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Journal of Food, Agriculture & Environment Vol.8 (3&4): 572-576. 2010

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In vitro plant regeneration and bulblet formation of Tunceli garlic (Allium tuncelianum (Kollman) zhatay, Matthew, Siraneci) by shoot and root culture
Ruhsar Yanmaz 1, Ezgi Yazar 2, K. Yaprak Kantolu 3* and Asl Alper 2
1

Ankara University, Faculty of Agriculture, Department of Horticulture, 06110, Ankara, Turkey. 2 Republic of Turkey Ministry of Agricultural and Rural Affairs, Ankara, Turkey. 3 Turkish Atomic Energy Authority Saraykoy Nuclear Research and Training Center, Ankara, Turkey. *e-mails: yaprak.taner@taek.gov.tr, kayaprakta@yahoo.com

Received 30 July 2010, accepted 9 November 2010.

Abstract
Tunceli garlic (Allium tuncelianum (Kollman) zhatay, Matthews, Siraneci) is an endemic garlic species located in the Eastern part of Turkey and used as cultivated garlic (Allium sativum). Allium tuncelianum is endangered because of insensible harvesting of the local people. This research was carried out to determine and adapt the ways of in vitro micropropagation methods for Tunceli garlic. A novel micropropagation method was developed for Tunceli garlic by root and shoot culture techniques. The different combinations of 2,4-dichlorophenoxyacetic acid (2,4-D), naphthalene acetic acid (NAA) (for root culture) and NAA, indole-3-acetic acid (IAA) with benzyladenin (BA) (0, 0.1, 1.0 mgl-1) (for shoot culture experiment) were used in Murashige & Skoog (MS) medium which were supplemented with 30 g l-1 sucrose and 0.8 mg l-1 agar. According to results, the root tip culture was not found as a proper method for shoot proliferation like Allium sativum. On the contrary, shoot culture was found effective on shoot formation. As an average, 1 or 2 shoots obtained per explant and lower doses of IAA and BA (0.1 mg l-1, 0.1 mg l-1) had an important effect on it. The proliferated shoots of Tunceli garlic produced bulblets by culture on auxin free MS medium; also subculture number relatively had an important effect on bulblet formation. Key words: Allium tuncelianum, garlic, shoot culture, root culture, bulblet.

Introduction The species of Allium, especially A. cepa and A. sativum of Alliaceae are important vegetable crops worldwide. There are nearly 500 Allium species in the world and 164 of these originated in Turkey. Forty percent of Allium species found in Turkey is endemic 1. Tunceli garlic (Allium tuncelianum) is one of the endemic Alliums in the world and only grown in Tunceli province and Munzur mountain at the height of 1100-1200 m, Erzincan and Sivas provinces in Turkey. It was classified as a sub species of A. macrochaetum in 1983, after that, it was defined as a different species and classified as A. tuncelianum 2, and also according to diversity studies, the genetic difference of Tunceli garlic was determined by molecular analysis 3. Morphologically Tunceli garlic (Allium tuncelianum) plants look like Allium sativum plants. Its leaves are broader and leaf length is around 35-40 cm. It has only one round clove which is in 2.5-3.5 cm height and 2.8-3.5 cm diameter. The bulb formation starts after the 6th leaf formation and bulb has only 2-3 leaf sheets. There are 1-4 bulblets between leaf sheets. After bulb formation, plant forms a flower stalk which is 80-135 cm length in the same vegetation year. The colour of flower is purple and there are 300-500 flowers in one flower set. Pollination procedure, seed formation and the seeds are like onion. The 1000 seed weight is 3.2 g 4. Tunceli garlic can be propagated by seed or bulblets; over-exploitation of the plant associated with poor seed set and germination has made it an endangered species. Allium tuncelianum is collected and consumed locally, and it is under
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government protection in Turkey, but people collect the bulbs from mountains and sell in the market. However, the next usable heads are produced at the end of third year 5. It is therefore under risk and getting looser the day by day. The continuous collection from the wild has led to concerns its survival as a wild species. To overcome this circumstance, in 2002 the United Nations Development Program awarded a grant to the Accessible Life Association (UYD) to enable them to determine the feasibility of the commercial cultivation of the plant in field conditions. The cultivation trials were carried out on a piece of land bought by 120 local farmers and was successfully completed. According to their results, a new rapid micropropagation method must be developed for Tunceli garlic 6. Improvement of garlic (Allium sativum L.) through classical breeding techniques is not possible because cultivated garlic is sexually sterile. For this reason, in vitro techniques have been developed for garlic. Rapid in vitro micropropagation techniques have advantages for producing vegetatively propagated plants. There are many researches as shoot and root tip culture 7-17, stem and leaf culture 18-23, embryo culture 24, 25, seed culture 26, embryogenic callus culture 27 and anther culture 28 have been reported for Allium species. Shoot culture is mainly used and preferred in Allium sativum for producing virus free plants. Also plant tissue culture has become a powerful tool to develop micropropagation methods for Tunceli garlic. In micropropagation

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of Allium spp., regeneration and multiplication of plants and bulbils are affected by explant types, combination of plant growth regulators and composition of culture medium. There are so many studies on Allium sativum by using in vitro techniques, but only two studies carried out on national garlic cultivars and Tunceli garlic 9, 29. In addition, there is no published result to use these methods for commercial production of Tunceli garlic. Therefore, this research is conducted with the aim of the determination and adaptation of rapid micropropagation technique for Tunceli garlic via in vitro root and shoot culture. In this paper, we describe a novel micropropagation method for Allium tuncelianum that result in plant propagation via shoot culture. Materials and Methods Plant material: Tunceli garlic cloves were collected from Munzur Mountain at Tunceli in the eastern part of Turkey and cultivated in the collection garden at Ankara University, Faculty of Agriculture, and Department of Horticulture. Garlic heads of 2-2.5 cm diameter were used as a source of explant 12. At the beginning of this research, a lot of explants were cancelled out because of the infection. For this reason, a series of sterilization experiments were also employed. According to the results, twice sterilization treatments were found effective. Bulbs with cover leaves were surface disinfected by soaking in 10% sodium hypochlorite solution for 10 min and rinsed with sterile water. After this treatment, heads were soaked in 70% ethyl alcohol for 30 s. Subsequently cover leaves were removed, materials were immersed to 25% sodium hypochlorite for 25 min and rinsed in three times with sterile water. Methods were developed for the rapid micropropagation of Allium tuncelianum through direct regeneration from root and shoot explants and in vitro bulblet formation. Shoot and root tips were placed on MS 30 medium supplemented with 30 g l-1 sucrose, 6 g l-1 agar with pH adjusted to 5.6, and 250 mg l-1 penicillin and 250 mg l-1 streptomycin mixture was added to each medium. Root culture: Root tips were obtained from 18 days old plantlets which were grown at in vitro conditions. To determine the best combinations of the growth regulators; 2,4-D and NAA (0, 1.0, 2.0 mg l-1) were used as auxin and BA (0, 0.1, 1.0 mg l-1) as cytokinin 31, 32 . Five Petri dishes were used for combinations. Four or five root tips were placed on each Petri dish and cultured at 251C under dark conditions. After four weeks, cultures were transferred to the 16/8 h (light/dark) photoperiod condition, which had fluorescent illumination 3000 lux 7. Shoot culture: Isolated shoot apexes, which were 1.0 cm in length and contained 1-2 leaf tasks, were placed aseptically on MS medium supplemented with different types of plant growth regulators such as IAA and NAA as auxin and BA as cytokinin presented in Table 1. In shoot culture treatments, 8 shoot apexes were used for each combination and every treatment replicated twice. After four weeks, shoots were transferred onto fresh proliferation medium, and cultures were subcultured in every two weeks. Multiplication rates were determined as number of shoot masses subcultured per multiple shoot. In vitro bulblet formation: Individual shoots were transferred to the MS medium containing NAA (0, 0.1 mg l-1) for bulblet formation. Numbers of bulblet per shoot mass were recorded after four weeks

Table 1. Plant growth regulator combinations for shoot culture.

1st experiment (mgl-1) BA NAA 0.00 0.00 0.05 0.10 0.10 0.50 IAA 0.00 0.10 0.50 2nd experiment (mgl-1) BA NAA 0.00 0.00 0.01 0.10 0.025 0.50 IAA 0.00 0.10 0.50

of in vitro bulblet formation. In this regard, shoots with bulblet were transferred to MS medium containing NAA (0, 0.5, 1.0, 2.0 mg l-1). Rooted shoots were planted to the pots containing turf, and the temperature was 25C except where stated. Light of 8000 lux was provided by white fluorescent tubes for 16 h daily. After four weeks, pots were transferred to the open field condition. Statistical analysis: Experiments were set up in a completely randomized way. Data on shoot formation frequency rate, average shoot number per explant, bulblet formation rate, number of rooted plants and rooting ratio were evaluated by SPSS statistical program and were determined different groups with Duncans test at p<0.01 level. Results and Discussion Tunceli garlic (Allium tunceliannum) is an endemic plant for Turkey, which is under protection of government to preserve the natural sources and illegal harvesting. For preserving the natural sources, emerging techniques must be applied. Therefore, this study was designed to determine proper and an alternative in vitro micropropagation method for Tunceli garlic. In this designed method, the objective of experiments was to obtain uniform and vigorous shoots. For this aim, root and shoot culture techniques were applied. Disinfection procedure caused problem at the beginning of this study, because raw material of Tunceli garlic had a disinfection problem. To overcome this problem, a series of disinfection experiments were carried out. According to results, twice sterilization treatments were found effective for Tunceli garlic. Root culture: To determine the effective micropropagation method for Tunceli garlic firstly, root tip culture experiments were carried out. However, direct shoot formation wasnt observed significantly from root tip explants under any plant growth regulator combinations tested. Only callus formation was obtained in 0.1 mg l-1 BA + 1.0 mg l-1 NAA combination. On the other hand, stimulative effect on regeneration capacity of root explants was not determined by using root media containing different levels of 2,4-D. Therefore any results were not presented on 2,4-D in this paper. Shoot culture and bulblet formation: When the first shoot regeneration trials via root tip culture failed, the experiments were focused on the shoot regeneration via shoot culture. In this regard, different plant growth regulators and subculture periods were examined, and obtained data on induced shoot regeneration in Tunceli garlic via shoot culture experiments are presented in Tables 2 and 3. As could be seen in Table 2, lower doses of auxins and cytokinin were equally good in induced shoot regeneration while the use of higher doses of auxins and cytokinin was less effective. Meanly one shoot was obtained per explant at 0.1 mg l-1 BA+0.5 mg l-1 IAA, 0.1 mg l-1 BA+ 0.1 mg l-1 IAA, 0.0 mg l-1 NAA+ 0.1 mg l-1 BA and 0.0 mg l-1 IAA+0.1 mg l-1 BA combinations. Proliferation 573

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frequency increased with the use of lower concentration of IAA and BA. According to our observations, higher doses of NAA application had no stimulative effect on shoot formation ratio, and statistically there wasnt any difference between the treatments. However, multiple shoots were not produced during the culture period of initial shoot culture. There was a statistical difference (p<0.01) value between the 0.1 BA mg l-1+0.5 mg l-1 NAA and 0.1 mg l-1 BA+ 0.0 mg l-1 NAA combinations on shoot formation frequency (Table 2). On the other hand, 0.1 mg l-1 BA and 0.5 mg l-1 BA treatments had no particular effect on it. When the plant growth regulators were investigated in detailed, it had another important influence on induction of multiple shoot formation from shoot explants. Multiple shoot formation ratio at shoot culture experiments was highest at 0.5 mg l-1 IAA+0.1 mg l-1 BA combinations, 0.1 mg l-1 BA+ 0.0 mg l-1 NAA, 0.0 mg l-1 IAA+0.1 mg l-1 BA, and 0.1 mg l-1 IAA+ 0.1 mg l-1 BA combinations followed it relatively (Table 2). The difference on multiple shoot proliferation was significant (p<0.01) between 0.5 mg l-1 BA+0.1 mg l-1 IAA and 0.5 mg l-1 IAA+ 0.1 mg l-1 BA combinations. The same result was also noted both for 0.0 mg l-1 NAA+0.1 mg l-1 BA and 0.1 mg l-1 NAA+0.1 mg l-1 BA treatments. The difference was statistically significant (p<0.01) on multiple shoot formation, and the data indicated that IAA treatment gave better response than NAA application. Another step of this research was to determine the effects of different subculture periods on shoot formation. For this aim, four subculture periods were examined and obtained data on shoot regeneration in Tunceli garlic via shoot culture experiments are presented in Table 3. According to results, there was a negative correlation between subculture time and shoot regeneration ratio. The results showed that the increasing subculture number promoted the growth of shoots. Regarding to visual observations, at first subculture 0.05

mg l-1 BA dose gave the highest shoot proliferation ratio and also lower dose of IAA (0.05 mg l-1) application was effective on it. This treatment was statistically significant (p<0.01) on shoot formation frequency. Conversely at 0.1 mg l-1 BA+0.1 mg l-1 IAA combination had the lowest regeneration effect at all subculture periods. In NAA free medias the rate was found as high as in all subcultures consequently NAA had no influence on shoot formation ability. On the contrary NAA had a drastic effect on bulblet formation. In medium containing NAA, bulblet formation period was found to be six weeks. In addition to these experiments, a new set of experiment was carried out to determine the bulblet formation under the lower doses of IAA (0.025 mg l-1) and BA (0.025 mg l-1), but any significant result was not obtained. Efficiency of bulblet formation from shoot explants was also influenced by subculture times. Subculture number relatively had an important effect on bulblet formation. This allowed in vitro bulblet formation from individual shoot at second subculture, so that bulblet formation ratio was increased by rising subculture times. According to Table 4, bulblet formation rate was 100% in medium absence of auxin and also 0.1 mg l-1 NAA (80%) treatment. Shoots did not form bulblet placed in both IAA and BA combinations. At fourth subculture IAA application gave best result on bulblet formation, but the quality of bulblets was poor. There was a difference between the NAA treatments on bulblet formation determined by statistical analysis. Bulblet initiation started after second subculture in cytokinin free medium containing 0.1 mg l-1 NAA. This result was in accordance with the results of Nagakuba et al. 33 and Roksana et al. 16 in cultivated garlic. Although bulblet formation ratio was low (30%), shoots did not form bulblet placed on lower doses (0.1 mg l-1) of NAA or auxin free medium. All of the shoots formed bulblet after one month, 37% of shoots started the rooting in auxin free medium and 40% of them were rooted in 0.1 mg l -1 NAA concentration at fourth subculture cycle. Rooting ratio was found to be low in this medium. This ratio increased Table 2. Effects of plant growth regulators on shoot and multiple shoot formation when unrooted shoots were transferred to ratio for Tunceli garlic (*p <0.01). the medium supplemented with higher doses BA (mgl-1) 0 0.1 0.5 IAA (mgl-1) 0 0.1 0.5 0 0.1 0.5 0 0.1 0.5 of NAA (2.0 mg l-1) at second subculture. SN 4 1 8 16 17 26 6 11 7 The highest rooting ratio was 33% in 2.0 SR% 0 0 38 abc 56 ab *76 a 65 ab 67 ab 27cd 29cd mg l-1 NAA and 0.5 mg l-1 NAA treatment MN 0 0 3 15 16 26 4 3 2 (17%) followed it. When rooted and unrooted MMSN 0 0 0.38bc *0.94a *0.94a *1.0a 0.66ab 0.27cd 0.29cd garlic bulblets were transferred to pots, NAA (mgl-1) 0 0.1 0.5 0 0.1 0.5 0 0.1 0.5 SN 4 5 4 16 11 5 6 9 7 plants died after second or third leaves were SR% 0 0 2cd 56 ab 36 abc 20 de 67 ab 67 ab 12 de generated. However, these garlic plantlets MN 0 0 1 15 5 1 4 6 2 produced small bulblets. This was normal for MMSN 0 0 0.25cd *0.94a 0.45bc 0.20de 0.67ab 0.67ab 0.12de SN Shoot number, SR Shoot formation ratio, MN Multiple shoot number, MMSN Mean multiple shoot number. Tunceli garlic 23. Table 3. Effects of subculture numbers on multiple shoot formation (*p < 0.01) and results of Duncans test.
(mgl-1) BA 0.0 IAA 0.1 NAA 0.5 NAA 0.1 IAA 0.5 IAA SN MSR% SN MSR% SN SR% SN MSR% SN MSR% 1st subculture 0.0 0.05 0.1 0 9 4 0 56 ab 67 ab 0 4 6 0 36 bc 67 ab 1 1 2 25 cd 20 de 12 de 0 13 3 0 *76 a 27 cd 3 17 2 38 bc 65 ab 29 cd 2nd subculture 0.0 0.05 0.1 0 4 1 0 57 ab 17 de 0 2 2 0 33 bc 25 cd 1 0 1 17 de 0 17 de 0 2 2 0 33 bc 25 cd 1 2 1 14 de 29 cd 14 de 3rd subculture 0.0 0.05 0.1 0 0 1 0 0 20 de 0 0 1 0 0 13 de 0 0 1 0 0 17 de 0 4 1 0 33 bc 10 de 0 7 0 0 44 ab 0 4th subculture 0.0 0.05 0.1 0 2 0 0 13 de 0 0 0 1 0 0 11 de 0 0 0 0 0 0 0 4 0 0 24 de 0 0 3 0 0 12 de 0

SN Shoot number, MSR Multiple shoot formation ratio.

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Table 4. The results of bulblet formation from shoot explants at different subculture stages (*p<0.01) and results of Duncans test.
(mgl-1) BA 0.0 NAA 0.1 NAA 0.5 NAA 0.1 IAA 0.1 IAA BN BF% BN BF% BN BF% BN BF% BN BF% 1st subculture 0.0 0.05 0.1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2nd subculture 0.0 0.05 0.1 4 0 0 *100 a 0 0 3 0 0 *60 b 0 0 0 0 2 0 0 33 cd 0 0 0 0 0 0 0 0 0 0 0 0 3rd subculture 0.0 0.05 0.1 0 0 0 0 0 0 1 0 0 20 d 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 4th subculture 0.0 0.05 0.1 0 0 3 0 0 50 bc 0 5 0 0 45 bc 0 2 2 3 50 bc 40 bc 29 d 0 2 5 0 12 ef 45 bc 4 3 2 50 bc 13 ef 14 ef 0.0 100 a 80 ab 50 bc 0 0 Mean 0.05 0 45 bc 40 bc 12 ef 12 ef 0.1 50 bc 0 71 ab 45 bc 71 ab

BN Bulblet number, BF Bulblet formation rate.

The rooted or unrooted shoots were planted to the pots containing turf and placed to climate room, unfortunately plants died after producing two leaves. However, these plants produced small bulbils. These bulbils could be used as propagation material of Tunceli garlic 4. According to field observations, over wintered garlic bulbils had a potential to produce bigger bulbs. This result was very important for cultivation of this species, because after in vitro micropropagation step adaptation of in vitro plants, their growth ability, adaptation capacity and survival rate have become more significant for the commercial production. Conclusions Classical propagation methods of Tunceli garlic has been carried out by bulbs, bulbils, bulblets and seed. However, the propagation rate and number of plantlets produced were not so high for practical propagation. We report here that Tunceli garlic was propagated by shoot culture and in vitro bulblet formation. The data obtained through this research showed that lower auxin and cytokinin concentrations were effective for shoot induction. In medium, 0.1 mg l-1 BA and 0.1 mg l-1 IAA were sufficient doses for Tunceli garlic. These findings were similar to Glen et al. 9 and Garcia et al. 14. According to results, reproduction capacity of Tunceli garlic was not as high as that of cultivated garlic Allium sativum. Although, generally only one shoot was obtained per explant, but in some combinations 2-3 shoots could be seen. Two types of explants were used in each individual experiment, and the results showed that explant type highly affected the response on shoot formation. Shoot explants gave the best result in this study and this result was the same with the results of Nagakuba et al. 33 and Chen et al. 34. When shoots which formed bulblet were transferred to the rooting medium that contained different doses of NAA (0.0, 0.5, 1.0, 2.0 mg l-1), rooting ratio was not as high as expected. In this research, when in vitro plants were transferred to field condition, over wintered Tunceli garlic bulbils had a potential to produce bigger bulbs than unwintered materials. These bulbils had a significant effect on uniform plant formation. For this reason, this finding was important for commercial production. The present study was significant in showing proper micropropagation method for our native species of Tunceli vicinity. At the end of this research, it was determined that Tunceli garlic could be propagated by the use of lower doses of plant growth regulators such of IAA and BA (0.1 mg l-1, 0.1 mg l-1) in vitro shoot culture.

Acknowledgements The authors acknowledge Ankara University, Biotechnology Institute and Turkish Atomic Energy Authority for financial assistance through the project coded V-E.03.TAEK.2.09. We thank Dr. Omer Kantoglu for English review of this paper.

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