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Improved In vitro regeneration and

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

Improved in vitro regeneration and propagation of Tunceli garlic

(Allium tuncelianum L.)
By S. KIZIL1*, D. Y. ICGIL1 and K. M. KHAWAR2
Department of Field Crops, Faculty of Agriculture, Dicle University, Main Campus,
21280 Diyarbakir, Turkey
2
Department of Field Crops, Faculty of Agriculture, Ankara University, Diskapi, 06110 Ankara,
Turkey
(e-mail: suleymankizil@gmail.com)
(Accepted 6 March 2014)

SUMMARY
Over-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 culture
protocol be developed for its conservation. Leaf tips, the middle portions of leaves, leaf bases, vertically-sectioned
halved or quartered bulbs, horizontally-sectioned upper and lower bulb halves, and root tip explants were cultured on
1.0
Murashige and Skoog (MS) medium containing 1.0, 2.0, 3.0, 4.0, or 5.0 mg l1 2,4-dichlorophenoxyacetic acid (2,4D) 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). The
results indicated that root tip explants were most suitable for bulblet regeneration on 1.0
MS medium containing 5.0
mg 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 growth
chamber conditions, then transferred to pots containing peat-moss in a greenhouse. The results will be important for
garlic breeders and researchers.

unceli garlic [Allium tuncelianum (Kollman) N.

Ozhatay, B. Mathew & Siraneci; Syn; A.
macrochaetum Boiss. and Hausskn. subsp. tuncelianum
Kollman] with white-to-purple flowered inflorescences
on single-cloved, cream-white bulbs is endemic to
Eastern Turkey, particularly the Province of Tunceli and
the surrounding Munzur mountains (Baktir, 2005;
Yanmaz et al., 2010). Tunceli garlic bears fertile black
seeds that can be used for propagation (Arslan, 2013).
The bulbs are produced asexually as propagules (i.e.,
vegetatively produced bulblets attached to the main
body of the mother bulb); however, their propagation
and proliferation are relatively slow. Propagation by seed
can produce mature plants after 2 3 years. Tunceli garlic
contains the biologically-active, organic sulphur
compound, allicin (thio-2-propene-1-sulphinic acid Sallyl ester). Allicin has been reported to have anticoagulant, anti-hypertensive, anti-microbial, anti-biotic,
anti-parasitic, anti-mycotic, anti-viral, anti-tumor, antioxidant, and anti-ageing activities (Jacob, 2006; Ozkan
et al., 2013). Allicin is also known to detoxify heavy
metals, be hypo-lipidaemic (i.e., lipid-lowering), anticarcinogenic, and anti-mutagenic (Munchberg et al.,
2007; Iciek et al., 2009; Ozkan et al., 2013).
Although the collection of endangered geophytes is
banned in Turkey under the Convention on the
International Trade in Endangered Species (CITES;
Arslan et al., 2002; Parmaksiz and Khawar, 2006; Ozel
et al., 2008; 2009; Gurbuz et al., 2009), approx. 15 25
metric tonnes (MT) of Tunceli garlic are collected from
the wild for household purposes each year (Arslan, 2013;
*Author for correspondence.

Yanmaz et al., 2010). Rapid urbanisation is also affecting

populations of this endangered species, and threatening
its existence. There is, therefore, an urgent need to
introduce Tunceli garlic into other areas by improving
agronomic techniques for the cultivation of plants, and
by applying non-conventional methods such as
micropropagation to conserve the species (SGP-UNDP,
2009).
Previous studies have reported micropropagation
techniques for in vitro regeneration of common garlic
from root tip explants (Shuto et al., 1993), the basal parts
of 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 leaves
within cloves (Abo El-Nil, 1977). However, there have
only been two reports on the in vitro micropropagation
of Tunceli garlic (Baktir, 2005; Yanmaz et al., 2010), both
of which showed poor levels of regeneration. This
indicated a need to develop an improved tissue culture
protocol to conserve this important species. The present
investigation was undertaken to optimise a protocol for
rapid bulblet propagation, rooting, and acclimatisation in
Tunceli garlic.

MATERIALS AND METHODS

A genetically mixed population of Tunceli garlic was
obtained from the experimental gardens of the
Department of Field Crops, Dicle University, Diyarbakir,
Turkey. The experimental material consisted of 2 3 cmdiameter bulbs (n = 60). The bulbs were washed in slowflowing tap water to remove all dirt and soil, then dried
at room temperature (24 1C) for 24 h. The bulbs were

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

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 removing
any residual roots. The bulbs were then rinsed threetimes, 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 screen
for possible fungal and/or bacterial contamination.
Experiments were then performed using each of the
following tissues or explants to achieve regeneration.
Experiment 1: The screened bulbs were cultured on 1.0

MS medium without any added plant growth regulator
(PGR) to promote the growth of leaves. After approx. 21
d, each bulb developed 4 - 5 cm-long leaves.
Leaf tips, the middle portion of the leaf, or the leaf
base (n = 60 of each) were excised aseptically and
cultured 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 into
two or four equal parts, each consisting of a half or a
quarter of the base plate, the bulb tissue, the tender
green leaves originating from the base plate (hereafter
called tender green leaves) in the central cavity within
the bulb, the cavity within the bulb, and the bulb tip (i.e.,
the portion above the cavity in bulb; Figure 1 A D).

409

Experiment 3: Each experimental treatment, including

the controls, used 60 explants, divided into six replicate
groups of ten. The bulbs were sectioned horizontally into
the proximal half (consisting of the fleshy bulb tissue, the
upper part of the tender green leaves in the central cavity
within the bulb, and the bulb-tip) and the distal half
(consisting of the base plate, the fleshy bulb tissue, and
the lower part of the tender green leaves arising from the
base plate in the central cavity).
Experiment 4: Single roots or roots attached to distal
half-bulbs (as described above), or roots attached to the
bulb were prepared.
Sixty explants of each type described above were used
for each experimental treatment, divided into six
replications. Explants were cultured on PGR-free 1.0

MS 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, or
containing 1.0, 2.0, 3.0, 4.0, or 5.0 mg l1 2,4dichlorophenoxyacetic acid (2,4-D), plus PGR-free
controls, all supplemented with 3.0% (w/v) sucrose and
solidified with 0.65% (w/v) agar for 56 d.
The developing bulblets were then rooted on 1.0
MS
medium containing 3.0% (w/v) sucrose. The pH of all
culture and rooting media was adjusted to 5.7 0.1 using
0.1 M NaOH or 0.1 M HC1 before autoclaving. All
media were autoclaved at 118 kPa and 121C for 20 min.
All cultures were incubated in a growth chamber (SGC
120; Fitotron, Loughborough, UK) at 24 1C with a
16 h light photoperiod at a photosynthetic photon flux
density (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 and
80.0% relative humidity in a growth-room under
controlled environmental conditions for 10 d to begin
photosynthesising and to acclimatise, followed by
transfer to a greenhouse.

STATISTICAL ANALYSIS
All data were subjected to one-way ANOVA using the
F-test in IBM - SPSS Statistics Version 20 software for
Windows (http://www-01.ibm.com/support/ docview.
wss?uid=swg24029274). Data in percentages were
subjected to arcsine square-root transformation
(Snedecor and Cochran, 1967) before statistical analysis.
The post hoc test was performed using Duncans
Multiple Range Test (DMRT) to compare differences
among treatment means at P 0.05 or P 0.01.
RESULTS

FIG. 1
Sectioning of Tunceli garlic bulbs to produce explants. Verticalsectioning (Panel A) into two equal halves showing (Panel B) half of (i)
the base plate, (ii) fleshy bulb tissue, (iii) tender green leaves
originating 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) tender
green leaves originating from the base plate, (iv) the cavity, and (v) the
bulb tip. Scale bars = 0.3 cm.

Optimisation of surface sterilisation of Allium

tuncelianum bulbs using 100.0% (v/v) commercial
bleach for different durations
No fungal or bacterial contamination was recorded
after 7 d on 1.0
MS medium after 15 or 30 min of
sterilisation in commercial bleach. Ten, 20, or 25 min of
sterilisation gave 16.7 83.3% fungal contamination. The
result showed that 15 or 30 min of surface-sterilisation in
100.0% (v/v) commercial bleach was effective. Due to
the ambiguous 15 min pattern of success in these

410

In vitro propogation of Tunceli garlic

TABLE I
Effect of 6-benzylaminopurine (BAP) plus 0.5 mg l1 -naphthaleneacetic
acid (NAA) on bulblet regeneration from leaf bases of A. tuncelianum
BAP (mg l1)
1.0
2.0
3.0
4.0
5.0
Mean

Shoot regeneration (%)

Mean number of
shoots per explant

0.0 c
13.3 a
6.7 b
6.7 b
0.0 c
5.3

0.0 c
1.0 a
1.0 a
0.7 b
0.0 c
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-case
letter are statistically different at P 0.01 by Duncans multiple range
test.

sterilisation experiments, we treated all explants with

100.0% (v/v) commercial bleach for 30 min in all
subsequent experiments
Effects of BAP plus 0.5 mg l1 NAA on bulblet
regeneration from leaf tips, the middle portion of leaves,
or leaf bases of A. tuncelianum
Bulblet regeneration data after 28 d in culture showed
no regeneration or callusing on leaf tips, or on the middle
portion 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 varying
extents. No bulblet regeneration was induced from leaf
bases on 1.0
MS medium containing 1.0 mg l1 BAP
plus 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 was
recorded on 1.0
MS medium containing 1.0 mg l1 BAP
plus 0.5 mg l1 NAA. All other culture media (those
containing 3.0 mg l1 BAP plus 0.5 mg l1 NAA or 4.0 mg
l1 BAP plus 0.5 mg l1 NAA) showed low regeneration
percentages (6.7% each; Figure 2). Mean values of 1.0,
1.0, and 0.7 bulblets per leaf base were recorded on 1.0

MS 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 leaf
regeneration from vertically-sectioned halves or quarters
of bulbs of A. tuncelianum
No bulblet regeneration was recorded on bulb tissue

from any vertically-sectioned half or quartered bulb

explant. However, a statistically significant effect of 2,4D concentration was recorded on the regeneration
percentage and the mean number of tender green
leaves arising from the base plate in the central cavity of
each explant. No tender green leaf regeneration was
noted on the base plates of any vertically-sectioned half
bulb explant on 1.0
MS medium containing 1.0 mg l1
2,4-D (Table II). Vertically-sectioned half and quarter
bulb explants showed 8.3 83.3% and 16.7 66.7%
regeneration of tender green leaves, respectively. Their
numbers ranged from 0.5 2.0 and 0.6 1.6 per explant.
The maximum tender green leaf regeneration
percentage, and the highest number of tender green
leaves per explant were noted on vertically-sectioned
half bulbs on 1.0
MS medium containing 2.0 mg l1 2,4D. Similarly, the maximum tender green leaf
regeneration percentage was noted on verticallysectioned quarter bulbs on 1.0
MS medium containing
1.0, 2.0, or 3.0 mg l1 2, 4-D. The base plates of these
explants induced the maximum number of 1.6 tender
green leaves on 1.0
MS medium containing 1.0 or 3.0
mg l1 2,4-D. These explants (vertically-sectioned half or
quartered bulbs) induced pseudo-bulblets with roots
coming out of the tender green leaves (Figure 2B). The
adjacent fleshy bulb tissue on both half and quartered
bulb explants changed from creamy-white to green,
increased in size, and swelled to form hard-textured bulb
tissue.
Effects of BAP plus NAA, or 2,4-D on bulblet and
tender green leaf regeneration from horizontallysectioned upper or lower half-bulb explants of A.
tuncelianum
Our results showed that both portions of horizontallysectioned bulb explants were ineffective at regenerating
new bulblets. Instead, the fleshy bulb tissue on both
horizontally-sectioned upper and lower half-bulbs
increased in size and became hard in texture at any
concentration of BAP plus NAA, or 2,4-D. The tender
green leaves grew and protruded by 2 3 cm from the
central cavity of both types of explant. The fleshy bulb
tissue of both types of explant increased in size and
swelled to form a hard-textured body with a light-green

FIG. 2
Bulblet 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)
1
plus 0.5 mg l -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

TABLE II
Effect 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
vertically-sectioned halved or quartered-Tunceli garlic bulbs
2,4-D (mg l1)
1
2
3
4
5
Mean

Number of tender green leaves in the central cavity of

vertically-sectioned halved or quartered-Tunceli garlic bulbs

Half-bulbs

Quarter-bulbs

Half-bulbs

Quarter-bulbs

0.0 e
83.3 a
8.3 d
41.7 c
58.3 b
38.3

66.7 a
66.7 a
66.7 a
41.7 ab
16.7 a
51.6

0.0 b
2.0 a
0.3 b
0.5 b
1.0 ab
1.6

1.6 ns
1.3 ns
1.6 ns
1.0 ns
0.6 ns
1.3

Each value is the mean of 60 explants (six replicates of ten explants) and mean values in each column followed by different lower-case letters are
statistically different at P 0.01 by Duncans multiple range test. ns, not significant.

colour. However, only horizontally-sectioned lower halfbulbs generated roots (0.3 1.3 roots per explant) at
various concentrations of BAP plus NAA (Table III).
These results suggest that both horizontally-sectioned
upper and lower half-bulb explants were unsuitable for
the regeneration of new bulblets.
Effects of 2,4-D or BAP plus NAA on bulblet
regeneration from root tip explants of A. tuncelianum
Regeneration percentages from root tip explants on
1.0
MS medium containing 2,4-D, or BAP plus NAA
were compared. These PGRs have different modes of
action. Root tips of Tunceli garlic roots showed swelling,
followed by browning and necrosis when the root tip
explants were used singly or still attached to the bulbs
and cultured on any concentration of 2,4-D (Figure 3A).
The results showed a significant effect (P 0.05) of
1.0
MS medium containing different concentrations of
BAP plus NAA on bulblet regeneration percentages
(with a range of 13.3 100.0%) and on the mean number
of bulblets per root tip explant (with a range of 0.1 1.0;
Table IV). Bulblet induction started as a swelling at the
root tip after approx. 24 26 d in culture. Underdeveloped bulblets were induced on 1.0
MS medium
containing 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 5.0
mg l1 BAP plus 0.5 mg l1 NAA. Most under-developed
bulblets did not root and developed 1.0 2.0 cm-long
twisted leaves or no leaves.
Root tip explants cultured on 1.0
MS medium
containing 4.0 mg l1 or 5.0 mg l1 BAP plus 0.5 mg l1
NAA showed a white-coloured swelling at their tip after
19 21 d in culture. This grew to a 0.2 0.3 cm-long green
coloured 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 these
regenerated bulblets induced roots and matured on 1.0

MS medium containing 3.0% (w/v) sucrose.

TABLE III
Effect of 6-benzylaminopurine (BAP) plus 0.5 mg l1 -naphthaleneacetic
acid (NAA) on rooting from horizontally-sectioned lower-half bulb
explants of A. tuncelianum
BAP (mg l1)
1.0
2.0
3.0
Mean

Mean number of roots per explant

1.3 a
0.7 b
0.3 c
0.7

Values are the means of 60 explants (six replicates of ten explants) and
values followed by a different lower-case letter are statistically different
at P 0.01 by Duncans multiple range test.

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 the
basal half of horizontally-sectioned or (iii) unsectioned
A. tuncelianum bulbs was achieved by culturing them on
1.0
MS medium containing 5.0 mg l1 BAP plus 0.5 mg
l1 NAA. These results showed that it was possible to
regenerate bulblets from roots bearing one root node
plus a root tip cultured on 1.0
MS medium containing
5.0 mg l1 BAP plus 0.5 mg l1 NAA (Table IV; Figure
3C). Bulblet induction started as a swelling at both the
root node and root tip after approx. 18 19 d of culture.
These bulblets were then excised from the root explants
and cultured on 1.0
MS medium containing 3.0% (w/v)
sucrose for maturation and proliferation.
The results of this study showd that regeneration of
bulblets on root tips of horizontally-sectioned or
unsectioned bulb explants was possible on 1.0
MS
medium containing 5.0 mg l1 BAP plus 0.5 mg l1 NAA
(Figure 3D E). After approx. 30 d in culture, the
developing bulblets were excised and cultured on 1.0

MS medium containing 3.0% (w/v) sucrose for
development, maturation, and growth. All of them
rooted and proliferated shoots from the bulb tip. These
were pre-hardened in pots for 14 d, followed by transfer
to open fields for acclimatisation (Figure 3F). Plants
acclimatised in 28 d, with visible signs of growth.

DISCUSSION
Tunceli garlic, an important endemic plant in Turkey,
multiplies naturally by seed or vegetatively by newlyregenerated bulb propagules attached to mother bulbs.
However, the percentage of new regenerated bulbs and
the number of plantlets per bulb is too low for practical
regeneration purposes (Yanmaz et al., 2010). Although
TABLE IV
Effect of 6-benzylaminopurine (BAP) plus 0.5 mg l1 -naphthaleneacetic
acid (NAA) on bulblet regeneration from root tip explants of A.
tuncelianum
BAP (mg l1)
1.0
2.0
3.0
4.0
5.0
Mean

Bulblet induction (%)

Mean number of
bulblets per explant

46.7 bc
33.4 bc
13.3 c
60.0 ab
100.0 a
50.7

0.5 bc
0.3 bc
0.1 c
0.6 ab
1.0 a
0.5

Values are the means of 60 explants (six replicates of ten explants) and
values in each column followed by different lower-case letters are
statistically different at P 0.01 by Duncans multiple range test.

412

In vitro propogation of Tunceli garlic

FIG. 3
Bulblet regeneration from root tip explants. Root tip explants showing little swelling followed by browning on 1.0
MS medium containing 5.0 mg
1
l 2,4-D (Panel A). Bulblet regeneration from root tips (Panel B). Bulblet regeneration on root tips of a horizontally-sectioned lower-half bulb (Panel
C). 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).

considerable efforts have been made to develop a

sustainable method of propagation, a reliable protocol
for the efficient propagation of bulbs has yet to be

developed (Halkinsesi On-Line Newspaper, 2013). This

study examined the possibility of using alternative
propagation methods via in vitro tissue culture.

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

Optimisation of surface sterilisation of A. tuncelianum
bulbs using 100% (v/v) commercial bleach for different
durations
Optimisation of the surface-sterilisation protocol is
important and a key factor in eliminating bacterial and/or
fungal contamination. The success of sterilisation
depends on the type of sterilising agent, its concentration,
and the duration of surface-sterilisation. As the plant
material used for sterilisation was collected from the
field, it was expected to have a high amount of microbial
contamination. Even a low percentage of fungal
contamination in a tissue culture box contaminates the
whole culture. Our results showed that 10 min was too
brief for surface sterilisation of the bulbs. Longer periods
of sterilisation were only partially effective, depending on
the extent of contamination of the material. Although 15
or 30 min in 100% commercial bleach resulted in
effective sterilisation in this experiment, unsuccessful
sterilisation results from 20 or 25 min emphasised that
sterilisation depended more on the selection of the field
material. Therefore, it was considered safest to sterilise
the Tunceli garlic bulbs for 30 min.
Effects of BAP plus 0.5 mg l1 NAA on bulblet
regeneration from leaf tips, the middle portion of leaves,
or leaf bases of A. tuncelianum
The results showed that leaf tips and the middle
portion of leaf blades were unsuitable for bulblet
regeneration as they lacked shoot meristems and could
not induce shoot meristems at the concentrations of
BAP plus NAA used in this experiment. The results also
showed that leaf bases were only partially competent for
regeneration, especially the distal ends. This might be
due to the presence of meristems or the induction of
meristems under the influence of PGRs, in agreement
with Hartmann et al. (2011). These workers showed
activation of meristem activity and growth on leaf bases
and found that cytokinins could be used to re-activate
meristem activity. In summary, these data suggest that
BAP plus NAA could stimulate meristematic activity in
leaf bases and regenerate bulblets.
Effects of 2,4-D or BAP plus NAA on regeneration
from tender green leaves in vertically or horizontallysectioned half or quarter bulb explants of A.
tuncelianum
Various concentrations of 2,4-D, or of BAP plus NAA
had variable effects on bulblet regeneration from
vertically-sectioned half or quarter bulb explants. Each
PGR induced hardening of the explants irrespective of
concentration.The PGRs stimulated the base plate, which
resulted in variable growth or induction and development
of new tender green leaves depending on the
concentration of the PGR. Tender green leaf explants
became shaped like pseudo-bulblets, with roots, on all
culture media, in agreement with Taylor (1997) and
Francis and Sorrell (2001). These results did not agree
with Yanmaz et al. (2010), who reported that 1.0
MS
medium containing 0.1 mg l1 BAP and 0.1 mg l1
indoleacetic acid (IAA) was sufficient for shoot induction
in A. tuncelianum. They found that shoots did not form
bulblets on combinations of IAA and BAP, and that IAA
resulted in the best bulblet regeneration after the fourth
sub-culture. They also noted that bulblet initiation started

413

after the second sub-culture or cytokinin-free 1.0
MS

medium containing 0.1 mg l1 NAA. These researchers
may have confused rooted pseudo-bulb formation from
tender green leaves arising from the base plate in the
central cavity with true bulbs. The results of this study
clearly demonstrate that vertically-sectioned half or
quarter-bulbs did not induce true bulblets.
Werner et al. (2001) also observed that cytokinins were
able to stimulate plant cell division in vivo and in vitro.
Tender green leaves in the central cavity of horizontallysectioned upper or lower-half bulbs grew inconsistently
and were not competent for true bulblet regeneration
using 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 from
garlic callus induced from base-plate cultures for somatic
embryogenesis and plant regeneration.
Effects of 2,4-D or BAP plus NAA on bulblet
regeneration from root tip explants of A. tuncelianum
It was possible to regenerate bulblets from single root
tip explants or from one root node and root tip
explants. Bulblet regeneration was also observed on the
tips of roots that had not been detached from the basal
half of horizontally-sectioned bulbs, or on unsectioned
whole bulbs of A. tuncelianum cultured on 1.0
MS
medium containing 5.0 mg l1 BAP plus 0.5 mg l1 NAA.
This is the first report of bulblet regeneration from
root tip explants of Tunceli garlic. The in vitroregenerated bulblets could be rooted and acclimatised
without difficulty on 1.0
MS medium, in agreement
with Zheng et al. (2003). These results are also in
agreement with (Ozel et al., 2008; 2009) who rooted other
important bulbous geophytes such as Ornithogalum
ulophyllum and Muscari macrocarpum on 1.0
MS
medium. 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 2.0
mg l1 NAA, respectively. Rooted bulbs acclimatised
successfully in pots under greenhouse conditions and
under 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 or
third leaf.
The results of this study emphasise that the
concentration of a combination of BAP and NAA are
important for bulblet regeneration from root tips or leaf
bases, which were the most competent tissues to
regenerate new bulblets. The results further suggest that
the correct selection of PGR and type of explant are
important factors for bulblet regeneration in Tunceli
garlic.

CONCLUSIONS
The present study has established an efficient protocol
for in vitro propagation of clonally-uniform Tunceli
garlic plants using root tip explants or the basal portion
of leaves. There is now a need to extend this technique
for breeding and propagation purposes.
This work was supported by a grant from the Scientific
and Technical Research Council of Turkey (TUBITAK;
Project No. 110 O 703).

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In vitro propogation of Tunceli garlic

REFERENCES

ABO EL-NIL, M. M. (1977). Organogenesis and embryogenesis in

callus cultures of garlic (Allium sativum L.). Plant Science
Letters, 9, 259264.
ARSLAN, N. (2013). Tunceli sarimsagi (Allium tuncelianum).
http://www.gidahatti.com/k-sa-k-sa-duenyadan-arsivi/tuncelisar-msag-allium-tuncelianum. 1 March 2014. (In Turkish.)
ARSLAN, N., GURBUZ, B., GUMUSCU, A., OZCAN, S., MIRICI, S. and
KHAWAR, K. M. (2002). Cultivation of Sternbergia fischeriana
(Herbert) Rupr. and a study on its morphological characteristics. Pakistan Journal of Botany, 34, 411418.
BAKTIR, I. (2005). In vitro micropropagation of Allium tuncelianum.
In: Proceedings of the GAP IV Agriculture Congress. Sanliurfa,
Turkey. 206208. (In Turkish.)
BHOJWANI, S. S., COHEN, D. and FRY, P. R. (1982). Production of
virus-free garlic and field performance of micropropagated
plants. Scientia Horticulturae, 18, 3943.
FRANCIS, D. and SORRELL, D. A. (2001). The interface between the
cell cycle and plant growth regulators: a mini review. Plant
Growth Regulation, 33, 112.
GURBUZ, B., ARSLAN, N., KHAWAR, K.M., IPEK, A., SARIHAN, E. O.,
OZCAN, S., PARMAKSIZ, I. and MIRICI, S. (2009). Adaptation of
endemic Mediterranean Sternbergia candida Mathew et T.
Baytop in the continental climate of Central Anatolia. Scientia
Horticulturae, 123, 99103.
HALKINSESI ON-LINE NEWSPAPER (2013). http://www.tunc elihalkinsesi.com/yerel/tunceli-sarimsaginin-korunmasi-ve-gelistirilmesi-projesi-kapsaminda-bilgilendirme-top-lantilari-h3425.
Html. 7 March 2013. (In Turkish.)
HARTMANN, A., SENNING, M., HEDDEN, P., SONNEWALD, U. and SONNEWALD, S. (2011). Reactivation of meristem activity and sprout
growth in potato tubers require both cytokinin and gibberellins.
Plant Physiology, 155, 776796.
ICIEK, M., KWIECIEN, I. and WLODEK, L. (2009). Biological properties of garlic and garlic-derived organosulfur compounds.
Environmental and Molecular Mutagenesis, 50, 247265.
LETHAM, D. S. and PALNI, L. M. S. (1983). The biosynthesis and
metabolism of cytokinins. Annual Review of Plant Physiology,
34, 163197.
MASUDA, K., HATAKEYAMA, E., ITO, A., TAKAHASHI, S. and INOUE, M.
(1994). Micropropagation of garlic (Allium sativum L.). Bulletin
of the Akita Prefectural College of Agriculture, 20, 4348.
MUNCHBERG, U., ANWAR, A., MECKLENBURG, S. and JACOB, C.
(2007). Polysulfides as biologically active ingredients of garlic.
Organic and Biomolecular Chemistry, 5, 15051518.
MURASHIGE, T. and SKOOG, F. (1962). A revised medium for rapid
growth and bioassays with tobacco tissue cultures. Physiologia
Plantarum, 15, 473497.
NAGAKUBO, T., NAGASAWA, A. and OHKAWA, H. (1993).
Micropropagation of garlic through in vitro bulblet formation.
Plant Cell, Tissue and Organ Culture, 32, 175183.

OZEL, C. A., KHAWAR, K. M., KARAMAN, S., ATES, M. A. and

ARSLAN, O. (2008). Efficient in vitro multiplication in
Ornithogalum ulophyllum Hand Mazz from twin scales.
Scientia Horticulturae, 116, 109112.
OZEL, C. A., KHAWAR, K. M., ARSLAN, O. and UNAL, F. (2009). In
vitro propagation of the golden grape hyacinth (Muscari
macrocarpum Sweet) from twin-scale explants. Propagation of
Ornamental Plants, 9, 169175.
OZKAN, O., GUL, S., KART, A., CICEK, B. A. and KILIC, K. (2013). In
vitro antimutagenicity of Allium tuncelianum ethanol extract
against induction of chromosome aberration by the mutagenic
agent mitomycin C. Journal of the Faculty of Veterinary
Medicine, Kafkas University, 19, 259262.
PARMAKSIZ, I. and KHAWAR, K. M. (2006). Plant regeneration by
somatic embryogenesis from immature seeds of Sternbergia
candida Mathew et T. Baytop, an endangenred endemic plant of
Turkey. Propagation of Ornamental Plants, 6, 128133.
SGP - UNDP (2009). www.sgp.undp.org/web/projects/8149.
SHUTO, H., ABE, T. and SASAHARA, T. (1993). In vitro propagation of
plants from root apex-derived calli in Chinese chive (Allium
tuberosum Rottler) and garlic (Allium sativum L.). Japanese
Journal of Breeding, 43, 349354.
SNEDECOR, G. W. and COCHRAN, W. G. (1967). Statistical Methods.
The Iowa State University Press, Ames, IA, USA. 327-329.
TAYLOR, C. B. (1997). Plant vegetative development: from seed and
embryo to shoot and root. Plant Cell, 9, 981988.
VERBEEK, M., VAN DIJK, P. and VAN WELL, P. M. A. (1995).
Efficiency of eradication of four viruses from garlic (Allium
sativum) by meristem tip culture. European Journal of Plant
Pathology, 101, 231239.
WERNER, T., MOTYKA, V., STRNAD, M. and SCHMULLING, T. (2001).
Regulation of plant growth by cytokinin. Proceedings of the
National Academy of Science of the USA, 98, 1048710492.
XUE, H. M., ARAKI, H and YAKUWA, T. (1991). Varietal difference of
embryonic callus induction and plant regeneration in garlic
(Allium sativum L.). Plant Tissue Culture Letters, 8, 166170.
YANMAZ, R., EZGI, Y. E., KANTOGLU, K. Y. and ALPER, A. (2010). In
vitro plant regeneration and bulblet formation of Tunceli garlic
(Allium tuncelianum (Kollman) Ozhatay, Matthew, Siraneci) by
shoot and root culture. Journal of Food, Agriculture and
Environment, 8, 572576.
ZHANG, R., ZHANG, X., WANG, J., LETHAM, D. S., MCKINNEY, S. A.
and HIGGINS, T. J. (1995). The effect of auxin on cytokinin levels
and metabolism in transgenic tobacco tissue expressing an Ipt
gene. Planta, 196, 8494.
ZHENG, S. J., HENKEN, B., KRENS, F. A. and KIK, C. (2003). The development of an efficient cultivar independent plant regeneration
system from callus derived from both apical and non-apical
root segments of garlic (Allium sativum L.). In Vitro Cellular &
Developmental Biology Plant, 39, 288-292.