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Improved in vitro regeneration and propagation of Tunceli garlic(Allium tuncelianum L.)

By S. KIZIL1*, D. Y. ICGIL1 and K. M. KHAWAR21Department of Field Crops, Faculty of Agriculture, Dicle University, Main Campus,21280 Diyarbakir, Turkey2Department of Field Crops, Faculty of Agriculture, Ankara University, Diskapi, 06110 Ankara,Turkey(e-mail: suleymankizil@gmail.com) (Accepted 6 March 2014)

SUMMARYOver-exploitation of endemic and threatened Tunceli garlic [Allium tuncelianum (Kollman) N. Ozhatay, B. Mathew &Siraneci] for household purposes has threatened the species and requires that a reliable, improved tissue cultureprotocol be developed for its conservation. Leaf tips, the middle portions of leaves, leaf bases, vertically-sectionedhalved or quartered bulbs, horizontally-sectioned upper and lower bulb halves, and root tip explants were cultured on1.0 Murashige and Skoog (MS) medium containing 1.0, 2.0, 3.0, 4.0, or 5.0 mg l1 2,4-dichlorophenoxyacetic acid (2,4-D) or 1.0, 2.0, 3.0, 4.0, or 5.0 mg l1 6-benzylaminopurine (BAP) plus 0.5 mg l1 -naphthaleneacetic acid (NAA). Theresults indicated that root tip explants were most suitable for bulblet regeneration on 1.0 MS medium containing 5.0mg l1 BAP plus 0.5 mg l1 NAA.All other explants failed to regenerate on different concentrations of BAP plus NAA,or on 2,4-D. The regenerated bulblets were acclimatised at 24 1C and 80.0% relative humidity under growthchamber conditions, then transferred to pots containing peat-moss in a greenhouse. The results will be important forgarlic breeders and researchers.

Tunceli garlic [Allium tuncelianum (Kollman) N.Ozhatay, B. Mathew & Siraneci; Syn; A.macrochaetum Boiss. and Hausskn. subsp. tuncelianumKollman] with white-to-purple flowered inflorescenceson single-cloved, cream-white bulbs is endemic toEastern Turkey, particularly the Province of Tunceli andthe surrounding Munzur mountains (Baktir, 2005;Yanmaz et al., 2010). Tunceli garlic bears fertile blackseeds that can be used for propagation (Arslan, 2013).The bulbs are produced asexually as propagules (i.e.,vegetatively produced bulblets attached to the mainbody of the mother bulb); however, their propagationand proliferation are relatively slow. Propagation by seedcan produce mature plants after 2 3 years.Tunceli garliccontains the biologically-active, organic sulphurcompound, allicin (thio-2-propene-1-sulphinic acid S-allyl ester). Allicin has been reported to have anti-coagulant, anti-hypertensive, anti-microbial, anti-biotic,anti-parasitic, anti-mycotic, anti-viral, anti-tumor, anti-oxidant, and anti-ageing activities (Jacob, 2006; Ozkanet al., 2013). Allicin is also known to detoxify heavymetals, be hypo-lipidaemic (i.e., lipid-lowering), anti-carcinogenic, and anti-mutagenic (Munchberg et al.,2007; Iciek et al., 2009; Ozkan et al., 2013).

Although the collection of endangered geophytes isbanned in Turkey under the Convention on theInternational Trade in Endangered Species (CITES;Arslan et al., 2002; Parmaksiz and Khawar, 2006; Ozelet al., 2008; 2009; Gurbuz et al., 2009), approx. 15 25metric tonnes (MT) of Tunceli garlic are collected fromthe wild for household purposes each year (Arslan, 2013;

Yanmaz et al., 2010). Rapid urbanisation is also affectingpopulations of this endangered species, and threateningits existence. There is, therefore, an urgent need tointroduce Tunceli garlic into other areas by improvingagronomic techniques for the cultivation of plants, andby applying non-conventional methods such asmicropropagation to conserve the species (SGP-UNDP,2009).

Previous studies have reported micropropagationtechniques for in vitro regeneration of common garlicfrom root tip explants (Shuto et al., 1993), the basal partsof bulblets (Masuda et al., 1994), scape tips (Xue et al.,1991), shoot tips (Abo El-Nil, 1977; Bhojwani et al., 1982;Nagakubo et al., 1993; Verbeek et al., 1995), or leaveswithin cloves (Abo El-Nil, 1977). However, there haveonly been two reports on the in vitro micropropagationof Tunceli garlic (Baktir, 2005; Yanmaz et al., 2010), bothof which showed poor levels of regeneration. Thisindicated a need to develop an improved tissue cultureprotocol to conserve this important species. The presentinvestigation was undertaken to optimise a protocol forrapid bulblet propagation, rooting, and acclimatisation inTunceli garlic.

MATERIALS AND METHODSA genetically mixed population of Tunceli garlic was

obtained from the experimental gardens of theDepartment of Field Crops, Dicle University, Diyarbakir,Turkey. The experimental material consisted of 2 3 cm-diameter bulbs (n = 60). The bulbs were washed in slow-flowing tap water to remove all dirt and soil, then driedat room temperature (24 1C) for 24 h.The bulbs were*Author for correspondence.

Journal of Horticultural Science & Biotechnology (2014) 89 (4) 408414

S. KIZIL, D. Y. ICGIL and K. M. KHAWAR 409

surface-sterilised using 100.0% (v/v) domestic bleach(Domestos; Unilever, Istanbul, Turkey) containing 5.0%(v/v) NaOCl for 10, 15, 20, 25, or 30 min after removingany residual roots. The bulbs were then rinsed three-times, for 3 min each, in sterilised double-distilled water.The bulbs were cultured on 1.0 MS medium(Murashige and Skoog, 1962) supplemented with 3.0%(w/v) sucrose and solidified with 0.62% (w/v) agar(Duchefa, Haarlem, The Netherlands) for 7 d to screenfor possible fungal and/or bacterial contamination.Experiments were then performed using each of thefollowing tissues or explants to achieve regeneration.

Experiment 1: The screened bulbs were cultured on 1.0MS medium without any added plant growth regulator(PGR) to promote the growth of leaves. After approx. 21d, each bulb developed 4 - 5 cm-long leaves.

Leaf tips, the middle portion of the leaf, or the leafbase (n = 60 of each) were excised aseptically andcultured on 1.0 MS medium containing 1.0, 2.0, 3.0, 4.0,or 5.0 mg l1 6-benzylaminopurine (BAP) plus 0.5 mg l1

-naphthaleneacetic acid (NAA).

Experiment 2: Single bulbs were sectioned vertically intotwo or four equal parts, each consisting of a half or aquarter of the base plate, the bulb tissue, the tendergreen leaves originating from the base plate (hereaftercalled tender green leaves) in the central cavity withinthe bulb, the cavity within the bulb, and the bulb tip (i.e.,the portion above the cavity in bulb; Figure 1 A D).

Experiment 3: Each experimental treatment, includingthe controls, used 60 explants, divided into six replicategroups of ten. The bulbs were sectioned horizontally intothe proximal half (consisting of the fleshy bulb tissue, theupper part of the tender green leaves in the central cavitywithin the bulb, and the bulb-tip) and the distal half(consisting of the base plate, the fleshy bulb tissue, andthe lower part of the tender green leaves arising from thebase plate in the central cavity).

Experiment 4: Single roots or roots attached to distalhalf-bulbs (as described above), or roots attached to thebulb were prepared.

Sixty explants of each type described above were usedfor each experimental treatment, divided into sixreplications. Explants were cultured on PGR-free 1.0MS medium or on 1.0 MS medium containing 1.0, 2.0,3.0, 4.0, or 5.0 mg l1 BAP plus 0.5 mg l1 NAA, orcontaining 1.0, 2.0, 3.0, 4.0, or 5.0 mg l1 2,4-dichlorophenoxyacetic acid (2,4-D), plus PGR-freecontrols, all supplemented with 3.0% (w/v) sucrose andsolidified with 0.65% (w/v) agar for 56 d.

The developing bulblets were then rooted on 1.0 MSmedium containing 3.0% (w/v) sucrose. The pH of allculture and rooting media was adjusted to 5.7 0.1 using0.1 M NaOH or 0.1 M HC1 before autoclaving. Allmedia were autoclaved at 118 kPa and 121C for 20 min.All cultures were incubated in a growth chamber (SGC120; Fitotron, Loughborough, UK) at 24 1C with a16 h light photoperiod at a photosynthetic photon fluxdensity (PPFD) of 35 mol m2 s1. After 56 d in culture,the rooted bulblets were transferred to 5 l pots (n = 8)containing 4.5 l of peat-moss and grown at 24 1C and80.0% relative humidity in a growth-room undercontrolled environmental conditions for 10 d to beginphotosynthesising and to acclimatise, followed bytransfer to a greenhouse.

STATISTICAL ANALYSISAll data were subjected to one-way ANOVA using the

F-test in IBM - SPSS Statistics Version 20 software forWindows (http://www-01.ibm.com/support/ docview.wss?uid=swg24029274). Data in percentages weresubjected to arcsine square-root transformation(Snedecor and Cochran, 1967) before statistical analysis.The post hoc test was performed using DuncansMultiple Range Test (DMRT) to compare differencesamong treatment means at P 0.05 or P 0.01.

RESULTS

Optimisation of surface sterilisation of Alliumtuncelianum bulbs using 100.0% (v/v) commercialbleach for different durations

No fungal or bacterial contamination was recordedafter 7 d on 1.0 MS medium after 15 or 30 min ofsterilisation in commercial bleach. Ten, 20, or 25 min ofsterilisation gave 16.7 83.3% fungal contamination.Theresult showed that 15 or 30 min of surface-sterilisation in100.0% (v/v) commercial bleach was effective. Due tothe ambiguous 15 min pattern of success in these

FIG. 1Sectioning of Tunceli garlic bulbs to produce explants. Vertical-sectioning (Panel A) into two equal halves showing (Panel B) half of (i)the base plate, (ii) fleshy bulb tissue, (iii) tender green leavesoriginating from the base plate, (iv) the cavity, and (v) the bulb tip.Vertical-sectioning (Panel C) into four equal halves showing (Panel D)one quarter of (i) the base plate, (ii) the fleshy bulb tissue, (iii) tendergreen leaves originating from the base plate, (iv) the cavity, and (v) the

bulb tip. Scale bars = 0.3 cm.

In vitro propogation of Tunceli garlic410

sterilisation experiments, we treated all explants with100.0% (v/v) commercial bleach for 30 min in allsubsequent experiments

Effects of BAP plus 0.5 mg l1 NAA on bulbletregeneration from leaf tips, the middle portion of leaves,or leaf bases of A. tuncelianum

Bulblet regeneration data after 28 d in culture showedno regeneration or callusing on leaf tips, or on the middleportion of leaves on 1.0 MS medium containing 0.5 1.0 mg l1 BAP plus 0.5 mg l1 NAA (five combinations).These explants increased in area and swelled to varyingextents. No bulblet regeneration was induced from leafbases on 1.0 MS medium containing 1.0 mg l1 BAPplus 0.5 mg l1 NAA, or 5.0 mg l1 BAP plus 0.5 mg l1

NAA (Table I). A maximum of 13.3% regeneration wasrecorded on 1.0 MS medium containing 1.0 mg l1 BAPplus 0.5 mg l1 NAA. All other culture media (thosecontaining 3.0 mg l1 BAP plus 0.5 mg l1 NAA or 4.0 mgl1 BAP plus 0.5 mg l1 NAA) showed low regenerationpercentages (6.7% each; Figure 2). Mean values of 1.0,1.0, and 0.7 bulblets per leaf base were recorded on 1.0MS medium containing 2.0 mg l1 BAP plus 0.5 mg l1

NAA (Figure 2A), 3.0 mg l1 BAP plus 0.5 mg l1 NAA,or 4.0 mg l1 BAP plus 0.5 mg l1 NAA, respectively.

Effect of 2,4-D on bulblet and tender green leafregeneration from vertically-sectioned halves or quartersof bulbs of A. tuncelianum

No bulblet regeneration was recorded on bulb tissue

from any vertically-sectioned half or quartered bulbexplant. However, a statistically significant effect of 2,4-D concentration was recorded on the regenerationpercentage and the mean number of tender greenleaves arising from the base plate in the central cavity ofeach explant. No tender green leaf regeneration wasnoted on the base plates of any vertically-sectioned halfbulb explant on 1.0 MS medium containing 1.0 mg l1

2,4-D (Table II). Vertically-sectioned half and quarterbulb explants showed 8.3 83.3% and 16.7 66.7%regeneration of tender green leaves, respectively.Theirnumbers ranged from 0.5 2.0 and 0.6 1.6 per explant.The maximum tender green leaf regenerationpercentage, and the highest number of tender greenleaves per explant were noted on vertically-sectionedhalf bulbs on 1.0 MS medium containing 2.0 mg l1 2,4-D. Similarly, the maximum tender green leafregeneration percentage was noted on vertically-sectioned quarter bulbs on 1.0 MS medium containing1.0, 2.0, or 3.0 mg l1 2, 4-D. The base plates of theseexplants induced the maximum number of 1.6 tendergreen leaves on 1.0 MS medium containing 1.0 or 3.0mg l1 2,4-D. These explants (vertically-sectioned half orquartered bulbs) induced pseudo-bulblets with rootscoming out of the tender green leaves (Figure 2B). Theadjacent fleshy bulb tissue on both half and quarteredbulb explants changed from creamy-white to green,increased in size, and swelled to form hard-textured bulbtissue.

Effects of BAP plus NAA, or 2,4-D on bulblet andtender green leaf regeneration from horizontally-sectioned upper or lower half-bulb explants of A.tuncelianum

Our results showed that both portions of horizontally-sectioned bulb explants were ineffective at regeneratingnew bulblets. Instead, the fleshy bulb tissue on bothhorizontally-sectioned upper and lower half-bulbsincreased in size and became hard in texture at anyconcentration of BAP plus NAA, or 2,4-D. The tendergreen leaves grew and protruded by 2 3 cm from thecentral cavity of both types of explant. The fleshy bulbtissue of both types of explant increased in size andswelled to form a hard-textured body with a light-green

TABLE IEffect of 6-benzylaminopurine (BAP) plus 0.5 mg l1 -naphthaleneaceticacid (NAA) on bulblet regeneration from leaf bases of A. tuncelianum

Mean number ofBAP (mg l1) Shoot regeneration (%) shoots per explant

1.0 0.0 c 0.0 c 2.0 13.3 a 1.0 a3.0 6.7 b 1.0 a4.0 6.7 b 0.7 b5.0 0.0 c 0.0 cMean 5.3 0.5 Values are the means of 60 explants (six replicates each of ten explants)and mean values in each column followed by a different lower-caseletter are statistically different at P 0.01 by Duncans multiple rangetest.

FIG. 2Bulblet regeneration from the lower portion of a leaf blade of A. tuncelianum on 1.0 MS medium containing 1.0 mg l1 6-benzylaminopurine (BAP)plus 0.5 mg l1 -naphthaleneacetic acid (NAA; Panel A). Development of a pseudo-bulblet and roots on 1.0 MS medium containing 5.0 mg l1

2,4-D (Panel B). Growth of tender green leaves from the base plate of swollen, hard-textured bulb tissue (Panel C). Scale bars = 0.4 cm (Panel A),0.5 cm (Panel B), and 0.6 cm (Panel C).

S. KIZIL, D. Y. ICGIL and K. M. KHAWAR 411

colour. However, only horizontally-sectioned lower half-bulbs generated roots (0.3 1.3 roots per explant) atvarious concentrations of BAP plus NAA (Table III).These results suggest that both horizontally-sectionedupper and lower half-bulb explants were unsuitable forthe regeneration of new bulblets.

Effects of 2,4-D or BAP plus NAA on bulbletregeneration from root tip explants of A. tuncelianum

Regeneration percentages from root tip explants on1.0 MS medium containing 2,4-D, or BAP plus NAAwere compared. These PGRs have different modes ofaction. Root tips of Tunceli garlic roots showed swelling,followed by browning and necrosis when the root tipexplants were used singly or still attached to the bulbsand cultured on any concentration of 2,4-D (Figure 3A).

The results showed a significant effect (P 0.05) of1.0 MS medium containing different concentrations ofBAP plus NAA on bulblet regeneration percentages(with a range of 13.3 100.0%) and on the mean numberof bulblets per root tip explant (with a range of 0.1 1.0;Table IV). Bulblet induction started as a swelling at theroot tip after approx. 24 26 d in culture. Under-developed bulblets were induced on 1.0 MS mediumcontaining 1.0, 2.0, or 3.0 mg l1 BAP plus 0.5 mg l1 NAA,while fully-developed bulblets were induced on 4.0 or 5.0mg l1 BAP plus 0.5 mg l1 NAA. Most under-developedbulblets did not root and developed 1.0 2.0 cm-longtwisted leaves or no leaves.

Root tip explants cultured on 1.0 MS mediumcontaining 4.0 mg l1 or 5.0 mg l1 BAP plus 0.5 mg l1

NAA showed a white-coloured swelling at their tip after19 21 d in culture.This grew to a 0.2 0.3 cm-long greencoloured single micro-bulblet after 30 - 31 d (Figure 3B).Their diameter increased from 0.4 0.5 cm after 45 d,and to 0.7 0.8 cm after 55 65 d in culture. All of theseregenerated bulblets induced roots and matured on 1.0MS medium containing 3.0% (w/v) sucrose.

In another experiment, bulblet regeneration from (i)root explants bearing one root node plus a root tip, or(ii) root tip explants that were not detached from thebasal half of horizontally-sectioned or (iii) unsectionedA. tuncelianum bulbs was achieved by culturing them on1.0 MS medium containing 5.0 mg l1 BAP plus 0.5 mgl1 NAA. These results showed that it was possible toregenerate bulblets from roots bearing one root nodeplus a root tip cultured on 1.0 MS medium containing5.0 mg l1 BAP plus 0.5 mg l1 NAA (Table IV; Figure3C). Bulblet induction started as a swelling at both theroot node and root tip after approx. 18 19 d of culture.These bulblets were then excised from the root explantsand cultured on 1.0 MS medium containing 3.0% (w/v)sucrose for maturation and proliferation.

The results of this study showd that regeneration ofbulblets on root tips of horizontally-sectioned orunsectioned bulb explants was possible on 1.0 MSmedium containing 5.0 mg l1 BAP plus 0.5 mg l1 NAA(Figure 3D E). After approx. 30 d in culture, thedeveloping bulblets were excised and cultured on 1.0MS medium containing 3.0% (w/v) sucrose fordevelopment, maturation, and growth. All of themrooted and proliferated shoots from the bulb tip. Thesewere pre-hardened in pots for 14 d, followed by transferto open fields for acclimatisation (Figure 3F). Plantsacclimatised in 28 d, with visible signs of growth.

DISCUSSIONTunceli garlic, an important endemic plant in Turkey,

multiplies naturally by seed or vegetatively by newly-regenerated bulb propagules attached to mother bulbs.However, the percentage of new regenerated bulbs andthe number of plantlets per bulb is too low for practicalregeneration purposes (Yanmaz et al., 2010). Although

TABLE IIEffect of 2,4-dichlorophenoxyacetic acid (2,4-D) on the regeneration of tender green leaves originating from the base plate from vertically-sectioned

half or quarter-bulbs of A. tuncelianum

Regeneration percentage (%)of tender green leaves in the central cavity of Number of tender green leaves in the central cavity of

vertically-sectioned halved or quartered-Tunceli garlic bulbs vertically-sectioned halved or quartered-Tunceli garlic bulbs

2,4-D (mg l1) Half-bulbs Quarter-bulbs Half-bulbs Quarter-bulbs

1 0.0 e 66.7 a 0.0 b 1.6 ns2 83.3 a 66.7 a 2.0 a 1.3 ns3 8.3 d 66.7 a 0.3 b 1.6 ns4 41.7 c 41.7 ab 0.5 b 1.0 ns5 58.3 b 16.7 a 1.0 ab 0.6 nsMean 38.3 51.6 1.6 1.3Each value is the mean of 60 explants (six replicates of ten explants) and mean values in each column followed by different lower-case letters arestatistically different at P 0.01 by Duncans multiple range test. ns, not significant.

TABLE IIIEffect of 6-benzylaminopurine (BAP) plus 0.5 mg l1 -naphthaleneaceticacid (NAA) on rooting from horizontally-sectioned lower-half bulb

explants of A. tuncelianum

BAP (mg l1) Mean number of roots per explant

1.0 1.3 a

2.0 0.7 b3.0 0.3 cMean 0.7Values are the means of 60 explants (six replicates of ten explants) andvalues followed by a different lower-case letter are statistically differentat P 0.01 by Duncans multiple range test.

TABLE IVEffect of 6-benzylaminopurine (BAP) plus 0.5 mg l1 -naphthaleneaceticacid (NAA) on bulblet regeneration from root tip explants of A.

tuncelianum

Mean number of BAP (mg l1) Bulblet induction (%) bulblets per explant

1.0 46.7 bc 0.5 bc2.0 33.4 bc 0.3 bc3.0 13.3 c 0.1 c4.0 60.0 ab 0.6 ab5.0 100.0 a 1.0 aMean 50.7 0.5Values are the means of 60 explants (six replicates of ten explants) andvalues in each column followed by different lower-case letters arestatistically different at P 0.01 by Duncans multiple range test.

In vitro propogation of Tunceli garlic412

considerable efforts have been made to develop asustainable method of propagation, a reliable protocolfor the efficient propagation of bulbs has yet to be

developed (Halkinsesi On-Line Newspaper, 2013). Thisstudy examined the possibility of using alternativepropagation methods via in vitro tissue culture.

FIG. 3Bulblet regeneration from root tip explants. Root tip explants showing little swelling followed by browning on 1.0 MS medium containing 5.0 mgl1 2,4-D (Panel A). Bulblet regeneration from root tips (Panel B). Bulblet regeneration on root tips of a horizontally-sectioned lower-half bulb (PanelC). Bulblet development on roots tips on non-sectioned bulbs on 1.0 MS medium containing 5.0 mg l1 BAP plus 0.5 mg l1 NAA (Panel D).Developing bulblets induced on root tips (Panel E). Tissue-cultured bulbs of A. tuncelianum in pots in a greenhouse after 28 d (Panel F). Scale bars

= 0.5 cm (Panel AC), 0.7 cm (Panel DE), and 3.5 cm (Panel F).

S. KIZIL, D. Y. ICGIL and K. M. KHAWAR 413

Optimisation of surface sterilisation of A. tuncelianumbulbs using 100% (v/v) commercial bleach for differentdurations

Optimisation of the surface-sterilisation protocol isimportant and a key factor in eliminating bacterial and/orfungal contamination. The success of sterilisationdepends on the type of sterilising agent, its concentration,and the duration of surface-sterilisation. As the plantmaterial used for sterilisation was collected from thefield, it was expected to have a high amount of microbialcontamination. Even a low percentage of fungalcontamination in a tissue culture box contaminates thewhole culture. Our results showed that 10 min was toobrief for surface sterilisation of the bulbs. Longer periodsof sterilisation were only partially effective, depending onthe extent of contamination of the material. Although 15or 30 min in 100% commercial bleach resulted ineffective sterilisation in this experiment, unsuccessfulsterilisation results from 20 or 25 min emphasised thatsterilisation depended more on the selection of the fieldmaterial. Therefore, it was considered safest to sterilisethe Tunceli garlic bulbs for 30 min.

Effects of BAP plus 0.5 mg l1 NAA on bulbletregeneration from leaf tips, the middle portion of leaves,or leaf bases of A. tuncelianum

The results showed that leaf tips and the middleportion of leaf blades were unsuitable for bulbletregeneration as they lacked shoot meristems and couldnot induce shoot meristems at the concentrations ofBAP plus NAA used in this experiment. The results alsoshowed that leaf bases were only partially competent forregeneration, especially the distal ends. This might bedue to the presence of meristems or the induction ofmeristems under the influence of PGRs, in agreementwith Hartmann et al. (2011). These workers showedactivation of meristem activity and growth on leaf basesand found that cytokinins could be used to re-activatemeristem activity. In summary, these data suggest thatBAP plus NAA could stimulate meristematic activity inleaf bases and regenerate bulblets.

Effects of 2,4-D or BAP plus NAA on regeneration from tender green leaves in vertically or horizontally-sectioned half or quarter bulb explants of A.tuncelianum

Various concentrations of 2,4-D, or of BAP plus NAAhad variable effects on bulblet regeneration fromvertically-sectioned half or quarter bulb explants. EachPGR induced hardening of the explants irrespective ofconcentration.The PGRs stimulated the base plate, whichresulted in variable growth or induction and developmentof new tender green leaves depending on theconcentration of the PGR. Tender green leaf explantsbecame shaped like pseudo-bulblets, with roots, on allculture media, in agreement with Taylor (1997) andFrancis and Sorrell (2001). These results did not agreewith Yanmaz et al. (2010), who reported that 1.0 MSmedium containing 0.1 mg l1 BAP and 0.1 mg l1

indoleacetic acid (IAA) was sufficient for shoot inductionin A. tuncelianum. They found that shoots did not formbulblets on combinations of IAA and BAP, and that IAAresulted in the best bulblet regeneration after the fourthsub-culture.They also noted that bulblet initiation started

after the second sub-culture or cytokinin-free 1.0 MSmedium containing 0.1 mg l1 NAA. These researchersmay have confused rooted pseudo-bulb formation fromtender green leaves arising from the base plate in thecentral cavity with true bulbs. The results of this studyclearly demonstrate that vertically-sectioned half orquarter-bulbs did not induce true bulblets.

Werner et al. (2001) also observed that cytokinins wereable to stimulate plant cell division in vivo and in vitro.Tender green leaves in the central cavity of horizontally-sectioned upper or lower-half bulbs grew inconsistentlyand were not competent for true bulblet regenerationusing PGRs. These results agree with Letham and Palni,(1983) and Zhang et al. (1995), but not with Xue et al.(1991) who reported improved shoot regeneration fromgarlic callus induced from base-plate cultures for somaticembryogenesis and plant regeneration.

Effects of 2,4-D or BAP plus NAA on bulbletregeneration from root tip explants of A. tuncelianum

It was possible to regenerate bulblets from single roottip explants or from one root node and root tipexplants. Bulblet regeneration was also observed on thetips of roots that had not been detached from the basalhalf of horizontally-sectioned bulbs, or on unsectionedwhole bulbs of A. tuncelianum cultured on 1.0 MSmedium containing 5.0 mg l1 BAP plus 0.5 mg l1 NAA.

This is the first report of bulblet regeneration fromroot tip explants of Tunceli garlic. The in vitro-regenerated bulblets could be rooted and acclimatisedwithout difficulty on 1.0 MS medium, in agreementwith Zheng et al. (2003). These results are also inagreement with (Ozel et al., 2008; 2009) who rooted otherimportant bulbous geophytes such as Ornithogalumulophyllum and Muscari macrocarpum on 1.0 MSmedium. The results do not agree with Yanmaz et al.(2010), who found the highest rooting percentages(17.0% and 33.0%) in Tunceli garlic on 0.5 mg l1 or 2.0mg l1 NAA, respectively. Rooted bulbs acclimatisedsuccessfully in pots under greenhouse conditions andunder field conditions. In contrast, Yanmaz et al. (2010)found that rooted or un-rooted Tunceli garlic bulbs,transferred to pots, died after generation of the second orthird leaf.

The results of this study emphasise that theconcentration of a combination of BAP and NAA areimportant for bulblet regeneration from root tips or leafbases, which were the most competent tissues toregenerate new bulblets. The results further suggest thatthe correct selection of PGR and type of explant areimportant factors for bulblet regeneration in Tunceligarlic.

CONCLUSIONSThe present study has established an efficient protocol

for in vitro propagation of clonally-uniform Tunceligarlic plants using root tip explants or the basal portionof leaves. There is now a need to extend this techniquefor breeding and propagation purposes.

This work was supported by a grant from the Scientificand Technical Research Council of Turkey (TUBITAK;Project No. 110 O 703).

In vitro propogation of Tunceli garlic414

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