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ISSN : 1598-5504(Print)
ISSN : 2383-8272(Online)
Journal of Agriculture & Life Science Vol.52 No.2 pp.73-80
DOI : https://doi.org/10.14397/jals.2018.52.2.73

in vitro Culture and Shoot Proliferation of Wild Garlic(Allium ochotense Prokh.)

Cheul Ho Lee, Mi Jin Jeong*
Plant Conservation Division, Korea National Arboretum, Pocheon, 11186, Korea
Corresponding author: Mi Jin Jeong Tel: +82-31-540-1054 Fax: +82-31-540-1060mjjeong121@korea.kr
February 25, 2018 March 26, 2018 March 26, 2018

Abstract


An efficient method for in vitro propagation and growth of the wild garlic(Allium ochotense Prokh.) was established. Bulbs of wild garlic were collected from Ullung Island, Korea, and the growth pattern of plantlets on various culture media was observed. High growth of shoot was obtained on LP, NN and N6 medium. The growth media supplemented with 3%(w/v) sucrose was found optimal for shoot growth. After 10 weeks multiple shoots were observed in the plantlets growing on the medium containing 1.0 mg/l of zeatine and 0.1 mg/l NAA. Roots were induced directly at the base of the shoot in all treatments. The medium with 2.0 mg/l of IBA proved to be the best rooting medium. The studies of this kind may be used to develop strategies for large-scale propagation of elite wild garlic.



초록


    Introduction

    Allium ochotense Prokh.(Liliaceae) commonly known as wild galric, great onion or wild onion, is distributed mainly in northern hemisphere, Asia including Korea, China, and Europe. Their leaves and bulbs have been used not only as wild-edible herbs but also as functional foods for the control of gastritis and heart failures(Moon, 1984). It was reported that the leaves contains 2-3% of carbohydrate and ascorbic acid whereas the bulbs possesses organosulfuric compounds. This species has the activities of anti-cancer(Lee et al., 2001), ani-oxidant(Shirataki et al., 2001), and antiatherogenic(Kim et al., 2000).

    The propagation of wild garlic is difficult, and its cultivation is not advanced. Shigeru et al.(2003) examined the cultivation of the plants with seed propagation and vegetative propagation(tillering, adventive bud) using seeds. However, propagation of wild garlic is very difficult due to its low germination capacity and low efficiency of seed and rootstock sprouting. Conventional method like use of seed bulb is the only way for cultivation of wild garlic. For each plant one seed bulb is needed. The lack of availability of seed bulbs is the limitation for its large scale propagation.

    Micropropagation has been evaluated as an alternative to conventional vegetative propagation by several previous research groups(Ishikawa et al., 1997;Park et al., 2004). However, the reports on the rapid propagation of wild garlic through in vitro methods are still inadequate.

    In micropropagation of Allium spp., regeneration and multiplication of plantlets is affected by composition of the culture medium, combination of growth regulators used and the kind of explants(Novak, 1990). Thus, this study has been conducted to determine efficient method in vitro propagation of wild garlic.

    Materials and methods

    1. Plant material and in vitro culture

    Healthy 2-year-old wild garlic was collected from Ullung Island, Korea. After the detachment of leaves, garlic bulbs were washed with 0.05% Tween-20 for 10 min, rinsed three times with distilled water, and sterilized with ethanol(70% v/v) for about 3 min. Bulbs were surface-disinfested with NaClO(6% v/v) for 10 min and rinsed five more times with sterile distilled water.

    The sterilized bulbs obtained from above steps were inoculated into culture bottle containing 50 ml of sterile MS(Murashige & Skoog, 1962) medium of pH 5.7, supplemented with sucrose(3% w/v) and gerlite(0.4% w/v). The cultures were incubated under a photoperiod of 16 h illumination with a light intensity of 25 μmol m−2 s−1 and 8 h dark at 25±1℃.

    2. Determination of optimal culture media

    The plantlets were cultured on several basal media such as MS, LP(Quoirin & Lepoivre, 1977), WPM (Lloyed & McCown, 1981), SH(Schenk & Hildebrandt, 1972), NN(Nitsch & Nitsch, 1969), LS(Linsmaier & Skoog, 1965), B5(Gamborg et al., 1968) and White (White, 1963) medium in order to determine the ideal growth medium. All media chosen contained 3%(w/v) sucrose as carbon source and 0.4%(w/v) gelrite. In all cases, the medium was adjusted to pH of 5.7 before autoclaving at 121℃ for 15 min.

    The effect of three carbon sources on optimal shoot growth was investigated by placing the plantlet on LP solid medium supplemented with 1, 3 and 5%(w/v) of sucrose, glucose or fructose. The plantlets were incubated as above.

    The shoot growth and growth of bulblet were measured 7-day-intervals for 5 weeks. The growth rate of shoots was represented by growth index(GI) which is calculated using the equation;

    GI=(final shoot length-inoculated shoot length) / inoculated shoot length

    3. Shoot proliferation through shoot tip

    For explants establishment, shoot tips of 1 cm length were transferred to LP medium supplemented with 0.1 mg/l NAA(Sigma, St. Louis, MO, USA) and two cytokinins(Zeatine and BAP, Sigma, St. Louis, MO, USA) 30 g/l sucrose, and 4.0 g/l gerlite for shoot induction. Individual shoots were cut from the multiple shoot clusters proliferated through the culturing on the basal LP solid medium without any plant growth regulators for 5 weeks. All cultures were maintained under a 16 h light/8 h dark photoperiod, in a growth chamber fitted with a cool fluorescent light emitting 25μmol m-2 s-1 of photosynthetically active radiation(PAR).

    4. Shoot elongation and in vitro rooting

    In vitro propagated shoots were collected and transferred to LP basal medium supplemented with 3%(w/v) sucrose. The shoot elongation was carried out on LP medium containing 3%(w/v) sucrose without growth regulators for eight weeks of culture. The experimental data were collected after 4 and 8 weeks of culturing.

    The in vitro rooting of the shoots carried out with LP medium supplemented various concentrations of two-auxins; IAA(0.5, 1.0, 2.0 and 5.0 mg/l) and IBA(0.5, 1.0, 2.0 and 5.0 mg/l). The elongated shoots were cut(3 cm long) and inoculated into test-tube containing 10 mL solid culture media. The root numbers and growth were counted after 4 weeks of culture under 3000 lux light condition.

    5. Acclimatization of in vitro plantlets

    Plantlets with roots transferred from the culture vessels, washed gently under running tap water and transplanted to pots containing autoclaved peat moss, sand and vermiculite mixture(1:1:1, w/w/w). The pots were covered with polythene bags. The plants were irrigated daily with tap water. The pots were placed in a growth chamber maintained at 25±1℃ with fluorescent lighting for 4 weeks before they were transferred to soil in the greenhouse. Survival rate was measured for 5 months of acclimatization.

    6. Statistical analysis

    The experiments were repeated for a minimum of five times. Data was subjected to statistical analysis by using SAS for Windows Version 6.12.

    Results and discussion

    1. Effect of culture media on shoot growth

    The shoot growth of A. ochotense was different significantly based on various culture media(Fig. 1). In our experiments, we observed that growth of wild garlic plantlets was very slow than garlic plant growth. Among seven basal media used in this study, higher growth of shoot was obtained on LP, NN and, N6 medium, while SH, White, 1/2MS, B5 and MS medium were poor for shoot growth. In special, LP medium yielded a higher growth rate(GI=0.6) than those of the others. The major differences in macronutrients among these media are in ammonium and nitrate ion concentrations and total ion concentration. Full-strength MS is high in ammonium(20.6 mM) and nitrate ions(39.4 mM), while LP is a low ammonium medium(5 mM). In addition, LP uses calcium nitrate as a nitrogen source. LP medium culture has also significantly affected the length of the sprouts and this after two successive subcultures of wild cherry trees(Mansseri-Lamrioui et al., 2009). Wang(1991) observed a higher degree of multiple shoot formation of the P. communis L. rootstock BP10030 on WPM and LP than MS in a double-phase culture system consisting of a liquid medium overlaid on semisolid medium.

    2. Effect of various carbon sources on plant growth

    Carbone sources in a culture influenced shoot and bulb growth(Fig. 2). The results show that the higher sucrose concentration in LP medium contributed to increased shoot growth. Among various sucrose concentrations, the highest growth was appeared from the 3%(w/v) and the lowest growth was obtained from the 1%(w/v) sucrose. Culture media from the inclusion of glucose was showed little shoot growth. Fructose at a concentration of 1%(w/v) grew significantly higher than at any other high concentration tested. The effects of 3%(w/v) sucrose and 1%(w/v) fructose on the growth of bulb and leaf were investigated(Fig. 3). Shoot growth of the inclusion of 3%(w/v) sucrose was significantly expressed by the significant increase in bulb diameter, leaf length, and diameter than another. Medium supplemented with 3%(w/v) sucrose was found optimal for shoot growth; GI of shoot, bulb, and leaf was also higher. Sucrose is converted into glucose and fructose within a few days after the start of culture(Ono et al., 1998). Reducing sugars such as glucose and fructose are readily taken up by tissue acting as an alternative carbohydrate source(Katase, 1993). Sucrose is another important factor which induces garlic bulb formation in vitro. It was demonstrated that 12% sucrose promotes bulb induction in garlic (Nagakubo et al., 1993) and also in onion and leek(Keller, 1993).

    3. Shoot proliferation of wild garlic through shoot tip

    Adventitious shoot bud formation appeared through shoot-tip culture(Fig. 4). It is also noticed that no shoot proliferation was observed in medium without any growth regulators. Therefore, it is proved that the use of phytohormone is essential for shoot-root multiplication and proliferation. The shoot clumps which had well-developed roots and shoots were treated with different combinations of cytokinins (Zeatine and BAP) and auxin(NAA) for shoot multiplication(Fig. 4). The shoot clumps were subcultured after every 21 days interval. The multiplied shoots were observed after 10 weeks in medium containing 1.0 mg/l of zeatine and 0.1 mg/l NAA. In case of other media more subcultures (20 weeks of cultures) were needed for multiple shoot formation.

    The concentrations of exogenous cytokinin appear to be the main factor affecting shoot multiplication. Our experimental results indicated that zeatine concentration significantly influenced shoot multiplication and elongation. This result was in not agreement with that reported in garlic(Roksana et al., 2002) in which 0.5 mg/l 2ip+0.25 mg/l NAA was most effective among cytokinins in inducing bud break and shoot proliferation in the explants.

    In this study, the number of multiplication per explant is low compared to garlic. The highest multiplication rate of garlic was(135 bulblets/explant) found when explants were cultured in the bulbing medium. In this study, multiple shoots per explants firstly measured for 5 weeks of cultures. If multiplied shoot clump re-cultured on cytokinin containing a medium, numerous shoots are inducible. Also, multiple shoots induced from the selected line would more effective on shoot multiplication.

    4. In vitro rooting and acclimation

    Multiplied wild garlic shoots were cultured on LP medium containing 3%(w/v) sucrose without growth regulators. The shoot could be elongated after 4 weeks of culture.

    The shoots with about 1.5 cm long were excised and placed on the rooting medium. In the experiments on root induction, different concentrations of IBA and IAA influenced the time of root formation, bulb and shoot growth(Table 1). The culture of IBA treatment was better than IAA treatment in terms of the number of roots. Roots were induced directly at the base of the shoot at all treatments. 2.0 mg /l of IBA, proved to be the best rooting medium. However, the growth of shoot and bulb was not parallel with IBA and IAA concentrations(Table 1).

    These plantlets with roots were successfully transferred to pots for 4 weeks hardening process(Fig. 5A and B). The resultant plantlets were gradually decreased survival rates with more acclimatization period to reach 55% after 5 months(Fig. 5A). However, the growth of plantlet was gradually increased with acclimatization period(Fig. 5B). The growth of bulb was also increased with acclimatization period. These rooted plantlets were successfully transferred to pots for 5 weeks hardening process.

    In the present protocol, we have established a rapid in vitro propagation system of plantlets for wild garlic. This rapid propagation rate could only be achieved using micropropagation rather than the conventional in vivo propagation method. The regenerated plants grown in the net house do not exhibit noticeable morphological variations in comparison to donor plants. The studies of this kind may be used to develop strategies for large-scale propagation of elite wild garlic.

    Figure

    JALS-52-73_F1.gif

    Effect of culture media on the shoot growth of wild garlic. Shoot growth was compared among the eight basal culture media. All experiments were conducted in five replications.

    JALS-52-73_F2.gif

    Effect of carbohydrates on the shoot growth of wild garlic. Shoots were cultured on modified LP medium supplemented with various concentrations of sucrose(S), glucose(G) and fructose(F).

    JALS-52-73_F3.gif

    Effect of carbohydrates on the bulb and leaf growth of wild garlic. Shoots were cultured on modified LP medium supplemented with various concentrations of sucrose, glucose and fructose. (A) bulb diameter, (B) leaf length, (C) leaf diameter and (D) T/R ratio.

    JALS-52-73_F4.gif

    Shoot proliferation of wild garlic through shoot-tip. The developmental stages were cultured on LP medium supplemented with combined PRGs. (A) 0.1 mg/L zeatine and 0.1 mg/L BA during 6 weeks (bar=0.5 cm). (B) 1.0 mg/L zeatine and 0.1 mg/L NAA during 10 weeks(bar=0.5 cm). (C) during 12 weeks(bar=0.5 cm). (D) during 16 weeks(bar=1.0 cm). (E) 0.1 mg/L BA and 0.1 mg/L NAA within 20 weeks(bar=1.0 cm).

    JALS-52-73_F5.gif

    Survival and shoot growth in vitro cultivated shoot based on acclimatization periods. (A) Survival rate of plant based on acclimatization periods and (B) Growth of shoot and bulb based on acclimatization periods.

    Table

    Effects of IAA and IBA concentration on root induction, shoot and bulb growth

    Shoots were cultured on LP medium supplemented with various concentrations of IAA and IBA, and 3% sucrose. All experiments were conducted in five replications. Value represents the mean±SE. Means following the same letter within columns are not significantly different, according to Duncan’s multiple range(p≤0.05) test.

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