophyllus using mist culture system BioTech 7(10) 697-702

Y983137983140983137983158 M K G983137983157983154 AK amp G983137983154983137983143 G K 2003 Development osuitable protocol to overcome hyperhydricity in carnationduring micropropagation Plant Cell Tissue Organ Culture 72 153-156

R egeneracija biljaka je postignuta u kulturi vršnih i nodalnih segmenata na podlozi MS2 Umnožavanje izdanaka

je bilo uspešno na istoj podlozi preko aksilarnih izdanaka Multiplikacioni indeks (MI) izdanaka vršnih

segmenta razlikovao se od MI izdanaka nodalnih segmenata Izdanci poreklom od nodalnih segmenata imali su

bolji MI (D ciliatus ssp dalmaticus = 786 D giganteus ssp croaticus = 068) od apikalnih segmenata (D ciliatus

ssp dalmaticus = 694 D giganteus ssp croaticus = 050) Kod obe vrste izdanci su bili ožiljavani na MS0 podlozi

bez hormona i sa hormonima na MS3 i MS

4 Adventivni pupoljci (AB) i strukture slične embrionu (ES) su bili

ormirani posle prenošenja žuto-zelenog kalusa sa podloge MS5 na podlogu MS

6 Dalje razviće i umnožavanje (AB)

i (ES) odvijalo se na podlozi MS7 Formiranje biljaka je postignuto mikroprpagacijom izdanaka organogenezom

iili somatskom embriogenezom In vitro biljčice karan1047297la obe vrste su bile prenete u alpinetum Botaničke bašte

lsquorsquoJevremovacrsquorsquo u Beogradu gde su cvetale U budućnosti ove biljke mogu se reintrodukovati u prirodno stanište

Ključne reči Dianthus ciliatus ssp dalmaticus Dianthus giganteus ssp croaticus adventivni pupoljci strukture

slične embrionu organogeneza kultura segmenata stabla

In vitro umnožavanje u kulturi segmenata stabla ofDianthus ciliatus ssp dalmaticus and D giganteus ssp croaticus (Caryophyllaceae)

Ljiljana R983137983140983151983146983141983158983145983303 Dušica Ćalić-D983154983137983143983151983155983137983158983137983139 Jovanka Š983152983145983154983145983303Branka S983156983141983158983137983150983151983158983145983303 i Vladimir S983156983141983158983137983150983151983158983145983303

REZIME

7212019 Dianthus


154 vol 34 (2)

In the Balkan Peninsula there were recorded about 170species o which almost 100 are endemics Tey inhabit

various types o geological substrata (sand loess limedolomite silicate serpentinite) in open habitats such assands steppes hilly and mountainous meadows and stony

grounds rom lowland regions to mountain tops over 2500m Te carnation plants preer neutral to slightly alkalinewell-drained soil Tey grow mostly in ull sun but alsotolerate light shade

Many species and more than 100 hybrids o the genusDianthus are used as ornamental plants o rocky gardensowing to its remarkably beautiul ragrant blossoms thatappear during the spring and summer and ofen deepinto the autumn Besides carnation plants are ofenused as cut-1047298owers and given that 1047298ower colour o dryplants is retained they are useul or pressed 1047298ower crafsand arrangements For 1047298ower industryhorticulture oparticular signi1047297cance are species having beautiul large

1047298owers o different shades o pink to red which aredistinguished by long blossoming period thriving onpoor acid soils and drought resistance (S983152983145983150983155983147983145 et al 1974C983144983151983157983139983144 amp 983158983137983150 S983156983137983140983141983150 1993 R983137983140983151983146983141983158983145983303 et al 2010)Such species and hybrids are common in parks especiallyin rocky gardens as 1047298oral edges o seedbeds or as coverso barren sites in settlements (R983137983140983151983146983141983158983145983303 2007)

Te endemic species are o particular interest bearingin mind the need o protection and preservation o speciesand genetic diversity o these more or less threatenedplants as well as their ornamental characteristics In theregion o the Balkan Peninsula to endemic carnationsbelong Dianthus ciliatus Guss and D giganteus Dum-Urville

D ciliatus ssp dalmaticus (Celak) Hayek is an endemicspecies spread in the Mediterranean and Submediterraneanzone o the Adriatic coast in Dalmatia (Croatia) andMontenegro (Fig1a) It is low semi-woody shrub

D giganteus ssp croaticus (Borbaacutes) utin is asubendemic species distributed in the western part o theBalkan Peninsula rom Slovenia eastward to Serbia (Fig1b) It is a perennial herb orming loose cushions

Hitherto only several papers concerned withconservative micropropagation o endemic species ocarnation were published (R983137983140983151983146983141983158983145983303 et al 1997 B983141983154983137983154983140983145

et al 2004 C983154983145983155983156983141983137 et al 2004 J983137983145983150 et al 2006 S983137983154983137983155983137983150et al 2006)Te aim o the present study was to orm a great

number o in vitro plants by applying stem segmentculture both or the purpose o preserving genopool othese species and or setting up an important resourceor the horticultural application o endemic carnations asornamental plants According to available data species Dciliatus ssp dalmaticus and D giganteus ssp croaticus tillnow have not been introduced to in vitro culture

MAERIAL AND MEHODS

Seed germination and seedling formationIn vitro culturewas initiated with seeds o D ciliatus ssp dalmaticus romMt Lovcen (Montenegro) and D gigantheus ssp croaticus rom Vlasina Lake (Serbia) Te seeds were washed withplenty o running water (2 h) and then immersed in 20 commercial bleach by modi1047297ed procedure o R983137983140983151983146983141983158983145983303et al (1990) or 20 min rinsed with sterile distilled waterand cultivated in Petri dishes (10 seeds per dish) containing

sterile 1047297lter paper with distilled water or germinationunder dark (10 days) Petri dishes were sealed withPara1047297lm (Pechiney Plastic Packing Chicago IL USA)Non-contaminated germinated seeds (Plate I Fig 1)reed rom seed coat were transerred in new Petri dishescontaining the MS

1=MS

0+ GA

3 (10 mg L-1) medium or

seedlings ormation and elongation (Plate I Fig 2)

Culture media Initial apical and nodal segments (about3-4 mm long) were cultivated on MS

2=MS

0+ NAA 10 +

Fig 1 Distribution o D ciliatus ssp dalmaticus (a) distri-bution o D giganteus ssp croaticus (b) (according to J983137983148983137983155 983078

S983157983151983149983145983150983141983150 1986)

7212019 Dianthus




2



2medium



hormones MS3 and MS

4= MS

0+ IBA (05-10 mg L-1

respectively) media




6=MS

0+ 24-D 10 + BAP 10 + NAA 01



0+ 24-D


All media (MS1-MS











149 MS + 3 sucrose + GA310 105 7046a


158 MS + 3 sucrose + GA310 66 4177b






08022007MS

027 867 plusmn 035 733 plusmn 158a

MS3


29032007MS

038 885 plusmn 076 752 plusmn 100a

MS3



26062006 MS0

15 566 plusmn 089 300 plusmn 043b

MS3

30 822 plusmn 096 785 plusmn 091a

16082006MS

015 260 plusmn 035 0

MS3



7212019 Dianthus


156 vol 34 (2)



3 in vitro plants




7212019 Dianthus






3




2










0 MS

3 and MS





3 medium (able 3







7212019 Dianthus


158 vol 34 (2)







7212019 Dianthus






5 and afer on MS

6 medium


5 to MS








CONCLUSIONS





Species

Segments

Medium



II III IV


AS 15

MS2

058 535 694

NS 126 137 741 786


AS 18 016 035 050

NS 34 032 050 068

7212019 Dianthus


160 vol 34 (2)

REFERENCES



































7212019 Dianthus

























REZIME

7212019 Dianthus




2



2medium



hormones MS3 and MS

4= MS

0+ IBA (05-10 mg L-1

respectively) media




6=MS

0+ 24-D 10 + BAP 10 + NAA 01



0+ 24-D


All media (MS1-MS











149 MS + 3 sucrose + GA310 105 7046a


158 MS + 3 sucrose + GA310 66 4177b






08022007MS

027 867 plusmn 035 733 plusmn 158a

MS3


29032007MS

038 885 plusmn 076 752 plusmn 100a

MS3



26062006 MS0

15 566 plusmn 089 300 plusmn 043b

MS3

30 822 plusmn 096 785 plusmn 091a

16082006MS

015 260 plusmn 035 0

MS3



7212019 Dianthus


156 vol 34 (2)



3 in vitro plants




7212019 Dianthus






3




2










0 MS

3 and MS





3 medium (able 3







7212019 Dianthus


158 vol 34 (2)







7212019 Dianthus






5 and afer on MS

6 medium


5 to MS








CONCLUSIONS





Species

Segments

Medium



II III IV


AS 15

MS2

058 535 694

NS 126 137 741 786


AS 18 016 035 050

NS 34 032 050 068

7212019 Dianthus


160 vol 34 (2)

REFERENCES



































7212019 Dianthus

























REZIME

7212019 Dianthus


156 vol 34 (2)



3 in vitro plants




7212019 Dianthus






3




2










0 MS

3 and MS





3 medium (able 3







7212019 Dianthus


158 vol 34 (2)







7212019 Dianthus






5 and afer on MS

6 medium


5 to MS








CONCLUSIONS





Species

Segments

Medium



II III IV


AS 15

MS2

058 535 694

NS 126 137 741 786


AS 18 016 035 050

NS 34 032 050 068

7212019 Dianthus


160 vol 34 (2)

REFERENCES



































7212019 Dianthus

























REZIME

7212019 Dianthus






3




2










0 MS

3 and MS





3 medium (able 3







7212019 Dianthus


158 vol 34 (2)







7212019 Dianthus






5 and afer on MS

6 medium


5 to MS








CONCLUSIONS





Species

Segments

Medium



II III IV


AS 15

MS2

058 535 694

NS 126 137 741 786


AS 18 016 035 050

NS 34 032 050 068

7212019 Dianthus


160 vol 34 (2)

REFERENCES



































7212019 Dianthus

























REZIME

7212019 Dianthus


158 vol 34 (2)







7212019 Dianthus






5 and afer on MS

6 medium


5 to MS








CONCLUSIONS





Species

Segments

Medium



II III IV


AS 15

MS2

058 535 694

NS 126 137 741 786


AS 18 016 035 050

NS 34 032 050 068

7212019 Dianthus


160 vol 34 (2)

REFERENCES



































7212019 Dianthus

























REZIME

7212019 Dianthus






5 and afer on MS

6 medium


5 to MS








CONCLUSIONS





Species

Segments

Medium



II III IV


AS 15

MS2

058 535 694

NS 126 137 741 786


AS 18 016 035 050

NS 34 032 050 068

7212019 Dianthus


160 vol 34 (2)

REFERENCES



































7212019 Dianthus

























REZIME

7212019 Dianthus


160 vol 34 (2)

REFERENCES



































7212019 Dianthus

























REZIME

7212019 Dianthus

























REZIME

Documents

Dianthus